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Sample records for distemper virus detection

  1. Detection of Arctic and European cluster of canine distemper virus in north and center of Iran.

    PubMed

    Namroodi, Somayeh; Rostami, Amir; Majidzadeh-Ardebili, Keyvan; Ghalyanchi Langroudi, Arash; Morovvati, Abbas

    2015-01-01

    Canine distemper virus (CDV) creates a very contagious viral multi-systemic canine distemper (CD) disease that affects most species of Carnivora order. The virus is genetically heterogeneous, particularly in section of the hemagglutinin (H) gene. Sequence analysis of the H gene can be useful to investigate distinction of various lineages related to geographical distribution and CDV molecular epidemiology. Since vaccination program is conducted only in large cities of Iran, CD still remains as one of the major causes of death in dogs in this country. In order to monitor H gene, CDV has been detected in 14 out of 19 sampled dogs through the amplification of nucleoprotein (NP) gene in nested-PCR assay. In the next step 665 bp of H gene was amplified in 9 out of 14 NP-gene positive dogs. Phylogenetic analysis distinguished two distinct CDV genotypes in Iran. JN941238 has been embedded in European cluster and JN941239 has been embedded in Arctic cluster. Nucleic analysis has been shown high difference among both Iranian CDV lineages with CDV vaccine strains. PMID:26893808

  2. Detection of Arctic and European cluster of canine distemper virus in north and center of Iran

    PubMed Central

    Namroodi, Somayeh; Rostami, Amir; Majidzadeh-Ardebili, Keyvan; Ghalyanchi Langroudi, Arash; Morovvati, Abbas

    2015-01-01

    Canine distemper virus (CDV) creates a very contagious viral multi-systemic canine distemper (CD) disease that affects most species of Carnivora order. The virus is genetically heterogeneous, particularly in section of the hemagglutinin (H) gene. Sequence analysis of the H gene can be useful to investigate distinction of various lineages related to geographical distribution and CDV molecular epidemiology. Since vaccination program is conducted only in large cities of Iran, CD still remains as one of the major causes of death in dogs in this country. In order to monitor H gene, CDV has been detected in 14 out of 19 sampled dogs through the amplification of nucleoprotein (NP) gene in nested-PCR assay. In the next step 665 bp of H gene was amplified in 9 out of 14 NP-gene positive dogs. Phylogenetic analysis distinguished two distinct CDV genotypes in Iran. JN941238 has been embedded in European cluster and JN941239 has been embedded in Arctic cluster. Nucleic analysis has been shown high difference among both Iranian CDV lineages with CDV vaccine strains. PMID:26893808

  3. Sandwich-dot enzyme-linked immunosorbent assay for the detection of canine distemper virus

    PubMed Central

    Li, Zhi; Zhang, Yanlong; Wang, Huiguo; Jin, Jinhua; Li, Wenzhe

    2013-01-01

    A sandwich-dot enzyme-linked immunosorbent assay (dot ELISA) was developed for the detection of canine distemper virus (CDV). In 56 dogs suspected to have CD the rates of detection of CDV antigen in samples of blood lymphocytes and palpebral conjunctiva by dot ELISA and ELISA were, respectively, 91% (49/54) and 81% (44/54) for the lymphocyte samples and 88% (28/32) and 75% (24/32) for the conjunctival samples. The CDV detection limits were 10 ng/50 μL for dot ELISA and 40 ng/50 μL for ELISA. The reliability of dot ELISA relative to electron microscopy was 96% with 22 samples: all 21 samples in which CDV particles were observed by electron microscopy yielded positive results with dot ELISA; the single sample in which particles were not observed yielded false-positive results with dot ELISA. The results indicate that the dot ELISA developed can serve as a reliable rapid diagnostic test in suspected cases of CD and also be useful for epidemiologic surveillance of the disease. PMID:24124274

  4. Detection and differentiation of wild-type and vaccine strains of canine distemper virus by a duplex reverse transcription polymerase chain reaction.

    PubMed

    Dong, X Y; Li, W H; Zhu, J L; Liu, W J; Zhao, M Q; Luo, Y W; Chen, J D

    2015-01-01

    Canine distemper virus (CDV) is the cause of canine distemper (CD) which is a severe and highly contagious disease in dogs. In the present study, a duplex reverse transcription polymerase chain reaction (RT-PCR) method was developed for the detection and differentiation of wild-type and vaccine strains of CDV. Four primers were designed to detect and discriminate the two viruses by generating 638- and 781-bp cDNA products, respectively. Furthermore, the duplex RT-PCR method was used to detect 67 field samples suspected of CD from Guangdong province in China. Results showed that, 33 samples were to be wild-type-like. The duplex RT-PCR method exhibited high specificity and sensitivity which could be used to effectively detect and differentiate wild-type and vaccine CDV, indicating its use for clinical detection and epidemiological surveillance. PMID:27175171

  5. Detection and differentiation of wild-type and vaccine strains of canine distemper virus by a duplex reverse transcription polymerase chain reaction

    PubMed Central

    Dong, X. Y.; Li, W. H.; Zhu, J. L.; Liu, W. J.; Zhao, M. Q.; Luo, Y. W.; Chen, J. D.

    2015-01-01

    Canine distemper virus (CDV) is the cause of canine distemper (CD) which is a severe and highly contagious disease in dogs. In the present study, a duplex reverse transcription polymerase chain reaction (RT-PCR) method was developed for the detection and differentiation of wild-type and vaccine strains of CDV. Four primers were designed to detect and discriminate the two viruses by generating 638- and 781-bp cDNA products, respectively. Furthermore, the duplex RT-PCR method was used to detect 67 field samples suspected of CD from Guangdong province in China. Results showed that, 33 samples were to be wild-type-like. The duplex RT-PCR method exhibited high specificity and sensitivity which could be used to effectively detect and differentiate wild-type and vaccine CDV, indicating its use for clinical detection and epidemiological surveillance. PMID:27175171

  6. Retrospective differentiation of canine distemper virus and phocine distemper virus in phocids.

    PubMed

    Stanton, James B; Brown, Corrie C; Poet, Steven; Lipscomb, Thomas P; Saliki, Jeremiah; Frasca, Salvatore

    2004-01-01

    Formalin-fixed paraffin-embedded tissues from one Caspian seal (Phoca caspica), one harp seal (Phoca groenlandica), one hooded seal (Cystophora cristata), and one harbor seal (Phoca vitulina vitulina) were used to compare the utility of immunohistochemistry (IHC) versus that of a novel seminested reverse transcriptase polymerase chain reaction (RT-PCR) to detect and differentiate canine distemper virus (CDV) and phocine distemper virus (PDV). Four antibodies made against PDV were able to detect both viruses. Two antibodies made against cetacean morbillivirus (CMV) did not label antigens from either CDV or PDV. A third anti-CMV antibody inconsistently stained CDV antigens but did not label PDV antigens. The seminested RT-PCR was able to detect RNA of the phosphoprotein gene in all positive cases. Nucleotide sequence analyses of seminested RT-PCR products were used to differentiate CDV RNA from PDV RNA. From these data, it was determined that IHC using antibodies generated against PDV provided a rapid means of detection for both CDV and PDV antigens; however, differentiation between CDV and PDV was achieved only with the RT-PCR assay. PMID:15137488

  7. Phylogenetic analysis of the haemagglutinin gene of canine distemper virus strains detected from giant panda and raccoon dogs in China

    PubMed Central

    2013-01-01

    Background Canine distemper virus (CDV) infects a variety of carnivores, including wild and domestic Canidae. In this study, we sequenced and phylogenetic analyses of the hemagglutinin (H) genes from eight canine distemper virus (CDV) isolates obtained from seven raccoon dogs (Nyctereutes procyonoides) and a giant panda (Ailuropoda melanoleuca) in China. Results Phylogenetic analysis of the partial hemagglutinin gene sequences showed close clustering for geographic lineages, clearly distinct from vaccine strains and other wild-type foreign CDV strains, all the CDV strains were characterized as Asia-1 genotype and were highly similar to each other (91.5-99.8% nt and 94.4-99.8% aa). The giant panda and raccoon dogs all were 549Y on the HA protein in this study, irrespective of the host species. Conclusions These findings enhance our knowledge of the genetic characteristics of Chinese CDV isolates, and may facilitate the development of effective strategies for monitoring and controlling CDV for wild canids and non-cainds in China. PMID:23566727

  8. Establishment of reverse transcription loop-mediated isothermal amplification for rapid detection and differentiation of canine distemper virus infected and vaccinated animals.

    PubMed

    Liu, Da-Fei; Liu, Chun-Guo; Tian, Jin; Jiang, Yi-Tong; Zhang, Xiao-Zhan; Chai, Hong-Liang; Yang, Tian-Kuo; Yin, Xiu-Chen; Zhang, Hong-Ying; Liu, Ming; Hua, Yu-Ping; Qu, Lian-Dong

    2015-06-01

    Although widespread vaccination against canine distemper virus (CDV) has been conducted for many decades, several canine distemper outbreaks in vaccinated animals have been reported frequently. In order to detect and differentiate the wild-type and vaccine strains of the CDV from the vaccinated animals, a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was developed. A set of four primers-two internal and two external-were designed to target the H gene for the specific detection of wild-type CDV variants. The CDV-H RT-LAMP assay rapidly amplified the target gene, within 60 min, using a water bath held at a constant temperature of 65°C. The assay was 100-fold more sensitive than conventional RT-PCR, with a detection limit of 10(-1)TCID50ml(-1). The system showed a preference for wild-type CDV, and exhibited less sensitivity to canine parvovirus, canine adenovirus type 1 and type 2, canine coronavirus, and canine parainfluenza virus. The assay was validated using 102 clinical samples obtained from vaccinated dog farms, and the results were comparable to a multiplex nested RT-PCR assay. The specific CDV-H RT-LAMP assay provides a simple, rapid, and sensitive tool for the detection of canines infected with wild-type CDV from canines vaccinated with attenuated vaccine. PMID:25769803

  9. Immunohistochemical detection of autophagy-related microtubule-associated protein 1 light chain 3 (LC3) in the cerebellums of dogs naturally infected with canine distemper virus.

    PubMed

    Kabak, Y B; Sozmen, M; Yarim, M; Guvenc, T; Karayigit, M O; Gulbahar, M Y

    2015-01-01

    We investigated the expression of microtubule-associated protein 1 light chain 3 (LC3) protein in the cerebellums of dogs infected with canine distemper virus (CDV) using immunohistochemistry to detect autophagy. The cerebellums of 20 dogs infected with CDV were used. Specimens showing demyelination of white matter were considered to have an acute infection, whereas specimens showing signs of severe perivascular cuffing and demyelination of white matter were classified as having chronic CDV. Cerebellar sections were immunostained with CDV and LC3 antibodies. The cytoplasm of Purkinje cells, granular layer cells, motor neurons in large cerebellar ganglia and some neurons in white matter were positive for the LC3 antibody in both the control and CDV-infected dogs. In the infected cerebellums, however, white matter was immunostained more intensely, particularly the neurons and gemistocytic astrocytes in the demyelinated areas, compared to controls. Autophagy also was demonstrated in CDV-positive cells using double immunofluorescence staining. Our findings indicate that increased autophagy in the cerebellum of dogs naturally infected with CDV may play a role in transferring the virus from cell to cell. PMID:26179070

  10. Detection of IgM antibodies against a recombinant nucleocapsid protein of canine distemper virus in dog sera using a dot-blot assay.

    PubMed

    Barben, G; Stettler, M; Jaggy, A; Vandevelde, M; Zurbriggen, A

    1999-03-01

    A dot-blot assay for the detection of IgM antibodies (ABs) against canine distemper virus (CDV) in canine serum is described. The diagnostic potential of this technique was evaluated by analysing sera from three test groups: (i) specific pathogen-free (SPF) beagle dogs experimentally infected with virulent CDV; (ii) SPF dogs immunized with a combined vaccine containing CDV, and (iii) SPF dogs immunized with a CDV-free vaccine. As antigen for the dot-blot assay we used the recombinant nucleocapsid protein (N protein) of the virulent A75/17 CDV strain. All 12 dogs of group 1, infected with virulent CDV, showed detectable CDV-specific IgM levels in their serum. All dogs of group 2 were also positive for anti-CDV IgM after the first immunization with the CDV-containing vaccine. The four dogs immunized with a CDV-free vaccine (group iii) remained negative throughout the course of the experiment. From these results, we conclude that the IgM detection test, which requires only a single serum sample, is a useful method for diagnosing current or recent CDV infection in CDV-infected or CDV-immunized dogs under experimental conditions. PMID:10216448

  11. Detection by RT-PCR and genetic characterization of canine distemper virus from vaccinated and non-vaccinated dogs in Argentina.

    PubMed

    Calderon, Marina Gallo; Remorini, Patricia; Periolo, Osvaldo; Iglesias, Marcela; Mattion, Nora; La Torre, José

    2007-12-15

    RT-PCR was used to detect canine distemper virus (CDV) RNA in clotted blood from Argentine domestic dogs. The NP gene was detected in 73 out of 99 blood samples analyzed. The deduced amino acid sequence of these gene fragments showed 100% identity with the sequence of other wild-type and vaccine strains. A fragment of the hemagglutinin gene was amplified from 24 (32.9%) of the NP-RNA-positive clinical specimens. These H fragments were further analyzed by restriction fragment length polymorphism (RFLP) and sequencing. A single NdeI site was detected in all 24 wild-type strains but was absent in the vaccine strains. Phylogenetic analysis of the partial hemagglutinin amino acid sequences showed close clustering for local strains, clearly distinct from vaccine strains and other wild-type foreign CDV strains. One of the local strains, Arg 23, branched out of the root of the Argentine clade, close to the European strains, suggesting that two different pathogenic CDV genotypes are currently circulating in Argentina, one of them clearly predominant. PMID:17628358

  12. Recombinant canine distemper virus serves as bivalent live vaccine against rabies and canine distemper.

    PubMed

    Wang, Xijun; Feng, Na; Ge, Jinying; Shuai, Lei; Peng, Liyan; Gao, Yuwei; Yang, Songtao; Xia, Xianzhu; Bu, Zhigao

    2012-07-20

    Effective, safe, and affordable rabies vaccines are still being sought. Attenuated live vaccine has been widely used to protect carnivores from canine distemper. In this study, we generated a recombinant canine distemper virus (CDV) vaccine strain, rCDV-RVG, expressing the rabies virus glycoprotein (RVG) by using reverse genetics. The recombinant virus rCDV-RVG retained growth properties similar to those of vector CDV in Vero cell culture. Animal studies demonstrated that rCDV-RVG was safe in mice and dogs. Mice inoculated intracerebrally or intramuscularly with rCDV-RVG showed no apparent signs of disease and developed a strong rabies virus (RABV) neutralizing antibody response, which completely protected mice from challenge with a lethal dose of street virus. Canine studies showed that vaccination with rCDV-RVG induced strong and long-lasting virus neutralizing antibody responses to RABV and CDV. This is the first study demonstrating that recombinant CDV has the potential to serve as bivalent live vaccine against rabies and canine distemper in animals. PMID:22698451

  13. Recombinant rabies virus expressing the H protein of canine distemper virus protects dogs from the lethal distemper challenge.

    PubMed

    Wang, Feng-Xue; Zhang, Shu-Qin; Zhu, Hong-Wei; Yang, Yong; Sun, Na; Tan, Bin; Li, Zhen-Guang; Cheng, Shi-Peng; Fu, Zhen F; Wen, Yong-Jun

    2014-12-01

    The rabies virus (RV) vector LBNSE expressing foreign antigens have shown considerable promise as vaccines against viral and bacteria diseases, which is effective and safe. We produced a new RV-based vaccine vehicle expressing 1.824 kb hemagglutinin (H) gene of the canine distemper virus (CDV) by reverse genetics technology. The recombinant virus LBNSE-CDV-H retained growth properties similar to those of vector LBNSE both in BSR and mNA cell culture. The H gene of CDV was expressed and detected by immunostaining. To compare the immunogenicity of LBNSE-CDV-H, dogs were immunized with each of these recombinant viruses by intramuscular (i.m.). The dogs were bled at third weeks after the immunization for the measurement of virus neutralizing antibody (VNA) and then challenged with virulent virus (ZJ 7) at fourth weeks. The parent virus (LBNSE) without expression of any foreign molecules was included for comparison. Dogs inoculated with LBNSE-CDV-H showed no any signs of disease and exhibited seroconversion against both RV and CDV H protein. The LBNSE-CDV-H did not cause disease in dogs and conferred protection from challenge with a lethal wild type CDV strain, demonstrating its potential value for wildlife conservation efforts. Together, these studies suggest that recombinant RV expressing H protein from CDV stimulated high levels of adaptive immune responses (VNA), and protected all dogs challenge infection. PMID:25465178

  14. 9 CFR 113.201 - Canine Distemper Vaccine, Killed Virus.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... distemper to determine susceptibility. A constant virus-varying serum neutralization test in cell culture... neutralization at a 1:2 final serum dilution. (i) The 20 dogs used as vaccinates shall be injected with one dose... neutralization test. Bulk or final container samples of completed product shall be tested for potency using...

  15. 9 CFR 113.201 - Canine Distemper Vaccine, Killed Virus.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... distemper to determine susceptibility. A constant virus-varying serum neutralization test in cell culture... neutralization at a 1:2 final serum dilution. (i) The 20 dogs used as vaccinates shall be injected with one dose... neutralization test. Bulk or final container samples of completed product shall be tested for potency using...

  16. 9 CFR 113.201 - Canine Distemper Vaccine, Killed Virus.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... distemper to determine susceptibility. A constant virus-varying serum neutralization test in cell culture... neutralization at a 1:2 final serum dilution. (i) The 20 dogs used as vaccinates shall be injected with one dose... neutralization test. Bulk or final container samples of completed product shall be tested for potency using...

  17. 9 CFR 113.201 - Canine Distemper Vaccine, Killed Virus.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... distemper to determine susceptibility. A constant virus-varying serum neutralization test in cell culture... neutralization at a 1:2 final serum dilution. (i) The 20 dogs used as vaccinates shall be injected with one dose... neutralization test. Bulk or final container samples of completed product shall be tested for potency using...

  18. 9 CFR 113.201 - Canine Distemper Vaccine, Killed Virus.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... distemper to determine susceptibility. A constant virus-varying serum neutralization test in cell culture... neutralization at a 1:2 final serum dilution. (i) The 20 dogs used as vaccinates shall be injected with one dose... neutralization test. Bulk or final container samples of completed product shall be tested for potency using...

  19. Epizootic canine distemper virus infection among wild mammals.

    PubMed

    Kameo, Yuki; Nagao, Yumiko; Nishio, Yohei; Shimoda, Hiroshi; Nakano, Hitoshi; Suzuki, Kazuo; Une, Yumi; Sato, Hiroshi; Shimojima, Masayuki; Maeda, Ken

    2012-01-27

    In the spring of 2007, seven raccoon dogs and a weasel were captured near the city of Tanabe in Wakayama prefecture, Japan. The causative agent of the animals' death 1-2 days after capture was identified as canine distemper virus (CDV) by virus isolation, immunostaining with an anti-CDV polyclonal antibody, and a commercially available CDV antigen-detection kit. Sequence analysis of hemagglutinin genes indicated the isolated viruses belong to genotype Asia-1 and possess the substitution from tyrosine (Y) to histidine (H) at position 549 that is associated with the spread of CDV to non-canine hosts. A serosurvey for CDV was then conducted among wild animals in the region. The animals assayed consisted of 104 raccoons, 41 wild boars, 19 raccoon dogs, five Sika deer, two badgers, one weasel, one marten, one Siberian weasel and one fox. Virus-neutralization (VN) tests showed that, except for fox and weasel, all of the species assayed had VN antibodies to CDV. Interestingly, 11 of the 41 wild boars (27%) and two of the five Sika deer assayed possessed VN antibodies to CDV. These findings indicate that CDV infection was widespread among wild mammals during this epizootic. PMID:21840141

  20. Immunopathogenic and Neurological Mechanisms of Canine Distemper Virus

    PubMed Central

    Carvalho, Otávio Valério; Botelho, Clarisse Vieira; Ferreira, Caroline Gracielle Torres; Scherer, Paulo Oldemar; Soares-Martins, Jamária Adriana Pinheiro; Almeida, Márcia Rogéria; Silva Júnior, Abelardo

    2012-01-01

    Canine distemper is a highly contagious viral disease caused by the canine distemper virus (CDV), which is a member of the Morbillivirus genus, Paramyxoviridae family. Animals that most commonly suffer from this disease belong to the Canidae family; however, the spectrum of natural hosts for CDV also includes several other families of the order Carnivora. The infectious disease presents worldwide distribution and maintains a high incidence and high levels of lethality, despite the availability of effective vaccines, and no specific treatment. CDV infection in dogs is characterized by the presentation of systemic and/or neurological courses, and viral persistence in some organs, including the central nervous system (CNS) and lymphoid tissues. An elucidation of the pathogenic mechanisms involved in canine distemper disease will lead to a better understanding of the injuries and clinical manifestations caused by CDV. Ultimately, further insight about this disease will enable the improvement of diagnostic methods as well as therapeutic studies. PMID:23193403

  1. Canine Distemper Virus (CDV) in Another Big Cat: Should CDV Be Renamed Carnivore Distemper Virus?

    PubMed Central

    Terio, Karen A.; Craft, Meggan E.

    2013-01-01

    ABSTRACT One of the greatest threats to the conservation of wild cat populations may be dogs or, at least, one of their viruses. Canine distemper virus (CDV), a single-stranded RNA virus in the Paramyxoviridae family and genus Morbillivirus, infects and causes disease in a variety of species, not just canids. An outbreak of CDV in wild lions in the Serengeti, Tanzania, in 1994 was a wake-up call for conservationists, as it demonstrated that an infectious disease could swiftly impact a previously healthy felid population. To understand how this virus causes disease in noncanid hosts, researchers have focused on specific mutations in the binding site of the CDV hemagglutinin gene. Now, Seimon et al. provide information on CDV in its latest feline victim, the endangered wild Amur tiger (Panthera tigris altaica) [T. A. Seimon et al., mBio 4(4):e00410-13, 2013, doi:10.1128/mBio.00410-13]. Their findings of CDV strains infecting tigers, in combination with recent information from other felids, paints a different picture, one in which CDV strains from a variety of geographic lineages and with a variety of amino acid residues in the hemagglutinin gene binding site can infect cats and cause disease. Although CDV has been known as a multihost disease since its discovery in domestic dogs in 1905, perhaps it is time to reconsider whether these noncanid species are not just incidental or “spillover” hosts but, rather, a normal part of the complex ecology of this infectious disease. PMID:24045642

  2. Canine distemper virus (CDV) in another big cat: should CDV be renamed carnivore distemper virus?

    PubMed

    Terio, Karen A; Craft, Meggan E

    2013-01-01

    One of the greatest threats to the conservation of wild cat populations may be dogs or, at least, one of their viruses. Canine distemper virus (CDV), a single-stranded RNA virus in the Paramyxoviridae family and genus Morbillivirus, infects and causes disease in a variety of species, not just canids. An outbreak of CDV in wild lions in the Serengeti, Tanzania, in 1994 was a wake-up call for conservationists, as it demonstrated that an infectious disease could swiftly impact a previously healthy felid population. To understand how this virus causes disease in noncanid hosts, researchers have focused on specific mutations in the binding site of the CDV hemagglutinin gene. Now, Seimon et al. provide information on CDV in its latest feline victim, the endangered wild Amur tiger (Panthera tigris altaica) [T. A. Seimon et al., mBio 4(4):e00410-13, 2013, doi:10.1128/mBio.00410-13]. Their findings of CDV strains infecting tigers, in combination with recent information from other felids, paints a different picture, one in which CDV strains from a variety of geographic lineages and with a variety of amino acid residues in the hemagglutinin gene binding site can infect cats and cause disease. Although CDV has been known as a multihost disease since its discovery in domestic dogs in 1905, perhaps it is time to reconsider whether these noncanid species are not just incidental or "spillover" hosts but, rather, a normal part of the complex ecology of this infectious disease. PMID:24045642

  3. Selective spread and reduced virus release leads to canine distemper virus persistence in the nervous system.

    PubMed

    Zurbriggen, A; Graber, H U; Vandevelde, M

    1995-05-01

    In primary dog brain cell cultures (DBCC) the attenuated canine distemper virus (CDV) is cytolytic, whereas virulent CDV is not. Thus, the question why cytolysis does or does not occur appears to be intimately associated with the mechanism of persistence. Persistence is most likely related to the way these viruses replicate. In the present study we used morphological and immunocytochemical approaches to compare several aspects of virus replication between a cytolytic and a virulent CDV strain using DBCC to study both strains. Quantitative measurements did not detect a difference in the rate of virus protein synthesis at the level of the single cell, between the two types of infection. Electron microscopical results and virus titration experiments showed marked differences in virus spread and virus release. Immunocytochemical studies showed differences in the distribution of the nucleocapsid and matrix proteins between the two infections. Budding and cytolysis are strongly limited in the virulent A75/17-CDV infection as compared to attenuated viruses. This is probably due to structural changes in the virus proteins leading to modifications of virus assembly. Thus the present study supports a mechanism of CDV persistence through a type of virus maturation and spread by which very little virus is released outside of the cell. PMID:8588323

  4. Measles vaccine in dogs: efficacy against aerosol challenge with virulent canine distemper virus.

    PubMed

    Strating, A

    1975-07-01

    Fifty young Beagle pups were used in studies on the efficacy of measles virus vaccine in providing protection against virulent canine distemper (CD) virus given intranasally. Among 29 dogs vaccinated with measles virus vaccine and subsequently exposed to virulent CD virus, 1 died, 7 developed relatively severe signs of CD, 15 had mild signs of distemper, and 6 remained clinically normal. Of 15 unvaccinated dogs similarly exposed to virulent CD virus, 11 succumbed to distemper. Six pups vaccinated with modified live-virus (MLV) CD virus vaccine remained clinically normal following immunity challenge. PMID:1150494

  5. Vaccination of mice against canine distemper virus-induced encephalitis with vaccinia virus recombinants encoding measles or canine distemper virus antigens.

    PubMed

    Wild, T F; Bernard, A; Spehner, D; Villeval, D; Drillien, R

    1993-01-01

    Measles and canine distemper are caused by serologically related viruses. Although dogs immunized with measles virus (MV) do not elicit canine distemper virus (CDV) neutralizing antibodies, they are protected against the fatal disease. To investigate the potential role of the MV antigens in protection against CDV, we have immunized mice with vaccinia virus (VV) recombinants expressing the MV haemagglutinin (HA), fusion (F), nucleoprotein (NP) and matrix (M) antigens and challenged them with CDV. A partial protection was observed with the VV recombinants expressing the F, NP and M antigens, but not the HA. In contrast, immunization with a VV recombinant expressing the CDV F protein completely protected mice from CDV. PMID:8470428

  6. Isolation and phylogenetic characterization of Canine distemper virus from India.

    PubMed

    Swati; Deka, Dipak; Uppal, Sanjeev Kumar; Verma, Ramneek

    2015-09-01

    Canine distemper (CD), caused by canine distemper virus (CDV) is a highly contagious disease that infects a variety of carnivores. Sequence analysis of CDVs from different geographical areas has shown a lot of variation in the genome of the virus especially in haemagglutinin gene which might be one of the causes of vaccine failure. In this study, we isolated the virus (place: Ludhiana, Punjab; year: 2014) and further cloned, sequenced and analyzed partial haemagglutinin (H) gene and full length genes for fusion protein (F), phosphoprotein (P) and matrix protein (M) from an Indian wild-type CDV. Higher sequence homology was observed with the strains from Switzerland, Hungary, Germany; and lower with the vaccine strains like Ondersteport, CDV3, Convac for all the genes. The multiple sequence alignment showed more variation in partial H (45 nucleotide and 5 amino acid substitutions) and complete F (79 nucleotide and 30 amino acid substitutions) than in complete P (44 nucleotide and 22 amino acid substitutions) and complete M (22 nucleotide and 4 amino acid substitutions) gene/protein. Predicted potential N-linked glycosylation sites in H, F, M and P proteins were similar to the previously known wild-type CDVs but different from the vaccine strains. The Indian CDV formed a distinct clade in the phylogenetic tree clearly separated from the previously known wild-type and vaccine strains. PMID:26396979

  7. Canine distemper virus persistence in the nervous system is associated with noncytolytic selective virus spread.

    PubMed Central

    Zurbriggen, A; Graber, H U; Wagner, A; Vandevelde, M

    1995-01-01

    Canine distemper virus (CDV), a negative-strand RNA morbillivirus, causes a progressive demyelinating disease in which virus persistence plays an essential role. The antiviral immune response leads to virus clearance in the inflammatory lesions. However, CDV can replicate and persist outside these inflammatory lesions within the brain. How CDV is capable of persisting in the presence of an effective antiviral immune response is poorly understood. In the present investigation, we studied several aspects of virus replication in primary dog brain cell cultures (DBCC), comparing an attenuated CDV strain and a virulent CDV strain. Confluent DBCC were infected with either virulent A75/17-CDV or attenuated Onderstepoort-CDV and monitored for 60 days. Persistence was not associated with defective virus production, because all mRNAs and corresponding proteins were continuously expressed in the noncytolytic infection. Quantitative measurements did not detect a difference between the two types of infection in the rate of virus transcription and protein synthesis at the level of the single cell. However, electron microscopy and virus titration experiments showed that in the persistent CDV infection virus budding is strongly limited compared with that of the attenuated virus. Morphometry and immunocytochemistry showed profound differences in the way the two viruses spread in the culture. The attenuated CDV spread randomly to immediately adjacent cells, whereas persistent CDV spread selectively to more-distant cells by way of cell processes. In conclusion, the present study supports a mechanism of CDV persistence through selective spread by way of cell processes, enabling virulent CDV to invade the central nervous system without the need of releasing much virus into the extracellular space. PMID:7853504

  8. Canine distemper virus persistence in the nervous system is associated with noncytolytic selective virus spread.

    PubMed

    Zurbriggen, A; Graber, H U; Wagner, A; Vandevelde, M

    1995-03-01

    Canine distemper virus (CDV), a negative-strand RNA morbillivirus, causes a progressive demyelinating disease in which virus persistence plays an essential role. The antiviral immune response leads to virus clearance in the inflammatory lesions. However, CDV can replicate and persist outside these inflammatory lesions within the brain. How CDV is capable of persisting in the presence of an effective antiviral immune response is poorly understood. In the present investigation, we studied several aspects of virus replication in primary dog brain cell cultures (DBCC), comparing an attenuated CDV strain and a virulent CDV strain. Confluent DBCC were infected with either virulent A75/17-CDV or attenuated Onderstepoort-CDV and monitored for 60 days. Persistence was not associated with defective virus production, because all mRNAs and corresponding proteins were continuously expressed in the noncytolytic infection. Quantitative measurements did not detect a difference between the two types of infection in the rate of virus transcription and protein synthesis at the level of the single cell. However, electron microscopy and virus titration experiments showed that in the persistent CDV infection virus budding is strongly limited compared with that of the attenuated virus. Morphometry and immunocytochemistry showed profound differences in the way the two viruses spread in the culture. The attenuated CDV spread randomly to immediately adjacent cells, whereas persistent CDV spread selectively to more-distant cells by way of cell processes. In conclusion, the present study supports a mechanism of CDV persistence through selective spread by way of cell processes, enabling virulent CDV to invade the central nervous system without the need of releasing much virus into the extracellular space. PMID:7853504

  9. Canine distemper virus antigen detection in external epithelia of recently vaccinated, sick dogs by fluorescence microscopy is a valuable prognostic indicator.

    PubMed

    Kapil, Sanjay; Neel, Tina

    2015-02-01

    Currently, there are no reliable predictors of the clinical outcomes of domesticated dogs that have been recently vaccinated against canine distemper virus (CDV) and develop respiratory disease. In this study, vaccinated dogs from Oklahoma City that were showing clinical signs of respiratory disease were evaluated for CDV antigen using a direct fluorescent antibody test (FAT). Clinical outcomes after standard symptomatic therapy for respiratory disease were recorded, and a statistical analysis of the results was performed. We present our study showing that CDV FAT results were predictive of clinical recovery (prognostic indicator, prospects of clinical recovery) among vaccinated dogs showing clinical signs of respiratory disease. Negative CDV FAT results equated to 80% chances of recovery after symptomatic therapy, compared to 55% chances of recovery when the CDV FAT results were positive. Based on the results of this study, we show that veterinarians can make better informed decisions about the clinical outcomes of suspected CDV cases, with 2-h turnaround times, by using the CDV FAT. Thus, antemortem examination with the CDV FAT on external epithelia of recently vaccinated, sick dogs is a clinically useful diagnostic test and valuable prognostic indicator for veterinarians. Application of the CDV FAT to these samples avoids unnecessary euthanasia of dogs with suspected CDV. PMID:25428156

  10. Canine Distemper Virus Antigen Detection in External Epithelia of Recently Vaccinated, Sick Dogs by Fluorescence Microscopy Is a Valuable Prognostic Indicator

    PubMed Central

    Neel, Tina

    2014-01-01

    Currently, there are no reliable predictors of the clinical outcomes of domesticated dogs that have been recently vaccinated against canine distemper virus (CDV) and develop respiratory disease. In this study, vaccinated dogs from Oklahoma City that were showing clinical signs of respiratory disease were evaluated for CDV antigen using a direct fluorescent antibody test (FAT). Clinical outcomes after standard symptomatic therapy for respiratory disease were recorded, and a statistical analysis of the results was performed. We present our study showing that CDV FAT results were predictive of clinical recovery (prognostic indicator, prospects of clinical recovery) among vaccinated dogs showing clinical signs of respiratory disease. Negative CDV FAT results equated to 80% chances of recovery after symptomatic therapy, compared to 55% chances of recovery when the CDV FAT results were positive. Based on the results of this study, we show that veterinarians can make better informed decisions about the clinical outcomes of suspected CDV cases, with 2-h turnaround times, by using the CDV FAT. Thus, antemortem examination with the CDV FAT on external epithelia of recently vaccinated, sick dogs is a clinically useful diagnostic test and valuable prognostic indicator for veterinarians. Application of the CDV FAT to these samples avoids unnecessary euthanasia of dogs with suspected CDV. PMID:25428156

  11. Rapid and sensitive detection of immunoglobulin M (IgM) and IgG antibodies against canine distemper virus by a new recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay.

    PubMed

    von Messling, V; Harder, T C; Moennig, V; Rautenberg, P; Nolte, I; Haas, L

    1999-04-01

    Canine distemper morbillivirus (CDV) infection causes a frequently fatal systemic disease in a broad range of carnivore species, including domestic dogs. In CDV infection, classical serology provides data of diagnostic and prognostic values (kinetics of seroconversion) and is also used to predict the optimal vaccination age of pups. Routine CDV serology is still based on time- and cost-intensive virus neutralization assays (V-NA). Here, we describe a new capture-sandwich enzyme-linked immunosorbent assay (ELISA) that uses recombinant baculovirus-expressed nucleocapsid (N) protein of a recent CDV wild-type isolate (2544/Han95) for the detection of CDV-specific antibodies in canine sera. Recombinant antigen was produced with high efficacy in Heliothis virescens larvae. The capture-sandwich ELISA enabled a clear-cut qualitative evaluation of the CDV-specific immunoglobulin G (IgG) and IgM serostatuses of 196 and 35 dog sera, respectively. Inter-rater agreement analysis (kappa = 0.988) indicated that the ELISA can be used unrestrictedly as a substitute for the V-NA for the qualitative determination of CDV-specific IgG serostatus. In an attempt to semiquantify N-specific antibodies, a one-step-dilution (alpha method) IgG-specific ELISA was implemented. Alpha values of >/=50% showed very good inter-rater agreement (kappa = 0.968) with V-NA titers of >/=1/100 50% neutralizing dose (ND50) as measured against the central European CDV wild-type isolate 2544/Han95 in canine sera originating from northern Germany. An ND50 titer of 1/100 is considered a threshold, and titers of >/=1/100 indicate a resilient, protective immunity. CDV N-specific antibodies of the IgM class were detected by the newly developed ELISA in 9 of 15 sera obtained from dogs with symptoms of acute distemper. In leucocytes of 5 of the 15 dogs (all of which were also IgM positive) CDV RNA was detected by reverse transcription (RT)-PCR. The recombinant capture-sandwich ELISA detecting N-specific antibodies

  12. Rapid and Sensitive Detection of Immunoglobulin M (IgM) and IgG Antibodies against Canine Distemper Virus by a New Recombinant Nucleocapsid Protein-Based Enzyme-Linked Immunosorbent Assay

    PubMed Central

    von Messling, Veronika; Harder, Timm C.; Moennig, Volker; Rautenberg, Peter; Nolte, Ingo; Haas, Ludwig

    1999-01-01

    Canine distemper morbillivirus (CDV) infection causes a frequently fatal systemic disease in a broad range of carnivore species, including domestic dogs. In CDV infection, classical serology provides data of diagnostic and prognostic values (kinetics of seroconversion) and is also used to predict the optimal vaccination age of pups. Routine CDV serology is still based on time- and cost-intensive virus neutralization assays (V-NA). Here, we describe a new capture-sandwich enzyme-linked immunosorbent assay (ELISA) that uses recombinant baculovirus-expressed nucleocapsid (N) protein of a recent CDV wild-type isolate (2544/Han95) for the detection of CDV-specific antibodies in canine sera. Recombinant antigen was produced with high efficacy in Heliothis virescens larvae. The capture-sandwich ELISA enabled a clear-cut qualitative evaluation of the CDV-specific immunoglobulin G (IgG) and IgM serostatuses of 196 and 35 dog sera, respectively. Inter-rater agreement analysis (κ = 0.988) indicated that the ELISA can be used unrestrictedly as a substitute for the V-NA for the qualitative determination of CDV-specific IgG serostatus. In an attempt to semiquantify N-specific antibodies, a one-step-dilution (alpha method) IgG-specific ELISA was implemented. Alpha values of ≥50% showed very good inter-rater agreement (κ = 0.968) with V-NA titers of ≥1/100 50% neutralizing dose (ND50) as measured against the central European CDV wild-type isolate 2544/Han95 in canine sera originating from northern Germany. An ND50 titer of 1/100 is considered a threshold, and titers of ≥1/100 indicate a resilient, protective immunity. CDV N-specific antibodies of the IgM class were detected by the newly developed ELISA in 9 of 15 sera obtained from dogs with symptoms of acute distemper. In leucocytes of 5 of the 15 dogs (all of which were also IgM positive) CDV RNA was detected by reverse transcription (RT)-PCR. The recombinant capture-sandwich ELISA detecting N-specific antibodies of the

  13. Canine Distemper Virus Strains Circulating among North American Dogs▿

    PubMed Central

    Kapil, Sanjay; Allison, Robin W.; Johnston, Larry; Murray, Brandy L.; Holland, Steven; Meinkoth, Jim; Johnson, Bill

    2008-01-01

    Canine distemper virus (CDV) is a highly contagious virus that causes multisystemic disease in dogs. We received seven samples from dogs with CD from the United States during 2007. CDV isolates from these samples formed large, multinucleated syncytia in a Vero cell line expressing canine signaling lymphocyte activation molecule (SLAM). Based on the hemagglutinin gene sequences, the CDV isolates from three states (California, Missouri, and Oklahoma) formed two CDV genetic groups: group I (major; six of seven isolates) consisted of CDV isolates closely related to the European wildlife lineage of CDV, and group II (minor; one of seven isolates) was genetically related to the Arctic-like lineage of CDV. However, both CDV groups were genetically different from the current vaccine strains that belong to the American-1 lineage of the old (1930 to 1950) CDV isolates. PMID:18256210

  14. Establishment of a Rescue System for Canine Distemper Virus

    PubMed Central

    Gassen, Uta; Collins, Fergal M.; Duprex, W. Paul; Rima, Bert K.

    2000-01-01

    Canine distemper virus (CDV) has been rescued from a full-length cDNA clone. Besides Measles virus (MV) and Rinderpest virus, a third morbillivirus is now available for genetic analysis using reverse genetics. A plasmid p(+)CDV was constructed by sequential cloning using the Onderstepoort vaccine strain large-plaque-forming variant. The presence of a T7 promoter allowed transcription of full-length antigenomic RNA by a T7 RNA polymerase, which was provided by a host range mutant of vaccinia virus (MVA-T7). Plasmids expressing the nucleocapsid protein, the phosphoprotein, and the viral RNA-dependent RNA polymerase, also under control of a T7 promoter, have been generated. Infection of HeLa cells with MVA-T7 and subsequent transfection of p(+)CDV plus the helper plasmids led to syncytium formation and release of infectious recombinant (r) CDV. Comparison of the rescued virus with the parental virus revealed no major differences in the progression of infection or in the shape and size of syncytia. A genetic tag, consisting of two nucleotide changes within the coding region of the L protein, has been identified in the rCDV genome. Expression by rCDV of all the major viral structural proteins has been demonstrated by immunofluorescence. PMID:11044118

  15. Fatal canine distemper virus infection of giant pandas in China.

    PubMed

    Feng, Na; Yu, Yicong; Wang, Tiecheng; Wilker, Peter; Wang, Jianzhong; Li, Yuanguo; Sun, Zhe; Gao, Yuwei; Xia, Xianzhu

    2016-01-01

    We report an outbreak of canine distemper virus (CDV) infection among endangered giant pandas (Ailuropoda melanoleuca). Five of six CDV infected giant pandas died. The surviving giant panda was previously vaccinated against CDV. Genomic sequencing of CDV isolated from one of the infected pandas (giant panda/SX/2014) suggests it belongs to the Asia-1 cluster. The hemagglutinin protein of the isolated virus and virus sequenced from lung samples originating from deceased giant pandas all possessed the substitutions V26M, T213A, K281R, S300N, P340Q, and Y549H. The presence of the Y549H substitution is notable as it is found at the signaling lymphocytic activation molecule (SLAM) receptor-binding site and has been implicated in the emergence of highly pathogenic CDV and host switching. These findings demonstrate that giant pandas are susceptible to CDV and suggest that surveillance and vaccination among all captive giant pandas are warranted to support conservation efforts for this endangered species. PMID:27310722

  16. Fatal canine distemper virus infection of giant pandas in China

    PubMed Central

    Feng, Na; Yu, Yicong; Wang, Tiecheng; Wilker, Peter; Wang, Jianzhong; Li, Yuanguo; Sun, Zhe; Gao, Yuwei; Xia, Xianzhu

    2016-01-01

    We report an outbreak of canine distemper virus (CDV) infection among endangered giant pandas (Ailuropoda melanoleuca). Five of six CDV infected giant pandas died. The surviving giant panda was previously vaccinated against CDV. Genomic sequencing of CDV isolated from one of the infected pandas (giant panda/SX/2014) suggests it belongs to the Asia-1 cluster. The hemagglutinin protein of the isolated virus and virus sequenced from lung samples originating from deceased giant pandas all possessed the substitutions V26M, T213A, K281R, S300N, P340Q, and Y549H. The presence of the Y549H substitution is notable as it is found at the signaling lymphocytic activation molecule (SLAM) receptor-binding site and has been implicated in the emergence of highly pathogenic CDV and host switching. These findings demonstrate that giant pandas are susceptible to CDV and suggest that surveillance and vaccination among all captive giant pandas are warranted to support conservation efforts for this endangered species. PMID:27310722

  17. Phocine Distemper Virus: Current Knowledge and Future Directions

    PubMed Central

    Duignan, Pádraig J.; Van Bressem, Marie-Françoise; Baker, Jason D.; Barbieri, Michelle; Colegrove, Kathleen M.; De Guise, Sylvain; de Swart, Rik L.; Di Guardo, Giovanni; Dobson, Andrew; Duprex, W. Paul; Early, Greg; Fauquier, Deborah; Goldstein, Tracey; Goodman, Simon J.; Grenfell, Bryan; Groch, Kátia R.; Gulland, Frances; Hall, Ailsa; Jensen, Brenda A.; Lamy, Karina; Matassa, Keith; Mazzariol, Sandro; Morris, Sinead E.; Nielsen, Ole; Rotstein, David; Rowles, Teresa K.; Saliki, Jeremy T.; Siebert, Ursula; Waltzek, Thomas; Wellehan, James F.X.

    2014-01-01

    Phocine distemper virus (PDV) was first recognized in 1988 following a massive epidemic in harbor and grey seals in north-western Europe. Since then, the epidemiology of infection in North Atlantic and Arctic pinnipeds has been investigated. In the western North Atlantic endemic infection in harp and grey seals predates the European epidemic, with relatively small, localized mortality events occurring primarily in harbor seals. By contrast, PDV seems not to have become established in European harbor seals following the 1988 epidemic and a second event of similar magnitude and extent occurred in 2002. PDV is a distinct species within the Morbillivirus genus with minor sequence variation between outbreaks over time. There is now mounting evidence of PDV-like viruses in the North Pacific/Western Arctic with serological and molecular evidence of infection in pinnipeds and sea otters. However, despite the absence of associated mortality in the region, there is concern that the virus may infect the large Pacific harbor seal and northern elephant seal populations or the endangered Hawaiian monk seals. Here, we review the current state of knowledge on PDV with particular focus on developments in diagnostics, pathogenesis, immune response, vaccine development, phylogenetics and modeling over the past 20 years. PMID:25533658

  18. Phocine distemper virus: current knowledge and future directions.

    PubMed

    Duignan, Pádraig J; Van Bressem, Marie-Françoise; Baker, Jason D; Barbieri, Michelle; Colegrove, Kathleen M; De Guise, Sylvain; de Swart, Rik L; Di Guardo, Giovanni; Dobson, Andrew; Duprex, W Paul; Early, Greg; Fauquier, Deborah; Goldstein, Tracey; Goodman, Simon J; Grenfell, Bryan; Groch, Kátia R; Gulland, Frances; Hall, Ailsa; Jensen, Brenda A; Lamy, Karina; Matassa, Keith; Mazzariol, Sandro; Morris, Sinead E; Nielsen, Ole; Rotstein, David; Rowles, Teresa K; Saliki, Jeremy T; Siebert, Ursula; Waltzek, Thomas; Wellehan, James F X

    2014-12-01

    Phocine distemper virus (PDV) was first recognized in 1988 following a massive epidemic in harbor and grey seals in north-western Europe. Since then, the epidemiology of infection in North Atlantic and Arctic pinnipeds has been investigated. In the western North Atlantic endemic infection in harp and grey seals predates the European epidemic, with relatively small, localized mortality events occurring primarily in harbor seals. By contrast, PDV seems not to have become established in European harbor seals following the 1988 epidemic and a second event of similar magnitude and extent occurred in 2002. PDV is a distinct species within the Morbillivirus genus with minor sequence variation between outbreaks over time. There is now mounting evidence of PDV-like viruses in the North Pacific/Western Arctic with serological and molecular evidence of infection in pinnipeds and sea otters. However, despite the absence of associated mortality in the region, there is concern that the virus may infect the large Pacific harbor seal and northern elephant seal populations or the endangered Hawaiian monk seals. Here, we review the current state of knowledge on PDV with particular focus on developments in diagnostics, pathogenesis, immune response, vaccine development, phylogenetics and modeling over the past 20 years. PMID:25533658

  19. The Hemagglutinin of Canine Distemper Virus Determines Tropism and Cytopathogenicity

    PubMed Central

    von Messling, Veronika; Zimmer, Gert; Herrler, Georg; Haas, Ludwig; Cattaneo, Roberto

    2001-01-01

    Canine distemper virus (CDV) and measles virus (MV) cause severe illnesses in their respective hosts. The viruses display a characteristic cytopathic effect by forming syncytia in susceptible cells. For CDV, the proficiency of syncytium formation varies among different strains and correlates with the degree of viral attenuation. In this study, we examined the determinants for the differential fusogenicity of the wild-type CDV isolate 5804Han89 (CDV5804), the small- and large-plaque-forming variants of the CDV vaccine strain Onderstepoort (CDVOS and CDVOL, respectively), and the MV vaccine strain Edmonston B (MVEdm). The cotransfection of different combinations of fusion (F) and hemagglutinin (H) genes in Vero cells indicated that the H protein is the main determinant of fusion efficiency. To verify the significance of this observation in the viral context, a reverse genetic system to generate recombinant CDVs was established. This system is based on a plasmid containing the full-length antigenomic sequence of CDVOS. The coding regions of the H proteins of all CDV strains and MVEdm were introduced into the CDV and MV genetic backgrounds, and recombinant viruses rCDV-H5804, rCDV-HOL, rCDV-HEdm, rMV-H5804, rMV-HOL, and rMV-HOS were recovered. Thus, the H proteins of the two morbilliviruses are interchangeable and fully functional in a heterologous complex. This is in contrast with the glycoproteins of other members of the family Paramyxoviridae, which do not function efficiently with heterologous partners. The fusogenicity, growth characteristics, and tropism of the recombinant viruses were examined and compared with those of the parental strains. All these characteristics were found to be predominantly mediated by the H protein regardless of the viral backbone used. PMID:11413309

  20. A recombinant canine distemper virus expressing a modified rabies virus glycoprotein induces immune responses in mice.

    PubMed

    Li, Zhili; Wang, Jigui; Yuan, Daoli; Wang, Shuang; Sun, Jiazeng; Yi, Bao; Hou, Qiang; Mao, Yaping; Liu, Weiquan

    2015-06-01

    Canine distemper virus (CDV) and rabies virus (RV) are two important pathogens of the dog. CDV, a member of the morbillivirus genus, has shown promise as an expression vector. The glycoprotein from RV is a main contributor to protective immunity and capable of eliciting the production of virus-neutralizing antibodies. In this study, we recovered an attenuated strain of canine distemper virus and constructed a recombinant virus, rCDV-RV-G, expressing a modified (R333Q) rabies virus glycoprotein (RV-G) of RV Flury strain LEP. RV-G expression by the recombinant viruses was confirmed. Furthermore, G was proved to be incorporated into the surface of CDV particles. While replication of the recombinant virus was slightly reduced compared with the parental CDV, it stably expressed the RV-G over ten serial passages. Inoculation of mice induced specific neutralizing antibodies against both RV-G and CDV. Therefore, the rCDV-RV-G has the potential as a vaccine that may be used to control rabies virus infection in dogs and other animals. PMID:25764477

  1. Phylogenetic analysis of canine distemper virus in domestic dogs in Nanjing, China.

    PubMed

    Bi, Zhenwei; Wang, Yongshan; Wang, Xiaoli; Xia, Xingxia

    2015-02-01

    Canine distemper virus (CDV) infects a broad range of carnivores, including wild and domestic Canidae. The hemagglutinin gene, which encodes the attachment protein that determines viral tropism, has been widely used to determine the relationship between CDV strains of different lineages circulating worldwide. We determined the full-length H gene sequences of seven CDV field strains detected in domestic dogs in Nanjing, China. A phylogenetic analysis of the H gene sequences of CDV strains from different geographic regions and vaccine strains was performed. Four of the seven CDV strains were grouped in the same cluster of the Asia-1 lineage to which the vast majority of Chinese CDV strains belong, whereas the other three were clustered within the Asia-4 lineage, which has never been detected in China. This represents the first record of detection of strains of the Asia-4 lineage in China since this lineage was reported in Thailand in 2013. PMID:25416856

  2. Fatal vaccine-induced canine distemper virus infection in black-footed ferrets

    USGS Publications Warehouse

    Carpenter, J.W.; Appel, M.J.G.; Erickson, R.C.; Novilla, M.N.

    1976-01-01

    Four black-footed ferrets that were live-trapped in South Dakota and transported to the Patuxent Wildlife Research Center died within 21 days after vaccination with modified live canine distemper virus. Immunofluorescence, European ferret inoculation, virus isolation attempts, and serum-neutralization tests indicated insufficient attenuation of the vaccine for this species.

  3. Canine Distemper Virus in Wild Felids of Costa Rica.

    PubMed

    Avendaño, Roberto; Barrueta, Flor; Soto-Fournier, Sofía; Chavarría, Max; Monge, Otto; Gutiérrez-Espeleta, Gustavo A; Chaves, Andrea

    2016-04-28

    Several highly infectious diseases can be transmitted through feces and cause elevated mortality among carnivore species. One such infectious agent, canine distemper virus (CDV; Paramyxoviridae: Morbillivirus), has been reported to affect wild carnivores, among them several felid species. We screened free-ranging and captive wild carnivores in Costa Rica for CDV. Between 2006 and 2012, we collected 306 fecal samples from 70 jaguars (Panther onca), 71 ocelots ( Leopardus pardalis ), five jaguarundis (Puma yaguaroundi), 105 pumas ( Puma concolor ), five margays ( Leopardus wiedii ), 23 coyotes ( Canis latrans ), and 27 undetermined Leopardus spp. We found CDV in six individuals: one captive jaguarundi (rescued in 2009), three free-ranging ocelots (samples collected in 2012), and two free-ranging pumas (samples collected in 2007). Phylogenetic analyses were performed using sequences of the phosphoprotein (P) gene. We provide evidence of CDV in wild carnivores in Costa Rica and sequence data from a Costa Rican CDV isolate, adding to the very few sequence data available for CDV isolates from wild Central American carnivores. PMID:26967127

  4. A canine distemper virus epidemic in Serengeti lions (Panthera leo).

    PubMed

    Roelke-Parker, M E; Munson, L; Packer, C; Kock, R; Cleaveland, S; Carpenter, M; O'Brien, S J; Pospischil, A; Hofmann-Lehmann, R; Lutz, H; Mwamengele, G L; Mgasa, M N; Machange, G A; Summers, B A; Appel, M J

    1996-02-01

    Canine distemper virus (CDV) is thought to have caused several fatal epidemics in canids within the Serengeti-Mara ecosystem of East Africa, affecting silver-backed jackals (Canis mesomelas) and bat-eared foxes (Otocyon megalotis) in 1978 (ref. 1), and African wild dogs (Lycaon pictus) in 1991 (refs 2, 3). The large, closely monitored Serengeti lion population was not affected in these epidemics. However, an epidemic caused by a morbillivirus closely related to CDV emerged abruptly in the lion population of the Serengeti National Park, Tanzania, in early 1994, resulting in fatal neurological disease characterized by grand mal seizures and myoclonus; the lions that died had encephalitis and pneumonia. Here we report the identification of CDV from these lions, and the close phylogenetic relationship between CDV isolates from lions and domestic dogs. By August 1994, 85% of the Serengeti lion population had anti-CDV antibodies, and the epidemic spread north to lions in the Maasai Mara National reserve, Kenya, and uncounted hyaenas, bat-eared foxes, and leopards were also affected. PMID:8559247

  5. Measles Vaccination of Nonhuman Primates Provides Partial Protection against Infection with Canine Distemper Virus

    PubMed Central

    de Vries, Rory D.; Ludlow, Martin; Verburgh, R. Joyce; van Amerongen, Geert; Yüksel, Selma; Nguyen, D. Tien; McQuaid, Stephen; Osterhaus, Albert D. M. E.; Duprex, W. Paul

    2014-01-01

    ABSTRACT Measles virus (MV) is being considered for global eradication, which would likely reduce compliance with MV vaccination. As a result, children will grow up without MV-specific immunity, creating a potential niche for closely related animal morbilliviruses such as canine distemper virus (CDV). Natural CDV infection causing clinical signs has never been reported in humans, but recent outbreaks in captive macaques have shown that CDV can cause disease in primates. We studied the virulence and tropism of recombinant CDV expressing enhanced green fluorescent protein in naive and measles-vaccinated cynomolgus macaques. In naive animals CDV caused viremia and fever and predominantly infected CD150+ lymphocytes and dendritic cells. Virus was reisolated from the upper and lower respiratory tracts, but infection of epithelial or neuronal cells was not detectable at the time points examined, and the infections were self-limiting. This demonstrates that CDV readily infects nonhuman primates but suggests that additional mutations are necessary to achieve full virulence in nonnatural hosts. Partial protection against CDV was observed in measles-vaccinated macaques, as demonstrated by accelerated control of virus replication and limited shedding from the upper respiratory tract. While neither CDV infection nor MV vaccination induced detectable cross-reactive neutralizing antibodies, MV-specific neutralizing antibody levels of MV-vaccinated macaques were boosted by CDV challenge infection, suggesting that cross-reactive VN epitopes exist. Rapid increases in white blood cell counts in MV-vaccinated macaques following CDV challenge suggested that cross-reactive cellular immune responses were also present. This study demonstrates that zoonotic morbillivirus infections can be controlled by measles vaccination. IMPORTANCE Throughout history viral zoonoses have had a substantial impact on human health. Given the drive toward global eradication of measles, it is essential to

  6. Vaccinia virus recombinants expressing either the measles virus fusion or hemagglutinin glycoprotein protect dogs against canine distemper virus challenge.

    PubMed

    Taylor, J; Pincus, S; Tartaglia, J; Richardson, C; Alkhatib, G; Briedis, D; Appel, M; Norton, E; Paoletti, E

    1991-08-01

    cDNA clones of the genes encoding either the hemagglutinin (HA) or fusion (F) proteins of the Edmonston strain of measles virus (MV) were expressed in vaccinia virus recombinants. Immunofluorescence analysis detected both proteins on the plasma membranes of unfixed cells as well as internally in fixed cells. Immunoprecipitation of metabolically radiolabeled infected-cell extracts by using specific sera demonstrated a 76-kDa HA polypeptide and gene products of 60, 44, and 23 kDa which correspond to a MV F precursor and cleavage products F0, F1, and F2, respectively. Neither recombinant induced cell fusion of Vero cells when inoculated individually, but efficient cell fusion was readily observed upon coinfection of cells with both recombinants. Inoculation of dogs with the vaccinia virus-MV F recombinant (VV-MVF) did not give rise to detectable MV-neutralizing antibody. Inoculation of dogs with the vaccinia virus-MV HA recombinant (VV-MVHA) or coinoculation with both recombinants (VV-MVF and VV-MVHA) induced significant MV-neutralizing titers that were increased following a booster inoculation. Inoculation of dogs with the vaccinia virus recombinants or with MV failed to induce canine distemper virus (CDV)-neutralizing antibodies. Upon challenge with a lethal dose of virulent CDV, signs of infection were observed in dogs inoculated with (VV-MVF). No symptoms of disease were observed in dogs that had been vaccinated with VV-MVHA or with VV-MVHA and VV-MVF and then challenged with CDV. All dogs vaccinated with the recombinant viruses as well as those inoculated with MV or a vaccine strain of CDV survived CDV challenge. PMID:1830113

  7. Vaccinia virus recombinants expressing either the measles virus fusion or hemagglutinin glycoprotein protect dogs against canine distemper virus challenge.

    PubMed Central

    Taylor, J; Pincus, S; Tartaglia, J; Richardson, C; Alkhatib, G; Briedis, D; Appel, M; Norton, E; Paoletti, E

    1991-01-01

    cDNA clones of the genes encoding either the hemagglutinin (HA) or fusion (F) proteins of the Edmonston strain of measles virus (MV) were expressed in vaccinia virus recombinants. Immunofluorescence analysis detected both proteins on the plasma membranes of unfixed cells as well as internally in fixed cells. Immunoprecipitation of metabolically radiolabeled infected-cell extracts by using specific sera demonstrated a 76-kDa HA polypeptide and gene products of 60, 44, and 23 kDa which correspond to a MV F precursor and cleavage products F0, F1, and F2, respectively. Neither recombinant induced cell fusion of Vero cells when inoculated individually, but efficient cell fusion was readily observed upon coinfection of cells with both recombinants. Inoculation of dogs with the vaccinia virus-MV F recombinant (VV-MVF) did not give rise to detectable MV-neutralizing antibody. Inoculation of dogs with the vaccinia virus-MV HA recombinant (VV-MVHA) or coinoculation with both recombinants (VV-MVF and VV-MVHA) induced significant MV-neutralizing titers that were increased following a booster inoculation. Inoculation of dogs with the vaccinia virus recombinants or with MV failed to induce canine distemper virus (CDV)-neutralizing antibodies. Upon challenge with a lethal dose of virulent CDV, signs of infection were observed in dogs inoculated with (VV-MVF). No symptoms of disease were observed in dogs that had been vaccinated with VV-MVHA or with VV-MVHA and VV-MVF and then challenged with CDV. All dogs vaccinated with the recombinant viruses as well as those inoculated with MV or a vaccine strain of CDV survived CDV challenge. Images PMID:1830113

  8. Phocine distemper virus (PDV) seroprevalence as predictor for future outbreaks in harbour seals.

    PubMed

    Ludes-Wehrmeister, Eva; Dupke, Claudia; Harder, Timm C; Baumgärtner, Wolfgang; Haas, Ludwig; Teilmann, Jonas; Dietz, Rune; Jensen, Lasse F; Siebert, Ursula

    2016-02-01

    Phocine distemper virus (PDV) infections caused the two most pronounced mass mortalities in marine mammals documented in the past century. During the two outbreaks, 23,000 and 30,000 harbour seals (Phoca vitulina), died in 1988/1989 and 2002 across populations in the Wadden Sea and adjacent waters, respectively. To follow the mechanism and development of disease spreading, the dynamics of Morbillivirus-specific antibodies in harbour seal populations in German and Danish waters were examined. 522 serum samples of free-ranging harbour seals of different ages were sampled between 1990 and 2014. By standard neutralisation assays, Morbillivirus-specific antibodies were detected, using either the PDV isolate 2558/Han 88 or the related canine distemper virus (CDV) strain Onderstepoort. A total of 159 (30.5%) of the harbour seals were seropositive. Annual seroprevalence rates showed an undulating course: Peaks were seen in the post-epidemic years 1990/1991 and 2002/2003. Following each PDV outbreak, seroprevalence decreased and six to eight years after the epidemics samples were tested seronegative, indicating that the populations are now again susceptible to new PDV outbreak. After the last outbreak in 2002, the populations grew steadily to an estimated maximum (since 1975) of about 39,100 individuals in the Wadden Sea in 2014 and about 23,540 harbour seals in the Kattegat area in 2013. A re-appearence of PDV would presumably result in another epizootic with high mortality rates as encountered in the previous outbreaks. The current high population density renders harbour seals vulnerable to rapid spread of infectious agents including PDV and the recently detected influenza A virus. PMID:26790934

  9. CANINE DISTEMPER VIRUS ANTIBODY TITERS IN DOMESTIC CATS AFTER DELIVERY OF A LIVE ATTENUATED VIRUS VACCINE.

    PubMed

    Ramsay, Edward; Sadler, Ryan; Rush, Robert; Seimon, Tracie; Tomaszewicz, Ania; Fleetwood, Ellen A; McAloose, Denise; Wilkes, Rebecca P

    2016-06-01

    Three methods for delivering a live attenuated canine distemper virus (CDV) vaccine to domestic cats ( Felis catus ) were investigated, as models for developing vaccination protocols for tigers (Panthera tigris). Twenty domestic cats were randomly divided into four treatment groups: saline injection (negative controls); and oral, intranasal, and subcutaneous vaccinates. Cats were injected with saline or a CDV vaccine (Nobivac DP, Merck) at wk 0 and 4. Blood and nasal swabs were collected at wk 0 (prior to the initial vaccination) and weekly thereafter for 9 wk. Urine samples were collected on wk 1 to 9 after initial vaccination. Forty-nine weeks following the initial vaccination series, three cats from the subcutaneous group and three cats from the intranasal group were revaccinated. Blood was collected immediately prior, and 7 and 21 days subsequent to revaccination. Nasal swabs and urine samples were collected from each cat prior to wk 49 revaccination and daily for 7 days thereafter. Nasal swabs and urine were analyzed by quantitative PCR for vaccine virus presence. Sera were tested for CDV antibodies by virus neutralization. All cats were sero-negative for CDV antibodies at the beginning of the study, and saline-injected cats remained sero-negative throughout the study. A dramatic anamnestic response was seen following wk 4 subcutaneous vaccinations, with titers peaking at wk 6 (geometric mean = 2,435.5). Following wk 49 revaccination, subcutaneous vaccinates again mounted impressive titers (wk 52 geometric mean = 2,048). Revaccination of the intranasal group cats at wk 49 produced a small increase in titers (wk 52 geometric mean = 203). CDV viral RNA was detected in six nasal swabs but no urine samples, demonstrating low viral shedding postvaccination. The strong antibody response to subcutaneous vaccination and the lack of adverse effects suggest this vaccine is safe and potentially protective against CDV infection in domestic cats. PMID:27468028

  10. SLAM- and Nectin-4-Independent Noncytolytic Spread of Canine Distemper Virus in Astrocytes

    PubMed Central

    Alves, Lisa; Khosravi, Mojtaba; Avila, Mislay; Ader-Ebert, Nadine; Bringolf, Fanny; Zurbriggen, Andreas; Vandevelde, Marc

    2015-01-01

    ABSTRACT Measles and canine distemper viruses (MeV and CDV, respectively) first replicate in lymphatic and epithelial tissues by using SLAM and nectin-4 as entry receptors, respectively. The viruses may also invade the brain to establish persistent infections, triggering fatal complications, such as subacute sclerosis pan-encephalitis (SSPE) in MeV infection or chronic, multiple sclerosis-like, multifocal demyelinating lesions in the case of CDV infection. In both diseases, persistence is mediated by viral nucleocapsids that do not require packaging into particles for infectivity but are directly transmitted from cell to cell (neurons in SSPE or astrocytes in distemper encephalitis), presumably by relying on restricted microfusion events. Indeed, although morphological evidence of fusion remained undetectable, viral fusion machineries and, thus, a putative cellular receptor, were shown to contribute to persistent infections. Here, we first showed that nectin-4-dependent cell-cell fusion in Vero cells, triggered by a demyelinating CDV strain, remained extremely limited, thereby supporting a potential role of nectin-4 in mediating persistent infections in astrocytes. However, nectin-4 could not be detected in either primary cultured astrocytes or the white matter of tissue sections. In addition, a bioengineered “nectin-4-blind” recombinant CDV retained full cell-to-cell transmission efficacy in primary astrocytes. Combined with our previous report demonstrating the absence of SLAM expression in astrocytes, these findings are suggestive for the existence of a hitherto unrecognized third CDV receptor expressed by glial cells that contributes to the induction of noncytolytic cell-to-cell viral transmission in astrocytes. IMPORTANCE While persistent measles virus (MeV) infection induces SSPE in humans, persistent canine distemper virus (CDV) infection causes chronic progressive or relapsing demyelination in carnivores. Common to both central nervous system (CNS

  11. CD9, a tetraspan transmembrane protein, renders cells susceptible to canine distemper virus.

    PubMed Central

    Löffler, S; Lottspeich, F; Lanza, F; Azorsa, D O; ter Meulen, V; Schneider-Schaulies, J

    1997-01-01

    Canine distemper virus (CDV), a lymphotropic and neurotropic negative-stranded RNA virus of the Morbillivirus genus, causes a life-threatening disease in several carnivores, including domestic dogs. To identify the cellular receptor(s) involved in the uptake of CDV by susceptible cells, we isolated a monoclonal antibody (MAb K41) which binds to the cell surface and inhibits the CDV infection of several cell lines from various species. Pretreatment of cells with MAb K41 reduces the number of infectious centers and the size of the syncytia. Using affinity chromatography with MAb K41, we purified from HeLa and Vero cell extracts a 26-kDa protein which contained the amino acid sequence TKDEPQRETLK of human CD9, a member of the tetraspan transmembrane or transmembrane 4 superfamily of cell surface proteins. Transfection of NIH 3T3 or MDBK cells with a CD9 expression plasmid rendered these cells permissive for viral infection and raised virus production by a factor of 10 to 100. The mechanism involved is still unclear, since we were unable to detect direct binding of CDV to CD9 by using immunoprecipitation and a virus overlay protein binding assay. These findings indicate that human CD9 and its homologs in other species are necessary factors for the uptake of CDV by target cells, the formation of syncytia, and the production of progeny virus. PMID:8985321

  12. Histopathology and immunohistochemistry of canine distemper virus-induced footpad hyperkeratosis (hard Pad disease) in dogs with natural canine distemper.

    PubMed

    Koutinas, A F; Baumgärtner, W; Tontis, D; Polizopoulou, Z; Saridomichelakis, M N; Lekkas, S

    2004-01-01

    Hard pad disease represents an uncommon manifestation of canine distemper virus (CDV) infection with a still uncertain pathogenesis. To study the pathogenesis of this uncommon, virally induced cutaneous lesion, the footpads of 19 dogs with naturally occurring distemper were investigated for histologic changes and distribution pattern of CDV antigen. All dogs displayed clinical signs of distemper, which had lasted from 10 to 75 days. Overt digital hyperkeratosis was observed in 12 animals (group A), whereas the footpads of the remaining seven dogs appeared normal macroscopically (group B). Orthokeratotic hyperkeratosis (12/12; 100%), irregular acanthosis (11/12; 92%), thickened rete ridges (10/12; 83%), and mild mononuclear perivascular (10/ 12; 83%) and periadnexal (7/12; 58%) dermatitis were the most common findings in dogs with hard pad disease. Surprisingly, orthokeratotic hyperkeratosis (5/7; 71%), irregular acanthosis (5/7; 71%), and thickened rete ridges (4/7; 57%) were also seen in the dogs without clinical evidence of digital hyperkeratosis. CDV-specific inclusion bodies and ballooning degeneration were not observed in the footpad epidermis of the 19 dogs. Immunohis-tochemistry revealed that CDV antigen was most frequently found in the stratum spinosum and granulosum and in the epithelial cells of the eccrine sweat glands and only rarely in the basal layer. Fibroblasts, pericytes, endothelial cells, and hair follicles were also positive in some animals. Despite the obvious difference regarding the macroscopic picture, the microscopic changes were less prominent between the animal groups. The selective infection of keratinocytes in the stratum spinosum might be the key event for the development of hard pad disease in the dog. PMID:14715962

  13. Characteristics of a Canine Distemper Virus Outbreak in Dichato, Chile Following the February 2010 Earthquake

    PubMed Central

    Garde, Elena; Pérez, Guillermo; Acosta-Jamett, Gerardo; Bronsvoort, Barend Mark

    2013-01-01

    Simple Summary A disease outbreak in domestic dogs was reported by a coastal Chilean community following the February 2010 earthquake and tsunami. Using clinical exams and diagnostic testing, canine distemper virus was confirmed. Most dogs seen had never been vaccinated, and the majority of those with positive results were recorded in dogs less than two years of age. There were no facilities to contain or treat dogs locally, and no plan to shelter free-roaming dogs. This observational study demonstrates the great need for disaster preparedness planning in developing countries that includes companion animals. Abstract Following the earthquake and tsunami disaster in Chile in February 2010, residents of Dichato reported high morbidity and mortality in dogs, descriptions of which resembled canine distemper virus (CDV). To assess the situation, free vaccine clinics were offered in April and May. Owner information, dog history and signalment were gathered; dogs received physical examinations and vaccines protecting against CDV, and other common canine pathogens. Blood was collected to screen for IgM antibodies to CDV. In total, 208 dogs received physical exams and vaccines were given to 177. IgM antibody titres to CDV were obtained for 104 dogs. Fifty-four dogs (51.9%) tested positive for CDV at the cut off titre of >1:50, but a total of 91.4% of dogs had a detectable titre >1:10. Most of the positive test results were in dogs less than 2 years of age; 33.5% had been previously vaccinated against CDV, and owners of 84 dogs (42.2%) reported clinical signs characteristic of CDV in their dogs following the disaster. The presence of endemic diseases in dog populations together with poor pre-disaster free-roaming dog management results in a potential for widespread negative effects following disasters. Creation of preparedness plans that include animal welfare, disease prevention and mitigation should be developed. PMID:26479537

  14. Unusual Necrotizing Encephalitis in Raccoons and Skunks Concurrently Infected With Canine Distemper Virus and Sarcocystis sp.

    PubMed

    Kubiski, S V; Sisó, S; Church, M E; Cartoceti, A N; Barr, B; Pesavento, P A

    2016-05-01

    Canine distemper virus commonly infects free-ranging, terrestrial mesopredators throughout the United States. Due to the immunosuppressive effects of the virus, concurrent opportunistic infections are also common. Among these, secondary systemic protozoal infections have been described in a number of species. We report an unusual presentation of necrotizing encephalitis associated withSarcocystissp in four raccoons and one skunk concurrently infected with canine distemper virus. Lesions were characterized by variably sized necrotizing cavitations composed of abundant mineral admixed with inflammatory cells and protozoa.Sarcocystissp was confirmed via immunohistochemistry using a monoclonal antibody toSarcocystis neurona The pathologic changes are similar to lesions in human AIDS patients infected withToxoplasma gondii. PMID:26374278

  15. Canine distemper virus infection among wildlife before and after the epidemic

    PubMed Central

    SUZUKI, Junko; NISHIO, Yohei; KAMEO, Yuki; TERADA, Yutaka; KUWATA, Ryusei; SHIMODA, Hiroshi; SUZUKI, Kazuo; MAEDA, Ken

    2015-01-01

    In 2007–2008, a canine distemper virus (CDV) epidemic occurred among wild animals in Wakayama Prefecture, Japan, and many mammals, including the wild boar and deer, were infected. In this study, CDV prevalence among wild animals was surveyed before and after the epidemic. At first, an enzyme-linked immunosorbent assay (ELISA) with horseradish peroxidase-conjugated protein A/G was established to detect CDV antibodies in many mammalian species. This established ELISA was available for testing dogs, raccoons and raccoon dogs as well as virus-neutralization test. Next, a serological survey of wild mammalians was conducted, and it was indicated that many wild mammalians, particularly raccoons, were infected with CDV during the epidemic, but few were infected before and after the epidemic. On the other hand, many raccoon dogs died during the epidemic, but CDV remained prevalent in the remaining population, and a small epidemic occurred in raccoon dogs in 2012–2013. These results indicated that the epidemic of 2007–2008 may have been intensified by transmission to raccoons. PMID:26074342

  16. Canine distemper virus infection among wildlife before and after the epidemic.

    PubMed

    Suzuki, Junko; Nishio, Yohei; Kameo, Yuki; Terada, Yutaka; Kuwata, Ryusei; Shimoda, Hiroshi; Suzuki, Kazuo; Maeda, Ken

    2015-11-01

    In 2007-2008, a canine distemper virus (CDV) epidemic occurred among wild animals in Wakayama Prefecture, Japan, and many mammals, including the wild boar and deer, were infected. In this study, CDV prevalence among wild animals was surveyed before and after the epidemic. At first, an enzyme-linked immunosorbent assay (ELISA) with horseradish peroxidase-conjugated protein A/G was established to detect CDV antibodies in many mammalian species. This established ELISA was available for testing dogs, raccoons and raccoon dogs as well as virus-neutralization test. Next, a serological survey of wild mammalians was conducted, and it was indicated that many wild mammalians, particularly raccoons, were infected with CDV during the epidemic, but few were infected before and after the epidemic. On the other hand, many raccoon dogs died during the epidemic, but CDV remained prevalent in the remaining population, and a small epidemic occurred in raccoon dogs in 2012-2013. These results indicated that the epidemic of 2007-2008 may have been intensified by transmission to raccoons. PMID:26074342

  17. Antibody study in canine distemper virus nucleocapsid protein gene-immunized mice.

    PubMed

    Yuan, B; Li, X Y; Zhu, T; Yuan, L; Hu, J P; Chen, J; Gao, W; Ren, W Z

    2015-01-01

    The gene for the nucleocapsid (N) protein of canine distemper virus was cloned into the pMD-18T vector, and positive recombinant plasmids were obtained by enzyme digestion and sequencing. After digestion by both EcoRI and KpnI, the plasmid was directionally cloned into the eukaryotic expression vector pcDNA; the positive clone pcDNA-N was screened by electrophoresis and then transfected into COS-7 cells. Immunofluorescence analysis results showed that the canine distemper virus N protein was expressed in the cytoplasm of transfected COS-7 cells. After emulsification in Freund's adjuvant, the recombinant plasmid pcDNA-N was injected into the abdominal cavity of 8-week-old BABL/c mice, with the pcDNA original vector used as a negative control. Mice were immunized 3 times every 2 weeks. The blood of immunized mice was drawn 2 weeks after completing the immunizations to measure titer levels. The antibody titer in the pcDNA-N test was 10(1.62 ± 0.164), while in the control group this value was 10(0.52 ± 0.56), indicating that specific humoral immunity was induced in canine distemper virus nucleocapsid protein-immunized mice. PMID:25966074

  18. Duration of Maternal Antibodies against Canine Distemper Virus and Hendra Virus in Pteropid Bats

    PubMed Central

    Zambrana-Torrelio, Carlos; Middleton, Deborah; Barr, Jennifer A.; DuBovi, Edward; Boyd, Victoria; Pope, Brian; Todd, Shawn; Crameri, Gary; Walsh, Allyson; Pelican, Katey; Fielder, Mark D.; Davies, Angela J.; Wang, Lin-Fa; Daszak, Peter

    2013-01-01

    Old World frugivorous bats have been identified as natural hosts for emerging zoonotic viruses of significant public health concern, including henipaviruses (Nipah and Hendra virus), Ebola virus, and Marburg virus. Epidemiological studies of these viruses in bats often utilize serology to describe viral dynamics, with particular attention paid to juveniles, whose birth increases the overall susceptibility of the population to a viral outbreak once maternal immunity wanes. However, little is understood about bat immunology, including the duration of maternal antibodies in neonates. Understanding duration of maternally derived immunity is critical for characterizing viral dynamics in bat populations, which may help assess the risk of spillover to humans. We conducted two separate studies of pregnant Pteropus bat species and their offspring to measure the half-life and duration of antibodies to 1) canine distemper virus antigen in vaccinated captive Pteropus hypomelanus; and 2) Hendra virus in wild-caught, naturally infected Pteropus alecto. Both of these pteropid bat species are known reservoirs for henipaviruses. We found that in both species, antibodies were transferred from dam to pup. In P. hypomelanus pups, titers against CDV waned over a mean period of 228.6 days (95% CI: 185.4–271.8) and had a mean terminal phase half-life of 96.0 days (CI 95%: 30.7–299.7). In P. alecto pups, antibodies waned over 255.13 days (95% CI: 221.0–289.3) and had a mean terminal phase half-life of 52.24 days (CI 95%: 33.76–80.83). Each species showed a duration of transferred maternal immunity of between 7.5 and 8.5 months, which was longer than has been previously estimated. These data will allow for more accurate interpretation of age-related Henipavirus serological data collected from wild pteropid bats. PMID:23826322

  19. Duration of Maternal Antibodies against Canine Distemper Virus and Hendra Virus in Pteropid Bats.

    PubMed

    Epstein, Jonathan H; Baker, Michelle L; Zambrana-Torrelio, Carlos; Middleton, Deborah; Barr, Jennifer A; Dubovi, Edward; Boyd, Victoria; Pope, Brian; Todd, Shawn; Crameri, Gary; Walsh, Allyson; Pelican, Katey; Fielder, Mark D; Davies, Angela J; Wang, Lin-Fa; Daszak, Peter

    2013-01-01

    Old World frugivorous bats have been identified as natural hosts for emerging zoonotic viruses of significant public health concern, including henipaviruses (Nipah and Hendra virus), Ebola virus, and Marburg virus. Epidemiological studies of these viruses in bats often utilize serology to describe viral dynamics, with particular attention paid to juveniles, whose birth increases the overall susceptibility of the population to a viral outbreak once maternal immunity wanes. However, little is understood about bat immunology, including the duration of maternal antibodies in neonates. Understanding duration of maternally derived immunity is critical for characterizing viral dynamics in bat populations, which may help assess the risk of spillover to humans. We conducted two separate studies of pregnant Pteropus bat species and their offspring to measure the half-life and duration of antibodies to 1) canine distemper virus antigen in vaccinated captive Pteropus hypomelanus; and 2) Hendra virus in wild-caught, naturally infected Pteropus alecto. Both of these pteropid bat species are known reservoirs for henipaviruses. We found that in both species, antibodies were transferred from dam to pup. In P. hypomelanus pups, titers against CDV waned over a mean period of 228.6 days (95% CI: 185.4-271.8) and had a mean terminal phase half-life of 96.0 days (CI 95%: 30.7-299.7). In P. alecto pups, antibodies waned over 255.13 days (95% CI: 221.0-289.3) and had a mean terminal phase half-life of 52.24 days (CI 95%: 33.76-80.83). Each species showed a duration of transferred maternal immunity of between 7.5 and 8.5 months, which was longer than has been previously estimated. These data will allow for more accurate interpretation of age-related Henipavirus serological data collected from wild pteropid bats. PMID:23826322

  20. Characteristics of a Canine Distemper Virus Outbreak in Dichato, Chile Following the February 2010 Earthquake.

    PubMed

    Garde, Elena; Pérez, Guillermo; Acosta-Jamett, Gerardo; Bronsvoort, Barend Mark

    2013-01-01

    Following the earthquake and tsunami disaster in Chile in February 2010, residents of Dichato reported high morbidity and mortality in dogs, descriptions of which resembled canine distemper virus (CDV). To assess the situation, free vaccine clinics were offered in April and May. Owner information, dog history and signalment were gathered; dogs received physical examinations and vaccines protecting against CDV, and other common canine pathogens. Blood was collected to screen for IgM antibodies to CDV. In total, 208 dogs received physical exams and vaccines were given to 177. IgM antibody titres to CDV were obtained for 104 dogs. Fifty-four dogs (51.9%) tested positive for CDV at the cut off titre of >1:50, but a total of 91.4% of dogs had a detectable titre >1:10. Most of the positive test results were in dogs less than 2 years of age; 33.5% had been previously vaccinated against CDV, and owners of 84 dogs (42.2%) reported clinical signs characteristic of CDV in their dogs following the disaster. The presence of endemic diseases in dog populations together with poor pre-disaster free-roaming dog management results in a potential for widespread negative effects following disasters. Creation of preparedness plans that include animal welfare, disease prevention and mitigation should be developed. PMID:26479537

  1. Prevalence of antibodies to canine parvovirus and distemper virus in wolves in the Canadian Rocky Mountains.

    PubMed

    Nelson, Brynn; Hebblewhite, Mark; Ezenwa, Vanessa; Shury, Todd; Merrill, Evelyn H; Paquet, Paul C; Schmiegelow, Fiona; Seip, Dale; Skinner, Geoff; Webb, Nathan

    2012-01-01

    Wild carnivores are often exposed to diseases via contact with peridomestic host species that travel through the wildland-urban interfaces. To determine the antibody prevalences and relationships to human activity for two common canid pathogens, we sampled 99 wolves (Canis lupus) from 2000 to 2008 for antibodies to canine parvovirus (CPV) and canine distemper virus (CDV) in Banff and Jasper National Parks and surrounding areas of the Canadian Rockies. This population was the source for wolves reintroduced into the Northern Rockies of the US. Of 99 wolves sampled, 94 had detectable antibody to CPV (95%), 24 were antibody-positive for CDV (24%), and 24 had antibodies to both pathogens (24%). We tested whether antibody prevalences for CPV and CDV were higher closer to human activity (roads, town sites, First Nation reserves) and as a function of sex and age class. Wolves ≥2 yr old were more likely to be have antibodies to CPV. For CDV, male wolves, wolves ≥2 yr, and those closer to First Nation reserves were more likely to have antibodies. Overall, however, we found minimal support for human influence on antibody prevalence for CDV and CPV. The similarity between our antibody prevalence results and results from recent studies in Yellowstone National Park suggests that at least in the case of CDV, and perhaps CPV, these could be important pathogens with potential effects on wolf populations. PMID:22247375

  2. Low Titers of Canine Distemper Virus Antibody in Wild Fishers (Martes pennanti) in the Eastern USA.

    PubMed

    Peper, Steven T; Peper, Randall L; Mitcheltree, Denise H; Kollias, George V; Brooks, Robert P; Stevens, Sadie S; Serfass, Thomas L

    2016-01-01

    Canine distemper virus (CDV) infects species in the order Carnivora. Members of the family Mustelidae are among the species most susceptible to CDV and have a high mortality rate after infection. Assessing an animal's pathogen or disease load prior to any reintroduction project is important to help protect the animal being reintroduced, as well as the wildlife and livestock in the area of relocation. We screened 58 fishers for CDV antibody prior to their release into Pennsylvania, US, as part of a reintroduction program. Five of the 58 (9%) fishers had a weak-positive reaction for CDV antibody at a dilution of 1:16. None of the fishers exhibited any clinical sign of canine distemper while being held prior to release. PMID:26555109

  3. Canine distemper virus infection in fennec fox (Vulpes zerda).

    PubMed

    Woo, Gye-Hyeong; Jho, Yeon-Sook; Bak, Eun-Jung

    2010-08-01

    Fifteen 8-month-old fennec foxes imported from Sudan showed fever, mucopurulent ocular discharge, diarrhea, severe emaciation, seizures, and generalized ataxia, and died. Three of the 15 animals were presented for diagnostic investigation. Severe dehydration, brain congestion, and gastric ulcers were observed in all animals. In one animal, the lungs had failed to collapse and were multifocally dark red in appearance. Histopathologically, there were lymphohistiocytic meningoencephalitis with malacia, mild interstitial pneumonia, lymphoid depletion of lymphoid tissues and organs, and intestinal villous atrophy with intralesional coccidia. There were many intracytoplasmic and/or intranuclear inclusion bodies in the epithelial cells of the medullary velum, lungs, liver, kidneys, trachea, pancreas, stomach, gall bladder, urinary bladder, and ureters, and in macrophages of malacia foci and lymphocytes and macrophages of lymphoid organs. Additionally, intestinal coccidia were confirmed to be Isospora species by a fecal test. To our knowledge, this is the first report of canine distemper with intestinal coccidiosis in fennec fox. PMID:20299771

  4. EVALUATION OF TWO CANINE DISTEMPER VIRUS VACCINES IN CAPTIVE TIGERS (PANTHERA TIGRIS).

    PubMed

    Sadler, Ryan A; Ramsay, Edward; McAloose, Denise; Rush, Robert; Wilkes, Rebecca P

    2016-06-01

    Canine distemper virus (CDV) has caused clinical disease and death in nondomestic felids in both captive settings and in the wild. Outbreaks resulting in high mortality rates in tigers (Panthera tigris) have prompted some zoos to vaccinate tigers for CDV. In this study, six tigers received a recombinant canarypox-vectored CDV vaccine (1 ml s.c.) and were revaccinated with 3 ml s.c. (mean) 39 days later. Blood collection for CDV antibody detection via serum neutralization was performed on (mean) days 0, 26, and 66 post-initial vaccination. No tigers had detectable antibodies at days 0 or 26, and only two tigers had low (16 and 32) antibody titers at day 66. Eight additional tigers received a live, attenuated CDV vaccine (1 ml s.c.) on day 0 and were revaccinated with 1 ml s.c. (mean) 171 days later. Blood collection for CDV antibody detection via serum neutralization was performed on (mean) days 0, 26, 171, and 196. Seven of eight tigers receiving the live, attenuated vaccine had no detectable titers prior to vaccination, but all animals had titers of >128 (range 128-1,024) at day 26. At 171 days, all tigers still had detectable titers (geometric mean 69.8, range 16-256), and at 196 days (2 wk post-revaccination) all but two showed an increase to >128 (range 32-512). To determine safety, an additional 41 tigers were vaccinated with 2 ml of a recombinant vaccine containing only CDV components, and an additional 38 tigers received 1 ml of the live, attenuated vaccine, administered either subcutaneously or intramuscularly; no serious adverse effects were noted. Although both vaccines appear safe, the live, attenuated vaccine produced a stronger and more consistent serologic response in tigers. PMID:27468029

  5. Cloning and characterization of DNA complementary to the canine distemper virus mRNA encoding matrix, phosphoprotein, and nucleocapsid protein

    SciTech Connect

    Rozenblatt, S.; Eizenberg, O.; Englund, G.; Bellini, W.J.

    1985-02-01

    Double-stranded cDNA synthesized from total polyadenylate-containing mRNA, extracted from monkey kidney cells infected with canine distemper virus (CDV), has been cloned into the PstI site of Escherichia coli plasmid pBR322. Clones containing canine distemper virus DNA were identified by hybridization to a canine distemper virus-specific, /sup 32/P-labeled cDNA. Four specific clones containing different classes of sequences have been identified. The cloned plasmids contain inserts of 800 (clone 44-80), 960 (clone 74-16), 1700 (clone 364), and 950 (clone 40-9) base pairs. The sizes of the mRNA species complementary to these inserts are 1500, 1850, 1850 and 2500 nucleotides, respectively, as determined by the Northern technique. Three of the cloned DNA fragments were further identified as the reverse transcripts of the mRNA coding for the matrix, phosphoprotein, and nucleocapsid protein of CDV.

  6. Pathogenesis of canine distemper virus in experimentally infected raccoon dogs, foxes, and minks.

    PubMed

    Zhao, Jianjun; Shi, Ning; Sun, Yangang; Martella, Vito; Nikolin, Veljko; Zhu, Chunsheng; Zhang, Hailing; Hu, Bo; Bai, Xue; Yan, Xijun

    2015-10-01

    Canine distemper virus (CDV) infects a broad range of carnivores and causes a highly contagious disease with severe immunosuppression. The disease severity markedly varies in different species. To investigate the pathogenesis of CDV in raccoon dog (Nyctereutes procyonoides), fox (Vulpes vulpes) and mink (Neovison vison) species, three groups of CDV sero-negative animals were infected with CDV strain LN(10)1. This CDV strain belongs to the Asia-1 genotype, which is epidemiologically predominant in carnivores in China. CDV infection provoked marked differences in virulence in the three species that were studied. Raccoon dogs developed fever, severe conjunctivitis, and pathological lesions, with 100% (5/5) mortality and with high viral RNA loads in organs within 15 days post infection (dpi). In infected foxes, the onset of the disease was delayed, with 40% (2/5) mortality by 21 dpi. Infected minks developed only mild clinical signs and pathological lesions, and mortality was not observed. Raccoon dogs and foxes showed more severe immune suppression (lymphopenia, decreased lymphocyte proliferation, viremia and low-level virus neutralizing antibodies) than minks. We also observed a distinct pattern of cytokine mRNA transcripts at different times after infection. Decreased IFN-γ and IL-4 mRNA responses were evident in the animals with fatal disease, while up-regulation of these cytokines was observed in the animals surviving the infection. Increased TNF-α response was detected in animals with mild or severe clinical signs. Based on the results, we could distinguish three different patterns of disease after experimental CDV infection, e.g. a mild form in minks, a moderate form in foxes and a severe disease in raccoon dogs. The observed differences in susceptibility to CDV could be related to distinct host cytokine profiles. Comparative evaluation of CDV pathogenesis in various animal species is pivotal to generate models suitable for the evaluation of CDV

  7. Characterization of a novel Canine distemper virus causing disease in wildlife.

    PubMed

    Pope, Jenny P; Miller, Debra L; Riley, Matthew C; Anis, Eman; Wilkes, Rebecca P

    2016-09-01

    Canine distemper virus (CDV) is a common cause of a multisystemic disease in both domestic dogs and wildlife species, including raccoons and foxes. Outbreaks of CDV in domestic dogs in eastern Tennessee have occurred since 2012, and it was determined that these outbreaks resulted from a novel genotype of CDV. We hypothesized that this virus is also infecting area wildlife and may be a source of the virus for these outbreaks in dogs. From 2013 to 2014, autopsies were performed and tissues collected from raccoons (Procyon lotor; n = 50) and gray foxes (Urocyon cinereoargenteus; n = 8) for CDV testing. A real-time reverse transcription PCR was used to document the presence of CDV in tissue samples, and a portion of the virus was subsequently sequenced for phylogenetic analysis. A high percentage of wildlife, both with (86%) and without (55%) clinical signs, tested positive for CDV, with the majority (77%) testing positive for the novel genotype. Microscopic findings, including syncytia in the lungs and viral inclusion bodies in urothelium, astrocytes, neurons, and bronchiolar epithelium, were also consistent with canine distemper. Minimal inflammation in the central nervous system of affected animals was indicative of the acute neurologic form of the disease. Pneumonia and parasitism were also commonly found in CDV-infected animals. Based on these results, CDV appears to be prevalent in eastern Tennessee wildlife. Subclinical or clinically recovered shedders are a potential source of this novel genotype for domestic dogs, and this genotype is genetically distinct from vaccine strains. PMID:27400957

  8. Fatal combined infection with canine distemper virus and orthopoxvirus in a group of Asian marmots (Marmota caudata).

    PubMed

    Origgi, F C; Sattler, U; Pilo, P; Waldvogel, A S

    2013-09-01

    A fatal combined infection with canine distemper virus (CDV) and orthopoxvirus (OPXV) in Asian marmots (Marmota caudata) is reported in this article. A total of 7 Asian marmots from a small zoological garden in Switzerland were found dead in hibernation during a routine check in the winter of 2011. The marmots died in February 2011. No clinical signs of disease were observed at any time. The viruses were detected in all individuals for which the tissues were available (n = 3). Detection of the viruses was performed by reverse transcription polymerase chain reaction. The most consistent gross lesion was a neck and thorax edema. A necrotizing pharyngitis and a multifocal necrotizing pneumonia were observed histologically. Numerous large intracytoplasmic eosinophilic inclusions were seen in the epithelial cells of the pharynx, of the airways, and in the skin keratinocytes. Brain lesions were limited to mild multifocal gliosis. Phylogenetic analysis revealed that the marmot CDV strain was closely related to the clusters of CDVs detected in Switzerland in wild carnivores during a local outbreak in 2002 and the 2009-2010 nationwide epidemic, suggesting a spillover of this virus from wildlife. The OPXV was most closely related to a strain of cowpoxvirus, a poxvirus species considered endemic in Europe. This is the first reported instance of CDV infection in a rodent species and of a combined CDV and OPXV infection. PMID:23381928

  9. Use of serologic tests to predict resistance to Canine distemper virus-induced disease in vaccinated dogs.

    PubMed

    Jensen, Wayne A; Totten, Janet S; Lappin, Michael R; Schultz, Ronald D

    2015-09-01

    The objective of the current study was to determine whether detection of Canine distemper virus (CDV)-specific serum antibodies correlates with resistance to challenge with virulent virus. Virus neutralization (VN) assay results were compared with resistance to viral challenge in 2 unvaccinated Beagle puppies, 9 unvaccinated Beagle dogs (4.4-7.2 years of age), and 9 vaccinated Beagle dogs (3.7-4.7 years of age). Eight of 9 (89%) unvaccinated adult dogs exhibited clinical signs after virus challenge, and 1 (13%) dog died. As compared to adult dogs, the 2 unvaccinated puppies developed more severe clinical signs and either died or were euthanized after challenge. In contrast, no clinical signs were detected after challenge of the 9 adult vaccinated dogs with post-vaccination intervals of up to 4.4 years. In vaccinated dogs, the positive and negative predictive values of VN assay results for resistance to challenge were 100% and 0%, respectively. Results indicate that dogs vaccinated with modified live CDV can be protected from challenge for ≤4.4 years postvaccination and that detection of virus-specific antibodies is predictive of whether dogs are resistant to challenge with virulent virus. Results also indicate that CDV infection in unvaccinated dogs results in age-dependent morbidity and mortality. Knowledge of age-dependent morbidity and mortality, duration of vaccine-induced immunity, and the positive and negative predictive values of detection of virus-specific serum antibodies are useful in development of rational booster vaccination intervals for the prevention of CDV-mediated disease in adult dogs. PMID:26330396

  10. Rabies virus and canine distemper virus in wild and domestic carnivores in Northern Kenya: are domestic dogs the reservoir?

    PubMed

    Prager, K C; Mazet, Jonna A K; Dubovi, Edward J; Frank, Laurence G; Munson, Linda; Wagner, Aaron P; Woodroffe, Rosie

    2012-12-01

    Rabies virus (RV) and canine distemper virus (CDV) can cause significant mortality in wild carnivore populations, and RV threatens human lives. We investigated serological patterns of exposure to CDV and RV in domestic dogs (Canis familiaris), African wild dogs (Lycaon pictus), black-backed jackals (Canis mesomelas), spotted hyenas (Crocuta crocuta), striped hyenas (Hyaena hyaena) and African lions (Panthera leo), over a 10-year period, in a Kenyan rangeland to assess the role domestic dogs may play in the transmission dynamics of these two important canid pathogens. Observed patterns of RV exposure suggested that repeated introduction, rather than maintenance, occurred in the wild carnivore species studied. However, RV appeared to have been maintained in domestic dogs: exposure was more likely in domestic dogs than in the wild carnivores; was detected consistently over time without variation among years; and was detected in juveniles (≤1-year-old) as well as adults (>1-year-old). We conclude that this domestic dog population could be a RV reservoir. By contrast, the absence of evidence of CDV exposure for each carnivore species examined in the study area, for specific years, suggested repeated introduction, rather than maintenance, and that CDV may require a larger reservoir population than RV. This reservoir could be a larger domestic dog population; another wildlife species; or a "metareservoir" consisting of multiple interconnected carnivore populations. Our findings suggest that RV risks to people and wild carnivores might be controlled by domestic dog vaccination, but that CDV control, if required, would need to target the species of concern. PMID:23459924

  11. Canine distemper virus neutralization activity is low in human serum and it is sensitive to an amino acid substitution in the hemagglutinin protein

    SciTech Connect

    Zhang, Xinsheng; Wallace, Olivia L.; Domi, Arban; Wright, Kevin J.; Driscoll, Jonathan; Anzala, Omu; Sanders, Eduard J.; Kamali, Anatoli; Allen, Susan; Fast, Pat; Gilmour, Jill; Price, Matt A.; Parks, Christopher L.

    2015-08-15

    Serum was analyzed from 146 healthy adult volunteers in eastern Africa to evaluate measles virus (MV) and canine distemper virus (CDV) neutralizing antibody (nAb) prevalence and potency. MV plaque reduction neutralization test (PRNT) results indicated that all sera were positive for MV nAbs. Furthermore, the 50% neutralizing dose (ND50) for the majority of sera corresponded to antibody titers induced by MV vaccination. CDV nAbs titers were low and generally were detected in sera with high MV nAb titers. A mutant CDV was generated that was less sensitive to neutralization by human serum. The mutant virus genome had 10 nucleotide substitutions, which coded for single amino acid substitutions in the fusion (F) and hemagglutinin (H) glycoproteins and two substitutions in the large polymerase (L) protein. The H substitution occurred in a conserved region involved in receptor interactions among morbilliviruses, implying that this region is a target for cross-reactive neutralizing antibodies. - Highlights: • Screened 146 serum samples for measles virus (MV) and canine distemper virus (CDV) neutralizing antibody (nAb). • MV nAb is prevalent in the sera. • CDV neutralizing activity is generally low or absent and when detected it is present in sera with high MV nAb titers. • A neutralization-resistant CDV mutant was isolated using human serum selection. • A mutation was identified in the receptor-binding region of CDV hemagglutinin protein that confers the neutralization resistance.

  12. Canine Distemper Virus: an Emerging Disease in Wild Endangered Amur Tigers (Panthera tigris altaica)

    PubMed Central

    Seimon, Tracie A.; Miquelle, Dale G.; Chang, Tylis Y.; Newton, Alisa L.; Korotkova, Irina; Ivanchuk, Galina; Lyubchenko, Elena; Tupikov, Andre; Slabe, Evgeny; McAloose, Denise

    2013-01-01

    ABSTRACT Fewer than 500 Amur tigers (Panthera tigris altaica) remain in the wild. Due to low numbers and their solitary and reclusive nature, tiger sightings across their range in the Russian Far East and China are rare; sightings of sick tigers are rarer still. Serious neurologic disease observed in several wild tigers since 2001 suggested disease emergence in this endangered species. To investigate this possibility, histology, immunohistochemistry (IHC), in situ hybridization (ISH), and reverse transcription-PCR (RT-PCR) were performed on tissues from 5 affected tigers that died or were destroyed in 2001, 2004, or 2010. Our results reveal canine distemper virus (CDV) infection as the cause of neurologic disease in two tigers and definitively establish infection in a third. Nonsuppurative encephalitis with demyelination, eosinophilic nuclear viral inclusions, and positive immunolabeling for CDV by IHC and ISH were present in the two tigers with available brain tissue. CDV phosphoprotein (P) and hemagglutinin (H) gene products were obtained from brains of these two tigers by RT-PCR, and a short fragment of CDV P gene sequence was detected in lymph node tissue of a third tiger. Phylogenetically, Amur tiger CDV groups with an Arctic-like strain in Baikal seals (Phoca siberica). Our results, which include mapping the location of positive tigers and recognition of a cluster of cases in 2010, coupled with a lack of reported CDV antibodies in Amur tigers prior to 2000 suggest wide geographic distribution of CDV across the tiger range and recent emergence of CDV as a significant infectious disease threat to endangered Amur tigers in the Russian Far East. PMID:23943758

  13. A comparison of the immune responses of dogs exposed to canine distemper virus (CDV) — Differences between vaccinated and wild-type virus exposed dogs

    PubMed Central

    Perrone, Danielle; Bender, Scott; Niewiesk, Stefan

    2010-01-01

    Canine distemper virus (CDV)-specific immune response was measured in different dog populations. Three groups of vaccinated or wild-type virus exposed dogs were tested: dogs with a known vaccination history, dogs without a known vaccination history (shelter dogs), and dogs with potential exposure to wild-type CDV. The use of a T-cell proliferation assay demonstrated a detectable CDV-specific T-cell response from both spleen and blood lymphocytes of dogs. Qualitatively, antibody assays [enzyme-linked immunosorbent assay (ELISA) and neutralization assay] predicted the presence of a T-cell response well, although quantitatively neither antibody assays nor the T-cell assay correlated well with each other. An interesting finding from our study was that half of the dogs in shelters were not vaccinated (potentially posing a public veterinary health problem) and that antibody levels in dogs living in an environment with endemic CDV were lower than in vaccinated animals. PMID:20885846

  14. Coinfection with Hepatozoon sp. and Canine Distemper Virus in a Yellow-throated Marten ( Martes flavigula koreana) in Korea.

    PubMed

    Park, Surim; Choi, Ul Soo; Kim, Eun Ju; Lee, Jong Hyun; Lee, Hae Beom; Cho, Ho Seong; Kim, Wonil; Lim, Chae Woong; Kim, Bumseok

    2016-04-28

    We describe coinfection with Hepatozoon sp. and canine distemper virus (CDV) in a yellow-throated marten ( Martes flavigula koreana). We found Hepatozoon cysts in muscular tissue and viral inclusion bodies in the brain. Hepatozoon sp., and CDV was confirmed in blood and brain, respectively, by PCR. PMID:27054471

  15. Canine Distemper Virus Epithelial Cell Infection Is Required for Clinical Disease but Not for Immunosuppression

    PubMed Central

    Sawatsky, Bevan; Wong, Xiao-Xiang; Hinkelmann, Sarah; Cattaneo, Roberto

    2012-01-01

    To characterize the importance of infection of epithelial cells for morbillivirus pathogenesis, we took advantage of the severe disease caused by canine distemper virus (CDV) in ferrets. To obtain a CDV that was unable to enter epithelial cells but retained the ability to enter immune cells, we transferred to its attachment (H) protein two mutations shown to interfere with the interaction of measles virus H with its epithelial receptor, human nectin-4. As expected for an epithelial receptor (EpR)-blind CDV, this virus infected dog and ferret epithelial cells inefficiently and did not cause cell fusion or syncytium formation. On the other hand, the EpR-blind CDV replicated in cells expressing canine signaling lymphocyte activation molecule (SLAM), the morbillivirus immune cell receptor, with similar kinetics to those of wild-type CDV. While ferrets infected with wild-type CDV died within 12 days after infection, after developing severe rash and fever, animals infected with the EpR-blind virus showed no clinical signs of disease. Nevertheless, both viruses spread rapidly and efficiently in immune cells, causing similar levels of leukopenia and inhibition of lymphocyte proliferation activity, two indicators of morbillivirus immunosuppression. Infection was documented for airway epithelia of ferrets infected with wild-type CDV but not for those of animals infected with the EpR-blind virus, and only animals infected with wild-type CDV shed virus. Thus, epithelial cell infection is necessary for clinical disease and efficient virus shedding but not for immunosuppression. PMID:22278252

  16. In-vitro antiviral efficacy of ribavirin and interferon-alpha against canine distemper virus

    PubMed Central

    Carvalho, Otávio V.; Saraiva, Giuliana L.; Ferreira, Caroline G.T.; Felix, Daniele M.; Fietto, Juliana L.R.; Bressan, Gustavo C.; Almeida, Márcia R.; Silva Júnior, Abelardo

    2014-01-01

    Canine distemper is a highly contagious disease with high incidence and lethality in the canine population. The objective of this study was to evaluate the efficacy of antiviral action with ribavirin (RBV), interferon-alpha (IFNα), and combinations of RBV and IFNα against canine distemper virus (CDV). Vero cells inoculated with CDV were treated with RBV, IFNα, and combinations of these drugs. The efficacy to inhibit viral replication was evaluated by adding the compounds at different times to determine which step of the viral replicative process was affected. Both drugs were effective against CDV in vitro. The IFNα was the most active compound, with an average IC50 (50% inhibitory concentration) value lower than the IC50 of the RBV. Ribavirin (RBV) was more selective than IFNα, however, and neither drug showed extracellular antiviral activity. The combination of RBV and IFNα exhibited antiviral activity for the intra- and extracellular stages of the replicative cycle of CDV, although the intracellular viral inhibition was higher. Both RBV and IFNα showed high antiviral efficacy against CDV, and furthermore, RBV + IFNα combinations have shown greater interference range in viral infectivity. These compounds could potentially be used to treat clinical disease associated with CDV infection. PMID:25355997

  17. Expression of a foreign gene by recombinant canine distemper virus recovered from cloned DNAs.

    PubMed

    Parks, Christopher L; Wang, Hai-Ping; Kovacs, Gerald R; Vasilakis, Nikos; Kowalski, Jacek; Nowak, Rebecca M; Lerch, Robert A; Walpita, Pramila; Sidhu, Mohinderjit S; Udem, Stephen A

    2002-02-26

    A canine distemper virus (CDV) genomic cDNA clone and expression plasmids required to establish a CDV rescue system were generated from a laboratory-adapted strain of the Onderstepoort vaccine virus. In addition, a CDV minireplicon was prepared and used in transient expression studies performed to identify optimal virus rescue conditions. Results from the transient expression experiments indicated that minireplicon-encoded reporter gene activity was increased when transfected cell cultures were maintained at 32 rather than 37 degrees C, and when the cellular stress response was induced by heat shock. Applying these findings to rescue of recombinant CDV (rCDV) resulted in efficient recovery of virus after transfected HEp2 or A549 cells were co-cultured with Vero cell monolayers. Nucleotide sequence determination and analysis of restriction site polymorphisms confirmed that rescued virus was rCDV. A rCDV strain also was engineered that contained the luciferase gene inserted between the P and M genes; this virus directed high levels of luciferase expression in infected cells. PMID:11864746

  18. Viral Oncolysis — Can Insights from Measles Be Transferred to Canine Distemper Virus?

    PubMed Central

    Lapp, Stefanie; Pfankuche, Vanessa M.; Baumgärtner, Wolfgang; Puff, Christina

    2014-01-01

    Neoplastic diseases represent one of the most common causes of death among humans and animals. Currently available and applied therapeutic options often remain insufficient and unsatisfactory, therefore new and innovative strategies and approaches are highly needed. Periodically, oncolytic viruses have been in the center of interest since the first anecdotal description of their potential usefulness as an anti-tumor treatment concept. Though first reports referred to an incidental measles virus infection causing tumor regression in a patient suffering from lymphoma several decades ago, no final treatment concept has been developed since then. However, numerous viruses, such as herpes-, adeno- and paramyxoviruses, have been investigated, characterized, and modified with the aim to generate a new anti-cancer treatment option. Among the different viruses, measles virus still represents a highly interesting candidate for such an approach. Numerous different tumors of humans including malignant lymphoma, lung and colorectal adenocarcinoma, mesothelioma, and ovarian cancer, have been studied in vitro and in vivo as potential targets. Moreover, several concepts using different virus preparations are now in clinical trials in humans and may proceed to a new treatment option. Surprisingly, only few studies have investigated viral oncolysis in veterinary medicine. The close relationship between measles virus (MV) and canine distemper virus (CDV), both are morbilliviruses, and the fact that numerous tumors in dogs exhibit similarities to their human counterpart, indicates that both the virus and species dog represent a highly interesting translational model for future research in viral oncolysis. Several recent studies support such an assumption. It is therefore the aim of the present communication to outline the mechanisms of morbillivirus-mediated oncolysis and to stimulate further research in this potentially expanding field of viral oncolysis in a highly suitable

  19. Cytotoxic T-lymphocyte activity specific for hemagglutinin (H) protein of canine distemper virus in dogs.

    PubMed

    Hirama, Kyoko; Togashi, Ken-ichi; Wakasa, Chiaki; Yoneda, Misako; Nishi, Toshiya; Endo, Yasuyuki; Miura, Ryuichi; Tsukiyama-Kohara, Kyoko; Kai, Chieko

    2003-01-01

    Cytotoxic T-lymphocyte (CTL) responses to hemagglutinin (H) protein of canine distemper virus (CDV) were evaluated in dogs using the replication-deficient adenovirus protein expression system. Skin fibroblasts were isolated from two dogs and were infected with recombinant adenovirus bearing the CDV-H gene (Ade-CDVH). CTL assay was performed using fibroblasts expressing CDV-H protein as target cells and peripheral blood lymphocytes (PBL) collected from the same dogs one week after immunization of CDV as effector cells. Specific cytotoxic activity was observed against autologous but not heterologous fibroblasts expressing CDV-H protein. These results indicate that the CTL epitope(s) were localized in the H protein. PMID:12576714

  20. Canine distemper virus neutralization activity is low in human serum and it is sensitive to an amino acid substitution in the hemagglutinin protein.

    PubMed

    Zhang, Xinsheng; Wallace, Olivia L; Domi, Arban; Wright, Kevin J; Driscoll, Jonathan; Anzala, Omu; Sanders, Eduard J; Kamali, Anatoli; Karita, Etienne; Allen, Susan; Fast, Pat; Gilmour, Jill; Price, Matt A; Parks, Christopher L

    2015-08-01

    Serum was analyzed from 146 healthy adult volunteers in eastern Africa to evaluate measles virus (MV) and canine distemper virus (CDV) neutralizing antibody (nAb) prevalence and potency. MV plaque reduction neutralization test (PRNT) results indicated that all sera were positive for MV nAbs. Furthermore, the 50% neutralizing dose (ND50) for the majority of sera corresponded to antibody titers induced by MV vaccination. CDV nAbs titers were low and generally were detected in sera with high MV nAb titers. A mutant CDV was generated that was less sensitive to neutralization by human serum. The mutant virus genome had 10 nucleotide substitutions, which coded for single amino acid substitutions in the fusion (F) and hemagglutinin (H) glycoproteins and two substitutions in the large polymerase (L) protein. The H substitution occurred in a conserved region involved in receptor interactions among morbilliviruses, implying that this region is a target for cross-reactive neutralizing antibodies. PMID:25880113

  1. Molecular phylogeography of canine distemper virus: Geographic origin and global spreading.

    PubMed

    Panzera, Yanina; Sarute, Nicolás; Iraola, Gregorio; Hernández, Martín; Pérez, Ruben

    2015-11-01

    Canine distemper virus (CDV) (Paramyxoviridae-Morbillivirus) is a worldwide spread virus causing a fatal systemic disease in a broad range of carnivore hosts. In this study we performed Bayesian inferences using 208 full-length hemagglutinin gene nucleotide sequences isolated in 16 countries during 37 years (1975-2011). The estimated time to the most recent common ancestor suggested that current CDV strains emerged in the United States in the 1880s. This ancestor diversified through time into two ancestral clades, the current America 1 lineage that recently spread to Asia, and other ancestral clade that diversified and spread worldwide to originate the remaining eight lineages characterized to date. The spreading of CDV was characterized by several migratory events with posterior local differentiation, and expansion of the virus host range. A significant genetic flow between domestic and wildlife hosts is displayed; being domestic hosts the main viral reservoirs worldwide. This study is an extensive and integrative description of spatio/temporal population dynamics of CDV lineages that provides a novel evolutionary paradigm about the origin and dissemination of the current strains of the virus. PMID:26151219

  2. Arctic Lineage-Canine Distemper Virus as a Cause of Death in Apennine Wolves (Canis lupus) in Italy

    PubMed Central

    Di Sabatino, Daria; Lorusso, Alessio; Di Francesco, Cristina E.; Gentile, Leonardo; Di Pirro, Vincenza; Bellacicco, Anna Lucia; Giovannini, Armando; Di Francesco, Gabriella; Marruchella, Giuseppe; Marsilio, Fulvio; Savini, Giovanni

    2014-01-01

    Canine distemper virus (CDV) infection is a primary threat affecting a wide number of carnivore species, including wild animals. In January 2013, two carcasses of Apennine wolves (Canis lupus) were collected in Ortona dei Marsi (L'Aquila province, Italy) by the local Veterinary Services. CDV was immediately identified either by RT-PCR or immunohistochemistry in lung and central nervous tissue samples. At the same time, severe clinical signs consistent with CDV infection were identified and taped (Videos S1–S3) from three wolves rescued in the areas surrounding the National Parks of the Abruzzi region by the Veterinary Services. The samples collected from these symptomatic animals also turned out CDV positive by RT-PCR. So far, 30 carcasses of wolves were screened and CDV was detected in 20 of them. The sequencing of the haemagglutinin gene and subsequent phylogenetic analysis demonstrated that the identified virus belonged to the CDV Arctic lineage. Strains belonging to this lineage are known to circulate in Italy and in Eastern Europe amongst domestic dogs. To the best of our knowledge this is the first report of CDV Arctic lineage epidemics in the wild population in Europe. PMID:24465373

  3. Use of hydrophilic extra-viral domain of canine distemper virus H protein for enzyme-linked immunosorbent assay development

    PubMed Central

    Cho, Ki-hyun; Kim, Jeongmi; Yoo, Hyun-ah; Kim, Dae-hee; Park, Seung-yong; Song, Chang-seon; Choi, In-soo

    2014-01-01

    Simple methods for measuring the levels of serum antibody against canine distemper virus (CDV) would assist in the effective vaccination of dogs. To develop an enzyme-linked immunosorbent assay (ELISA) specific for CDV, we expressed hydrophilic extra-viral domain (HEVD) protein of the A75/17-CDV H gene in a pET 28a plasmid-based Escherichia (E.) coli vector system. Expression was confirmed by dot and Western blotting. We proposed that detection of E. coli-expressed H protein might be conformation-dependent because intensities of the reactions observed with these two methods varied. The H gene HEVD protein was further purified and used as an antigen for an ELISA. Samples from dogs with undetectable to high anti-CDV antibody titers were analyzed using this HEVD-specific ELISA and a commercial CDV antibody detection kit (ImmunoComb). Levels of HEVD antigenicity measured with the assays and immunochromatography correlated. These data indicated that the HEDV protein may be used as antigen to develop techniques for detecting antibodies against CDV. PMID:25234325

  4. Use of hydrophilic extra-viral domain of canine distemper virus H protein for enzyme-linked immunosorbent assay development.

    PubMed

    Cho, Ki-hyun; Kim, Jeongmi; Yoo, Hyun-ah; Kim, Dae-hee; Park, Seung-yong; Song, Chang-seon; Choi, In-soo; Lee, Joong-bok

    2014-12-01

    Simple methods for measuring the levels of serum antibody against canine distemper virus (CDV) would assist in the effective vaccination of dogs. To develop an enzyme-linked immunosorbent assay (ELISA) specific for CDV, we expressed hydrophilic extra-viral domain (HEVD) protein of the A75/17-CDV H gene in a pET 28a plasmid-based Escherichia (E.) coli vector system. Expression was confirmed by dot and Western blotting. We proposed that detection of E. coli-expressed H protein might be conformation- dependent because intensities of the reactions observed with these two methods varied. The H gene HEVD protein was further purified and used as an antigen for an ELISA. Samples from dogs with undetectable to high anti-CDV antibody titers were analyzed using this HEVD-specific ELISA and a commercial CDV antibody detection kit (ImmunoComb). Levels of HEVD antigenicity measured with the assays and immunochromatography correlated. These data indicated that the HEDV protein may be used as antigen to develop techniques for detecting antibodies against CDV. PMID:25234325

  5. Canine Distemper Viruses Expressing a Hemagglutinin without N-Glycans Lose Virulence but Retain Immunosuppression▿

    PubMed Central

    Sawatsky, Bevan; von Messling, Veronika

    2010-01-01

    Paramyxovirus glycoproteins are posttranslationally modified by the addition of N-linked glycans, which are often necessary for correct folding, processing, and cell surface expression. To establish the contribution of N glycosylation to morbillivirus attachment (H) protein function and overall virulence, we first determined the use of the potential N-glycosylation sites in the canine distemper virus (CDV) H proteins. Biochemical characterization revealed that the three sites conserved in all strains were N glycosylated, whereas only two of the up to five additional sites present in wild-type strains are used. A wild-type virus with an H protein reproducing the vaccine strain N-glycosylation pattern remained lethal in ferrets but with a prolonged course of disease. In contrast, introduction of the vaccine H protein in the wild-type context resulted in complete attenuation. To further characterize the role of N glycosylation in CDV pathogenesis, the N-glycosylation sites of wild-type H proteins were successively deleted, including a nonstandard site, to ultimately generate a nonglycosylated H protein. Despite reduced expression levels, this protein remained fully functional. Recombinant viruses expressing N-glycan-deficient H proteins no longer caused disease, even though their immunosuppressive capacities were retained, indicating that reduced N glycosylation contributes to attenuation without affecting immunosuppression. PMID:20042514

  6. Canine distemper virus DNA vaccination of mink can overcome interference by maternal antibodies.

    PubMed

    Jensen, Trine Hammer; Nielsen, Line; Aasted, Bent; Pertoldi, Cino; Blixenkrone-Møller, Merete

    2015-03-10

    Canine distemper virus (CDV) is highly contagious and can cause severe disease against which conventional live vaccines are ineffective in the presence of maternal antibodies. Vaccination in the presences of maternal antibodies was challenged by vaccination of 5 days old and 3 weeks old mink kits with CDV DNA vaccines. Virus neutralising (VN) antibody responses were induced in mink kits vaccinated with a plasmid encoding the haemaglutinin protein (H) of CDV (n=5, pCDV-H) or a combination of the H, fusion (F) and nucleoprotein (N) of CDV (n=5, pCDV-HFN). These DNA vaccinated kits were protected against virulent experimental infection with field strains of CDV. The pCDV-H was more efficient in inducing protective immunity in the presence of maternal antibodies compared to the pCDV-HFN. The results show that DNA vaccination with the pCDV-H or pCDV-HFN (n=4) only given once at 5 days of age induces virus specific immune response in neonatal mink and protection against virulent CDV exposure later in life. PMID:25637861

  7. The V domain of dog PVRL4 (nectin-4) mediates canine distemper virus entry and virus cell-to-cell spread

    SciTech Connect

    Delpeut, Sebastien; Noyce, Ryan S.; Richardson, Christopher D.

    2014-04-15

    The entry of canine distemper virus (CDV) is a multistep process that involves the attachment of CDV hemagglutinin (H) to its cellular receptor, followed by fusion between virus and cell membranes. Our laboratory recently identified PVRL4 (nectin-4) to be the epithelial receptor for measles and canine distemper viruses. In this study, we demonstrate that the V domain of PVRL4 is critical for CDV entry and virus cell-to-cell spread. Furthermore, four key amino acid residues within the V domain of dog PVRL4 and two within the CDV hemagglutinin were shown to be essential for receptor-mediated virus entry. - Highlights: • PVRL4 (nectin-4) is the epithelial cell receptor for measles and canine distemper viruses. • V domain of PVRL4 is critical for CDV entry, cell-to-cell spread, and syncytia formation. • Chimeric PVRL1 backbone substituted with the V domain of PVRL4 can function as a receptor. • Amino acids (F132/P133/A134/G135) within the V domain are essential for PVRL4 receptor activity. • Amino acids (P493/Y539) within CDV H protein are essential for PVRL4 receptor interaction.

  8. Inhibition of viral RNA synthesis in canine distemper virus infection by proanthocyanidin A2.

    PubMed

    Gallina, Laura; Dal Pozzo, Fabiana; Galligioni, Viola; Bombardelli, Ezio; Scagliarini, Alessandra

    2011-12-01

    Canine distemper virus (CDV) is a contagious and multisystemic viral disease that affects domestic and wild canines as well as other terrestrial and aquatic carnivores. The disease in dogs is often fatal and no specific antiviral therapy is currently available. In this study, we evaluated the in vitro antiviral activity against CDV of proanthocyanidin A2 (PA2), a phenolic dimer belonging to the class of condensed tannins present in plants. Our results showed that PA2 exerted in vitro antiviral activity against CDV with a higher selectivity index compared to ribavirin, included in our study for the previously tested anti-CDV activity. The time of addition assay led us to observe that PA2 was able to decrease the viral RNA synthesis and to reduce progeny virus liberation, at different times post infection suggesting multiple mechanisms of action including inhibition of viral replicative complex and modulation of the redox milieu. These data suggest that PA2, isolated from the bark of Aesculus hippocastanum, has potential usefulness as an anti-CDV compound inhibiting viral replication. PMID:22020306

  9. Development and characterization of neutralizing monoclonal antibodies against canine distemper virus hemagglutinin protein.

    PubMed

    Bi, Zhenwei; Xia, Xingxia; Wang, Yongshan; Mei, Yongjie

    2015-04-01

    Canine distemper virus (CDV) causes a serious multisystemic disease in dogs and other carnivora. Hemagglutinin (H) protein-specific antibodies are mainly responsible for protective immunity against CDV infection. In the present study, six neutralizing MAbs to the H protein of CDV were newly obtained and characterized by immunizing BALB/c mice with a recent Chinese field isolate. Competitive binding inhibition assay revealed that they recognized four distinct antigenic regions of the H protein. Immunofluorescence assay and western blotting showed that all MAbs recognize the conformational rather than the linear epitopes of the H protein. Furthermore, in immunofluorescence and virus neutralization assays, two of the MAbs were found to react only with the recent Chinese field isolate and not with older CDV strains, including vaccine strain Onderstepoort, indicating there are neutralization-related antigenic variations between the recent Chinese field isolate and the older CDV strains examined in this study. The newly established MAbs are useful for differentiating the expanding CDV strains and could be used in immunotherapy and immunodiagnosis against infection with CDV. PMID:25644427

  10. Canine Distemper

    MedlinePlus

    Although this brochure provides basic information about canine distemper, your veterinarian is always your best source of health information. Consult your veterinarian for more information about canine distemper and its prevention. ...

  11. Antiviral antibodies stimulate production of reactive oxygen species in cultured canine brain cells infected with canine distemper virus.

    PubMed Central

    Bürge, T; Griot, C; Vandevelde, M; Peterhans, E

    1989-01-01

    Canine distemper is characterized mainly by respiratory, enteric, and nervous symptoms. Infection of the central nervous system results in demyelination, to which inflammation has been shown to contribute significantly. It has been proposed that macrophages play a major role as effector cells in this process. We report that cultured dog brain cells contain a population of macrophages capable of producing reactive oxygen species as measured by luminol-dependent chemiluminescence. In cultures infected with canine distemper virus, a burst of reactive oxygen is triggered by antiviral antibody. This response depends on the presence of viral antigens on the surfaces of infected cells and is mediated by the interaction of antigen-bound antibody with Fc receptors on the macrophages. Since there is no evidence in vitro or in vivo that oligodendrocytes, the cells forming myelin, are infected, our observation supports the hypothesis that "innocent bystander killing" is important in demyelination caused by canine distemper virus. Reactive oxygen species released from macrophages may contribute to destruction of myelin. Images PMID:2724413

  12. Matrix genes of measles virus and canine distemper virus: cloning, nucleotide sequences, and deduced amino acid sequences.

    PubMed Central

    Bellini, W J; Englund, G; Richardson, C D; Rozenblatt, S; Lazzarini, R A

    1986-01-01

    The nucleotide sequences encoding the matrix (M) proteins of measles virus (MV) and canine distemper virus (CDV) were determined from cDNA clones containing these genes in their entirety. In both cases, single open reading frames specifying basic proteins of 335 amino acid residues were predicted from the nucleotide sequences. Both viral messages were composed of approximately 1,450 nucleotides and contained 400 nucleotides of presumptive noncoding sequences at their respective 3' ends. MV and CDV M-protein-coding regions were 67% homologous at the nucleotide level and 76% homologous at the amino acid level. Only chance homology was observed in the 400-nucleotide trailer sequences. Comparisons of the M protein sequences of MV and CDV with the sequence reported for Sendai virus (B. M. Blumberg, K. Rose, M. G. Simona, L. Roux, C. Giorgi, and D. Kolakofsky, J. Virol. 52:656-663; Y. Hidaka, T. Kanda, K. Iwasaki, A. Nomoto, T. Shioda, and H. Shibuta, Nucleic Acids Res. 12:7965-7973) indicated the greatest homology among these M proteins in the carboxyterminal third of the molecule. Secondary-structure analyses of this shared region indicated a structurally conserved, hydrophobic sequence which possibly interacted with the lipid bilayer. Images PMID:3754588

  13. Efficacy of Recombinant Canine Distemper Virus Expressing Leishmania Antigen against Leishmania Challenge in Dogs.

    PubMed

    Miura, Ryuichi; Kooriyama, Takanori; Yoneda, Misako; Takenaka, Akiko; Doki, Miho; Goto, Yasuyuki; Sanjoba, Chizu; Endo, Yasuyuki; Fujiyuki, Tomoko; Sugai, Akihiro; Tsukiyama-Kohara, Kyoko; Matsumoto, Yoshitsugu; Sato, Hiroki; Kai, Chieko

    2015-01-01

    Canine distemper virus (CDV) vaccination confers long-term protection against CDV reinfection. To investigate the utility of CDV as a polyvalent vaccine vector for Leishmania, we generated recombinant CDVs, based on an avirulent Yanaka strain, that expressed Leishmania antigens: LACK, TSA, or LmSTI1 (rCDV-LACK, rCDV-TSA, and rCDV-LmSTI1, respectively). Dogs immunized with rCDV-LACK were protected against challenge with lethal doses of virulent CDV, in the same way as the parental Yanaka strain. To evaluate the protective effects of the recombinant CDVs against cutaneous leishmaniasis in dogs, dogs were immunized with one recombinant CDV or a cocktail of three recombinant CDVs, before intradermal challenge (in the ears) with infective-stage promastigotes of Leishmania major. Unvaccinated dogs showed increased nodules with ulcer formation after 3 weeks, whereas dogs immunized with rCDV-LACK showed markedly smaller nodules without ulceration. Although the rCDV-TSA- and rCDV-LmSTI1-immunized dogs showed little protection against L. major, the cocktail of three recombinant CDVs more effectively suppressed the progression of nodule formation than immunization with rCDV-LACK alone. These results indicate that recombinant CDV is suitable for use as a polyvalent live attenuated vaccine for protection against both CDV and L. major infections in dogs. PMID:26162094

  14. Efficacy of Recombinant Canine Distemper Virus Expressing Leishmania Antigen against Leishmania Challenge in Dogs

    PubMed Central

    Yoneda, Misako; Takenaka, Akiko; Doki, Miho; Goto, Yasuyuki; Sanjoba, Chizu; Endo, Yasuyuki; Fujiyuki, Tomoko; Sugai, Akihiro; Tsukiyama-Kohara, Kyoko; Matsumoto, Yoshitsugu; Sato, Hiroki; Kai, Chieko

    2015-01-01

    Canine distemper virus (CDV) vaccination confers long-term protection against CDV reinfection. To investigate the utility of CDV as a polyvalent vaccine vector for Leishmania, we generated recombinant CDVs, based on an avirulent Yanaka strain, that expressed Leishmania antigens: LACK, TSA, or LmSTI1 (rCDV–LACK, rCDV–TSA, and rCDV–LmSTI1, respectively). Dogs immunized with rCDV-LACK were protected against challenge with lethal doses of virulent CDV, in the same way as the parental Yanaka strain. To evaluate the protective effects of the recombinant CDVs against cutaneous leishmaniasis in dogs, dogs were immunized with one recombinant CDV or a cocktail of three recombinant CDVs, before intradermal challenge (in the ears) with infective-stage promastigotes of Leishmania major. Unvaccinated dogs showed increased nodules with ulcer formation after 3 weeks, whereas dogs immunized with rCDV–LACK showed markedly smaller nodules without ulceration. Although the rCDV–TSA- and rCDV–LmSTI1-immunized dogs showed little protection against L. major, the cocktail of three recombinant CDVs more effectively suppressed the progression of nodule formation than immunization with rCDV–LACK alone. These results indicate that recombinant CDV is suitable for use as a polyvalent live attenuated vaccine for protection against both CDV and L. major infections in dogs. PMID:26162094

  15. Canine distemper virus as a threat to wild tigers in Russia and across their range.

    PubMed

    Gilbert, Martin; Soutyrina, Svetlana V; Seryodkin, Ivan V; Sulikhan, Nadezhda; Uphyrkina, Olga V; Goncharuk, Mikhail; Matthews, Louise; Cleaveland, Sarah; Miquelle, Dale G

    2015-07-01

    Canine distemper virus (CDV) has recently been identified in populations of wild tigers in Russia and India. Tiger populations are generally too small to maintain CDV for long periods, but are at risk of infections arising from more abundant susceptible hosts that constitute a reservoir of infection. Because CDV is an additive mortality factor, it could represent a significant threat to small, isolated tiger populations. In Russia, CDV was associated with the deaths of tigers in 2004 and 2010, and was coincident with a localized decline of tigers in Sikhote-Alin Biosphere Zapovednik (from 25 tigers in 2008 to 9 in 2012). Habitat continuity with surrounding areas likely played an important role in promoting an ongoing recovery. We recommend steps be taken to assess the presence and the impact of CDV in all tiger range states, but should not detract focus away from the primary threats to tigers, which include habitat loss and fragmentation, poaching and retaliatory killing. Research priorities include: (i) recognition and diagnosis of clinical cases of CDV in tigers when they occur; and (ii) collection of baseline data on the health of wild tigers. CDV infection of individual tigers need not imply a conservation threat, and modeling should complement disease surveillance and targeted research to assess the potential impact to tiger populations across the range of ecosystems, population densities and climate extremes occupied by tigers. Describing the role of domestic and wild carnivores as contributors to a local CDV reservoir is an important precursor to considering control measures. PMID:25939829

  16. Canine Distemper Virus Fusion Activation: Critical Role of Residue E123 of CD150/SLAM

    PubMed Central

    Khosravi, Mojtaba; Bringolf, Fanny; Röthlisberger, Silvan; Bieringer, Maria; Schneider-Schaulies, Jürgen; Zurbriggen, Andreas; Origgi, Francesco

    2015-01-01

    ABSTRACT Measles virus (MeV) and canine distemper virus (CDV) possess tetrameric attachment proteins (H) and trimeric fusion proteins, which cooperate with either SLAM or nectin 4 receptors to trigger membrane fusion for cell entry. While the MeV H-SLAM cocrystal structure revealed the binding interface, two distinct oligomeric H assemblies were also determined. In one of the conformations, two SLAM units were sandwiched between two discrete H head domains, thus spotlighting two binding interfaces (“front” and “back”). Here, we investigated the functional relevance of both interfaces in activating the CDV membrane fusion machinery. While alanine-scanning mutagenesis identified five critical regulatory residues in the front H-binding site of SLAM, the replacement of a conserved glutamate residue (E at position 123, replaced with A [E123A]) led to the most pronounced impact on fusion promotion. Intriguingly, while determination of the interaction of H with the receptor using soluble constructs revealed reduced binding for the identified SLAM mutants, no effect was recorded when physical interaction was investigated with the full-length counterparts of both molecules. Conversely, although mutagenesis of three strategically selected residues within the back H-binding site of SLAM did not substantially affect fusion triggering, nevertheless, the mutants weakened the H-SLAM interaction recorded with the membrane-anchored protein constructs. Collectively, our findings support a mode of binding between the attachment protein and the V domain of SLAM that is common to all morbilliviruses and suggest a major role of the SLAM residue E123, located at the front H-binding site, in triggering the fusion machinery. However, our data additionally support the hypothesis that other microdomain(s) of both glycoproteins (including the back H-binding site) might be required to achieve fully productive H-SLAM interactions. IMPORTANCE A complete understanding of the measles virus

  17. Evaluation of Trapper-Collected Nobuto Filter-Paper Blood Samples for Distemper and Parvovirus Antibody Detection in Coyotes (Canis latrans) and Raccoons (Procyon lotor).

    PubMed

    Kamps, Amanda J; Dubay, Shelli A; Langenberg, Julie; Maes, Roger K

    2015-07-01

    Blood samples are often collected from free-ranging wildlife for antibody detection. However, filter-paper (FP) strips are more cost efficient and easy to collect and store. We evaluated trapper-collected FP strips and body-cavity blood for canine distemper (CDV) and parvovirus (CPV-2) antibody detection in raccoons (Procyon lotor) and coyotes (Canis latrans). From 2008 to 2010, licensed trappers near Madison and Milwaukee, Wisconsin, US collected paired samples from harvested animals. Canine distemper antibodies were detected using virus neutralization and parvovirus antibodies were detected using hemagglutination inhibition. Titers ≥ 1:32 for CDV and ≥ 1:25 for CPV-2 were considered evidence of exposure. Using Cohen's kappa test of agreement, FP strip titers agreed with sera for CDV in coyotes (n = 28, K = 0.772) and raccoons (n = 29, K = 0.858) and for CPV-2 in coyotes (n = 40, K = 0.775) and raccoons (n = 70, K = 0.646). However, raccoons determined to be exposed to CPV-2 from sera were unexposed by FP strips in 35% of the samples. Titer results may be affected by quality and volume of blood samples, interval between collection and processing, small sample sizes, and diagnostic testing procedures. Filter-paper strips can be useful for detecting CDV and CPV-2 exposure in coyotes and raccoons with correct field sample collection and appropriate diagnostic testing procedures. PMID:25973631

  18. Molecular Determinants Defining the Triggering Range of Prefusion F Complexes of Canine Distemper Virus

    PubMed Central

    Avila, Mislay; Alves, Lisa; Khosravi, Mojtaba; Ader-Ebert, Nadine; Origgi, Francesco; Schneider-Schaulies, Jürgen; Zurbriggen, Andreas; Plemper, Richard K.

    2014-01-01

    ABSTRACT The morbillivirus cell entry machinery consists of a fusion (F) protein trimer that refolds to mediate membrane fusion following receptor-induced conformational changes in its binding partner, the tetrameric attachment (H) protein. To identify molecular determinants that control F refolding, we generated F chimeras between measles virus (MeV) and canine distemper virus (CDV). We located a central pocket in the globular head domain of CDV F that regulates the stability of the metastable, prefusion conformational state of the F trimer. Most mutations introduced into this “pocket'” appeared to mediate a destabilizing effect, a phenotype associated with enhanced membrane fusion activity. Strikingly, under specific triggering conditions (i.e., variation of receptor type and H protein origin), some F mutants also exhibited resistance to a potent morbillivirus entry inhibitor, which is known to block F triggering by enhancing the stability of prefusion F trimers. Our data reveal that the molecular nature of the F stimulus and the intrinsic stability of metastable prefusion F both regulate the efficiency of F refolding and escape from small-molecule refolding blockers. IMPORTANCE With the aim to better characterize the thermodynamic basis of morbillivirus membrane fusion for cell entry and spread, we report here that the activation energy barrier of prefusion F trimers together with the molecular nature of the triggering “stimulus” (attachment protein and receptor types) define a “triggering range,” which governs the initiation of the membrane fusion process. A central “pocket” microdomain in the globular F head contributes substantially to the regulation of the conformational stability of the prefusion complexes. The triggering range also defines the mechanism of viral escape from entry inhibitors and describes how the cellular environment can affect membrane fusion efficiency. PMID:24371057

  19. Susceptibility of carnivore hosts to strains of canine distemper virus from distinct genetic lineages.

    PubMed

    Nikolin, Veljko M; Wibbelt, Gudrun; Michler, Frank-Uwe F; Wolf, Peter; East, Marion L

    2012-04-23

    Using the complete haemagglutinin (HA) gene and partial phosphoprotein (P) gene we investigated the genotype of canine distemper virus (CDV) strains recovered from two wildlife species in Mecklenburg-Vorpommern, Germany. Phylogenetic analyses demonstrated significant differences between the strains from raccoons Procyon lotor (family Procyonidae) obtained in 2007 and strains from red foxes Vulpes vulpes (family Canidae) obtained in 2008. The raccoon strains belonged to the CDV European wildlife lineage whereas the red fox strains belonged to the CDV Europe lineage. We combined our genetic sequence data with published data from 138 CDV stains worldwide to investigate the proposed importance of amino acid substitutions in the SLAM binding region of the CDV HA protein at position 530 (G/E to R/D/N) and 549 (Y to H) to the spread of domestic dog-adapted CDV strains to other carnivores. We found no evidence that amino acid 530 was strongly affected by host species. Rather, site 530 was conserved within CDV lineages, regardless of host species. Contrary to expectation, strains from non-dog hosts did not exhibit a bias towards the predicted substitution Y549H. Wild canid hosts were more frequently infected by strains with 549Y, a pattern similar to domestic dogs. Non-canid strains showed no significant bias towards either H or Y at site 549, although there was a trend towards 549H. Significant differences between the prevalence of 549Y and 549H in wild canid strains and non-canid strains suggests a degree of virus adaptation to these categories of host. PMID:22024346

  20. In Vitro Exposure of Harbor Seal Immune Cells to Aroclor 1260 Alters Phocine Distemper Virus Replication.

    PubMed

    Bogomolni, Andrea; Frasca, Salvatore; Levin, Milton; Matassa, Keith; Nielsen, Ole; Waring, Gordon; De Guise, Sylvain

    2016-01-01

    In the last 30 years, several large-scale marine mammal mortality events have occurred, often in close association with highly polluted regions, leading to suspicions that contaminant-induced immunosuppression contributed to these epizootics. Some of these recent events also identified morbillivirus as a cause of or contributor to death. The role of contaminant exposures regarding morbillivirus mortality is still unclear. The results of this study aimed to address the potential for a mixture of polychlorinated biphenyls (PCBs), specifically Aroclor 1260, to alter harbor seal T-lymphocyte proliferation and to assess if exposure resulted in increased likelihood of phocine distemper virus (PDV USA 2006) to infect susceptible seals in an in vitro system. Exposure of peripheral blood mononuclear cells to Aroclor 1260 did not significantly alter lymphocyte proliferation (1, 5, 10, and 20 ppm). However, using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), lymphocytes exposed to 20 ppm Aroclor 1260 exhibited a significant decrease in PDV replication at day 7 and a significant increase at day 11 compared with unexposed control cells. Similar and significant differences were apparent on exposure to Aroclor 1260 in monocytes and supernatant. The results here indicate that in harbor seals, Aroclor 1260 exposure results in a decrease in virus early during infection and an increase during late infection. The consequences of this contaminant-induced infection pattern in a highly susceptible host could result in a greater potential for systemic infection with greater viral load, which could explain the correlative findings seen in wild populations exposed to a range of persistent contaminants that suffer from morbillivirus epizootics. PMID:26142119

  1. The anti-canine distemper virus activities of ex vivo-expanded canine natural killer cells.

    PubMed

    Park, Ji-Yun; Shin, Dong-Jun; Lee, Soo-Hyeon; Lee, Je-Jung; Suh, Guk-Hyun; Cho, Duck; Kim, Sang-Ki

    2015-04-17

    Natural killer (NK) cells play critical roles in induction of antiviral effects against various viruses of humans and animals. However, few data on NK cell activities during canine distemper virus (CDV) infections are available. Recently, we established a culture system allowing activation and expansion of canine non-B, non-T, large granular NK lymphocytes from PBMCs of normal dogs. In the present study, we explored the ability of such expanded NK cells to inhibit CDV infection in vitro. Cultured CD3-CD5-CD21- NK cells produced large amounts of IFN-γ, exhibited highly upregulated expression of mRNAs encoding NK-cell-associated receptors, and demonstrated strong natural killing activity against canine tumor cells. Although the expanded NK cells were dose-dependently cytotoxic to both normal and CDV-infected Vero cells, CDV infection rendered Vero cells more susceptible to NK cells. Pretreatment with anti-CDV serum from hyperimmunized dogs enhanced the antibody-dependent cellular cytotoxicity (ADCC) of NK cells against CDV-infected Vero cells. The culture supernatants of NK cells, added before or after infection, dose-dependently inhibited both CDV replication and development of CDV-induced cytopathic effects (CPEs) in Vero cells. Anti-IFN-γ antibody neutralized the inhibitory effects of NK cell culture supernatants on CDV replication and CPE induction in Vero cells. Such results emphasize the potential significance of NK cells in controlling CDV infection, and indicate that NK cells may play roles both during CDV infection and in combating such infections, under certain conditions. PMID:25680810

  2. Isolation and sequence analysis of a canine distemper virus from a raccoon dog in Jilin Province, China.

    PubMed

    Cheng, Yuening; Wang, Jianke; Zhang, Miao; Zhao, Jianjun; Shao, Xiqun; Ma, Zengjun; Zhao, Hang; Lin, Peng; Wu, Hua

    2015-10-01

    Canine distemper virus (CDV) is a major pathogen not only in raccoon dogs but also in a variety of carnivorous animals, including domesticated animals, particularly if they have not been vaccinated. In this study, a wild-type strain of CDV was isolated from lung tissue from a raccoon dog kept at a fur farm in Jilin Province, China. Cytopathic effects typical of CDV infection were observed after three blind passages in Vero cells, yielding a virus titer of 10(4.6) TCID50/mL. Virus identification was carried out by RT-PCR, immunofluorescence, electron microscopy, and genome sequencing. The results showed that the isolated virus, termed the SY strain, corresponded to the Asia-1 genotype of CDV and has a genome of 15,690 nucleotides. This represents the first complete nucleotide sequence of a CDV strain circulating in raccoon dogs in China. PMID:26265248

  3. Canine distemper virus (CDV) infection of ferrets as a model for testing Morbillivirus vaccine strategies: NYVAC- and ALVAC-based CDV recombinants protect against symptomatic infection.

    PubMed

    Stephensen, C B; Welter, J; Thaker, S R; Taylor, J; Tartaglia, J; Paoletti, E

    1997-02-01

    Canine distemper virus (CDV) infection of ferrets causes an acute systemic disease involving multiple organ systems, including the respiratory tract, lymphoid system, and central nervous system (CNS). We have tested candidate CDV vaccines incorporating the fusion (F) and hemagglutinin (HA) proteins in the highly attenuated NYVAC strain of vaccinia virus and in the ALVAC strain of canarypox virus, which does not productively replicate in mammalian hosts. Juvenile ferrets were vaccinated twice with these constructs, or with an attenuated live-virus vaccine, while controls received saline or the NYVAC and ALVAC vectors expressing rabies virus glycoprotein. Control animals did not develop neutralizing antibody and succumbed to distemper after developing fever, weight loss, leukocytopenia, decreased activity, conjunctivitis, an erythematous rash typical of distemper, CNS signs, and viremia in peripheral blood mononuclear cells (as measured by reverse transcription-PCR). All three CDV vaccines elicited neutralizing titers of at least 1:96. All vaccinated ferrets survived, and none developed viremia. Both recombinant vaccines also protected against the development of symptomatic distemper. However, ferrets receiving the live-virus vaccine lost weight, became lymphocytopenic, and developed the erythematous rash typical of CDV. These data show that ferrets are an excellent model for evaluating the ability of CDV vaccines to protect against symptomatic infection. Because the pathogenesis and clinical course of CDV infection of ferrets is quite similar to that of other Morbillivirus infections, including measles, this model will be useful in testing new candidate Morbillivirus vaccines. PMID:8995676

  4. Canine distemper virus (CDV) infection of ferrets as a model for testing Morbillivirus vaccine strategies: NYVAC- and ALVAC-based CDV recombinants protect against symptomatic infection.

    PubMed Central

    Stephensen, C B; Welter, J; Thaker, S R; Taylor, J; Tartaglia, J; Paoletti, E

    1997-01-01

    Canine distemper virus (CDV) infection of ferrets causes an acute systemic disease involving multiple organ systems, including the respiratory tract, lymphoid system, and central nervous system (CNS). We have tested candidate CDV vaccines incorporating the fusion (F) and hemagglutinin (HA) proteins in the highly attenuated NYVAC strain of vaccinia virus and in the ALVAC strain of canarypox virus, which does not productively replicate in mammalian hosts. Juvenile ferrets were vaccinated twice with these constructs, or with an attenuated live-virus vaccine, while controls received saline or the NYVAC and ALVAC vectors expressing rabies virus glycoprotein. Control animals did not develop neutralizing antibody and succumbed to distemper after developing fever, weight loss, leukocytopenia, decreased activity, conjunctivitis, an erythematous rash typical of distemper, CNS signs, and viremia in peripheral blood mononuclear cells (as measured by reverse transcription-PCR). All three CDV vaccines elicited neutralizing titers of at least 1:96. All vaccinated ferrets survived, and none developed viremia. Both recombinant vaccines also protected against the development of symptomatic distemper. However, ferrets receiving the live-virus vaccine lost weight, became lymphocytopenic, and developed the erythematous rash typical of CDV. These data show that ferrets are an excellent model for evaluating the ability of CDV vaccines to protect against symptomatic infection. Because the pathogenesis and clinical course of CDV infection of ferrets is quite similar to that of other Morbillivirus infections, including measles, this model will be useful in testing new candidate Morbillivirus vaccines. PMID:8995676

  5. Black-backed jackal exposure to rabies virus, canine distemper virus, and Bacillus anthracis in Etosha National Park, Namibia.

    PubMed

    Bellan, Steve E; Cizauskas, Carrie A; Miyen, Jacobeth; Ebersohn, Karen; Küsters, Martina; Prager, K C; Van Vuuren, Moritz; Sabeta, Claude; Getz, Wayne M

    2012-04-01

    Canine distemper virus (CDV) and rabies virus (RABV) occur worldwide in wild carnivore and domestic dog populations and pose threats to wildlife conservation and public health. In Etosha National Park (ENP), Namibia, anthrax is endemic and generates carcasses frequently fed on by an unusually dense population of black-backed jackals (Canis mesomelas). Using serology, phylogenetic analyses (on samples obtained from February 2009-July 2010), and historical mortality records (1975-2011), we assessed jackal exposure to Bacillus anthracis (BA; the causal bacterial agent of anthrax), CDV, and RABV. Prevalence of antibodies against BA (95%, n = 86) and CDV (71%, n = 80) was relatively high, while that of antibodies against RABV was low (9%, n = 81). Exposure to BA increased significantly with age, and all animals >6 mo old were antibody-positive. As with BA, prevalence of antibodies against CDV increased significantly with age, with similar age-specific trends during both years of the study. No significant effect of age was found on the prevalence of antibodies against RABV. Three of the seven animals with antibodies against RABV were monitored for more than 1 yr after sampling and showed no signs of active infection. Mortality records revealed that rabid animals are destroyed nearly every year inside the ENP tourist camps. Phylogenetic analyses demonstrated that jackal RABV in ENP is part of the same transmission cycle as other dog-jackal RABV cycles in Namibia. PMID:22493112

  6. Recombinant Newcastle disease viral vector expressing hemagglutinin or fusion of canine distemper virus is safe and immunogenic in minks.

    PubMed

    Ge, Jinying; Wang, Xijun; Tian, Meijie; Gao, Yuwei; Wen, Zhiyuan; Yu, Guimei; Zhou, Weiwei; Zu, Shulong; Bu, Zhigao

    2015-05-15

    Canine Distemper Virus (CDV) infects many carnivores and cause several high-mortality disease outbreaks. The current CDV live vaccine cannot be safely used in some exotic species, such as mink and ferret. Here, we generated recombinant lentogenic Newcastle disease virus (NDV) LaSota expressing either envelope glycoproyein, heamagglutinine (H) or fusion protein (F), named as rLa-CDVH and rLa-CDVF, respectively. The feasibility of these recombinant NDVs to serve as live virus-vectored CD vaccine was evaluated in minks. rLa-CDVH induced significant neutralization antibodies (NA) to CDV and provided solid protection against virulent CDV challenge. On the contrast, rLa-CDVF induced much lower NA to CDV and fail to protected mink from virulent CDV challenge. Results suggest that recombinant NDV expressing CDV H is safe and efficient candidate vaccine against CDV in mink, and maybe other host species. PMID:25865465

  7. Phylogenetic features of hemagglutin gene in canine distemper virus strains from different genetic lineages.

    PubMed

    Liao, Peng; Guo, Li; Wen, Yongjun; Yang, Yangling; Cheng, Shipeng

    2015-01-01

    In the present study, the genotype of two Canine distemper virus (CDV) strains, namely, ZJJ-SD and ZJJ-LN, were investigated, based on the whole hemagglutinin (HA) gene. The CDV strains were obtained from two foxes in Shandong Province and Liaoning Province in 2011. Phylogenetic analyses were carried out for 260 CDV strains worldwide, and a statistical analysis was performed in the amino acid substitutions at positions 530 and 549 of the HA protein. Phylogenetic analyses revealed that the two strains, ZJJ-SD and ZJJ-LN, belonged to the CDV Asia I lineage. Site 530 of HA protein was found to be relatively conserved within CDV lineages in different host species by combining the genetic sequence data with the published data from 260 CDV strains worldwide. The data analysis showed a bias toward the predicted substitution Y549H for the non-dog strains in Asia I and Europe lineages. The ratio of site 549 genetic drift in the HA gene were significantly different between dogs and non-dogs in the two lineages. The strain ZJJ-SD, from wild canid, has an Y549H substitution. It is one of three Y549H substitution for wild canids in Asia I lineages. Site 530 of HA protein was not immediately relative to CDV genetic drift from dogs to non-dogs. Statistical analysis indicated that non-dog strains have a high probability to contain Y549H than dog strains in Asia I and Europe lineages. Thus, site 549 is considered important in genetic drift from dogs to non-dogs, at least in Asia I and Europe lineages. PMID:26131292

  8. Complement-mediated neutralization of canine distemper virus in vitro: cross-reaction between vaccine Onderstepoort and field KDK-1 strains with different hemagglutinin gene characteristics.

    PubMed

    Mochizuki, Masami; Motoyoshi, Megumi; Maeda, Ken; Kai, Kazunari

    2002-07-01

    The properties of neutralization of antigens of canine distemper virus Onderstepoort and a recent field isolate, KDK-1, were investigated with strain-specific dog sera. A conventional neutralization assay indicated antigenic dissimilarity between the strains; however, when guinea pig complement was included in the reaction mixture, the strains were neutralized with not only the homologous but also the heterologous antibodies. PMID:12093697

  9. Genetically distant American Canine distemper virus lineages have recently caused epizootics with somewhat different characteristics in raccoons living around a large suburban zoo in the USA

    PubMed Central

    Lednicky, John A; Dubach, Jean; Kinsel, Michael J; Meehan, Thomas P; Bocchetta, Maurizio; Hungerford, Laura L; Sarich, Nicolene A; Witecki, Kelley E; Braid, Michael D; Pedrak, Casandra; Houde, Christiane M

    2004-01-01

    Background Mortality rates have differed during distemper outbreaks among free-ranging raccoons (Procyon lotor) living around a large Chicago-area zoo, and appeared higher in year 2001 than in 1998 and 2000. We hypothesized that a more lethal variant of the local Canine distemper virus (CDV) lineage had emerged in 2001, and sought the genetic basis that led to increased virulence. However, a more complex model surfaced during preliminary analyses of CDV genomic sequences in infected tissues and of virus isolated in vitro from the raccoons. Results Phylogenetic analyses of subgenomic CDV fusion (F) -, phosphoprotein (P) -, and complete hemagglutinin (H) – gene sequences indicated that distinct American CDV lineages caused the distemper epizootics. The 1998 outbreak was caused by viruses that are likely from an old CDV lineage that includes CDV Snyder Hill and Lederle, which are CDV strains from the early 1950's. The 2000 and 2001 viruses appear to stem from the lineage of CDV A75/17, which was isolated in the mid 1970's. Only the 2001 viruses formed large syncytia in brain and/or lung tissue, and during primary isolation in-vitro in Vero cells, demonstrating at least one phenotypic property by which they differed from the other viruses. Conclusions Two different American CDV lineages caused the raccoon distemper outbreaks. The 1998 viruses are genetically distant to the 2000/2001 viruses. Since CDV does not cause persistent infections, the cycling of different CDV lineages within the same locale suggests multiple reintroductions of the virus to area raccoons. Our findings establish a precedent for determining whether the perceived differences in mortality rates are actual and attributable in part to inherent differences between CDV strains arising from different CDV lineages. PMID:15507154

  10. The humoral immune response to recombinant nucleocapsid antigen of canine distemper virus in dogs vaccinated with attenuated distemper virus or DNA encoding the nucleocapsid of wild-type virus.

    PubMed

    Griot-Wenk, M E; Cherpillod, P; Koch, A; Zurbriggen, R; Bruckner, L; Wittek, R; Zurbriggen, A

    2001-06-01

    This study compared the humoral immune response against the nucleocapsid-(N) protein of canine distemper virus (CDV) of dogs vaccinated with a multivalent vaccine against parvo-, adeno-, and parainfluenza virus and leptospira combined with either the attenuated CDV Onderstepoort strain (n = 15) or an expression plasmid containing the N-gene of CDV (n = 30). The vaccinations were applied intramuscularly three times at 2-week intervals beginning at the age of 6 weeks. None of the pre-immune sera recognized the recombinant N-protein, confirming the lack of maternal antibodies at this age. Immunization with DNA vaccine for CDV resulted in positive serum N-specific IgG response. However, their IgG (and IgA) titres were lower than those of CDV-vaccinated dogs. Likewise, DNA-vaccinated dogs did not show an IgM peak. There was no increase in N-specific serum IgE titres in either group. Serum titres to the other multivalent vaccine components were similar in both groups. PMID:11475904

  11. Virological and serological findings in dogs with naturally occurring distemper.

    PubMed

    Elia, Gabriella; Camero, Michele; Losurdo, Michele; Lucente, Maria Stella; Larocca, Vittorio; Martella, Vito; Decaro, Nicola; Buonavoglia, Canio

    2015-03-01

    Canine distemper virus (CDV) is the cause of a severe and highly contagious disease in dogs. The unpredictable and variable course of CDV-related disease may hamper correct diagnosis of infection and makes it crucial the collection of samples suitable for laboratory confirmation. In the present study we were able to follow the disease in two dogs infected naturally, collecting different biological matrices during the entire period of infection. By real time RT-PCR, viral RNA was detected and quantified, suggesting that urine and rectal swabs would be useful for ante-mortem diagnosis of distemper in dogs, regardless of the clinical stage and form of the illness. PMID:25512131

  12. Persistence of canine distemper virus in the Greater Yellowstone ecosystem's carnivore community.

    PubMed

    Almberg, Emily S; Cross, Paul C; Smith, Douglas W

    2010-10-01

    Canine distemper virus (CDV) is an acute, highly immunizing pathogen that should require high densities and large populations of hosts for long-term persistence, yet CDV persists among terrestrial carnivores with small, patchily distributed groups. We used CDV in the Greater Yellowstone ecosystem's (GYE) wolves (Canis lupus) and coyotes (Canis latrans) as a case study for exploring how metapopulation structure, host demographics, and multi-host transmission affect the critical community size and spatial scale required for CDV persistence. We illustrate how host spatial connectivity and demographic turnover interact to affect both local epidemic dynamics, such as the length and variation in inter-epidemic periods, and pathogen persistence using stochastic, spatially explicit susceptible-exposed-infectious-recovered simulation models. Given the apparent absence of other known persistence mechanisms (e.g., a carrier or environmental state, densely populated host, chronic infection, or a vector), we suggest that CDV requires either large spatial scales or multi-host transmission for persistence. Current GYE wolf populations are probably too small to support endemic CDV. Coyotes are a plausible reservoir host, but CDV would still require 50000-100000 individuals for moderate persistence (> 50% over 10 years), which would equate to an area of 1-3 times the size of the GYE (60000-200000 km2). Coyotes, and carnivores in general, are not uniformly distributed; therefore, this is probably a gross underestimate of the spatial scale of CDV persistence. However, the presence of a second competent host species can greatly increase the probability of long-term CDV persistence at much smaller spatial scales. Although no management of CDV is currently recommended for the GYE, wolf managers in the region should expect periodic but unpredictable CDV-related population declines as often as every 2-5 years. Awareness and monitoring of such outbreaks will allow corresponding adjustments

  13. Persistence of canine distemper virus in the Greater Yellowstone Ecosystem's carnivore community

    USGS Publications Warehouse

    Almberg, E.S.; Cross, P.C.; Smith, D.W.

    2010-01-01

    Canine distemper virus (CDV) is an acute, highly immunizing pathogen that should require high densities and large populations of hosts for long-term persistence, yet CDV persists among terrestrial carnivores with small, patchily distributed groups. We used CDV in the Greater Yellowstone ecosystem's (GYE) wolves (Canis lupus) and coyotes (Canis latrans) as a case study for exploring how metapopulation structure, host demographics, and multi-host transmission affect the critical community size and spatial scale required for CDV persistence. We illustrate how host spatial connectivity and demographic turnover interact to affect both local epidemic dynamics, such as the length and variation in inter-epidemic periods, and pathogen persistence using stochastic, spatially explicit susceptible-exposed-infectious-recovered simulation models. Given the apparent absence of other known persistence mechanisms (e.g., a carrier or environmental state, densely populated host, chronic infection, or a vector), we suggest that CDV requires either large spatial scales or multi-host transmission for persistence. Current GYE wolf populations are probably too small to support endemic CDV. Coyotes are a plausible reservoir host, but CDV would still require 50 000-100 000 individuals for moderate persistence (>50% over 10 years), which would equate to an area of 1-3 times the size of the GYE (60000-200000 km2). Coyotes, and carnivores in general, are not uniformly distributed; therefore, this is probably a gross underestimate of the spatial scale of CDV persistence. However, the presence of a second competent host species can greatly increase the probability of long-term CDV persistence at much smaller spatial scales. Although no management of CDV is currently recommended for the GYE, wolf managers in the region should expect periodic but unpredictable CDV-related population declines as often as every 2-5 years. Awareness and monitoring of such outbreaks will allow corresponding

  14. Genotypes of Canine Distemper Virus Determined by Analysis of the Hemagglutinin Genes of Recent Isolates from Dogs in Japan

    PubMed Central

    Mochizuki, Masami; Hashimoto, Michiru; Hagiwara, Shigeyuki; Yoshida, Yoshio; Ishiguro, Shinryo

    1999-01-01

    Canine distemper of domestic dogs is caused by canine distemper virus (CDV), a member of the morbilliviruses. It has been a highly contagious disease of great veterinary importance for centuries, but for the last several decades it has been controlled satisfactorily by modified live vaccines. In the 1990s, however, it was described that CDV strains genetically different from vaccine strains may have caused the disease in vaccinated dogs. The highest antigenic variation is found in the H protein. Therefore, in the present study, hemagglutinin (H) genes obtained from current vaccines and field isolates and amplified directly from clinical specimens were genetically analyzed by restriction fragment length polymorphism assay and sequencing. Phylogenetic analysis of the H-gene amino acid sequences revealed that at least two CDV genotypes are circulating among dogs in Japan; one is a genotype to which almost all Japanese CDV isolates belong and the other has not been previously described. Both are separate and independent from the other lineages or genotypes of vaccine strains, as well as European and U.S. CDV isolates. The results suggest that CDV has also evolved in Japan, and further studies will be needed for an evaluation and possible improvement of the efficacies of current CDV vaccines. PMID:10449479

  15. Three-year duration of immunity in dogs vaccinated with a canarypox-vectored recombinant canine distemper virus vaccine.

    PubMed

    Larson, L J; Schultz, R D

    2007-01-01

    Two studies evaluated the duration of serologic response to the recombinant, canarypox-vectored canine distemper virus vaccine (Recombitek, Merial). Serologic duration of immunity was shown to be at least 36 months. Thus, Recombitek provides protection when administered less frequently than the manufacturer's label. After the initial vaccination protocol of two or more doses administered approximately 4 weeks apart, with the last dose given at 12 to 16 weeks of age or older, and re-vaccination at 1 year of age, Recombitek can confidently be readministered every 3 years with assurance of protection in immunocompetent dogs. This allows the vaccine to be administered in accordance with the recommendations of the American Animal Hospital Association Canine Vaccine Task Force and others. PMID:17616944

  16. Molecular typing of canine distemper virus strains reveals the presence of a new genetic variant in South America.

    PubMed

    Sarute, Nicolás; Pérez, Ruben; Aldaz, Jaime; Alfieri, Amauri A; Alfieri, Alice F; Name, Daniela; Llanes, Jessika; Hernández, Martín; Francia, Lourdes; Panzera, Yanina

    2014-06-01

    Canine distemper virus (CDV, Paramyxoviridae, Morbillivirus) is the causative agent of a severe infectious disease affecting terrestrial and marine carnivores worldwide. Phylogenetic relationships and the genetic variability of the hemagglutinin (H) protein and the fusion protein signal-peptide (Fsp) allow for the classification of field strains into genetic lineages. Currently, there are nine CDV lineages worldwide, two of them co-circulating in South America. Using the Fsp-coding region, we analyzed the genetic variability of strains from Uruguay, Brazil, and Ecuador, and compared them with those described previously in South America and other geographical areas. The results revealed that the Brazilian and Uruguayan strains belong to the already described South America lineage (EU1/SA1), whereas the Ecuadorian strains cluster in a new clade, here named South America 3, which may represent the third CDV lineage described in South America. PMID:24647552

  17. 9 CFR 113.306 - Canine Distemper Vaccine.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Live Virus Vaccines § 113.306 Canine Distemper Vaccine. Canine Distemper Vaccine shall be prepared from virus-bearing cell culture fluids or embryonated chicken eggs. Only Master Seed Virus which has been...

  18. 9 CFR 113.302 - Distemper Vaccine-Mink.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Live Virus Vaccines § 113.302 Distemper Vaccine—Mink. Distemper Vaccine—Mink shall be prepared from virus-bearing cell culture fluids. Only Master Seed Virus which has been established as pure, safe,...

  19. 9 CFR 113.306 - Canine Distemper Vaccine.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Live Virus Vaccines § 113.306 Canine Distemper Vaccine. Canine Distemper Vaccine shall be prepared from virus-bearing cell culture fluids or embryonated chicken eggs. Only Master Seed Virus which has been...

  20. 9 CFR 113.302 - Distemper Vaccine-Mink.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Live Virus Vaccines § 113.302 Distemper Vaccine—Mink. Distemper Vaccine—Mink shall be prepared from virus-bearing cell culture fluids. Only Master Seed Virus which has been established as pure, safe,...

  1. 9 CFR 113.302 - Distemper Vaccine-Mink.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Live Virus Vaccines § 113.302 Distemper Vaccine—Mink. Distemper Vaccine—Mink shall be prepared from virus-bearing cell culture fluids. Only Master Seed Virus which has been established as pure, safe,...

  2. 9 CFR 113.306 - Canine Distemper Vaccine.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Live Virus Vaccines § 113.306 Canine Distemper Vaccine. Canine Distemper Vaccine shall be prepared from virus-bearing cell culture fluids or embryonated chicken eggs. Only Master Seed Virus which has been...

  3. 9 CFR 113.302 - Distemper Vaccine-Mink.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Live Virus Vaccines § 113.302 Distemper Vaccine—Mink. Distemper Vaccine—Mink shall be prepared from virus-bearing cell culture fluids. Only Master Seed Virus which has been established as pure, safe,...

  4. 9 CFR 113.306 - Canine Distemper Vaccine.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Live Virus Vaccines § 113.306 Canine Distemper Vaccine. Canine Distemper Vaccine shall be prepared from virus-bearing cell culture fluids or embryonated chicken eggs. Only Master Seed Virus which has been...

  5. Serologic responses after vaccination of fennec foxes (Vulpes zerda) and meerkats (Suricata suricatta) with a live, canarypox-vectored canine distemper virus vaccine.

    PubMed

    Coke, Rob L; Backues, Kay A; Hoover, John P; Saliki, Jeremiah T; Ritchey, Jerry W; West, Gary D

    2005-06-01

    Fennec foxes (Vulpes zerda) and meerkats (Suricata suricatta) are considered to be susceptible to canine distemper virus (CDV) infection. Although no definitive clinical cases of natural CDV infections have been reported, mortalities due to CDV have been suspected and are reported in other closely related species. A commercially available monovalent, live, canarypox-vectored CDV vaccine induced neutralizing antibody titers that were maintained for at least a year in both fennec foxes and meerkats. PMID:17323579

  6. Construction of an Expression System for Bioactive IL-18 and Generation of Recombinant Canine Distemper Virus Expressing IL-18

    PubMed Central

    LIU, Yuxiu; SATO, Hiroki; HAMANA, Masahiro; MOONAN, Navita Anisia; YONEDA, Misako; XIA, Xianzhu; KAI, Chieko

    2014-01-01

    ABSTRACT Interleukin 18 (IL-18) plays an important role in the T-helper-cell type 1 immune response against intracellular parasites, bacteria and viral infections. It has been widely used as an adjuvant for vaccines and as an anticancer agent. However, IL-18 protein lacks a typical signal sequence and requires cleavage into its mature active form by caspase 1. In this study, we constructed mammalian expression vectors carrying cDNA encoding mature canine IL-18 (cIL-18) or mouse IL-18 (mIL-18) fused to the human IL-2 (hIL-2) signal sequence. The expressed proIL-18 proteins were processed to their mature forms in the cells. The supernatants of cells transfected with these plasmids induced high interferon-γ production in canine peripheral blood mononuclear cells or mouse splenocytes, respectively, indicating the secretion of bioactive IL-18. Using reverse genetics, we also generated a recombinant canine distemper virus that expresses cIL-18 or mIL-18 fused to the hIL-2 signal sequence. As expected, both recombinant viruses produced mature IL-18 in the infected cells, which secreted bioactive IL-18. These results indicate that the signal sequence from hIL-2 is suitable for the secretion of mature IL-18. These recombinant viruses can also potentially be used as immunoadjuvants and agents for anticancer therapies in vivo. PMID:24898077

  7. Construction of an expression system for bioactive IL-18 and generation of recombinant canine distemper virus expressing IL-18.

    PubMed

    Liu, Yuxiu; Sato, Hiroki; Hamana, Masahiro; Moonan, Navita Anisia; Yoneda, Misako; Xia, Xianzhu; Kai, Chieko

    2014-09-01

    Interleukin 18 (IL-18) plays an important role in the T-helper-cell type 1 immune response against intracellular parasites, bacteria and viral infections. It has been widely used as an adjuvant for vaccines and as an anticancer agent. However, IL-18 protein lacks a typical signal sequence and requires cleavage into its mature active form by caspase 1. In this study, we constructed mammalian expression vectors carrying cDNA encoding mature canine IL-18 (cIL-18) or mouse IL-18 (mIL-18) fused to the human IL-2 (hIL-2) signal sequence. The expressed proIL-18 proteins were processed to their mature forms in the cells. The supernatants of cells transfected with these plasmids induced high interferon-γ production in canine peripheral blood mononuclear cells or mouse splenocytes, respectively, indicating the secretion of bioactive IL-18. Using reverse genetics, we also generated a recombinant canine distemper virus that expresses cIL-18 or mIL-18 fused to the hIL-2 signal sequence. As expected, both recombinant viruses produced mature IL-18 in the infected cells, which secreted bioactive IL-18. These results indicate that the signal sequence from hIL-2 is suitable for the secretion of mature IL-18. These recombinant viruses can also potentially be used as immunoadjuvants and agents for anticancer therapies in vivo. PMID:24898077

  8. Pathogenesis and phylogenetic analyses of canine distemper virus strain ZJ7 isolate from domestic dogs in China

    PubMed Central

    2011-01-01

    A new isolate of canine distemper virus (CDV), named ZJ7, was isolated from lung tissues of a dog suspected with CDV infection using MDCK cells. The ZJ7 isolate induced cytopathogenic effects of syncytia in MDCK cell after six passages. In order to evaluate pathogenesis of ZJ7 strain, three CDV sero-negative dogs were intranasally inoculated with its virus suspension. All infected dogs developed clinical signs of severe bloody diarrhea, conjunctivitis, ocular discharge, nasal discharge and coughing, fever and weight loss at 21 dpi, whereas the mock group infected with DMEM were normal. The results demonstrated that CDV-ZJ7 strain isolated by MDCK cell was virulent, and the nucleotide and amino acid sequences of strain ZJ7 had no change after isolation by MDCK cell when compared with the original virus from the fresh tissues. Molecular and phylogenetic analyses for the nucleocapsid (N), phosphoprotein (P) and receptor binding haemagglutinin (H) gene of the ZJ7 isolate clearly showed it is joins to the Asia 1 group cluster of CDV strains, the predominant genotype in China. PMID:22087872

  9. Phylogenetic analysis of canine distemper virus in South America clade 1 reveals unique molecular signatures of the local epidemic.

    PubMed

    Fischer, Cristine D B; Gräf, Tiago; Ikuta, Nilo; Lehmann, Fernanda K M; Passos, Daniel T; Makiejczuk, Aline; Silveira, Marcos A T; Fonseca, André S K; Canal, Cláudio W; Lunge, Vagner R

    2016-07-01

    Canine distemper virus (CDV) is a highly contagious pathogen for domestic dogs and several wild carnivore species. In Brazil, natural infection of CDV in dogs is very high due to the large non-vaccinated dog population, a scenario that calls for new studies on the molecular epidemiology. This study investigates the phylodynamics and amino-acid signatures of CDV epidemic in South America by analyzing a large dataset compiled from publicly available sequences and also by collecting new samples from Brazil. A population of 175 dogs with canine distemper (CD) signs was sampled, from which 89 were positive for CDV, generating 42 new CDV sequences. Phylogenetic analysis of the new and publicly available sequences revealed that Brazilian sequences mainly clustered in South America 1 (SA1) clade, which has its origin estimated to the late 1980's. The reconstruction of the demographic history in SA1 clade showed an epidemic expanding until the recent years, doubling in size every nine years. SA1 clade epidemic distinguished from the world CDV epidemic by the emergence of the R580Q strain, a very rare and potentially detrimental substitution in the viral genome. The R580Q substitution was estimated to have happened in one single evolutionary step in the epidemic history in SA1 clade, emerging shortly after introduction to the continent. Moreover, a high prevalence (11.9%) of the Y549H mutation was observed among the domestic dogs sampled here. This finding was associated (p<0.05) with outcome-death and higher frequency in mixed-breed dogs, the later being an indicator of a continuous exchange of CDV strains circulating among wild carnivores and domestic dogs. The results reported here highlight the diversity of the worldwide CDV epidemic and reveal local features that can be valuable for combating the disease. PMID:27060756

  10. Antibodies to CD9, a Tetraspan Transmembrane Protein, Inhibit Canine Distemper Virus-Induced Cell-Cell Fusion but Not Virus-Cell Fusion

    PubMed Central

    Schmid, Erik; Zurbriggen, Andreas; Gassen, Uta; Rima, Bert; ter Meulen, Volker; Schneider-Schaulies, Jürgen

    2000-01-01

    Canine distemper virus (CDV) causes a life-threatening disease in several carnivores including domestic dogs. Recently, we identified a molecule, CD9, a member of the tetraspan transmembrane protein family, which facilitates, and antibodies to which inhibit, the infection of tissue culture cells with CDV (strain Onderstepoort). Here we describe that an anti-CD9 monoclonal antibody (MAb K41) did not interfere with binding of CDV to cells and uptake of virus. In addition, in single-step growth experiments, MAb K41 did not induce differences in the levels of viral mRNA and proteins. However, the virus release of syncytium-forming strains of CDV, the virus-induced cell-cell fusion in lytically infected cultures, and the cell-cell fusion of uninfected with persistently CDV-infected HeLa cells were strongly inhibited by MAb K41. These data indicate that anti-CD9 antibodies selectively block virus-induced cell-cell fusion, whereas virus-cell fusion is not affected. PMID:10906209

  11. Effect of vaccination with recombinant canine distemper virus vaccine immediately before exposure under shelter-like conditions.

    PubMed

    Larson, L J; Schultz, R D

    2006-01-01

    Vaccination with modified-live virus (MLV) canine distemper virus (CDV) vaccine has historically been recommended for animals in high-risk environments because of the rapid onset of immunity following vaccination. Recombinant CDV (rCDV) vaccine was deemed a suitable alternative to MLV-CDV vaccination in pet dogs, but insufficient data precluded its use where CDV was a serious threat to puppies, such as in shelters, kennels, and pet stores. In this study, dogs experimentally challenged hours after a single dose of rCDV or MLV vaccine became sick but recovered, whereas unvaccinated dogs became sick and died. Dogs vaccinated with a single dose of rCDV or MLV vaccine 1 week before being experimentally challenged remained healthy and showed no clinical signs. Dogs given one dose of rCDV vaccine hours before being placed in a CDV-contaminated environment did not become sick. These findings support the hypothesis that rCDV vaccine has a similar time-to-immunity as MLV-CDV vaccines and can likewise protect dogs in high-risk environments after one dose. PMID:16871493

  12. The fusion protein of wild-type canine distemper virus is a major determinant of persistent infection

    SciTech Connect

    Plattet, Philippe; Rivals, Jean-Paul; Zuber, BenoIt; Brunner, Jean-Marc; Zurbriggen, Andreas; Wittek, Riccardo . E-mail: Riccardo.Wittek@unil.ch

    2005-07-05

    The wild-type A75/17 canine distemper virus (CDV) strain induces a persistent infection in the central nervous system but infects cell lines very inefficiently. In contrast, the genetically more distant Onderstepoort CDV vaccine strain (OP-CDV) induces extensive syncytia formation. Here, we investigated the roles of wild-type fusion (F{sub WT}) and attachment (H{sub WT}) proteins in Vero cells expressing, or not, the canine SLAM receptor by transfection experiments and by studying recombinants viruses expressing different combinations of wild-type and OP-CDV glycoproteins. We show that low fusogenicity is not due to a defect of the envelope proteins to reach the cell surface and that H{sub WT} determines persistent infection in a receptor-dependent manner, emphasizing the role of SLAM as a potent enhancer of fusogenicity. However, importantly, F{sub WT} reduced cell-to-cell fusion independently of the cell surface receptor, thus demonstrating that the fusion protein of the neurovirulent A75/17-CDV strain plays a key role in determining persistent infection.

  13. Experimental Adaptation of Wild-Type Canine Distemper Virus (CDV) to the Human Entry Receptor CD150

    PubMed Central

    Bieringer, Maria; Han, Jung Woo; Kendl, Sabine; Khosravi, Mojtaba; Plattet, Philippe; Schneider-Schaulies, Jürgen

    2013-01-01

    Canine distemper virus (CDV), a close relative of measles virus (MV), is widespread and well known for its broad host range. When the goal of measles eradication may be achieved, and when measles vaccination will be stopped, CDV might eventually cross the species barrier to humans and emerge as a new human pathogen. In order to get an impression how fast such alterations may occur, we characterized required adaptive mutations to the human entry receptors CD150 (SLAM) and nectin-4 as first step to infect human target cells. Recombinant wild-type CDV-A75/17red adapted quickly to growth in human H358 epithelial cells expressing human nectin-4. Sequencing of the viral attachment proteins (hemagglutinin, H, and fusion protein, F) genes revealed that no adaptive alteration was required to utilize human nectin-4. In contrast, the virus replicated only to low titres (102 pfu/ml) in Vero cells expressing human CD150 (Vero-hSLAM). After three passages using these cells virus was adapted to human CD150 and replicated to high titres (105 pfu/ml). Sequence analyses revealed that only one amino acid exchange in the H-protein at position 540 Asp→Gly (D540G) was required for functional adaptation to human CD150. Structural modelling suggests that the adaptive mutation D540G in H reflects the sequence alteration from canine to human CD150 at position 70 and 71 from Pro to Leu (P70L) and Gly to Glu (G71E), and compensates for the gain of a negative charge in the human CD150 molecule. Using this model system our data indicate that only a minimal alteration, in this case one adaptive mutation, is required for adaptation of CDV to the human entry receptors, and help to understand the molecular basis why this adaptive mutation occurs. PMID:23554862

  14. RT-PCR and sequence analysis of the full-length fusion protein of Canine Distemper Virus from domestic dogs.

    PubMed

    Romanutti, Carina; Gallo Calderón, Marina; Keller, Leticia; Mattion, Nora; La Torre, José

    2016-02-01

    During 2007-2014, 84 out of 236 (35.6%) samples from domestic dogs submitted to our laboratory for diagnostic purposes were positive for Canine Distemper Virus (CDV), as analyzed by RT-PCR amplification of a fragment of the nucleoprotein gene. Fifty-nine of them (70.2%) were from dogs that had been vaccinated against CDV. The full-length gene encoding the Fusion (F) protein of fifteen isolates was sequenced and compared with that of those of other CDVs, including wild-type and vaccine strains. Phylogenetic analysis using the F gene full-length sequences grouped all the Argentinean CDV strains in the SA2 clade. Sequence identity with the Onderstepoort vaccine strain was 89.0-90.6%, and the highest divergence was found in the 135 amino acids corresponding to the F protein signal-peptide, Fsp (64.4-66.7% identity). In contrast, this region was highly conserved among the local strains (94.1-100% identity). One extra putative N-glycosylation site was identified in the F gene of CDV Argentinean strains with respect to the vaccine strain. The present report is the first to analyze full-length F protein sequences of CDV strains circulating in Argentina, and contributes to the knowledge of molecular epidemiology of CDV, which may help in understanding future disease outbreaks. PMID:26611227

  15. Identification of a novel canine distemper virus B-cell epitope using a monoclonal antibody against nucleocapsid protein.

    PubMed

    Yi, Li; Cheng, Yuening; Zhang, Miao; Cao, Zhigang; Tong, Mingwei; Wang, Jianke; Zhao, Hang; Lin, Peng; Cheng, Shipeng

    2016-02-01

    Canine distemper virus (CDV) is a member of the genus Morbillivirus within the family Paramyxoviridae and has caused severe economic losses in China. Nucleocapsid protein (N) is the major structural viral protein and can be used to diagnose CDV and other morbilliviruses. In this study, a specific monoclonal antibody, 1N8, was produced against the CDV N protein (amino acids 277-471). A linear N protein epitope was identified by subjecting a series of partially overlapping synthesized peptides to enzyme-linked immunosorbent assay (ELISA) analysis. The results indicated that (350)LNFGRSYFDPA(360) was the minimal linear epitope that could be recognized by mAb 1N8. ELISA assays revealed that mouse anti-CDV sera could also recognize the minimal linear epitope. Alignment analysis of the amino acid sequences indicated that the epitope was highly conserved among CDV strains. Furthermore, the epitope was conserved among other morbilliviruses, which was confirmed with PRRV using western blotting. Taken together, the results of this study may have potential applications in the development of suitable diagnostic techniques for CDV or other morbilliviruses. PMID:26514066

  16. Parasites in harbour seals ( Phoca vitulina) from the German Wadden Sea between two Phocine Distemper Virus epidemics

    NASA Astrophysics Data System (ADS)

    Lehnert, K.; Raga, J. A.; Siebert, U.

    2007-12-01

    Parasites were collected from 107 harbour seals ( Phoca vitulina) found on the coasts of Schleswig-Holstein, Germany, between 1997 and 2000. The prevalence of the parasites and their associated pathology were investigated. Eight species of parasites, primarily nematodes, were identified from the examined organs: two anisakid nematodes ( Pseudoterranova decipiens (sensu lato) , Contracaecum osculatum (sensu lato)) from the stomach, Otostrongylus circumlitus (Crenosomatidae) and Parafilaroides gymnurus (Filaroididae) from the respiratory tract, one filarioid nematode ( Acanthocheilonema spirocauda) from the heart, two acanthocephalans, Corynosoma strumosum and C. semerme (Polymorphidae), from the intestine and an ectoparasite, Echinophthirius horridus (Anoplura, Insecta). Lungworm infection was the most prominent parasitological finding and secondary bacterial bronchopneumonia the most pathogenic lesion correlated with the parasites. Heavy nematode burdens in the respiratory tract were highly age-related and more frequent in young seals. A positive correlation was observed between high levels of pulmonary infection and severity of bronchopneumonia. The prevalence of lungworms in this study was higher than in seals that died during the 1988/1989 Phocine Distemper Virus epidemic, and the prevalence of acanthocephalans and heartworms had decreased compared to findings from the first die-off.

  17. Two Domains of the V Protein of Virulent Canine Distemper Virus Selectively Inhibit STAT1 and STAT2 Nuclear Import▿

    PubMed Central

    Röthlisberger, Anne; Wiener, Dominique; Schweizer, Matthias; Peterhans, Ernst; Zurbriggen, Andreas; Plattet, Philippe

    2010-01-01

    Canine distemper virus (CDV) causes in dogs a severe systemic infection, with a high frequency of demyelinating encephalitis. Among the six genes transcribed by CDV, the P gene encodes the polymerase cofactor protein (P) as well as two additional nonstructural proteins, C and V; of these V was shown to act as a virulence factor. We investigated the molecular mechanisms by which the P gene products of the neurovirulent CDV A75/17 strain disrupt type I interferon (IFN-α/β)-induced signaling that results in the establishment of the antiviral state. Using recombinant knockout A75/17 viruses, the V protein was identified as the main antagonist of IFN-α/β-mediated signaling. Importantly, immunofluorescence analysis illustrated that the inhibition of IFN-α/β-mediated signaling correlated with impaired STAT1/STAT2 nuclear import, whereas the phosphorylation state of these proteins was not affected. Coimmunoprecipitation assays identified the N-terminal region of V (VNT) responsible for STAT1 targeting, which correlated with its ability to inhibit the activity of the IFN-α/β-mediated antiviral state. Conversely, while the C-terminal domain of V (VCT) could not function autonomously, when fused to VNT it optimally interacted with STAT2 and subsequently efficiently suppressed the IFN-α/β-mediated signaling pathway. The latter result was further supported by a single mutation at position 110 within the VNT domain of CDV V protein, resulting in a mutant that lost STAT1 binding while retaining a partial STAT2 association. Taken together, our results identified the CDV VNT and VCT as two essential modules that complement each other to interfere with the antiviral state induced by IFN-α/β-mediated signaling. Hence, our experiments reveal a novel mechanism of IFN-α/β evasion among the morbilliviruses. PMID:20427537

  18. Estimating the potential impact of canine distemper virus on the Amur tiger population (Panthera tigris altaica) in Russia.

    PubMed

    Gilbert, Martin; Miquelle, Dale G; Goodrich, John M; Reeve, Richard; Cleaveland, Sarah; Matthews, Louise; Joly, Damien O

    2014-01-01

    Lethal infections with canine distemper virus (CDV) have recently been diagnosed in Amur tigers (Panthera tigris altaica), but long-term implications for the population are unknown. This study evaluates the potential impact of CDV on a key tiger population in Sikhote-Alin Biosphere Zapovednik (SABZ), and assesses how CDV might influence the extinction potential of other tiger populations of varying sizes. An individual-based stochastic, SIRD (susceptible-infected-recovered/dead) model was used to simulate infection through predation of infected domestic dogs, and/or wild carnivores, and direct tiger-to-tiger transmission. CDV prevalence and effective contact based on published and observed data was used to define plausible low- and high-risk infection scenarios. CDV infection increased the 50-year extinction probability of tigers in SABZ by 6.3% to 55.8% compared to a control population, depending on risk scenario. The most significant factors influencing model outcome were virus prevalence in the reservoir population(s) and its effective contact rate with tigers. Adjustment of the mortality rate had a proportional impact, while inclusion of epizootic infection waves had negligible additional impact. Small populations were found to be disproportionately vulnerable to extinction through CDV infection. The 50-year extinction risk in populations consisting of 25 individuals was 1.65 times greater when CDV was present than that of control populations. The effects of density dependence do not protect an endangered population from the impacts of a multi-host pathogen, such as CDV, where they coexist with an abundant reservoir presenting a persistent threat. Awareness of CDV is a critical component of a successful tiger conservation management policy. PMID:25354196

  19. Estimating the Potential Impact of Canine Distemper Virus on the Amur Tiger Population (Panthera tigris altaica) in Russia

    PubMed Central

    Gilbert, Martin; Miquelle, Dale G.; Goodrich, John M.; Reeve, Richard; Cleaveland, Sarah; Matthews, Louise; Joly, Damien O.

    2014-01-01

    Lethal infections with canine distemper virus (CDV) have recently been diagnosed in Amur tigers (Panthera tigris altaica), but long-term implications for the population are unknown. This study evaluates the potential impact of CDV on a key tiger population in Sikhote-Alin Biosphere Zapovednik (SABZ), and assesses how CDV might influence the extinction potential of other tiger populations of varying sizes. An individual-based stochastic, SIRD (susceptible-infected-recovered/dead) model was used to simulate infection through predation of infected domestic dogs, and/or wild carnivores, and direct tiger-to-tiger transmission. CDV prevalence and effective contact based on published and observed data was used to define plausible low- and high-risk infection scenarios. CDV infection increased the 50-year extinction probability of tigers in SABZ by 6.3% to 55.8% compared to a control population, depending on risk scenario. The most significant factors influencing model outcome were virus prevalence in the reservoir population(s) and its effective contact rate with tigers. Adjustment of the mortality rate had a proportional impact, while inclusion of epizootic infection waves had negligible additional impact. Small populations were found to be disproportionately vulnerable to extinction through CDV infection. The 50-year extinction risk in populations consisting of 25 individuals was 1.65 times greater when CDV was present than that of control populations. The effects of density dependence do not protect an endangered population from the impacts of a multi-host pathogen, such as CDV, where they coexist with an abundant reservoir presenting a persistent threat. Awareness of CDV is a critical component of a successful tiger conservation management policy. PMID:25354196

  20. Water system virus detection

    NASA Technical Reports Server (NTRS)

    Fraser, A. S.; Wells, A. F.; Tenoso, H. J.

    1975-01-01

    A monitoring system developed to test the capability of a water recovery system to reject the passage of viruses into the recovered water is described. A nonpathogenic marker virus, bacteriophage F2, is fed into the process stream before the recovery unit and the reclaimed water is assayed for its presence. Detection of the marker virus consists of two major components, concentration and isolation of the marker virus, and detection of the marker virus. The concentration system involves adsorption of virus to cellulose acetate filters in the presence of trivalent cations and low pH with subsequent desorption of the virus using volumes of high pH buffer. The detection of the virus is performed by a passive immune agglutination test utilizing specially prepared polystyrene particles. An engineering preliminary design was performed as a parallel effort to the laboratory development of the marker virus test system. Engineering schematics and drawings of a fully functional laboratory prototype capable of zero-G operation are presented. The instrument consists of reagent pump/metering system, reagent storage containers, a filter concentrator, an incubation/detector system, and an electronic readout and control system.

  1. The hemagglutinin envelope protein of canine distemper virus (CDV) confers cell tropism as illustrated by CDV and measles virus complementation analysis.

    PubMed Central

    Stern, L B; Greenberg, M; Gershoni, J M; Rozenblatt, S

    1995-01-01

    Measles virus (MV) and canine distemper virus (CDV) are morbilliviruses that cause acute illnesses and several persistent central nervous system infections in humans and in dogs, respectively. Characteristically, the cytopathic effect of these viruses is the formation of syncytia in permissive cells. In this study, a vaccinia virus expression system was used to express MV and CDV hemagglutinin (HA) and fusion (F) envelope proteins. We found that cotransfecting F and HA genes of MV or F and HA genes of CDV resulted in extensive syncytium formation in permissive cells while transfecting either F or HA alone did not. Similar experiments with heterologous pairs of proteins, CDV-F with MV-HA or MV-F with CDV-HA, caused significant cell fusion in both cases. These results indicate that in this expression system, cell fusion requires both F and HA; however, the functions of these proteins are interchangeable between the two types of morbilliviruses. Human-mouse somatic hybrids were used to determine the human chromosome conferring susceptibility to either MV and CDV. Of the 12 hybrids screened, none were sensitive to MV. Two of the hybrids containing human chromosome 19 formed syncytia following CDV infection. In addition, these two hybrids underwent cell fusion when cotransfected with CDV-F and CDV-HA (but not MV-F and MV-HA) glycoproteins by using the vaccinia virus expression system. To discover the viral component responsible for cell specificity, complementation experiments coexpressing CDV-HA with MV-F or CDV-F with MV-HA in the CDV-sensitive hybrids were performed. We found that syncytia were formed only in the presence of CDV-HA. These results support the idea that the HA protein is responsible for cell tropism. Furthermore, while the F protein is necessary for the fusion process, it is interchangeable with the F protein from other morbilliviruses. PMID:7853502

  2. Dynamics of a morbillivirus at the domestic–wildlife interface: Canine distemper virus in domestic dogs and lions

    PubMed Central

    Viana, Mafalda; Matthiopoulos, Jason; Halliday, Jo; Packer, Craig; Craft, Meggan E.; Hampson, Katie; Czupryna, Anna; Dobson, Andrew P.; Dubovi, Edward J.; Ernest, Eblate; Fyumagwa, Robert; Hoare, Richard; Hopcraft, J. Grant C.; Horton, Daniel L.; Kaare, Magai T.; Kanellos, Theo; Lankester, Felix; Mentzel, Christine; Mlengeya, Titus; Mzimbiri, Imam; Takahashi, Emi; Willett, Brian; Haydon, Daniel T.; Lembo, Tiziana

    2015-01-01

    Morbilliviruses cause many diseases of medical and veterinary importance, and although some (e.g., measles and rinderpest) have been controlled successfully, others, such as canine distemper virus (CDV), are a growing concern. A propensity for host-switching has resulted in CDV emergence in new species, including endangered wildlife, posing challenges for controlling disease in multispecies communities. CDV is typically associated with domestic dogs, but little is known about its maintenance and transmission in species-rich areas or about the potential role of domestic dog vaccination as a means of reducing disease threats to wildlife. We address these questions by analyzing a long-term serological dataset of CDV in lions and domestic dogs from Tanzania’s Serengeti ecosystem. Using a Bayesian state–space model, we show that dynamics of CDV have changed considerably over the past three decades. Initially, peaks of CDV infection in dogs preceded those in lions, suggesting that spill-over from dogs was the main driver of infection in wildlife. However, despite dog-to-lion transmission dominating cross-species transmission models, infection peaks in lions became more frequent and asynchronous from those in dogs, suggesting that other wildlife species may play a role in a potentially complex maintenance community. Widespread mass vaccination of domestic dogs reduced the probability of infection in dogs and the size of outbreaks but did not prevent transmission to or peaks of infection in lions. This study demonstrates the complexity of CDV dynamics in natural ecosystems and the value of long-term, large-scale datasets for investigating transmission patterns and evaluating disease control strategies. PMID:25605919

  3. Multiplex Amplification Refractory Mutation System Polymerase Chain Reaction (ARMS-PCR) for diagnosis of natural infection with canine distemper virus

    PubMed Central

    2010-01-01

    Background Canine distemper virus (CDV) is present worldwide and produces a lethal systemic infection of wild and domestic Canidae. Pre-existing antibodies acquired from vaccination or previous CDV infection might interfere the interpretation of a serologic diagnosis method. In addition, due to the high similarity of nucleic acid sequences between wild-type CDV and the new vaccine strain, current PCR derived methods cannot be applied for the definite confirmation of CD infection. Hence, it is worthy of developing a simple and rapid nucleotide-based assay for differentiation of wild-type CDV which is a cause of disease from attenuated CDVs after vaccination. High frequency variations have been found in the region spanning from the 3'-untranslated region (UTR) of the matrix (M) gene to the fusion (F) gene (designated M-F UTR) in a few CDV strains. To establish a differential diagnosis assay, an amplification refractory mutation analysis was established based on the highly variable region on M-F UTR and F regions. Results Sequences of frequent polymorphisms were found scattered throughout the M-F UTR region; the identity of nucleic acid between local strains and vaccine strains ranged from 82.5% to 93.8%. A track of AAA residue located 35 nucleotides downstream from F gene start codon highly conserved in three vaccine strains were replaced with TGC in the local strains; that severed as target sequences for deign of discrimination primers. The method established in the present study successfully differentiated seven Taiwanese CDV field isolates, all belonging to the Asia-1 lineage, from vaccine strains. Conclusions The method described herein would be useful for several clinical applications, such as confirmation of nature CDV infection, evaluation of vaccination status and verification of the circulating viral genotypes. PMID:20534175

  4. The Role of Canine Distemper Virus and Persistent Organic Pollutants in Mortality Patterns of Caspian Seals (Pusa caspica)

    PubMed Central

    Wilson, Susan C.; Eybatov, Tariel M.; Amano, Masao; Jepson, Paul D.; Goodman, Simon J.

    2014-01-01

    Persistent organic pollutants are a concern for species occupying high trophic levels since they can cause immunosuppression and impair reproduction. Mass mortalities due to canine distemper virus (CDV) occurred in Caspian seals (Pusa caspica), in spring of 1997, 2000 and 2001, but the potential role of organochlorine exposure in these epizootics remains undetermined. Here we integrate Caspian seal mortality data spanning 1971–2008, with data on age, body condition, pathology and blubber organochlorine concentration for carcases stranded between 1997 and 2002. We test the hypothesis that summed PCB and DDT concentrations contributed to CDV associated mortality during epizootics. We show that age is the primary factor explaining variation in blubber organochlorine concentrations, and that organochlorine burden, age, sex, and body condition do not account for CDV infection status (positive/negative) of animals dying in epizootics. Most animals (57%, n = 67) had PCB concentrations below proposed thresholds for toxic effects in marine mammals (17 µg/g lipid weight), and only 3 of 67 animals had predicted TEQ values exceeding levels seen to be associated with immune suppression in harbour seals (200 pg/g lipid weight). Mean organonchlorine levels were higher in CDV-negative animals indicating that organochlorines did not contribute significantly to CDV mortality in epizootics. Mortality monitoring in Azerbaijan 1971–2008 revealed bi-annual stranding peaks in late spring, following the annual moult and during autumn migrations northwards. Mortality peaks comparable to epizootic years were also recorded in the 1970s–1980s, consistent with previous undocumented CDV outbreaks. Gompertz growth curves show that Caspian seals achieve an asymptotic standard body length of 126–129 cm (n = 111). Males may continue to grow slowly throughout life. Mortality during epizootics may exceed the potential biological removal level (PBR) for the population, but the low

  5. Dog overpopulation and burden of exposure to canine distemper virus and other pathogens on Santa Cruz Island, Galapagos.

    PubMed

    Diaz, Nicole M; Mendez, Gabriella S; Grijalva, C Jaime; Walden, Heather S; Cruz, Marilyn; Aragon, Eduardo; Hernandez, Jorge A

    2016-01-01

    Dog overpopulation and diseases are hazards to native island species and humans on the Galapagos. Vaccination and importation of dogs are prohibited on the Galapagos. Risk management of these hazards requires the use of science-based risk assessment and risk communication. The objectives of the study reported here were (i) to estimate the human:dog ratio and (ii) the prevalence of and identify exposure factors associated with positive antibody titers to canine distemper virus (CDV) and other pathogens, as well as infection with intestinal parasites in owned dogs on Santa Cruz Island, Galapagos in September 2014. The observed human:dog ratio was 6.148:1 which extrapolates to 2503 dogs (two times more than a recent dog count conducted by Galapagos Biosecurity Agency in March 2014). The proportion of spayed female dogs (50%) was higher, compared to neutered male dogs (30%) (p=0.04). Prevalence of dogs with positive antibody titers to CDV was 36% (95% CI=26, 46%), to canine parvovirus was 89% (95% CI=82, 95%), and to canine adenovirus was 40% (95% CI=30, 51%). The frequency of seropositive dogs to CDV was lower in urban dogs (26%), compared to rural dogs (53%) (p<0.05). A positive interaction effect between rural residence and spay/neuter status on seropositivity to CDV was observed, which we discuss in this report. Because vaccination is prohibited, the dog population on Santa Cruz is susceptible to an outbreak of CDV (particularly among urban dogs) with potential spill over to marine mammals. Dog's age (1-2 or 3-14 years old, compared to younger dogs), and residence (rural, urban) were associated with positive antibody titers to parvovirus, adenovirus, Ehrlichia spp., or Anaplasma spp., as well as infection with Ancylostoma spp., an intestinal parasite in dogs that can be transmitted to humans, particularly children. These results provide the most comprehensive assessment of dog overpopulation and exposure to CDV and other pathogens on the Galapagos to date. PMID

  6. Dynamics of a morbillivirus at the domestic-wildlife interface: Canine distemper virus in domestic dogs and lions.

    PubMed

    Viana, Mafalda; Cleaveland, Sarah; Matthiopoulos, Jason; Halliday, Jo; Packer, Craig; Craft, Meggan E; Hampson, Katie; Czupryna, Anna; Dobson, Andrew P; Dubovi, Edward J; Ernest, Eblate; Fyumagwa, Robert; Hoare, Richard; Hopcraft, J Grant C; Horton, Daniel L; Kaare, Magai T; Kanellos, Theo; Lankester, Felix; Mentzel, Christine; Mlengeya, Titus; Mzimbiri, Imam; Takahashi, Emi; Willett, Brian; Haydon, Daniel T; Lembo, Tiziana

    2015-02-01

    Morbilliviruses cause many diseases of medical and veterinary importance, and although some (e.g., measles and rinderpest) have been controlled successfully, others, such as canine distemper virus (CDV), are a growing concern. A propensity for host-switching has resulted in CDV emergence in new species, including endangered wildlife, posing challenges for controlling disease in multispecies communities. CDV is typically associated with domestic dogs, but little is known about its maintenance and transmission in species-rich areas or about the potential role of domestic dog vaccination as a means of reducing disease threats to wildlife. We address these questions by analyzing a long-term serological dataset of CDV in lions and domestic dogs from Tanzania's Serengeti ecosystem. Using a Bayesian state-space model, we show that dynamics of CDV have changed considerably over the past three decades. Initially, peaks of CDV infection in dogs preceded those in lions, suggesting that spill-over from dogs was the main driver of infection in wildlife. However, despite dog-to-lion transmission dominating cross-species transmission models, infection peaks in lions became more frequent and asynchronous from those in dogs, suggesting that other wildlife species may play a role in a potentially complex maintenance community. Widespread mass vaccination of domestic dogs reduced the probability of infection in dogs and the size of outbreaks but did not prevent transmission to or peaks of infection in lions. This study demonstrates the complexity of CDV dynamics in natural ecosystems and the value of long-term, large-scale datasets for investigating transmission patterns and evaluating disease control strategies. PMID:25605919

  7. The role of canine distemper virus and persistent organic pollutants in mortality patterns of Caspian seals (Pusa caspica).

    PubMed

    Wilson, Susan C; Eybatov, Tariel M; Amano, Masao; Jepson, Paul D; Goodman, Simon J

    2014-01-01

    Persistent organic pollutants are a concern for species occupying high trophic levels since they can cause immunosuppression and impair reproduction. Mass mortalities due to canine distemper virus (CDV) occurred in Caspian seals (Pusa caspica), in spring of 1997, 2000 and 2001, but the potential role of organochlorine exposure in these epizootics remains undetermined. Here we integrate Caspian seal mortality data spanning 1971-2008, with data on age, body condition, pathology and blubber organochlorine concentration for carcases stranded between 1997 and 2002. We test the hypothesis that summed PCB and DDT concentrations contributed to CDV associated mortality during epizootics. We show that age is the primary factor explaining variation in blubber organochlorine concentrations, and that organochlorine burden, age, sex, and body condition do not account for CDV infection status (positive/negative) of animals dying in epizootics. Most animals (57%, n = 67) had PCB concentrations below proposed thresholds for toxic effects in marine mammals (17 µg/g lipid weight), and only 3 of 67 animals had predicted TEQ values exceeding levels seen to be associated with immune suppression in harbour seals (200 pg/g lipid weight). Mean organonchlorine levels were higher in CDV-negative animals indicating that organochlorines did not contribute significantly to CDV mortality in epizootics. Mortality monitoring in Azerbaijan 1971-2008 revealed bi-annual stranding peaks in late spring, following the annual moult and during autumn migrations northwards. Mortality peaks comparable to epizootic years were also recorded in the 1970s-1980s, consistent with previous undocumented CDV outbreaks. Gompertz growth curves show that Caspian seals achieve an asymptotic standard body length of 126-129 cm (n = 111). Males may continue to grow slowly throughout life. Mortality during epizootics may exceed the potential biological removal level (PBR) for the population, but the low frequency of

  8. Water system virus detection

    NASA Technical Reports Server (NTRS)

    Fraser, A. S.; Wells, A. F.; Tenoso, H. J. (Inventor)

    1978-01-01

    The performance of a waste water reclamation system is monitored by introducing a non-pathogenic marker virus, bacteriophage F2, into the waste-water prior to treatment and, thereafter, testing the reclaimed water for the presence of the marker virus. A test sample is first concentrated by absorbing any marker virus onto a cellulose acetate filter in the presence of a trivalent cation at low pH and then flushing the filter with a limited quantity of a glycine buffer solution to desorb any marker virus present on the filter. Photo-optical detection of indirect passive immune agglutination by polystyrene beads indicates the performance of the water reclamation system in removing the marker virus. A closed system provides for concentrating any marker virus, initiating and monitoring the passive immune agglutination reaction, and then flushing the system to prepare for another sample.

  9. Nucleotide and deduced amino acid sequences of the nucleocapsid protein of the virulent A75/17-CDV strain of canine distemper virus.

    PubMed

    Stettler, M; Zurbriggen, A

    1995-05-01

    Virus persistence is essential in the chronic inflammatory canine distemper virus (CDV)-induced demyelinating disease. In the case of CDV there is a close association between persistence and virulence. Virulent CDV isolated from dogs with distemper shows immediate persistence in primary dog brain cell cultures (DBCC) and in different cell lines. We have evidence that the nucleocapsid (NP) protein plays an important role in the development of persistence. The NP-protein, the most abundant structural virus protein, also influences virus assembly and has some regulatory functions in virus transcription and replication. In this study we compared the nucleotide and deduced amino acid sequence of a virulent CDV strain (A75/17-CDV) to a culture-attenuated non-virulent strain (OP-CDV). Viral RNA was extracted from DBCC infected with virulent CDV. Virulent CDV retains its in vivo properties, such as virulence and ability to cause demyelination, when propagated in these DBCC. The viral RNA was reverse transcribed and the resulting cDNA amplified by polymerase chain reaction for subsequent cloning. The nucleotide sequences of these clones were determined by the dideoxy chain termination method. The number of nucleotides and the putative NP-protein of the virulent strain matched the attenuated CDV strain. We observed a total of 105 nucleotide differences. Three were localised within the 3' and five within the 5' non-coding region of the NP-gene. The 97 nucleotide changes within the coding region resulted in 22 amino acid differences. 10 of these amino acid (AA) modifications were within the N-terminal region (AA 1 to 159) and 12 within the C-terminal area (AA 351 to 523).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8588315

  10. In vitro cytokine responses of peripheral blood mononuclear cells from healthy dogs to distemper virus, Malassezia and Toxocara.

    PubMed

    Valli, J L; Williamson, A; Sharif, S; Rice, J; Shewen, P E

    2010-04-15

    Naïve CD4+ T cells may differentiate into a number of subsets including T helper 1 (Th1) Th2, Th3, Th17 and T regulatory (Treg) cells depending on the type of antigen they encounter. These CD4+ families have been defined based on the array of cytokines they produce and the effects they have on adaptive immune responses. CD4+ subsets are cross regulatory and at times cooperative. The study of these adaptive immune modulators has revealed the important role that cytokines play in mounting effective as well as detrimental immune responses to pathogens. Examining the cytokine responses of lymphocytes in culture can provide important understanding of how immune responses to pathogens are orchestrated. For this purpose the in vitro cytokine production of peripheral blood mononuclear cells (PBMC) from healthy dogs was examined in response to stimulation with antigens from a common canine virus (canine distemper virus, CDV), a commensal skin yeast of dogs (Malassezia pachydermatis) and a common canine helminth (Toxocara canis (T. canis)). Cell culture supernatants were removed from antigen stimulated and unstimulated control PBMC after 4, 24, 48 and 72 h and the concentration of Th1 type cytokines (IL-2, IFN-gamma, TNF-alpha) and Th2 type cytokines (IL-4, IL-5, IL-10) was determined using sandwich ELISA assays. CDV induced low levels of cytokine production initially with a predominance of IL-10 at 24h and a balanced response at 48 h of incubation. Malassezia antigen stimulated an early type 2 cytokine response with dramatic production of IL-4 at 24h of incubation compared to the other stimulants examined. By 48 h of incubation, however, the cytokine mix in response to Malassezia had also moved toward a Th1 type response. T. canis induced early production of Th2 type cytokines with IL-5 predominating; however, with longer incubation (48-72 h) there was a switch to a balanced Th1/Th2 response. In conclusion, the cytokines produced in vitro by canine PBMC in response to

  11. Vaccination against canine distemper virus infection in infant ferrets with and without maternal antibody protection, using recombinant attenuated poxvirus vaccines.

    PubMed

    Welter, J; Taylor, J; Tartaglia, J; Paoletti, E; Stephensen, C B

    2000-07-01

    Canine distemper virus (CDV) infection of ferrets is clinically and immunologically similar to measles, making this a useful model for the human disease. The model was used to determine if parenteral or mucosal immunization of infant ferrets at 3 and 6 weeks of age with attenuated vaccinia virus (NYVAC) or canarypox virus (ALVAC) vaccine strains expressing the CDV hemagglutinin (H) and fusion (F) protein genes (NYVAC-HF and ALVAC-HF) would induce serum neutralizing antibody and protect against challenge infection at 12 weeks of age. Ferrets without maternal antibody that were vaccinated parenterally with NYVAC-HF (n = 5) or ALVAC-HF (n = 4) developed significant neutralizing titers (log(10) inverse mean titer +/- standard deviation of 2.30 +/- 0.12 and 2.20 +/- 0.34, respectively) by the day of challenge, and all survived with no clinical or virologic evidence of infection. Ferrets without maternal antibody that were vaccinated intranasally (i.n.) developed lower neutralizing titers, with NYVAC-HF producing higher titers at challenge (1.11 +/- 0.57 versus 0.40 +/- 0.37, P = 0.02) and a better survival rate (6/7 versus 0/5, P = 0.008) than ALVAC-HF. Ferrets with maternal antibody that were vaccinated parenterally with NYVAC-HF (n = 7) and ALVAC-HF (n = 7) developed significantly higher antibody titers (1.64 +/- 0. 54 and 1.28 +/- 0.40, respectively) than did ferrets immunized with an attenuated CDV vaccine (0.46 +/- 0.59; n = 7) or the recombinant vectors expressing rabies glycoprotein (RG) (0.19 +/- 0.32; n = 8, P = 7 x 10(-6)). The NYVAC vaccine also protected against weight loss, and both the NYVAC and attenuated CDV vaccines protected against the development of some clinical signs of infection, although survival in each of the three vaccine groups was low (one of seven) and not significantly different from the RG controls (none of eight). Combined i.n.-parenteral immunization of ferrets with maternal antibody using NYVAC-HF (n = 9) produced higher titers (1

  12. Vaccination against Canine Distemper Virus Infection in Infant Ferrets with and without Maternal Antibody Protection, Using Recombinant Attenuated Poxvirus Vaccines

    PubMed Central

    Welter, Janet; Taylor, Jill; Tartaglia, James; Paoletti, Enzo; Stephensen, Charles B.

    2000-01-01

    Canine distemper virus (CDV) infection of ferrets is clinically and immunologically similar to measles, making this a useful model for the human disease. The model was used to determine if parenteral or mucosal immunization of infant ferrets at 3 and 6 weeks of age with attenuated vaccinia virus (NYVAC) or canarypox virus (ALVAC) vaccine strains expressing the CDV hemagglutinin (H) and fusion (F) protein genes (NYVAC-HF and ALVAC-HF) would induce serum neutralizing antibody and protect against challenge infection at 12 weeks of age. Ferrets without maternal antibody that were vaccinated parenterally with NYVAC-HF (n = 5) or ALVAC-HF (n = 4) developed significant neutralizing titers (log10 inverse mean titer ± standard deviation of 2.30 ± 0.12 and 2.20 ± 0.34, respectively) by the day of challenge, and all survived with no clinical or virologic evidence of infection. Ferrets without maternal antibody that were vaccinated intranasally (i.n.) developed lower neutralizing titers, with NYVAC-HF producing higher titers at challenge (1.11 ± 0.57 versus 0.40 ± 0.37, P = 0.02) and a better survival rate (6/7 versus 0/5, P = 0.008) than ALVAC-HF. Ferrets with maternal antibody that were vaccinated parenterally with NYVAC-HF (n = 7) and ALVAC-HF (n = 7) developed significantly higher antibody titers (1.64 ± 0.54 and 1.28 ± 0.40, respectively) than did ferrets immunized with an attenuated CDV vaccine (0.46 ± 0.59; n = 7) or the recombinant vectors expressing rabies glycoprotein (RG) (0.19 ± 0.32; n = 8, P = 7 × 10−6). The NYVAC vaccine also protected against weight loss, and both the NYVAC and attenuated CDV vaccines protected against the development of some clinical signs of infection, although survival in each of the three vaccine groups was low (one of seven) and not significantly different from the RG controls (none of eight). Combined i.n.-parenteral immunization of ferrets with maternal antibody using NYVAC-HF (n = 9) produced higher titers (1.63 ± 0

  13. Sequence Analysis and Expression of the Attachment and Fusion Proteins of Canine Distemper Virus Wild-Type Strain A75/17

    PubMed Central

    Cherpillod, Pascal; Beck, Karin; Zurbriggen, Andreas; Wittek, Riccardo

    1999-01-01

    The biological properties of wild-type A75/17 and cell culture-adapted Onderstepoort canine distemper virus differ markedly. To learn more about the molecular basis for these differences, we have isolated and sequenced the protein-coding regions of the attachment and fusion proteins of wild-type canine distemper virus strain A75/17. In the attachment protein, a total of 57 amino acid differences were observed between the Onderstepoort strain and strain A75/17, and these were distributed evenly over the entire protein. Interestingly, the attachment protein of strain A75/17 contained an extension of three amino acids at the C terminus. Expression studies showed that the attachment protein of strain A75/17 had a higher apparent molecular mass than the attachment protein of the Onderstepoort strain, in both the presence and absence of tunicamycin. In the fusion protein, 60 amino acid differences were observed between the two strains, of which 44 were clustered in the much smaller F2 portion of the molecule. Significantly, the AUG that has been proposed as a translation initiation codon in the Onderstepoort strain is an AUA codon in strain A75/17. Detailed mutation analyses showed that both the first and second AUGs of strain A75/17 are the major translation initiation sites of the fusion protein. Similar analyses demonstrated that, also in the Onderstepoort strain, the first two AUGs are the translation initiation codons which contribute most to the generation of precursor molecules yielding the mature form of the fusion protein. PMID:9971809

  14. Computer Viruses: Pathology and Detection.

    ERIC Educational Resources Information Center

    Maxwell, John R.; Lamon, William E.

    1992-01-01

    Explains how computer viruses were originally created, how a computer can become infected by a virus, how viruses operate, symptoms that indicate a computer is infected, how to detect and remove viruses, and how to prevent a reinfection. A sidebar lists eight antivirus resources. (four references) (LRW)

  15. Development of a challenge-protective vaccine concept by modification of the viral RNA-dependent RNA polymerase of canine distemper virus.

    PubMed

    Silin, D; Lyubomska, O; Ludlow, M; Duprex, W P; Rima, B K

    2007-12-01

    We demonstrate that insertion of the open reading frame of enhanced green fluorescent protein (EGFP) into the coding sequence for the second hinge region of the viral L (large) protein (RNA-dependent RNA polymerase) attenuates a wild-type canine distemper virus. Moreover, we show that single intranasal immunization with this recombinant virus provides significant protection against challenge with the virulent parental virus. Protection against wild-type challenge was gained either after recovery of cellular immunity postimmunization or after development of neutralizing antibodies. Insertion of EGFP seems to result in overattenuation of the virus, while our previous experiments demonstrated that the insertion of an epitope tag into a similar position did not affect L protein function. Thus, a desirable level of attenuation could be reached by manipulating the length of the insert (in the second hinge region of the L protein), providing additional tools for optimization of controlled attenuation. This strategy for controlled attenuation may be useful for a "quick response" in vaccine development against well-known and "new" viral infections and could be combined efficiently with other strategies of vaccine development and delivery systems. PMID:17898047

  16. Vaccination of puppies born to immune dams with a canine adenovirus-based vaccine protects against a canine distemper virus challenge.

    PubMed

    Fischer, Laurent; Tronel, Jean Phillipe; Pardo-David, Camilla; Tanner, Patrick; Colombet, Guy; Minke, Jules; Audonnet, Jean-Christophe

    2002-10-01

    None of the currently available distemper vaccines provides a satisfactory solution for the immunization of very young carnivores in the face of maternal-derived immunity. Since mucosal immunization with replication-competent adenovirus-based vaccines has been proven effective in the face of passive immunity against the vector, it has the potential to provide a solution for the vaccination of young puppies born to canine distemper virus (CDV)-immune dams. We report the engineering and the characterization of two replication-competent canine adenovirus type 2 (CAV2)-based vaccines expressing, respectively, the CDV hemagglutinin (HA) and fusion (F) antigens. We first demonstrated that the intranasal vaccination with a mixture of both recombinant CAV2s provides an excellent level of protection in seronegative puppies, confirming the value of replication-competent adenovirus-based vectors for mucosal vaccination. In contrast, intranasal immunization with the same vaccine of puppies born to CDV- and CAV2-immune dams, failed to activate specific and protective immune responses. We hypothesized that an active CAV2 infection occurred while puppies were in close contact with the vaccinated dams in the breeding units and that the resulting active mucosal immunity interfered with the intranasal administration of CAV2-based CDV vaccine. However, when puppies born to CDV- and CAV2-immune dams were vaccinated subcutaneously with the CAV2-based CDV vaccine, significant seroconversion and solid protective immunity were triggered despite pre-existing systemic immunity to the vector. This latter result is surprising and suggests that subcutaneous vaccination with a replication-competent recombinant CAV2 may be an efficient strategy to overcome both passive and active adenovirus specific immunity in the dog. From a practical point of view, this could pave the way for an original strategy to vaccinate young puppies in the face of maternal-derived immunity. PMID:12297394

  17. Characterization of two recent Japanese field isolates of canine distemper virus and examination of the avirulent strain utility as an attenuated vaccine.

    PubMed

    Takenaka, Akiko; Yoneda, Misako; Seki, Takahiro; Uema, Masashi; Kooriyama, Takanori; Nishi, Toshiya; Fujita, Kentaro; Miura, Ryuichi; Tsukiyama-Kohara, Kyoko; Sato, Hiroki; Kai, Chieko

    2014-12-01

    Recently, several new strains of canine distemper virus (CDV) have been isolated in Japan. To investigate their pathogenesis in dogs, the Yanaka and Bunkyo-K strains were investigated by infecting dogs and determining clinical signs, amount of virus, and antibody responses. The Yanaka strain is avirulent and induced an antibody response. The Bunkyo-K strain induced typical CDV clinical signs in infected dogs and virulence was enhanced by brain passage. Molecular and phylogenetic analyses of H genes demonstrated the Bunkyo-K strains were of a different lineage from Asia-1 group including the Yanaka strain and Asia-2 group that contain recent Japanese isolates, which were recently identified as major prevalent strains worldwide but distinct from old vaccine strains. Based on these data, we tested the ability of the Yanaka strain for vaccination. Inoculation with the Yanaka strain efficiently induced CDV neutralizing antibodies with no clinical signs, and the protection effects against challenge with either old virulent strain or Bunkyo-K strain were equal or greater when compared with vaccination by an original vaccine strain. Thus, the Yanaka strain is a potential vaccine candidate against recent prevalent CDV strains. PMID:25465179

  18. Development of a duplex real-time RT-qPCR assay to monitor genome replication, gene expression and gene insert stability during in vivo replication of a prototype live attenuated canine distemper virus vector encoding SIV gag.

    PubMed

    Coleman, John W; Wright, Kevin J; Wallace, Olivia L; Sharma, Palka; Arendt, Heather; Martinez, Jennifer; DeStefano, Joanne; Zamb, Timothy P; Zhang, Xinsheng; Parks, Christopher L

    2015-03-01

    Advancement of new vaccines based on live viral vectors requires sensitive assays to analyze in vivo replication, gene expression and genetic stability. In this study, attenuated canine distemper virus (CDV) was used as a vaccine delivery vector and duplex 2-step quantitative real-time RT-PCR (RT-qPCR) assays specific for genomic RNA (gRNA) or mRNA have been developed that concurrently quantify coding sequences for the CDV nucleocapsid protein (N) and a foreign vaccine antigen (SIV Gag). These amplicons, which had detection limits of about 10 copies per PCR reaction, were used to show that abdominal cavity lymphoid tissues were a primary site of CDV vector replication in infected ferrets, and importantly, CDV gRNA or mRNA was undetectable in brain tissue. In addition, the gRNA duplex assay was adapted for monitoring foreign gene insert genetic stability during in vivo replication by analyzing the ratio of CDV N and SIV gag genomic RNA copies over the course of vector infection. This measurement was found to be a sensitive probe for assessing the in vivo genetic stability of the foreign gene insert. PMID:25486083

  19. Display of neutralizing epitopes of Canine parvovirus and a T-cell epitope of the fusion protein of Canine distemper virus on chimeric tymovirus-like particles and its use as a vaccine candidate both against Canine parvo and Canine distemper.

    PubMed

    Chandran, Dev; Shahana, Pallichera Vijayan; Rani, Gudavelli Sudha; Sugumar, Parthasarthy; Shankar, Chinchkar Ramchandra; Srinivasan, Villuppanoor Alwar

    2009-12-10

    Expression of Physalis mottle tymovirus coat protein in Escherichia coli was earlier shown to self-assemble into empty capsids that were nearly identical to the capsids formed in vivo. Amino acid substitutions were made at the N-terminus of wild-type Physalis mottle virus coat protein with neutralizing epitopes of Canine parvovirus containing the antigenic sites 1-2, 4 and 6-7 and T-cell epitope of the fusion protein of Canine distemper virus in various combinations to yield PhMV1, PhMV2, PhMV3, PhMV4 and PhMV5. These constructs were cloned and expressed in E. coli. The chimeric proteins self-assembled into chimeric tymovirus-like particles (TVLPs) as determined by electron microscopy. The TVLPs were purified by ultracentrifugation and injected into guinea pigs and dogs to determine their immunogenicity. Initial immunogenicity studies in guinea pigs indicated that PhMV3 gave a higher response in comparison to the other TVLPs for both CPV and CDV and hence all further experiments in dogs were done with PhMV3. HI was done against different isolates obtained from various parts of the country. Protective titres indicated the broad spectrum of the vaccine. In conclusion the study indicated that the above chimeric VLP based vaccine could be used in dogs to generate a protective immune response against diseases caused by both Canine parvo and Canine distemper virus. PMID:19818723

  20. Canine Distemper Virus Infection Leads to an Inhibitory Phenotype of Monocyte-Derived Dendritic Cells In Vitro with Reduced Expression of Co-Stimulatory Molecules and Increased Interleukin-10 Transcription

    PubMed Central

    Herder, Vanessa; Stein, Veronika M.; Tipold, Andrea; Urhausen, Carola; Günzel-Apel, Anne-Rose; Rohn, Karl; Baumgärtner, Wolfgang; Beineke, Andreas

    2014-01-01

    Canine distemper virus (CDV) exhibits a profound lymphotropism that causes immunosuppression and increased susceptibility of affected dogs to opportunistic infections. Similar to human measles virus, CDV is supposed to inhibit terminal differentiation of dendritic cells (DCs), responsible for disturbed repopulation of lymphoid tissues and diminished antigen presenting function in dogs. In order to testify the hypothesis that CDV-infection leads to an impairment of professional antigen presenting cells, canine DCs have been generated from peripheral blood monocytes in vitro and infected with CDV. Virus infection was confirmed and quantified by transmission electron microscopy, CDV-specific immunofluorescence, and virus titration. Flow cytometric analyses revealed a significant down-regulation of the major histocompatibility complex class II and co-stimulatory molecules CD80 and CD86 in CDV-infected DCs, indicative of disturbed antigen presenting capacity. Molecular analyses revealed an increased expression of the immune inhibitory cytokine interleukin-10 in DCs following infection. Results of the present study demonstrate that CDV causes phenotypical changes and altered cytokine expression of DCs, which represent potential mechanisms to evade host immune responses and might contribute to immune dysfunction and virus persistence in canine distemper. PMID:24769532

  1. Induction of castration by immunization of male dogs with recombinant gonadotropin-releasing hormone (GnRH)-canine distemper virus (CDV) T helper cell epitope p35.

    PubMed

    Jung, Mi-Jeong; Moon, Young-Chan; Cho, Ik-Hyun; Yeh, Jung-Yong; Kim, Sun-Eui; Chang, Wha-Seok; Park, Seung-Young; Song, Chang-Seon; Kim, Hwi-Yool; Park, Keun-Kyu; McOrist, Steven; Choi, In-Soo; Lee, Joong-Bok

    2005-03-01

    Immunocastration is a considerable alternative to a surgical castration method especially in male animal species for alleviating unwanted male behaviors and characteristics. Induction of high titer of antibody specific for gonadotropin-releasing hormone (GnRH) correlates with the regression of testes. Fusion proteins composed of canine GnRH and T helper (Th) cell epitope p35 originated from canine distemper virus (CDV) F protein and goat rotavirus VP6 protein were produced in E. coli. When these fusion proteins were injected to male dogs which were previously immunized with CDV vaccine, the fusion protein of GnRH-CDV Th cell epitope p35 induced much higher antibody than that of GnRH-rotavirus VP6 protein or GnRH alone. The degeneration of spermatogenesis was also verified in the male dogs immunized with the fusion protein of GnRH-CDV Th cell epitope p35. These results indicate that canine GnRH conjugated to CDV Th cell epitope p35 acted as a strong immunogen and the antibody to GnRH specifically neutralized GnRH in the testes. This study also implies a potential application of GnRH-based vaccines for immunocastration of male pets. PMID:15785119

  2. The Fusion Protein Signal-Peptide-Coding Region of Canine Distemper Virus: A Useful Tool for Phylogenetic Reconstruction and Lineage Identification

    PubMed Central

    Sarute, Nicolás; Calderón, Marina Gallo; Pérez, Ruben; La Torre, José; Hernández, Martín; Francia, Lourdes; Panzera, Yanina

    2013-01-01

    Canine distemper virus (CDV; Paramyxoviridae, Morbillivirus) is the etiologic agent of a multisystemic infectious disease affecting all terrestrial carnivore families with high incidence and mortality in domestic dogs. Sequence analysis of the hemagglutinin (H) gene has been widely employed to characterize field strains, permitting the identification of nine CDV lineages worldwide. Recently, it has been established that the sequences of the fusion protein signal-peptide (Fsp) coding region are extremely variable, suggesting that analysis of its sequence might be useful for strain characterization studies. However, the divergence of Fsp sequences among worldwide strains and its phylogenetic resolution has not yet been evaluated. We constructed datasets containing the Fsp-coding region and H gene sequences of the same strains belonging to eight CDV lineages. Both datasets were used to evaluate their phylogenetic resolution. The phylogenetic analysis revealed that both datasets clustered the same strains into eight different branches, corresponding to CDV lineages. The inter-lineage amino acid divergence was fourfold greater for the Fsp peptide than for the H protein. The likelihood mapping revealed that both datasets display strong phylogenetic signals in the region of well-resolved topologies. These features indicate that Fsp-coding region sequence analysis is suitable for evolutionary studies as it allows for straightforward identification of CDV lineages. PMID:23675493

  3. Possible lin between elevated accumulation of trace elements and canine distemper virus infection in the Caspian seals (Phoca caspica) stranded in 2000 and 2001

    NASA Astrophysics Data System (ADS)

    Shinsuke, T.; Takashi, K.; Yasumi, A.; Tokutaka, I.; Reiji, K.

    2003-05-01

    In the Caspian Sea, a die-off of thousands of Caspian seals (Phoca caspica) occurred in 1997 and 2000. While a direct cause for these deaths seems to be canine distemper virus (CDV) infection, immunosuppression due to environmental pollutants is considered as one of the possible explanations for the development of the disease. The purpose of this work is to examine whether exposure to trace metals could be one of the factors involved in the mass mortality of Caspian seals. Concentrations of 13 trace elements weredetermined in liver, kidney and muscle of Caspian seals found stranded along the coasts of the Caspian Sea in 2000 and 2001. Concentrations of toxic elemen ts (Ag, Cd, Hg, Tl and Pb) in the Caspian seals collected in 2000 and 2001 were comparable to or lower than those in healthy Caspian seals collected in 1993 and 1998 and in seals from other regions, suggesting that these elements would not be the causative agent for the death of Caspian seals. In contrast, Zn and Fe concentrations in the stranded Caspian seals were apparently higher than those in seals from other locations. These results suggest the disturbance in homeostatic control and nutritional statu s of essential elements in the stranded Caspian seals.

  4. Canine Distemper Virus Infects Canine Keratinocytes and Immune Cells by Using Overlapping and Distinct Regions Located on One Side of the Attachment Protein▿

    PubMed Central

    Langedijk, Johannes P. M.; Janda, Jozef; Origgi, Francesco C.; Örvell, Claes; Vandevelde, Marc; Zurbriggen, Andreas; Plattet, Philippe

    2011-01-01

    The morbilliviruses measles virus (MeV) and canine distemper virus (CDV) both rely on two surface glycoproteins, the attachment (H) and fusion proteins, to promote fusion activity for viral cell entry. Growing evidence suggests that morbilliviruses infect multiple cell types by binding to distinct host cell surface receptors. Currently, the only known in vivo receptor used by morbilliviruses is CD150/SLAM, a molecule expressed in certain immune cells. Here we investigated the usage of multiple receptors by the highly virulent and demyelinating CDV strain A75/17. We based our study on the assumption that CDV-H may interact with receptors similar to those for MeV, and we conducted systematic alanine-scanning mutagenesis on CDV-H throughout one side of the β-propeller documented in MeV-H to contain multiple receptor-binding sites. Functional and biochemical assays performed with SLAM-expressing cells and primary canine epithelial keratinocytes identified 11 residues mutation of which selectively abrogated fusion in keratinocytes. Among these, four were identical to amino acids identified in MeV-H as residues contacting a putative receptor expressed in polarized epithelial cells. Strikingly, when mapped on a CDV-H structural model, all residues clustered in or around a recessed groove located on one side of CDV-H. In contrast, reported CDV-H mutants with SLAM-dependent fusion deficiencies were characterized by additional impairments to the promotion of fusion in keratinocytes. Furthermore, upon transfer of residues that selectively impaired fusion induction in keratinocytes into the CDV-H of the vaccine strain, fusion remained largely unaltered. Taken together, our results suggest that a restricted region on one side of CDV-H contains distinct and overlapping sites that control functional interaction with multiple receptors. PMID:21849439

  5. 9 CFR 113.306 - Canine Distemper Vaccine.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Canine Distemper Vaccine. 113.306 Section 113.306 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Live Virus Vaccines § 113.306 Canine...

  6. 9 CFR 113.302 - Distemper Vaccine-Mink.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Distemper Vaccine-Mink. 113.302 Section 113.302 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Live Virus Vaccines § 113.302...

  7. Combined distemper-adenoviral pneumonia in a dog

    PubMed Central

    Rodriguez-Tovar, Luis E.; Ramírez-Romero, Rafael; Valdez-Nava, Yazel; Nevárez-Garza, Alicia M.; Zárate-Ramos, Juan J.; López, Alfonso

    2007-01-01

    A 3 1/2-month-old pug with oculonasal discharge and seizures was submitted for postmortem examination. Grossly, the lungs had cranioventral consolidation, and microscopically, 2 distinct types of inclusion bodies compatible with Canine distemper virus and Canine adenovirus type 2. Presence of both viruses was confirmed via immunohistochemical staining. PMID:17616064

  8. Combined distemper-adenoviral pneumonia in a dog.

    PubMed

    Rodriguez-Tovar, Luis E; Ramírez-Romero, Rafael; Valdez-Nava, Yazel; Nevárez-Garza, Alicia M; Zárate-Ramos, Juan J; López, Alfonso

    2007-06-01

    A 3 1/2-month-old pug with oculonasal discharge and seizures was submitted for postmortem examination. Grossly, the lungs had cranioventral consolidation, and microscopically, 2 distinct types of inclusion bodies compatible with Canine distemper virus and Canine adenovirus type 2. Presence of both viruses was confirmed via immunohistochemical staining. PMID:17616064

  9. Canine distemper spillover in domestic dogs from urban wildlife.

    PubMed

    Kapil, Sanjay; Yeary, Teresa J

    2011-11-01

    Canine distemper virus (CDV) causes a major disease of domestic dogs that develops as a serious systemic infection in unvaccinated or improperly vaccinated dogs. Domesticated dogs are the main reservoir of CDV, a multihost pathogen. This virus of the genus Morbillivirus in the family Paramyxoviridae occurs in other carnivorous species including all members of the Canidae and Mustelidae families and in some members of the Procyonidae, Hyaenidae, Ursidae, and Viverridae families. Canine distemper also has been reported in the Felidae family and marine mammals. The spread and incidences of CDV epidemics in dogs and wildlife here and worldwide are increasing. PMID:22041204

  10. Interaction of a synthetic peptide corresponding to the N-terminus of canine distemper virus fusion protein with phospholipid vesicles: a biophysical study.

    PubMed

    Aranda, Francisco J; Teruel, José A; Ortiz, Antonio

    2003-12-01

    The F protein of canine distemper virus (CDV) is a classic type I glycoprotein formed by two polypeptides, F1 and F2. The N-terminal regions of the F1 polypeptides of CDV, measles virus and other paramyxoviruses present moderate to high homology, supporting the existence of a high conservation within these structures, which emphasises its role in viral-host cell membrane fusion. This N-terminal polypeptide is usually termed the fusion peptide. The fusion peptides of most viral fusion-mediating glycoproteins contain a high proportion of hydrophobic amino acids, which facilitates its insertion into target membranes during fusion. In this work we report on the interaction of a 31-residue synthetic peptide (FP31) corresponding to the N terminus of CDV F1 protein with phospholipid membranes composed of various phospholipids, as studied by means of various biophysical techniques. FTIR investigation of FP31 secondary structure in aqueous medium and in membranes resulted in a major proportion of alpha-helical structure which increased upon membrane insertion. Differential scanning calorimetry (DSC) showed that the presence of concentrations of FP31 as low as 0.1 mol%, in mixtures with L-alpha-dimyristoylphosphatidylcholine (DMPC), L-alpha-dipalmitoylphosphatidylcholine (DPPC) and L-alpha-distearoylphosphatidylcholine (DSPC), already affected the thermotropic properties of the gel to liquid-crystalline phase transition. In mixtures with the three lipids, increasing the concentration of peptide made the pretransition to disappear, and lowered and broadened the main transition. This effect was slightly stronger as the acyl chain length of the phospholipid grew larger. In the corresponding partial phase diagrams, no immiscibilities or critical points were observed. FTIR showed that incorporation of 1 mol% of peptide in DPPC shifted the antisymmetric and symmetric CH2 stretching bands to higher values, indicating the existence of an additional disordering of the acyl chain

  11. Use of quantitative real-time RT-PCR to investigate the correlation between viremia and viral shedding of canine distemper virus, and infection outcomes in experimentally infected dogs

    PubMed Central

    SEHATA, Go; SATO, Hiroaki; ITO, Toshihiro; IMAIZUMI, Yoshitaka; NORO, Taichi; OISHI, Eiji

    2015-01-01

    We used real-time RT-PCR and virus titration to examine canine distemper virus (CDV) kinetics in peripheral blood and rectal and nasal secretions from 12 experimentally infected dogs. Real-time RT-PCR proved extremely sensitive, and the correlation between the two methods for rectal and nasal (r=0.78, 0.80) samples on the peak day of viral RNA was good. Although the dogs showed diverse symptoms, viral RNA kinetics were similar; the peak of viral RNA in the symptomatic dogs was consistent with the onset of symptoms. These results indicate that real-time RT-PCR is sufficiently sensitive to monitor CDV replication in experimentally infected dogs regardless of the degree of clinical manifestation and suggest that the peak of viral RNA reflects active CDV replication. PMID:25728411

  12. Use of quantitative real-time RT-PCR to investigate the correlation between viremia and viral shedding of canine distemper virus, and infection outcomes in experimentally infected dogs.

    PubMed

    Sehata, Go; Sato, Hiroaki; Ito, Toshihiro; Imaizumi, Yoshitaka; Noro, Taichi; Oishi, Eiji

    2015-07-01

    We used real-time RT-PCR and virus titration to examine canine distemper virus (CDV) kinetics in peripheral blood and rectal and nasal secretions from 12 experimentally infected dogs. Real-time RT-PCR proved extremely sensitive, and the correlation between the two methods for rectal and nasal (r=0.78, 0.80) samples on the peak day of viral RNA was good. Although the dogs showed diverse symptoms, viral RNA kinetics were similar; the peak of viral RNA in the symptomatic dogs was consistent with the onset of symptoms. These results indicate that real-time RT-PCR is sufficiently sensitive to monitor CDV replication in experimentally infected dogs regardless of the degree of clinical manifestation and suggest that the peak of viral RNA reflects active CDV replication. PMID:25728411

  13. Jaundice and bilirubinemia as manifestations of canine distemper in raccoons and ferrets

    USGS Publications Warehouse

    Kilham, L.; Habermann, R.T.; Herman, C.M.

    1956-01-01

    1) Two strains of distemper virus have been isolated from wild raccoons and one strain from ferrets. 2) All strains isolated have induced bilirubinemia in raccoons and ferrets. Many raccoons with bilirubinemia also had jaundice. 3) Identification of these strains as members of the canine distemper virus complex has been by clinical and pathological findings consistent with this diagnosis as well as by cross-immunity tests.

  14. Control of canine distemper.

    PubMed

    Chappuis, G

    1995-05-01

    Control of canine distemper can realistically only be achieved by the use of vaccination. The types of vaccine in current use are described, together with some of the problems encountered such as interference by maternal antibodies, and usage in species other than dogs. Modified live viral vaccines, as used for more than thirty years, have proved very effective. Nevertheless there is scope for some improvement in vaccine efficacy and recent developments in genetic recombinant methods are described. PMID:8588329

  15. New Aspects of the Pathogenesis of Canine Distemper Leukoencephalitis

    PubMed Central

    Lempp, Charlotte; Spitzbarth, Ingo; Puff, Christina; Cana, Armend; Kegler, Kristel; Techangamsuwan, Somporn; Baumgärtner, Wolfgang; Seehusen, Frauke

    2014-01-01

    Canine distemper virus (CDV) is a member of the genus morbillivirus, which is known to cause a variety of disorders in dogs including demyelinating leukoencephalitis (CDV-DL). In recent years, substantial progress in understanding the pathogenetic mechanisms of CDV-DL has been made. In vivo and in vitro investigations provided new insights into its pathogenesis with special emphasis on axon-myelin-glia interaction, potential endogenous mechanisms of regeneration, and astroglial plasticity. CDV-DL is characterized by lesions with a variable degree of demyelination and mononuclear inflammation accompanied by a dysregulated orchestration of cytokines as well as matrix metalloproteinases and their inhibitors. Despite decades of research, several new aspects of the neuropathogenesis of CDV-DL have been described only recently. Early axonal damage seems to represent an initial and progressive lesion in CDV-DL, which interestingly precedes demyelination. Axonopathy may, thus, function as a potential trigger for subsequent disturbed axon-myelin-glia interactions. In particular, the detection of early axonal damage suggests that demyelination is at least in part a secondary event in CDV-DL, thus challenging the dogma of CDV as a purely primary demyelinating disease. Another unexpected finding refers to the appearance of p75 neurotrophin (NTR)-positive bipolar cells during CDV-DL. As p75NTR is a prototype marker for immature Schwann cells, this finding suggests that Schwann cell remyelination might represent a so far underestimated endogenous mechanism of regeneration, though this hypothesis still remains to be proven. Although it is well known that astrocytes represent the major target of CDV infection in CDV-DL, the detection of infected vimentin-positive astrocytes in chronic lesions indicates a crucial role of this cell population in nervous distemper. While glial fibrillary acidic protein represents the characteristic intermediate filament of mature astrocytes

  16. Encephalitis in dogs associated with a batch of canine distemper (Rockborn) vaccine.

    PubMed

    Cornwell, H J; Thompson, H; McCandlish, I A; Macartney, L; Nash, A S

    1988-01-16

    During a period of seven months in 1982-83 cases of postvaccinal encephalitis were recorded in dogs in various parts of Britain after the administration of a particular batch of combined distemper/hepatitis vaccine. Detailed investigations of one of these cases revealed that the distemper component was responsible and the vaccine virus was recovered from the brain of an affected dog. PMID:2895528

  17. Biosensors for waterborne viruses: Detection and removal.

    PubMed

    Altintas, Zeynep; Gittens, Micah; Pocock, Jack; Tothill, Ibtisam E

    2015-08-01

    Detection of waterborne viruses is important to eliminate and control their harmful effect as pathogens. Hence, the use of rapid and sensitive detection technologies is critically important as they can aid in investigating outbreaks and help in developing prevention strategies. To date range of viruses can contaminate drinking water sources, causing illnesses such as diarrhoea, pneumonia and gastroenteritis which can result in death. Due to their small size (nm) their complete removal from water can be difficult with current water treatment processes while being resistant to disinfectants. Available techniques for virus detection include filtration technologies, enzyme-linked immunosorbent assays and polymerase chain reaction. Although each technique has limitations, the use of biosensor technology with smart affinity materials and nanomaterials can show great potential in sensing viruses in water samples. This review reports on the latest technologies used for waterborne virus removal and detection with focus on rapid detection using biosensors. PMID:26005094

  18. Detection of Lassa Virus, Mali

    PubMed Central

    Safronetz, David; Lopez, Job E.; Sogoba, Nafomon; Traore’, Sékou F.; Raffel, Sandra J.; Fischer, Elizabeth R.; Ebihara, Hideki; Branco, Luis; Garry, Robert F.; Schwan, Tom G.

    2010-01-01

    To determine whether Lassa virus was circulating in southern Mali, we tested samples from small mammals from 3 villages, including Soromba, where in 2009 a British citizen probably contracted a lethal Lassa virus infection. We report the isolation and genetic characterization of Lassa virus from an area previously unknown for Lassa fever. PMID:20587185

  19. Canine Distemper in Endangered Ethiopian Wolves

    PubMed Central

    Gordon, Christopher H.; Hussein, Alo; Laurenson, M. Karen; Malcolm, James R.; Marino, Jorgelina; Regassa, Fekede; Stewart, Anne-Marie E.; Fooks, Anthony R.; Sillero-Zubiri, Claudio

    2015-01-01

    The Ethiopian wolf (Canis simensis) is the world’s rarest canid; ≈500 wolves remain. The largest population is found within the Bale Mountains National Park (BMNP) in southeastern Ethiopia, where conservation efforts have demonstrated the negative effect of rabies virus on wolf populations. We describe previously unreported infections with canine distemper virus (CDV) among these wolves during 2005–2006 and 2010. Death rates ranged from 43% to 68% in affected subpopulations and were higher for subadult than adult wolves (83%–87% vs. 34%–39%). The 2010 CDV outbreak started 20 months after a rabies outbreak, before the population had fully recovered, and led to the eradication of several focal packs in BMNP’s Web Valley. The combined effect of rabies and CDV increases the chance of pack extinction, exacerbating the typically slow recovery of wolf populations, and represents a key extinction threat to populations of this highly endangered carnivore. PMID:25898177

  20. Canine distemper in endangered Ethiopian wolves.

    PubMed

    Gordon, Christopher H; Banyard, Ashley C; Hussein, Alo; Laurenson, M Karen; Malcolm, James R; Marino, Jorgelina; Regassa, Fekede; Stewart, Anne-Marie E; Fooks, Anthony R; Sillero-Zubiri, Claudio

    2015-05-01

    The Ethiopian wolf (Canis simensis) is the world's rarest canid; ≈500 wolves remain. The largest population is found within the Bale Mountains National Park (BMNP) in southeastern Ethiopia, where conservation efforts have demonstrated the negative effect of rabies virus on wolf populations. We describe previously unreported infections with canine distemper virus (CDV) among these wolves during 2005-2006 and 2010. Death rates ranged from 43% to 68% in affected subpopulations and were higher for subadult than adult wolves (83%-87% vs. 34%-39%). The 2010 CDV outbreak started 20 months after a rabies outbreak, before the population had fully recovered, and led to the eradication of several focal packs in BMNP's Web Valley. The combined effect of rabies and CDV increases the chance of pack extinction, exacerbating the typically slow recovery of wolf populations, and represents a key extinction threat to populations of this highly endangered carnivore. PMID:25898177

  1. Method for detecting viruses in aerosols.

    PubMed

    Wallis, C; Melnick, J L; Rao, V C; Sox, T E

    1985-11-01

    A simple method with poliovirus as the model was developed for recovering human enteric viruses from aerosols. Filterite filters (pore size, 0.45 micron; Filterite Corp., Timonium, Md.) moistened with glycine buffer (pH 3.5) were used for adsorbing the aerosolized virus. No virus passed the filter, even with air flow rates of 100 liters/min. Virus recovery from the filter was achieved by rapid elution with 800 ml of glycine buffer, pH 10. The virus in the primary eluate was reconcentrated by adjusting the pH to 3.5, adding AlCl3 to 0.0005 M, collecting the virus on a 0.25-micron-pore Filerite disk (diameter, 25 mm) and and eluting with 6 ml of buffer, pH 10. With this method, virus could be detected regularly in aerosols produced by flushing when 3 X 10(8) PFU of poliovirus were present in the toilet bowl. Poliovirus-containing fecal material from two of four infants who had recently received oral polio vaccine also yielded virus in the aerosols when feces containing 2.4 X 10(7) to 4.5 X 10(7) PFU of virus had been added to the toilet bowl. Persons infected with a variety of natural enteric viruses are known to excrete this amount of virus in their daily stools. PMID:3004329

  2. Method for detecting viruses in aerosols.

    PubMed Central

    Wallis, C; Melnick, J L; Rao, V C; Sox, T E

    1985-01-01

    A simple method with poliovirus as the model was developed for recovering human enteric viruses from aerosols. Filterite filters (pore size, 0.45 micron; Filterite Corp., Timonium, Md.) moistened with glycine buffer (pH 3.5) were used for adsorbing the aerosolized virus. No virus passed the filter, even with air flow rates of 100 liters/min. Virus recovery from the filter was achieved by rapid elution with 800 ml of glycine buffer, pH 10. The virus in the primary eluate was reconcentrated by adjusting the pH to 3.5, adding AlCl3 to 0.0005 M, collecting the virus on a 0.25-micron-pore Filerite disk (diameter, 25 mm) and and eluting with 6 ml of buffer, pH 10. With this method, virus could be detected regularly in aerosols produced by flushing when 3 X 10(8) PFU of poliovirus were present in the toilet bowl. Poliovirus-containing fecal material from two of four infants who had recently received oral polio vaccine also yielded virus in the aerosols when feces containing 2.4 X 10(7) to 4.5 X 10(7) PFU of virus had been added to the toilet bowl. Persons infected with a variety of natural enteric viruses are known to excrete this amount of virus in their daily stools. Images PMID:3004329

  3. Virus Detection and Field Management

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There are two primary pathogens of Oat – Crown Rust and the Luteoviruses Barley and Cereal Yellow Dwarf Virus (BYDV and CYDV). These viruses can only be transmitted by aphids, are limited to the vascular system in infected plants and can lead to severe stunting and a concomitant yield loss. In 200...

  4. Differentiation of canine distemper virus isolates in fur animals from various vaccine strains by reverse transcription-polymerase chain reaction-restriction fragment length polymorphism according to phylogenetic relations in china

    PubMed Central

    2011-01-01

    In order to effectively identify the vaccine and field strains of Canine distemper virus (CDV), a new differential diagnostic test has been developed based on reverse transcription-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP). We selected an 829 bp fragment of the nucleoprotein (N) gene of CDV. By RFLP analysis using BamHI, field isolates were distinguishable from the vaccine strains. Two fragments were obtained from the vaccine strains by RT-PCR-RFLP analysis while three were observed in the field strains. An 829 nucleotide region of the CDV N gene was analyzed in 19 CDV field strains isolated from minks, raccoon dogs and foxes in China between 2005 and 2007. The results suggest this method is precise, accurate and efficient. It was also determined that three different genotypes exist in CDV field strains in fur animal herds of the north of China, most of which belong to Asian type. Mutated field strains, JSY06-R1, JSY06-R2 and JDH07-F1 also exist in Northern China, but are most closely related to the standard virulent strain A75/17, designated in Arctic and America-2 genetype in the present study, respectively. PMID:21352564

  5. Specific detection of peste des petits ruminants virus antibodies in sheep and goat sera by the luciferase immunoprecipitation system.

    PubMed

    Berguido, Francisco J; Bodjo, Sanne Charles; Loitsch, Angelika; Diallo, Adama

    2016-01-01

    Peste des petits ruminants (PPR) is a contagious and often fatal transboundary animal disease affecting mostly sheep, goats and wild small ruminants. This disease is endemic in most of Africa, the Middle, Near East, and large parts of Asia. The causal agent is peste des petits ruminants virus (PPRV), which belongs to the genus Morbillivirus in the family Paramyxoviridae. This genus also includes measles virus (MV), canine distemper virus (CDV) and rinderpest virus (RPV). All are closely related viruses with serological cross reactivity. In this study, we have developed a Luciferase Immunoprecipitation System (LIPS) for the rapid detection of antibodies against PPRV in serum samples and for specific differentiation from antibodies against RPV. PPR and rinderpest (RP) serum samples were assayed by PPR-LIPS and two commercially available PPR cELISA tests. The PPR-LIPS showed high sensitivity and specificity for the samples tested and showed no cross reactivity with RPV unlike the commercial PPR cELISA tests which did cross react with RPV. Based on the results shown in this study, PPR-LIPS is presented as a good candidate for the specific serosurveillance of PPR. PMID:26506137

  6. DNA Microarray for Detection of Gastrointestinal Viruses

    PubMed Central

    Martínez, Miguel A.; Soto-del Río, María de los Dolores; Gutiérrez, Rosa María; Chiu, Charles Y.; Greninger, Alexander L.; Contreras, Juan Francisco; López, Susana; Arias, Carlos F.

    2014-01-01

    Gastroenteritis is a clinical illness of humans and other animals that is characterized by vomiting and diarrhea and caused by a variety of pathogens, including viruses. An increasing number of viral species have been associated with gastroenteritis or have been found in stool samples as new molecular tools have been developed. In this work, a DNA microarray capable in theory of parallel detection of more than 100 viral species was developed and tested. Initial validation was done with 10 different virus species, and an additional 5 species were validated using clinical samples. Detection limits of 1 × 103 virus particles of Human adenovirus C (HAdV), Human astrovirus (HAstV), and group A Rotavirus (RV-A) were established. Furthermore, when exogenous RNA was added, the limit for RV-A detection decreased by one log. In a small group of clinical samples from children with gastroenteritis (n = 76), the microarray detected at least one viral species in 92% of the samples. Single infection was identified in 63 samples (83%), and coinfection with more than one virus was identified in 7 samples (9%). The most abundant virus species were RV-A (58%), followed by Anellovirus (15.8%), HAstV (6.6%), HAdV (5.3%), Norwalk virus (6.6%), Human enterovirus (HEV) (9.2%), Human parechovirus (1.3%), Sapporo virus (1.3%), and Human bocavirus (1.3%). To further test the specificity and sensitivity of the microarray, the results were verified by reverse transcription-PCR (RT-PCR) detection of 5 gastrointestinal viruses. The RT-PCR assay detected a virus in 59 samples (78%). The microarray showed good performance for detection of RV-A, HAstV, and calicivirus, while the sensitivity for HAdV and HEV was low. Furthermore, some discrepancies in detection of mixed infections were observed and were addressed by reverse transcription-quantitative PCR (RT-qPCR) of the viruses involved. It was observed that differences in the amount of genetic material favored the detection of the most abundant

  7. Severe canine distemper outbreak in unvaccinated dogs in Mozambique.

    PubMed

    Zacarias, Julieta; Dimande, Alberto; Achá, Sara; Dias, Paula T; Leonel, Elisa M; Messa, Aurora; Macucule, Baltazar; Júnior, José L; Bila, Custódio G

    2016-01-01

    Although significant animal suffering caused by preventable diseases is frequently seen in developing countries, reports of this are scarce. This report describes avoidable animal suffering owing to a suspected canine distemper (CD) outbreak in unvaccinated dogs owned by low-income families in Mozambique that killed approximately 200 animals. Affected dogs exhibited clinical signs, and gross and microscopic lesions compatible with CD. Immunohistochemical staining confirmed the presence of canine distemper virus (CDV) in the kidney of one dog from the cohort. This brief communication again illustrates that large outbreaks of CDV in unvaccinated dogs occur and that large-scale avoidable suffering and threats to the health of dogs and wild canines continue. Mass vaccination supported by government and non-government organisations is recommended. PMID:27543040

  8. Uncommon acute neurologic presentation of canine distemper in 4 adult dogs

    PubMed Central

    Galán, Alba; Gamito, Araceli; Carletti, Beatrice E.; Guisado, Alicia; de las Mulas, Juana Martín; Pérez, José; Martín, Eva M.

    2014-01-01

    Four uncommon cases of canine distemper (CD) were diagnosed in vaccinated adult dogs. All dogs had acute onset of neurologic signs, including seizures, abnormal mentation, ataxia, and proprioceptive deficits. Polymerase chain reaction for CD virus was positive on cerebrospinal fluid in 2 cases. Due to rapid deterioration the dogs were euthanized and CD was confirmed by postmortem examination. PMID:24688139

  9. Uncommon acute neurologic presentation of canine distemper in 4 adult dogs.

    PubMed

    Galán, Alba; Gamito, Araceli; Carletti, Beatrice E; Guisado, Alicia; de las Mulas, Juana Martín; Pérez, José; Martín, Eva M

    2014-04-01

    Four uncommon cases of canine distemper (CD) were diagnosed in vaccinated adult dogs. All dogs had acute onset of neurologic signs, including seizures, abnormal mentation, ataxia, and proprioceptive deficits. Polymerase chain reaction for CD virus was positive on cerebrospinal fluid in 2 cases. Due to rapid deterioration the dogs were euthanized and CD was confirmed by postmortem examination. PMID:24688139

  10. Detection of DNA viruses in prostate cancer

    PubMed Central

    Smelov, Vitaly; Bzhalava, Davit; Arroyo Mühr, Laila Sara; Eklund, Carina; Komyakov, Boris; Gorelov, Andrey; Dillner, Joakim; Hultin, Emilie

    2016-01-01

    We tested prostatic secretions from men with and without prostate cancer (13 cases and 13 matched controls) or prostatitis (18 cases and 18 matched controls) with metagenomic sequencing. A large number (>200) of viral reads was only detected among four prostate cancer cases (1 patient each positive for Merkel cell polyomavirus, JC polyomavirus and Human Papillomavirus types 89 or 40, respectively). Lower numbers of reads from a large variety of viruses were detected in all patient groups. Our knowledge of the biology of the prostate may be furthered by the fact that DNA viruses are commonly shed from the prostate and can be readily detected by metagenomic sequencing of expressed prostate secretions. PMID:27121729

  11. 9 CFR 113.34 - Detection of hemagglutinating viruses.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Detection of hemagglutinating viruses..., DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Standard Procedures § 113.34 Detection of hemagglutinating viruses. The test for detection...

  12. 9 CFR 113.34 - Detection of hemagglutinating viruses.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Detection of hemagglutinating viruses..., DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Standard Procedures § 113.34 Detection of hemagglutinating viruses. The test for detection...

  13. 9 CFR 113.34 - Detection of hemagglutinating viruses.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Detection of hemagglutinating viruses..., DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Standard Procedures § 113.34 Detection of hemagglutinating viruses. The test for detection...

  14. 9 CFR 113.34 - Detection of hemagglutinating viruses.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Detection of hemagglutinating viruses..., DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Standard Procedures § 113.34 Detection of hemagglutinating viruses. The test for detection...

  15. 9 CFR 113.34 - Detection of hemagglutinating viruses.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Detection of hemagglutinating viruses..., DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Standard Procedures § 113.34 Detection of hemagglutinating viruses. The test for detection...

  16. Detection, isolation, and persistence of viruses within bivalve mollusks

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Norovirus (NV), hepatitis A virus (HAV), and other virus transmission by molluscan shellfish is a significant issue. Research at the ARS-Dover DE laboratory has led to the development of improved methods for detecting these viruses. To identify pathogenic viruses within mollusks, a rapid highly-se...

  17. Detection of virus in shrimp using digital color correlation

    NASA Astrophysics Data System (ADS)

    Alvarez-Borrego, Josue; Chavez-Sanchez, Cristina; Bueno-Ibarra, Mario A.

    1999-07-01

    Detection of virus in shrimp tissue using digital color correlation is presented. Phase filters in three channels (red, green and blue) were used in order to detect HPV virus like target. These first results obtained showed that is possible to detect virus in shrimp tissue. More research must be made with color correlation in order to consider natural morphology of the virus, color, scale and rotation and noise in the samples.

  18. The Unknown Computer Viruses Detection Based on Similarity

    NASA Astrophysics Data System (ADS)

    Liu, Zhongda; Nakaya, Naoshi; Koui, Yuuji

    New computer viruses are continually being generated and they cause damage all over the world. In general, current anti-virus software detects viruses by matching a pattern based on the signature; thus, unknown viruses without any signature cannot be detected. Although there are some static analysis technologies that do not depend on signatures, virus writers often use code obfuscation techniques, which make it difficult to execute a code analysis. As is generally known, unknown viruses and known viruses share a common feature. In this paper we propose a new static analysis technology that can circumvent code obfuscation to extract the common feature and detect unknown viruses based on similarity. The results of evaluation experiments demonstrated that this technique is able to detect unknown viruses without false positives.

  19. Biosensors for hepatitis B virus detection.

    PubMed

    Yao, Chun-Yan; Fu, Wei-Ling

    2014-09-21

    A biosensor is an analytical device used for the detection of analytes, which combines a biological component with a physicochemical detector. Recently, an increasing number of biosensors have been used in clinical research, for example, the blood glucose biosensor. This review focuses on the current state of biosensor research with respect to efficient, specific and rapid detection of hepatitis B virus (HBV). The biosensors developed based on different techniques, including optical methods (e.g., surface plasmon resonance), acoustic wave technologies (e.g., quartz crystal microbalance), electrochemistry (amperometry, voltammetry and impedance) and novel nanotechnology, are also discussed. PMID:25253948

  20. Microbiology--Detection, Occurrence, and Removal of Viruses.

    ERIC Educational Resources Information Center

    Berg, Gerald

    1978-01-01

    Presents a literature review of the detection, survival, and destruction of viruses in wastewater sludges, covering publications of 1976-77. This review includes these topics: (1) detection methodology; (2) viruses in sludge, shellfish, and aerosols; and (3) indicators of viruses. A list of 59 references is also presented. (HM)

  1. Porous silicon biosensor for detection of viruses.

    PubMed

    Rossi, Andrea M; Wang, Lili; Reipa, Vytas; Murphy, Thomas E

    2007-12-15

    There is a growing need for virus sensors with improved sensitivity and dynamic range, for applications including disease diagnosis, pharmaceutical research, agriculture and homeland security. We report here a new method for improving the sensitivity for detection of the bacteriophage virus MS2 using thin films of nanoporous silicon. Porous silicon is an easily fabricated material that has extremely high surface area to volume ratio, making it an ideal platform for surface based sensors. We have developed and evaluated two different methods for covalent bioconjugation of antibodies inside of porous silicon films, and we show that the pore penetration and binding efficiency depend on the wettability of the porous surface. The resulting films were used to selectively capture dye-labeled MS2 viruses from solution, and a viral concentration as low as 2 x 10(7) plaque-forming units per mL (pfu/mL) was detectable by measuring the fluorescence from the exposed porous silicon film. The system exhibits sensitivity and dynamic range similar to the Luminex liquid array-based assay while outperforming protein micro-array methods. PMID:17723292

  2. Development of a two-step SYBR Green I based real time RT-PCR assay for detecting and quantifying peste des petits ruminants virus in clinical samples.

    PubMed

    Abera, Tsegalem; Thangavelu, Ardhanary

    2014-12-01

    A two-step SYBR Green I based real time RT-PCR targeting the matrix (M) gene of Peste des petits ruminants virus (PPRV) was developed. The specificity of the assay was assessed against viral nucleic acid extracted from a range of animal viruses of clinical and structural similarities to PPRV including canine distemper virus, measles virus, bluetongue virus and Newcastle disease virus. But none of the viruses and no template control showed an amplification signal. Sensitivity of the same assay was assessed based on plasmid DNA copy number and with respect to infectivity titre. The lower detection limit achieved was 2.88 plasmid DNA copies/μl with corresponding Ct value of 35.93. Based on tissue culture infectivity titre the lower detection limits were 0.0001TCID50/ml and 1TCID50/ml for the SYBR green I based real time RT-PCR and conventional RT-PCR, respectively. The calculated coefficient of variations values for intra- and inter-assay variability were low, ranging from 0.21% to 1.83% and 0.44% to 1.97%, respectively. The performance of newly developed assay was evaluated on a total of 36 clinical samples suspected of PPR and compared with conventional RT-PCR. The SYBR Green I based real time RT-PCR assay detected PPRV in 32 (88.8%) of clinical samples compared to 19 (52.7%) by conventional RT-PCR. Thus, the two-step SYBR Green I based real time RT-PCR assay targeting the M gene of PPRV reported in this study was highly sensitive, specific and reproducible for detection and quantitation of PPRV nucleic acids. PMID:25194891

  3. Rapid Detection of the Varicella Zoster Virus

    NASA Technical Reports Server (NTRS)

    Lewis, Michelle P.; Harding, Robert

    2011-01-01

    1.Technology Description-Researchers discovered that when the Varicella Zoster Virus (VZV) reactivates from latency in the body, the virus is consistently present in saliva before the appearance of skin lesions. A small saliva sample is mixed with a specialized reagent in a test kit. If the virus is present in the saliva sample, the mixture turns a red color. The sensitivity and specificity emanates from an antibody-antigen reaction. This technology is a rapid, non-invasive, point of-of-care testing kit for detecting the virus from a saliva sample. The device is easy to use and can be used in clinics and in remote locations to quickly detect VZV and begin treatment with antiviral drugs. 2.Market Opportunity- RST Bioscience will be the first and only company to market a rapid, same day test kit for the detection of VZV in saliva. The RST detection test kit will have several advantages over existing, competitive technology. The test kit is self contained and laboratory equipment is not required for analysis of the sample. Only a single saliva sample is required to be taken instead of blood or cerebral spinal fluid. The test kit is portable, sterile and disposable after use. RST detection test kits require no electrical power or expensive storage equipment and can be used in remote locations. 3.Market Analysis- According to the CDC, it is estimated that 1 million cases of shingles occur each year in the U.S. with more than half over the age of sixty. There is a high demand for rapid diagnostics by the public. The point-of-care testing (POCT) market is growing faster than other segments of in vitro diagnostics. According to a July 2007 InteLab Corporation industry report the overall market for POCT was forecast to increase from $10.3 billion in 2005 to $18.7 billion by 2011. The market value of this test kit has not been determined. 4.Competition- The VZV vaccine prevents 50% of cases and reduces neuralgia by 66%. The most popular test detects VZV-specific IgM antibody

  4. Real-Time Detection of a Virus Using Detection Dogs.

    PubMed

    Angle, T Craig; Passler, Thomas; Waggoner, Paul L; Fischer, Terrence D; Rogers, Bart; Galik, Patricia K; Maxwell, Herris S

    2015-01-01

    Viral infections are ubiquitous in humans, animals, and plants. Real-time methods to identify viral infections are limited and do not exist for use in harsh or resource-constrained environments. Previous research identified that tissues produce unique volatile organic compounds (VOC) and demonstrated that VOC concentrations change during pathologic states, including infection, neoplasia, or metabolic disease. Patterns of VOC expression may be pathogen specific and may be associated with an odor that could be used for disease detection. We investigated the ability of two trained dogs to detect cell cultures infected with bovine viral diarrhea virus (BVDV) and to discriminate BVDV-infected cell cultures from uninfected cell cultures and from cell cultures infected with bovine herpes virus 1 (BHV 1) and bovine parainfluenza virus 3 (BPIV 3). Dogs were trained to recognize cell cultures infected with two different biotypes of BVDV propagated in Madin-Darby bovine kidney cells using one of three culture media. For detection trials, one target and seven distractors were presented on a scent wheel by a dog handler unaware of the location of targets and distractors. Detection of BVDV-infected cell cultures by Dog 1 had a diagnostic sensitivity of 0.850 (95% CI: 0.701-0.942), which was lower than Dog 2 (0.967, 95% CI: 0.837-0.994). Both dogs exhibited very high diagnostic specificity (0.981, 95% CI: 0.960-0.993) and (0.993, 95% CI: 0.975-0.999), respectively. These findings demonstrate that trained dogs can differentiate between cultured cells infected with BVDV, BHV1, and BPIV3 and are a realistic real-time mobile pathogen sensing technology for viral pathogens. The ability to discriminate between target and distractor samples plausibly results from expression of unique VOC patterns in virus-infected and -uninfected cells. PMID:26779494

  5. Real-Time Detection of a Virus Using Detection Dogs

    PubMed Central

    Angle, T. Craig; Passler, Thomas; Waggoner, Paul L.; Fischer, Terrence D.; Rogers, Bart; Galik, Patricia K.; Maxwell, Herris S.

    2016-01-01

    Viral infections are ubiquitous in humans, animals, and plants. Real-time methods to identify viral infections are limited and do not exist for use in harsh or resource-constrained environments. Previous research identified that tissues produce unique volatile organic compounds (VOC) and demonstrated that VOC concentrations change during pathologic states, including infection, neoplasia, or metabolic disease. Patterns of VOC expression may be pathogen specific and may be associated with an odor that could be used for disease detection. We investigated the ability of two trained dogs to detect cell cultures infected with bovine viral diarrhea virus (BVDV) and to discriminate BVDV-infected cell cultures from uninfected cell cultures and from cell cultures infected with bovine herpes virus 1 (BHV 1) and bovine parainfluenza virus 3 (BPIV 3). Dogs were trained to recognize cell cultures infected with two different biotypes of BVDV propagated in Madin–Darby bovine kidney cells using one of three culture media. For detection trials, one target and seven distractors were presented on a scent wheel by a dog handler unaware of the location of targets and distractors. Detection of BVDV-infected cell cultures by Dog 1 had a diagnostic sensitivity of 0.850 (95% CI: 0.701–0.942), which was lower than Dog 2 (0.967, 95% CI: 0.837–0.994). Both dogs exhibited very high diagnostic specificity (0.981, 95% CI: 0.960–0.993) and (0.993, 95% CI: 0.975–0.999), respectively. These findings demonstrate that trained dogs can differentiate between cultured cells infected with BVDV, BHV1, and BPIV3 and are a realistic real-time mobile pathogen sensing technology for viral pathogens. The ability to discriminate between target and distractor samples plausibly results from expression of unique VOC patterns in virus-infected and -uninfected cells. PMID:26779494

  6. Simultaneous multiplex PCR detection of seven cucurbit-infecting viruses.

    PubMed

    Kwon, Ji Yeon; Hong, Jin Sung; Kim, Min Jea; Choi, Sun Hee; Min, Byeong Eun; Song, Eun Gyeong; Kim, Hyun Hee; Ryu, Ki Hyun

    2014-09-01

    Two multiplex polymerase chain reaction (PCR) systems using dual priming oligonucleotide (DPO) primers were developed for the simultaneous detection of seven cucurbit-infecting viruses. One system allows for the detection of papaya ringspot virus, watermelon mosaic virus, and zucchini yellow mosaic virus, whereas the other permits the detection of cucumber green mottle mosaic virus, cucumber fruit mottle mosaic virus, kyuri green mottle mosaic virus, and zucchini green mottle mosaic virus. Viral species-specific DPO primers developed in this study detected as little as 10 fg/μl of viral RNA under monoplex conditions and 10 pg/μl of viral RNA under multiplex conditions. Multiplex PCR using the DPO primer sets was capable of amplifying viral genes at annealing temperatures ranging from 53 °C to 63 °C. Whereas the use of conventional primers gave rise to non-specific bands, the DPO primers detected target viral genes in the absence of non-specific amplification. When these DPO multiplex primer sets were applied to virus-infected cucurbit samples obtained in the field, multiple infection as well as single infection was accurately identified. This novel approach could also detect multiple viruses in infected seeds. The reliability of multiplex PCR systems using DPO primers for plant virus detection is discussed. PMID:24937806

  7. Development of a rapid detection method for Yellow Dwarf Viruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Barley and Cereal yellow dwarf viruses (B/CYDVs), constitute the most economically important group of oat viruses. A multiplex reverse transcription polymerase chain reaction method was developed for the simultaneous detection and discrimination of five B/CYDVs viruses. The protocol uses specific pr...

  8. Transgenic cell lines for detection of animal viruses.

    PubMed Central

    Olivo, P D

    1996-01-01

    Rapid diagnostic assays based on direct detection of viral antigen or nucleic acid are being used with increasing frequency in clinical virology laboratories. Virus culture, however, remains the only way to detect infectious virus and to analyze clinically relevant viral phenotypes, such as drug resistance. Growth of viruses in cell culture is labor intensive and time-consuming and requires the use of many different cell lines. Transgenic technology, together with increasing knowledge of the molecular pathways of virus replication, offers the possibility of using genetically modified cell lines to improve virus growth in cell culture and to facilitate detection of virus-infected cells. Genetically modifying cells so that they express a reporter gene only after infection with a specific virus can allow the detection of infectious virus by rapid and simple enzyme assays such as beta-galactosidase assays without the need for antibodies. Although transgenic cells have recently been successfully used for herpes simplex virus detection, much more work needs to be done to adapt this technology to other human viral pathogens such as cytomegalovirus and respiratory viruses. This review offers some strategies for applying this technology to a wide spectrum of animal viruses. PMID:8809463

  9. Detection of influenza C virus but not influenza D virus in Scottish respiratory samples

    PubMed Central

    Smith, Donald B.; Gaunt, Eleanor R.; Digard, Paul; Templeton, Kate; Simmonds, Peter

    2016-01-01

    Background A newly proposed genus of influenza virus (influenza D) is associated with respiratory disease in pigs and cattle. The novel virus is most closely related to human influenza C virus and can infect ferrets but infection has not been reported in humans. Objectives To ascertain if influenza D virus can be detected retrospectively in patient respiratory samples. Study design 3300 human respiratory samples from Edinburgh, Scotland, covering the period 2006–2008, were screened in pools of 10 by RT-PCR using primers capable of detecting both influenza C and D viruses. Results Influenza D was not detected in any sample. Influenza C was present in 6 samples (0.2%), compared with frequencies of 3.3% and 0.9% for influenza A and B viruses from RT-PCR testing of respiratory samples over the same period. Influenza C virus was detected in samples from individuals <2 years or >45 years old, with cases occurring throughout the year. Phylogenetic analysis of nearly complete sequences of all seven segments revealed the presence of multiple, reassortant lineages. Conclusion We were unable to detect viruses related to influenza D virus in human respiratory samples. Influenza C virus was less prevalent than influenza A and B viruses, was associated with mild disease in the young (<2 years) and old (>45 years) and comprised multiple, reassortant lineages. Inclusion of influenza C virus as part of a diagnostic testing panel for respiratory infections would be of limited additional value. PMID:26655269

  10. Molecular and serological surveillance of canine enteric viruses in stray dogs from Vila do Maio, Cape Verde

    PubMed Central

    2014-01-01

    Background Infections caused by canine parvovirus, canine distemper virus and canine coronavirus are an important cause of mortality and morbidity in dogs worldwide. Prior to this study, no information was available concerning the incidence and prevalence of these viruses in Cape Verde archipelago. Results To provide information regarding the health status of the canine population in Vila do Maio, Maio Island, Cape Verde, 53 rectal swabs were collected from 53 stray dogs during 2010 and 93 rectal swabs and 88 blood samples were collected from 125 stray dogs in 2011. All rectal swabs (2010 n = 53; 2011 n = 93) were analysed for the presence of canine parvovirus, canine distemper virus and canine coronavirus nucleic acids by quantitative PCR methods. Specific antibodies against canine distemper virus and canine parvovirus were also assessed (2011 n = 88). From the 2010 sampling, 43.3% (23/53) were positive for canine parvovirus DNA, 11.3% (6/53) for canine distemper virus RNA and 1.9% (1/53) for canine coronavirus RNA. In 2011, the prevalence values for canine parvovirus and canine coronavirus were quite similar to those from the previous year, respectively 44.1% (41/93), and 1.1% (1/93), but canine distemper virus was not detected in any of the samples analysed (0%, 0/93). Antibodies against canine parvovirus were detected in 71.6% (63/88) blood samples and the seroprevalence found for canine distemper virus was 51.1% (45/88). Conclusions This study discloses the data obtained in a molecular and serological epidemiological surveillance carried out in urban populations of stray and domestic animals. Virus transmission and spreading occurs easily in large dog populations leading to high mortality rates particularly in unvaccinated susceptible animals. In addition, these animals can act as disease reservoirs for wild animal populations by occasional contact. Identification of susceptible wildlife of Maio Island is of upmost importance to evaluate the risk

  11. Detection of Pathogenic Viruses in Sewage Provided Early Warnings of Hepatitis A Virus and Norovirus Outbreaks

    PubMed Central

    Hellmér, Maria; Paxéus, Nicklas; Magnius, Lars; Enache, Lucica; Arnholm, Birgitta; Johansson, Annette; Bergström, Tomas

    2014-01-01

    Most persons infected with enterically transmitted viruses shed large amounts of virus in feces for days or weeks, both before and after onset of symptoms. Therefore, viruses causing gastroenteritis may be detected in wastewater, even if only a few persons are infected. In this study, the presence of eight pathogenic viruses (norovirus, astrovirus, rotavirus, adenovirus, Aichi virus, parechovirus, hepatitis A virus [HAV], and hepatitis E virus) was investigated in sewage to explore whether their identification could be used as an early warning of outbreaks. Samples of the untreated sewage were collected in proportion to flow at Ryaverket, Gothenburg, Sweden. Daily samples collected during every second week between January and May 2013 were pooled and analyzed for detection of viruses by concentration through adsorption to milk proteins and PCR. The largest amount of noroviruses was detected in sewage 2 to 3 weeks before most patients were diagnosed with this infection in Gothenburg. The other viruses were detected at lower levels. HAV was detected between weeks 5 and 13, and partial sequencing of the structural VP1protein identified three different strains. Two strains were involved in an ongoing outbreak in Scandinavia and were also identified in samples from patients with acute hepatitis A in Gothenburg during spring of 2013. The third strain was unique and was not detected in any patient sample. The method used may thus be a tool to detect incipient outbreaks of these viruses and provide early warning before the causative pathogens have been recognized in health care. PMID:25172863

  12. Porcupine quills in raccoons as an indicator of rabies, distemper, or both diseases: disease management implications.

    PubMed

    Rosatte, Rick; Wandeler, Alex; Muldoon, Frances; Campbell, Doug

    2007-03-01

    A relationship was detected between the presence of embedded porcupine quills and the diagnosis of rabies in raccoons in eastern Canada during 1999-2004. No relationship was found between the presence of quills in raccoons and the diagnosis of canine distemper. Raccoons with embedded quills should be submitted for rabies testing. PMID:17436909

  13. DETECTION OF NORWALK VIRUS IN STOOLS BY ENZYME IMMUNOASSAY

    EPA Science Inventory

    The development of a solid-phase microtiter enzyme immunoassay (EIA) for detection of Norwalk virus antigen in stool samples is described. The EIA was compared with a previously developed radioimmunoassay (RIA) for detection of Norwalk virus antigen in stools obtained from 30 vol...

  14. Simultaneous detection of bee viruses by multiplex PCR.

    PubMed

    Sguazza, Guillermo Hernán; Reynaldi, Francisco José; Galosi, Cecilia Mónica; Pecoraro, Marcelo Ricardo

    2013-12-01

    Honey bee mortality is a serious problem that beekeepers in Argentina have had to face during the last 3 years. It is known that the consequence of the complex interactions between environmental and beekeeping parameters added to the effect of different disease agents such as viruses, bacteria, fungi and parasitic mites may result in a sudden collapse of the colony. In addition, multiple viral infections are detected frequently concomitantly in bee colonies. The aim of this study was to establish a multiplex polymerase chain reaction method for rapid and simultaneous detection of the most prevalent bee viruses. This multiplex PCR assay will provide specific, rapid and reliable results and allow for the cost effective detection of a particular virus as well as multiple virus infections in a single reaction tube. This method could be a helpful tool in the surveillance of the most frequently found bee viruses and to study the dynamics and the interactions of the virus populations within colonies. PMID:23948157

  15. Canine distemper epizootic in lions, tigers, and leopards in North America.

    PubMed

    Appel, M J; Yates, R A; Foley, G L; Bernstein, J J; Santinelli, S; Spelman, L H; Miller, L D; Arp, L H; Anderson, M; Barr, M

    1994-07-01

    Canine distemper virus (CDV) infection occurred in captive leopards (Panthera pardus), tigers (Panthera tigris), lions (Panthera leo), and a jaguar (Panthera onca) in 1991 and 1992. An epizootic affected all 4 types of cats at the Wildlife Waystation, San Fernando, California, with 17 mortalities. CDV-infected raccoons were thought to be the source of infection in these cats. Two black leopards died at the Naibi Zoo, Coal Valley, Illinois, and 2 tigers died at the Shambala Preserve, Acton, California. Initial clinical signs were anorexia with gastrointestinal and/or respiratory disease followed by seizures. Canine distemper virus was isolated from 3 leopards, 3 tigers, and 3 lions that died or were euthanized when moribund. Monoclonal antibody testing identified the virus isolates as CDV. Gross and histopathologic findings were similar to those found in canids with distemper with a few exceptions. There were fewer lesions in the brain, and there was a pronounced type 2 cell proliferation in the lung, with inclusion bodies and CDV antigen demonstrated by immunohistology. Neutralizing antibody to CDV was found in high titers in serum from most animals but was absent or was found only in low titers in some cats that succumbed after CDV infection. There was a marked difference in neutralizing antibody titers when tests were done with different strains of CDV. PMID:7948195

  16. Detection and Diagnosis of Plant Viruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Diseases caused by viruses are a constant and major problem for crop production worldwide. Human interest in plant genetic resources and globalization of human activities has inadvertently contributed to the spread of viruses throughout the world, increasing the frequency of plant virus outbreaks. C...

  17. Virus detection and quantification using electrical parameters

    PubMed Central

    Ahmad, Mahmoud Al; Mustafa, Farah; Ali, Lizna M.; Rizvi, Tahir A.

    2014-01-01

    Here we identify and quantitate two similar viruses, human and feline immunodeficiency viruses (HIV and FIV), suspended in a liquid medium without labeling, using a semiconductor technique. The virus count was estimated by calculating the impurities inside a defined volume by observing the change in electrical parameters. Empirically, the virus count was similar to the absolute value of the ratio of the change of the virus suspension dopant concentration relative to the mock dopant over the change in virus suspension Debye volume relative to mock Debye volume. The virus type was identified by constructing a concentration-mobility relationship which is unique for each kind of virus, allowing for a fast (within minutes) and label-free virus quantification and identification. For validation, the HIV and FIV virus preparations were further quantified by a biochemical technique and the results obtained by both approaches corroborated well. We further demonstrate that the electrical technique could be applied to accurately measure and characterize silica nanoparticles that resemble the virus particles in size. Based on these results, we anticipate our present approach to be a starting point towards establishing the foundation for label-free electrical-based identification and quantification of an unlimited number of viruses and other nano-sized particles. PMID:25355078

  18. Detection of Multiple Potato Viruses in the Field Suggests Synergistic Interactions among Potato Viruses in Pakistan.

    PubMed

    Hameed, Amir; Iqbal, Zafar; Asad, Shaheen; Mansoor, Shahid

    2014-12-01

    Viral diseases have been a major limiting factor threating sustainable potato (Solanum tuberosum L.) production in Pakistan. Surveys were conducted to serologically quantify the incidence of RNA viruses infecting potato; Potato virus X (PVX), Potato virus Y (PVY), Potato virus S (PVS), Potato virus A (PVA), Potato virus M (PVM) and Potato leaf roll virus (PLRV) in two major potato cultivars (Desiree and Cardinal). The results suggest the prevalence of multiple viruses in all surveyed areas with PVY, PVS and PVX dominantly widespread with infection levels of up to 50% in some regions. Co-infections were detected with the highest incidence (15.5%) for PVX and PVS. Additionally the data showed a positive correlation between co-infecting viruses with significant increase in absorbance value (virus titre) for at least one of the virus in an infected plant and suggested a synergistic interaction. To test this hypothesis, glasshouse grown potato plants were challenged with multiple viruses and analyzed for systemic infections and symptomology studies. The results obtained conclude that multiple viral infections dramatically increase disease epidemics as compared to single infection and an effective resistance strategy in targeting multiple RNA viruses is required to save potato crop. PMID:25506305

  19. Microcavity single virus detection and sizing with molecular sensitivity

    NASA Astrophysics Data System (ADS)

    Dantham, V. R.; Holler, S.; Kolchenko, V.; Wan, Z.; Arnold, S.

    2013-02-01

    We report the label-free detection and sizing of the smallest individual RNA virus, MS2 by a spherical microcavity. Mass of this virus is ~6 ag and produces a theoretical resonance shift ~0.25 fm upon adsorbing an individual virus at the equator of the bare microcavity, which is well below the r.m.s background noise of 2 fm. However, detection was accomplished with ease (S/N = 8, Q = 4x105) using a single dipole stimulated plasmonic-nanoshell as a microcavity wavelength shift enhancer. Analytical expressions based on the "reactive sensing principle" are developed to extract the radius of the virus from the measured signals. Estimated limit of detection for these experiments was ~0.4 ag or 240 kDa below the size of all known viruses, largest globular and elongated proteins [Phosphofructokinase (345 kDa) and Fibrinogen (390 kDa), respectively].

  20. DETECTION OF AVIAN INFLUENZA VIRUS USING AN INTERFEROMETRIC BIOSENSOR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An optical interferometric waveguide immunoassay for direct and label-less detection of avian influenza virus is described. The assay response is based on index of refraction changes that occur upon binding of virus particles to antigen (hemagglutinin) specific antibodies on the waveguide surface. ...

  1. Label-free virus detection using silicon photonic microring resonators

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Viruses represent a continual threat to humans through a number of mechanisms, which include disease, bioterrorism, and destruction of both plant and animal food resources. Many contemporary techniques used for the detection of viruses and viral infections suffer from limitations such as the need fo...

  2. Detection of enteric viruses in shellfish

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Norovirus and hepatitis A virus contamination are significant threats to the safety of shellfish and other foods. Methods for the extraction and assay of these viruses from shellfish are complex, time consuming, and technically challenging. Here, we itemize some of the salient points in extracting...

  3. Novel methods for detection of foodborne viruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enteric viruses such as norovirus are the number one cause of foodborne illness. Bivalve shellfish such as oysters efficiently bioconcentrate and retain theses pathogens, making raw shellfish consumption a significant risk factor for acquisition of these viruses. Recent ARS research indicates...

  4. Immunological-based assays for specific detection of shrimp viruses

    PubMed Central

    Chaivisuthangkura, Parin; Longyant, Siwaporn; Sithigorngul, Paisarn

    2014-01-01

    Among shrimp viral pathogens, white spot syndrome virus (WSSV) and yellow head virus (YHV) are the most lethal agents, causing serious problems for both the whiteleg shrimp, Penaeus (Litopenaeus) vannamei, and the black tiger shrimp, Penaeus (Penaeus) monodon. Another important virus that infects P. vannamei is infectious myonecrosis virus (IMNV), which induces the white discoloration of affected muscle. In the cases of taura syndrome virus and Penaeus stylirostris densovirus (PstDNV; formerly known as infectious hypodermal and hematopoietic necrosis virus), their impacts were greatly diminished after the introduction of tolerant stocks of P. vannamei. Less important viruses are Penaeus monodon densovirus (PmDNV; formerly called hepatopancreatic parvovirus), and Penaeus monodon nucleopolyhedrovirus (PemoNPV; previously called monodon baculovirus). For freshwater prawn, Macrobrachium rosenbergii nodavirus and extra small virus are considered important viral pathogens. Monoclonal antibodies (MAbs) specific to the shrimp viruses described above have been generated and used as an alternative tool in various immunoassays such as enzyme-linked immunosorbent assay, dot blotting, Western blotting and immunohistochemistry. Some of these MAbs were further developed into immunochromatographic strip tests for the detection of WSSV, YHV, IMNV and PemoNPV and into a dual strip test for the simultaneous detection of WSSV/YHV. The strip test has the advantages of speed, as the result can be obtained within 15 min, and simplicity, as laboratory equipment and specialized skills are not required. Therefore, strip tests can be used by shrimp farmers for the pond-side monitoring of viral infection. PMID:24567913

  5. Immunological-based assays for specific detection of shrimp viruses.

    PubMed

    Chaivisuthangkura, Parin; Longyant, Siwaporn; Sithigorngul, Paisarn

    2014-02-12

    Among shrimp viral pathogens, white spot syndrome virus (WSSV) and yellow head virus (YHV) are the most lethal agents, causing serious problems for both the whiteleg shrimp, Penaeus (Litopenaeus) vannamei, and the black tiger shrimp, Penaeus (Penaeus) monodon. Another important virus that infects P. vannamei is infectious myonecrosis virus (IMNV), which induces the white discoloration of affected muscle. In the cases of taura syndrome virus and Penaeus stylirostris densovirus (PstDNV; formerly known as infectious hypodermal and hematopoietic necrosis virus), their impacts were greatly diminished after the introduction of tolerant stocks of P. vannamei. Less important viruses are Penaeus monodon densovirus (PmDNV; formerly called hepatopancreatic parvovirus), and Penaeus monodon nucleopolyhedrovirus (PemoNPV; previously called monodon baculovirus). For freshwater prawn, Macrobrachium rosenbergii nodavirus and extra small virus are considered important viral pathogens. Monoclonal antibodies (MAbs) specific to the shrimp viruses described above have been generated and used as an alternative tool in various immunoassays such as enzyme-linked immunosorbent assay, dot blotting, Western blotting and immunohistochemistry. Some of these MAbs were further developed into immunochromatographic strip tests for the detection of WSSV, YHV, IMNV and PemoNPV and into a dual strip test for the simultaneous detection of WSSV/YHV. The strip test has the advantages of speed, as the result can be obtained within 15 min, and simplicity, as laboratory equipment and specialized skills are not required. Therefore, strip tests can be used by shrimp farmers for the pond-side monitoring of viral infection. PMID:24567913

  6. Newcastle disease virus detection and differentiation from avian influenza.

    PubMed

    Miller, Patti J; Torchetti, Mia Kim

    2014-01-01

    Newcastle disease (ND) is a contagious and often fatal disease that affects over 250 bird species worldwide, and is caused by infection with virulent strains of avian paramyxovirus-1 (APMV-1) of the family Paramyxoviridae, genus Avulavirus. Infections of poultry with virulent strains of APMV-1 (Newcastle disease virus) are reportable to the World Organization for Animal Health (OIE). Vaccination of poultry species is a key measure in the control of ND. Other APMV-1 viruses of low virulence, which are not used as vaccines, are also often isolated from wild bird species. The APMV-1 virus, like avian influenza virus (AIV), is a hemagglutinating virus (HA) and able to agglutinate chicken red blood cells (RBC). Because the clinical presentation of ND can be difficult to distinguish from disease caused by AIV, techniques for differential diagnosis are essential, as well as the ability to detect mixed infections. When an HA positive virus is detected from virus isolation, additional assays can be performed to determine which virus is present. Both antigenic and molecular methods are necessary as some virulent ND viruses from cormorants in the USA after 2002 have lost their ability to hemagglutinate chicken RBC and molecular methods are needed for identification. PMID:24899433

  7. Detection and molecular characterization of Egyptian isolates of grapevine viruses.

    PubMed

    Fattouh, F; Ratti, C; El-Ahwany, A M D; Aleem, E Abdel; Babini, A R; Autonell, C Rubies

    2014-01-01

    Selected commercial and/or local vineyards and nurseries in three different governorates of Egypt (Alexandria, El-Beheira and El-Menofia) were surveyed for symptoms indicative of infection by grapevine viruses. Leaf samples from red-fruited and white-fruited Vitis vinefera were tested for grapevine leafroll associated viruses (GLRaV-1, GLRaV-2, and GLRaV-3), grapevine viruses A and B (GVA, GVB), grapevine rupestris stem pitting virus (GRSPaV), grapevine fanleaf virus (GFLV), and grapevine fleck virus (GFKV) from early April to late October 2010. Incidence of these viruses was assessed by RT-PCR in 60 different samples. Selected amplicons were sequenced. While GVA was the most wide spread (30%), GLRaV-1, GVB, GFLV, and GFKV were not detected during the survey. However, GVA, GLRaV-2, GLRaV-3, and GRSPaV were detected in the form of single infection or in mixed infections of 2 to 4 viruses. Phylogenetic analysis was performed on all Egyptian isolates of GLRaV-2 (4), GLRaV-3 (7), GVA (3), and GRSPaV (6). GRSPaV was detected for the first time in Egypt. Phylogenetic analysis provided insights into the evolutionary relationship between the reported Egyptian isolates and other previously reported isolates. PMID:24957718

  8. Using Small RNA Deep Sequencing Data to Detect Human Viruses

    PubMed Central

    Wang, Fang; Sun, Yu; Ruan, Jishou; Chen, Rui; Chen, Xin; Chen, Chengjie; Kreuze, Jan F.; Fei, ZhangJun; Zhu, Xiao

    2016-01-01

    Small RNA sequencing (sRNA-seq) can be used to detect viruses in infected hosts without the necessity to have any prior knowledge or specialized sample preparation. The sRNA-seq method was initially used for viral detection and identification in plants and then in invertebrates and fungi. However, it is still controversial to use sRNA-seq in the detection of mammalian or human viruses. In this study, we used 931 sRNA-seq runs of data from the NCBI SRA database to detect and identify viruses in human cells or tissues, particularly from some clinical samples. Six viruses including HPV-18, HBV, HCV, HIV-1, SMRV, and EBV were detected from 36 runs of data. Four viruses were consistent with the annotations from the previous studies. HIV-1 was found in clinical samples without the HIV-positive reports, and SMRV was found in Diffuse Large B-Cell Lymphoma cells for the first time. In conclusion, these results suggest the sRNA-seq can be used to detect viruses in mammals and humans. PMID:27066498

  9. Using Small RNA Deep Sequencing Data to Detect Human Viruses.

    PubMed

    Wang, Fang; Sun, Yu; Ruan, Jishou; Chen, Rui; Chen, Xin; Chen, Chengjie; Kreuze, Jan F; Fei, ZhangJun; Zhu, Xiao; Gao, Shan

    2016-01-01

    Small RNA sequencing (sRNA-seq) can be used to detect viruses in infected hosts without the necessity to have any prior knowledge or specialized sample preparation. The sRNA-seq method was initially used for viral detection and identification in plants and then in invertebrates and fungi. However, it is still controversial to use sRNA-seq in the detection of mammalian or human viruses. In this study, we used 931 sRNA-seq runs of data from the NCBI SRA database to detect and identify viruses in human cells or tissues, particularly from some clinical samples. Six viruses including HPV-18, HBV, HCV, HIV-1, SMRV, and EBV were detected from 36 runs of data. Four viruses were consistent with the annotations from the previous studies. HIV-1 was found in clinical samples without the HIV-positive reports, and SMRV was found in Diffuse Large B-Cell Lymphoma cells for the first time. In conclusion, these results suggest the sRNA-seq can be used to detect viruses in mammals and humans. PMID:27066498

  10. First detection of bluetongue virus serotype 14 in Poland.

    PubMed

    Orłowska, Anna; Trębas, Paweł; Smreczak, Marcin; Marzec, Anna; Żmudziński, Jan F

    2016-07-01

    Here, we present the first detected cases of bluetongue virus (BTV) in native cattle from Poland. The virus was found in animals located near the Polish-Belarusian and Polish-Lithuanian borders. The positive animals were detected through an official epidemiological surveillance program. A combination of type-specific real-time RT-PCR and phylogenetic tests revealed the presence of BTV serotype 14 (BTV-14). This serotype is highly homologous to the vaccine strain and BTV-14 present in Russia, Lithuania, and Spain (from an animal imported from Lithuania). The most probable route of virus introduction to Poland was transmission through midges. All of the cases were subclinical. PMID:27068167

  11. Towards detecting the human immunodeficiency virus using microcantilever sensors

    NASA Astrophysics Data System (ADS)

    Alodhayb, Abdullah; Brown, Nicole; Saydur Rahman, S. M.; Harrigan, Richard; Beaulieu, L. Y.

    2013-04-01

    Detecting the human immunodeficiency virus (HIV) is difficult because the virus is prone to mutations and is in low concentrations in the body. Inside the HIV virion are two well characterized single stranded (ss) RNA molecules (viral genome) that feature both variable regions and regions that are conserved under virus mutation. In this work, microcantilever sensors have been employed as potential HIV detectors by targeting a conserved sequence of the viral genome by attempting to detect target ssDNA and ssRNA molecules that are significantly longer than the ssDNA molecules functionalized on the cantilever.

  12. Magnetic protein microbead-aided indirect fluoroimmunoassay for the determination of canine virus specific antibodies.

    PubMed

    Wang, Xueqin; Ren, Li; Tu, Qin; Wang, Jianchun; Zhang, Yanrong; Li, Manlin; Liu, Rui; Wang, Jinyi

    2011-03-15

    Rabies, canine distemper, and canine parvovirus are common contagious viral diseases of dogs and many other carnivores, and pose a severe threat to the population dynamics of wild carnivores, as well as endangering carnivore conservation. However, clinical diagnosis of these diseases, especially canine distemper and canine parvovirus, is difficult because of the broad spectrum of symptoms that may be confused with other respiratory and enteric diseases of dogs. The most frequently used and proven techniques for diagnosing viral diseases include the conventional enzyme-linked immunosorbent assay (ELISA), rapid fluorescent focus inhibition test (RFFIT), mouse neutralisation test (MNT), and fluorescent antibody virus neutralization (FAVN) test. However, these methods still have some inherent limitations. In this study, a magnetic protein microbead-aided indirect fluoroimmunoassay was developed to detect canine virus specific antibodies, human rabies immunoglobulin, CDV McAbs, and CPV McAbs. In this assay, an avidin-biotin system was employed to combine magnetic microbeads and virus antigens (rabies virus, canine distemper virus, and canine parvovirus). Quantification of the targeted virus antibodies was analyzed through indirect fluoroimmunoassay using the specific antigen-antibody reaction, as well as their corresponding FITC-labeled detection antibodies (mouse anti-human IgG/FITC conjugate or rabbit anti-dog IgG/FITC conjugate). The results indicated that the fluorescence intensity increased when a higher concentration of the targeted analyte was used, but the control had almost no fluorescence, much like the conventional ELISA. For human rabies immunoglobulin, CDV McAbs, and CPV McAbs, the minimum detectable concentrations were 0.2 IU/mL, 0.3 ng/mL, and 0.5 ng/mL, respectively. All of these results indicate that this assay can be employed to determine the presence of canine virus specific antibodies. In addition, the method devised here can be utilized as a general

  13. EPA METHODS FOR VIRUS DETECTION IN WATER

    EPA Science Inventory

    A number of different types of human enteric viruses cause waterborne outbreaks when individuals are exposed to contaminated drinking and recreational waters. Members of the enterovirus group cause numerous diseases, including gastroenteritis, encephalitis, meningitis, myocard...

  14. Single virus particle mass detection using microresonators with nanoscale thickness

    NASA Astrophysics Data System (ADS)

    Gupta, A.; Akin, D.; Bashir, R.

    2004-03-01

    In this letter, we present the microfabrication and application of arrays of silicon cantilever beams as microresonator sensors with nanoscale thickness to detect the mass of individual virus particles. The dimensions of the fabricated cantilever beams were in the range of 4-5 μm in length, 1-2 μm in width and 20-30 nm in thickness. The virus particles we used in the study were vaccinia virus, which is a member of the Poxviridae family and forms the basis of the smallpox vaccine. The frequency spectra of the cantilever beams, due to thermal and ambient noise, were measured using a laser Doppler vibrometer under ambient conditions. The change in resonant frequency as a function of the virus particle mass binding on the cantilever beam surface forms the basis of the detection scheme. We have demonstrated the detection of a single vaccinia virus particle with an average mass of 9.5 fg. These devices can be very useful as components of biosensors for the detection of airborne virus particles.

  15. Optofluidic wavelength division multiplexing for single-virus detection.

    PubMed

    Ozcelik, Damla; Parks, Joshua W; Wall, Thomas A; Stott, Matthew A; Cai, Hong; Parks, Joseph W; Hawkins, Aaron R; Schmidt, Holger

    2015-10-20

    Optical waveguides simultaneously transport light at different colors, forming the basis of fiber-optic telecommunication networks that shuttle data in dozens of spectrally separated channels. Here, we reimagine this wavelength division multiplexing (WDM) paradigm in a novel context--the differentiated detection and identification of single influenza viruses on a chip. We use a single multimode interference (MMI) waveguide to create wavelength-dependent spot patterns across the entire visible spectrum and enable multiplexed single biomolecule detection on an optofluidic chip. Each target is identified by its time-dependent fluorescence signal without the need for spectral demultiplexing upon detection. We demonstrate detection of individual fluorescently labeled virus particles of three influenza A subtypes in two implementations: labeling of each virus using three different colors and two-color combinatorial labeling. By extending combinatorial multiplexing to three or more colors, MMI-based WDM provides the multiplexing power required for differentiated clinical tests and the growing field of personalized medicine. PMID:26438840

  16. Rapid detection of measles virus in skin rashes by immunofluroescence.

    PubMed

    Olding-Stenkvist, E; Bjorvatn, B

    1976-11-01

    Specific immunofluorescence was used to detect measles virus antigen in skin rashes. Cryostat sections of punch-biopsy specimens of the skin were stained with use of hyperimmune rabbit and horse antisera to measles virus, also conjugated with fluorescein isothiocyanate, served as controls. Measles virus was specifically demonstrated in 20 of 21 biopsy specimens taken within four days after the onset of exanthema. Measles virus antigen was also found in three of five biopsy specimens from nonexanthematous skin during the first four days of the exanthematous phase. The viral antigen was found in single cells or in clusters of cells in the surface epithelium, skin appendages, and corium. No viral antigen was detected in biopsy specimens taken five to six days after the onset of rash. PMID:792356

  17. Controversial results of therapy with mesenchymal stem cells in the acute phase of canine distemper disease.

    PubMed

    Pinheiro, A O; Cardoso, M T; Vidane, A S; Casals, J B; Passarelli, D; Alencar, A L F; Sousa, R L M; Fantinato-Neto, P; Oliveira, V C; Lara, V M; Ambrósio, C E

    2016-01-01

    Distemper disease is an infectious disease reported in several species of domestic and wild carnivores. The high mortality rate of animals infected with canine distemper virus (CDV) treated with currently available therapies has driven the study of new efficacious treatments. Mesenchymal stem cell (MSC)-based therapy is a promising therapeutic option for many degenerative, hereditary, and inflammatory diseases. Therefore, the aim of this study was to characterize stem cells derived from the canine fetal olfactory epithelium and to assess the systemic response of animals infected with CDV to symptomatic therapy and treatment with MSCs. Eight domestic mongrel dogs (N = 8) were divided into two groups: support group (SG) (N = 5) and support group + cell therapy (SGCT) (N = 3), which were monitored over 15 days. Blood samples were collected on days 0, 6, 9, 12, and 15 to assess blood count and serum biochemistry (urea, creatinine, alanine transferase, alkaline phosphatase, gamma-glutamyl transferase, total protein, albumin, and globulin), and urine samples were obtained on days 0 and 15 for urinary evaluation (urine I). The results showed a high mortality rate (SG = 4 and SGCT = 2), providing inadequate data on the clinical course of CDV infection. MSC therapy resulted in no significant improvement when administered during the acute phase of canine distemper disease, and a prevalence of animals with high mortality rate was found in both groups due to the severity of symptoms. PMID:27323085

  18. The 2000 canine distemper epidemic in Caspian seals (Phoca caspica): pathology and analysis of contributory factors.

    PubMed

    Kuiken, T; Kennedy, S; Barrett, T; Van de Bildt, M W G; Borgsteede, F H; Brew, S D; Codd, G A; Duck, C; Deaville, R; Eybatov, T; Forsyth, M A; Foster, G; Jepson, P D; Kydyrmanov, A; Mitrofanov, I; Ward, C J; Wilson, S; Osterhaus, A D M E

    2006-05-01

    More than 10,000 Caspian seals (Phoca caspica) were reported dead in the Caspian Sea during spring and summer 2000. We performed necropsies and extensive laboratory analyses on 18 seals, as well as examination of the pattern of strandings and variation in weather in recent years, to identify the cause of mortality and potential contributory factors. The monthly stranding rate in 2000 was up to 2.8 times the historic mean. It was preceded by an unusually mild winter, as observed before in mass mortality events of pinnipeds. The primary diagnosis in 11 of 13 seals was canine distemper, characterized by broncho-interstitial pneumonia, lymphocytic necrosis and depletion in lymphoid organs, and the presence of typical intracytoplasmic inclusion bodies in multiple epithelia. Canine distemper virus infection was confirmed by phylogenetic analysis of reverse transcriptase-polymerase chain reaction products. Organochlorine and zinc concentrations in tissues of seals with canine distemper were comparable to those of Caspian seals in previous years. Concurrent bacterial infections that may have contributed to the mortality of the seals included Bordetella bronchiseptica (4/8 seals), Streptococcus phocae (3/8), Salmonella dublin (1/8), and S. choleraesuis (1/8). A newly identified bacterium, Corynebacterium caspium, was associated with balanoposthitis in one seal. Several infectious and parasitic organisms, including poxvirus, Atopobacter phocae, Eimeria- and Sarcocystis-like organisms, and Halarachne sp. were identified in Caspian seals for the first time. PMID:16672579

  19. IMPROVING DETECTION METHODS FOR ENTERIC WATERBORNE VIRUSES

    EPA Science Inventory

    Waterborne viruses are a significant cause of illness, both within the US and worldwide. These illnesses can occur as the result of outbreaks, potentially affecting hundreds or thousands of people, or as a part of a background level of endemic infection. While many of these out...

  20. Detection of Strawberry Viruses in Egypt

    Technology Transfer Automated Retrieval System (TEKTRAN)

    As part of a USAID-MERC funded project, ‘Disease-indexing and mass propagation of superior strawberry cultivars’, an effort was made to evaluate the virus status of strawberries in Egypt. Diagnostic reverse transcription-polymerase chain reaction (RT-PCR) tests for Strawberry mottle, Strawberry cri...

  1. Laboratory Detection of Respiratory Viruses by Automated Techniques

    PubMed Central

    Pérez-Ruiz, Mercedes; Pedrosa-Corral, Irene; Sanbonmatsu-Gámez, Sara; Navarro-Marí, José-María

    2012-01-01

    Advances in clinical virology for detecting respiratory viruses have been focused on nucleic acids amplification techniques, which have converted in the reference method for the diagnosis of acute respiratory infections of viral aetiology. Improvements of current commercial molecular assays to reduce hands-on-time rely on two strategies, a stepwise automation (semi-automation) and the complete automation of the whole procedure. Contributions to the former strategy have been the use of automated nucleic acids extractors, multiplex PCR, real-time PCR and/or DNA arrays for detection of amplicons. Commercial fully-automated molecular systems are now available for the detection of respiratory viruses. Some of them could convert in point-of-care methods substituting antigen tests for detection of respiratory syncytial virus and influenza A and B viruses. This article describes laboratory methods for detection of respiratory viruses. A cost-effective and rational diagnostic algorithm is proposed, considering technical aspects of the available assays, infrastructure possibilities of each laboratory and clinic-epidemiologic factors of the infection PMID:23248735

  2. A rapid immunochromatographic test strip for detecting rabies virus antibody.

    PubMed

    Wang, Hualei; Feng, Na; Yang, Songtao; Wang, Chengyu; Wang, Tiecheng; Gao, Yuwei; Su, Jianqing; Zheng, Xuexing; Hou, Xiaoqiang; Huang, Hainan; Yang, Ruimei; Zou, Xiaohuan; Huang, Geng; Xia, Xianzhu

    2010-12-01

    An immunochromatographic test strip (ICTS) for detecting antibodies to rabies virus was developed, using colloidal gold particles labeled with rabies virus glycoprotein as the tracer. The assay was evaluated using sera from dogs immunized with various commercial rabies vaccines, or from dogs in the clinics and sera from dogs immunized with vaccines against pathogens other than rabies virus, and negative sera from a wide variety of animal sources, including dogs, mice, and cats which had never been vaccinated. The ICTS was found to be highly specific for antibodies against rabies virus, with a detection limit of 0.5IU/ml as measured by the fluorescent antibody virus neutralization (FAVN) test. Compared with the FAVN test, the specificity and sensitivity of ICTS were 98.2% and 90.4%, respectively. There was an excellent agreement between results obtained by the ICTS and FAVN tests (kappa=0.888). Strips stored at 4°C in a plastic bag with a desiccant retained their specificity and sensitivity for at least 15 months, and strips stored at ambient temperature remained stable for 12 months. The immunochromatographic test strip may therefore be useful for clinical laboratories lacking specialized equipment and for diagnosis in the field for rapid detection of rabies virus-specific antibodies. PMID:20837065

  3. New viruses in veterinary medicine, detected by metagenomic approaches.

    PubMed

    Belák, Sándor; Karlsson, Oskar E; Blomström, Anne-Lie; Berg, Mikael; Granberg, Fredrik

    2013-07-26

    In our world, which is faced today with exceptional environmental changes and dramatically intensifying globalisation, we are encountering challenges due to many new factors, including the emergence or re-emergence of novel, so far "unknown" infectious diseases. Although a broad arsenal of diagnostic methods is at our disposal, the majority of the conventional diagnostic tests is highly virus-specific or is targeted entirely towards a limited group of infectious agents. This specificity complicates or even hinders the detection of new or unexpected pathogens, such as new, emerging or re-emerging viruses or novel viral variants. The recently developed approaches of viral metagenomics provide an effective novel way to screen samples and detect viruses without previous knowledge of the infectious agent, thereby enabling a better diagnosis and disease control, in line with the "One World, One Health" principles (www.oneworldonehealth.org). Using metagenomic approaches, we have recently identified a broad variety of new viruses, such as novel bocaviruses, Torque Teno viruses, astroviruses, rotaviruses and kobuviruses in porcine disease syndromes, new virus variants in honeybee populations, as well as a range of other infectious agents in further host species. These findings indicate that the metagenomic detection of viral pathogens is becoming now a powerful, cultivation-independent, and useful novel diagnostic tool in veterinary diagnostic virology. PMID:23428379

  4. Old diseases for new nightmares: distemper strikes back in Italy.

    PubMed

    Lorusso, Alessio; Savini, Giovanni

    2014-01-01

    This article analyses the distemper outbreak that affected the population of Apennine wolves (Canis lupus) in Italy during 2013. Distemper, as rabies, is a well-known viral infectious disease that concerns the canine population worldwide and represents a threat for wild species too. Implementation of vaccination and legislation for compulsory vaccination strategies should be achieved in areas with endangered wild species. PMID:24817331

  5. Rapid and selective detection of viruses using virus-imprinted polymer films

    NASA Astrophysics Data System (ADS)

    Karthik, A.; Margulis, K.; Ren, K.; Zare, R. N.; Leung, L. W.

    2015-11-01

    We prepared a nanopatterned polymer film of polydimethylsiloxane (PDMS) via virus imprinting. The imprinted surface exhibited nanoscale cavities with the mean size of 120 +/- 4 nm. These cavities demonstrated the ability to preferentially capture a target virus from an aqueous suspension of ultralow volume (5 μL) after only 1 minute of contact. Two inactivated viruses with similar shape, Influenza A (HK68) and Newcastle Disease Virus (NDV), were employed as model pathogens. The polymer film, which was first imprinted with HK68 and exposed sequentially to suspensions containing fluorescently labeled NDV and HK68, was able to preferentially bind HK68 at a capture ratio of 1 : 8.0. When we reversed the procedure and imprinted with NDV, the capture ratio was 1 : 7.6. These results were obtained within 20 minutes of static exposure. The suspensions contained viruses at concentrations close to those occurring physiologically in influenza infections. The limit of detection was approximately 8 fM. Production of virus-imprinted films can be readily scaled to large quantities and yields a disposable, simple-to-use device that allows for rapid detection of viruses.

  6. A SYBR Green I based real time RT-PCR assay for specific detection and quantitation of Peste des petits ruminants virus

    PubMed Central

    2014-01-01

    Background Peste des petits ruminants (PPR) is an economically important disease of small ruminants such as sheep and goats. The disease is characterized by severe pyrexia, oculo-nasal discharge, pneumonia, necrosis and ulceration of the mucous membrane and inflammation of the gastro-intestinal tract leading to severe diarrhea. A SYBR Green I based real time RT-PCR targeting the N gene of PPRV has not been established for PPRV detection. Thus, the objective of present study was to develop highly sensitive N gene target SYBR Green I real time RT-PCR for specific detection and quantification of PPRV in clinical samples. A set of primers was designed to detect the nucleocapsid (N) gene of PPRV. Results The assay exhibited high specificity as all the viruses which have clinical and structural similarities to PPRV including Canine distemper virus (CDV), Measles virus (MV), Bluetongue virus (BTV) and Newcastle disease virus (NDV) failed to show an amplification signal. The lower detection limit of the assay was 5.11 copies/μl (Ct value of 33.67 ± 0.5) and 0.001 TCID50/ml (Ct value of 34.7 ± 0.5) based on plasmid copy number and tissue culture infectivity titre. The assay was 3-log more sensitive than the conventional RT-PCR. The coefficient of variation (CV) values for intra- and inter-assay variability were low, ranging from 0.32% - 2.31%, and 0.71% - 5.32%, respectively. To evaluate the performance of the newly developed assay, a total of 36 clinical samples suspected of PPR were screened for the presence of PPRV in parallel with conventional RT-PCR. The real time RT-PCR assay detected PPRV in 30 (83.3%) of clinical samples compared to 16 (44.4%) by conventional RT-PCR. Conclusions The two-step SYBR Green I based real time RT-PCR assay reported here is highly sensitive, specific, reproducible and rapid for detection and quantification of PPRV nucleic acids. PMID:24423231

  7. Progress in development of a Universal Plant Virus Micrarray for the detection and identification of plant viruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microarrays based on oligonucleotides representing sequences conserved at the level of viral species, genera, and families are able to detect and identify both characterized and previously uncharacterized viruses infecting mammals and birds. Software initially developed for these animal virus microa...

  8. Dog distemper: imported into Europe from South America?.

    PubMed

    Blancou, Jean

    2004-01-01

    According to Charles Frédéric Heusinger (1853), dog distemper had been imported from Peru into Spain in the course of the 17th century. The disease was well described in 1746 by Ulloa in his work Relación histórica del viaje a la América meridional. During the course of the 1760s, the disease was reported in Spain, followed by England, Italy (1764) and Russia (1770). In 1763, 900 dogs died in a single day in Madrid. In 1844, Karle succeeded in the first experimental transmission of the disease by brushing the lips of young dogs with the discharge from sick animals. The causal agent of the disease was only discovered in 1905, when the virus was isolated by Henri Carré. In the meantime, Edward Jenner, who thought that the disease was a pox-like affection, claimed that it could be prevented by inoculation of the vaccinia virus. PMID:15376360

  9. Single-Reaction Multiplex Reverse Transcription PCR for Detection of Zika, Chikungunya, and Dengue Viruses.

    PubMed

    Waggoner, Jesse J; Gresh, Lionel; Mohamed-Hadley, Alisha; Ballesteros, Gabriela; Davila, Maria Jose Vargas; Tellez, Yolanda; Sahoo, Malaya K; Balmaseda, Angel; Harris, Eva; Pinsky, Benjamin A

    2016-07-01

    Clinical manifestations of Zika virus, chikungunya virus, and dengue virus infections can be similar. To improve virus detection, streamline molecular workflow, and decrease test costs, we developed and evaluated a multiplex real-time reverse transcription PCR for these viruses. PMID:27184629

  10. Single-Reaction Multiplex Reverse Transcription PCR for Detection of Zika, Chikungunya, and Dengue Viruses

    PubMed Central

    Waggoner, Jesse J.; Gresh, Lionel; Mohamed-Hadley, Alisha; Ballesteros, Gabriela; Davila, Maria Jose Vargas; Tellez, Yolanda; Sahoo, Malaya K.; Balmaseda, Angel; Harris, Eva

    2016-01-01

    Clinical manifestations of Zika virus, chikungunya virus, and dengue virus infections can be similar. To improve virus detection, streamline molecular workflow, and decrease test costs, we developed and evaluated a multiplex real-time reverse transcription PCR for these viruses. PMID:27184629

  11. Detection of Entebbe Bat Virus After 54 Years.

    PubMed

    Kading, Rebekah C; Kityo, Robert; Nakayiki, Teddie; Ledermann, Jeremy; Crabtree, Mary B; Lutwama, Julius; Miller, Barry R

    2015-09-01

    Entebbe bat virus (ENTV; Flaviviridae: Flavivirus), closely related to yellow fever virus, was first isolated from a little free-tailed bat (Chaerephon pumilus) in Uganda in 1957, but was not detected after that initial isolation. In 2011, we isolated ENTV from a little free-tailed bat captured from the attic of a house near where it had originally been found. Infectious virus was recovered from the spleen and lung, and the viral RNA was sequenced and compared with that of the original isolate. Across the polypeptide sequence, there were 76 amino acid substitutions, resulting in 97.8% identity at the amino acid level between the 1957 and 2011 isolates. Further study of this virus would provide valuable insights into the ecological and genetic factors governing the evolution and transmission of bat- and mosquito-borne flaviviruses. PMID:26101270

  12. Nano-mechanical Resonantor Sensors for Virus Detection

    NASA Astrophysics Data System (ADS)

    Bashir, Rashid

    2005-03-01

    Micro and nanoscale cantilever beams can be used as highly sensitive mass detectors. Scaling down the area of the cantilever allows a decrease in minimum detectable mass limit while scaling down the thickness allows the resonant frequencies to be within measurable range. We have fabricated arrays of silicon cantilever beams as nanomechanical resonant sensors to detect the mass of individual virus particles. The dimensions of the fabricated cantilever beams were in the range of 4-5 μm in length, 1-2 μm in width and 20-30 nm in thickness. The virus particles we used in the study were vaccinia virus, which is a member of the Poxviridae family and forms the basis of the smallpox vaccine. The frequency spectra of the cantilever beams, due to thermal and ambient noise, were measured using a laser Doppler vibrometer under ambient conditions. The change in resonant frequency as a function of the virus particle mass binding on the cantilever beam surface forms the basis of the detection scheme. We have demonstrated the detection of a single vaccinia virus particle with an average mass of 9.5 fg. Specific capture of the antigens requires attachment of antibodies, which can be in the same range of thickness as these cantilever sensors, and can alter their mechanical properties. We have attached protein layers on both sides of 30nm thick cantilever beams and we show that the resonant frequencies can increase or decrease upon the attachment of protein layers to the cantilevers. In certain cases, the increase in spring constant out-weighs the increase in mass and the resonant frequencies can increase upon the attachment of the protein layers. These devices can be very useful as components of biosensors for the detection of air-borne virus particles.

  13. Detection of viral hemorrhagic septicemia virus

    USGS Publications Warehouse

    Winton, James; Kurath, Gael; Batts, William

    2007-01-01

    Viral hemorrhagic septicemia virus (VHSV) is considered to be one of the most important viral pathogens of finfish and is listed as reportable by many nations and international organizations (Office International des Epizooties 2006). Prior to 1988, VHSV was thought to be limited to Europe (Wolf 1988; Smail 1999). Subsequently, it was shown that the virus is endemic among many marine and anadromous fish species in both the Pacific and Atlantic Oceans (Meyers and Winton 1995; Skall et al. 2005). Genetic analysis reveals that isolates of VHSV can be divided into four genotypes that generally correlate with geographic location with the North American isolates generally falling into VHSV Genotype IV (Snow et al. 2004). In 2005-2006, reports from the Great Lakes region indicated that wild fish had experienced disease or, in some cases, very large die-offs from VHSV (Elsayed et al. 2006, Lumsden et al. 2007). The new strain from the Great Lakes, now identified as VHSV Genotype IVb, appears most closely related to isolates of VHSV from mortalities that occurred during 2000-2004 in rivers and near-shore areas of New Brunswick and Nova Scotia, Canada (Gagne et al. 2007). The type IVb isolate found in the Great Lakes region is the only strain outside of Europe that has been associated with significant mortality in freshwater species.

  14. Detection of Chikungunya Virus in Nepal.

    PubMed

    Pandey, Basu Dev; Neupane, Biswas; Pandey, Kishor; Tun, Mya Myat Ngwe; Morita, Kouichi

    2015-10-01

    Chikungunya virus (CHIKV) is an emerging alphaviral disease and a public health problem in South Asia including Nepal in recent years. In this study, sera were collected from patients presenting with fever, headache, muscular pain, fatigue, and joint pain of both upper and lower extremities. A total of 169 serum samples were tested for CHIKV and dengue virus (DENV) by using Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibody using enzyme-linked immunosorbent assay (ELISA) method during August to November 2013. Results showed that 3.6% and 27.8% samples were positive for CHIKV and DENV IgM positive, respectively. Similarly, results of IgG showed 3.0% samples were positive for CHIKV IgG and 29.0% were for DENV IgG positive. Further, a 50% focal reduction neutralization test (FRNT50) was performed to confirm the presence of CHIKV, which demonstrated that 8.9% of CHIKV IgM and/or IgG ELISA positive possessed neutralizing anti-CHIK antibodies. To our knowledge, this is the first report in which the presence of CHIKV is confirmed in Nepalese patients by FRNT50. Basic scientists and clinicians need to consider CHIKV as a differential diagnosis in febrile Nepalese patients, and policy makers should consider appropriate surveillance and actions for control strategies. PMID:26195462

  15. Comparison of different methods for detecting human immune deficiency virus in human immunodeficiency virus-seropositive hemophiliacs.

    PubMed

    Schneweis, K E; Ackermann, A; Friedrich, A; Kleim, J P; Kornau, K; Ruff, R; Siefer-Wippermann, B

    1989-10-01

    Since the detection of antibodies against the human immune deficiency virus (HIV) does not definitely prove HIV infection in hemophiliacs, virus detection was attempted by virus isolation from the peripheral blood monocytes (PBL), by demonstration of p24 antigen and decline of p24 antibody, and by detection of viral DNA by the polymerase chain reaction (PCR). Virus isolation was optimized by immediate coculture of PBL and by replacement of the reverse transcriptase test by the p24 antigen test, whereas the elimination of CD8+ lymphocytes proved to be unnecessary. Virus detection was dependent on the clinical stage of the illness. Virus isolation in 70 of 211 patients (33%) was more sensitive than detection of p24 antigen or decline of p24 antibody. PCR was performed in 25 patients and indicated infection in all of 15 isolation-positive cases and in 6 of 10 patients from whom virus was not isolated. Changes from negative to positive virus culture and from a weakly fusiogenic to a highly fusiogenic isolate were often accompanied by a progression of the disease. The results suggest that reactivation of HIV occurs when immune deficiency has become manifest. Apparently virus isolation detects only the virus already reactivated in vivo, whereas the PCR may also detect latent virus. PMID:2689596

  16. Nanofluidic devices for rapid detection of virus particles.

    SciTech Connect

    Gourley, Paul Lee; McDonald, Anthony Eugene; Hendricks, Judy K.

    2005-01-01

    Technologies that could quickly detect and identify virus particles would play a critical role in fighting bioterrorism and help to contain the rapid spread of disease. Of special interest is the ability to detect the presence and movement of virions without chemically modifying them by attaching molecular probes. This would be useful for rapid detection of pathogens in food or water supplies without the use of expensive chemical reagents. Such detection requires new devices to quickly screen for the presence of tiny pathogens. To develop such a device, we fabricated nanochannels to transport virus particles through ultrashort laser cavities and measured the lasing output as a sensor for virions. To understand this transduction mechanism, we also investigated light scattering from virions, both to determine the magnitude of the scattered signal and to use it to investigate the motion of virions.

  17. Detection of Waterborne Viruses Using High Affinity Molecularly Imprinted Polymers.

    PubMed

    Altintas, Zeynep; Gittens, Micah; Guerreiro, Antonio; Thompson, Katy-Anne; Walker, Jimmy; Piletsky, Sergey; Tothill, Ibtisam E

    2015-07-01

    Molecularly imprinted polymers (MIPs) are artificial receptor ligands which can recognize and specifically bind to a target molecule. They are more resistant to chemical and biological damage and inactivation than antibodies. Therefore, target specific-MIP nanoparticles are aimed to develop and implemented to biosensors for the detection of biological toxic agents such as viruses, bacteria, and fungi toxins that cause many diseases and death due to the environmental contamination. For the first time, a molecularly imprinted polymer (MIP) targeting the bacteriophage MS2 as the template was investigated using a novel solid-phase synthesis method to obtain the artificial affinity ligand for the detection and removal of waterborne viruses through optical-based sensors. A high affinity between the artificial ligand and the target was found, and a regenerative MIP-based virus detection assay was successfully developed using a new surface plasmon resonance (SPR)-biosensor which provides an alternative technology for the specific detection and removal of waterborne viruses that lead to high disease and death rates all over the world. PMID:26008649

  18. Assays to Detect West Nile Virus in Dead Birds

    PubMed Central

    Therrien, Joseph E.; Benson, Robert; Kramer, Laura; Kauffman, Elizabeth B.; Eidson, Millicent; Campbell, Scott

    2005-01-01

    Using oral swab samples to detect West Nile virus in dead birds, we compared the Rapid Analyte Measurement Platform (RAMP) assay with VecTest and real-time reverse-transcriptase–polymerase chain reaction. The sensitivities of RAMP and VecTest for testing corvid species were 91.0% and 82.1%, respectively. PMID:16318736

  19. Multiplex detection of Solenopsis invicta viruses -1, -2, and -3

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A multiplex polymerase chain reaction (PCR) method was developed to detect simultaneously Solenopsis invicta viruses -1, -2, and -3 in their host, the red imported fire ant, Solenopsis invicta. cDNA synthesis was conducted in a single reaction containing three oligonucleotide primers specific for ...

  20. The use of a handheld Raman system for virus detection

    NASA Astrophysics Data System (ADS)

    Song, Chunyuan; Driskell, Jeremy D.; Tripp, Ralph A.; Cui, Yiping; Zhao, Yiping

    2012-06-01

    The combination of surface enhanced Raman spectroscopy (SERS) with a handheld Raman system would lead to a powerful portable device for defense and security applications. The Thermo Scientific FirstDefender RM instrument is a 785-nm handheld Raman spectrometer intended for rapid field identification of unknown solid and liquid samples. Its sensitivity and effectiveness for SERS-based detection was initially confirmed by evaluating detection of 1,2-di(4- pyridyl)ethylene as a reporter molecule on a silver nanorod (AgNR) substrate, and the results are comparable to those from a confocal Bruker Raman system. As avian influenza A viruses (AIV) are recognized as an important emerging threat to public health, this portable handheld Raman spectrometer is used, for the first time, to detect and identify avian influenza A viruses using a multi-well AgNR SERS chip. The SERS spectra obtained had rich peaks which demonstrated that the instrument can be effectively used for SERS-based influenza virus detection. According to the SERS spectra, these different influenza viruses were distinguished from the negative control via the principal component analysis and by partial least squares-discriminate analysis. Together, these results show that the combination effective SERS substrates when combined with a portable Raman spectrometer provides a powerful field device for chemical and biological sensing.

  1. Using Fluorescent Viruses for Detecting Bacteria in Water

    NASA Technical Reports Server (NTRS)

    Tabacco, Mary Beth; Qian, Xiaohua; Russo, Jaimie A.

    2009-01-01

    A method of detecting water-borne pathogenic bacteria is based partly on established molecular-recognition and fluorescent-labeling concepts, according to which bacteria of a species of interest are labeled with fluorescent reporter molecules and the bacteria can then be detected by fluorescence spectroscopy. The novelty of the present method lies in the use of bacteriophages (viruses that infect bacteria) to deliver the fluorescent reporter molecules to the bacteria of the species of interest.

  2. Detection of West Nile virus in large pools of mosquitoes.

    PubMed

    Sutherland, Genevieve L; Nasci, Roger S

    2007-12-01

    We conducted a laboratory evaluation of the ability of commercial antigen-capture assays, the Rapid Analyte Measurement Platform (RAMP) and the VecTest wicking assay, as well as Real Time reverse transcriptase polymerase chain reaction (RT-PCR, Taqman) and Vero cell plaque assay to detect West Nile virus (WNV) in large mosquito pools. Real-Time PCR (Taqman) was the most sensitive, detecting WNV ribonucleic acid (RNA) in 100% of samples containing a single infected mosquito in pool sizes of up to 500 mosquitoes. Mosquito body tissues minimally impacted the ability of Real Time RT-PCR to detect WNV in a pool size of 500, reducing sensitivity by 0.6 log10 plaque-forming units (PFU)/ml. Vero cell plaque assay detected live virus from a single infected mosquito in 100% of pools containing up to 200 mosquitoes, but was unreliable at larger pool sizes. VecTest detected 100% of positive pools containing 50 mosquitoes with 5.8 log10 PFU/ml virus, 100 mosquitoes with 5.9 log10 PFU/ml, and 200 mosquitoes with 5.2 log10 PFU/ ml. The RAMP assay detected 100% of positive pools containing 50 mosquitoes with 3.3 log10 PFU/ml virus, 100 mosquitoes with 3.7 log10 PFU/ml, and 200 mosquitoes with 4.0 log10 PFU/ml. Results indicate that WNV can be reliably detected by all 4 assays in pools of mosquitoes exceeding 50 specimens, though there is some loss of sensitivity with very large pool sizes. PMID:18240515

  3. Serum virus neutralization assay for detection and quantitation of serum neutralizing antibodies to influenza A virus in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The serum virus neutralization (SVN) assay is a serological test to detect the presence and magnitude of functional systemic antibodies that prevent infectivity of a virus. The SVN assay is a highly sensitive and specific test that may be applied to influenza A viruses (IAV) in swine to measure the ...

  4. [Simultaneous detection of respiratory viruses and influenza A virus subtypes using multiplex PCR].

    PubMed

    Ciçek, Candan; Bayram, Nuri; Anıl, Murat; Gülen, Figen; Pullukçu, Hüsnü; Saz, Eylem Ulaş; Telli, Canan; Cok, Gürsel

    2014-10-01

    This study was conducted to investigate the respiratory viruses and subtyping of influenza A virus when positive by multiplex PCR in patients with flu-like symptoms, after the pandemic caused by influenza A (H1N1)pdm09. Nasopharyngeal swab samples collected from 700 patients (313 female, 387 male; age range: 24 days-94 yrs, median age: 1 yr) between December 2010 - January 2013 with flu-like symptoms including fever, headache, sore throat, rhinitis, cough, myalgia as defined by the World Health Organization were included in the study. Nucleic acid extractions (Viral DNA/RNA Extraction Kit, iNtRON, South Korea) and cDNA synthesis (RevertAid First Strand cDNA Synthesis Kits, Fermentas, USA) were performed according to the manufacturer's protocol. Multiplex amplification of nucleic acids was performed using DPO (dual priming oligonucleotide) primers and RV5 ACE Screening Kit (Seegene, South Korea) in terms of the presence of influenza A (INF-A) virus, influenza B (INF-B) virus, respiratory syncytial virus (RSV), and the other respiratory viruses. PCR products were detected by automated polyacrylamide gel electrophoresis using Screen Tape multiple detection system. Specimens which were positive for viral nucleic acids have been further studied by using specific DPO primers, FluA ACE Subtyping and RV15 Screening (Seegene, South Korea) kits. Four INF-A virus subtypes [human H1 (hH1), human H3 (hH3), swine H1 (sH1), avian H5 (aH5)] and 11 other respiratory viruses [Adenovirus, parainfluenza virus (PIV) types 1-4, human bocavirus (HBoV), human metapneumovirus (HMPV), rhinovirus types A and B, human coronaviruses (HCoV) OC43, 229E/NL63] were investigated with those tests. In the study, 53.6% (375/700) of the patients were found to be infected with at least one virus and multiple respiratory virus infections were detected in 15.7% (59/375) of the positive cases, which were mostly (49/59, 83%) in pediatric patients. RSV and rhinovirus coinfections were the most prevalent (18

  5. Detection of Human Enteric Viruses in Freshwater from European Countries.

    PubMed

    D'Ugo, Emilio; Marcheggiani, Stefania; Fioramonti, Ilaria; Giuseppetti, Roberto; Spurio, Roberto; Helmi, Karim; Guillebault, Delphine; Medlin, Linda K; Simeonovski, Ivan; Boots, Bas; Breitenbach, Ulrich; Koker, Latife; Albay, Meric; Mancini, Laura

    2016-09-01

    The transmission of water-borne pathogens typically occurs by a faecal-oral route, through inhalation of aerosols, or by direct or indirect contact with contaminated water. Previous molecular-based studies have identified viral particles of zoonotic and human nature in surface waters. Contaminated water can lead to human health issues, and the development of rapid methods for the detection of pathogenic microorganisms is a valuable tool for the prevention of their spread. The aims of this work were to determine the presence and identity of representative human pathogenic enteric viruses in water samples from six European countries by quantitative polymerase chain reaction (q-PCR) and to develop two quantitative PCR methods for Adenovirus 41 and Mammalian Orthoreoviruses. A 2-year survey showed that Norovirus, Mammalian Orthoreovirus and Adenoviruses were the most frequently identified enteric viruses in the sampled surface waters. Although it was not possible to establish viability and infectivity of the viruses considered, the detectable presence of pathogenic viruses may represent a potential risk for human health. The methodology developed may aid in rapid detection of these pathogens for monitoring quality of surface waters. PMID:27117764

  6. Standardized RT-PCR conditions for detection and identification of eleven viruses of potato and Potato spindle tuber viroid

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Standardized RT-PCR procedures were developed and validated for detection of Alfalfa mosaic virus (AMV), Impatiens necrotic spot virus (INSV), Tobacco rattle virus (TRV), Tomato spotted wilt virus (TSWV), Potato leaf roll virus (PLRV), Potato mop top virus (PMTV), Potato virus A (PVA), Potato viru...

  7. Direct detection of influenza virus antigen in nasopharyngeal specimens by direct enzyme immunoassay in comparison with quantitating virus shedding.

    PubMed Central

    Döller, G; Schuy, W; Tjhen, K Y; Stekeler, B; Gerth, H J

    1992-01-01

    We developed a direct enzyme immunoassay [EIA; Enzygnost Influenza A(Ag) and Enzygnost Influenza B(Ag)] for the direct detection of influenza A and B virus antigens in nasopharyngeal secretion specimens (NPS). The test is performed without sonification of specimens, and results are obtained within 4 h. A direct comparison between direct EIA and quantitation of virus shedding for influenza A and B virus antigen detection was carried out. A total of 210 NPS and 98 nasopharyngeal wash specimens (NPW) were investigated. We isolated influenza A viruses from 79 (37.6%) of 210 NPS; of these 79 cell-culture-positive NPS, 70 (88.6%) were also positive by direct EIA. Of 29 (13.8%) NPS from which influenza B virus was isolated, 24 (82.8%) NPS were positive by direct EIA. Virus shedding was determined quantitatively in 48 NPS from patients with influenza A and in 24 NPS from patients with influenza B. Only a crude correlation between optical density values and virus concentrations was observed. Detection of influenza virus antigens in NPS by direct EIA showed sensitivities of 89.7% for influenza A virus and 87.9% for influenza B virus and specificities of 99.3% for influenza A virus and 100% for influenza B virus. With direct EIA, all NPW were negative for influenza A virus, although virus was isolated from 21 (21.4%) NPW. Of 15 NPW from which influenza B virus was isolated, 7 showed positive results in direct EIA. In addition, direct EIA is suitable for detecting influenza A and B viruses in cell cultures before the appearance of any cytopathic effects and can be used as a cell culture confirmation test. PMID:1572972

  8. Optofluidic wavelength division multiplexing for single-virus detection

    PubMed Central

    Ozcelik, Damla; Parks, Joshua W.; Wall, Thomas A.; Stott, Matthew A.; Cai, Hong; Parks, Joseph W.; Hawkins, Aaron R.; Schmidt, Holger

    2015-01-01

    Optical waveguides simultaneously transport light at different colors, forming the basis of fiber-optic telecommunication networks that shuttle data in dozens of spectrally separated channels. Here, we reimagine this wavelength division multiplexing (WDM) paradigm in a novel context––the differentiated detection and identification of single influenza viruses on a chip. We use a single multimode interference (MMI) waveguide to create wavelength-dependent spot patterns across the entire visible spectrum and enable multiplexed single biomolecule detection on an optofluidic chip. Each target is identified by its time-dependent fluorescence signal without the need for spectral demultiplexing upon detection. We demonstrate detection of individual fluorescently labeled virus particles of three influenza A subtypes in two implementations: labeling of each virus using three different colors and two-color combinatorial labeling. By extending combinatorial multiplexing to three or more colors, MMI-based WDM provides the multiplexing power required for differentiated clinical tests and the growing field of personalized medicine. PMID:26438840

  9. Detection method for avian influenza viruses in water.

    PubMed

    Rönnqvist, Maria; Ziegler, Thedi; von Bonsdorff, Carl-Henrik; Maunula, Leena

    2012-03-01

    Recent events have shown that humans may become infected with some pathogenic avian influenza A viruses (AIV). Since soil and water, including lakes, rivers, and seashores, may be contaminated by AIV excreted by birds, effective methods are needed for monitoring water for emerging viruses. Combining water filtration with molecular methods such as PCR is a fast and effective way for detecting viruses. The objective of this study was to apply a convenient method for the detection of AIV in natural water samples. Distilled water and lake, river, and seawater were artificially contaminated with AIV (H5N3) and passed through a filter system. AIV was detected from filter membrane by real-time RT-PCR. The performance of Zetapor, SMWP, and Sartobind D5F membranes in recovering influenza viruses was first evaluated using contaminated distilled water. SWMP, which gave the highest virus recoveries, was then compared with a pre-filter combined GF/F filter membrane in a trial using natural water samples. In this study, the cellulose membrane SMWP was found to be practical for recovery of AIVs in water. Viral yields varied between 62.1 and 65.9% in distilled water and between 1 and 16.7% in natural water samples. The borosilicate glass membrane GF/F combined with pre-filter was also feasible in filtering natural water samples with viral yields from 1.98 to 7.33%. The methods described can be used for monitoring fresh and seawater samples for the presence of AIV and to determine the source of AIV transmission in an outbreak situation. PMID:23412765

  10. First evidence of canine distemper in Brazilian free-ranging felids.

    PubMed

    Nava, Alessandra Ferreira Dales; Cullen, Laury; Sana, Dênis Aléssio; Nardi, Marcello Schiavo; Filho, José Domingues Ramos; Lima, Thiago Ferraz; Abreu, Kauê Cachuba; Ferreira, Fernando

    2008-12-01

    Serum samples from 19 jaguars (Panthera onca), nine pumas (Puma concolor), and two ocelots (Leopardus pardalis) were collected between January 1999 and March of 2005 and tested for presence of canine distemper virus (CDV). All cats were free-ranging animals living in two protected areas in the Brazilian Atlantic Forest. In addition, 111 domestic dogs from nearby areas were sampled for CDV. Our results show the first evidence of CDV exposure in Brazilian free-ranging felids. From the 30 samples analyzed, six jaguars and one puma were tested seropositive for CDV. All seropositive large felids were from Ivinhema State Park, resulting in 31.5% of the sampled jaguars or 60% of the total jaguar population in Ivinhema State Park, and 11.28% of the sampled pumas. From the total 111 domestic dogs sampled, 45 were tested seropositive for CDV. At Morro do Diabo State Park, 34.6% of the dogs sampled were positive for CDV, and 100% at Ivinhema State Park. Canine distemper virus in wild felids seems to be related with home range use and in close association with domestic dogs living in nearby areas. PMID:19259737

  11. Bioluminescent detection probe for tick-borne encephalitis virus immunoassay.

    PubMed

    Burakova, Ludmila P; Kudryavtsev, Alexander N; Stepanyuk, Galina A; Baykov, Ivan K; Morozova, Vera V; Tikunova, Nina V; Dubova, Maria A; Lyapustin, Victor N; Yakimenko, Valeri V; Frank, Ludmila A

    2015-07-01

    To facilitate the detection of the tick-borne encephalitis virus (TBEV), the causative agent of one of the most severe human neuroinfections, we have developed an immunoassay based on bioluminescent hybrid protein 14D5a-Rm7 as a detection probe. The protein containing Renilla luciferase as a reporter and a single-chain variable fragment (scFv) of murine immunoglobulin to TBEV as a recognition element was constructed, produced by bacterial expression, purified, and tested. Both domains were shown to reveal their specific biological properties-affinity to the target antigen and bioluminescent activity. Hybrid protein was applied as a label for solid-phase immunoassay of the antigens, associated with the tick-borne encephalitis virus (native glycoprotein E or extracts of the infected strain of lab ticks). The assay demonstrates high sensitivity (0.056 ng of glycoprotein E; 10(4)-10(5) virus particles or 0.1 pg virions) and simplicity and is competitive with conventional methods for detection of TBEV. PMID:25925861

  12. Multiplex nested RT-PCR for detecting avian influenza virus, infectious bronchitis virus and Newcastle disease virus.

    PubMed

    Nguyen, Thanh Trung; Kwon, Hyuk-Joon; Kim, Il-Hwan; Hong, Seung-Min; Seong, Won-Jin; Jang, Jin-Wook; Kim, Jae-Hong

    2013-03-01

    In this study, multiplex nested RT-PCR (mnRT-PCR) was applied to simultaneous detect multiplex PCR with the higher sensitivity of nested PCR that is required for avian influenza, infectious bronchitis and Newcastle disease virus using two steps of amplification. For the first PCR, primers that were specific for each virus were newly designed from the nucleoprotein gene of AIV, the nucleocapsid protein gene of IBV and the fusion protein gene of NDV to amplify products of 665, 386 and 236 nucleotides, respectively. The multiplex PCR step provides mass amplification using common primers, which increased markedly the sensitivity of the test. Non-specific reactions were not observed when other viruses and bacteria were used for evaluating the mnRT-PCR. As a field application, 172 samples were tested by RT-PCR and mnRT-PCR. Among these samples, the concordance rates for mnRT-PCR and the single conventional RT-PCR showed 98.9% (kappa=0.98) and 98.8% (kappa=0.96) similarity for IBV and AIV, respectively. As a result, it is recommended the multiplex nested PCR as an effective tool for detecting and studying the molecular epidemiology of various mixed infections of one or more of these viruses in poultry. PMID:23261801

  13. 9 CFR 113.47 - Detection of extraneous viruses by the fluorescent antibody technique.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Detection of extraneous viruses by the... INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Standard Procedures § 113.47 Detection of extraneous viruses by...

  14. 9 CFR 113.47 - Detection of extraneous viruses by the fluorescent antibody technique.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Detection of extraneous viruses by the... INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Standard Procedures § 113.47 Detection of extraneous viruses by...

  15. Hendra virus detection using Loop-Mediated Isothermal Amplification.

    PubMed

    Foord, Adam J; Middleton, Deborah; Heine, Hans G

    2012-04-01

    Hendra virus (HeV) is a zoonotic paramyxovirus endemic in Australian Pteropus bats (fruit bats or flying foxes). Although bats appear to be unaffected by the virus, HeV can spread from fruit bats to horses, causing severe disease. Human infection results from close contact with the blood, body fluids and tissues of infected horses. HeV is a biosecurity level 4 (BSL-4) pathogen, with a high case-fatality rate in humans and horses. Current assays for HeV detection require complex instrumentation and are generally time consuming. The aim of this study was to develop a Loop-Mediated Isothermal Amplification (LAMP) assay to detect nucleic acid from all known HeV strains in horses without the requirement for complex laboratory equipment. A LAMP assay targeting a conserved region of the HeV P-gene was combined with a Lateral Flow Device (LFD) for detection of amplified product. All HeV isolates, the original HeV isolated in 1994 as well as the most recent isolates from 2011 were detected. Analytical sensitivity and specificity of the HeV-LAMP assay was equal to a TaqMan assay developed previously. Significantly, these assays detected HeV in horses before clinical signs were observed. The combined LAMP-LFD procedure is a sensitive method suitable for HeV diagnosis in a resource-limited situation or where rapid test results are critical. PMID:22327143

  16. Specific qPCR assays for the detection of orf virus, pseudocowpox virus and bovine papular stomatitis virus.

    PubMed

    Zhao, Hui; Wilkins, Kimberly; Damon, Inger K; Li, Yu

    2013-12-01

    The genus Parapoxvirus (PAPV) is comprised traditionally of orf virus (ORFV), pseudocowpox virus (PCPV) and bovine papular stomatitis virus (BPSV), which cause infections of ruminants and their handlers in the U.S. and worldwide. Unlike orthopoxvirus infections, which can cause systemic or localized infections, PAPV infections present normally as benign, self-limited and localized skin lesions; infections do not confer lifelong immunity. In recent years, related potentially to enhanced awareness and the availability of diagnostic methods, there has been an observed increase in reported cases of PAPV in animals and humans. This study describes TaqMan based real-time PCR assays for both generic and specific detection of PAPV species for surveillance and outbreak investigations. These assays target highly conserved PAPV RNA polymerase gene sequences and are capable of detecting three known species of PAPVs (ORFV, PCPV, and BPSV). The assays were evaluated using a panel of PAPV DNA derived from human infections or animal specimen remainders. The sensitivities of all four assays were determined using droplet digital PCR; fewer than 10 copies of clinical PAPV DNA can be detected consistently. These assays provide a reliable and sensitive method for rapid confirmation and characterization PAPV infections with varying clinical presentations. PMID:24035807

  17. Detection of RNA viruses: current technologies and future perspectives.

    PubMed

    Cella, Lakshmi N; Blackstock, Daniel; Yates, Marylynn A; Mulchandani, Ashok; Chen, Wilfred

    2013-01-01

    RNA viruses constitute one of the major classes of pathogenic organisms causing human diseases, with varying degrees of severity. This review summarizes the conventional and emerging technologies that are available for the detection of these organisms. Cell culture-based techniques for viral detection have been popular since their inception and continue to be the gold standard against which all other techniques. Over many years, these techniques have undergone some radical changes, reducing the total time needed for detection and improving sensitivity, although even with their reliability and improved features they are being slowly replaced by nucleic acid-based technologies. These molecular detection techniques have revolutionized the area of viral detection by their high sensitivity, selectivity, and short detection time. The majority of nucleic acid-based techniques depend on amplifying viral RNA; however, there are some newer emerging techniques that detect viral RNA in live cells using various configurations of florescent probes. In addition, nucleic acid-based technology has made it possible for multiviral detection with either multiplex polymerase chain reaction assays or microarrays. Every technique described in this review has its own unique abilities, making them indispensable for viral detection. However, we believe that nucleic acid-based technologies will find widespread use after being standardized, limiting other technologies to very specific uses. PMID:23582035

  18. Effect of recombinant canine distemper vaccine on antibody titers in previously vaccinated dogs.

    PubMed

    Larson, L J; Hageny, T L; Haase, C J; Schultz, R D

    2006-01-01

    Two canine distemper virus (CDV) vaccine types are currently commercially available: modified-live virus (MLV) vaccines and a canarypox recombinant CDV (rCDV) vaccine (Recombitek, Merial). This study compared the ability of the rCDV vaccine and MLV vaccines to significantly enhance (boost) the antibody response of previously immunized adult and juvenile dogs. A significant (fourfold or greater) increase in titer occurred in significantly more dogs revaccinated with Recombitek C-4 or Recombitek C-6 than with the MLV-CDV vaccines. This study demonstrates that Recombitek, the only vaccine for dogs containing rCDV, is more likely to significantly boost the CDV antibody response in previously vaccinated dogs than are the MLV-CDV vaccines. Because rCDV vaccine can boost the antibody titer of dogs previously vaccinated with an MLV vaccine, it can and should be used when core vaccines are readministered. PMID:16871492

  19. The Microbial Detection Array for Detection of Emerging Viruses in Clinical Samples - A Useful Panmicrobial Diagnostic Tool

    PubMed Central

    Rosenstierne, Maiken W.; McLoughlin, Kevin S.; Olesen, Majken Lindholm; Papa, Anna; Gardner, Shea N.; Engler, Olivier; Plumet, Sebastien; Mirazimi, Ali; Weidmann, Manfred; Niedrig, Matthias; Fomsgaard, Anders; Erlandsson, Lena

    2014-01-01

    Emerging viruses are usually endemic to tropical and sub-tropical regions of the world, but increased global travel, climate change and changes in lifestyle are believed to contribute to the spread of these viruses into new regions. Many of these viruses cause similar disease symptoms as other emerging viruses or common infections, making these unexpected pathogens difficult to diagnose. Broad-spectrum pathogen detection microarrays containing probes for all sequenced viruses and bacteria can provide rapid identification of viruses, guiding decisions about treatment and appropriate case management. We report a modified Whole Transcriptome Amplification (WTA) method that increases unbiased amplification, particular of RNA viruses. Using this modified WTA method, we tested the specificity and sensitivity of the Lawrence Livermore Microbial Detection Array (LLMDA) against a wide range of emerging viruses present in both non-clinical and clinical samples using two different microarray data analysis methods. PMID:24963710

  20. The microbial detection array for detection of emerging viruses in clinical samples--a useful panmicrobial diagnostic tool.

    PubMed

    Rosenstierne, Maiken W; McLoughlin, Kevin S; Olesen, Majken Lindholm; Papa, Anna; Gardner, Shea N; Engler, Olivier; Plumet, Sebastien; Mirazimi, Ali; Weidmann, Manfred; Niedrig, Matthias; Fomsgaard, Anders; Erlandsson, Lena

    2014-01-01

    Emerging viruses are usually endemic to tropical and sub-tropical regions of the world, but increased global travel, climate change and changes in lifestyle are believed to contribute to the spread of these viruses into new regions. Many of these viruses cause similar disease symptoms as other emerging viruses or common infections, making these unexpected pathogens difficult to diagnose. Broad-spectrum pathogen detection microarrays containing probes for all sequenced viruses and bacteria can provide rapid identification of viruses, guiding decisions about treatment and appropriate case management. We report a modified Whole Transcriptome Amplification (WTA) method that increases unbiased amplification, particular of RNA viruses. Using this modified WTA method, we tested the specificity and sensitivity of the Lawrence Livermore Microbial Detection Array (LLMDA) against a wide range of emerging viruses present in both non-clinical and clinical samples using two different microarray data analysis methods. PMID:24963710

  1. Detection of viruses and body fluids which may contain viruses in the domestic environment.

    PubMed

    Bellamy, K; Laban, K L; Barrett, K E; Talbot, D C

    1998-12-01

    The domestic environment was investigated for the presence of viruses and body fluids that may contain viruses. A range of surfaces in 39 homes (17 visited on 2 occasions) were sampled by swabbing and analysed using cell culture, reverse transcription polymerase chain reaction for enteroviral RNA, haemoglobin as a marker for blood, amylase as an indicator of urine, saliva and sweat, and protein as an indicator of general hygiene. Haemoglobin was found on 1.9% of surfaces sampled and of the positive samples 30% were from articles frequently handled. Amylase (> 5 U/l) was found in 29.3% of samples tested. Protein was found in 97.8% of samples tested. Enteroviral RNA, indicating the presence of virus, was detected in 3 out of 448 samples tested; they were from a tap handle, telephone handpiece and a toilet bowl. No viruses were isolated in cell culture, however significant problems were encountered with bacterial and fungal contamination. This work demonstrates that only testing environmental samples for bacteria and ATP may not give a total view of the microbiological problem in the home. A range of test methods is useful to gain a broad view of the problems of hygiene in the home and to allow comparative studies of specific areas such as the kitchen and bathroom. PMID:10030717

  2. Detection of viruses and body fluids which may contain viruses in the domestic environment.

    PubMed Central

    Bellamy, K.; Laban, K. L.; Barrett, K. E.; Talbot, D. C.

    1998-01-01

    The domestic environment was investigated for the presence of viruses and body fluids that may contain viruses. A range of surfaces in 39 homes (17 visited on 2 occasions) were sampled by swabbing and analysed using cell culture, reverse transcription polymerase chain reaction for enteroviral RNA, haemoglobin as a marker for blood, amylase as an indicator of urine, saliva and sweat, and protein as an indicator of general hygiene. Haemoglobin was found on 1.9% of surfaces sampled and of the positive samples 30% were from articles frequently handled. Amylase (> 5 U/l) was found in 29.3% of samples tested. Protein was found in 97.8% of samples tested. Enteroviral RNA, indicating the presence of virus, was detected in 3 out of 448 samples tested; they were from a tap handle, telephone handpiece and a toilet bowl. No viruses were isolated in cell culture, however significant problems were encountered with bacterial and fungal contamination. This work demonstrates that only testing environmental samples for bacteria and ATP may not give a total view of the microbiological problem in the home. A range of test methods is useful to gain a broad view of the problems of hygiene in the home and to allow comparative studies of specific areas such as the kitchen and bathroom. PMID:10030717

  3. Detection of sweet potato viruses in Yunnan and genetic diversity analysis of the common viruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two hundred seventy-nine samples with virus-like symptoms collected from 16 regions in Yunnan Province were tested by RT-PCR/PCR using virus-specific primers for 8 sweet potato viruses. Six viruses, Sweet potato chlorotic fleck virus (SPCFV), Sweet Potato feathery mottle virus (SPFMV), Sweet potato ...

  4. Nanoplasmonic Quantitative Detection of Intact Viruses from Unprocessed Whole Blood

    PubMed Central

    Inci, Fatih; Tokel, Onur; Wang, ShuQi; Gurkan, Umut Atakan; Tasoglu, Savas; Kuritzkes, Daniel R.; Demirci, Utkan

    2013-01-01

    Infectious diseases such as HIV and Hepatitis B infection pose an omnipresent threat to global health. Reliable, fast, accurate and sensitive platforms that can be deployed at the point-of-care (POC) in multiple settings, such as airports and offices for detection of infectious pathogens are essential for the management of epidemics and possible biological attacks. To the best of our knowledge, no viral load technology adaptable to the POC settings exists today due to critical technical and biological challenges. Here, we present for the first time a broadly applicable technology for quantitative, nanoplasmonic-based intact virus detection at clinically relevant concentrations. The sensing platform is based on unique nanoplasmonic properties of nanoparticles utilizing immobilized antibodies to selectively capture rapidly evolving viral subtypes. We demonstrate the capture, detection and quantification of multiple HIV subtypes (A, B, C, D, E, G, and subtype panel) with high repeatability, sensitivity and specificity down to 98 ± 39 copies/mL (i.e., subtype D) using spiked whole blood samples and clinical discarded HIV-infected patient whole blood samples validated by the gold standard, i.e., RT-qPCR. This platform technology offers an assay time of 1 hour and 10 minutes (1 hour for capture, 10 minutes for detection and data analysis). The presented platform is also able to capture intact viruses at high efficiency using immuno-surface chemistry approaches directly from whole blood samples without any sample preprocessing steps such as spin-down or sorting. Evidence is presented showing the system to be accurate, repeatable and reliable. Additionally, the presented platform technology can be broadly adapted to detect other pathogens having reasonably well-described biomarkers by adapting the surface chemistry. Thus, this broadly applicable detection platform holds great promise to be implemented potentially at POC settings, hospital and primary care settings. PMID

  5. Detection of hepatitis B virus infection: A systematic review

    PubMed Central

    Ghosh, Mallika; Nandi, Srijita; Dutta, Shrinwanti; Saha, Malay Kumar

    2015-01-01

    AIM: To review published methods for detection of hepatitis B virus (HBV) infection. METHODS: A thorough search on Medline database was conducted to find original articles describing different methods or techniques of detection of HBV, which are published in English in last 10 years. Articles outlining methods of detection of mutants or drug resistance were excluded. Full texts and abstracts (if full text not available) were reviewed thoroughly. Manual search of references of retrieved articles were also done. We extracted data on different samples and techniques of detection of HBV, their sensitivity (Sn), specificity (Sp) and applicability. RESULTS: A total of 72 studies were reviewed. HBV was detected from dried blood/plasma spots, hepatocytes, ovarian tissue, cerumen, saliva, parotid tissue, renal tissue, oocytes and embryos, cholangiocarcinoma tissue, etc. Sensitivity of dried blood spot for detecting HBV was > 90% in all the studies. In case of seronegative patients, HBV DNA or serological markers have been detected from hepatocytes or renal tissue in many instances. Enzyme linked immunosorbent assay and Chemiluminescent immunoassay (CLIA) are most commonly used serological tests for detection. CLIA systems are also used for quantitation. Molecular techniques are used qualitatively as well as for quantitative detection. Among the molecular techniques version 2.0 of the CobasAmpliprep/CobasTaqMan assay and Abbott’s real time polymerase chain reaction kit were found to be most sensitive with a lower detection limit of only 6.25 IU/mL and 1.48 IU/mL respectively. CONCLUSION: Serological and molecular assays are predominant and reliable methods for HBV detection. Automated systems are highly sensitive and quantify HBV DNA and serological markers for monitoring. PMID:26483870

  6. Detection of dengue group viruses by fluorescence in situ hybridization

    PubMed Central

    2012-01-01

    Background Dengue fever (DF) and dengue hemorrhagic fever (DHF) represent a global challenge in public health. It is estimated that 50 to 100 million infections occur each year causing approximately 20,000 deaths that are usually linked to severe cases like DHF and dengue shock syndrome. The causative agent of DF is dengue virus (genus Flavivirus) that comprises four distinct serotypes (DENV-1 to DENV-4). Fluorescence in situ hybridization (FISH) has been used successfully to detect pathogenic agents, but has not been implemented in detecting DENV. To improve our understanding of DENV infection and dissemination in host tissues, we designed specific probes to detect DENV in FISH assays. Methods Oligonucleotide probes were designed to hybridize with RNA from the broadest range of DENV isolates belonging to the four serotypes, but not to the closest Flavivirus genomes. Three probes that fit the criteria defined for FISH experiments were selected, targeting both coding and non-coding regions of the DENV genome. These probes were tested in FISH assays against the dengue vector Aedes albopictus (Diptera: Culicidae). The FISH experiments were led in vitro using the C6/36 cell line, and in vivo against dissected salivary glands, with epifluorescence and confocal microscopy. Results The three 60-nt oligonucleotides probes DENV-Probe A, B and C cover a broad range of DENV isolates from the four serotypes. When the three probes were used together, specific fluorescent signals were observed in C6/36 infected with each DENV serotypes. No signal was detected in either cells infected with close Flavivirus members West Nile virus or yellow fever virus. The same protocol was used on salivary glands of Ae. albopictus fed with a DENV-2 infectious blood-meal which showed positive signals in the lateral lobes of infected samples, with no significant signal in uninfected mosquitoes. Conclusion Based on the FISH technique, we propose a way to design and use oligonucleotide probes to

  7. Design of different strategies of multivalent DNA-based vaccination against rabies and canine distemper in mice and dogs

    PubMed Central

    2012-01-01

    Background During the vaccination campaigns, puppies younger than 3 months old are not targeted and remain unvaccinated for at least the first year of their lives. Almost half of the reported rabid dogs are 6 months or younger. Hence, we should recommend the vaccination against rabies of young puppies. Unfortunately, owing to the exposure of puppies to infections with either canine parvovirus (CPV) or distemper virus (CDV) after the intervention of the vaccinators, owners are reluctant to vaccinate puppies against rabies. Therefore, it is necessary to include the CPV and CDV valences in the vaccine against rabies. Multivalent DNA-based vaccination in dogs, including rabies and distemper valences, could help in raising vaccine coverage. Methods We have designed monovalent and multivalent DNA-based vaccine candidates for in vitro and in vivo assays. These plasmids encode to the rabies virus glycoprotein and/or the canine distemper virus hemagglutinin. The first strategy of multivalent DNA-based vaccination is by mixing plasmids encoding to a single antigen each. The second is by simply fusing the genes of the antigens together. The third is by adding the foot and mouth disease virus (FMDV) 2A oligopeptide gene into the antigen genes. The last strategy is by the design and use of a bicistronic plasmid with an “Internal Ribosome Entry Site” (IRES) domain. Results The monovalent construct against canine distemper was efficiently validated by inducing higher humoral immune responses compared to cell-culture-derived vaccine both in mice and dogs. All multivalent plasmids efficiently expressed both valences after in vitro transfection of BHK-21 cells. In BALB/c mice, the bicistronic IRES-dependant construct was the most efficient inducer of virus-neutralizing antibodies against both valences. It was able to induce better humoral immune responses compared to the administration of either cell-culture-derived vaccines or monovalent plasmids. The FMDV 2A was also efficient

  8. Insect-specific flaviviruses, a worldwide widespread group of viruses only detected in insects.

    PubMed

    Calzolari, Mattia; Zé-Zé, Líbia; Vázquez, Ana; Sánchez Seco, Mari Paz; Amaro, Fátima; Dottori, Michele

    2016-06-01

    Several flaviviruses are important pathogens for humans and animals (Dengue viruses, Japanese encephalitis virus, Yellow-fever virus, Tick-borne encephalitis virus, West Nile virus). In recent years, numerous novel and related flaviviruses without known pathogenic capacity have been isolated worldwide in the natural mosquito population. However, phylogenetic studies have shown that genomic sequences of these viruses diverge from other flaviviruses. Moreover, these viruses seem to be exclusive of insects (they do not seem to grow on vertebrate cell lines), and were already defined as mosquito-only flaviviruses or insect-specific flaviviruses. At least eleven of these viruses were isolated worldwide, and sequences ascribable to other eleven putative viruses were detected in several mosquito species. A large part of the cycle of these viruses is not well known, and their persistence in the environment is poorly understood. These viruses are detected in a wide variety of distinct mosquito species and also in sandflies and chironomids worldwide; a single virus, or the genetic material ascribable to a virus, was detected in several mosquito species in different countries, often in different continents. Furthermore, some of these viruses are carried by invasive mosquitoes, and do not seem to have a depressive action on their fitness. The global distribution and the continuous detection of new viruses in this group point out the likely underestimation of their number, and raise interesting issues about their possible interactions with the pathogenic flaviviruses, and their influence on the bionomics of arthropod hosts. Some enigmatic features, as their integration in the mosquito genome, the recognition of their genetic material in DNA forms in field-collected mosquitoes, or the detection of the same virus in both mosquitoes and sandflies, indicate that the cycle of these viruses has unknown characteristics that could be of use to reach a deeper understanding of the cycle

  9. Rapid Detection of Herpes Viruses for Clinical Applications

    NASA Technical Reports Server (NTRS)

    Pierson, Duane; Mehta, Satish

    2013-01-01

    There are eight herpes viruses that infect humans, causing a wide range of diseases resulting in considerable morbidity and associated costs. Varicella zoster virus (VZV) is a human herpes virus that causes chickenpox in children and shingles in adults. Approximately 1,000,000 new cases of shingles occur each year; post-herpetic neuralgia (PHN) follows shingles in 100,000 to 200,000 people annually. PHN is characterized by debilitating, nearly unbearable pain for weeks, months, and even years. The onset of shingles is characterized by pain, followed by the zoster rash, leading to blisters and severe pain. The problem is that in the early stages, shingles can be difficult to diagnose; chickenpox in adults can be equally difficult to diagnose. As a result, both diseases can be misdiagnosed (false positive/negative). A molecular assay has been adapted for use in diagnosing VZV diseases. The polymerase chain reaction (PCR) assay is a non-invasive, rapid, sensitive, and highly specific method for VZV DNA detection. It provides unequivocal results and can effectively end misdiagnoses. This is an approximately two-hour assay that allows unequivocal diagnosis and rapid antiviral drug intervention. It has been demonstrated that rapid intervention can prevent full development of the disease, resulting in reduced likelihood of PHN. The technology was extended to shingles patients and demonstrated that VZV is shed in saliva and blood of all shingles patients. The amount of VZV in saliva parallels the medical outcome.

  10. Rapid detection of pseudorabies virus by the shell vial technique.

    PubMed

    Tahir, R A; Goyal, S M

    1995-04-01

    Conventional 24-well microtiter plates and shell vials were seeded with pig kidney (PK-15) and bovine turbinate (BT) cells. The monolayers were inoculated with 244 clinical specimens from pigs suspected of having pseudorabies virus (PRV) infection. The results of a shell vial assay (SVA) were compared with those obtained in a 24-well plate cell culture assay in terms of sensitivity and speed of virus isolation. All samples were passaged only once in cell cultures in both assays. Samples producing cytopathic effects (cpe) in 1 or both assay systems and showing positive fluorescence in a direct fluorescent antibody assay were considered to be positive for PRV. Of the 244 samples examined, 118 (48.4%) and 121 (49.2%) were positive by the 24-well plate assay and SVA, respectively. Of the 118 samples positive in 24-well plates, 113 (95.8%) were positive in BT cells and 117 (99.2%) were positive in PK-15 cells. The SVA detected 121 positive samples of which 121 (100%) were positive in PK-15 cells and 113 (93.4%) were positive in BT cells. Virus-specific cpe appeared earlier in the SVA than in the 24-well assay. At 24 hours postinoculation, 91 (75.2%) samples were cpe positive by SVA, whereas only 15 (12.7%) were positive in 24-well plates. All but 2 of the 121 (98.3%) SVA-positive samples were positive within 48 hours postinoculation, whereas only 56 of 118 (47.5%) were positive in 24-well plates during the same time period. These results indicate that the SVA is comparable in sensitivity to 24-well plate assay but yields virus isolation results more quickly. Also, PK-15 cells appeared to be more sensitive than BT cells for PRV isolation. PMID:7619897

  11. West Nile virus detection in nonvascular feathers from avian carcasses.

    PubMed

    Nemeth, Nicole M; Young, Ginger R; Burkhalter, Kristen L; Brault, Aaron C; Reisen, William K; Komar, Nicholas

    2009-09-01

    West Nile virus (WNV) is a public health threat and has caused the death of thousands of North American birds. As such, surveillance for WNV has been ongoing, utilizing numerous biological specimens and testing methods. Nonvascular (i.e., fully grown) feathers would provide a simple method of collection from either dead or live birds of all ages and molt cycles, with presumably less biosafety risk compared with other specimen types, including feather pulp. The current study evaluates WNV detection in nonvascular feathers removed from naturally infected avian carcasses of several species groups. Feathers of corvid passeriforms had the highest sensitivity of detection (64%), followed by noncorvid passeriforms (43%), columbiforms (33%), and falconiforms (31%). Storing feathers for 1 year at -20 degrees C or at ambient room temperature resulted in detection rates of infectious WNV of 16% and zero, respectively, but had no effect on detection rates of WNV RNA in a subset of matched feather pairs (47% for both storage temperatures). The efficacy of WNV detection in nonvascular feathers is greatly enhanced by testing multiple feathers. The advantages of using nonvascular feathers over other tissues may outweigh the relatively low detectability of WNV RNA in certain situations such as remote areas lacking resources for acquiring other types of samples or maintaining the cold chain. PMID:19737756

  12. ESTABLISH AND STANDARDIZE METHODOLOGY FOR DETECTION OF WATERBORNE VIRUSES FROM HUMAN SOURCES

    EPA Science Inventory

    Research is conducted to develop and standardize methods to detect and measure occurrence of human enteric viruses that cause waterborne disease. The viruses of concern include the emerging pathogens--hepatitis E virus and group B rotaviruses. Also of concern are the coxsackiev...

  13. IMPROVED DETECTION OF HUMAN ENTERIC VIRUSES IN FOODS BY RT-PCR. (R826139)

    EPA Science Inventory

    Human enteric viruses (including hepatitis A virus (HAV) and Norwalk-like viruses (NLVs)) are now recognized as common causes of foodborne disease. While methods to detect these agents in clinical specimens have improved significantly over the last 10 years, applications to fo...

  14. Rapid and quantitative detection of hepatitis B virus

    PubMed Central

    Liu, Yue-Ping; Yao, Chun-Yan

    2015-01-01

    Despite availability of a universal vaccine, hepatitis B virus (HBV) infection has a huge impact on public health worldwide. Accurate and timely diagnosis of HBV infection is needed. Rapid developments have been made in the diagnostic and monitoring methods for HBV infection, including serological and molecular assays. In clinical practice, qualitative hepatitis B surface antigen (HBsAg) testing has long served as a diagnostic marker for individuals infected with HBV. More recently, HBsAg level has been used to predict treatment outcome when determined early during treatment or at baseline. However, identification of HBV DNA positive cases that do not have detectable HBsAg has encouraged the application of molecular tests. Hence, combination of quantitative detection of HBV DNA and HBsAg can be used to discriminate patients during the course of HBV infection and to monitor therapy. This article reviews the most commonly used quantitative methods for HBsAg and HBV DNA. PMID:26576084

  15. Antibody Derived Peptides for Detection of Ebola Virus Glycoprotein

    PubMed Central

    López-Pacheco, Felipe; Pérez-Chavarría, Roberto; González-Vázquez, Juan Carlos; González-González, Everardo; Trujillo-de Santiago, Grissel; Ponce-Ponce de León, César Alejandro; Zhang, Yu Shrike; Dokmeci, Mehmet Remzi; Khademhosseini, Ali; Alvarez, Mario Moisés

    2015-01-01

    Background Current Ebola virus (EBOV) detection methods are costly and impractical for epidemic scenarios. Different immune-based assays have been reported for the detection and quantification of Ebola virus (EBOV) proteins. In particular, several monoclonal antibodies (mAbs) have been described that bind the capsid glycoprotein (GP) of EBOV GP. However, the currently available platforms for the design and production of full-length mAbs are cumbersome and costly. The use of antibody fragments, rather than full-length antibodies, might represent a cost-effective alternative for the development of diagnostic and possibly even therapeutic alternatives for EBOV. Methods/Principal Findings We report the design and expression of three recombinant anti-GP mAb fragments in Escherichia coli cultures. These fragments contained the heavy and light variable portions of the three well-studied anti-GP full-length mAbs 13C6, 13F6, and KZ52, and are consequently named scFv-13C6, scFv-13F6, and Fab-KZ52, respectively. All three fragments exhibited specific anti-GP binding activity in ELISA experiments comparable to that of full-length anti-GP antibodies (i.e., the same order of magnitude) and they are easily and economically produced in bacterial cultures. Conclusion/Significance Antibody fragments might represent a useful, effective, and low cost alternative to full-length antibodies in Ebola related capture and diagnostics applications. PMID:26489048

  16. Detection and differentiation of Newcastle disease virus and influenza virus by using duplex real-time PCR.

    PubMed

    Nidzworski, Dawid; Wasilewska, Edyta; Smietanka, Krzysztof; Szewczyk, Bogusław; Minta, Zenon

    2013-01-01

    Newcastle disease virus (NDV), member of the Paramyxoviridae family and avian influenza virus (AIV), member of the Orthomyxoviridae family, are two main avian pathogens causing serious economic problems in poultry health. Both are enveloped, single-stranded, negative-sense RNA viruses and cause similar symptoms, ranging from sub-clinical infections to severe diseases, including decrease in egg production, acute respiratory syndrome, and high mortality. Similar symptoms hinder the differentiation of infection with the two viruses by standard veterinary procedures like clinical examination or necropsy. To overcome this problem, we have developed a new duplex real-time PCR assay for the detection and differentiation of these two viruses. Eighteen NDV strains, fourteen AIV strains, and twelve other (negative control) strains viruses were isolated from allantoic fluids of specific pathogen-free (SPF), embryonated eggs. Four-weeks-old SPF chickens were co-infected with both viruses (NDV - LaSota and AIV - H7N1). Swabs from cloaca and trachea were collected and examined. The results obtained in this study show that by using duplex real-time PCR, it was possible to detect and distinguish both viruses within less than three hours and with high sensitivity, even in case a bird was co-infected. Additionally, the results show the applicability of the real-time PCR assay in laboratory practice for the identification and differentiation of Newcastle disease and influenza A viruses in birds. PMID:24040628

  17. Detection of West Nile virus in mosquitoes by RT-PCR.

    PubMed

    Hadfield, T L; Turell, M; Dempsey, M P; David, J; Park, E J

    2001-06-01

    A reverse transcriptase-polymerase chain reaction (RT-PCR) assay employing detection technology was developed to identify West Nile virus in experimentally infected mosquitoes. The specificity of the assay was evaluated with the following viruses: eastern equine encephalitis, Ilheus, West Nile and yellow fever viruses. The limits of detection were determined using West Nile viral RNA extracted from serial dilutions of virus culture in infected mosquitoes. Limit of detection was 5 PFU from extracted mosquitoes. We were able to detect the presence of one infected mosquito in a pool of 50 repeatedly. When the RT-PCR was used with coded samples of intrathoracically-infected and uninfected mosquitoes, the assay detected the virus in all infected mosquitoes. Analytic sensitivity and specificity were 100%. This assay offers an efficient and rapid method of identifying West Nile virus in infected mosquitoes or cell culture. PMID:11352595

  18. A novel multiplex isothermal amplification method for rapid detection and identification of viruses

    PubMed Central

    Nyan, Dougbeh-Chris; Swinson, Kevin L.

    2015-01-01

    A rapid multiplex isothermal amplification assay has been developed for detection and identification of multiple blood-borne viruses that infect millions of people world-wide. These infections may lead to chronic diseases or death if not diagnosed and treated in a timely manner. Sets of virus-specific oligonucleotides and oligofluorophores were designed and used in a reverse-transcription loop-mediated multiplexed isothermal amplification reaction for detection and gel electrophoretic identification of human Immunodeficiency virus (HIV), hepatitis-B virus (HBV), hepatitis-C virus (HCV), hepatitis-E virus (HEV), dengue virus (DENV), and West Nile (WNV) virus infection in blood plasma. Amplification was catalyzed with two thermostable enzymes for 30–60 minutes under isothermal condition, utilizing a simple digital heat source. Electrophoretic analysis of amplified products demonstrated simultaneous detection of 6 viruses that were distinctly identified by unique ladder-like banding patterns. Naked-eye fluorescent visualization of amplicons revealed intensely fluorescing products that indicated positive detection. The test demonstrated a 97% sensitivity and a 100% specificity, with no cross-reaction with other viruses observed. This portable detection tool may have clinical and field utility in the developing and developed world settings. This may enable rapid diagnosis and identification of viruses for targeted therapeutic intervention and prevention of disease transmission. PMID:26643761

  19. Optimizing a custom tiling microarray for low input detection and identification of unamplified virus targets.

    PubMed

    Yu, Christine; Wales, Samantha Q; Mammel, Mark K; Hida, Kaoru; Kulka, Michael

    2016-08-01

    Viruses are major pathogens causing foodborne illnesses and are often present at low levels in foods, thus requiring sensitive techniques for their detection in contaminated foods. The lack of efficient culture methods for many foodborne viruses and the potential for multi-species viral contamination have driven investigation toward non-amplification based methods for virus detection and identification. A custom DNA microarray (FDA_EVIR) was assessed for its sensitivity in the detection and identification of low-input virus targets, human hepatitis A virus, norovirus, and coxsackievirus, individually and in combination. Modifications to sample processing were made to accommodate low input levels of unamplified virus targets, which included addition of carrier cDNA, RNase treatment, and optimization of DNase I-mediated target fragmentation. Amplification-free detection and identification of foodborne viruses were achieved in the range of 250-500 copies of virus RNA. Alternative data analysis methods were employed to distinguish the genotypes of the viruses particularly at lower levels of target input and the single probe-based analysis approach made it possible to identify a minority species in a multi-virus complex. The oligonucleotide array is shown to be a promising platform to detect foodborne viruses at low levels close to what are anticipated in food or environmental samples. PMID:27033182

  20. F F{sub 1}-ATPase as biosensor to detect single virus

    SciTech Connect

    Liu, XiaoLong; Zhang, Yun; Yue, JiaChang . E-mail: yuejc@sun5.ibp.ac.cn; Jiang, PeiDong; Zhang, ZhenXi

    2006-04-21

    F F{sub 1}-ATPase within chromatophore was constructed as a biosensor (immuno-rotary biosensor) for the purpose of capturing single virus. Capture of virus was based on antibody-antigen reaction. The detection of virus based on proton flux change driven by ATP-synthesis of F F{sub 1}-ATPase, which was indicated by F1300, was directly observed by a fluorescence microscope. The results demonstrate that the biosensor loading of virus particles has remarkable signal-to-noise ratio (3.8:1) compared to its control at single molecular level, and will be convenient, quick, and even super-sensitive for detecting virus particles.

  1. Rapid detection of hendra virus using magnetic particles and quantum dots.

    PubMed

    Lisi, Fabio; Falcaro, Paolo; Buso, Dario; Hill, Anita J; Barr, Jennifer A; Crameri, Gary; Nguyen, Tich-Lam; Wang, Lin-Fa; Mulvaney, Paul

    2012-09-01

    A proof-of-concept for the development of a fast and portable Hendra virus biosensor is presented. Hendra virus, a deadly emerging pathogen in Australia, can be co-localized, concentrated and revealed using simultaneously magnetic and luminescent functional particles. This method should be applicable for the early detection of any other virus by targeting the specific virus with the corresponding antibody. PMID:23184798

  2. Charge detection mass spectrometry: Instrumentation & applications to viruses

    NASA Astrophysics Data System (ADS)

    Pierson, Elizabeth E.

    For over three decades, electrospray ionization (ESI) has been used to ionize non-covalent complexes and subsequently transfer the intact ion into the gas phase for mass spectrometry (MS) analysis. ESI generates a distribution of multiple charged ions, resulting in an m/z spectrum comprised of a series of peaks, known as a charge state envelope. To obtain mass information, the number of charges for each peak must be deduced. For smaller biological analytes like peptides, the charge states are sufficiently resolved and this process is straightforward. For macromolecular complexes exceeding ~100 kDa, this process is complicated by the broadening and shifting of charge states due to incomplete desolvation, salt adduction, and inherent mass heterogeneity. As the analyte mass approaches the MDa regime, the m/z spectrum is often comprised of a broad distribution of unresolved charge states. In such cases, mass determination is precluded. Charge detection mass spectrometry (CDMS) is an emerging MS technique for determining the masses of heterogeneous, macromolecular complexes. In CDMS, the m/z and z of single ions are measured concurrently so that mass is easily calculated. With this approach, deconvolution of an m/z spectrum is unnecessary. This measurement is carried out by passing macroions through a conductive cylinder. The induced image charge on the cylindrical detector provides information about m/z and z: the m/z is related to its time-of-flight through the detector, and the z is related to the intensity of the image charge. We have applied CDMS to study the self-assembly of virus capsids. Late-stage intermediates in the assembly of hepatitis B virus, a devastating human pathogen, have been identified. This is the first time that such intermediates have been detected and represent a significant advancement towards understanding virus capsid assembly. CDMS has also been used to identify oversized, non-icosahedral polymorphs in the assembly of woodchuck hepatitis

  3. Rapid Detection and Quantification of RNA of Ebola and Marburg Viruses, Lassa Virus, Crimean-Congo Hemorrhagic Fever Virus, Rift Valley Fever Virus, Dengue Virus, and Yellow Fever Virus by Real-Time Reverse Transcription-PCR

    PubMed Central

    Drosten, Christian; Göttig, Stephan; Schilling, Stefan; Asper, Marcel; Panning, Marcus; Schmitz, Herbert; Günther, Stephan

    2002-01-01

    Viral hemorrhagic fevers (VHFs) are acute infections with high case fatality rates. Important VHF agents are Ebola and Marburg viruses (MBGV/EBOV), Lassa virus (LASV), Crimean-Congo hemorrhagic fever virus (CCHFV), Rift Valley fever virus (RVFV), dengue virus (DENV), and yellow fever virus (YFV). VHFs are clinically difficult to diagnose and to distinguish; a rapid and reliable laboratory diagnosis is required in suspected cases. We have established six one-step, real-time reverse transcription-PCR assays for these pathogens based on the Superscript reverse transcriptase-Platinum Taq polymerase enzyme mixture. Novel primers and/or 5′-nuclease detection probes were designed for RVFV, DENV, YFV, and CCHFV by using the latest DNA database entries. PCR products were detected in real time on a LightCycler instrument by using 5′-nuclease technology (RVFV, DENV, and YFV) or SybrGreen dye intercalation (MBGV/EBOV, LASV, and CCHFV). The inhibitory effect of SybrGreen on reverse transcription was overcome by initial immobilization of the dye in the reaction capillaries. Universal cycling conditions for SybrGreen and 5′-nuclease probe detection were established. Thus, up to three assays could be performed in parallel, facilitating rapid testing for several pathogens. All assays were thoroughly optimized and validated in terms of analytical sensitivity by using in vitro-transcribed RNA. The ≥95% detection limits as determined by probit regression analysis ranged from 1,545 to 2,835 viral genome equivalents/ml of serum (8.6 to 16 RNA copies per assay). The suitability of the assays was exemplified by detection and quantification of viral RNA in serum samples of VHF patients. PMID:12089242

  4. Vy-PER: eliminating false positive detection of virus integration events in next generation sequencing data

    PubMed Central

    Forster, Michael; Szymczak, Silke; Ellinghaus, David; Hemmrich, Georg; Rühlemann, Malte; Kraemer, Lars; Mucha, Sören; Wienbrandt, Lars; Stanulla, Martin; Franke, Andre

    2015-01-01

    Several pathogenic viruses such as hepatitis B and human immunodeficiency viruses may integrate into the host genome. These virus/host integrations are detectable using paired-end next generation sequencing. However, the low number of expected true virus integrations may be difficult to distinguish from the noise of many false positive candidates. Here, we propose a novel filtering approach that increases specificity without compromising sensitivity for virus/host chimera detection. Our detection pipeline termed Vy-PER (Virus integration detection bY Paired End Reads) outperforms existing similar tools in speed and accuracy. We analysed whole genome data from childhood acute lymphoblastic leukemia (ALL), which is characterised by genomic rearrangements and usually associated with radiation exposure. This analysis was motivated by the recently reported virus integrations at genomic rearrangement sites and association with chromosomal instability in liver cancer. However, as expected, our analysis of 20 tumour and matched germline genomes from ALL patients finds no significant evidence for integrations by known viruses. Nevertheless, our method eliminates 12,800 false positives per genome (80× coverage) and only our method detects singleton human-phiX174-chimeras caused by optical errors of the Illumina HiSeq platform. This high accuracy is useful for detecting low virus integration levels as well as non-integrated viruses. PMID:26166306

  5. Vy-PER: eliminating false positive detection of virus integration events in next generation sequencing data.

    PubMed

    Forster, Michael; Szymczak, Silke; Ellinghaus, David; Hemmrich, Georg; Rühlemann, Malte; Kraemer, Lars; Mucha, Sören; Wienbrandt, Lars; Stanulla, Martin; Franke, Andre

    2015-01-01

    Several pathogenic viruses such as hepatitis B and human immunodeficiency viruses may integrate into the host genome. These virus/host integrations are detectable using paired-end next generation sequencing. However, the low number of expected true virus integrations may be difficult to distinguish from the noise of many false positive candidates. Here, we propose a novel filtering approach that increases specificity without compromising sensitivity for virus/host chimera detection. Our detection pipeline termed Vy-PER (Virus integration detection bY Paired End Reads) outperforms existing similar tools in speed and accuracy. We analysed whole genome data from childhood acute lymphoblastic leukemia (ALL), which is characterised by genomic rearrangements and usually associated with radiation exposure. This analysis was motivated by the recently reported virus integrations at genomic rearrangement sites and association with chromosomal instability in liver cancer. However, as expected, our analysis of 20 tumour and matched germline genomes from ALL patients finds no significant evidence for integrations by known viruses. Nevertheless, our method eliminates 12,800 false positives per genome (80× coverage) and only our method detects singleton human-phiX174-chimeras caused by optical errors of the Illumina HiSeq platform. This high accuracy is useful for detecting low virus integration levels as well as non-integrated viruses. PMID:26166306

  6. Distemper in raccoons and foxes suspected of having rabies

    USGS Publications Warehouse

    Habermann, R.T.; Herman, C.M.; Williams, F.P., Jr.

    1958-01-01

    1) Twenty-one raccoons and 3 red foxes were collected from areas where suspected rabies occurred. All were found to be nonrabid. 2) Distemper was diagnosed in 14 of the 21 raccoons by demonstrating intracytoplasmic and intranuclear inclusions in the brain and visceral tissues. Two of the 3 foxes were considered to have distemper; the clinical signs were typical and mouse inoculation tests were negative for rabies. 3) Deaths of the other 7 raccoons were attributed to: leishmaniasis 1, gastritis 1, bronchopneumonia 1, parasitism 2, car injury 1; 1 showed no significant lesions. The death of 1 fox was attributed to parasitism. 4) Distemper may be a frequent cause of death in raccoons and foxes, in epizootics which simulate rabies.

  7. Accumulation of Extracellular Matrix in Advanced Lesions of Canine Distemper Demyelinating Encephalitis.

    PubMed

    Seehusen, Frauke; Al-Azreg, Seham A; Raddatz, Barbara B; Haist, Verena; Puff, Christina; Spitzbarth, Ingo; Ulrich, Reiner; Baumgärtner, Wolfgang

    2016-01-01

    In demyelinating diseases, changes in the quality and quantity of the extracellular matrix (ECM) may contribute to demyelination and failure of myelin repair and axonal sprouting, especially in chronic lesions. To characterize changes in the ECM in canine distemper demyelinating leukoencephalitis (DL), histochemical and immunohistochemical investigations of formalin-fixed paraffin-embedded cerebella using azan, picrosirius red and Gomori`s silver stain as well as antibodies directed against aggrecan, type I and IV collagen, fibronectin, laminin and phosphacan showed alterations of the ECM in CDV-infected dogs. A significantly increased amount of aggrecan was detected in early and late white matter lesions. In addition, the positive signal for collagens I and IV as well as fibronectin was significantly increased in late lesions. Conversely, the expression of phosphacan was significantly decreased in early and more pronounced in late lesions compared to controls. Furthermore, a set of genes involved in ECM was extracted from a publically available microarray data set and was analyzed for differential gene expression. Gene expression of ECM molecules, their biosynthesis pathways, and pro-fibrotic factors was mildly up-regulated whereas expression of matrix remodeling enzymes was up-regulated to a relatively higher extent. Summarized, the observed findings indicate that changes in the quality and content of ECM molecules represent important, mainly post-transcriptional features in advanced canine distemper lesions. Considering the insufficiency of morphological regeneration in chronic distemper lesions, the accumulated ECM seems to play a crucial role upon regenerative processes and may explain the relatively small regenerative potential in late stages of this disease. PMID:27441688

  8. Accumulation of Extracellular Matrix in Advanced Lesions of Canine Distemper Demyelinating Encephalitis

    PubMed Central

    Seehusen, Frauke; Al-Azreg, Seham A.; Raddatz, Barbara B.; Haist, Verena; Puff, Christina; Spitzbarth, Ingo; Ulrich, Reiner; Baumgärtner, Wolfgang

    2016-01-01

    In demyelinating diseases, changes in the quality and quantity of the extracellular matrix (ECM) may contribute to demyelination and failure of myelin repair and axonal sprouting, especially in chronic lesions. To characterize changes in the ECM in canine distemper demyelinating leukoencephalitis (DL), histochemical and immunohistochemical investigations of formalin-fixed paraffin-embedded cerebella using azan, picrosirius red and Gomori`s silver stain as well as antibodies directed against aggrecan, type I and IV collagen, fibronectin, laminin and phosphacan showed alterations of the ECM in CDV-infected dogs. A significantly increased amount of aggrecan was detected in early and late white matter lesions. In addition, the positive signal for collagens I and IV as well as fibronectin was significantly increased in late lesions. Conversely, the expression of phosphacan was significantly decreased in early and more pronounced in late lesions compared to controls. Furthermore, a set of genes involved in ECM was extracted from a publically available microarray data set and was analyzed for differential gene expression. Gene expression of ECM molecules, their biosynthesis pathways, and pro-fibrotic factors was mildly up-regulated whereas expression of matrix remodeling enzymes was up-regulated to a relatively higher extent. Summarized, the observed findings indicate that changes in the quality and content of ECM molecules represent important, mainly post-transcriptional features in advanced canine distemper lesions. Considering the insufficiency of morphological regeneration in chronic distemper lesions, the accumulated ECM seems to play a crucial role upon regenerative processes and may explain the relatively small regenerative potential in late stages of this disease. PMID:27441688

  9. High-throughput detection method for influenza virus.

    PubMed

    Kumar, Pawan; Bartoszek, Allison E; Moran, Thomas M; Gorski, Jack; Bhattacharyya, Sanjib; Navidad, Jose F; Thakar, Monica S; Malarkannan, Subramaniam

    2012-01-01

    Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture (1,2). Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus (3) to test the efficacy of this technique using MDCK cells (4). MDCK cells (10(4) or 5 x 10(3) per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 (5) and hemagglutinin (1) are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 10(2)-10(5) PFU could be consistently observed. Minimal but detectable positivity consistently seen with 10(2)-10(3) PFU PR8 viral titers demonstrated the high

  10. High-throughput Detection Method for Influenza Virus

    PubMed Central

    Kumar, Pawan; Bartoszek, Allison E.; Moran, Thomas M.; Gorski, Jack; Bhattacharyya, Sanjib; Navidad, Jose F.; Thakar, Monica S.; Malarkannan, Subramaniam

    2012-01-01

    Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2. Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near

  11. Nanoscale optofluidic sensor arrays for Dengue virus detection

    NASA Astrophysics Data System (ADS)

    Mandal, Sudeep; Akhmechet, Roman; Chen, Likun; Nugen, Sam; Baeumner, Antje; Erickson, David

    2007-09-01

    Here we present our work towards the development of Nanoscale Optofluidic Sensor Arrays (NOSA), which is an optofluidic architecture for performing label free, highly parallel, detections of biomolecular interactions. The approach is based on the use of optically resonant devices whose resonant wavelength is shifted due to a local change in refractive index caused by a positive binding event between a surface bound molecule and it solution phase target. A special two stage micro-/nanofluidics architecture is used to first functionalize the devices and then to deliver the targets. Two variants of the NOSA will be presented here. The first approach utilizes a 1D resonant cavity in a 1D silicon-on-insulator (SOI) waveguide with a unique differential size functionalization approach. This approach allows binding events at one or at a combination of the many sensing sites which causes a unique shift in the output resonator spectrum. The latter approach consists of a SOI waveguide evanescently coupled to multiple 1-D photonic crystal resonators of different sizes along the length, each of which is functionalized with a different oligonucleotide probe. These devices have an extremely low limit of detection and are compatible with aqueous environments. The primary advantage of these devices over existing technology is that it combines the sensitivity (limit of detection) of nanosensor technology with the parallelism of the microarray type format. Our initial application is in the detection of viral RNA of Dengue virus.

  12. Detection of Persistent Chikungunya Virus RNA but not Infectious Virus in Experimental Vertical Transmission in Aedes aegypti from Malaysia.

    PubMed

    Wong, Hui Vern; Vythilingam, Indra; Sulaiman, Wan Yusof Wan; Lulla, Aleksei; Merits, Andres; Chan, Yoke Fun; Sam, I-Ching

    2016-01-01

    Vertical transmission may contribute to the maintenance of arthropod-borne viruses, but its existence in chikungunya virus (CHIKV) is unclear. Experimental vertical transmission of infectious clones of CHIKV in Aedes aegypti mosquitoes from Malaysia was investigated. Eggs and adult progeny from the second gonotrophic cycles of infected parental mosquitoes were tested. Using polymerase chain reaction (PCR), 56.3% of pooled eggs and 10% of adult progeny had detectable CHIKV RNA, but no samples had detectable infectious virus by plaque assay. Transfected CHIKV RNA from PCR-positive eggs did not yield infectious virus in BHK-21 cells. Thus, vertical transmission of viable CHIKV was not demonstrated. Noninfectious CHIKV RNA persists in eggs and progeny of infected Ae. aegypti, but the mechanism and significance are unknown. There is insufficient evidence to conclude that vertical transmission exists in CHIKV, as positive results reported in previous studies were almost exclusively based only on viral RNA detection. PMID:26598564

  13. Rapid Detection of the Varicella Zoster Virus in Saliva

    NASA Technical Reports Server (NTRS)

    Pierson, Duane L.; Mehta, Satish K.; Cohrs, Randall J.; Gilden, Don H.; Harding, Robert E.

    2011-01-01

    Varicella zoster virus (VZV) causes chicken pox on first exposure (usually in children), and reactivates from latency causing shingles (usually in adults). Shingles can be extremely painful, causing nerve damage, organ damage, and blindness in some cases. The virus can be life-threatening in immune-compromised individuals. The virus is very difficult to culture for diagnosis, requiring a week or longer. This invention is a rapid test for VZV from a saliva sample and can be performed in a doctor s office. The kit is small, compact, and lightweight. Detec tion is sensitive, specific, and noninvasive (no needles); only a saliva sample is required. The test provides results in minutes. The entire test is performed in a closed system, with no exposure to infectious materials. The components are made mostly of inexpensive plastic injection molded parts, many of which can be purchased off the shelf and merely assembled. All biological waste is contained for fast, efficient disposal. This innovation was made possible because of discovery of a NASA scientists flight experiment showing the presence of VZV in saliva during high stress periods and disease. This finding enables clinicians to quickly screen patients for VZV and treat the ones that show positive results with antiviral medicines. This promotes a rapid recovery, easing of pain and symptoms, and reduces chances of complications from zoster. Screening of high-risk patients could be incorporated as part of a regular physical exam. These patients include the elderly, pregnant women, and immune-compromised individuals. In these patients, VZV can be a life-threatening disease. In both high- and low-risk patients, early detection and treatment with antiviral drugs can dramatically decrease or even eliminate the clinical manifestation of disease.

  14. Classical swine fever virus Strain 'C'. How long is it detectable after oral vaccination?

    PubMed

    Kaden, V; Lange, E; Riebe, R; Lange, B

    2004-08-01

    To determine the persistence period of C-strain vaccine virus in immunized animals, domestic pigs and wild boars were vaccinated orally and killed on different days post vaccinationem (dpv). Tissue samples were taken at necropsy from both species for detection of C-strain virus. From domestic pigs nasal swabs and faeces were also collected. During the investigation period (2-12 dpv) vaccine virus could never be detected in nasal secretions and in faeces of vaccinated domestic pigs. In contrast, C-strain virus was found in organs until day 8 pv in domestic pigs and until day 9 pv in wild boars. Whereas in domestic pigs virus was detected in tonsils, Ln. mandibularis or in spleen, in wild boar it only was found in tonsils. We conclude that C-strain vaccine virus is not detectable in wild boars longer than 10-12 days after intake of the vaccine baits. PMID:15458487

  15. Detection and identification of infectious bronchitis virus by RT-PCR in Iran.

    PubMed

    Homayounimehr, Alireza; Pakbin, Ahmad; Momayyez, Reza; Fatemi, Seyyedeh Mahsa Rastegar

    2016-06-01

    Infectious bronchitis virus (IBV) causes severe diseases in poultry with significant economic consequences to the poultry industry in Iran. The aim of this study was the detection and identification of IBV by reverse transcription(RT)-PCR in Iran. Ten IB virus strains were detected by testing trachea, cecal tonsil, and kidney tissues collected from broiler and layer farms in Iran. In order to detect infectious bronchitis virus, an optimized RT-PCR was used. Primers targeting the conserved region of known IBV serotypes were used in the RT-PCR assay. Primers selectively detecting Massachusetts and 793/B type IB viruses were designed to amplify the S1 gene of the virus and used in the nested PCR test. Our findings indicate the circulation of at least three genotypes of IB viruses (Massachusetts, 793/B, and variant 2) among poultry flocks. PMID:27010714

  16. Comparison of oral and intramuscular recombinant canine distemper vaccination in African wild dogs (Lycaon pictus).

    PubMed

    Connolly, Maren; Thomas, Patrick; Woodroffe, Rosie; Raphael, Bonnie L

    2013-12-01

    A series of three doses of recombinant canary-pox-vectored canine distemper virus vaccine was administered at 1-mo intervals, orally (n = 8) or intramuscularly (n = 13), to 21 previously unvaccinated juvenile African wild dogs (Lycaon pictus) at the Wildlife Conservation Society's Bronx Zoo. Titers were measured by serum neutralization at each vaccination and at intervals over a period of 3.5-21.5 mo after the initial vaccination. All postvaccination titers were negative for orally vaccinated animals at all sampling time points. Of the animals that received intramuscular vaccinations, 100% had presumed protective titers by the end of the course of vaccination, but only 50% of those sampled at 6.5 mo postvaccination had positive titers. None of the three animals sampled at 21.5 mo postvaccination had positive titers. PMID:24450046

  17. Multi-Gene Detection and Identification of Mosquito-Borne RNA Viruses Using an Oligonucleotide Microarray

    PubMed Central

    Grubaugh, Nathan D.; McMenamy, Scott S.; Turell, Michael J.; Lee, John S.

    2013-01-01

    Background Arthropod-borne viruses are important emerging pathogens world-wide. Viruses transmitted by mosquitoes, such as dengue, yellow fever, and Japanese encephalitis viruses, infect hundreds of millions of people and animals each year. Global surveillance of these viruses in mosquito vectors using molecular based assays is critical for prevention and control of the associated diseases. Here, we report an oligonucleotide DNA microarray design, termed ArboChip5.1, for multi-gene detection and identification of mosquito-borne RNA viruses from the genera Flavivirus (family Flaviviridae), Alphavirus (Togaviridae), Orthobunyavirus (Bunyaviridae), and Phlebovirus (Bunyaviridae). Methodology/Principal Findings The assay utilizes targeted PCR amplification of three genes from each virus genus for electrochemical detection on a portable, field-tested microarray platform. Fifty-two viruses propagated in cell-culture were used to evaluate the specificity of the PCR primer sets and the ArboChip5.1 microarray capture probes. The microarray detected all of the tested viruses and differentiated between many closely related viruses such as members of the dengue, Japanese encephalitis, and Semliki Forest virus clades. Laboratory infected mosquitoes were used to simulate field samples and to determine the limits of detection. Additionally, we identified dengue virus type 3, Japanese encephalitis virus, Tembusu virus, Culex flavivirus, and a Quang Binh-like virus from mosquitoes collected in Thailand in 2011 and 2012. Conclusions/Significance We demonstrated that the described assay can be utilized in a comprehensive field surveillance program by the broad-range amplification and specific identification of arboviruses from infected mosquitoes. Furthermore, the microarray platform can be deployed in the field and viral RNA extraction to data analysis can occur in as little as 12 h. The information derived from the ArboChip5.1 microarray can help to establish public health

  18. Microarray platform for the detection of a range of plant viruses and viroids.

    PubMed

    Adams, Ian; Harrison, Catherine; Tomlinson, Jenny; Boonham, Neil

    2015-01-01

    Diagnostic microarrays are a useful tool for the simultaneous detection of multiple targets. In this chapter we describe the use of a simple tube-based microarray platform for the detection of plant infecting viruses and viroids. PMID:25981261

  19. New Approaches for Virus Detection through Multidisciplinary Partnerships.

    PubMed

    Fawcett, Helen; Ünlü, M Selim; Connor, John H

    2016-06-10

    A critical requirement for controlling outbreaks of viral infection is sensitive and accurate diagnostics, which can be expensive and are frequently located in resource-intensive clinical laboratories. Outbreaks of many viral infections occur in countries where healthcare resources are limited and clinical laboratories scarce. This creates a fulfillment gap, one that could be filled through the development of inexpensive, sensitive, easy to use, and portable diagnostics. Here we describe our efforts to develop a diagnostic technology that detects viruses without needing to label the particle directly. Our approach has the advantage of speed and assay simplicity while maintaining high sensitivity. Essential in this approach has been the assembly of an integrated, diverse, and interdisciplinary team that worked together to evaluate technologies, spin-out a company, and produce a product for infectious disease diagnostics. The synergy of different individuals with complementary skills has been critical for the development of our transformative technology. PMID:27627625

  20. High sensitive method detection of plant RNA viruses by electrochemiluminescence reverse transcription PCR

    NASA Astrophysics Data System (ADS)

    Tang, Ya-bing; Xing, Da; Zhu, De-bin; Zhou, Xiao-ming

    2007-05-01

    It is well known that plant and animal viruses had widely spread the whole of world, and made a big loss in farming and husbandry. It is necessary that a highly efficient and accurate virus's detection method was developed. This research combines reverse transcription polymerase chain reaction (RT-PCR) technique with electrochemiluminescence method, to detect plant RNA viruses for the first time. Biotin-probe hybridizes with PCR product to specific select the target for detection, thus can avoid pseudo-positive result. TBR-probe hybridizes with PCR product to emit light for ECL detection. Specific nucleic acid sequences (20bp) were added to 5' terminal all of the primers, which can improve the chance of hybridization between TBR-probe and PCR product. At the same time, one of the PCR product chain can hybridize two Ru-probes, the ECL signal is intensified. The method was used to detect Odntoglossum ringspot virus ORSV, Sugarcane mosaic virus ScMV, Sorghum mosaic virus SrMV, and Maize dwarf mosaic virus MDMV, the experiment results show that this method could reliably identity virus infected plant samples. In a word, this method has higher sensitivity and lower cost than others. It can effectively detect the plant viruses with simplicity, stability, and high sensitivity.

  1. Detection and Isolation of H5N1 Influenza virus from Large Volumes of Natural Water

    PubMed Central

    Khalenkov, Alexey; Laver, W. Graeme; Webster, Robert G.

    2009-01-01

    Various species of aquatic or wetlands birds can be the natural reservoir of avian influenza A viruses of all hemagglutinin (HA) subtypes. Shedding of the virus into water leads to transmission between waterfowl and is a major threat for epidemics in poultry and pandemics in humans. Concentrations of the influenza virus in natural water reservoirs are often too low to be detected by most methods. The procedure was designed to detect low concentrations of the influenza virus in large volumes of water without the need for costly installations and reagents. The virus was adsorbed onto formalin-fixed erythrocytes and subsequently isolated in chicken embryos. Sensitivity of the method was determined using a reverse-genetic H5N1 virus. A concentration as low as 0.03 of the 50% egg infection dose per milliliter (EID50/ml) of the initial volume of water was effectively detected. The probability of detection was ∼13%, which is comparable to that of detecting the influenza virus M-gene by PCR amplification. The method can be used by field workers, ecologists, ornithologists, and researchers who need a simple method to isolate H5N1 influenza virus from natural reservoirs. The detection and isolation of virus in embryonated chicken eggs may help epidemiologic, genetic, and vaccine studies. PMID:18325605

  2. Canine distemper infections, with special reference to South Africa, with a review of the literature.

    PubMed

    Leisewitz, A L; Carter, A; van Vuuren, M; van Blerk, L

    2001-09-01

    Canine distemper virus is a member of the genus Morbillivirus of the family Paramyxoviridae that causes severe disease in dogs and a range of wild mammals. The clinical signs relate essentially to the respiratory, gastrointestinal and central nervous systems. In South Africa, infection with Ehrlichia canis and canine parvovirus may present similarly Many dogs will initially present with a wide range of central nervous system signs without any history of systemic disease. A recent South African study evaluating ante mortem diagnosis highlighted the importance of recognising clinical signs, cerebrospinal fluid IgG titres, serum IgM titres and immunocytochemistry of epithelial tissue. A 2-year retrospective evaluation of cerebrospinal fluid samples collected from dogs presented to the Onderstepoort Veterinary Academic Hospital indicates that distemper infection is common, and this disease should routinely be suspected in cases of diverse neurological disease in dogs. The South African dog population is specifically at high risk for the disease because of the large pool of unvaccinated, reproductively-active dogs that expose the wildlife resources of the country to risk of fatal disease. Outbreaks of disease in dogs continue to occur in developed and developing communities in both vaccinated and unvaccinated dogs worldwide, and have also been described in a wide range of free-ranging wildlife, including seals, dolphins and lions, and in endangered zoo animals. Modified live virus vaccines have contributed markedly to disease control in the dog population but have caused mortality in some wild carnivores. New recombinant vaccines are being developed that will be safe in wild animals. The pathogenesis of CNS demyelination has been compared to various important demyelinating diseases in humans and, amongst other things, relates to down-regulation of the oligodendrocyte gene coding for myelin synthesis and non-immunocyte CNS cell expression of type II major

  3. Efficacy of two canine distemper vaccines in wild Nearctic river otters (Lontra canadensis).

    PubMed

    Peper, Steven T; Peper, Randall L; Kollias, George V; Brooks, Robert P; Stevens, Sadie S; Serfass, Thomas L

    2014-09-01

    Canine distemper virus (CDV), a contagious morbillivirus, infects families in the order Carnivora, including Nearctic river otters (Lontra canadensis). As a preventative measure, vaccinations against CDV are frequently given to mustelids in captive environments. The Pennsylvania River Otter Reintroduction Project (PRORP) used wild-caught river otters to evaluate the efficacy and need for vaccinations against CDV as part of any reintroduction project. The objectives of this study were to: 1) evaluate the prevalence of exposure to CDV in wild river otters, 2) determine the immunologic response of river otters (i.e., seroconversion) after vaccination with a single (primary) vaccine dose compared to a second (booster) dose of Galaxy-D, a modified live-virus canine distemper (CD) vaccine (MLV CDV), and 3) determine the immunologic response after being vaccinated with a primary vaccination compared to a booster dose of Fervac-D, an MLV CDV. River otters were injected subcutaneously in the nape of the neck with their designated vaccine. Timeframes for collection of blood samples and/or injection of booster vaccines varied depending on the parameters of PRORP. Ten of the 22 river otters had positive prevaccination titer levels to CD. Both vaccines, Galaxy-D and Fervac-D, produced sufficient seroconversion or rise of titer levels (86% and 57%, respectively) to recommend the use of vaccines in wild river otters. Future studies are recommended to evaluate currently produced CD vaccines. Future research should also focus on the number of days required between administration of primary and booster vaccines to achieve sufficient immune response. If only a primary dose is required, then hard-release reintroduction projects for river otters could be recommended. If primary and booster vaccines are required then soft-release reintroduction projects should be recommended. Soft-release projects should include captive management periods that allow for appropriate vaccination intervals

  4. Pathologic and Molecular Virologic Characterization of a Canine Distemper Outbreak in Farmed Civets.

    PubMed

    Techangamsuwan, S; Banlunara, W; Radtanakatikanon, A; Sommanustweechai, A; Siriaroonrat, B; Lombardini, E D; Rungsipipat, A

    2015-07-01

    In October 2011, a fatal disease outbreak occurred in 3 civet species farmed for their use in the coffee industry in Thailand. The disease quickly killed 20 animals in a mixed population of Asian palm civets (Paradoxurus hermaphroditus; n = 18), a masked palm civet (Paguma larvata; n = 1), and small Indian civet (Viverricula indica; n = 1). Clinical signs consisted of severe lethargy, weakness, vomiting, and diarrhea with associated dehydration, dyspnea, nasal and footpad hyperkeratosis, and seizures. All civets were positive for canine morbillivirus using the commercial canine distemper virus (CDV) antigen test kit. Consistently observed necropsy findings consisted of severe pneumonia and hemorrhagic enteritis. Microscopic examination revealed severe gastroenteritis, bronchointerstitial pneumonia, lymphadenitis, necrotizing dermatitis, nonsuppurative polioencephalitis, and characteristic intranuclear/intracytoplasmic eosinophilic viral inclusions in multiple tissues. Immunohistochemical analysis revealed immunoreactivity of varying intensity, while virus isolation demonstrated typical cytopathic effects. To confirm CDV infection, reverse transcription-polymerase chain reaction against fusion (F), phosphoprotein (P), and hemagglutinin (H) genes showed bands of expected size using conjunctival swabs (9 civets, 1 dog [Canis lupus familiaris] living on the farm). Phylogenetic analyses and restriction fragment length polymorphism results indicated that the civets were infected by the Asia-1 strain of CDV commonly found in dogs in Thailand. The deduced amino acid sequences of the signaling lymphocyte activation molecule binding region of the CDV-H proteins revealed a Y549H mutation in both CDV-infected Asian palm civets (n = 4) and a co-located dog. We report a canine distemper outbreak in a civet colony with lineage classification and a Y549H mutation in noncanid species in Thailand. PMID:25253065

  5. Bioaerosol sampling for the detection of aerosolized influenza virus

    PubMed Central

    Blachere, Francoise M.; Lindsley, William G.; Slaven, James E.; Green, Brett J.; Anderson, Stacey E.; Chen, Bean T.; Beezhold, Don H.

    2007-01-01

    Background Influenza virus was used to characterize the efficacy of a cyclone‐based, two‐stage personal bioaerosol sampler for the collection and size fractionation of aerosolized viral particles. Methods A Collison single‐jet nebulizer was used to aerosolize the attenuated FluMist® vaccine into a calm‐air settling chamber. Viral particles were captured with bioaerosol samplers that utilize 2 microcentrifuge tubes to collect airborne particulates. The first tube (T1) collects particles greater than 1.8 μm in diameter, while the second tube (T2) collects particles between 1.0 and 1.8 μm, and the back‐up filter (F) collects submicron particles. Following aerosolization, quantitative PCR was used to detect and quantify H1N1 and H3N2 influenza strains. Results Based on qPCR results, we demonstrate that aerosolized viral particles were efficiently collected and separated according to aerodynamic size using the two‐stage bioaerosol sampler. Most viral particles were collected in T2 (1‐1.8 μm) and on the back‐up filter (< 1 μm) of the bioaerosol sampler. Furthermore, we found that the detection of viral particles with the two‐stage sampler was directly proportional to the collection time. Consequently, viral particle counts were significantly greater at 40 minutes in comparison to 5, 10 and 20 minute aerosol collection points. Conclusions Due to a lack of empirical data, aerosol transmission of influenza is often questioned. Using FluMist®, we demonstrated that a newly developed bioaerosol sampler is able to recover and size fractionate aerosolized viral particles. This sampler should be an important tool for studying viral transmission in clinical settings and may significantly contribute towards understanding the modes of influenza virus transmission. PMID:19453416

  6. Antiviral Activity of Carbobenzoxy Di- and Tripeptides on Measles Virus

    PubMed Central

    Miller, F. A.; Dixon, G. J.; Arnett, G.; Dice, J. R.; Rightsel, W. A.; Schabel, F. M.; Mclean, I. W.

    1968-01-01

    A series of simple carbobenzoxy peptides showed high and consistent antiviral chemotherapeutic activity in cell culture. In general, greatest activity was found against the measles-distemper or herpesvirus groups, or both, but various representatives of the series had quantitatively and qualitatively different antiviral activities. Several of the compounds, showing the highest antimeasles activity, were investigated extensively. In human cell culture plaque assays, these compounds were active against measles virus at levels of from 15 to 500 μg/ml. At single doses of about 250 to 500 mg/kg, orally in three animal species, significant serum levels of drugs were detected in virus cell culture assays. The mode of action appeared to be therapeutic, as an effect was seen in cell systems infected for at least 24 hr before treatment. PMID:4971720

  7. Multiplex biomarker analysis biosensor for detection of hepatitis B virus.

    PubMed

    Xu, Hua; Gu, Dayong; He, Jian'an; Shi, Lei; Yao, Jingyu; Liu, Chunxiao; Zhao, Chunzhong; Xu, Yunqing; Jiang, Shengyang; Long, Jun

    2015-01-01

    In this paper, we report the development of a protein microarray-based biosensor for the detection of the hepatitis B virus (HBV) serological markers using surface plasmon resonance (SPR Printing buffer, protein immobilization time and concentration of the capture protein were optimized systematically to determine the best performance of the biosensor. Under optimal conditions, five hepatitis B markers in 20 μL human serum can be simultaneously detected within 30 minutes, whereas other methods such as ELISA and PCR can detect only one marker within four hours. This platform has been validated by analysis of 35 patients known to have hepatitis B, with 85% agreement between the test platform and analysis by commercial enzyme-linked immunosorbent assay (ELISA) kits. The results demonstrate that the protein microarray with SPR displayed a sensitivity of 0.1 ng mL(-1) for HBsAg. In addition to high sensitivity, it also shows excellent specificity, reproducibility and stability. This integrated protein microarray technique combined with SPR is a promising candidate for hepatitis B diagnosis with high-throughput. PMID:26405988

  8. Highly Specific Detection of Five Exotic Quarantine Plant Viruses using RT-PCR.

    PubMed

    Choi, Hoseong; Cho, Won Kyong; Yu, Jisuk; Lee, Jong-Seung; Kim, Kook-Hyung

    2013-03-01

    To detect five plant viruses (Beet black scorch virus, Beet necrotic yellow vein virus, Eggplant mottled dwarf virus, Pelargonium zonate spot virus, and Rice yellow mottle virus) for quarantine purposes, we designed 15 RT-PCR primer sets. Primer design was based on the nucleotide sequence of the coat protein gene, which is highly conserved within species. All but one primer set successfully amplified the targets, and gradient PCRs indicated that the optimal temperature for the 14 useful primer sets was 51.9°C. Some primer sets worked well regardless of annealing temperature while others required a very specific annealing temperature. A primer specificity test using plant total RNAs and cDNAs of other plant virus-infected samples demonstrated that the designed primer sets were highly specific and generated reproducible results. The newly developed RT-PCR primer sets would be useful for quarantine inspections aimed at preventing the entry of exotic plant viruses into Korea. PMID:25288934

  9. Evaluation of Commercially Available Chikungunya Virus Immunoglobulin M Detection Assays.

    PubMed

    Johnson, Barbara W; Goodman, Christin H; Holloway, Kimberly; de Salazar, P Martinez; Valadere, Anne M; Drebot, Michael A

    2016-07-01

    Commercial chikungunya virus (CHIKV)-specific IgM detection kits were evaluated at the Centers for Disease Control and Prevention (CDC), the Public Health Agency of Canada National Microbiology Laboratory, and the Caribbean Public Health Agency (CARPHA). The Euroimmun Anti-CHIKV IgM ELISA kit had ≥ 95% concordance with all three reference laboratory results. The limit of detection for low CHIK IgM+ samples, as measured by serial dilution of seven sera up to 1:12,800 ranged from 1:800 to 1:3,200. The Euroimmun IIFT kit evaluated at CDC and CARPHA performed well, but required more retesting of equivocal results. The InBios CHIKjj Detect MAC-ELISA had 100% and 98% concordance with CDC and CARPHA results, respectively, and had equal sensitivity to the CDC MAC-ELISA to 1:12,800 dilution in serially diluted samples. The Abcam Anti-CHIKV IgM ELISA had high performance at CARPHA, but at CDC, performance was inconsistent between lots. After replacement of the biotinylated IgM antibody controls with serum containing CHIKV-specific IgM and additional quality assurance/control measures, the Abcam kit was rereleased and reevaluated at CDC. The reformatted Abcam kit had 97% concordance with CDC results and limit of detection of 1:800 to 1:3,200. Two rapid tests and three other CHIKV MAC-ELISAs evaluated at CDC had low sensitivity, as the CDC CHIKV IgM in-house positive controls were below the level of detection. In conclusion, laboratories have options for CHIKV serological diagnosis using validated commercial kits. PMID:26976887

  10. Evaluation of Commercially Available Chikungunya Virus Immunoglobulin M Detection Assays

    PubMed Central

    Johnson, Barbara W.; Goodman, Christin H.; Holloway, Kimberly; de Salazar, P. Martinez; Valadere, Anne M.; Drebot, Michael A.

    2016-01-01

    Commercial chikungunya virus (CHIKV)–specific IgM detection kits were evaluated at the Centers for Disease Control and Prevention (CDC), the Public Health Agency of Canada National Microbiology Laboratory, and the Caribbean Public Health Agency (CARPHA). The Euroimmun Anti-CHIKV IgM ELISA kit had ≥ 95% concordance with all three reference laboratory results. The limit of detection for low CHIK IgM+ samples, as measured by serial dilution of seven sera up to 1:12,800 ranged from 1:800 to 1:3,200. The Euroimmun IIFT kit evaluated at CDC and CARPHA performed well, but required more retesting of equivocal results. The InBios CHIKjj Detect MAC-ELISA had 100% and 98% concordance with CDC and CARPHA results, respectively, and had equal sensitivity to the CDC MAC-ELISA to 1:12,800 dilution in serially diluted samples. The Abcam Anti-CHIKV IgM ELISA had high performance at CARPHA, but at CDC, performance was inconsistent between lots. After replacement of the biotinylated IgM antibody controls with serum containing CHIKV-specific IgM and additional quality assurance/control measures, the Abcam kit was rereleased and reevaluated at CDC. The reformatted Abcam kit had 97% concordance with CDC results and limit of detection of 1:800 to 1:3,200. Two rapid tests and three other CHIKV MAC-ELISAs evaluated at CDC had low sensitivity, as the CDC CHIKV IgM in-house positive controls were below the level of detection. In conclusion, laboratories have options for CHIKV serological diagnosis using validated commercial kits. PMID:26976887

  11. The influenza virus nucleoprotein synthesized from cloned DNA in a simian virus 40 vector is detected in the nucleus.

    PubMed Central

    Lin, B C; Lai, C J

    1983-01-01

    We obtained DNA sequences coding for the nucleoprotein (NP) of an influenza A virus by reverse transcription of virion RNA with synthetic oligonucleotide primers. Terminal sequence analysis showed that the cloned gene contained a full-length copy of the virion RNA segment. The NP-specific DNA was inserted into the late region of a simian virus 40 vector, and the DNA recombinant was propagated in the presence of an early simian virus 40 temperature-sensitive mutant helper. Infection of African green monkey kidney cells with the recombinant produced a polypeptide immunoprecipitable with NP-specific antisera. The polypeptide product had a molecular weight of 56,000, identical to that of the nucleoprotein of influenza virus as estimated on polyacrylamide gels. The putative NP was detected in the nucleus of infected primate cells by an immunofluorescence assay. This nuclear localization of NP from recombinant DNA was similar to that seen during influenza virus infection. Images PMID:6296449

  12. THE APPLICATION OF EMERGING TECHNOLOGIES TO VIRUS DETECTION IN WATER

    EPA Science Inventory

    Human enteric viruses belonging to many different viral genera cause waterborne disease when susceptible individuals are exposed to contaminated drinking and recreational waters. Diseases resulting from infection with these viruses include gastroenteritis, hepatitis, encephali...

  13. DETECTION AND INACTIVATION OF ENTERIC VIRUSES IN WASTEWATER

    EPA Science Inventory

    This report covers studies on the development and evaluation of methods for concentrating and assaying low levels of viruses in large volumes of water as well as studies on the use of ozone in inactivating viruses in water and wastewater. Of the eight virus concentration methods ...

  14. Cucurbit yellow stunting disorder virus detected in pigweed in Florida

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Samples were collected from pigweed, a species of Amaranth, that were in proximity to virus-infected watermelons in south Florida. No symptoms of virus infection were observed on the weed samples; however, testing by two independent methods found infection by Cucurbit yellow stunting disorder virus...

  15. DETECTION OF VIRUSES IN ENVIRONMENTAL WATERS, SEWAGE AND SEWAGE SLUDGES

    EPA Science Inventory

    There are many different groups of viruses that will be found in environmental waters. These include the many types of viruses whose hosts are natural aquatic organisms. There are also groups of viruses present in environmental waters which represent exogenous contaminants, whose...

  16. Detection of C-type virus by immunoferritin technique in bat lung cell line chronically infected with bovine leucosis virus.

    PubMed

    Mihailescu, D; Patrascu, I V; Apostol, I; Mazilu, M

    1980-01-01

    Reported in this paper are morphological studies and tests for the detection of Type-C particles from a line of bat lung cells chronically infected with bovine leucosis virus. The immunoferritin technique was used. Ferritin labelling of Type-C particles was regularly accompanied by black-spot arrangement of ferritin around the virus envelope, which provided evidence to the specificity of this immunochemical technique. PMID:6260052

  17. Reverse Transcription Polymerase Chain Reaction-based System for Simultaneous Detection of Multiple Lily-infecting Viruses

    PubMed Central

    Kwon, Ji Yeon; Ryu, Ki Hyun; Choi, Sun Hee

    2013-01-01

    A detection system based on a multiplex reverse transcription (RT) polymerase chain reaction (PCR) was developed to simultaneously identify multiple viruses in the lily plant. The most common viruses infecting lily plants are the cucumber mosaic virus (CMV), lily mottle virus (LMoV), lily symptomless virus (LSV). Leaf samples were collected at lily-cultivation facilities located in the Kangwon province of Korea and used to evaluate the detection system. Simplex and multiplex RT-PCR were performed using virus-specific primers to detect single-or mixed viral infections in lily plants. Our results demonstrate the selective detection of 3 different viruses (CMV, LMoV and LSV) by using specific primers as well as the potential of simultaneously detecting 2 or 3 different viruses in lily plants with mixed infections. Three sets of primers for each target virus, and one set of internal control primers were used to evaluate the detection system for efficiency, reliability, and reproducibility. PMID:25288961

  18. Immunohistochemical detection of bluetongue virus in fixed tissue.

    PubMed

    Sánchez-Cordón, P J; Rodríguez-Sánchez, B; Risalde, M A; Molina, V; Pedrera, M; Sánchez-Vizcaíno, J M; Gómez-Villamandos, J C

    2010-07-01

    The VP7 structural protein is the most abundant of the major core proteins and is highly conserved in all serotypes of bluetongue virus (BTV). The aim of this study was to develop immunohistochemical techniques for the detection of BTV VP7 in Bouin's- and formalin-fixed and paraffin wax-embedded tissues from small ruminants (sheep and goats) naturally infected with BTV. Tissue samples were taken from animals in which BTV infection had been confirmed by reverse transcriptase polymerase chain reaction. Optimal results were obtained by incubation of monoclonal antibody 2E9 on samples fixed with Bouin's solution or neutral buffered formalin. Optimum antigen retrieval for Bouin's-fixed samples was by microwave heating (6 min) of tissue samples in citrate buffer (pH 6.0, 0.01 M), while for formalin-fixed samples a 30 min heating period in pH 9.0 buffer was required. In both species, BTV was mainly detected in the spleen, lymph nodes and lungs; specifically within the arteriolar and capillary endothelial cells, together with macrophages and lymphocytes. The immunohistochemical method described will be a useful tool for future research. PMID:20156627

  19. Detection and isolation of Bluetongue virus from commercial vaccine batches.

    PubMed

    Bumbarov, Velizar; Golender, Natalia; Erster, Oran; Khinich, Yevgeny

    2016-06-14

    In this report we describe the detection and identification of Bluetongue virus (BTV) contaminations in commercial vaccines. BTV RNA was detected in vaccine batches of Lumpy skin disease (LSD) and Sheep pox (SP) using quantitative PCR (qPCR) for VP1 and NS3 genes. Both batches were positive for VP1 and NS3 in qPCR. The LSD vaccine-derived sample was positive for VP1 and VP2 in conventional PCR. The SP vaccine-derived sample was examined by amplification of VP1, VP4, VP6, VP7, NS2 and NS3 gene segments in conventional PCR. The SP vaccine-derived sample was further propagated in embryonated chicken eggs (ECE) and Vero cells. Preliminary sequence analysis showed that the LSD vaccine-derived sequence was 98-99% similar to BTV9. Analysis of the six genomic segments from the SP vaccine-derived isolate showed the highest similarity to BTV26 (66.3-97.8%). These findings are particularly important due to the effect of BTV on cattle and sheep, for which the vaccines are intended. They also demonstrate the necessity of rigorous vaccine inspection and strict vaccine production control. PMID:27171751

  20. Viruses and Human Cancer: From Detection to Causality

    PubMed Central

    Sarid, Ronit; Gao, Shou-Jiang

    2010-01-01

    The study of cancer is incomplete without taking into consideration of tumorigenic viruses. Initially, searches for human cancer viruses were fruitless despite an expansion of our knowledge in the same period concerning acute-transforming retroviruses in animals. However, over the last 40 years, we have witnessed rapid progress in the tumor virology field. Currently, acknowledged human cancer viruses include Epstein-Barr virus, Hepatitis B virus, Hepatitis C virus, High-risk human papilloma viruses, Human T-cell lymphotropic virus type 1 and Kaposi’s sarcoma-associated herpesvirus. Extensive epidemiological and mechanistic studies have led to the development of novel preventive and therapeutic approaches for managing some of these infections and associated cancers. In addition, recent advances in molecular technologies have enabled the discovery of a new potential human tumor virus, Merkel cell polyomavirus, but its association with cancer remains to be validated. It is anticipated that in the next few decades many additional human cancer viruses will be discovered and the mechanisms underlying viral oncogenesis delineated. Thus, it can be expected that better tools for preventing and treating virus-associated cancer will be available in the near future. PMID:20971551

  1. Polymerase chain reaction for detection of herpesvirus simiae (B virus) in clinical specimens.

    PubMed

    Slomka, M J; Brown, D W; Clewley, J P; Bennett, A M; Harrington, L; Kelly, D C

    1993-01-01

    A polymerase chain reaction (PCR) was designed which is specific to Macaca fascicularis (cynomolgus monkey) isolates of B virus. The PCR primers produced the expected 188 basepair product from the Cyno 2 strain and seven other cynomolgus monkey isolates of B virus. Oligomer hybridization with a 31-mer oligonucleotide was used to confirm the origin of this product. The PCR failed to amplify DNA of Epstein-Barr virus, cytomegalovirus, varicella-zoster virus, and other alphaherpesviruses (herpes simplex virus types 1 and 2, four SA 8 isolates and three rhesus isolates of B virus). PCR testing of swabs obtained from four orally-infected cynomolgus monkeys confirmed the presence of B virus DNA in samples previously shown to be positive by culture. In addition, PCR detected B virus in several swabs from infected monkeys that were culture negative. Total DNA extracts from the trigeminal and sacral ganglia of these animals were tested by nested PCR and B virus DNA was detected in the trigeminal ganglia of 3 of the 4 orally-infected cynomolgus monkeys. Nested PCR did not detect B virus DNA in total DNA extracts obtained from the brains of the four monkeys. PMID:8392323

  2. Detection of airborne viruses using electro-aerodynamic deposition and a field-effect transistor

    NASA Astrophysics Data System (ADS)

    Park, Kyu-Tae; Cho, Dong-Guk; Park, Ji-Woon; Hong, Seunghun; Hwang, Jungho

    2015-12-01

    We report a technique for the detection of aerosolized viruses. Conventional field-effect-transistor (FET)-based techniques use solution-based processes, thus require antibody binding to the detection region of the FET prior to the supply of the analyte. With the method described here, virus-antibody-bound particles are delivered to the FET during detection; therefore, neither a pre-treatment antibody binding step on the FET channel nor washing process for virus-antibody-binding are necessary. Our method is based on the concept that virus-antibody-bound particles are larger than the virus or antibody alone, and thus have larger charge numbers following aerosol charging. When these particles are charged by negative ions and electro-aerodynamically deposited on a substrate, there exists a location on the substrate where neither lone virus nor antibody particles land, and where only virus-antibody-bound particles are deposited. If this location coincides with the channel of the FET, the resulting variation in the current can be used to indicate the existence of a virus. By aerosolizing a mixed solution of the virus and the antibody, only the virus-antibody-bound particles were transported to the swCNT-FET, and the electric current in the swCNT-FET decreased to 30% of that measured with no deposited particles.

  3. Finite Element Analysis on Nanomechanical Detection of Small Particles: Toward Virus Detection

    PubMed Central

    Imamura, Gaku; Shiba, Kota; Yoshikawa, Genki

    2016-01-01

    Detection of small particles, including viruses and particulate matter (PM), has been attracting much attention in light of increasing need for environmental monitoring. Owing to their high versatility, a nanomechanical sensor is one of the most promising sensors which can be adapted to various monitoring systems. In this study, we present an optimization strategy to efficiently detect small particles with nanomechanical sensors. Adsorption of particles on the receptor layer of nanomechanical sensors and the resultant signal are analyzed using finite element analysis (FEA). We investigate the effect of structural parameters (e.g., adsorption position and embedded depth of a particle and thickness of the receptor layer) and elastic properties of the receptor layer (e.g., Young's modulus and Poisson's ratio) on the sensitivity. It is found that a membrane-type surface stress sensors (MSS) has the potential for robust detection of small particles. PMID:27148181

  4. Detection of enteric viruses in activated sludge by feasible concentration methods

    PubMed Central

    Prado, Tatiana; Gaspar, Ana Maria Coimbra; Miagostovich, Marize Pereira

    2014-01-01

    Human enteric viruses are responsible to cause several diseases, including gastroenteritis and hepatitis, and can be present in high amounts in sewage sludge. This study compared virus recovery efficiency of two feasible concentration methods used for detecting human adenovirus (HAdV), rotavirus species A (RV-A), norovirus genogroup II (NoV GII) and hepatitis A virus (HAV) in sewage sludge from an activated sludge process. Twelve sewage sludge samples were collected bi-monthly from January to July, 2011. Ultracentrifugation was compared with a simplified protocol based on beef extract elution for recovering enteric viruses. Viruses were quantified by quantitative real-time PCR assays and virus recovery efficiency and limits of detection were determined. Methods showed mean recovery rates lower than 7.5%, presenting critical limits of detection (higher than 102 – 103 genome copies - GC L−1 for all viruses analyzed). Nevertheless, HAdV were detected in 90% of the analyzed sewage sludge samples (range: 1.8 × 104 to 1.1 × 105 GC L−1), followed by RV-A and NoV (both in 50%) and HAV (8%). Results suggesting that activated sludge is contaminated with high viral loads and HAdV are widely disseminated in these samples. The low virus recovery rates achieved, especially for HAV, indicate that other feasible concentration methods could be developed to improve virus recovery efficiency in these environmental matrices. PMID:24948954

  5. New perspectives on virus detection in shellfish: hemocytes as a source of concentrated virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    USDA ARS research indicates that circulating phagocytic cells (hemocytes) within oysters retain virus particles. We find that persistence of hepatitis A virus (HAV) within oyster hemocytes correlates with the presence of virus within whole oysters. Since bivalve shellfish have no self-nonself immun...

  6. Spiranthes Mosaic Virus 3 and Bidens Mottle Virus,Two Potyviruses Detected in Phlox divaricata

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This is the first report of Spiranthes mosaic virus in Florida and the first report of Bidens mottle virus in Phlox divaricata. This report provides an overview of this virus for growers, extension workers, crop consultants and research and regulatory scientists....

  7. Multiplex Real Time PCR For Detection of Wheat Streak Mosaic Virus and Triticum Mosaic Virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TRIMV) are widespread throughout the southwestern Great Plains states. Using conventional diagnostics such as Enzyme-Linked Immunosorbent Assays (ELISA), these two viruses are commonly found together in infected wheat samples. Methods for m...

  8. Simultaneous detection of three lily-infecting viruses using a multiplex Luminex bead array.

    PubMed

    Lim, Mi Sang; Kim, Su Min; Choi, Sun Hee

    2016-05-01

    A Luminex bead array was applied to detect multiple-virus coinfection in lily plants exhibiting typical symptoms, and the efficiency of this detection system was assessed. Specific primer sets for the simultaneous detection of 4 targets in virus-infected lily plants were constructed and used for reverse transcription (RT)-polymerase chain reaction (PCR), and specific probes were used for Luminex-based assay. Each of the 4 targets was amplified, and the amplicons were used for Luminex bead array experiments. A Luminex bead array analysis of lily-infecting viruses was performed using the quadruplex RT-PCR products followed by hybridization between the biotinylated targets and anti-tagged microsphere beads. The hybridization products produced fluorescence signals that were detected by the Luminex system. Signal strengths were analyzed by their median fluorescence intensity (MFI) values. Detection of the different target elements was found to be very specific to the corresponding viruses in lilies, and coinfection with multiple viruses was specifically detected via the MFI signals. Therefore, the use of a Luminex bead array for the detection of co-infected multiple viruses in lily plants can be an improved system for screening and analyzing multiple-virus infection. PMID:26898956

  9. A virus-MIPs fluorescent sensor based on FRET for highly sensitive detection of JEV.

    PubMed

    Liang, Caishuang; Wang, Huan; He, Kui; Chen, Chunyan; Chen, Xiaoming; Gong, Hang; Cai, Changqun

    2016-11-01

    Major stumbling blocks in the recognition and detection of virus are the unstable biological recognition element or the complex detection means. Here a fluorescent sensor based on virus-molecular imprinted polymers (virus-MIPs) was designed for specific recognition and highly sensitive detection of Japanese encephalitis virus (JEV). The virus-MIPs were anchored on the surface of silica microspheres modified by fluorescent dye, pyrene-1-carboxaldehyde (PC). The fluorescence intensity of PC can be enhanced by the principle of fluorescence resonance energy transfer (FRET), where virus acted as energy donor and PC acted as energy acceptor. The enhanced fluorescence intensity was proportional to the concentration of virus in the range of 24-960pM, with a limit of detection (LOD, 3σ) of 9.6pM, and the relative standard deviation was 1.99%. In additional, the specificity study confirmed the resultant MIPs has high-selectivity for JEV. This sensor would become a new key for the detection of virus because of its high sensitive, simple operation, high stability and low cost. PMID:27591625

  10. Detection of virus-specific RNA in simian sarcoma-leukemia virus-infected cells in in situ hybridization to viral complementary DNA.

    PubMed Central

    Kaufman, S L; Gallo, R C; Miller, N R

    1979-01-01

    An in situ molecular hybridization system which will detect retrovirus RNA in the cytoplasm of individual virus-infected cells has been developed. The technique was applied to cells infected with simian sarcoma-leukemia virus, where the virus-specific RNA was detected by hybridization to simian sarcoma-leukemia virus 3H-labeled complementary DNA. The system is useful for detecting viral RNA-containing cells in the presence of an excess of virus-negative cells and for determining which type of cell in a heterogenous population is expressing viral RNA. Images PMID:224220

  11. Detecting the emergence of novel, zoonotic viruses pathogenic to humans

    PubMed Central

    2015-01-01

    RNA viruses, with their high potential for mutation and epidemic spread, are the most common class of pathogens found as new causes of human illness. Despite great advances made in diagnostic technology since the 1950s, the annual rate at which novel virulent viruses have been found has remained at 2–3. Most emerging viruses are zoonoses; they have jumped from mammal or bird hosts to humans. An analysis of virus discovery indicates that the small number of novel viruses discovered annually is an artifact of inadequate surveillance in tropical and subtropical countries, where even established endemic pathogens are often misdiagnosed. Many of the emerging viruses of the future are already infecting humans but remain to be uncovered by a strategy of disease surveillance in selected populations. PMID:25416679

  12. Inactivation of viruses by benzalkonium chloride.

    PubMed

    Armstrong, J A; Froelich, E J

    1964-03-01

    Benzalkonium chloride (as Roccal or Zephiran) was found to inactivate influenza, measles, canine distemper, rabies, fowl laryngotracheitis, vaccinia, Semliki Forest, feline pneumonitis, meningopneumonitis, and herpes simplex viruses after 10 min of exposure at 30 C or at room temperature. Poliovirus and encephalomyocarditis virus were not inactivated under the same conditions. It was concluded that all viruses tested were sensitive except members of the picorna group. The literature was reviewed. PMID:4288740

  13. Inactivation of Viruses by Benzalkonium Chloride

    PubMed Central

    Armstrong, J. A.; Froelich, E. J.

    1964-01-01

    Benzalkonium chloride (as Roccal or Zephiran) was found to inactivate influenza, measles, canine distemper, rabies, fowl laryngotracheitis, vaccinia, Semliki Forest, feline pneumonitis, meningopneumonitis, and herpes simplex viruses after 10 min of exposure at 30 C or at room temperature. Poliovirus and encephalomyocarditis virus were not inactivated under the same conditions. It was concluded that all viruses tested were sensitive except members of the picorna group. The literature was reviewed. PMID:4288740

  14. Detection of measles virus RNA in urine specimens from vaccine recipients.

    PubMed Central

    Rota, P A; Khan, A S; Durigon, E; Yuran, T; Villamarzo, Y S; Bellini, W J

    1995-01-01

    Analysis of urine specimens by using reverse transcriptase-PCR was evaluated as a rapid assay to identify individuals infected with measles virus. For the study, daily urine samples were obtained from either 15-month-old children or young adults following measles immunization. Overall, measles virus RNA was detected in 10 of 12 children during the 2-week sampling period. In some cases, measles virus RNA was detected as early as 1 day or as late as 14 days after vaccination. Measles virus RNA was also detected in the urine samples from all four of the young adults between 1 and 13 days after vaccination. This assay will enable continued studies of the shedding and transmission of measles virus and, it is hoped, will provide a rapid means to identify measles infection, especially in mild or asymptomatic cases. PMID:7494055

  15. Detection and transmission of infectious hematopoietic necrosis virus in rainbow trout

    USGS Publications Warehouse

    Amend, Donald F.

    1975-01-01

    Detection and transmission of Infectious Hematopoietic Necrosis Virus in rainbow trout (Salmo gairdneri) was studied at a commercial trout hatchery. Transmission of virus was demonstrated via water, feed and contaminated eggs. If eggs from carrier females were incubated several weeks in virus-free water, the resulting fry did not become infected. However, if fry subsequently became infected they were lifetime carriers. Infectious virus was readily detectable in most tissues of moribund fish; in carriers it was detected in sex products of spawning fish, and in samples from the intestine of post-spawning fish, but not in samples from blood, feces, kidney, or liver. The carrier rate was not significantly different between sexes. It was concluded that adult carriers are the reservoir of infection and that transmission occurs primarily when carriers shed virus and expose susceptible fish or eggs.

  16. Ultrasensitive detection of influenza viruses with a glycan-based impedimetric biosensor.

    PubMed

    Hushegyi, András; Pihíková, Dominika; Bertok, Tomas; Adam, Vojtech; Kizek, René; Tkac, Jan

    2016-05-15

    An ultrasensitive impedimetric glycan-based biosensor for reliable and selective detection of inactivated, but intact influenza viruses H3N2 was developed. Such glycan-based approach has a distinct advantage over antibody-based detection of influenza viruses since glycans are natural viral receptors with a possibility to selectively distinguish between potentially pathogenic influenza subtypes by the glycan-based biosensors. Build-up of the biosensor was carefully optimized with atomic force microscopy applied for visualization of the biosensor surface after binding of viruses with the topology of an individual viral particle H3N2 analyzed. The glycan biosensor could detect a glycan binding lectin with a limit of detection (LOD) of 5 aM. The biosensor was finally applied for analysis of influenza viruses H3N2 with LOD of 13 viral particles in 1 μl, what is the lowest LOD for analysis of influenza viral particles by the glycan-based device achieved so far. The biosensor could detect H3N2 viruses selectively with a sensitivity ratio of 30 over influenza viruses H7N7. The impedimetric biosensor presented here is the most sensitive glycan-based device for detection of influenza viruses and among the most sensitive antibody or aptamer based biosensor devices. PMID:26765527

  17. Real-Time PCR System for Detection of Orthopoxviruses and Simultaneous Identification of Smallpox Virus

    PubMed Central

    Olson, Victoria A.; Laue, Thomas; Laker, Miriam T.; Babkin, Igor V.; Drosten, Christian; Shchelkunov, Sergei N.; Niedrig, Matthias; Damon, Inger K.; Meyer, Hermann

    2004-01-01

    A screening assay for real-time LightCycler (Roche Applied Science, Mannheim, Germany) PCR identification of smallpox virus DNA was developed and compiled in a kit system under good manufacturing practice conditions with standardized reagents. In search of a sequence region unique to smallpox virus, the nucleotide sequence of the 14-kDa fusion protein gene of each of 14 variola virus isolates of the Russian World Health Organization smallpox virus repository was determined and compared to published sequences. PCR primers were designed to detect all Eurasian-African species of the genus Orthopoxvirus. A single nucleotide mismatch resulting in a unique amino acid substitution in smallpox virus was used to design a hybridization probe pair with a specific sensor probe that allows reliable differentiation of smallpox virus from other orthopoxviruses by melting-curve analysis. The applicability was demonstrated by successful amplification of 120 strains belonging to the orthopoxvirus species variola, vaccinia, camelpox, mousepox, cowpox, and monkeypox virus. The melting temperatures (Tms) determined for 46 strains of variola virus (Tms, 55.9 to 57.8°C) differed significantly (P = 0.005) from those obtained for 11 strains of vaccinia virus (Tms, 61.7 to 62.7°C), 15 strains of monkeypox virus (Tms, 61.9 to 62.2°C), 40 strains of cowpox virus (Tms, 61.3 to 63.7°C), 8 strains of mousepox virus (Tm, 61.9°C), and 8 strains of camelpox virus (Tms, 64.0 to 65.0°C). As most of the smallpox virus samples were derived from infected cell cultures and tissues, smallpox virus DNA could be detected in a background of human DNA. By applying probit regression analysis, the analytical sensitivity was determined to be 4 copies of smallpox virus target DNA per sample. The DNAs of several human herpesviruses as well as poxviruses other than orthopoxviruses were not detected by this method. The assay proved to be a reliable technique for the detection of orthopoxviruses, with the

  18. Canine distemper in an isolated population of fishers (Martes pennanti) from California.

    PubMed

    Keller, Stefan M; Gabriel, Mourad; Terio, Karen A; Dubovi, Edward J; VanWormer, Elizabeth; Sweitzer, Rick; Barret, Reginald; Thompson, Craig; Purcell, Kathryn; Munson, Linda

    2012-10-01

    Four fishers (Martes pennanti) from an insular population in the southern Sierra Nevada Mountains, California, USA died as a consequence of an infection with canine distemper virus (CDV) in 2009. Three fishers were found in close temporal and spatial relationship; the fourth fisher died 4 mo later at a 70 km distance from the initial group. Gross lesions were restricted to hyperkeratosis of periocular skin and ulceration of footpads. All animals had necrotizing bronchitis and bronchiolitis with syncytia and intracytoplasmic inclusion bodies. Inclusion bodies were abundant in the epithelia of urinary bladder and epididymis but were infrequent in the renal pelvis and the female genital epithelia. No histopathologic or immunohistochemical evidence for virus spread to the central nervous system was found. One fisher had encephalitis caused by Sarcocystis neurona and another had severe head trauma as a consequence of predation. The H gene nucleotide sequence of the virus isolates from the first three fishers was identical and was 99.6% identical to the isolate from the fourth fisher. Phylogenetically, the isolates clustered with other North American isolates separate from classical European wildlife lineage strains. These data suggest that the European wildlife lineage might consist of two separate subgroups that are genetically distinct and endemic in different geographic regions. The source of infection as well as pertinent transmission routes remained unclear. This is the first report of CDV in fishers and underscores the significance of CDV as a pathogen of management concern. PMID:23060505

  19. Evaluation of a commercial bELISA serologic assay for avian influenza virus detection in wild birds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Avian influenza (AI) virus surveillance in wild birds is predominately dependent on diagnostic assays that identify the virus, including reverse transcriptase polymerase chain reaction and virus isolation. A sensitive and specific assay to detect AI virus antibodies would complement existing survei...

  20. 9 CFR 113.55 - Detection of extraneous agents in Master Seed Virus.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... Master Seed Virus. 113.55 Section 113.55 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Ingredient Requirements § 113.55 Detection of extraneous agents in Master Seed...

  1. 9 CFR 113.55 - Detection of extraneous agents in Master Seed Virus.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Master Seed Virus. 113.55 Section 113.55 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Ingredient Requirements § 113.55 Detection of extraneous agents in Master Seed...

  2. 9 CFR 113.55 - Detection of extraneous agents in Master Seed Virus.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Master Seed Virus. 113.55 Section 113.55 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Ingredient Requirements § 113.55 Detection of extraneous agents in Master Seed...

  3. 9 CFR 113.55 - Detection of extraneous agents in Master Seed Virus.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Master Seed Virus. 113.55 Section 113.55 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Ingredient Requirements § 113.55 Detection of extraneous agents in Master Seed...

  4. 9 CFR 113.55 - Detection of extraneous agents in Master Seed Virus.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Master Seed Virus. 113.55 Section 113.55 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Ingredient Requirements § 113.55 Detection of extraneous agents in Master Seed...

  5. Detecting the Presence of Nora Virus in "Drosophila" Utilizing Single Fly RT-PCR

    ERIC Educational Resources Information Center

    Munn, Bethany; Ericson, Brad; Carlson, Darby J.; Carlson, Kimberly A.

    2015-01-01

    A single fly RT-PCR protocol has recently been developed to detect the presence of the persistent, horizontally transmitted Nora virus in "Drosophila." Wild-caught flies from Ohio were tested for the presence of the virus, with nearly one-fifth testing positive. The investigation presented can serve as an ideal project for biology…

  6. 9 CFR 113.47 - Detection of extraneous viruses by the fluorescent antibody technique.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Detection of extraneous viruses by the fluorescent antibody technique. 113.47 Section 113.47 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD...

  7. Double-Stranded RNA Is Detected by Immunofluorescence Analysis in RNA and DNA Virus Infections, Including Those by Negative-Stranded RNA Viruses

    PubMed Central

    Son, Kyung-No; Liang, Zhiguo

    2015-01-01

    ABSTRACT Early biochemical studies of viral replication suggested that most viruses produce double-stranded RNA (dsRNA), which is essential for the induction of the host immune response. However, it was reported in 2006 that dsRNA could be detected by immunofluorescence antibody staining in double-stranded DNA and positive-strand RNA virus infections but not in negative-strand RNA virus infections. Other reports in the literature seemed to support these observations. This suggested that negative-strand RNA viruses produce little, if any, dsRNA or that more efficient viral countermeasures to mask dsRNA are mounted. Because of our interest in the use of dsRNA antibodies for virus discovery, particularly in pathological specimens, we wanted to determine how universal immunostaining for dsRNA might be in animal virus infections. We have detected the in situ formation of dsRNA in cells infected with vesicular stomatitis virus, measles virus, influenza A virus, and Nyamanini virus, which represent viruses from different negative-strand RNA virus families. dsRNA was also detected in cells infected with lymphocytic choriomeningitis virus, an ambisense RNA virus, and minute virus of mice (MVM), a single-stranded DNA (ssDNA) parvovirus, but not hepatitis B virus. Although dsRNA staining was primarily observed in the cytoplasm, it was also seen in the nucleus of cells infected with influenza A virus, Nyamanini virus, and MVM. Thus, it is likely that most animal virus infections produce dsRNA species that can be detected by immunofluorescence staining. The apoptosis induced in several uninfected cell lines failed to upregulate dsRNA formation. IMPORTANCE An effective antiviral host immune response depends on recognition of viral invasion and an intact innate immune system as a first line of defense. Double-stranded RNA (dsRNA) is a viral product essential for the induction of innate immunity, leading to the production of type I interferons (IFNs) and the activation of hundreds

  8. Evidence of Endemic Hendra Virus Infection in Flying-Foxes (Pteropus conspicillatus)—Implications for Disease Risk Management

    PubMed Central

    Breed, Andrew C.; Breed, Martin F.; Meers, Joanne; Field, Hume E.

    2011-01-01

    This study investigated the seroepidemiology of Hendra virus in a spectacled flying-fox (Pteropus conspicillatus) population in northern Australia, near the location of an equine and associated human Hendra virus infection in late 2004. The pattern of infection in the population was investigated using a serial cross-sectional serological study over a 25-month period, with blood sampled from 521 individuals over six sampling sessions. Antibody titres to the virus were determined by virus neutralisation test. In contrast to the expected episodic infection pattern, we observed that seroprevalence gradually increased over the two years suggesting infection was endemic in the population over the study period. Our results suggested age, pregnancy and lactation were significant risk factors for a detectable neutralizing antibody response. Antibody titres were significantly higher in females than males, with the highest titres occurring in pregnant animals. Temporal variation in antibody titres suggests that herd immunity to the virus may wax and wane on a seasonal basis. These findings support an endemic infection pattern of henipaviruses in bat populations suggesting their infection dynamics may differ significantly from the acute, self limiting episodic pattern observed with related viruses (e.g. measles virus, phocine distemper virus, rinderpest virus) hence requiring a much smaller critical host population size to sustain the virus. These findings help inform predictive modelling of henipavirus infection in bat populations, and indicate that the life cycle of the reservoir species should be taken into account when developing risk management strategies for henipaviruses. PMID:22194920

  9. Detection and characterisation of novel bocavirus (genus Bocaparvovirus) and gastroenteritis viruses from asymptomatic pigs in Ireland

    PubMed Central

    Gunn, Lynda; Collins, Patrick James; Fanning, Séamus; McKillen, John; Morgan, John; Staines, Anthony; O'Shea, Helen

    2015-01-01

    Background Livestock animals have been the assumed source of several human epidemics in recent years, for example, influenza H1N1, rotavirus G8/G9, and MERS-CoV. Surveillance of novel viruses in animals is essential to evaluate the risk to human and animal health and to determine any economic impact, for example, failure to thrive. There is a paucity of data regarding detection and characterisation of gastroenteritis viruses, particularly novel viruses, in porcines in Ireland. Recently, a number of small novel porcine DNA viruses have emerged globally, for example, torque teno sus virus, porcine bocavirus, and parvoviruses 2 & 4, and little is known about the biology and potential pathogenicity of these viruses. Bocaparvovirus is a genetically distinct group of viruses which has been recently detected in humans and animals. Methods In this study, the presence of gastroenteritis viruses (rotavirus A, porcine circovirus, adenovirus, and porcine bocavirus) was investigated in a selection of archived faecal samples from asymptomatic piglets from a commercial farm in Ireland. A total of 104 specimens were pooled and screened using conventional molecular techniques (PCR and RT-PCR), a subset of specimens (n=44) were then examined individually. Viral diversity was then investigated using statistical and phylogenetic techniques. Results Initial screening showed a high prevalence of PBoV in this farm, with the formation of three distinct groups in phylogenetic analysis. Other viruses were also investigated in this study with the first report of PCV, PAdV and lineage I G5 RVA in Ireland. Some specimens contained >1 virus, with statistical analysis indicating a strong correlation for mixed infections of PBoV and PAdV on this farm. Conclusion Investigating the diversity of circulating enteric viruses on Irish porcine farms is important to improve the prophylactic tools available and to facilitate the early detection of changes in circulating viruses. PMID:26065833

  10. DEVELOPMENT OF MOLECULAR METHODS TO DETECT EMERGING VIRUSES

    EPA Science Inventory

    A large number of human enteric viruses are known to cause gastrointestinal illness and waterborne outbreaks. Many of these are emerging viruses that do not grow or grow poorly in cell culture and so molecular detectoin methods based on the polymerase chain reaction (PCR) are be...

  11. Type- and subtype-specific detection of influenza viruses in clinical specimens by rapid culture assay.

    PubMed

    Ziegler, T; Hall, H; Sánchez-Fauquier, A; Gamble, W C; Cox, N J

    1995-02-01

    A rapid culture assay which allows for the simultaneous typing and subtyping of currently circulating influenza A(H1N1), A(H3N2), and B viruses in clinical specimens was developed. Pools of monoclonal antibodies (MAbs) against influenza A and B viruses and MAbs HA1-71 and HA2-76, obtained by immunizing mice with the denatured hemagglutinin subfragments HA1 and HA2 of influenza virus A/Victoria/3/75, were used for immunoperoxidase staining of antigens in infected MDCK cells. MAb HA1-71 reacted exclusively with influenza A viruses of the H3 subtype, while MAb HA2-76 reacted with subtypes H1, H3, H4, H6, H8, H9, H10, H11, and H12, as determined with 78 human, 4 swine, and 10 avian influenza virus reference strains subtyped by the hemagglutination inhibition test. To determine if the technique can be used as a rapid diagnostic test, 263 known influenza virus-positive frozen nasal or throat swabs were inoculated into MDCK cells. After an overnight incubation, the cells were fixed and viral antigens were detected by immunoperoxidase staining. Influenza A viruses of the H1 and H3 subtypes were detected in 31 and 113 specimens, respectively. The subtypes of 10 influenza A virus-positive specimens could not be determined because they contained too little virus. Influenza B viruses were detected in 84 specimens, and 25 specimens were negative. We conclude that this assay is a rapid, convenient, non-labor-intensive, and relatively inexpensive test for detecting, typing, and subtyping influenza viruses in clinical specimens. PMID:7714186

  12. Rapid detection of Ebola virus with a reagent-free, point-of-care biosensor

    DOE PAGESBeta

    Baca, Justin T.; Severns, Virginia; Lovato, Debbie; Branch, Darren W.; Larson, Richard S.

    2015-04-14

    Surface acoustic wave (SAW) sensors can rapidly detect Ebola antigens at the point-of-care without the need for added reagents, sample processing, or specialized personnel. This preliminary study demonstrates SAW biosensor detection of the Ebola virus in a concentration-dependent manner. The detection limit with this methodology is below the average level of viremia detected on the first day of symptoms by PCR. We observe a log-linear sensor response for highly fragmented Ebola viral particles, with a detection limit corresponding to 1.9 × 10⁴ PFU/mL prior to virus inactivation. We predict greatly improved sensitivity for intact, infectious Ebola virus. This point-of-care methodologymore » has the potential to detect Ebola viremia prior to symptom onset, greatly enabling infection control and rapid treatment. This biosensor platform is powered by disposable AA batteries and can be rapidly adapted to detect other emerging diseases in austere conditions.« less

  13. Rapid detection of Ebola virus with a reagent-free, point-of-care biosensor

    SciTech Connect

    Baca, Justin T.; Severns, Virginia; Lovato, Debbie; Branch, Darren W.; Larson, Richard S.

    2015-04-14

    Surface acoustic wave (SAW) sensors can rapidly detect Ebola antigens at the point-of-care without the need for added reagents, sample processing, or specialized personnel. This preliminary study demonstrates SAW biosensor detection of the Ebola virus in a concentration-dependent manner. The detection limit with this methodology is below the average level of viremia detected on the first day of symptoms by PCR. We observe a log-linear sensor response for highly fragmented Ebola viral particles, with a detection limit corresponding to 1.9 × 10⁴ PFU/mL prior to virus inactivation. We predict greatly improved sensitivity for intact, infectious Ebola virus. This point-of-care methodology has the potential to detect Ebola viremia prior to symptom onset, greatly enabling infection control and rapid treatment. This biosensor platform is powered by disposable AA batteries and can be rapidly adapted to detect other emerging diseases in austere conditions.

  14. Neuraminidase Resistant Sialosides for the Detection of Influenza Viruses.

    PubMed

    He, Yun; Yang, Yang; Iyer, Suri S

    2016-06-15

    We report the synthesis of influenza virus neuraminidase (NA) resistant sialosides that include different glycoside linkages (C-, S-, and triazole). These unnatural sialosides were printed onto glass slides to generate a small focused microarray. We evaluated the binding affinity of multiple lectins and compared the stability of these sialosides with O-linked sialosides toward influenza virus neuraminidase and intact virus. We demonstrated the ability of these molecules to capture eight different strains of influenza virus at ambient temperature without the addition of NA inhibitors. The glycans capture extremely low, clinically relevant concentrations of viruses and each strain gives rise to a specific "fingerprint" binding pattern, which could potentially be used in rapid diagnostic tests. PMID:27139196

  15. Development of a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of Sugarcane mosaic virus and Sorghum mosaic virus in sugarcane

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting Sugarcane mosaic virus (SCMV) and Sorghum mosaic virus (SrMV) in sugarcane. Six sets of four primers corresponding to the conserved coat protein gene were designed for each virus and their succ...

  16. Real-time RT-PCR for detection of Raspberry bushy dwarf virus, Raspberry leaf mottle virus and characterizing synergistic interactions in mixed infections

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two TaqMan-based real-time One-Step RT-PCR assays were developed for the rapid and efficient detection of Raspberry bushy dwarf virus (RBDV) and Raspberry leaf mottle virus (RLMV), two of the most common raspberry viruses in North America and Europe. The primers and probes were designed from conser...

  17. Methodological Guidelines for Accurate Detection of Viruses in Wild Plant Species.

    PubMed

    Lacroix, Christelle; Renner, Kurra; Cole, Ellen; Seabloom, Eric W; Borer, Elizabeth T; Malmstrom, Carolyn M

    2016-03-01

    Ecological understanding of disease risk, emergence, and dynamics and of the efficacy of control strategies relies heavily on efficient tools for microorganism identification and characterization. Misdetection, such as the misclassification of infected hosts as healthy, can strongly bias estimates of disease prevalence and lead to inaccurate conclusions. In natural plant ecosystems, interest in assessing microbial dynamics is increasing exponentially, but guidelines for detection of microorganisms in wild plants remain limited, particularly so for plant viruses. To address this gap, we explored issues and solutions associated with virus detection by serological and molecular methods in noncrop plant species as applied to the globally important Barley yellow dwarf virus PAV (Luteoviridae), which infects wild native plants as well as crops. With enzyme-linked immunosorbent assays (ELISA), we demonstrate how virus detection in a perennial wild plant species may be much greater in stems than in leaves, although leaves are most commonly sampled, and may also vary among tillers within an individual, thereby highlighting the importance of designing effective sampling strategies. With reverse transcription-PCR (RT-PCR), we demonstrate how inhibitors in tissues of perennial wild hosts can suppress virus detection but can be overcome with methods and products that improve isolation and amplification of nucleic acids. These examples demonstrate the paramount importance of testing and validating survey designs and virus detection methods for noncrop plant communities to ensure accurate ecological surveys and reliable assumptions about virus dynamics in wild hosts. PMID:26773088

  18. Development and evaluation of a polydiacetylene based biosensor for the detection of H5 influenza virus.

    PubMed

    Jiang, Lixiang; Luo, Jing; Dong, Wenjie; Wang, Chengmin; Jin, Wen; Xia, Yuetong; Wang, Haijing; Ding, Hua; Jiang, Long; He, Hongxuan

    2015-07-01

    H5N1 avian influenza has caused serious economic losses as well as posed significant threats to public health, agriculture and wildlife. It is important to develop a rapid, sensitive and specific detection platform suitable for disease surveillance and control. In this study, a highly sensitive, specific and rapid biosensor based on polydiacetylene was developed for detecting H5 influenza virus. The polydiacetylene based biosensor was produced from an optimized ratio of 10,12-pentacosadiynoic acid and 1,2-dimyristoyl-sn-glycero-3-phosphocholine, with the anti-H5 influenza antibody embedded onto the vesicle surface. The optimized polydiacetylene vesicle could detect H5 influenza virus sensitively with a detection limit of 0.53 copies/μL, showing a dramatic blue-to-red color change that can be observed directly by the naked eye and recorded by a UV-vis spectrometer. The sensitivity, specificity and accuracy of the biosensor were also evaluated. The sensor could specifically differentiate H5 influenza virus from H3 influenza virus, Newcastle disease virus and porcine reproductive and respiratory syndrome virus. Detection using tracheal swabs was in accord with virus isolation results, and comparable to the RT-PCR method. These results offer the possibility and potential of simple polydiacetylene based bio-analytical method for influenza surveillance. PMID:25819686

  19. External Quality Assessment of Molecular Detection of Ebola Virus in China

    PubMed Central

    Wang, Guojing; Sun, Yu; Zhang, Kuo; Jia, Tingting; Hao, Mingju; Zhang, Dong; Chang, Le; Zhang, Lei; Zhang, Rui; Lin, Guigao; Peng, Rongxue; Li, Jinming

    2015-01-01

    In 2014, Ebola hemorrhagic fever broke out in West Africa. As contact between China and West Africa is frequent, the possibility that Ebola virus would enter China was high. Thus, an external assessment of the quality of Ebola virus detection was organized by the National Center for Clinical Laboratories in China. Virus-like particles encapsulating known sequences of epidemic strains of Ebola virus from 2014 were prepared as positive quality controls. The sample panel, which was composed of seven positive and three negative samples, was dispatched to 19 laboratories participating in this assessment of Ebola virus detection. Accurate detection was reported at 14 of the 19 participating laboratories, with a sensitivity of 91.43% and a specificity of 100%. Four participants (21.05%) reported false-negative results and were classified as “acceptable.” One participant (5.26%) did not detect any positive samples and was thus classified as “improvable.” Based on the results returned, the ability to detect weakly positive Ebola specimens should be improved. Furthermore, commercial assays and the standard primers offered by the Chinese Centers for Disease Control and Prevention were found to be most accurate and dependable for Ebola detection. A two-target detection approach is recommended for Ebola screening; this approach could reduce the probability of false-negative results. Additionally, standardization of operations and punctual adjustment of instruments are necessary for the control and prevention of Ebola virus. PMID:26177537

  20. Simple and Portable Magnetic Immunoassay for Rapid Detection and Sensitive Quantification of Plant Viruses

    PubMed Central

    Rettcher, Stefanie; Jungk, Felicitas; Kühn, Christoph; Krause, Hans-Joachim; Nölke, Greta; Commandeur, Ulrich; Fischer, Rainer; Schillberg, Stefan

    2015-01-01

    Plant pathogens cause major economic losses in the agricultural industry because late detection delays the implementation of measures that can prevent their dissemination. Sensitive and robust procedures for the rapid detection of plant pathogens are therefore required to reduce yield losses and the use of expensive, environmentally damaging chemicals. Here we describe a simple and portable system for the rapid detection of viral pathogens in infected plants based on immunofiltration, subsequent magnetic detection, and the quantification of magnetically labeled virus particles. Grapevine fanleaf virus (GFLV) was chosen as a model pathogen. Monoclonal antibodies recognizing the GFLV capsid protein were immobilized onto immunofiltration columns, and the same antibodies were linked to magnetic nanoparticles. GFLV was quantified by immunofiltration with magnetic labeling in a double-antibody sandwich configuration. A magnetic frequency mixing technique, in which a two-frequency magnetic excitation field was used to induce a sum frequency signal in the resonant detection coil, corresponding to the virus concentration within the immunofiltration column, was used for high-sensitivity quantification. We were able to measure GFLV concentrations in the range of 6 ng/ml to 20 μg/ml in less than 30 min. The magnetic immunoassay could also be adapted to detect other plant viruses, including Potato virus X and Tobacco mosaic virus, with detection limits of 2 to 60 ng/ml. PMID:25710366

  1. Detection of sweet potato virus C, sweet potato virus 2 and sweet potato feathery mottle virus in Portugal.

    PubMed

    Varanda, Carla M R; Santos, Susana J; Oliveira, Mônica D M; Clara, Maria Ivone E; Félix, Maria Rosário F

    2015-06-01

    Field sweet potato plants showing virus-like symptoms, as stunting, leaf distortion, mosaic and chlorosis, were collected in southwest Portugal and tested for the presence of four potyviruses, sweet potato virus C (SPVC), sweet potato virus 2 (SPV2), sweet potato feathery mottle virus (SPFMV), sweet potato virus G (SPVG), and the crinivirus sweet potato chlorotic stunt virus (SPCSV). DsRNA fractions were extracted from symptomatic leaves and used as templates in single and multiplex RT-PCR assays using previously described specific primers for each analyzed virus. The amplified reaction products for SPVC, SPV2 and SPFMV were of expected size, and direct sequencing of PCR products revealed that they correspond to the coat protein gene (CP) and showed 98%, 99% and 99% identity, respectively, to those viruses. Comparison of the CP genomic and amino acid sequences of the Portuguese viral isolates recovered here with those of ten other sequences of isolates obtained in different countries retrieved from the GenBank showed very few differences. The application of the RT-PCR assays revealed for the first time the presence of SPVC and SPFMV in the sweet potato crop in Portugal, the absence of SPVG and SPCSV in tested plants, as well as the occurrence of triple virus infections under field conditions. PMID:26104336

  2. The effects of pooling, swab type and transport conditions on avian influenza virus and Newcastle disease virus detection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A crucial aspect of achieving high diagnostic test accuracy is sample collection and transport. In order to optimize avian influenza virus (AIV) detection in poultry we have evaluated swab pooling, different swab material types, and transport conditions. Swab pooling was evaluated with Newcastle d...

  3. Single virus detection by means of atomic force microscopy in combination with advanced image analysis.

    PubMed

    Bocklitz, Thomas; Kämmer, Evelyn; Stöckel, Stephan; Cialla-May, Dana; Weber, Karina; Zell, Roland; Deckert, Volker; Popp, Jürgen

    2014-10-01

    In the present contribution virions of five different virus species, namely Varicella-zoster virus, Porcine teschovirus, Tobacco mosaic virus, Coliphage M13 and Enterobacteria phage PsP3, are investigated using atomic force microscopy (AFM). From the resulting height images quantitative features like maximal height, area and volume of the viruses could be extracted and compared to reference values. Subsequently, these features were accompanied by image moments, which quantify the morphology of the virions. Both types of features could be utilized for an automatic discrimination of the five virus species. The accuracy of this classification model was 96.8%. Thus, a virus detection on a single-particle level using AFM images is possible. Due to the application of advanced image analysis the morphology could be quantified and used for further analysis. Here, an automatic recognition by means of a classification model could be achieved in a reliable and objective manner. PMID:25196422

  4. Infectious hematopoietic necrosis virus detected by separation and incubation of cells from salmonid cavity fluid.

    USGS Publications Warehouse

    Mulcahy, D.; Batts, W.N.

    1987-01-01

    Infectious hematopoietic necrosis (IHN) virus is usually detected by inoculating susceptible cell cultures with cavity ("ovarian") fluid (CF) from spawning females. We identified additional adult carriers of virus in spawning populations of steelhead trout (Salmo gairdneri) and sockeye salmon (Oncorhynchus nerka) by collecting nonerythrocytic cells from CF samples by low-speed centrifugation, culturing the cells for at least 7 d at 15 °C, and then testing the culture medium for virus. Virus appeared in the cultured cells from some samples of CF that remained negative during incubation. In additional samples of CF from these species, the virus titer increased in cultured cells compared with the titer in the original CF sample. With chinook salmon (O.tshawytscha), no negative samples converted to positive during incubation, but the virus titer was retained in incubated CF cells, but not in cell-free CF.

  5. Development of SPR biosensor for simultaneous detection of multiplex respiratory viruses.

    PubMed

    Shi, Lei; Sun, Qiuxiang; He, Jian'an; Xu, Hua; Liu, Chunxiao; Zhao, Chunzhong; Xu, Yunqing; Wu, Changlin; Xiang, Junjian; Gu, Dayong; Long, Jun; Lan, Hekui

    2015-01-01

    A surface plasmon resonance (SPR)-based biosensor was developed for specific detection of nine common respiratory virus, including influenza A and influenza B, H1N1, respiratory syncytial virus (RSV), parainfluenza virus 1-3 (PIV1, 2, 3), adenovirus, and severe acute respiratory syndrome coronavirus (SARS). The SPR biosensor was developed by immobilizing nine respiratory virus-specific oligonucleotides in an SPR chip. To increase the biosensor sensitivity, biotin was used to label the PCR primer and further amplify the signal by introducing streptavidin after hybridization. Throat swab specimens representing nine common respiratory viruses were tested by the innovative SPR-based biosensor to evaluate the sensitivity, specificity and reproducibility of this method. Results suggest that this biosensor has the potential to simultaneously identify common respiratory viruses. PMID:26406000

  6. Host-Encoded Reporters for the Detection and Purification of Multiple Enveloped Viruses

    PubMed Central

    Ketteler, Robin; Tomov, Vesko; Neunkirchner, Alina; Xie, Qiang; Pickl, Winfried F.; Seed, Brian

    2010-01-01

    The identification of host cell factors for virus replication holds great promise for the development of new anti-viral therapies. Recently, high-throughput screening methods have emerged as powerful tools to identify candidate host factors for therapeutic intervention. The development of assay systems suitable for large-scale automated screening is of particular importance for novel viruses with high pathogenic potential for which limited biological information can be developed in a short period of time. This report presents a general enzymatic reporter system for the detection and characterization of multiple enveloped viruses that does not rely on engineering of the virus. Instead, reporter enzymes are incorporated into virus particles by targeting to lipid microdomains in producer cells. The approach allows a variety of human pathogenic enveloped viruses to be detected by sensitive, inexpensive and automatable enzymatic assays. Tagged viruses can be purified quickly and efficiently by a magnetic bead-based capture method. The method allows general detection of enveloped viruses without prior reference to their sequence. PMID:20399809

  7. Evaluation of the Broad-Range PCR-Electrospray Ionization Mass Spectrometry (PCR/ESI-MS) System and Virus Microarrays for Virus Detection

    PubMed Central

    Taliaferro, Lanyn P.; Galvin, Teresa A.; Ma, Hailun; Shaheduzzaman, Syed; Williams, Dhanya K.; Glasner, Dustin R.; Khan, Arifa S.

    2014-01-01

    Advanced nucleic acid-based technologies are powerful research tools for novel virus discovery but need to be standardized for broader applications such as virus detection in biological products and clinical samples. We have used well-characterized retrovirus stocks to evaluate the limit of detection (LOD) for broad-range PCR with electrospray ionization mass spectrometry (PCR/ESI-MS or PLEX-ID), RT-PCR assays, and virus microarrays. The results indicated that in the absence of background cellular nucleic acids, PLEX-ID and RT-PCR had a similar LOD for xenotropic murine retrovirus-related virus (XMRV; 3.12 particles per µL) whereas sensitivity of virus detection was 10-fold greater using virus microarrays. When virus was spiked into a background of cellular nucleic acids, the LOD using PLEX-ID remained the same, whereas virus detection by RT-PCR was 10-fold less sensitive, and no virus could be detected by microarrays. Expected endogenous retrovirus (ERV) sequences were detected in cell lines tested and known species-specific viral sequences were detected in bovine serum and porcine trypsin. A follow-up strategy was developed using PCR amplification, nucleotide sequencing, and bioinformatics to demonstrate that an RD114-like retrovirus sequence that was detected by PLEX-ID in canine cell lines (Madin-Darby canine kidney (MDCK) and Cf2Th canine thymus) was due to defective, endogenous gammaretrovirus-related sequences. PMID:24777034

  8. Detection of herpes simplex virus-specific DNA sequences in latently infected mice and in humans.

    PubMed Central

    Efstathiou, S; Minson, A C; Field, H J; Anderson, J R; Wildy, P

    1986-01-01

    Herpes simplex virus-specific DNA sequences have been detected by Southern hybridization analysis in both central and peripheral nervous system tissues of latently infected mice. We have detected virus-specific sequences corresponding to the junction fragment but not the genomic termini, an observation first made by Rock and Fraser (Nature [London] 302:523-525, 1983). This "endless" herpes simplex virus DNA is both qualitatively and quantitatively stable in mouse neural tissue analyzed over a 4-month period. In addition, examination of DNA extracted from human trigeminal ganglia has shown herpes simplex virus DNA to be present in an "endless" form similar to that found in the mouse model system. Further restriction enzyme analysis of latently infected mouse brainstem and human trigeminal DNA has shown that this "endless" herpes simplex virus DNA is present in all four isomeric configurations. Images PMID:3003377

  9. Detection of plant viruses in mixed infection by a macroarray-assisted method.

    PubMed

    Shimura, Hanako; Furuta, Kazuyoshi; Masuta, Chikara

    2015-01-01

    The protocol for a simple, sensitive, and specific method using a cDNA macroarray to detect multiple viruses is provided. The method can be used even at the production sites for crops, which need a reliable routine diagnosis for mixed infection of plant viruses. The method consists of three steps: RNA extraction, duplex RT-PCR, and "microtube hybridization" (MTH). Biotinylated cDNA probes are prepared using RT-PCR and used to hybridize a nylon membrane containing target viral cDNAs by MTH. Positive signals can be visualized by colorimetric reaction and judged by eyes. We here demonstrate this method to detect asparagus viruses (Asparagus virus 1 and Asparagus virus 2) from latently infected asparagus plants. PMID:25287491

  10. Respiratory viruses in children with cystic fibrosis: viral detection and clinical findings

    PubMed Central

    Burns, Jane L.; Emerson, Julia; Kuypers, Jane; Campbell, Angela P.; Gibson, Ronald L.; McNamara, Sharon; Worrell, Kelly; Englund, Janet A.

    2011-01-01

    Please cite this paper as: Burns et al. (2011) Respiratory viruses in children with cystic fibrosis: viral detection and clinical findings. Influenza and Other Respiratory Viruses 6(3), 218–223. Background  Viral detection from different respiratory sample types in children with cystic fibrosis (CF) is facilitated by available molecular methods, but optimum sampling strategies have not been identified. In addition, associations between viral detection and respiratory symptoms are not well described. Objectives  Study goals were to compare molecular detection of viruses from concurrent upper airway and sputum samples in children with CF and to describe relative frequency of respiratory viral infections and identify potential clinical associations. Methods  We conducted a 2‐year prospective surveillance study in 44 children with CF aged 6–18 years. Upper airway and sputum samples were collected quarterly and during pulmonary exacerbations and tested for respiratory syncytial virus (RSV), influenza viruses, parainfluenza viruses types 1–4, human metapneumovirus, coronaviruses, rhinoviruses, and adenoviruses. Physical exams and symptom surveys were used to identify respiratory signs and symptoms. Results  Upper airway samples were collected at 359 visits; concordance of PCR‐based viral detection was examined in a subset of paired upper airway and sputum samples from 21 participants at 92 visits. Rhinovirus was the most commonly detected virus (23·1% overall), and rhinovirus detection was the same for both sample types (21·7% each). Sensitivity and specificity for the detection of rhinovirus in sputum relative to upper airway sampling were 70% and 91·7%, respectively. Respiratory symptoms associated with rhinovirus detection included increased cough, increased nasal congestion, increased sputum production, and wheezing. Conclusions  A relatively high frequency of rhinovirus detection was observed by either upper airway or sputum samples, and

  11. Development of an antigen-capture ELISA for the detection of avian leukosis virus p27 antigen.

    PubMed

    Yun, Bingling; Li, Delong; Zhu, Haibo; Liu, Wen; Qin, Liting; Liu, Zaisi; Wu, Guan; Wang, Yongqiang; Qi, Xiaole; Gao, Honglei; Wang, Xiaomei; Gao, Yulong

    2013-02-01

    An antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) employing monoclonal and polyclonal antibodies against p27 was developed for the detection of the avian leukosis virus (ALV). The specificity of the optimized AC-ELISA was evaluated using avian leukosis virus subgroup J (ALV-J), avian leukosis virus subgroup A (ALV-A), avian leukosis virus subgroup B (ALV-B), avian infectious bronchitis virus (IBV), Marek's disease virus (MDV), avian infectious laryngotracheitis virus (ILTV), Fowlpox virus (FPV), infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), avian reovirus (ARV), reticuloendotheliosis virus (REV), avian influenza virus (AIV) and Escherichia coli. The only specimens that yielded a strong signal were ALV-J, ALV-A and ALV-B, indicating that this assay is suitable for the detection of ALV. The limit of detection of this assay was 1.25 ng/ml of rp27 protein and 10(1.79)TCID(50) units of HLJ09MDJ-1 (ALV-J). Moreover, this AC-ELISA can detect ALV in cloacal swabs of chickens experimentally infected as early as 12 days post-infection. The AC-ELISA detected the virus in the albumin and cloacal swabs of naturally infected chickens, and the results were confirmed by PCR, indicating that the AC-ELISA was a suitable method for the detection of ALV. This test is rapid and sensitive and could be convenient for epidemiological studies and eradication programs. PMID:23201286

  12. Detection of white spot syndrome virus (WSSV) of Penaeus chinensis by in situ hybridization

    NASA Astrophysics Data System (ADS)

    Zhan, Wen-Bin; Wang, Yuan-Hong; Zhang, Zhi-Dong; Hideo, Fukuda

    2000-09-01

    White Spot Syndrome Virus (WSSV) was purified from hemolymph of infected shrimp. After nucleic acid extraction from the purified virus particles, EcoR I-digested fragments of the WSSV genome were cloned; three of these fragments were used as non-radioactive probes labeled with DIG-11-dUTP. The probes hybridized in situ, with sections located in the nuclei of all WSSV-infected tissues. The virus was detected in the gill, stomach, epidermis, and connective tissue and so on, but not detected in healthy shrimp tissues and epithelial cells of hepatopancreatic tubules of diseased shrimp.

  13. Carbon Nanotube Thin Film Biosensors for Sensitive and Reproducible Whole Virus Detection

    PubMed Central

    Mandal, Himadri S.; Su, Zhengding; Ward, Andrew; Tang, Xiaowu (Shirley)

    2012-01-01

    Here, we report the label-free, sensitive, and real-time electrical detection of whole viruses using carbon nanotube thin film (CNT-TF) field effect devices. Selective detection of approximately 550 model viruses, M13-bacteriophage, is demonstrated using a simple two-terminal (no gate electrode) configuration. Chemical gating through specific antibody-virus binding on CNT surface is proposed to be the sensing mechanism. Compared to electrical impedance sensors with identical microelectrode dimensions (no CNT), the CNT-TF sensors exhibit sensitivity 5 orders higher. We believe the reported approach could lead to a reproducible and cost-effective solution for rapid viral identification. PMID:22448194

  14. Rabies, canine distemper, and canine parvovirus exposure in large carnivore communities from two Zambian ecosystems.

    PubMed

    Berentsen, Are R; Dunbar, Mike R; Becker, Matthew S; M'soka, Jassiel; Droge, Egil; Sakuya, Nicholas M; Matandiko, Wigganson; McRobb, Rachel; Hanlon, Cathleen A

    2013-09-01

    Disease transmission within and among wild and domestic carnivores can have significant impacts on populations, particularly for threatened and endangered species. We used serology to evaluate potential exposure to rabies virus, canine distemper virus (CDV), and canine parvovirus (CPV) for populations of African lions (Panthera leo), African wild dogs (Lycaon pictus), and spotted hyenas (Crocuta crocuta) in Zambia's South Luangwa National Park (SLNP) and Liuwa Plain National Park (LPNP) as well as community lands bordering these areas. In addition, domestic dogs in the study region were evaluated for exposure to CDV and rabies. We provide the first comprehensive disease exposure data for these species in these ecosystems. Twenty-one lions, 20 hyenas, 13 wild dogs, and 38 domestic dogs were sampled across both regions from 2009 to 2011. Laboratory results show 10.5% of domestic dogs, 5.0% of hyenas, and 7.7% of wild dogs sampled were positive for CDV exposure. All lions were negative. Exposure to CPV was 10.0% and 4.8% for hyenas and lions, respectively. All wild dogs were negative, and domestic dogs were not tested due to insufficient serum samples. All species sampled were negative for rabies virus neutralizing antibodies except lions. Forty percent of lions tested positive for rabies virus neutralizing antibodies. Because these lions appeared clinically healthy, this finding is consistent with seroconversion following exposure to rabies antigen. To our knowledge, this finding represents the first ever documentation of rabies virus neutralizing antibodies consistent with rabies exposure that did not lead to clinical disease in free-ranging African lions from this region. With ever-increasing human pressure on these ecosystems, understanding disease transmission dynamics is essential for proper management and conservation of these carnivore species. PMID:23805791

  15. Detection of West Nile virus and tick-borne encephalitis virus in birds in Slovakia, using a universal primer set.

    PubMed

    Csank, Tomáš; Bhide, Katarína; Bencúrová, Elena; Dolinská, Saskia; Drzewnioková, Petra; Major, Peter; Korytár, Ľuboš; Bocková, Eva; Bhide, Mangesh; Pistl, Juraj

    2016-06-01

    West Nile virus (WNV) is a mosquito-borne neurotropic pathogen that presents a major public health concern. Information on WNV prevalence and circulation in Slovakia is insufficient. Oral and cloacal swabs and bird brain samples were tested for flavivirus RNA by RT-PCR using newly designed generic primers. The species designation was confirmed by sequencing. WNV was detected in swab and brain samples, whereas one brain sample was positive for tick-borne encephalitis virus (TBEV). The WNV sequences clustered with lineages 1 and 2. These results confirm the circulation of WNV in birds in Slovakia and emphasize the risk of infection of humans and horses. PMID:27001305

  16. The modification of fluorescent antibody virus neutralization (FAVN) test for the detection of antibodies to rabies virus.

    PubMed

    Hostnik, P

    2000-08-01

    The fluorescent antibody virus neutralization test (FAVN) for the detection of antibodies against rabies virus was modified by using a monoclonal anti-rabies antibodies and peroxidase anti-mouse conjugate instead of a fluorescent anti-rabies conjugate. The results were read on an automatic multi-channel spectrophotometer. A total of 182 serum samples from dogs were tested by both the original and modified FAVN methods and the results were compared. Good correlation was found between the two tests. Practically, the modified FAVN test was quicker and could be used for a larger number of samples. PMID:11014062

  17. Synthetic sialylglycopolymer receptor for virus detection using cantilever-based sensors.

    PubMed

    Gorelkin, P V; Erofeev, A S; Kiselev, G A; Kolesov, D V; Dubrovin, E V; Yaminsky, I V

    2015-09-01

    We describe the rapid, label-free detection of Influenza A viruses using a cantilever transducer modified with a synthetic sialylglycopolymer receptor layer. Surface stresses induced by viruses binding to the receptor layer were used as the analytical signal. The synthetic sialylglycopolymer receptor layer can be used in nanoscale strain-gauge cantilever transducers for highly sensitive virus detection. Strain-gage transducers using such sensor layers exhibit long lifetimes, high sensitivities, and possible regeneration. Nanomechanical cantilever systems using optical detectors were used for the surface stress measurements. We demonstrated the positive, label-free detection of Influenza A at concentrations below 10(6) viruses per ml. In contrast to hemagglutination assays, cantilever sensors are label free, in situ, and rapid (less than 30 min), and they require minimal or nearly no sample preparation. PMID:26215598

  18. Detection of Acute Bee Paralysis Virus and Black Queen Cell Virus from Honeybees by Reverse Transcriptase PCR

    PubMed Central

    Benjeddou, Mongi; Leat, Neil; Allsopp, Mike; Davison, Sean

    2001-01-01

    A reverse transcriptase PCR (RT-PCR) assay was developed for the detection of acute bee paralysis virus (ABPV) and black queen cell virus (BQCV), two honeybee viruses. Complete genome sequences were used to design unique PCR primers within a 1-kb region from the 3′ end of both genomes to amplify a fragment of 900 bp from ABPV and 700 bp from BQCV. The combined guanidinium thiocyanate and silica membrane method was used to extract total RNA from samples of healthy and laboratory-infected bee pupae. In a blind test, RT-PCR successfully identified the samples containing ABPV and BQCV. Sensitivities were approximately 1,600 genome equivalents of purified ABPV and 130 genome equivalents of BQCV. PMID:11319129

  19. Relationship between airborne detection of influenza A virus and the number of infected pigs

    PubMed Central

    Corzo, Cesar A.; Romagosa, Anna; Dee, Scott; Gramer, Marie; Morrison, Robert B; Torremorell, Montserrat

    2012-01-01

    Influenza A virus infects a wide range of species including both birds and mammals (including humans). One of the key routes by which the virus can infect populations of animals is by aerosol transmission. This study explored the relationship between number of infected pigs and the probability of detecting influenza virus RNA in bioaerosols through the course of an acute infection. Bioaerosols were collected using a cyclonic collector in two groups of 7 week-old pigs that were experimentally infected by exposure with a contact infected pig (seeder pig). After contact exposure, individual pig nasal swab samples were collected daily and air samples were collected three times per day for 8 days. All samples were tested for influenza by real-time reverse transcriptase (RRT)-PCR targeting the influenza virus matrix gene. All pigs' nasal swabs became influenza virus RRT-PCR positive upon exposure to the infected seeder pig. Airborne influenza was detected in 28/43 (65%) air samples. The temporal dynamics of influenza virus detection in air samples was in close agreement with the nasal shedding pattern in the infected pigs. First detection of positive bioaerosols happened at 1 day post contact (DPC). Positive bioaerosols were consistently detected between 3 and 6 DPC, a time when most pigs were also shedding virus in nasal secretions. Overall, the odds of detecting a positive air sample increased 2.2 times for every additional nasal swab positive pig in the group. In summary, there was a strong relationship between the number of pigs shedding influenza virus in nasal secretions and the generation of bioaerosols during the course of an acute infection. PMID:23164957

  20. Fatal canine distemper infection in a pack of African wild dogs in the Serengeti ecosystem, Tanzania.

    PubMed

    Goller, Katja V; Fyumagwa, Robert D; Nikolin, Veljko; East, Marion L; Kilewo, Morris; Speck, Stephanie; Müller, Thomas; Matzke, Martina; Wibbelt, Gudrun

    2010-12-15

    In 2007, disease related mortality occurred in one African wild dog (Lycaon pictus) pack close to the north-eastern boundary of the Serengeti National Park, Tanzania. Histopathological examination of tissues from six animals revealed that the main pathologic changes comprised interstitial pneumonia and suppurative to necrotizing bronchopneumonia. Respiratory epithelial cells contained numerous eosinophilic intracytoplasmic inclusion bodies and multiple syncytial cells were found throughout the parenchymal tissue, both reacting clearly positive with antibodies against canine distemper virus (CDV) antigen. Phylogenetic analysis based on a 388 nucleotide (nt) fragment of the CDV phosphoprotein (P) gene revealed that the pack was infected with a CDV variant most closely related to Tanzanian variants, including those obtained in 1994 during a CDV epidemic in the Serengeti National Park and from captive African wild dogs in the Mkomazi Game Reserve in 2000. Phylogenetic analysis of a 335-nt fragment of the fusion (F) gene confirmed that the pack in 2007 was infected with a variant most closely related to one variant from 1994 during the epidemic in the Serengeti National Park from which a comparable fragment is available. Screening of tissue samples for concurrent infections revealed evidence of canine parvovirus, Streptococcus equi subsp. ruminatorum and Hepatozoon sp. No evidence of infection with Babesia sp. or rabies virus was found. Possible implications of concurrent infections are discussed. This is the first molecular characterisation of CDV in free-ranging African wild dogs and only the third confirmed case of fatal CDV infection in a free-ranging pack. PMID:20684868

  1. Molecular simultaneous detection of Cherry necrotic rusty mottle virus and Cherry green ring mottle virus by real-time RT-PCR and high resolution melting analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this study, real-time RT-PCR assays were combined with high resolution melting (HRM) analysis for the simultaneous detection of Cherry necrotic rusty mottle virus (CNRMV) and Cherry green ring mottle virus (CGRMV) infection in sweet cherry trees. Detection of CNRMV and CGRMV was performed using a...

  2. Development of a candidate reference material for adventitious virus detection in vaccine and biologicals manufacturing by deep sequencing

    PubMed Central

    Mee, Edward T.; Preston, Mark D.; Minor, Philip D.; Schepelmann, Silke; Huang, Xuening; Nguyen, Jenny; Wall, David; Hargrove, Stacey; Fu, Thomas; Xu, George; Li, Li; Cote, Colette; Delwart, Eric; Li, Linlin; Hewlett, Indira; Simonyan, Vahan; Ragupathy, Viswanath; Alin, Voskanian-Kordi; Mermod, Nicolas; Hill, Christiane; Ottenwälder, Birgit; Richter, Daniel C.; Tehrani, Arman; Jacqueline, Weber-Lehmann; Cassart, Jean-Pol; Letellier, Carine; Vandeputte, Olivier; Ruelle, Jean-Louis; Deyati, Avisek; La Neve, Fabio; Modena, Chiara; Mee, Edward; Schepelmann, Silke; Preston, Mark; Minor, Philip; Eloit, Marc; Muth, Erika; Lamamy, Arnaud; Jagorel, Florence; Cheval, Justine; Anscombe, Catherine; Misra, Raju; Wooldridge, David; Gharbia, Saheer; Rose, Graham; Ng, Siemon H.S.; Charlebois, Robert L.; Gisonni-Lex, Lucy; Mallet, Laurent; Dorange, Fabien; Chiu, Charles; Naccache, Samia; Kellam, Paul; van der Hoek, Lia; Cotten, Matt; Mitchell, Christine; Baier, Brian S.; Sun, Wenping; Malicki, Heather D.

    2016-01-01

    Background Unbiased deep sequencing offers the potential for improved adventitious virus screening in vaccines and biotherapeutics. Successful implementation of such assays will require appropriate control materials to confirm assay performance and sensitivity. Methods A common reference material containing 25 target viruses was produced and 16 laboratories were invited to process it using their preferred adventitious virus detection assay. Results Fifteen laboratories returned results, obtained using a wide range of wet-lab and informatics methods. Six of 25 target viruses were detected by all laboratories, with the remaining viruses detected by 4–14 laboratories. Six non-target viruses were detected by three or more laboratories. Conclusion The study demonstrated that a wide range of methods are currently used for adventitious virus detection screening in biological products by deep sequencing and that they can yield significantly different results. This underscores the need for common reference materials to ensure satisfactory assay performance and enable comparisons between laboratories. PMID:26709640

  3. Metal enhanced fluorescence on nanoporous gold leaf-based assay platform for virus detection.

    PubMed

    Ahmed, Syed Rahin; Hossain, Md Ashraf; Park, Jung Youn; Kim, Soo-Hyung; Lee, Dongyun; Suzuki, Tetsuro; Lee, Jaebeom; Park, Enoch Y

    2014-08-15

    In the present study, a rapid, sensitive and quantitative detection of influenza A virus targeting hemagglutinin (HA) was developed using hybrid structure of quantum dots (QDs) and nanoporous gold leaf (NPGL). NPGL film was prepared by dealloying bimetallic film where its surface morphology and roughness were fairly controlled. Anti-influenza A virus HA antibody (ab66189) was bound with NPGL and amine (-NH2) terminated QDs. These biofunctionalized NPGL and QDs formed a complex with the influenza virus A/Beijing/262/95 (H1N1) and the photoluminescence (PL) intensities of QDs were linearly correlated with the concentrations of the virus up to 1ng/mL while no PL was observed in the absence of the virus, or in bovine serum albumin (BSA, 1µg/mL) alone. In addition, it was demonstrated that this assay detected successfully influenza virus A/Yokohama/110/2009 (H3N2) that is isolated from a clinical sample, at a concentration of ca. 50 plaque forming units (PFU)/mL. This detection limit is 2-order more sensitive than a commercially available rapid influenza diagnostic test. From these results, the proposed assay may offer a new strategy to monitor influenza virus for public health. PMID:24607620

  4. Respiratory Virus Detection and Clinical Diagnosis in Children Attending Day Care

    PubMed Central

    Moe, Nina; Pedersen, Bård; Nordbø, Svein Arne; Skanke, Lars Høsøien; Krokstad, Sidsel; Smyrnaios, Anastasios; Døllner, Henrik

    2016-01-01

    Background Respiratory viruses often have been studied in children with respiratory tract infection (RTI), but less knowledge exists about viruses in asymptomatic children. We have studied the occurrence of a broad panel of respiratory viruses in apparently healthy children attending day care, taking into account the influence of possible confounding factors, such as age, clinical signs of respiratory tract infection (RTI), location (day-care section) and season. Methods We have studied 161 children in two day-care centers, each with separate sections for younger and older children, during four autumn and winter visits over a two-year period. A total of 355 clinical examinations were performed, and 343 nasopharyngeal samples (NPS) were analyzed by semi-quantitative, real-time, polymerase chain reaction (PCR) tests for 19 respiratory pathogens. Result Forty-three percent of all NPS were PCR-positive for ≥ 1 of 13 virus species, with high species variation during visits. Rhinovirus 26% (88/343 NPS), enterovirus 12% (40/343) and parechovirus 9% (30/343) were detected in every visit, and the rates varied in relation to age, day-care section and season. Ten other viruses were detected in ≤ 3% of the NPS. Generally, viruses occurred together in the NPS. In 24% (79/331) of the clinical examinations with available NPS, the children had clear signs of RTI, while in 41% (135/331) they had mild signs, and in 35% (117/331) the children had no signs of RTI. Moreover, viruses were found in 70% (55/79) of children with clear signs of RTI, in 41% (55/135) with mild signs and in 30% (35/117) without any signs of RTI (p < 0.001). Conclusions Positive PCR tests for respiratory viruses, particularly picornaviruses, were frequently detected in apparently healthy children attending day care. Virus detection rates were related to age, presence of clinical signs of RTI, location in day care and season. PMID:27433803

  5. Optimization of the virus concentration method using polyethyleneimine-conjugated magnetic beads and its application to the detection of human hepatitis A, B and C viruses.

    PubMed

    Uchida, Eriko; Kogi, Mieko; Oshizawa, Tadashi; Furuta, Birei; Satoh, Koei; Iwata, Akiko; Murata, Mitsuhiro; Hikata, Mikio; Yamaguchi, Teruhide

    2007-07-01

    To enhance the sensitivity of virus detection by polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR), a novel virus concentration method using polyethyleneimine (PEI)-conjugated magnetic beads was developed in our previous study. However, several viruses could not be concentrated by this method. In this paper, the conditions of virus concentration were optimized to concentrate a wide range of viruses more efficiently. The PEI beads adsorbed viruses more efficiently than other cationic polymers, and the optimum virus concentration was obtained under weak acidic conditions. Mass spectrometric analysis revealed that several serum proteins, such as complement type 3, complement type 4 and immunoglobulin M (IgM), were co-adsorbed by the PEI beads, suggesting that the beads may adsorb viruses not only by direct adsorption, but also via immune complex formation. This hypothesis was confirmed by the result that poliovirus, which PEI beads could not adsorb directly, could be concentrated by the beads via immune complex formation. On the other hand, hepatitis A (HAV) and hepatitis C (HCV) viruses were adsorbed directly by PEI beads almost completely. Like poliovirus, hepatitis B virus (HBV) was concentrated efficiently by the addition of anti-HBV IgM. In conclusion, virus concentration using PEI beads is a useful method to concentrate a wide range of viruses and can be used to enhance the sensitivity of detection of HAV, HBV and HCV. PMID:17433454

  6. Infectious laryngotracheitis virus (ILTV) vaccine intake evaluation by detection of virus amplification in feather pulps of vaccinated chickens.

    PubMed

    Davidson, I; Raibshtein, I; Altori, A; Elkin, N

    2016-03-18

    Infectious laryngotracheitis (ILT) is a respiratory disease of poultry caused by an alphaherpesvirus, ILTV. The live vaccine is applied worldwide by drinking water or by the respiratory route, and by the vent application in Israel. No system of direct evaluation of the efficacy of vaccination exists today, except of antibody elicitation, which is an indirect indication of vaccination intake and might happen due to environment exposure. We suggest for the first time an assay for evaluating the accuracy of the vaccination process by spotting the spread of the live vaccine systemically, namely by virus detection in the feather shafts of the vaccinated birds. The feathers are particularly beneficial as they are easy to collect, non-lethal for the bird, therefore advantageous for monitoring purposes. Moreover, the continuous survey of the vaccine virus unveiled the different kinetics of viremia by the different vaccination routes; while after the vent vaccination the systemic viremia peaks during the first week afterwards, after two consecutive vaccine administration by drinking water with 6 day interval, the vireamia peaks only after the second administration. A robust amplification was needed because the vaccine ILTV was present in the bird in minute quantities compared to the wild-type virus. For the vaccine virus identification in feather shafts a nested real-time PCR for the TK ILTV gene was developed. The sensitivity of detection of the nested rtPCR was greater by 1000 compared to conventional nested PCR and 10 times that real-time PCR. PMID:26784685

  7. Multiplex PCR for the detection of African cassava mosaic virus and East African cassava mosaic Cameroon virus in cassava.

    PubMed

    Alabi, Olufemi J; Kumar, P Lava; Naidu, Rayapati A

    2008-12-01

    A multiplex PCR was developed for simultaneous detection of African cassava mosaic virus (ACMV) and East African cassava mosaic Cameroon virus (EACMCV) in cassava affected with cassava mosaic disease (CMD). One set of three primers consisting of an upstream primer common for both viruses and two down stream virus-specific primers were designed for simultaneous amplification of 368 base pair (bp) and 650 bp DNA fragments specific to the replicase gene of ACMV and EACMCV, respectively. Similarly, a second set of three primers were designed for simultaneous amplification of 540 bp and 655 bp fragments specific to the coat protein gene of EACMCV and ACMV, respectively. Primers that can amplify a 171 bp fragment of the large subunit of ribulose bisphosphate carboxylase oxygenase L were included as an internal control in these assays to determine the reliability of multiplex PCR. A simplified, cost-effective and rapid sample preparation method was adapted in place of the conventional plant DNA extraction procedure for multiplex PCR detection of ACMV and EACMCV. The method was validated using CMD-infected cassava samples obtained from farmers' fields in Nigeria. The multiplex PCR is useful for reliable assessment of the prevalence of CMBs in epidemiological studies and for crop improvement and phytosanitary programs in African countries. PMID:18789974

  8. How Does Sampling Methodology Influence Molecular Detection and Isolation Success in Influenza A Virus Field Studies?

    PubMed

    Latorre-Margalef, Neus; Avril, Alexis; Tolf, Conny; Olsen, Björn; Waldenström, Jonas

    2016-02-01

    Wild waterfowl are important reservoir hosts for influenza A virus (IAV) and a potential source of spillover infections in other hosts, including poultry and swine. The emergence of highly pathogenic avian influenza (HPAI) viruses, such as H5N1 and H5N8, and subsequent spread along migratory flyways prompted the initiation of several programs in Europe, North America, and Africa to monitor circulation of HPAI and low-pathogenicity precursor viruses (low-pathogenicity avian influenza [LPAI] viruses). Given the costs of maintaining such programs, it is essential to establish best practice for field methodologies to provide robust data for epidemiological interpretation. Here, we use long-term surveillance data from a single site to evaluate the influence of a number of parameters on virus detection and isolation of LPAI viruses. A total of 26,586 samples (oropharyngeal, fecal, and cloacal) collected from wild mallards were screened by real-time PCR, and positive samples were subjected to isolation in embryonated chicken eggs. The LPAI virus detection rate was influenced by the sample type: cloacal/fecal samples showed a consistently higher detection rate and lower cycle threshold (Ct) value than oropharyngeal samples. Molecular detection was more sensitive than isolation, and virus isolation success was proportional to the number of RNA copies in the sample. Interestingly, for a given Ct value, the isolation success was lower in samples from adult birds than in those from juveniles. Comparing the results of specific real-time reverse transcriptase (RRT)-PCRs and of isolation, it was clear that coinfections were common in the investigated birds. The effects of sample type and detection methods warrant some caution in interpretation of the surveillance data. PMID:26655759

  9. How Does Sampling Methodology Influence Molecular Detection and Isolation Success in Influenza A Virus Field Studies?

    PubMed Central

    Avril, Alexis; Tolf, Conny; Olsen, Björn

    2015-01-01

    Wild waterfowl are important reservoir hosts for influenza A virus (IAV) and a potential source of spillover infections in other hosts, including poultry and swine. The emergence of highly pathogenic avian influenza (HPAI) viruses, such as H5N1 and H5N8, and subsequent spread along migratory flyways prompted the initiation of several programs in Europe, North America, and Africa to monitor circulation of HPAI and low-pathogenicity precursor viruses (low-pathogenicity avian influenza [LPAI] viruses). Given the costs of maintaining such programs, it is essential to establish best practice for field methodologies to provide robust data for epidemiological interpretation. Here, we use long-term surveillance data from a single site to evaluate the influence of a number of parameters on virus detection and isolation of LPAI viruses. A total of 26,586 samples (oropharyngeal, fecal, and cloacal) collected from wild mallards were screened by real-time PCR, and positive samples were subjected to isolation in embryonated chicken eggs. The LPAI virus detection rate was influenced by the sample type: cloacal/fecal samples showed a consistently higher detection rate and lower cycle threshold (Ct) value than oropharyngeal samples. Molecular detection was more sensitive than isolation, and virus isolation success was proportional to the number of RNA copies in the sample. Interestingly, for a given Ct value, the isolation success was lower in samples from adult birds than in those from juveniles. Comparing the results of specific real-time reverse transcriptase (RRT)-PCRs and of isolation, it was clear that coinfections were common in the investigated birds. The effects of sample type and detection methods warrant some caution in interpretation of the surveillance data. PMID:26655759

  10. Detection of Australasian Flavivirus encephalitic viruses using rapid fluorogenic TaqMan RT-PCR assays.

    PubMed

    Pyke, Alyssa T; Smith, Ina L; van den Hurk, Andrew F; Northill, Judith A; Chuan, Teck F; Westacott, Alan J; Smith, Greg A

    2004-05-01

    The development of single, sensitive, fluorogenic reverse transcriptase-polymerase chain reaction (TaqMan) assays were required for the rapid and specific detection of three encephalitic viruses found in the Australasian region, namely; Japanese encephalitis virus (JEV), Murray Valley encephalitis virus (MVEV), and Kunjin virus (KUNV). Primers and a fluorogenic probe were individually designed to be complementary to a nucleotide region encompassing the 3' terminus of the nonstructural (NS) 5 gene and a portion of the 3' untranslated region (NS5-3'UTR) of each of the viral genomes respectively. Synthetically produced primer and probe controls were developed to minimize the likelihood of contamination and generation of false positives. Viral RNA from singly infected mosquitoes could be detected in pools of 1000 mosquitoes and positive mosquito pools collected from the field have been identified using each assay, indicating a high level of sensitivity and suitability for use in mosquito surveillance programs. In addition, the JEV TaqMan assay has been used to detect successfully viral RNA in sentinel pig serum samples. These assays potentially offer superior and timely detection of encephalitic viruses from surveillance samples, which is essential for the rapid implementation of vector control measures and continued monitoring of virus activity in the Australasian region. PMID:15041213

  11. Ultrasensitive Detection of Ebola Virus Oligonucleotide Based on Upconversion Nanoprobe/Nanoporous Membrane System.

    PubMed

    Tsang, Ming-Kiu; Ye, WeiWei; Wang, Guojing; Li, Jingming; Yang, Mo; Hao, Jianhua

    2016-01-26

    Ebola outbreaks are currently of great concern, and therefore, development of effective diagnosis methods is urgently needed. The key for lethal virus detection is high sensitivity, since early-stage detection of virus may increase the probability of survival. Here, we propose a luminescence scheme of assay consisting of BaGdF5:Yb/Er upconversion nanoparticles (UCNPs) conjugated with oligonucleotide probe and gold nanoparticles (AuNPs) linked with target Ebola virus oligonucleotide. As a proof of concept, a homogeneous assay was fabricated and tested, yielding a detection limit at picomolar level. The luminescence resonance energy transfer is ascribed to the spectral overlapping of upconversion luminescence and the absorption characteristics of AuNPs. Moreover, we anchored the UCNPs and AuNPs on a nanoporous alumina (NAAO) membrane to form a heterogeneous assay. Importantly, the detection limit was greatly improved, exhibiting a remarkable value at the femtomolar level. The enhancement is attributed to the increased light-matter interaction throughout the nanopore walls of the NAAO membrane. The specificity test suggested that the nanoprobes were specific to Ebola virus oligonucleotides. The strategy combining UCNPs, AuNPs, and NAAO membrane provides new insight into low-cost, rapid, and ultrasensitive detection of different diseases. Furthermore, we explored the feasibility of clinical application by using inactivated Ebola virus samples. The detection results showed great potential of our heterogeneous design for practical application. PMID:26720408

  12. Detection of Nipah virus RNA in fruit bat (Pteropus giganteus) from India.

    PubMed

    Yadav, Pragya D; Raut, Chandrashekhar G; Shete, Anita M; Mishra, Akhilesh C; Towner, Jonathan S; Nichol, Stuart T; Mourya, Devendra T

    2012-09-01

    The study deals with the survey of different bat populations (Pteropus giganteus, Cynopterus sphinx, and Megaderma lyra) in India for highly pathogenic Nipah virus (NiV), Reston Ebola virus, and Marburg virus. Bats (n = 140) from two states in India (Maharashtra and West Bengal) were tested for IgG (serum samples) against these viruses and for virus RNAs. Only NiV RNA was detected in a liver homogenate of P. giganteus captured in Myanaguri, West Bengal. Partial sequence analysis of nucleocapsid, glycoprotein, fusion, and phosphoprotein genes showed similarity with the NiV sequences from earlier outbreaks in India. A serum sample of this bat was also positive by enzyme-linked immunosorbent assay for NiV-specific IgG. This is the first report on confirmation of Nipah viral RNA in Pteropus bat from India and suggests the possible role of this species in transmission of NiV in India. PMID:22802440

  13. Detection of Serum Antibodies to Borna Disease Virus in Patients with Psychiatric Disorders

    NASA Astrophysics Data System (ADS)

    Rott, R.; Herzog, S.; Fleischer, B.; Winokur, A.; Amsterdam, J.; Dyson, W.; Koprowski, H.

    1985-05-01

    Borna disease virus causes a rare meningoencephalitis in horses and sheep and has been shown to produce behavioral effects in some species. The possibility that the Borna virus is associated with mental disorders in humans was evaluated by examining serum samples from 979 psychiatric patients and 200 normal volunteers for the presence of Borna virus-specific antibodies. Antibodies were detected by the indirect immunofluorescence focus assay. Antibodies to the virus were demonstrated in 16 of the patients but none of the normal volunteers. The patients with the positive serum samples were characterized by having histories of affective disorders, particularly of a cyclic nature. Further studies are needed to define the possible involvement of Borna virus in human psychiatric disturbances.

  14. Detection of Epstein-Barr virus in leucoreduced blood products.

    PubMed

    Trottier, H; Delage, G; Hu, J; Robitaille, N; Buteau, C; Tucci, M; Lacroix, J; Alfieri, C

    2016-02-01

    This study examined the prevalence of three human herpesviruses (HHV), namely HHV-4 (Epstein-Barr virus/EBV), HHV-6b and HHV-7 in leucoreduced blood products obtained from the Sainte-Justine Hospital blood bank. A total of 100 specimens, including 34 red blood cell concentrates, 33 platelet bags and 33 plasma units, were collected and screened by a sensitive PCR assay using virus-specific primers. Positive units were then retested by quantitative PCR. Of the 100 specimens, one platelet unit tested positive for EBV. PMID:26383177

  15. Combining reverse-transcription multiplex PCR and microfluidic electrophoresis to simultaneously detect seven mosquito-transmitted zoonotic encephalomyelitis viruses.

    PubMed

    Wang, Yu; Ostlund, Eileen N; Jun, Yang; Nie, Fu-Ping; Li, Ying-Guo; Johnson, Donna J; Lin, Rui; Li, Zheng-Guo

    2016-06-01

    Several mosquito-transmitted viruses are causative agents for zoonotic encephalomyelitis. Rapid identification of these viruses in mosquito populations is an effective method for surveying these diseases. To detect multiple mosquito-transmitted viral agents, including West Nile virus, Saint Louis encephalitis virus, Venezuelan equine encephalomyelitis virus, Western equine encephalomyelitis virus, Eastern equine encephalomyelitis virus, Highlands J virus and Japanese encephalitis virus, an assay using multiplex reverse-transcription PCR combined with microfluidic electrophoresis was developed and evaluated. Tailed nested primers were used in the assay to amplify specific viral genomic segments, and products with specific length were further analyzed by using a microfluidic electrophoresis chip. The assay exhibited good specificity and analytical sensitivity (10(2) copies/µL). This technology can be helpful in the quarantine and surveillance of exotic encephalomyelitis viruses which are transmitted by mosquitoes. PMID:27256022

  16. SimulFluor Respiratory Screen for Rapid Detection of Multiple Respiratory Viruses in Clinical Specimens by Immunofluorescence Staining

    PubMed Central

    Landry, Marie L.; Ferguson, David

    2000-01-01

    A new rapid direct immunofluorescence assay (DFA) respiratory screen reagent for detection of seven common respiratory viruses (respiratory syncytial virus [RSV], influenza A and B viruses, parainfluenza virus types 1 to 3, and adenovirus) was compared with standard single or dual DFA reagents and culture. In total, 1,531 respiratory samples were adequate for testing with both SimulFluor Respiratory Screen (RS) reagent (Chemicon International, Temecula, Calif.) and single or dual DFA reagents. The RS DFA reagent detected 367 (98.4%) and single or dual DFA reagents detected 368 (98.7%) of 373 DFA-positive samples. In addition, the RS DFA reagent was equivalent to or better than culture for detection of all viruses except adenovirus. Only 15 of 799 (1.9%) RS-negative samples inoculated into cell cultures yielded respiratory virus isolates (one RSV, five influenza A virus, two influenza B virus, one parainfluenza virus, and six adenovirus). Sixty-six other virus isolates (13 rhinovirus, 24 cytomegalovirus, 28 herpes simplex virus type 1, and 1 enterovirus) were also recovered in culture. With cytospin preparation of slides, only 7.5% of samples submitted were deemed inadequate for DFA. The availability of a rapid DFA screening reagent for detection of multiple common respiratory viruses within 1 to 2 h of sample collection should be of great benefit in terms of patient management and infection control. PMID:10655371

  17. Application of Directigen FLU-A for the detection of influenza A virus in human and nonhuman specimens.

    PubMed Central

    Ryan-Poirier, K A; Katz, J M; Webster, R G; Kawaoka, Y

    1992-01-01

    Directigen FLU-A, a new enzyme immunoassay membrane test, rapidly detects influenza A virus antigen in specimens from patients. Nasopharyngeal washes and pharyngeal gargles were used to determine the effectiveness of the assay as applied to different types of routinely collected clinical samples. All specimens had been previously shown to contain influenza A virus by virus isolation in tissue culture. Directigen FLU-A was 90% sensitive (95% confidence interval, 56 to 99.7%) with nasopharyngeal washes but only 39% sensitive (95% confidence interval, 17 to 64%) with pharyngeal gargles (P = 0.018) when used with samples containing similar amounts of infectious virus (50% tissue culture infective dose, 1.0 to 4.5). The intensity of the positive reaction with Directigen FLU-A did not correlate with the amount of virus in the specimens. Directigen FLU-A was found to detect cell-associated antigen more readily than free virus; only 20 infected cells were required to identify cell-associated influenza A virus antigen, whereas the limit of detection for free virus was 1.63 x 10(3) infectious virus particles. These findings suggest that Directigen FLU-A detects the cell-associated antigen present in clinical specimens rather than free virus. In addition, Directigen FLU-A detected avian and swine influenza A viruses in both cloacal swabs (75% sensitivity) and swine lung homogenates (86% sensitivity), indicating its potential usefulness in the surveillance of nonhuman influenza A viruses. PMID:1583103

  18. Detection of West Nile virus RNA from the louse fly Icosta americana (Diptera: Hippoboscidae).

    PubMed

    Farajollahi, Ary; Crans, Wayne J; Nickerson, Diane; Bryant, Patricia; Wolf, Bruce; Glaser, Amy; Andreadis, Theodore G

    2005-12-01

    West Nile virus (WNV) was detected by Taqman reverse transcription-polymerase chain reaction in 4 of 85 (4.7%) blood-engorged (n = 2) and unengorged (n = 2) Icosta americana (Leach) hippoboscid flies that were collected from wild raptors submitted to a wildlife rehabilitation center in Mercer County, NJ, in 2003. This report represents an additional detection of WNV in a nonculicine arthropod in North America and the first documented detection of the virus in unengorged hippoboscid flies, further suggesting a possible role that this species may play in the transmission of WNV in North America. PMID:16506578

  19. Use of S1 nuclease in deep sequencing for detection of double-stranded RNA viruses.

    PubMed

    Shimada, Saya; Nagai, Makoto; Moriyama, Hiromitsu; Fukuhara, Toshiyuki; Koyama, Satoshi; Omatsu, Tsutomu; Furuya, Tetsuya; Shirai, Junsuke; Mizutani, Tetsuya

    2015-09-01

    Metagenomic approach using next-generation DNA sequencing has facilitated the detection of many pathogenic viruses from fecal samples. However, in many cases, majority of the detected sequences originate from the host genome and bacterial flora in the gut. Here, to improve efficiency of the detection of double-stranded (ds) RNA viruses from samples, we evaluated the applicability of S1 nuclease on deep sequencing. Treating total RNA with S1 nuclease resulted in 1.5-28.4- and 10.1-208.9-fold increases in sequence reads of group A rotavirus in fecal and viral culture samples, respectively. Moreover, increasing coverage of mapping to reference sequences allowed for sufficient genotyping using analytical software. These results suggest that library construction using S1 nuclease is useful for deep sequencing in the detection of dsRNA viruses. PMID:25843154

  20. Real-time RT-PCR Assay for Detection and Differentiation of Citrus Tristeza Virus Isolates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Multiplex one step real time RT-PCR assays using TaqMan probes were developed for detection and strain differentiation of Citrus tristeza virus (CTV). For broad spectrum CTV detection, a TaqMan primer and Cy5-labeled probe were designed using CP gene sequences. An internal control was developed us...

  1. Real-time RT-PCR assay for detection and differentiation of Citrus tristeza virus isolates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    For universal detection of Citrus tristeza virus (CTV) strains by real time RT-PCR, a protocol was developed based on a set of primers and a Cy5-labeled TaqMan probe. This test included primers and a TET-labeled TaqMan probe selected on the mitochondrial nad5 gene for the simultaneous detection of ...

  2. Genetic diversity and frequency of bovine viral diarrhea virus (BVDV) detected in cattle in Turkey

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rapid detection and culling of persistently infected animals and efficacious vaccination are key factors to control bovine viral diarrhea virus (BVDV) infections in cattle. The aim of this study was to investigate frequency of detection of persistently infected cattle and examine the diversity of bo...

  3. Simultaneous detection of papaya ringspot virus, papaya leaf distortion mosaic virus, and papaya mosaic virus by multiplex real-time reverse transcription PCR.

    PubMed

    Huo, P; Shen, W T; Yan, P; Tuo, D C; Li, X Y; Zhou, P

    2015-12-01

    Both the single infection of papaya ringspot virus (PRSV), papaya leaf distortion mosaic virus (PLDMV) or papaya mosaic virus (PapMV) and double infection of PRSV and PLDMV or PapMV which cause indistinguishable symptoms, threaten the papaya industry in Hainan Island, China. In this study, a multiplex real-time reverse transcription PCR (RT-PCR) was developed to detect simultaneously the three viruses based on their distinctive melting temperatures (Tms): 81.0±0.8°C for PRSV, 84.7±0.6°C for PLDMV, and 88.7±0.4°C for PapMV. The multiplex real-time RT-PCR method was specific and sensitive in detecting the three viruses, with a detection limit of 1.0×10(1), 1.0×10(2), and 1.0×10(2) copies for PRSV, PLDMV, and PapMV, respectively. Indeed, the reaction was 100 times more sensitive than the multiplex RT-PCR for PRSV, and 10 times more sensitive than multiplex RT-PCR for PLDMV. Field application of the multiplex real-time RT-PCR demonstrated that some non-symptomatic samples were positive for PLDMV by multiplex real-time RT-PCR but negative by multiplex RT-PCR, whereas some samples were positive for both PRSV and PLDMV by multiplex real-time RT-PCR assay but only positive for PLDMV by multiplex RT-PCR. Therefore, this multiplex real-time RT-PCR assay provides a more rapid, sensitive and reliable method for simultaneous detection of PRSV, PLDMV, PapMV and their mixed infections in papaya. PMID:26666186

  4. [Real-time PCR Detection Method for the Reston Subtype of the Ebola Virus].

    PubMed

    Xu, Lili; Bao, Linlin; Gu, Songzhi; Qin, Chuan

    2015-05-01

    We aimed to develop a real-time polymerase chain reaction (PCR) detection method for the Reston subtype of the Ebola virus. The NP gene of the Reston subtype of the Ebola virus was selected as the detection object. Sequences of different subtypes of Ebola viruses were aligned using Clustal W software. The most unique and conserved regions of the Reston subtype of the Ebola virus were recruited as candidate sequences for specific primers. Primer Express and Primer Premier 5. 0 software were used to filter the optimal pair of primers for detection. Real-time PCR was carried out using optimized parameters and positive DNA prepared by serial (tenfold) dilution of a recombinant plasmid and by plotting a standard curve. In addition, the reproducibility, accuracy, and specificity of the assay were tested. Results showed that the sensitivity of detection of the Reston subtype of the Ebola virus by real-time PCR could reached 10(2) copies/microL. The linear relationship (R2) reached 0.997, the slope of the standard curve was -0.3101, and amplification efficiency was 110.145%. A sharp and narrow melting peak appeared at 79.94 degrees C for all standards in different dilutions. In conclusion, a fast and sensitive real-time PCR detection system for the Reston subtype of the Ebola virus was developed. This system could be used as a supplementary diagnostic and monitoring approach for basic and clinical studies on the Reston subtype of the Ebola virus. The detection system does not require expensive technology or specialist operators. PMID:26470534

  5. Molecular Detection of Some Strawberry Viruses in Egypt

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Strawberry plants exhibiting distinct virus-like symptoms (stunting, mottling, yellowing, vein clearing, vein necrosis and vein banding) were collected from strawberry production fields and nurseries in Qalubia Governorate, Egypt (about 20 km north of Cairo). Plants of 'Festival' and 'Sweet Charlie'...

  6. Corvidae feather pulp and West Nile virus detection

    USGS Publications Warehouse

    Docherty, D.E.; Romaine Long, R.; Griffin, Katie M.; Saito, E.K.

    2004-01-01

    We evaluated cloacal swab, vascular pulp of flight feather, and kidney and spleen pool samples from carcasses of members of the family Corvidae as sources of West Nile virus (WNV). The cloacal swab, kidney and spleen pool, and feather pulp were the source of WNV in 38%, 43%, and 77%, respectively, of the carcasses.

  7. "Detection of bluetongue virus in Culicoides cell cultures"

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bluetongue virus (BTV) infects sheep, cattle and other ruminants and is transmitted by Culicoides spp. of biting midges. Cell lines derived from C. sonorensis have been developed at the Arthropod-borne Animal Diseases Research Laboratory. These cell lines have been shown to support BTV replication, ...

  8. DETECTION OF ENTERIC VIRUSES IN TREATED DRINKING WATER

    EPA Science Inventory

    The occurrence of viruses in conventionally treated drinking water derived from a heavily polluted source was evaluated by collecting and analyzing 38 large volume (65 to 756 liter) samples of water from a 9m3/sec (205 mgd) water treatment plant. Samples of raw, clarified, filter...

  9. Detection and survey of coffee ringspot virus in Brazil.

    PubMed

    Ramalho, T O; Figueira, A R; Wang, R; Jones, O; Harris, L E; Goodin, M M

    2016-02-01

    Coffee ringspot virus (CoRSV) a member of the proposed genus "Dichorhavirus", was surveyed on commercial and research farms spanning an area responsible for the majority of Coffea arabica production in Brazil. Virus-infected plants were found at one hundred percent of locations (n = 45) sampled. All cultivars, regardless of cherry color, were found to serve as hosts, suggesting that there is limited resistance in commercially employed germplasm. Reverse transcription PCR analysis revealed that the virus is contained within symptomatic lesions, with little systemic spread throughout leaves. Phylogenetic analysis based on the ORF1 (nucleocapsid) gene identified a strong geo-spatial relationship among isolates, which clustered into three clades. Despite low genetic diversity among isolates, variation in symptom expression was observed in the experimental host Chenopodium quinoa. Our analyses support the hypothesis that the spread of CoRSV is constrained by the clonal expansion of thelytokous populations of Brevipalpus phoenicis. The widespread occurrence of this virus suggests that it is much more prevalent than previously thought. PMID:26553342

  10. Detection of Cucurbit leaf crumple virus in Florida cucurbits

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cucurbit leaf crumple virus (CuLCrV) is a recently described begomovirus causing significant economic losses in cucurbit production in the western US. This research provides the first report of CuLCrV or any other begomovirus infecting cucurbits in Florida. ...

  11. New targets for expedient detection of viruses within shellfish

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previously our laboratory developed an expedient method for extraction of viral RNA from food-borne virus contaminated bivalve shellfish, termed the GPTT protocol. This protocol utilizes either whole shellfish or dissected digestive diverticula. This four step protocol utilizes a high pH glycine or...

  12. Potential virus detection and intervention methods for molluscan shellfish

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Norovirus is the number one cause of foodborne illness in the Unites States, causing an estimated 9 million cases/yr. Hepatitis A is uncommon in the US but can result in serious illness. Bivalve shellfish are efficient bioconcentrators of these viruses from contaminated growing waters. Consequentl...

  13. Development of a blocking latex agglutination test for the detection of antibodies to chicken anemia virus.

    PubMed

    Trinh, Dai Quang; Ogawa, Haruko; Bui, Vuong Nghia; Nguyen, Tham Thi Hong; Gronsang, Dulyatad; Baatartsogt, Tugsbaatar; Kizito, Mugimba Kahoza; AboElkhair, Mohammed; Yamaguchi, Shigeo; Nguyen, Viet Khong; Imai, Kunitoshi

    2015-09-01

    A blocking latex agglutination test (b-LAT) developed in this study was evaluated for the detection of antibodies against chicken anemia virus (CAV) in chickens. Polystyrene latex beads were coupled with a neutralizing monoclonal antibody (mAb) to CAV (mAb-beads). When mAb-beads were mixed with antigens prepared from the lysate of MDCC-MSB1 cells infected with CAV, agglutination occurred. A short pre-incubation of CAV antigens with CAV-specific antiserum inhibited the agglutination of mAb-beads. The test results were obtained within 5min. The specificity of b-LAT was evaluated using sera from specific pathogen-free chickens and sera containing antibodies to avian influenza virus, Newcastle disease virus, infectious bursal disease virus, and Marek's disease virus; nonspecific agglutination and cross-reactivity with antibodies to unrelated viruses were not observed. The examination of 94 serum samples collected from commercial breeder chickens of various ages (17-63 weeks) revealed good agreement (93.6%, Kappa value=0.82) between b-LAT and a virus neutralization test, known to be most sensitive and specific in the detection of antibodies to CAV. These results indicate that b-LAT, a simple and rapid test, is a useful and reliable tool in CAV serology. PMID:25952731

  14. Giant Magnetoresistance-based Biosensor for Detection of Influenza A Virus

    PubMed Central

    Krishna, Venkatramana D.; Wu, Kai; Perez, Andres M.; Wang, Jian-Ping

    2016-01-01

    We have developed a simple and sensitive method for the detection of influenza A virus based on giant magnetoresistance (GMR) biosensor. This assay employs monoclonal antibodies to viral nucleoprotein (NP) in combination with magnetic nanoparticles (MNPs). Presence of influenza virus allows the binding of MNPs to the GMR sensor and the binding is proportional to the concentration of virus. Binding of MNPs onto the GMR sensor causes change in the resistance of sensor, which is measured in a real time electrical readout. GMR biosensor detected as low as 1.5 × 102 TCID50/mL virus and the signal intensity increased with increasing concentration of virus up to 1.0 × 105 TCID50/mL. This study showed that the GMR biosensor assay is relevant for diagnostic application since the virus concentration in nasal samples of influenza virus infected swine was reported to be in the range of 103 to 105 TCID50/mL. PMID:27065967

  15. Detection of Evolutionarily Distinct Avian Influenza A Viruses in Antarctica

    PubMed Central

    Vijaykrishna, Dhanasekaran; Butler, Jeffrey; Baas, Chantal; Maurer-Stroh, Sebastian; Silva-de-la-Fuente, M. Carolina; Medina-Vogel, Gonzalo; Olsen, Bjorn; Kelso, Anne; Barr, Ian G.; González-Acuña, Daniel

    2014-01-01

    ABSTRACT Distinct lineages of avian influenza viruses (AIVs) are harbored by spatially segregated birds, yet significant surveillance gaps exist around the globe. Virtually nothing is known from the Antarctic. Using virus culture, molecular analysis, full genome sequencing, and serology of samples from Adélie penguins in Antarctica, we confirmed infection by H11N2 subtype AIVs. Their genetic segments were distinct from all known contemporary influenza viruses, including South American AIVs, suggesting spatial separation from other lineages. Only in the matrix and polymerase acidic gene phylogenies did the Antarctic sequences form a sister relationship to South American AIVs, whereas distant phylogenetic relationships were evident in all other gene segments. Interestingly, their neuraminidase genes formed a distant relationship to all avian and human influenza lineages, and the polymerase basic 1 and polymerase acidic formed a sister relationship to the equine H3N8 influenza virus lineage that emerged during 1963 and whose avian origins were previously unknown. We also estimated that each gene segment had diverged for 49 to 80 years from its most closely related sequences, highlighting a significant gap in our AIV knowledge in the region. We also show that the receptor binding properties of the H11N2 viruses are predominantly avian and that they were unable to replicate efficiently in experimentally inoculated ferrets, suggesting their continuous evolution in avian hosts. These findings add substantially to our understanding of both the ecology and the intra- and intercontinental movement of Antarctic AIVs and highlight the potential risk of an incursion of highly pathogenic AIVs into this fragile environment. PMID:24803521

  16. Detection and transmission of Carrot torrado virus, a novel putative member of the Torradovirus genus.

    PubMed

    Rozado-Aguirre, Zuriñe; Adams, Ian; Collins, Larissa; Fox, Adrian; Dickinson, Matthew; Boonham, Neil

    2016-09-01

    A new Torradovirus tentatively named Carrot torrado virus (CaTV) was an incidental finding following a next generation sequencing study investigating internal vascular necrosis in carrot. The closest related viruses are Lettuce necrotic leaf curl virus (LNLCV) found in the Netherlands in 2011 and Motherwort yellow mottle virus (MYMoV) found in Korea in 2014. Primers for reverse transcriptase-PCR (RT-PCR) and RT-qPCR were designed with the aim of testing for the presence of virus in plant samples collected from the field. Both methods successfully amplified the target from infected samples but not from healthy control samples. The specificity of the CaTV assay was also checked against other known carrot viruses and no cross-reaction was seen. A comparative study between methods showed RT-qPCR was the most reliable method, giving positive results in samples where RT-PCR fails. Evaluation of the Ct values following RT-qPCR and a direct comparison demonstrated this was due to improved sensitivity. The previous published Torradovirus genus specific RT-PCR primers were tested and shown to detect CaTV. Also, virus transmission experiments carried out suggest that unlike other species of the same genus, Carrot torrado virus could be aphid-transmitted. PMID:27260658

  17. Detection of infectious laryngotracheitis virus by real-time PCR in naturally and experimentally infected chickens.

    PubMed

    Zhao, Yan; Kong, Congcong; Cui, Xianlan; Cui, Hongyu; Shi, Xingming; Zhang, Xiaomin; Hu, Shunlei; Hao, Lianwei; Wang, Yunfeng

    2013-01-01

    Infectious laryngotracheitis (ILT) is an acute, highly contagious upper-respiratory infectious disease of chickens. In this study, a real-time PCR method was developed for fast and accurate detection and quantitation of ILTV DNA of chickens experimentally infected with ILTV strain LJS09 and naturally infected chickens. The detection lower limit of the assay was 10 copies of DNA. There were no cross reactions with the DNA and RNA of infectious bursal disease virus, chicken anemia virus, reticuloendotheliosis virus, avian reovirus, Newcastle disease virus, and Marek's disease virus. The real-time PCR was reproducible as the coefficients of variation of reproducibility of the intra-assay and the inter-assay were less than 2%. The real-time PCR was used to detect the levels of the ILTV DNA in the tissues of specific pathogen free (SPF) chickens infected with ILTV at different times post infection. ILTV DNA was detected by real-time PCR in the heart, liver, spleen, lung, kidney, larynx, tongue, thymus, glandular stomach, duodenum, pancreatic gland, small intestine, large intestine, cecum, cecal tonsil, bursa of Fabricius, and brain of chickens in the infection group and the contact-exposure group. The sensitivity, specificity, and reproducibility of the ILTV real-time PCR assay revealed its suitability for detection and quantitation of ILTV in the samples from clinically and experimentally ILTV infected chickens. PMID:23840745

  18. Detection of Infectious Laryngotracheitis Virus by Real-Time PCR in Naturally and Experimentally Infected Chickens

    PubMed Central

    Zhao, Yan; Kong, Congcong; Cui, Xianlan; Cui, Hongyu; Shi, Xingming; Zhang, Xiaomin; Hu, Shunlei; Hao, Lianwei; Wang, Yunfeng

    2013-01-01

    Infectious laryngotracheitis (ILT) is an acute, highly contagious upper-respiratory infectious disease of chickens. In this study, a real-time PCR method was developed for fast and accurate detection and quantitation of ILTV DNA of chickens experimentally infected with ILTV strain LJS09 and naturally infected chickens. The detection lower limit of the assay was 10 copies of DNA. There were no cross reactions with the DNA and RNA of infectious bursal disease virus, chicken anemia virus, reticuloendotheliosis virus, avian reovirus, Newcastle disease virus, and Marek's disease virus. The real-time PCR was reproducible as the coefficients of variation of reproducibility of the intra-assay and the inter-assay were less than 2%. The real-time PCR was used to detect the levels of the ILTV DNA in the tissues of specific pathogen free (SPF) chickens infected with ILTV at different times post infection. ILTV DNA was detected by real-time PCR in the heart, liver, spleen, lung, kidney, larynx, tongue, thymus, glandular stomach, duodenum, pancreatic gland, small intestine, large intestine, cecum, cecal tonsil, bursa of Fabricius, and brain of chickens in the infection group and the contact-exposure group. The sensitivity, specificity, and reproducibility of the ILTV real-time PCR assay revealed its suitability for detection and quantitation of ILTV in the samples from clinically and experimentally ILTV infected chickens. PMID:23840745

  19. Optical biosensor system for the quick and reliable detection of virus infections: VIROSENS

    NASA Astrophysics Data System (ADS)

    Proll, Günther; Hartjes, Anja; Sinclair, Alexander; Markovic, Goran; Pröll, Florian; Patel, Pranav; Niedrig, Matthias

    2014-10-01

    Viral infections are of special threat because they can induce severe courses of disease but only few medical treatments are available. Because of socio-economic and climate changes, increased worldwide mobility and population growth, the risk of newly occurring and quickly spreading viral pathogens has increased. A diagnosis of these diseases at an early stage is essential for a quick risk assessment and a proper health management as well as patient's treatment in an optimal way. Currently, the diagnosis of such diseases is based on time consuming and costly detection methods that can only be performed by specially trained personnel in laboratories at specific security levels. Aim of the project VIROSENS is the development of a biosensor platform that can specifically detect virus particles as well as virus-specific antibodies out of biological matrices like blood, serum, plasma and other body fluids. For this purpose, a disposable cartridge for such antibody- and virus-arrays is designed and developed within the project. The optical detection of viruses is performed with a portable device that will be benchmarked and evaluated concerning currently used standard detection methods in terms of its analytical performance. Within this project, a novel combination of serological tests and direct detection of virus particles will be developed, which will provide faster and more reliable results than presently available and used test systems.

  20. A Novel Optical Biosensing System Using Mach-Zehnder-Type Optical Waveguide for Influenza Virus Detection.

    PubMed

    Sakamoto, Hiroaki; Minpou, Yuma; Sawai, Takayuki; Enami, Yasufumi; Suye, Shin-Ichiro

    2016-02-01

    In order to minimize the damage from viral epidemics, early detection of the causative agent of a viral epidemic and prevention of its immediate spread are urgent social demands. Therefore, in this study, we evaluated the utility of a Mach-Zehnder-type optical waveguide as a sensing device for influenza virus detection. However, it is impossible to detect a 100-nm-size virus using a sol-gel optical biosensor because sol-gel glass has a pore size of only a few nanometers, which makes it impossible for the virus to diffuse into the silica thin film. In order to construct the influenza-specific Mach-Zehnder optical biosensor for influenza detection, a stable antibody immobilization method with resulting high density on the sol-gel surface is strongly required. In this study, the sol-gel glass surface was modified with amino and carboxyl groups, and an anti-H1N1/HA1 antibody was covalently immobilized using a cross-linking agent. We successfully prepared a carboxyl-modified sol-gel surface, using NHS/EDC as the cross-linker, for antibody immobilization, and confirmed the detection of influenza virus using the antibody-immobilized sol-gel glass. After treatment with a 100 μg/mL influenza virus solution for 15 min, a peak wavelength shift (~24 nm) was observed in the output light spectrum. PMID:26498024

  1. Detecting Protein-Protein Interactions in Vesicular Stomatitis Virus Using a Cytoplasmic Yeast Two Hybrid System

    PubMed Central

    Moerdyk-Schauwecker, Megan; DeStephanis, Darla; Hastie, Eric; Grdzelishvili, Valery Z.

    2011-01-01

    Summary Protein-protein interactions play an important role in many virus-encoded functions and in virus-host interactions. While a “classical” yeast two-hybrid system (Y2H) is one of the most common techniques to detect such interactions, it has a number of limitations, including a requirement for the proteins of interest to be relocated to the nucleus. Modified Y2H, such as the Sos recruitment system (SRS), which detect interactions occurring in the cytoplasm rather than the nucleus, allow proteins from viruses replicating in the cytoplasm to be tested in a more natural context. In this study, a SRS was used to detect interactions involving proteins from vesicular stomatitis virus (VSV), a prototypic non-segmented negative strand RNA (NNS) virus. All five full-length VSV proteins, as well as several truncated proteins, were screened against each other. Using the SRS, most interactions demonstrated previously involving VSV phosphoprotein, nucleocapsid (N) and large polymerase proteins were confirmed independently, while difficulties were encountered using the membrane associated matrix and glycoproteins. A human cDNA library was also screened against VSV N protein and one cellular protein, SFRS18, was identified which interacted with N in this context. The system presented can be redesigned easily for studies in other less tractable NNS viruses. PMID:21320532

  2. Organic Substances Interfere with Reverse Transcription-Quantitative PCR-Based Virus Detection in Water Samples

    PubMed Central

    Katayama, Hiroyuki; Furumai, Hiroaki

    2014-01-01

    Reverse transcription (RT)-PCR-based virus detection from water samples is occasionally hampered by organic substances that are coconcentrated during virus concentration procedures. To characterize these organic substances, samples containing commercially available humic acid, which is known to inhibit RT-PCR, and river water samples were subjected to adsorption-elution-based virus concentration using an electronegative membrane. In this study, the samples before, during, and after the concentration were analyzed in terms of organic properties and virus detection efficiencies. Two out of the three humic acid solutions resulted in RT-quantitative PCR (qPCR) inhibition that caused >3-log10-unit underestimation of spiked poliovirus. Over 60% of the organics contained in the two solutions were recovered in the concentrate, while over 60% of the organics in the uninhibited solution were lost during the concentration process. River water concentrates also caused inhibition of RT-qPCR. Organic concentrations in the river water samples increased by 2.3 to 3.9 times after the virus concentration procedure. The inhibitory samples contained organic fractions in the 10- to 100-kDa size range, which are suspected to be RT-PCR inhibitors. According to excitation-emission matrices, humic acid-like and protein-like fractions were also recovered from river water concentrates, but these fractions did not seem to affect virus detection. Our findings reveal that detailed organic analyses are effective in characterizing inhibitory substances. PMID:25527552

  3. Acanthamoeba polyphaga mimivirus Stability in Environmental and Clinical Substrates: Implications for Virus Detection and Isolation

    PubMed Central

    de Almeida, Gabriel M.; Campos, Rafael K.; Boratto, Paulo V. M.; Franco-Luiz, Ana P. M.; La Scola, Bernard; Ferreira, Paulo C. P.; Kroon, Erna G.; Abrahão, Jônatas S.

    2014-01-01

    Viruses are extremely diverse and abundant and are present in countless environments. Giant viruses of the Megavirales order have emerged as a fascinating research topic for virologists around the world. As evidence of their ubiquity and ecological impact, mimiviruses have been found in multiple environmental samples. However, isolation of these viruses from environmental samples is inefficient, mainly due to methodological limitations and lack of information regarding the interactions between viruses and substrates. In this work, we demonstrate the long-lasting stability of mimivirus in environmental (freshwater and saline water) and hospital (ventilator plastic device tube) substrates, showing the detection of infectious particles after more than 9 months. In addition, an enrichment protocol was implemented that remarkably increased mimivirus detection from all tested substrates, including field tests. Moreover, biological, morphological and genetic tests revealed that the enrichment protocol maintained mimivirus particle integrity. In conclusion, our work demonstrated the stability of APMV in samples of environmental and health interest and proposed a reliable and easy protocol to improve giant virus isolation. The data presented here can guide future giant virus detection and isolation studies. PMID:24498379

  4. Detection of human enteric viruses in stream water with RT-PCR and cell culture.

    USGS Publications Warehouse

    Denis-Mize, K.; Fout, G.S.; Dahling, D.R.; Francy, D.S.

    2004-01-01

    A multiplex RT-PCR method was used to measure virus occurrence at five stream water sites that span a range of hydroclimatic, water-quality, and land-use characteristics. The performance of the molecular method was evaluated in comparison with traditional cell culture and Escherichia coli membrane filtration assays. The study incorporated multiple quality controls and included a control for virus recovery during the sampling procedure as well as controls to detect potentially false-negative and false-positive data. Poliovirus recovery ranged from 16 to 65% and was variable, even in samples collected within the same stream. All five sites were positive for viruses by both molecular and cell culture-based virus assays. Enteroviruses, reoviruses, rotaviruses, and hepatitis A viruses were detected, but the use of the quality controls proved critical for interpretation of the molecular data. All sites showed evidence of faecal contamination, and culturable viruses were detected in four samples that would have met the US Environmental Protection Agency's recommended E. coli guideline for safe recreational water.

  5. A 1-Year Study on the Detection of Human Enteric Viruses in New Caledonia.

    PubMed

    Kaas, Laetitia; Gourinat, Ann-Claire; Urbès, Florence; Langlet, Jérémie

    2016-03-01

    Human enteric viruses occur in high concentrations in wastewater and can contaminate receiving environmental waters. Due to the lack of data on the prevalence of enteric viruses in New Caledonia, the presence and the concentrations of enteric viruses in wastewater and seawater were determined. Untreated wastewater and seawater samples were collected monthly for 1 year from a wastewater treatment plant (WWTP) and from the WWTP's outlet, located directly on a popular recreational beach. Samples were tested for norovirus genogroups I and II (NoV GI and GII), astroviruses (AsV), sapoviruses (SaV), enteroviruses (EV), hepatitis A viruses (HAV), rotaviruses (RoV), human adenoviruses (HAdV) and human polyomaviruses (HPyV). To support these data, faecal samples from cases of gastroenteritis were tested for the first time for NoV and detected in the population. NoV GI, NoV GII, EV, SaV, HAdV and HPyV were detected in all wastewaters, RoV in 75% and AsV in 67%. HAV were not detected in wastewater. Overall, 92% of seawater samples were positive for at least one virus. HPyV were detected most frequently in 92% of samples and at concentrations up to 7.7 × 10(3) genome copies/L. NoV GI, NoV GII, EV, SaV, RoV and HAdV were found in 33, 66, 41, 33, 16 and 66% of seawater samples, respectively. AsV were not detected in seawater. This study reports for the first time the presence of NoV and other enteric viruses in New Caledonia and highlights the year-round presence of enteric viruses in the seawater of a popular beach. PMID:26670603

  6. Canine distemper outbreak in raccoons suggests pathogen interspecies transmission amongst alien and native carnivores in urban areas from Germany.

    PubMed

    Rentería-Solís, Zaida; Förster, Christine; Aue, Angelika; Wittstatt, Ulrich; Wibbelt, Gudrun; König, Matthias

    2014-11-01

    From December 2012 to May 2013, an outbreak occurred among urban wild carnivores from Berlin. We collected 97 free-ranging raccoons from the city area. PCR assays, histopathology and immunohistochemistry confirmed canine distemper virus (CDV) infection in 74 raccoons. Phylogenetic analysis of haemagglutinin gene fragments (1767 nucleotides) of CDV isolated from four raccoons showed close relation to CDV isolates from foxes from Germany and a domestic dog from Hungary; all belonging to the "Europe" lineage of CDV. These study results suggest an inter-species transmission of CDV as the origin for the outbreak among the raccoon population. Implications for domestic pets and suggested interspecies transmission between urban wildlife and raccoons are discussed. This is the first major outbreak of CDV amongst free-ranging raccoons in Europe. PMID:25258173

  7. Targeted virus detection in next-generation sequencing data using an automated e-probe based approach.

    PubMed

    Visser, Marike; Burger, Johan T; Maree, Hans J

    2016-08-01

    The use of next-generation sequencing for plant virus detection is rapidly expanding, necessitating the development of bioinformatic pipelines to support analysis of these large datasets. Pipelines need to be easy implementable to mitigate potential insufficient computational infrastructure and/or skills. In this study user-friendly software was developed for the targeted detection of plant viruses based on e-probes. It can be used for both custom e-probe design, as well as screening preloaded probes against raw NGS data for virus detection. The pipeline was compared to de novo assembly-based virus detection in grapevine and produced comparable results, requiring less time and computational resources. The software, named Truffle, is available for the design and screening of e-probes tailored for user-specific virus species and data, along with preloaded probe-sets for grapevine virus detection. PMID:27209446

  8. Diversity in the enteric viruses detected in outbreaks of gastroenteritis from Mumbai, Western India.

    PubMed

    Chitambar, Shobha; Gopalkrishna, Varanasi; Chhabra, Preeti; Patil, Pooja; Verma, Harsha; Lahon, Anismrita; Arora, Ritu; Tatte, Vaishali; Ranshing, Sujata; Dhale, Ganesh; Kolhapure, Rajendra; Tikute, Sanjay; Kulkarni, Jagannath; Bhardwaj, Renu; Akarte, Sulbha; Pawar, Sashikant

    2012-03-01

    Faecal specimens collected from two outbreaks of acute gastroenteritis that occurred in southern Mumbai, India in March and October, 2006 were tested for seven different enteric viruses. Among the 218 specimens tested, 95 (43.6%) were positive, 73 (76.8%) for a single virus and 22 (23.2%) for multiple viruses. Single viral infections in both, March and October showed predominance of enterovirus (EV, 33.3% and 40%) and rotavirus A (RVA, 33.3% and 25%). The other viruses detected in these months were norovirus (NoV, 12.1% and 10%), rotavirus B (RVB, 12.1% and 10%), enteric adenovirus (AdV, 6.1% and 7.5%), Aichivirus (AiV, 3% and 7.5%) and human astrovirus (HAstV, 3% and 0%). Mixed viral infections were largely represented by two viruses (84.6% and 88.9%), a small proportion showed presence of three (7.7% and 11%) and four (7.7% and 0%) viruses in the two outbreaks. Genotyping of the viruses revealed predominance of RVA G2P[4], RVB G2 (Indian Bangladeshi lineage), NoV GII.4, AdV-40, HAstV-8 and AiV B types. VP1/2A junction region based genotyping showed presence of 11 different serotypes of EVs. Although no virus was detected in the tested water samples, examination of both water and sewage pipelines in gastroenteritis affected localities indicated leakages and possibility of contamination of drinking water with sewage water. Coexistence of multiple enteric viruses during the two outbreaks of gastroenteritis emphasizes the need to expand such investigations to other parts of India. PMID:22690171

  9. Diversity in the Enteric Viruses Detected in Outbreaks of Gastroenteritis from Mumbai, Western India

    PubMed Central

    Chitambar, Shobha; Gopalkrishna, Varanasi; Chhabra, Preeti; Patil, Pooja; Verma, Harsha; Lahon, Anismrita; Arora, Ritu; Tatte, Vaishali; Ranshing, Sujata; Dhale, Ganesh; Kolhapure, Rajendra; Tikute, Sanjay; Kulkarni, Jagannath; Bhardwaj, Renu; Akarte, Sulbha; Pawar, Sashikant

    2012-01-01

    Faecal specimens collected from two outbreaks of acute gastroenteritis that occurred in southern Mumbai, India in March and October, 2006 were tested for seven different enteric viruses. Among the 218 specimens tested, 95 (43.6%) were positive, 73 (76.8%) for a single virus and 22 (23.2%) for multiple viruses. Single viral infections in both, March and October showed predominance of enterovirus (EV, 33.3% and 40%) and rotavirus A (RVA, 33.3% and 25%). The other viruses detected in these months were norovirus (NoV, 12.1% and 10%), rotavirus B (RVB, 12.1% and 10%), enteric adenovirus (AdV, 6.1% and 7.5%), Aichivirus (AiV, 3% and 7.5%) and human astrovirus (HAstV, 3% and 0%). Mixed viral infections were largely represented by two viruses (84.6% and 88.9%), a small proportion showed presence of three (7.7% and 11%) and four (7.7% and 0%) viruses in the two outbreaks. Genotyping of the viruses revealed predominance of RVA G2P[4], RVB G2 (Indian Bangladeshi lineage), NoV GII.4, AdV-40, HAstV-8 and AiV B types. VP1/2A junction region based genotyping showed presence of 11 different serotypes of EVs. Although no virus was detected in the tested water samples, examination of both water and sewage pipelines in gastroenteritis affected localities indicated leakages and possibility of contamination of drinking water with sewage water. Coexistence of multiple enteric viruses during the two outbreaks of gastroenteritis emphasizes the need to expand such investigations to other parts of India. PMID:22690171

  10. Detection and typing of viruses using broadly sensitive cocktail-PCR and mass spectrometric cataloging: demonstration with dengue virus.

    PubMed

    Gijavanekar, Charul; Drabek, Rafal; Soni, Mithil; Jackson, George W; Strych, Ulrich; Fox, George E; Fofanov, Yuriy; Willson, Richard C

    2012-07-01

    Virus detection and taxonomic identification of serotypes, strains, or genotypes provide important information relevant for diagnosis, and for the epidemiological characterization and tracking of new strains in an endemic region. In the specific case of dengue virus, rapid serotype identification can also be useful in the treatment of secondary infections that may cause the more severe dengue hemorrhagic fever and dengue shock syndrome. In this work, dengue virus was used as a model to test a new approach of combining broadly sensitive RT-PCR amplification of nearly any virus strain with subsequent serotype- and finer-level identification by mass spectrometry. PCR primers were appended with promoter sequences, such that the resulting PCR products could be transcribed into RNA. RNA fragments generated by guanosine-specific RNase T(1) digestion were analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Viral serotypes were identified by comparing the pattern of observed fragment masses to a mass database. The database was created by computationally fragmenting 2517 dengue strains after each guanosine residue using the same primers. Computationally, all 2517 strains in the mass database were correctly identified at the serotype level from the predicted PCR product. The methodology was successfully demonstrated experimentally by identifying the serotypes of eight test strains using mosquito cell cultures infected with strains of all four serotypes and with full-length cDNA clones. PMID:22579629

  11. Detection of Sindbis and Inkoo Virus RNA in Genetically Typed Mosquito Larvae Sampled in Northern Sweden

    PubMed Central

    Tingström, Olov; Wesula Lwande, Olivia; Näslund, Jonas; Spyckerelle, Iris; Engdahl, Cecilia; Von Schoenberg, Pontus; Ahlm, Clas; Evander, Magnus

    2016-01-01

    Abstract Introduction: Mosquito-borne viruses have a widespread distribution across the globe and are known to pose serious threats to human and animal health. The maintenance and dissemination of these viruses in nature are driven through horizontal and vertical transmission. In the temperate climate of northern Sweden, there is a dearth of knowledge on whether mosquito-borne viruses that occur are transmitted transovarially. To gain a better understanding of mosquito-borne virus circulation and maintenance, mosquito larvae were sampled in northern Sweden during the first and second year after a large outbreak of Ockelbo disease in 2013 caused by Sindbis virus (SINV). Materials and Methods: A total of 3123 larvae were sampled during the summers of 2014 and 2015 at multiple sites in northern Sweden. The larvae were homogenized and screened for viruses using RT-PCR and sequencing. Species identification of selected larvae was performed by genetic barcoding targeting the cytochrome C oxidase subunit I gene. Results and Discussion: SINV RNA was detected in mosquito larvae of three different species, Ochlerotatus (Oc.) communis, Oc. punctor, and Oc. diantaeus. Inkoo virus (INKV) RNA was detected in Oc. communis larvae. This finding suggested that these mosquitoes could support transovarial transmission of SINV and INKV. Detection of virus in mosquito larva may serve as an early warning for emerging arboviral diseases and add information to epidemiological investigations before, during, and after outbreaks. Furthermore, our results demonstrated the relevance of genetic barcoding as an attractive and effective method for mosquito larva typing. However, further mosquito transmission studies are needed to ascertain the possible role of different mosquito species and developmental stages in the transmission cycle of arboviruses. PMID:27159120

  12. Performance of Various Commercial Assays for the Detection of Toscana Virus Antibodies

    PubMed Central

    Litzba, Nadine; Lo, Modou Moustapha; Aydoğan, Sibel; Saygan, Mehmet B.; Us, Dürdal; Weidmann, Manfred; Niedrig, Matthias

    2011-01-01

    Abstract Introduction Sandfly fever virus (SFV) serotypes sandfly fever Naples virus, sandfly fever Sicilian virus, and sandfly fever Cyprus virus cause febrile diseases, whereas Toscana virus (TOSV) is responsible for aseptic meningoencephalitis. Diagnosis and surveillance of TOSV depend heavily on virus serology, and various commercial assays utilizing various antigen sources and formats have been available. The aim of this study was to perform comparative evaluation of commercially available serological assays for anti-TOSV immunoglobulins. Materials and Methods A collection of 120 sera from healthy blood donors from an endemic region, previously identified to be reactive for antibodies against various SFV serotypes by indirect immunofluorescence test (IIFT), was reevaluated for IgG/IgM via IIFT, enzyme-linked immunosorbent assay, and an immunoblot assay manufactured by Euroimmun, Diesse, and Mikrogen, respectively. Virus neutralization test (VNT) was performed for 99 sera using standard TOSV, sandfly fever Sicilian virus, and sandfly fever Naples virus strains. Results A total of 89 samples (74.2%) were reactive for TOSV IgG in at least one of the commercial assays, and 31 samples (31.3%) were reactive in VNT for various SFV serotypes. Average percentage agreements among commercial assays and between VNT and the commercial assays were noted as 57.8% and 62.6%, respectively. No significant correlation between assay results and VNT titers was observed. SFV IgM antibodies were detected in a total of eight samples (6.7%) via IIFT, which were nonreactive in enzyme-linked immunosorbent assay and VNT. Discussion Commercial diagnostic immunoassays displayed slight to fair agreement for TOSV IgG as assessed via kappa and percentage agreement values. The results could only be confirmed via virus neutralization in a portion of the samples, and overall agreement between the commercial assays and VNT was slight. Commercial assays such as immunoblot can be used in addition

  13. Detection of Common Respiratory Viruses and Mycoplasma pneumoniae in Patient-Occupied Rooms in Pediatric Wards

    PubMed Central

    Wan, Gwo-Hwa; Huang, Chung-Guei; Chung, Fen-Fang; Lin, Tzou-Yien; Tsao, Kuo-Chien; Huang, Yhu-Chering

    2016-01-01

    Abstract Few studies have assessed viral contamination in the rooms of hospital wards. This cross-sectional study evaluated the air and objects in patient-occupied rooms in pediatric wards for the presence of common respiratory viruses and Mycoplasma pneumoniae. Air samplers were placed at a short (60–80 cm) and long (320 cm) distance from the head of the beds of 58 pediatric patients, who were subsequently confirmed to be infected with enterovirus (n = 17), respiratory syncytial virus (RSV) (n = 13), influenza A virus (n = 13), adenovirus (n = 9), or M pneumoniae (n = 6). Swab samples were collected from the surfaces of 5 different types of objects in the patients’ rooms. All air and swab samples were analyzed via real-time quantitative polymerase chain reaction assay for the presence of the above pathogens. All pathogens except enterovirus were detected in the air, on the objects, or in both locations in the patients’ rooms. The detection rates of influenza A virus, adenovirus, and M pneumoniae for the long distance air sampling were 15%, 67%, and 17%, respectively. Both adenovirus and M pneumoniae were detected at very high rates, with high concentrations, on all sampled objects. The respiratory pathogens RSV, influenza A virus, adenovirus, and M pneumoniae were detected in the air and/or on the objects in the pediatric ward rooms. Appropriate infection control measures should be strictly implemented when caring for such patients. PMID:27057827

  14. Detection of Common Respiratory Viruses and Mycoplasma pneumoniae in Patient-Occupied Rooms in Pediatric Wards.

    PubMed

    Wan, Gwo-Hwa; Huang, Chung-Guei; Chung, Fen-Fang; Lin, Tzou-Yien; Tsao, Kuo-Chien; Huang, Yhu-Chering

    2016-04-01

    Few studies have assessed viral contamination in the rooms of hospital wards. This cross-sectional study evaluated the air and objects in patient-occupied rooms in pediatric wards for the presence of common respiratory viruses and Mycoplasma pneumoniae.Air samplers were placed at a short (60-80 cm) and long (320 cm) distance from the head of the beds of 58 pediatric patients, who were subsequently confirmed to be infected with enterovirus (n = 17), respiratory syncytial virus (RSV) (n = 13), influenza A virus (n = 13), adenovirus (n = 9), or M pneumoniae (n = 6). Swab samples were collected from the surfaces of 5 different types of objects in the patients' rooms. All air and swab samples were analyzed via real-time quantitative polymerase chain reaction assay for the presence of the above pathogens.All pathogens except enterovirus were detected in the air, on the objects, or in both locations in the patients' rooms. The detection rates of influenza A virus, adenovirus, and M pneumoniae for the long distance air sampling were 15%, 67%, and 17%, respectively. Both adenovirus and M pneumoniae were detected at very high rates, with high concentrations, on all sampled objects.The respiratory pathogens RSV, influenza A virus, adenovirus, and M pneumoniae were detected in the air and/or on the objects in the pediatric ward rooms. Appropriate infection control measures should be strictly implemented when caring for such patients. PMID:27057827

  15. Development of methodologies for virus detection in soybean and wheat seeds.

    PubMed

    Botelho, Stephanie R A; Martins, Thais P; Duarte, Macária F; Barbosa, Andreza V; Lau, Douglas; Fernandes, Fernanda R; Sanches, Marcio M

    2016-01-01

    Seeds that contain large amounts of oil, starch, fibers and phenols are the most difficult tissues for RNA extraction. Currently, there are some reports of virus detection in seeds using commercial kits for RNA extraction. However, individual seeds were used, which may not be always suitable for analyses that deal with large amounts of seeds. Sangha [1] described a simple, quick and efficient protocol for RNA extraction and downstream applications in a group of seeds of jatropha (Jatropha curcas), mustard (Brassica sp.) and rice (Oryza sativa). We tested this protocol for soybean (Glycine max), maize (Zea mays), wheat (Triticum aestivum) and triticale (×Triticosecale) seeds and further reverse transcription PCR (RT-PCR)/quantitative real-time PCR (qPCR) in order to have a faster and more practical method for virus detection from seeds than the traditional scheme of seed planting and subsequent Elisa/RT-PCR from leaves. The essential points in the method are:•Some modifications in the protocol [1] were done in order to increase performance: Wheat and triticale seeds are incubated with water prior to maceration. An amount of 1.2 g of dry soybean seeds is used to maceration.•RT-PCR is used for detection of Wheat streak mosaic virus from wheat seeds and RT-qPCR for detection of Soybean mosaic virus from soybean seeds.•The method may be tested for other viruses, however, pre-validation will be needed. PMID:27408831

  16. Polythiophene nanofilms for sensitive fluorescence detection of viruses in drinking water.

    PubMed

    Wankar, Shashwati; Turner, Nicholas W; Krupadam, Reddithota J

    2016-08-15

    Molecular imprints of the tobacco necrosis virus (TNV) have been formed within polythiophene nanofilms with an approximate thickness of 200nm. These films have been electrochemically deposited onto conducting Au surfaces. Upon rebinding, the TNV-polythiophene complex changes the fluorescence intensity of the nanofilm. The fluorescence intensity at 410nm was observed to be proportional to the concentration of viruses in the range of 0.1-10ngL(-1) (0.15-15pg) with the lower calculated detection limit of 2.29ngL(-1) (3.4pg). The intensity of the fluorescence emission is not affected by the thickness of the polythiophene film and the nature of TNV specific binding sites. Kinetic data analyses showed that the nanofilm responds to TNV within 2min; and cross-selectivity studies with tobacco mosaic virus (TMV) showed an excellent specificity for the targeted TNV. These binding experiments demonstrate the potential of fluorescence emission for the specific, label free and rapid detection of viruses using nanofilm sensors. Taking into account the lower limit of detection, the fluorescence sensing reported here is reliable, simple to perform, rapid, cost-effective and offers a sensitive analytical method for virus detection in water resources. PMID:27031187

  17. Comparison of monoclonal antibody-based sandwich enzyme-linked immunosorbent assay and virus isolation for detection of peste des petits ruminants virus in goat tissues and secretions.

    PubMed Central

    Saliki, J T; House, J A; Mebus, C A; Dubovi, E J

    1994-01-01

    A monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (S-ELISA) was developed for specific detection of peste des petits ruminants virus. Compared with virus isolation in Vero cell cultures using 89 paired tissue and secretion samples from six experimentally infected goats, S-ELISA was significantly more sensitive (71.9% versus 65.2%; P < 0.05). The S-ELISA is a suitable alternative to virus isolation. PMID:8051266

  18. PCR and non-isotopic labeling techniques for plant virus detection.

    PubMed

    Fenby, N S; Scott, N W; Slater, A; Elliott, M C

    1995-07-01

    PCR technology permits the detection of viruses at levels several orders of magnitude lower than is possible by other methods. This high sensitivity facilitates detection of virus sequences during the early stages of infection of plants and in soil and vector samples. Early detection of beet necrotic yellow vein virus (BNYVV) in Beta vulgaris is an important part of the strategy for prevention of the spread of rhizomania, a commercially significant disease of sugar beet. A diagnostic test for BNYVV has been developed. This test involves amplification of the viral genome by PCR coupled with non-isotopic labeling and detection of specific sequences. The PCR amplification of BNYVV sequences has been optimized with respect to primer design, sample preparation and reaction conditions. Several non-isotopic labeling strategies for signal amplification have been compared. Hybridization with digoxigenin-labelled cDNA permits the most sensitive detection of PCR products and is the most appropriate method for routine diagnosis. These observations are discussed in the context of the application of PCR for detecting a wide range of viruses. PMID:7580844

  19. Towards detection and diagnosis of Ebola virus disease at point-of-care.

    PubMed

    Kaushik, Ajeet; Tiwari, Sneham; Dev Jayant, Rahul; Marty, Aileen; Nair, Madhavan

    2016-01-15

    Ebola outbreak-2014 (mainly Zaire strain related Ebola virus) has been declared most widely spread deadly persistent epidemic due to unavailability of rapid diagnostic, detection, and therapeutics. Ebola virus disease (EVD), a severe viral hemorrhagic fever syndrome caused by Ebola virus (EBOV) is transmitted by direct contact with the body fluids of infected person and objects contaminated with virus or infected animals. World Health Organization (WHO) has declared EVD epidemic as public health emergency of international concern with severe global economic burden. At fatal EBOV infection stage, patients usually die before the antibody response. Currently, rapid blood tests to diagnose EBOV infection include the antigen or antibodies capture using ELISA and RNA detection using RT/Q-PCR within 3-10 days after the onset of symptoms. Moreover, few nanotechnology-based colorimetric and paper-based immunoassay methods have been recently reported to detect Ebola virus. Unfortunately, these methods are limited to laboratory only. As state-of-the art (SoA) diagnostics time to confirm Ebola infection, varies from 6h to about 3 days, it causes delay in therapeutic approaches. Thus developing a cost-effective, rapid, sensitive, and selective sensor to detect EVD at point-of-care (POC) is certainly worth exploring to establish rapid diagnostics to decide therapeutics. This review highlights SoA of Ebola diagnostics and also a call to develop rapid, selective and sensitive POC detection of EBOV for global health care. We propose that adopting miniaturized electrochemical EBOV immunosensing can detect virus level at pM concentration within ∼40min compared to 3 days of ELISA test at nM levels. PMID:26319169

  20. Surveillance theory applied to virus detection: a case for targeted discovery

    USGS Publications Warehouse

    Bogich, Tiffany L.; Anthony, Simon J.; Nichols, James D.

    2013-01-01

    Virus detection and mathematical modeling have gone through rapid developments in the past decade. Both offer new insights into the epidemiology of infectious disease and characterization of future risk; however, modeling has not yet been applied to designing the best surveillance strategies for viral and pathogen discovery. We review recent developments and propose methods to integrate viral and pathogen discovery and mathematical modeling through optimal surveillance theory, arguing for a more targeted approach to novel virus detection guided by the principles of adaptive management and structured decision-making.

  1. Establishment and application of a multiplex PCR for rapid and simultaneous detection of six viruses in swine.

    PubMed

    Zeng, Zhiyong; Liu, Zhijie; Wang, Weicheng; Tang, Deyuan; Liang, Haiying; Liu, Zhao

    2014-11-01

    A multiplex PCR assay was developed and evaluated subsequently for its effectiveness in simultaneously detecting mixed viral infections of swine. Specific primers were designed and used for testing the six swine viruses: three DNA viruses, including pseudorabies virus (PRV), porcine parvovirus (PPV), and porcine circovirus type 2 (PCV2); three common RNA viruses, including porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), and Japanese encephalitis virus (JEV). This technique has shown to be highly sensitive in that the minimum detection amounts of nucleic acids from PRV, PPV, PCV2, PRRSV, CSFV, and JEV were 6.6, 96, 12.9, 10.5, 51, and 46 pg, respectively. It also was effective for detecting one or multiple viruses in the specimens, such as the lungs, spleens, lymph nodes, and tonsils collected from clinically ill pigs. The multiplex PCR method can detect simultaneously not only infection of the six viruses, but also other swine DNA and RNA viruses. Given its rapidity, specificity, and sensitivity, the multiplex PCR is a useful tool for diagnosing clinically the mixed infections of swine DNA and RNA viruses. PMID:25116201

  2. A multiple RT-PCR assay for simultaneous detection and differentiation of latent viruses and apscarviroids in apple trees.

    PubMed

    Hao, Lu; Xie, Jipeng; Chen, Shanyi; Wang, Shaojie; Gong, Zhuoqun; Ling, Kai-Shu; Guo, Liyun; Fan, Zaifeng; Zhou, Tao

    2016-08-01

    Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV), and Apple stem pitting virus (ASPV) are three latent viruses frequently occurring in apple trees worldwide. In field orchards, these viruses are frequently found in a mixed infection with viroids in the genus Apscarviroid, including Apple scar skin viroid, and Apple dimple fruit viroid. Together these viruses and viroids could cause serious damage to apple fruit production worldwide. Rapid and efficient detection methods are pivotal to identify and select the virus-free propagation material for healthy apple orchard management. In this study a multiplex Reverse Transcription-PCR (RT-PCR) was developed and optimized for simultaneous detection and differentiation of the three latent viruses and apscarviroids. With newly designed specific primers for ACLSV, ASGV, APSV, and EF-1α (as an internal control), and a pair of degenerate primers for apscarviroids, optimized parameters for multiplex RT-PCR were determined. The resulting PCR products from each target virus and viroid could be easily identified because their product sizes differ by at least a 100bp. The multiplex RT-PCR method is expected to detect different variants of the viruses as the test results showed that a variety of isolates from different regions in China gave positive results. To the best of our knowledge, this multiplex RT-PCR assay is the first to simultaneously detect multiple viruses and viroids infecting apple trees in a single reaction tube. This assay, therefore, offers a useful tool for routine certification and quarantine programs. PMID:27054889

  3. Development of single dilution immunoassay to detect E2 protein specific classical swine fever virus antibody.

    PubMed

    Kumar, Rakesh; Barman, Nagendra N; Khatoon, Elina; Kumar, Sachin

    2016-04-01

    Classical swine fever virus (CSFV) is the causative agent of a highly contagious disease in swine. The disease is endemic in different parts of the world and vaccination is the only way to protect pigs from CSFV infection. The virus surface protein E2 is the major immunogenic protein eliciting protective immunity against CSFV infection in swine. The whole virus antigen cannot differentiate CSFV from other pestiviruses as it cross reacts with border disease and bovine viral diarrhoea viruses. Commercial available ELISA is based on the whole CSFV particle and can lead to false positive results. Moreover, the available commercial ELISA is not cost effective. In the present study, a recombinant E2 protein based single serum dilution ELISA was developed which showed enhanced sensitivity, specificity and accuracy as compared to commercial CSFV detection ELISA. The recombinant E2 protein based ELISA could be an alternate to existing diagnostics against CSFV infection in pigs. PMID:27032503

  4. Detection of Corchorus golden mosaic virus Associated with Yellow Mosaic Disease of Jute (Corchorus capsularis).

    PubMed

    Ghosh, Raju; Palit, Paramita; Paul, Sujay; Ghosh, Subrata Kumar; Roy, Anirban

    2012-06-01

    Yellow mosaic disease, caused by a whitefly transmitted New World Begomovirus, named Corchorus golden mosaic virus (CoGMV), is emerging as a serious biotic constraint for jute fibre production in Asia. For rapid and sensitive diagnosis of the Begomovirus associated with this disease, a non-radiolabelled diagnostic probe, developed against the DNA A component of the east Indian isolate of CoGMV, detected the presence of the virus in infected plants and viruliferous whiteflies following Southern hybridization and nucleic acid spot hybridization tests. Presence of the virus was also confirmed when polymerase chain reaction amplification was performed using virus-specific primers on DNA templates isolated from infected plants and viruliferous whiteflies. PMID:23730007

  5. A prospective field evaluation of an enzyme immunoassay: Detection of eastern equine encephalomyelitis virus antigen in pools of Culiseta melanura

    USGS Publications Warehouse

    Scott, T.W.; Olson, J.G.; Lewis, T.E.; Carpenter, J.W.; Lorenz, L.H.; Lembeck, L.A.; Joseph, S.R.; Pagac, B.B.

    1987-01-01

    A prospective field study was conducted to determine the sensitivity and specificity of an enzyme immunoassay (EIA) compared to virus isolation in cell culture for the detection of eastern equine encephalomyelitis (EEE) virus in naturally infected mosquitoes. A total of 10,811 adult female Culiseta melanura were collected in light traps during 1985 from four locations in Maryland. Eastern equine encephalomyelitis virus was isolated from 5 of 495 mosquito pools in African green monkey kidney and baby hamster kidney cell cultures. All five virus-infected pools were detected by the EIA, and all 490 uninfected pools were correctly scored as not containing virus. The EIA did not produce false positive or false negative results. Results support the assertion of previous researchers that the antigen detection EIA is a rapid, sensitive, specific, and simple alternative to traditional bioassays for the detection of EEE virus in mosquitoes.

  6. Comparison of Detection of Bovine Virus Diarrhea Virus Antigen in Various Types of Tissue and Fluid Samples Collected from Persistently Infected Cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bovine viral diarrhea viruses are economically important pathogens of cattle. Most new infections are acquired from animals persistently infected with the virus. Surveillance programs rely on skin biopsies for detection of persistently infected cattle. The purpose of this study was to compare ant...

  7. False-Positive Transcription-Mediated Amplification Assay Detection of West Nile Virus in Blood from a Patient with Viremia Caused by an Usutu Virus Infection▿

    PubMed Central

    Gaibani, Paolo; Pierro, Anna Maria; Cavrini, Francesca; Rossini, Giada; Landini, Maria Paola; Sambri, Vittorio

    2010-01-01

    Detection of West Nile virus (WNV) by nucleic acid amplification technology (NAAT) is used widely to screen blood and organ donations in areas where WNV is endemic. We report a false-positive result of a WNV transcription-mediated amplification assay (TMA) in a patient with viremia that was caused by Usutu virus, a mosquito-borne flavivirus. PMID:20592138

  8. Detection of Zika Virus Infection in Thailand, 2012–2014

    PubMed Central

    Buathong, Rome; Hermann, Laura; Thaisomboonsuk, Butsaya; Rutvisuttinunt, Wiriya; Klungthong, Chonticha; Chinnawirotpisan, Piyawan; Manasatienkij, Wudtichai; Nisalak, Ananda; Fernandez, Stefan; Yoon, In-Kyu; Akrasewi, Passakorn; Plipat, Tanarak

    2015-01-01

    Zika virus (ZIKV) is an emerging mosquito-borne pathogen with reported cases in Africa, Asia, and large outbreaks in the Pacific. No autochthonous ZIKV infections have been confirmed in Thailand. However, there have been several cases reported in travelers returning from Thailand. Here we report seven cases of acute ZIKV infection in Thai residents across the country confirmed by molecular or serological testing including sequence data. These endemic cases, combined with previous reports in travelers, provide evidence that ZIKV is widespread throughout Thailand. PMID:26101272

  9. Henipavirus microsphere immuno-assays for detection of antibodies against Hendra virus.

    PubMed

    McNabb, Leanne; Barr, J; Crameri, G; Juzva, S; Riddell, S; Colling, A; Boyd, V; Broder, C; Wang, L-F; Lunt, R

    2014-05-01

    Hendra and Nipah viruses (HeV and NiV) are closely related zoonotic pathogens of the Paramyxoviridae family. Both viruses belong to the Henipavirus genus and cause fatal disease in animals and humans, though only HeV is endemic in Australia. In general and due to the acute nature of the disease, agent detection by PCR and virus isolation are the primary tools for diagnostic investigations. Assays for the detection of antibodies against HeV are fit more readily for the purpose of surveillance testing in disease epidemiology and to meet certification requirements in the international movement of horses. The first generation indirect ELISA has been affected by non-specific reactions which must be resolved using virus neutralisation serology conducted at laboratory bio-safety level 4 containment (PC4). Recent developments have enabled improvements in the available serology assays. The production of an expressed recombinant truncated HeV G protein has been utilised in ELISA and in Luminex-based multiplexed microsphere assays. In the latter format, two Luminex assays have been developed for use in henipavirus serology: a binding assay (designed for antibody detection and differentiation) and a blocking assay (designed as a surrogate for virus neutralisation). Equine and canine field sera were used to evaluate the two Luminex assays relative to ELISA and virus neutralisation serology. Results showed that Luminex assays can be effective as rapid, sensitive and specific tests for the detection of HeV antibody in horse and dog sera. The tests do not require PC4 containment and are appropriate for high throughput applications as might be required for disease investigations and other epidemiological surveillance. Also, the results show that the Luminex assays detect effectively HeV vaccine-induced antibodies. PMID:24508193

  10. Immunoaffinity concentration and purification of waterborne enteric viruses for detection by reverse transcriptase PCR.

    PubMed Central

    Schwab, K J; De Leon, R; Sobsey, M D

    1996-01-01

    To assess the risks from viral contamination of drinking-water supplies, there is a clear need for methods to directly detect viral pathogens. In this study, we developed a broad-spectrum immunocapture method for concentration and purification of enteric viruses. The method involved indirect antibody capture (AbCap) of intact viruses followed by release of virion genomic RNA and reverse transcriptase PCR for amplification and oligoprobe hybridization for detection. The procedure involved concentrating enteric viruses from large volumes of water by standard filtration-elution techniques with IMDS filters and 1 liter of 1% beef extract-0.05 M glycine (BE/G) as an eluate. The BE/G eluate was concentrated and purified by polyethylene glycol (PEG) precipitation, Pro-Cipitate (a commercially available protein precipitating reagent) precipitation, and a second PEG precipitation to a volume of approximately 500 mu l. Aliquots of the second PEG precipitate were further processed by RNA extraction, AbCap, or cell culture analysis for infectious viruses. The AbCap method was applied to 11 field samples of fecally contaminated surface water. Of the 11 samples, 9 were positive for enteric viruses by AbCap method 4 of 11 samples were positive for enteric viruses by direct RNA extraction of a small aliquot of the second PEG concentrate; and 4 of 11 samples were positive for enteric viruses by measurement of cell culture infectivity. The results of enteric viruses were compared with those for standard bacterial and coliphage indicators of fecal contamination. PMID:8787407

  11. Ultrasensitive Detection of RNA and DNA Viruses Simultaneously Using Duplex UNDP-PCR Assay

    PubMed Central

    Wang, Zengguo; Zhang, Xiujuan; Zhao, Xiaomin; Du, Qian; Chang, Lingling; Tong, Dewen

    2015-01-01

    Mixed infection of multiple viruses is common in modern intensive pig rearing. However, there are no methods available to detect DNA and RNA viruses in the same reaction system in preclinical level. In this study, we aimed to develop a duplex ultrasensitive nanoparticle DNA probe-based PCR assay (duplex UNDP-PCR) that was able to simultaneously detect DNA and RNA viruses in the same reaction system. PCV2 and TGEV are selected as representatives of the two different types of viruses. PCV2 DNA and TGEV RNA were simultaneously released from the serum sample by boiling with lysis buffer, then magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for TGEV and PCV2 were added to form a sandwich-like complex with nucleic acids released from viruses. After magnetic separation, DNA barcodes specific for PCV2 and TGEV were eluted using DTT and characterized by specific PCR assay for specific DNA barcodes subsequently. The duplex UNDP-PCR showed similar sensitivity as that of single UNDP-PCR and was able to detect 20 copies each of PCV2 and TGEV in the serum, showing approximately 250-fold more sensitivity than conventional duplex PCR/RT-PCR assays. No cross-reaction was observed with other viruses. The positive detection rate of single MMPs- and duplex MMPs-based duplex UNDP-PCR was identical, with 29.6% for PCV2, 9.3% for TGEV and 3.7% for PCV2 and TGEV mixed infection. This duplex UNDP-PCR assay could detect TGEV (RNA virus) and PCV2 (DNA virus) from large-scale serum samples simultaneously without the need for DNA/RNA extraction, purification and reverse transcription of RNA, and showed a significantly increased positive detection rate for PCV2 (29%) and TGEV (11.7%) preclinical infection than conventional duplex PCR/RT-PCR. Therefore, the established duplex UNDP-PCR is a rapid and economical detection method, exhibiting high sensitivity, specificity and reproducibility. PMID:26544710

  12. Combined DECS Analysis and Next-Generation Sequencing Enable Efficient Detection of Novel Plant RNA Viruses.

    PubMed

    Yanagisawa, Hironobu; Tomita, Reiko; Katsu, Koji; Uehara, Takuya; Atsumi, Go; Tateda, Chika; Kobayashi, Kappei; Sekine, Ken-Taro

    2016-03-01

    The presence of high molecular weight double-stranded RNA (dsRNA) within plant cells is an indicator of infection with RNA viruses as these possess genomic or replicative dsRNA. DECS (dsRNA isolation, exhaustive amplification, cloning, and sequencing) analysis has been shown to be capable of detecting unknown viruses. We postulated that a combination of DECS analysis and next-generation sequencing (NGS) would improve detection efficiency and usability of the technique. Here, we describe a model case in which we efficiently detected the presumed genome sequence of Blueberry shoestring virus (BSSV), a member of the genus Sobemovirus, which has not so far been reported. dsRNAs were isolated from BSSV-infected blueberry plants using the dsRNA-binding protein, reverse-transcribed, amplified, and sequenced using NGS. A contig of 4,020 nucleotides (nt) that shared similarities with sequences from other Sobemovirus species was obtained as a candidate of the BSSV genomic sequence. Reverse transcription (RT)-PCR primer sets based on sequences from this contig enabled the detection of BSSV in all BSSV-infected plants tested but not in healthy controls. A recombinant protein encoded by the putative coat protein gene was bound by the BSSV-antibody, indicating that the candidate sequence was that of BSSV itself. Our results suggest that a combination of DECS analysis and NGS, designated here as "DECS-C," is a powerful method for detecting novel plant viruses. PMID:27072419

  13. Combined DECS Analysis and Next-Generation Sequencing Enable Efficient Detection of Novel Plant RNA Viruses

    PubMed Central

    Yanagisawa, Hironobu; Tomita, Reiko; Katsu, Koji; Uehara, Takuya; Atsumi, Go; Tateda, Chika; Kobayashi, Kappei; Sekine, Ken-Taro

    2016-01-01

    The presence of high molecular weight double-stranded RNA (dsRNA) within plant cells is an indicator of infection with RNA viruses as these possess genomic or replicative dsRNA. DECS (dsRNA isolation, exhaustive amplification, cloning, and sequencing) analysis has been shown to be capable of detecting unknown viruses. We postulated that a combination of DECS analysis and next-generation sequencing (NGS) would improve detection efficiency and usability of the technique. Here, we describe a model case in which we efficiently detected the presumed genome sequence of Blueberry shoestring virus (BSSV), a member of the genus Sobemovirus, which has not so far been reported. dsRNAs were isolated from BSSV-infected blueberry plants using the dsRNA-binding protein, reverse-transcribed, amplified, and sequenced using NGS. A contig of 4,020 nucleotides (nt) that shared similarities with sequences from other Sobemovirus species was obtained as a candidate of the BSSV genomic sequence. Reverse transcription (RT)-PCR primer sets based on sequences from this contig enabled the detection of BSSV in all BSSV-infected plants tested but not in healthy controls. A recombinant protein encoded by the putative coat protein gene was bound by the BSSV-antibody, indicating that the candidate sequence was that of BSSV itself. Our results suggest that a combination of DECS analysis and NGS, designated here as “DECS-C,” is a powerful method for detecting novel plant viruses. PMID:27072419

  14. Development and characterization of polyclonal peptide antibodies for the detection of Yellow fever virus proteins.

    PubMed

    Stock, N K; Escadafal, C; Achazi, K; Cissé, M; Niedrig, M

    2015-09-15

    There is still a considerable need for development of new tools and methods detecting specific viral proteins for the diagnosis and pathogenesis study of the Yellow fever virus (YFV). This study aimed to develop and characterize polyclonal peptide antisera for detection of YFV-C and -NS1 proteins. The antisera were used further to investigate NS1 protein expression during YFV infection in mammalian cells. YFV target proteins were detected by all antisera in western blot and immunofluorescence assays. No cross-reactivity was observed with Dengue virus, West Nile virus, Tick-borne encephalitis virus and Japanese encephalitis virus. Nuclear localization of the YFV-C protein was demonstrated for the first time. Experiments investigating NS1 expression suggested a potential use of the YFV-NS1 antisera for development of diagnostic approaches targeting the secreted form of the NS1 protein. The antisera described in this study offer new possibilities for use in YFV research and for the development of novel diagnostic tests. PMID:26086983

  15. Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components.

    PubMed

    Pardee, Keith; Green, Alexander A; Takahashi, Melissa K; Braff, Dana; Lambert, Guillaume; Lee, Jeong Wook; Ferrante, Tom; Ma, Duo; Donghia, Nina; Fan, Melina; Daringer, Nichole M; Bosch, Irene; Dudley, Dawn M; O'Connor, David H; Gehrke, Lee; Collins, James J

    2016-05-19

    The recent Zika virus outbreak highlights the need for low-cost diagnostics that can be rapidly developed for distribution and use in pandemic regions. Here, we report a pipeline for the rapid design, assembly, and validation of cell-free, paper-based sensors for the detection of the Zika virus RNA genome. By linking isothermal RNA amplification to toehold switch RNA sensors, we detect clinically relevant concentrations of Zika virus sequences and demonstrate specificity against closely related Dengue virus sequences. When coupled with a novel CRISPR/Cas9-based module, our sensors can discriminate between viral strains with single-base resolution. We successfully demonstrate a simple, field-ready sample-processing workflow and detect Zika virus from the plasma of a viremic macaque. Our freeze-dried biomolecular platform resolves important practical limitations to the deployment of molecular diagnostics in the field and demonstrates how synthetic biology can be used to develop diagnostic tools for confronting global health crises. PAPERCLIP. PMID:27160350

  16. Detection of airborne viruses using electro-aerodynamic deposition and a field-effect transistor

    PubMed Central

    Park, Kyu-Tae; Cho, Dong-Guk; Park, Ji-Woon; Hong, Seunghun; Hwang, Jungho

    2015-01-01

    We report a technique for the detection of aerosolized viruses. Conventional field-effect-transistor (FET)-based techniques use solution-based processes, thus require antibody binding to the detection region of the FET prior to the supply of the analyte. With the method described here, virus–antibody-bound particles are delivered to the FET during detection; therefore, neither a pre-treatment antibody binding step on the FET channel nor washing process for virus–antibody-binding are necessary. Our method is based on the concept that virus–antibody-bound particles are larger than the virus or antibody alone, and thus have larger charge numbers following aerosol charging. When these particles are charged by negative ions and electro-aerodynamically deposited on a substrate, there exists a location on the substrate where neither lone virus nor antibody particles land, and where only virus–antibody-bound particles are deposited. If this location coincides with the channel of the FET, the resulting variation in the current can be used to indicate the existence of a virus. By aerosolizing a mixed solution of the virus and the antibody, only the virus–antibody-bound particles were transported to the swCNT-FET, and the electric current in the swCNT-FET decreased to 30% of that measured with no deposited particles. PMID:26642822

  17. Detection of strawberry vein banding virus by polymerase chain reaction and dot blot hybridization.

    PubMed

    Mráz, I; Petrzik, K; Fránová-Honetslegrová, J; Síp, M

    1997-08-01

    Strawberry vein banding virus (SVBV) is one of seventeen members of the family Caulimoviridae. Natural infection with the virus is known in Fragaria species only. Infections caused by SVBV are often symptomless (1), but their significance increases in mixed infections with strawberry crinkle or strawberry latent C viruses (2,3). This virus has been originally found on strawberries in USA and firstly described by Frazier (4), but it is probably world-wide distributed by planting or breeding materials. SVBV has been observed on cultivated strawberries in North America, Australia, Brazil, Japan (5) and recently in Europe (6,7). The concentration of SVBV in infected plants is usually very low. Its detection by ELISA is impossible because of lack of specific antibodies. Evidence of the caulimovirus nature of SVBV has been confirmed by its circular dsDNA genome, shape and size of viral particles (8), presence of cytoplasmic inclusion bodies typical for caulimoviruses, and distant serological relationship with cauliflower mosaic virus (CaMV, 9). In this paper we present detection of SVBV by combination of two detection methods--polymerase chain reaction (PCR) and dot blot hybridization with a non-radioactive probe. PMID:9391655

  18. Easy and inexpensive molecular detection of dengue, chikungunya and zika viruses in febrile patients.

    PubMed

    Calvo, Eliana P; Sánchez-Quete, Fernando; Durán, Sandra; Sandoval, Isabel; Castellanos, Jaime E

    2016-11-01

    Dengue (DENV), chikungunya (CHIKV) and zika (ZIKV) are arthropod-borne viruses (arboviruses) sharing a common vector, the mosquito Aedes aegypti. At initial stages, patients infected with these viruses have similar clinical manifestations, however, the outcomes and clinical management of these diseases are different, for this reason early and accurate identification of the causative virus is necessary. This paper reports the development of a rapid and specific nested-PCR for detection of DENV, CHIKV and ZIKV infection in the same sample. A set of six outer primers targeting the C-preM, E1, and E gene respectively was used in a multiplex one-step RT-PCR assay, followed by the second round of amplification with specific inner primers for each virus. The specificity of the present assay was validated with positive and negative serum samples for viruses and supernatants of infected cells. The assay was tested using clinical samples from febrile patients. In these samples, we detected mono and dual infections and a case of triple co-infection DENV-CHIKV-ZIKV. This assay might be a useful and an inexpensive tool for detection of these infections in regions where these arboviruses co-circulate. PMID:27477452

  19. Comparison of concentration methods for detection of hepatitis A virus in water samples.

    PubMed

    Qiao, Yuting; Sui, Zhiwei; Hu, Guoliang; Cao, Huabin; Yang, Guoxiang; Li, Yong; Lei, Yongsong; Zhao, Lihua; Chen, Quanjiao

    2016-08-01

    Hepatitis A virus is a pathogen associated with water pollution. Contaminated drinking water can cause hepatitis A outbreaks, lead to economic losses, and even threaten human lives. It is difficult to detect low levels of hepatitis A virus in water, so the virus must be concentrated in order to quantify it accurately. Here, we present a simple, rapid, efficient technique for the concentration and detection of hepatitis A virus in water. Our data showed that adding phosphate-buffered saline to the water, pre-filtering the water, and adding Trizol reagent directly to the filtration membrane can significantly improve concentration efficiency. Of three types of filtration membranes studied (mixed cellulose ester membrane, polyvinylidene fluoride membrane, and nylon membrane), the concentration efficiency using mixed cellulose ester membrane with a 0.1-μm pore size was the highest, reaching 92.62 ± 5.17%. This method was used to concentrate hepatitis A virus in water samples from Donghu Lake. Using SYBR Green real-time reverse transcription polymerase chain reaction analysis, the detection sensitivity of this method reached 10(1) copies/μL and its concentration efficiency reached 79.45 ± 9.88%. PMID:27535067

  20. Patterns of Exposure of Iberian Wolves (Canis lupus) to Canine Viruses in Human-Dominated Landscapes.

    PubMed

    Millán, Javier; López-Bao, José Vicente; García, Emilio J; Oleaga, Álvaro; Llaneza, Luis; Palacios, Vicente; de la Torre, Ana; Rodríguez, Alejandro; Dubovi, Edward J; Esperón, Fernando

    2016-03-01

    Wildlife inhabiting human-dominated landscapes is at risk of pathogen spill-over from domestic species. With the aim of gaining knowledge in the dynamics of viral infections in Iberian wolves (Canis lupus) living in anthropized landscapes of northern Spain, we analysed between 2010 and 2013 the samples of 54 wolves by serology and polymerase chain reaction (PCR) for exposure to four pathogenic canine viruses: canine distemper virus (CDV), canine parvovirus-2 (CPV), canine adenovirus 1 and 2 (CAV-1 and CAV-2) and canine herpesvirus. Overall, 76% of the studied wolves presented evidence of exposure to CPV (96% by HI, 66% by PCR) and 75% to CAV (75% by virus neutralization (VN), 76% by PCR, of which 70% CAV-1 and 6% CAV-2). This represents the first detection of CAV-2 infection in a wild carnivore. CPV/CAV-1 co-infection occurred in 51% of the wolves. The probability of wolf exposure to CPV was positively and significantly correlated with farm density in a buffer zone around the place where the wolf was found, indicating that rural dogs might be the origin of CPV infecting wolves. CPV and CAV-1 appear to be enzootic in the Iberian wolf population, which is supported by the absence of seasonal and inter-annual variations in the proportion of positive samples detected. However, while CPV may depend on periodical introductions by dogs, CAV-1 may be maintained within the wolf population. All wolves were negative for exposure to CDV (by VN and PCR) and CHV (by PCR). The absence of acquired immunity against CDV in this population may predispose it to an elevated rate of mortality in the event of a distemper spill-over via dogs. PMID:26589403

  1. Detection of Cardamom mosaic virus and Banana bract mosaic virus in cardamom using SYBR Green based reverse transcription-quantitative PCR.

    PubMed

    Siljo, A; Bhat, A I; Biju, C N

    2014-01-01

    Cardamom being perennial, propagated vegetatively, detecting viruses in planting material is important to check the spread of viruses through infected material. Thus development of effective and sensitive assay for detection of viruses is need of the time. In this view, assay for the detection of Cardamom mosaic virus (CdMV) and Banana bract mosaic virus (BBrMV), infecting cardamom was developed using SYBR Green one step reverse transcription-quantitative PCR (RT-qPCR). The RT-qPCR assay amplified all isolates of CdMV and BBrMV tested but no amplification was obtained with RNA of healthy plants. Recombinant plasmids carrying target virus regions corresponding to both viruses were quantified, serially diluted and used as standards in qPCR to develop standard curve to enable quantification. When tenfold serial dilutions of the total RNAs from infected plants were tested through RT-qPCR, the detection limit of the assay was estimated to be 16 copies for CdMV and 10 copies for BBrMV, which was approximately 1,000-fold higher than the conventional RT-PCR. The RT-qPCR assay was validated by testing field samples collected from different cardamom growing regions of India. This is the first report of RT-qPCR assay for the detection of CdMV and BBrMV in cardamom. PMID:24426323

  2. 81 FR 23723 - Authorization of Emergency Use of an In Vitro Diagnostic Device for Detection of Zika Virus...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2016-04-22

    ... Secretary was published in the Federal Register on March 2, 2016 (81 FR 10878). On March 14, 2016, CDC... Device for Detection of Zika Virus; Availability AGENCY: Food and Drug Administration, HHS. ACTION... Authorization (EUA) (the Authorization) for an in vitro diagnostic device for detection of Zika virus...

  3. Detecting Newcastle disease virus in combination of RT-PCR with red blood cell absorption.

    PubMed

    Yi, Jianzhong; Liu, Chengqian

    2011-01-01

    Reverse transcription-polymerase chain reaction (RT-PCR) has limited sensitivity when treating complicated samples, such as feces, waste-water in farms, and nucleic acids, protein rich tissue samples, all the factors may interfere with the sensitivity of PCR test or generate false results. In this study, we developed a sensitive RT-PCR, combination of red blood cell adsorption, for detecting Newcastle disease virus (NDV). One pair of primers which was highly homologous to three NDV pathotypes was designed according to the consensus nucleocapsid protein (NP) gene sequence. To eliminate the interfere of microbes and toxic substances, we concentrated and purified NDV from varied samples utilizing the ability of NDV binding red blood cells (RBCs). The RT-PCR coupled with red blood cell adsorption was much more sensitive in comparison with regular RT-PCR. The approach could also be used to detect other viruses with the property of hemagglutination, such as influenza viruses. PMID:21535888

  4. Detection of Viruses By Counting Single Fluorescent Genetically Biotinylated Reporter Immunophage Using a Lateral Flow Assay

    PubMed Central

    Kim, Jinsu; Adhikari, Meena; Dhamane, Sagar; Hagström, Anna E. V.; Kourentzi, Katerina; Strych, Ulrich; Willson, Richard C.; Conrad, Jacinta C.

    2015-01-01

    We demonstrated a lateral flow immunoassay (LFA) for detection of viruses using fluorescently-labeled M13 bacteriophage as reporters and single-reporter counting as the readout. AviTag-biotinylated M13 phage were functionalized with antibodies using avidin-biotin conjugation and fluorescently labeled with AlexaFluor 555. Individual phage bound to target viruses (here MS2 as a model) captured on an LFA membrane strip were imaged using epi-fluorescence microscopy. Using automated image processing, we counted the number of bound phage in micrographs as a function of target concentration. The resultant assay was more sensitive than enzyme-linked immunosorbent assays and traditional colloidal-gold nanoparticle LFAs for direct detection of viruses. PMID:25581289

  5. Detection of Hepatitis E Virus in Sewage After an Outbreak on a French Island.

    PubMed

    Miura, Takayuki; Lhomme, Sébastien; Le Saux, Jean-Claude; Le Mehaute, Philippe; Guillois, Yvonnick; Couturier, Elizabeth; Izopet, Jacques; Abranavel, Florence; Le Guyader, Françoise S

    2016-09-01

    A hepatitis E outbreak, which occurred on a small isolated island, provided an opportunity to evaluate the association between the number of hepatitis E cases in the community and the concentration of virus detected in sewage. Samples were collected from the different sewage treatment plants from the island and analyzed for the presence of hepatitis E (HEV) virus using real-time RT-PCR. We demonstrated that if 1-4 % of inhabitants connected to a WWTP were infected with HEV, raw sewage contained HEV at detectable levels. The finding that such a small number of infected people can contaminate municipal sewage works raises the potential of the further distribution of the virus. Indeed, investigating the routes of transmission of HEV, including the potential for sewage effluent to contain infectious HEV, may help us to better understand the epidemiology of this pathogen, which is considered to be an emerging concern in Europe. PMID:27165600

  6. Visual detection of murray valley encephalitis virus by reverse transcription loop-mediated isothermal amplification.

    PubMed

    Gong, Rui; Wang, Han Hua; Qin, Hong; Guo, Xiao Ping; Ma, Xue Jun

    2015-03-01

    A sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid visual detection of Murray valley encephalitis virus (MVEV) infection. The reaction was performed in one step in a single tube at 63 °C for 60 min with the addition of the hydroxynaphthol blue (HNB) dye prior to amplification. The detection limit of the RT-LAMP assay was 100 copies per reaction based on 10-fold dilutions of in vitro transcribed RNA derived from a synthetic MVEV DNA template. No cross-reaction was observed with other encephalitis-associated viruses. The assay was further evaluated using spiked cerebrospinal fluid sample with pseudotype virus containing the NS5 gene of MVEV. PMID:25800449

  7. Rapid detection of Newcastle disease virus replication in embryonated chicken eggs using quantitative real time polymerase chain reaction.

    PubMed

    Gopinath, V P; Raj, Gopal Dhinakar; Raja, A; Kumanan, K; Elankumaran, Subbiah

    2011-01-01

    Newcastle disease virus (NDV), an avian paramyxovirus, is an economically important disease of poultry globally. Rapid methods to detect and differentiate the virus are important to curtail the spread of this virus. Nucleic acid based detection methods are routinely employed for diagnosis that suffer from the disadvantage of failure to discriminate viable virus and non-infectious genome. However, virus isolation remains the gold standard for diagnosis of field outbreaks. The sensitivity of virus isolation was combined with nucleic acid based detection methods so that the time taken for confirmatory diagnosis could be considerably reduced while increasing sensitivity. Quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) and conventional RT-PCR techniques were compared for the detection of NDV genome replication in 9-11-day-old embryonated chicken eggs (ECE) using the nucleoprotein (NP) gene of the virus as a target. The results suggest that at least two to fourfold increase in cycle threshold (C(t)) values over the baseline C(t) value of samples lacking infectious virus, would indicate live NDV replication. The limit of detection of NDV replication using qRT-PCR was 1×10(4.0) mean embryo infective doses (EID(50)). The earliest time point when live virus replication was detectable by qRT-PCR or RT-PCR was 30h post-inoculation in ECE. PMID:20951166

  8. Evaluation of field and laboratory protocols used to detect avian influenza viruses in wild aquatic birds.

    PubMed

    Dormitorio, T V; Giambrone, J J; Guo, K; Hepp, G R

    2009-09-01

    Careful selection and observance of standard field and laboratory protocols are critical for successful detection and characterization of avian influenza viruses (AIV) from wild birds. Cloacal swabs were collected from hunter-killed or nesting waterfowl and shorebirds from wildlife refuges in Alabama, Georgia, and Florida during 2006 to 2008. Swab samples were inoculated into embryonated eggs followed by hemagglutination (HA) test to determine the presence of hemagglutinating agents. Antigen capture-ELISA (AC-ELISA) and real-time reverse transcription-PCR (RRT-PCR) were used to detect AIV from both allantoic fluids (AF) and swab specimens of HA-positive samples. Hemagglutination inhibition test was used to detect Newcastle disease virus, another hemagglutinating virus common in wild birds. The HA-positive AF were sent to the National Veterinary Services Laboratory for subtyping of the isolates. Out of 825 samples tested, 19 AIV and 3 avian paramyxovirus subtypes were identified by the National Veterinary Services Laboratory. Without egg passage, AC-ELISA did not detect virus, whereas matrix gene of 13 AIV were detected using RRT-PCR. When testing was done on AF, 14 were positive for influenza A by AC-ELISA and 20 by RRT-PCR. Antigen capture-ELISA did not detect influenza A when the HA titer was lower than 125, whereas RRT-PCR detected AIV from AF with HA titer as low as 4. The highest isolation rate was from Florida, where out of 109 samples analyzed, 14 AIV were detected by RRT-PCR from AF. Real-time reverse transcription-PCR was more sensitive, specific, and cost-effective than AC-ELISA. However, to avoid false-negative results, testing should be performed on AF and not directly from cloacal swabs. Our procedures to detect AIV directly from cloacal swabs need further optimization for improved sensitivity. PMID:19687266

  9. Complementary monoclonal antibody-based dot ELISA for universal detection of H5 avian influenza virus

    PubMed Central

    2010-01-01

    Background Rapid diagnosis and surveillance for H5 subtype viruses are critical for the control of H5N1 infection. Results In this study, H5 Dot ELISA, a rapid test for the detection of avian H5N1 influenza virus, was developed with two complementary H5 monoclonal antibodies. HA sequencing of escape mutants followed by epitope mapping revealed that the two Mabs target the epitope component (189th amino acid) on the HA protein but are specific for different amino acids (189Lys or 189Arg). Gene alignment indicated that these two amino acids are the most frequent types on this position among all of the H5 AIV reported in GeneBank. These two H5 Mabs were used together in a dot ELISA to detect H5 viral antigen. The detection limit of the developed test for multiple clades of H5N1 viruses, including clades 0, 1, 2.1, 2.2, 2.3, 4, 7, and 8, was less than 0.5 hemagglutinin units. The specificity of the optimized dot ELISA was examined by using 100 H5 strains, including H5N1 HPAI strains from multiple clades, 36 non-H5N1 viruses, and 4 influenza B viruses. No cross-reactivity was observed for any of the non-H5N1 viruses tested. Among 200 random poultry samples, the test gave 100% positive results for all of the twelve RT-PCR-positive samples. Conclusions Considering that the test is convenient for field use, this H5 Dot ELISA can be used for on-site detection of H5N1 infection in clinical or environmental specimens and facilitate the investigation of H5N1 influenza outbreaks and surveillance in poultry. PMID:21192824

  10. Highly Sensitive Multiplex Assay for Detection of Human Immunodeficiency Virus Type 1 and Hepatitis C Virus RNA

    PubMed Central

    Giachetti, C.; Linnen, J. M.; Kolk, D. P.; Dockter, J.; Gillotte-Taylor, K.; Park, M.; Ho-Sing-Loy, M.; McCormick, M. K.; Mimms, L. T.; McDonough, S. H.

    2002-01-01

    Various nucleic acid assays have been developed and implemented for diagnostics and therapeutic monitoring of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) infections. The high-throughput, semiautomated assays described here were developed to provide a method suitable for screening plasma specimens for the presence of HIV-1 and HCV RNAs. Three assays were developed: a multiplex HIV-1/HCV assay for simultaneous detection of HIV-1 and HCV, and discriminatory assays for specific detection of HIV-1 and HCV. The assay systems utilize three proprietary technologies: (i) target capture-based sample preparation, (ii) transcription-mediated amplification (TMA), and (iii) hybridization protection assay (HPA). An internal control is incorporated into each reaction to control for every step of the assay and identify random false-negative reactions. The assays demonstrated a sensitivity of at least 100 copies/ml for each target, and they detected with similar sensitivity all major variants of HCV and HIV-1, including HIV-1 group O strains. Assay sensitivity for one virus was not affected by the presence of the other. The specificity of these TMA-driven assays was ≥99.5% in both normal donor specimens and plasma containing potentially interfering substances or other blood-borne pathogens. Statistical receiver operating characteristic plots of 1 − specificity versus sensitivity data determined very wide analyte cutoff values for each assay at the point at which the assay specificity and sensitivity were both ≥99.5%. The sensitivity, specificity, and throughput capability predict that these assays will be valuable for large-volume plasma screening, either in a blood bank setting or in other diagnostic applications. PMID:12089255

  11. Detection of enteric viruses in oysters by using the polymerase chain reaction.

    PubMed Central

    Atmar, R L; Metcalf, T G; Neill, F H; Estes, M K

    1993-01-01

    A procedure for the detection of enteric viral nucleic acid in oysters by the polymerase chain reaction was developed. Known quantities of poliovirus type 1 were seeded into oysters. Virus was extracted and concentrated by using organic flocculation and polyethylene glycol precipitation. Inhibitors of reverse transcription-polymerase chain reaction were present in the oyster extracts, preventing amplification of target viral nucleic acid. The use of cetyltrimethylammonium bromide precipitation sufficiently removed inhibitors to allow detection of as few as 10 PFU of poliovirus. Norwalk virus also could be detected after being seeded into oysters. This methodology may be useful for the detection of these and other shellfish-borne viral pathogens. Images PMID:8382024

  12. Detection of sugarcane yellow leaf virus by direct antigen coated enzyme-linked immunosorbent assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The L9 (34) orthogonal diagram was applied to optimize detection conditions of direct antigen coated enzyme-linked immunosorbent assay (DAC-ELISA) for Sugarcane yellow leaf virus (SCYLV) in sugarcane. Statistic analyses indicated that 150 µL of SCYLV in juice and leaf crude extract antigens was the ...

  13. A MULTI-LABORATORY EVALUATION OF METHODS FOR DETECTING ENTERIC VIRUSES IN SOILS

    EPA Science Inventory

    Two candidate methods for the recovery and detection of viruses in soil were subjected to round robin comparative testing by members of the American Society for Testing and Materials D19:24:04:04 Subcommittee Task Group. Selection of the methods, designated “Berg” and “Goyal,” wa...

  14. Detection by Enzyme-Linked Immunosorbent Assay of Antibodies to West Nile virus in Birds

    PubMed Central

    Dupuis, Alan P.; Nicholas, David; Young, Donna; Maffei, Joseph; Kramer, Laura D.

    2002-01-01

    We adapted an indirect immunoglobulin G enzyme-linked immunosorbent assay to facilitate studies of West Nile virus (WNV) and evaluated its application to taxonomically diverse avian species. Anti-WNV antibodies were detected in 23 bird species, including many exotic species, demonstrating its value in studies of WNV epizootiology. PMID:12194778

  15. Heminested PCR assay for detection of six genotypes of rabies and rabies-related viruses.

    PubMed Central

    Heaton, P R; Johnstone, P; McElhinney, L M; Cowley, R; O'Sullivan, E; Whitby, J E

    1997-01-01

    A heminested reverse transcriptase PCR (hnRT-PCR) protocol which is rapid and sensitive for the detection of rabies virus and rabies-related viruses is described. Sixty isolates from six of the seven genotypes of rabies and rabies-related viruses were screened successfully by hnRT-PCR and Southern blot hybridization. Of the 60 isolates, 93% (56 of 60) were positive by external PCR, while all isolates were detected by heminested PCR and Southern blot hybridization. We also report on a comparison of the sensitivity of the standard fluorescent-antibody test (FAT) for rabies antigen and that of hnRT-PCR for rabies viral RNA with degraded tissue infected with a genotype 1 virus. Results indicated that FAT failed to detect viral antigen in brain tissue that was incubated at 37 degrees C for greater than 72 h, while hnRT-PCR detected viral RNA in brain tissue that was incubated at 37 degrees C for 360 h. PMID:9350729

  16. DETECTION AND QUANTITATION OF SOLENOPSIS INVICTA VIRUS IN FIRE ANTS BY REAL-TIME PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A quantitative real-time PCR (QPCR) method was developed to detect and quantify the amount of Solenopsis invicta virus (SINV) infecting individual ants of Solenopsis invicta. The two-step method utilized a gene-specific oligonucleotide primer targeting the SINV RNA-dependent RNA polymerase (RdRp) f...

  17. Concentration and detection of hepatitis A virus and its indicator from artificial seawater using zeolite.

    PubMed

    Cormier, Jiemin; Janes, Marlene

    2016-09-01

    Hepatitis A virus (HAV) infection is the leading worldwide cause of acute viral hepatitis, and outbreaks caused by this virus often occur in fecal polluted waters. Rapid concentration and detection of viral contamination in water environments can prevent economic loss and can identify the source of contamination within a short time. However, conventional methods for virus concentration are often laborious, time consuming, and subject to clogging. Furthermore, most methods require a secondary concentration step to reduce the final volume of samples. We developed a method to concentrate HAV from seawater using zeolite in aid of rapid detection. In this method,artificial seawater was inoculated with HAV (7-8 log TCID50) and filtered with zeolite. The viruses were then eluted from zeolite with sodium dodecyl sulfate and detected via real-time PCR (qPCR). Zeolite was able to concentrate HAV from artificial seawater with ∼99% efficiency in less than 5min and was more efficient in seawater than in fresh water. The entire concentration and detection can be done in approximately 2h. Compared to existing methods, this method eliminated the need for a secondary concentration step as well as the necessity to modify the pH or salinity of the seawater during concentration, and was simple and inexpensive. PMID:27150045

  18. Detection and Characterization of a Plant Virus in Wild Raspberry, Rubus idaeus L., in Alaska

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In 2008, mosaic leaf symptoms were detected on wild raspberry plants, Rubus idaeus L., in north central Alaska. They were growing on remnant patches within developing agricultural sites. Partially purified virus samples were obtained by differential centrifugation of homogenized leaves according to ...

  19. Sensitive detection of multiple hepatitis A virus genotypes with a single polony-based assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hepatitis A virus (HAV) is one of the major causes of non-bacterial gastroenteritis in humans worldwide. HAV is mostly transmitted via direct person-to-person contact, or by consumption of contaminated foods and water. Since only a few viral particles may cause disease, detection of low levels of HA...

  20. Lateral flow immunoassay for the rapid detection of citrus tristeza virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A lateral flow methodology was developed using gold nanoparticles for rapid detection of Citrus tristeza virus (CTV). The test strip was based on a sandwich immunoassay and could be accomplished within 10 minutes. A sample was considered negative for CTV when only the control line appeared; whereas,...