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Sample records for tristeza virus isolate

  1. Molecular Characterization of Citrus tristeza virus Isolates from Panama

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Twelve isolates of Citrus tristeza virus (CTV) were collected from the main citrus growing regions in Panama and characterized at the molecular level. The CTV coat protein gene (CPG) was amplified by RT-PCR, and the amplified PCR products were cloned and sequenced. The sequences analyses showed the ...

  2. CPm gene diversity in field isolates of Citrus tristeza virus from Colombia.

    PubMed

    Oliveros-Garay, Oscar Arturo; Martinez-Salazar, Natalhie; Torres-Ruiz, Yanneth; Acosta, Orlando

    2009-01-01

    The nucleotide sequence diversity of the CPm gene from 28 field isolates of Citrus tristeza virus (CTV) was assessed by SSCP and sequence analyses. These isolates showed two major shared haplotypes, which differed in distribution: A1 was the major haplotype in 23 isolates from different geographic regions, whereas R1 was found in isolates from a discrete region. Phylogenetic reconstruction clustered A1 within an independent group, while R1 was grouped with mild isolates T30 from Florida and T385 from Spain. Some isolates contained several minor haplotypes, which were very similar to, and associated with, the major haplotype. PMID:19882104

  3. Isolates of Citrus tristeza virus that overcome Poncirus trifoliata resistance comprise a novel strain.

    PubMed

    Harper, S J; Dawson, T E; Pearson, M N

    2010-04-01

    The economically important rootstock species Poncirus trifoliata is resistant to most isolates of Citrus tristeza virus (CTV), but not to members of the CTV resistance-breaking (RB) strain presently found in New Zealand. In this study, five known and suspected RB isolates were separated from field mixtures, and their genomes were sequenced in full. It was found that the RB isolates are members of a single phylogenetically distinct clade with an average of 90.3% genomic nucleotide sequence identity to the closest extant isolate, T36. These isolates also show evidence of multiple recombination events throughout their evolutionary history, with T36, T30 and VT-like isolates, and with each other. Finally, the genomic sequences of these isolates show that several genes contain unique polymorphisms that may or may not be involved in overcoming resistance. These data will aid in the understanding of host-virus interactions, and the mechanism of resistance in P. trifoliata. PMID:20352212

  4. Complete genome sequences of two distinct and diverse Citrus tristeza virus isolates from New Zealand.

    PubMed

    Harper, S J; Dawson, T E; Pearson, M N

    2009-01-01

    Two Citrus tristeza virus (CTV) isolates from New Zealand that display distinct phenotypes were isolated, examined and sequenced in full. The first isolate, NZ-M16, is largely asymptomatic and non-transmissible by the aphid vector Toxoptera citricida, while the second, NZ-B18, is highly transmissible and induces very severe symptoms on C. sinensis and C. aurantii. Phylogenetic analysis of the genome sequences showed that both isolates were approximately 90-93% similar to the VT and T318 isolates but possessed only 89% identity to one another. Based on sequence identity, both isolates are VT subtypes, with NZ-M16 being T3-like, while NZ-B18 is a member of a novel subtype with B165 from India. PMID:19629636

  5. Complete Nucleotide Sequence of a New Genotype of Citrus Tristeza Virus from an Isolate Having a Mixed Infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An isolate of Citrus tristeza virus (CTV) that causes severe stem pitting in grapefruits (# 3800) was used for sequencing. Analysis of the isolate revealed the presence of at least three different populations, one belonging to T30 genotype and the other two belonging to new genotypes, designated T2K...

  6. Molecular characterization of Citrus tristeza virus isolates from the Northeastern Himalayan region of India.

    PubMed

    Biswas, K K

    2010-06-01

    Citrus tristeza virus (CTV) isolates from the Darjeeling hills of the Northeastern Himalayan region of India were characterized by biological indexing, multiple molecular marker (MMM) analysis, heteroduplex mobility assay (HMA) and sequence analysis. Variability was studied using the CP gene and a 5' ORF1a fragment of the CTV genome. HMA and sequence analysis of the 5' ORF1a fragment classified Darjeeling isolates into two groups, whereas CP gene analysis provided evidence for three different groups. Darjeeling CTV isolates shared nucleotide sequence identities of 89-97 and 91-92% in the 5' ORF1a fragment and CP gene, respectively, suggesting extensive diversity among CTV isolates from this Indian region. PMID:20437063

  7. Genetic diversity and evolution of two capsid protein genes of citrus tristeza virus isolates from China.

    PubMed

    Wu, Guan-Wei; Tang, Min; Wang, Guo-Ping; Jin, Feng-Yin; Yang, Zuo-Kun; Cheng, Li-Jing; Hong, Ni

    2015-03-01

    The genetic diversity and population structure of citrus tristeza virus (CTV) isolates from China were investigated based on partial sequences spanning the C-terminal end of p61 and the complete sequences of the CPm and CP genes. Phylogenetic analysis revealed five known groups (RB, T30, T36, HA and VT) and one new group (VI) consisting of only Chinese CTV isolates. Incongruent phylogenetic trees coupled with recombination analysis suggested several recombination events in the CPm gene. Positive selection was detected at codon 9 of CPm and codons 31, 41 and 68 of CP. The widespread CTV subpopulation AT-1 found in China has a unique amino acid insertion at the C-terminus of p61, which could increase CTV population complexity with implications for the evolutionary history of the virus. Our results suggest relevant roles for gene flow, purifying selection and recombination in shaping the CTV population in China. PMID:25387862

  8. Molecular characterization of Cirus tristeza virus isolates associated with stem pitting CTV cross-protection in Peru

    Technology Transfer Automated Retrieval System (TEKTRAN)

    During the 1970s and early 1980s, the Peruvian citrus industry was destroyed by severe Citrus tristeza virus (CTV) strains spread by the brown citrus aphid. The Topara Nursery, located 180 km south of Lima Peru, selected and identified CTV isolates that confer cross-protection against virulent stem...

  9. Segregation of distinct variants from Citrus tristeza virus isolate SY568 using aphid transmission.

    PubMed

    Velazquez-Monreal, J J; Mathews, D M; Dodds, J A

    2009-10-01

    A well-studied severe isolate of Citrus tristeza virus (CTV) known as SY568 has previously been shown to contain multiple variants of the virus which differ in their genetic and biological characters. Aphid transmission was used in an attempt to segregate some of these variants for further characterization. Resulting infections gave symptoms which varied from asymptomatic to more severe than the inoculum source. RNase protection assays (RPAs) were used to compare nine regions of the CTV genome and determine whether unique strains could be identified. Five aphid-transmitted subcultures, with fingerprints that were different from those of the inoculum sources in at least one genomic area, were then cloned, sequenced, and compared with known isolates. An asymptomatic strain was shown to be different in every area of the CTV genome when examined by RPA and sequencing of selected regions. Mixed-infection studies using graft transmission of the asymptomatic subculture and two of the more severe aphid-transmitted subcultures showed that the mild strain was not able to compete well when in the presence of any of the severe variants tested, and its titer was significantly reduced from that seen in single infection. The mild strain and a selected severe strain were singly graft inoculated into five different citrus hosts (sweet orange, grapefruit, sour orange, lemon, and lime), where they maintained their distinct biological and genetic characteristics. PMID:19740030

  10. HISTOLOGY OF SWEET ORANGE STEM PITTING CAUSED BY AN AUSTRALIAN ISOLATE OF CITRUS TRISTEZA VIRUS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Some strains of the citrus tristeza virus (CTV) cause stem pitting in sweet orange (Citrus sinensis (L.) Osbeck). This abnormality causes tree decline and reduction in fruit size and yield of affected citrus trees. Stem-pitting symptoms can occur on trunks, on all sizes of limbs, and on the twigs ...

  11. Protein-protein interactions between proteins of Citrus tristeza virus isolates.

    PubMed

    Nchongboh, Chofong Gilbert; Wu, Guan-Wei; Hong, Ni; Wang, Guo-Ping

    2014-12-01

    Citrus tristeza virus (CTV) is one of the most devastating pathogens of citrus. Its genome is organized into 12 open reading frames (ORFs), of which ten ORFs located at the 3'-terminus of the genome have multiple biological functions. The ten genes at the 3'-terminus of the genome of a severe isolate (CTV-S4) and three ORFs (CP, CPm and p20) of three other isolates (N4, S45 and HB1) were cloned into pGBKT7 and pGADT7 yeast shuttle vectors. Yeast two-hybridization (Y2H) assays results revealed a strong self-interaction for CP and p20, and a unique interaction between the CPm of CTV-S4 (severe) and CP of CTV-N4 (mild) isolates. Bimolecular fluorescence complementation also confirmed these interactions. Analysis of the deletion mutants delineated the domains of CP and p20 self-interaction. Furthermore, the domains responsible for CP and p20 self-interactions were mapped at the CP amino acids sites 41-84 and p20 amino acids sites 1-21 by Y2H. This study provided new information on CTV protein interactions which will help for further understanding the biological functions. PMID:25064367

  12. Genome analysis of an orange stem pitting citrus tristeza virus isolate reveals a novel recombinant genotype.

    PubMed

    Roy, Avijit; Brlansky, R H

    2010-08-01

    An orange stem pitting citrus tristeza virus (CTV) isolate CTV-B165 was found to be symptomatically similar to other known CTV-VT isolates however molecular methods failed to classify it as an identifiable CTV genotype. The sequence variation of the Indian CTV-B165 isolate was compared to the three well known CTV genotypes, T36, T30, and VT. The genome of the predominant component of CTV-B165 was 19,247 nt in length with 12 open reading frames (ORFs) and was structurally identical to the other CTV isolates. All the completely sequenced CTV isolates except the VT isolate were 2-55 nt longer than the CTV-B165. In comparison to the other fully sequenced T36, T30 and VT genotypic isolates, CTV-B165 had nucleotide identity of 72-86% in ORF1 and 92-99% in ORFs 2-11. Sequence data of independent overlapping clones from the CTV-B165 genome showed highly divergent sequences of the overlapping region of 5'-UTR and ORF1a, the inter-domain region of ORF1a and the partial regions of ORF2. Phylogenetic analysis of five domains of ORF1a, ORF1b, and ORF2 revealed that CTV-B165 isolate distinctly segregates from the existing three genotypes in the dendrograms and was supported by high bootstrap values and robust tree topology. The PHYLPRO graphical analysis showed multiple recombination signals with significant correlation values. The precise detection of recombination sites for different genomic regions in CTV sequences was supported by several recombination-detecting methods. Collectively, the phylogenetic and recombination analyses suggest that the observed CTV-B165 genotype variation is an outcome of inter-genotype recombination. To determine the presence of the CTV-B165 genotype a pair of genome specific primers was designed and standardized for reliable detection of the novel CTV genotype by reverse-transcription polymerase chain reaction. PMID:20381550

  13. Characterization of Citrus Tristeza Virus Isolates by Single-strand Conformation Polymorphism Analysis of the Coat Protein Gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A method is needed to rapidly assess Citrus tristeza virus (CTV) strains and to identify mixed populations in tristeza-infected trees. Single-strand conformation polymorphism (SSCP) can detect point mutations in DNA fragments and determine the structure of viral populations. Previous reports utili...

  14. Complete 3' end genome analysis of the asymptomatic citrus tristeza virus isolate B192 and its eight single aphid transmitted subisolates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The most important viral disease of citrus is caused by Citrus tristeza virus (CTV). CTV infection often exists in field isolates as a complex of multiple genotypes. Aphid transmission is important for CTV dispersal. The complete 3' terminal half sequences of the asymptomatic CTV isolate B192 and it...

  15. The genotypes of citrus tristeza virus isolates from China revealed by sequence analysis of multiple molecular markers.

    PubMed

    Wu, Guan-Wei; Pan, Song; Wang, Guo-Ping; Tang, Min; Liu, Yong; Yang, Fan; Hong, Ni

    2013-01-01

    The genotypes of ten citrus tristeza virus (CTV) isolates from central China were determined by examining multiple molecular markers (MMMs) using 11 primer pairs. The results revealed that one isolate contained a single T30 genotype, two isolates contained a single VT genotype, and the other seven isolates were mixtures of two or more genotypes. Sequence analysis of amplified MMMs showed a high genetic diversity in Chinese CTV populations. The genotypes resembling T36, RB and B165 were identified from Chinese CTV isolates for the first time. Our results suggest that genotype assignment of CTV cannot be based solely on the amplification profiles of MMMs, and sequencing of MMMs is required. PMID:22987316

  16. Characterization of a novel citrus tristeza virus genotype within three cross-protecting source GFMS12 sub-isolates in South Africa by means of Illumina sequencing.

    PubMed

    Zablocki, Olivier; Pietersen, Gerhard

    2014-08-01

    Tristeza disease (caused by citrus tristeza virus, CTV) is currently controlled in South Africa by means of cross-protection. In this study, we characterized the CTV populations of three grapefruit mild strain 12 (GFMS12) single-aphid-transmission-derived sub-isolates at the whole-genome level using Illumina sequencing technology. A novel South African isolate (CT-ZA3, of the T68 genotype) was shown to be the dominant genotype in all GFMS12 sub-isolates tested, along with reads unique to various other genotypes occurring as minor components. Uncertainty remains as to the significance of these minor components. PMID:24623089

  17. Citrus tristeza virus: characterization of Texas isolates, studies on aphid transmission and pathogen-derived control strategies 

    E-print Network

    Herron, Caroline Mary

    2004-11-15

    Citrus tristeza virus (CTV), an economically important graft-transmissible pathogen of citrus, causes major global declines in citrus production. In the commercial citrus of the Lower Rio Grande Valley of Texas (LRGV), ...

  18. Polymorphism of a specific region in gene p23 of Citrus tristeza virus allows discrimination between mild and severe isolates.

    PubMed

    Sambade, A; López, C; Rubio, L; Flores, R; Guerri, J; Moreno, P

    2003-12-01

    The pathogenicity determinants of Citrus tristeza virus (CTV) are presently unknown, although transgenic Mexican limes over-expressing CTV p23, an RNA-binding protein involved in regulating the asymmetrical accumulation of viral RNA strands, display typical CTV symptoms. Here we compared the predominant sequence variants of gene p23 from 18 CTV isolates of different geographic origin and pathogenicity characteristics. Phylogenetic analysis of these sequences revealed three groups of isolates: i) mild, inducing only symptoms in lime and/or decline of citrus species grafted on sour orange rootstock, ii) severe, causing additionally stem pitting on sweet orange and/or grapefruit, and iii) an atypical group of isolates inciting variable symptoms. The sequences of the isolates located at the periphery of each group were recombinants. Pairwise comparisons of the predicted amino acid sequences showed that residues at positions 78-80 were characteristic of each group of isolates. Group-specific primers based on these differences allowed RT-PCR detection of each sequence type in dsRNA-rich preparations from infected tissues. While mild isolates contained only the sequence characteristic of this group, most severe isolates contained the sequences characteristic of their group, and additionally, sequences characteristic of the mild and/or the atypical groups, suggesting that the severe phenotype is associated with the presence of the severe and/or the atypical sequence types. This association can be exploited for quick detection of potentially damaging sequence variants and for monitoring cross protection. PMID:14648289

  19. Development and application of a hexaplex reverse transcription polymerase chain reaction for screening global Citrus tristeza virus isolates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The discovery of the diversity of Citrus tristeza virus (CTV) genotypes has complicated detection and diagnostic measures. To simplify the identification and differentiation of CTV genotypes, an efficient multiplex reverse transcription polymerase chain reaction (M-RT-PCR) technique for the screenin...

  20. Development and application of a hexaplex reverse transcription polymerase chain reaction for screening global citrus tristeza virus isolates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The discovery of the diversity of Citrus tristeza virus (CTV) genotypes has complicated detection and diagnostic measures. To simplify the identification and differentiation of CTV genotypes, an efficient multiplex reverse transcription polymerase chain reaction (M-RT-PCR) technique for the screenin...

  1. The First Identified Citrus tristeza virus Isolate of Turkey Contains a Mixture of Mild and Severe Strains.

    PubMed

    Cevik, Bayram; Yardimci, Nejla; Korkmaz, Sava?

    2013-03-01

    The presence of Citrus tristeza virus (CTV) has previously been reported in citrus growing regions of Turkey. All serologically and biologically characterized isolates including I?d?r, which was the first identified CTV isolates from Turkey, were considered mild isolates. In this study, molecular characteristics of the I?d?r isolate were determined by different methods. Analysis of the I?d?r isolate by western blot and BD-RT-PCR assays showed the presence of MCA13 epitope, predominantly found in severe isolates, in the I?d?r isolate revealing that it contains a severe component. For further characterization, the coat protein (CP) and the RNA-dependent RNA polymerase (RdRp) genes representing the 3' and 5' half of CTV genome, respectively, were amplified from dsRNA by RT-PCR. Both genes were cloned separately and two clones for each gene were sequenced. Comparisons of nucleotide and deduced amino acid sequences showed that while two CP gene sequences were identical, two RdRp clones showed only 90% and 91% sequence identity in their nucleotide and amino acid sequences, respectively, suggesting a mixed infection with different strains. Phylogenetic analyses of the CP and RdRp genes of I?d?r isolate with previously characterized CTV isolates from different citrus growing regions showed that the CP gene was clustered with NZRB-TH30, a resistance breaking isolate from New Zealand, clearly showing the presence of severe component. Furthermore, two different clones of the RdRp gene were clustered separately with different CTV isolates with a diverse biological activity. While the RdRp-1 was clustered with T30 and T385, two well-characterized mild isolates from Florida and Spain, respectively, the RdRp-2 was most closely related to NZRB-G90 and NZRB-TH30, two well-characterized resistance breaking and stem pitting (SP) isolates from New Zealand confirming the mixed infection. These results clearly demonstrated that the I?d?r isolate, which was previously described as biologically a mild isolate, actually contains a mixture of mild and severe strains. PMID:25288926

  2. Deep sequencing and analysis of small RNAs in sweet orange grafted on sour orange infected with two citrus tristeza virus isolates prevalent in Sicily.

    PubMed

    Licciardello, Grazia; Scuderi, Giuseppe; Ferraro, Rosario; Giampetruzzi, Annalisa; Russo, Marcella; Lombardo, Alessandro; Raspagliesi, Domenico; Bar-Joseph, Moshe; Catara, Antonino

    2015-10-01

    Two representative isolates of a citrus tristeza virus population in Sicily, SG29 (aggressive) and Bau282 (mild), were sequenced via viral small RNAs (vsRNA) produced in budlings of sweet orange grafted on sour orange. Phylogenetic relationships with Mediterranean and exotic isolates revealed that SG29 clustered within the "VT-Asian" subtype, whereas Bau282 belonged to the cluster T30. The study confirms that molecular data need to be integrated with bio-indexing in order to obtain adequate information for risk assessment. PMID:26175068

  3. Citrus tristeza virus-aphid interactions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A review chapter on aphid transmission of Citrus tristeza virus is provided for a book on “Vector-Mediated Transmission of Plant Pathogens”. Earliest uses of citrus goes back over two millennia as items of trade, gifts and medicinal compounds. Citrus propagation during this period was by seed and si...

  4. Citrus tristeza virus-host interactions

    PubMed Central

    Dawson, W. O.; Garnsey, S. M.; Tatineni, S.; Folimonova, S. Y.; Harper, S. J.; Gowda, S.

    2013-01-01

    Citrus tristeza virus (CTV) is a phloem-limited virus whose natural host range is restricted to citrus and related species. Although the virus has killed millions of trees, almost destroying whole industries, and continually limits production in many citrus growing areas, most isolates are mild or symptomless in most of their host range. There is little understanding of how the virus causes severe disease in some citrus and none in others. Movement and distribution of CTV differs considerably from that of well-studied viruses of herbaceous plants where movement occurs largely through adjacent cells. In contrast, CTV systemically infects plants mainly by long-distance movement with only limited cell-to-cell movement. The virus is transported through sieve elements and occasionally enters an adjacent companion or phloem parenchyma cell where virus replication occurs. In some plants this is followed by cell-to-cell movement into only a small cluster of adjacent cells, while in others there is no cell-to-cell movement. Different proportions of cells adjacent to sieve elements become infected in different plant species. This appears to be related to how well viral gene products interact with specific hosts. CTV has three genes (p33, p18, and p13) that are not necessary for infection of most of its hosts, but are needed in different combinations for infection of certain citrus species. These genes apparently were acquired by the virus to extend its host range. Some specific viral gene products have been implicated in symptom induction. Remarkably, the deletion of these genes from the virus genome can induce large increases in stem pitting (SP) symptoms. The p23 gene, which is a suppressor of RNA silencing and a regulator of viral RNA synthesis, has been shown to be the cause of seedling yellows (SY) symptoms in sour orange. Most isolates of CTV in nature are populations of different strains of CTV. The next frontier of CTV biology is the understanding how the virus variants in those mixtures interact with each other and cause diseases. PMID:23717303

  5. Citrus tristeza virus-host interactions.

    PubMed

    Dawson, W O; Garnsey, S M; Tatineni, S; Folimonova, S Y; Harper, S J; Gowda, S

    2013-01-01

    Citrus tristeza virus (CTV) is a phloem-limited virus whose natural host range is restricted to citrus and related species. Although the virus has killed millions of trees, almost destroying whole industries, and continually limits production in many citrus growing areas, most isolates are mild or symptomless in most of their host range. There is little understanding of how the virus causes severe disease in some citrus and none in others. Movement and distribution of CTV differs considerably from that of well-studied viruses of herbaceous plants where movement occurs largely through adjacent cells. In contrast, CTV systemically infects plants mainly by long-distance movement with only limited cell-to-cell movement. The virus is transported through sieve elements and occasionally enters an adjacent companion or phloem parenchyma cell where virus replication occurs. In some plants this is followed by cell-to-cell movement into only a small cluster of adjacent cells, while in others there is no cell-to-cell movement. Different proportions of cells adjacent to sieve elements become infected in different plant species. This appears to be related to how well viral gene products interact with specific hosts. CTV has three genes (p33, p18, and p13) that are not necessary for infection of most of its hosts, but are needed in different combinations for infection of certain citrus species. These genes apparently were acquired by the virus to extend its host range. Some specific viral gene products have been implicated in symptom induction. Remarkably, the deletion of these genes from the virus genome can induce large increases in stem pitting (SP) symptoms. The p23 gene, which is a suppressor of RNA silencing and a regulator of viral RNA synthesis, has been shown to be the cause of seedling yellows (SY) symptoms in sour orange. Most isolates of CTV in nature are populations of different strains of CTV. The next frontier of CTV biology is the understanding how the virus variants in those mixtures interact with each other and cause diseases. PMID:23717303

  6. Characterization of the mixture of genotypes of a Citrus tristeza virus isolate by reverse transcription-quantitative real-time PCR.

    PubMed

    Ananthakrishnan, G; Venkataprasanna, T; Roy, Avijit; Brlansky, R H

    2010-03-01

    A multiplex real-time PCR assay was developed to detect and quantify the Citrus tristeza virus (CTV) genotypic mixture present in infected plants. CTV isolate FS627, a complex Florida isolate containing T36, T30 and VT genotypes and its aphid transmitted subisolates was used. The relative quantitative assay was carried out using specific primers and probes developed from the genotypes of three CTV virus isolates and included the coat protein region of isolate T36 and the 5' end, ORF 1a and ORF 2 region of isolates T36, T30 and VT. Among the three genotypes present in the aphid transmitted subisolates, the T30 genotype showed higher overall relative quantitation in all specific regions compared to other isolates. The profiles of the some aphid transmitted subisolates were different from the parent source from which they transmitted. The 2(-DeltaDeltaCt) method (the amount of target, normalized to an endogenous control and relative to a calibrator) was used to analyze the relative titers of the three reference genotypes in the aphid transmitted plants infected with FS627. This protocol enabled assessments of CTV genetic diversity in the aphid transmitted subisolates. This simple quantitative assay was sensitive, efficient, and took less time than other existing methods. This relative quantitative assay will be a reliable tool for diagnosis, detection and genetic diversity studies on CTV. PMID:20005260

  7. Amplification of Citrus Tristeza Virus from a cDNA Clone and Infection of Citrus Trees T. Satyanarayana,*,1

    E-print Network

    Burns, Jacqueline K.

    Amplification of Citrus Tristeza Virus from a cDNA Clone and Infection of Citrus Trees T, Citrus Research and Education Center, Lake Alfred, Florida 33850; The Volcani Institute, Bet-Dagan 50250 for revision November 13, 2000; accepted November 22, 2000 Isolates of the Closterovirus, Citrus tristeza virus

  8. Evidence of Recombinant Citrus tristeza virus Isolate Occurring in Acid Lime cv. Pant Lemon Orchard in Uttarakhand Terai Region of Northern Himalaya in India.

    PubMed

    Singh, Jaywant Kumar; Tarafdar, Avijit; Sharma, Susheel Kumar; Biswas, Kajal Kumar

    2013-06-01

    The present study for the first time describes biological and molecular characterization of Citrus tristeza virus (CTV) occurring in the Terai area of Uttarakhand State in Northern Himalaya region of India. Direct antigen coated-ELISA and reverse transcriptase-polymerase chain reaction (RT-PCR) detected the CTV infection in Acid lime cv. Pant lemon (Citrus aurantifolia) orchards of Pantnagar with an estimated disease incidence of 16.6-20.5 %. To know the biological and genetic properties, an isolate, CTV Pant 4 was characterized. Isolate Pant 4 could be graft transmitted to Kinnow, Nagpur and Darjeeling mandarins, Mosambi sweet orange, Kagzi lime, Sweet lime, Sour orange but not to Rough lemon. The sequence analyses of the 5'ORF1a (3038 nucleotides) of LPro domain and 3'end (2058 nt) covering ORF7-ORF10 regions of the CTV genome revealed that Pant 4 was closely related to the previously reported Indian CTV isolate, Kpg3 from Northeastern Himalaya region with 97 and 98 % sequence identity, respectively. Whereas, it differed from the previously reported CTV isolate B165 from Southern India with 79 and 92 % identity, respectively for 5'ORF1a and 3' end regions. Recombination and SplitsTree decomposition analyses indicated that CTV isolate Pant 4 was a recombinant isolate originating from Kpg3 as a major and B165 as a minor donor. PMID:24426255

  9. Characterization of isolates of Citrus tristeza virus by sequential analyses of enzyme immunoassays and capillary electrophoresis-single-strand conformation polymorphisms.

    PubMed

    Licciardello, G; Raspagliesi, D; Bar-Joseph, M; Catara, A

    2012-05-01

    Citrus tristeza virus (CTV) is the causal agent of tristeza disease, which is one of the most devastating diseases of citrus crops worldwide. This paper describes a method for the rapid detection and genotyping of naturally spreading CTV isolates. This method uses ELISA or dot-blot immunological tests to detect trees infected with CTV. The reaction wells or membrane spots for which there is a positive reaction are sequentially treated by (i) washing and elution of viral RNA from the trapped samples, (ii) one-step synthesis of cDNA and PCR and (iii) automated fluorescence-based capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) analysis of amplification products. Comparative CE-SSCP results are presented for CTV RNA extracted directly from infected leaves and ELISA plates or from membranes. In the analyses of all of these RNA samples, the p18, p27 and p23 CTV genes were targeted for amplification. Specific profiles of forward and reverse strands were obtained from a group of eight CTV isolates collected in Sicily, each with distinct biological characteristics, which were analyzed using the conventional two-step procedure (immunological detection followed by CE-SSCP molecular characterization after RNA isolation) or in a continuous process of ELISA/CE-SSCP or dot-blot/CE-SSCP starting from infected plant material. The combined method is simple, highly sensitive and reproducible, thus allowing the processing of numerous field samples for a variety of epidemiological needs. The sequential processing of an ELISA or dot-blot/ELISA followed by CE-SSCP is expected to allow the rapid detection of recent CTV infections along with the simultaneous characterization of the genetic diversity and structure of the population of newly invading CTV. PMID:22305960

  10. Deep sequencing of viral small-RNAs of citrus tristeza virus (CTV) reveals genomic differences between two Italian isolates of CTV

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A recent Citrus tristeza virus (CTV) epidemic of quick decline (QD) killed many sweet orange trees grafted on sour orange rootstock in Sicily but left some asymptomatic trees in the same field. Recent reports indicated cross-protection involves exclusion of a severe CTV strain by a mild strain of th...

  11. Population dynamics of a Florida Citrus tristeza virus isolate and aphid-transmitted subisolates: identification of three genotypic groups and recombinants after aphid transmission.

    PubMed

    Roy, Avijit; Brlansky, R H

    2009-11-01

    Tristeza is an important citrus disease affecting the viability and productivity of citrus worldwide. The causal agent, Citrus tristeza virus (CTV), usually occurs as a mixture of genotypes in nature, with one of the genotypes often dominating the population. CTV has a monopartite, positive-sense RNA genome of approximately 19.3 kb and exhibits over 30% diversity in the 5' half and less than 10% in the 3' half among different genotypes. A Florida CTV isolate, FS627, was selected for this study. Isolate FS627 was analyzed by reverse-transcription polymerase chain reaction (RT-PCR) using primers to three regions: 788-bp region in the 5' (697 to 1,484 nucleotides), open reading frame (ORF)1a, 696 or 718 bp from the overlapping region of the RdRp (ORF1b) and p33 (ORF2) gene, and a 672-bp major coat protein gene (ORF7) in the 3' end of the CTV genome. The presence of T36, T30, and VT genotypes in isolate FS627 was confirmed utilizing the genotype specific overlapping region of RdRp primer pairs for RT-PCR amplification followed by cloning and sequence analysis. Analysis of single-strand conformational polymorphisms and sequences of RT-PCR-amplified products of the above regions were used to determine the presence of genotypes in both the parent and aphid-transmitted (AT) subisolates. Although the parent isolate had T36 as the major genotype, T30 was the major genotype in most of the AT subisolates. Some intermediate genotypes were detected that differed from the parental or AT subisolates. These intermediate genotypes were considered to be recombinants of the T30 and T36 genotypes and also were observed in the second level of AT subisolates generated from the of first-level AT subisolates of CTV-FS627. This work provides advance information on the population dynamics in CTV mixtures and the generation of virus recombinants after aphid transmission. PMID:19821734

  12. Development and application of a multiplex reverse-transcription polymerase chain reaction assay for screening a global collection of Citrus tristeza virus isolates.

    PubMed

    Roy, Avijit; Ananthakrishnan, G; Hartung, John S; Brlansky, R H

    2010-10-01

    The emerging diversity of Citrus tristeza virus (CTV) genotypes has complicated detection and diagnostic measures and prompted the search for new differentiation methods. To simplify the identification and differentiation of CTV genotypes, a multiplex reverse-transcription polymerase chain reaction (RT-PCR) technique for the screening of CTV isolates was developed. Variable regions within the open reading frame (ORF)-1a of diverse CTV genotypes were identified to develop first a simplex (S) and then a hexaplex (H) RT-PCR. CTV isolates have been grouped previously into five genotypes (namely, T3, T30, T36, VT, and B165) based on the nucleotide sequence comparisons and phylogenetic analyses. Nucleotide sequences from GenBank were used to design species and genotype-specific primers (GSPs). The GSPs were initially used for reliable detection of all CTV genotypes using S-RT-PCR. Furthermore, detection of all five recognized CTV genotypes was established using the H-RT-PCR. Six amplicons, one generic to all CTV isolates and one for each of the five recognized genotypes, were identified on the basis of their size and were confirmed by sequence analysis. In all, 175 CTV isolates from 29 citrus-growing countries were successfully analyzed by S- and H-RT-PCR. Of these, 97 isolates contained T36 genotypes, 95 contained T3 genotypes, 76 contained T30 genotypes, 71 contained VT genotypes, and 24 contained B165 genotype isolates. In total, 126 isolates contained mixed infections of 2 to 5 of the known CTV genotypes. Two of the CTV isolates could not be assigned to a known genotype. H-RT-PCR provides a sensitive, specific, reliable, and rapid way to screen for CTV genotypes compared with other methods for CTV genotype detection. Efficient identification of CTV genotypes will facilitate a better understanding of CTV isolates, including the possible interaction of different genotypes in causing or preventing diseases. The methods described can also be used in virus-free citrus propagation programs and in the development of CTV-resistant cultivars. PMID:20839943

  13. Genotype composition of populations of grapefruit-cross-protecting citrus tristeza virus strain GFMS12 in different host plants and aphid-transmitted sub-isolates.

    PubMed

    Scott, Katherine Anne; Hlela, Quinsile; Zablocki, Olivier; Read, David; van Vuuren, Stephanus; Pietersen, Gerhard

    2013-01-01

    Citrus tristeza virus (CTV) causes severe losses in grapefruit production in South Africa and requires mild-strain cross-protection to maintain production. Unfortunately, cross-protection breakdown of the pre-immunizing CTV grapefruit mild source GFMS12 is prevalent in grapefruit in South Africa. The CTV genotype composition of the GFMS12 population inoculated onto different hosts was determined by sequencing part of ORF1a and the p23 gene of multiple clones from each plant. Analysis of the GFMS12 population in Mexican lime and Marsh and Star Ruby grapefruit varieties revealed that at least four genotypes occur in the GFMS12 population and that genotype compositions differed amongst the populations in different host plants. Single-aphid-transmitted sub-isolates derived from the GFMS12 mother population on Mexican lime appeared to contain three populations of a mixture of VT-like and recombinant B165/VT-like genotypes; a mixture of recombinant RB/VT- and B165/VT-like genotypes; and a single recombinant B165/VT-like genotype. This study underlines the importance of determining the genotype composition of a potential CTV pre-immunizing source on a range of inoculated host species before utilization. PMID:22932923

  14. Production of Polyclonal Antibodies to the Recombinant Coat Protein of Citrus tristeza virus and Their Effectiveness for Virus Detection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The p25 coat protein gene of three Citrus tristeza virus (CTV) isolates, two from Mexico and one from India, was amplified by RT-PCR and further cloned and expressed in Escherichia coli cells. The recombinant coat protein (rCP) of the three CTV isolates was injected into rabbits and goats for antibo...

  15. Dramatic Change in Citrus tristeza virus populations in the Dominican Republic

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Citrus tristeza virus (CTV) is the most destructive viral pathogen of citrus and has been an important concern for the citrus industry in the Dominican Republic. Earlier studies documented widespread distribution of mild isolates of the T30 genotype, which caused no disease in the infected trees, an...

  16. Transgenic Resistance to Citrus tristeza virus in Grapefruit

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Grapefruit (Citrus paradisi) transgenic plants transformed with a variety of constructs derived from the Citrus tristeza virus (CTV) genome were tested for their resistance to the virus. Most transgenic lines were susceptible (27 lines), a few were partially resistant (6 lines) and only one line, tr...

  17. Emergence and phylodynamics of Citrus tristeza virus in Sicily, Italy.

    PubMed

    Davino, Salvatore; Willemsen, Anouk; Panno, Stefano; Davino, Mario; Catara, Antonino; Elena, Santiago F; Rubio, Luis

    2013-01-01

    Citrus tristeza virus (CTV) outbreaks were detected in Sicily island, Italy for the first time in 2002. To gain insight into the evolutionary forces driving the emergence and phylogeography of these CTV populations, we determined and analyzed the nucleotide sequences of the p20 gene from 108 CTV isolates collected from 2002 to 2009. Bayesian phylogenetic analysis revealed that mild and severe CTV isolates belonging to five different clades (lineages) were introduced in Sicily in 2002. Phylogeographic analysis showed that four lineages co-circulated in the main citrus growing area located in Eastern Sicily. However, only one lineage (composed of mild isolates) spread to distant areas of Sicily and was detected after 2007. No correlation was found between genetic variation and citrus host, indicating that citrus cultivars did not exert differential selective pressures on the virus. The genetic variation of CTV was not structured according to geographical location or sampling time, likely due to the multiple introduction events and a complex migration pattern with intense co- and re-circulation of different lineages in the same area. The phylogenetic structure, statistical tests of neutrality and comparison of synonymous and nonsynonymous substitution rates suggest that weak negative selection and genetic drift following a rapid expansion may be the main causes of the CTV variability observed today in Sicily. Nonetheless, three adjacent amino acids at the p20 N-terminal region were found to be under positive selection, likely resulting from adaptation events. PMID:23818960

  18. Emergence and Phylodynamics of Citrus tristeza virus in Sicily, Italy

    PubMed Central

    Davino, Salvatore; Willemsen, Anouk; Panno, Stefano; Davino, Mario; Catara, Antonino; Elena, Santiago F.; Rubio, Luis

    2013-01-01

    Citrus tristeza virus (CTV) outbreaks were detected in Sicily island, Italy for the first time in 2002. To gain insight into the evolutionary forces driving the emergence and phylogeography of these CTV populations, we determined and analyzed the nucleotide sequences of the p20 gene from 108 CTV isolates collected from 2002 to 2009. Bayesian phylogenetic analysis revealed that mild and severe CTV isolates belonging to five different clades (lineages) were introduced in Sicily in 2002. Phylogeographic analysis showed that four lineages co-circulated in the main citrus growing area located in Eastern Sicily. However, only one lineage (composed of mild isolates) spread to distant areas of Sicily and was detected after 2007. No correlation was found between genetic variation and citrus host, indicating that citrus cultivars did not exert differential selective pressures on the virus. The genetic variation of CTV was not structured according to geographical location or sampling time, likely due to the multiple introduction events and a complex migration pattern with intense co- and re-circulation of different lineages in the same area. The phylogenetic structure, statistical tests of neutrality and comparison of synonymous and nonsynonymous substitution rates suggest that weak negative selection and genetic drift following a rapid expansion may be the main causes of the CTV variability observed today in Sicily. Nonetheless, three adjacent amino acids at the p20 N-terminal region were found to be under positive selection, likely resulting from adaptation events. PMID:23818960

  19. Thirty years of citrus tristeza virus observations in Peru

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Peruvian citrus industry was devastated by epidemics of Citrus tristeza virus (CTV) decline (CTV-D) on sour orange rootstock between 1950 and 1965 and CTV stem pitting (SP) between 1965 and 1985. CTV-SP debilitates citrus and fruit production regardless of rootstock. Control of CTV-SP by mild st...

  20. Discrimination between mild and severe Citrus tristeza virus isolates with a rapid and highly specific real-time reverse transcription-polymerase chain reaction method using TaqMan LNA probes.

    PubMed

    Ruiz-Ruiz, S; Moreno, P; Guerri, J; Ambrós, S

    2009-03-01

    Severe isolates of Citrus tristeza virus (CTV) inducing seedling yellows (SY) and/or stem pitting (SP) in grapefruit or sweet orange are a major threat for the citrus industry worldwide. Identification of these CTV variants was achieved by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) using a general primer set and three TaqMan locked nucleic acids (LNA) probes targeting sequences characteristic of severe, mild (non-SY, non-SP), and T36-like isolates. Successful amplification was achieved from fresh or silica-desiccated CTV-infected samples and all isolates but one reacted with one or more probes. Standard curves using RNA transcripts homologous to the three probes allowed a reproducible quantitative assay, with a wide dynamic range of detection starting with 10(2) copies. RT-PCR assays with homologous and heterologous transcript RNA mixes demonstrated that each probe reacted only with its cognate sequence which was detected even at ratios below 2.5%. Analysis of 56 pathogenically distinct CTV isolates from 20 countries showed that mild isolates reacted only with the mild probe, whereas severe SP and SY isolates reacted with the severe-SP or the T36-like probes, respectively, and often with a second probe. This procedure can be useful to identify and control potentially dangerous CTV isolates in areas affected only by mild isolates. PMID:19203284

  1. Infection with strains of Citrus tristeza virus does not exclude superinfection by other strains of the virus.

    PubMed

    Folimonova, Svetlana Y; Robertson, Cecile J; Shilts, Turksen; Folimonov, Alexey S; Hilf, Mark E; Garnsey, Stephen M; Dawson, William O

    2010-02-01

    Superinfection exclusion or homologous interference, a phenomenon in which a primary viral infection prevents a secondary infection with the same or closely related virus, has been observed commonly for viruses in various systems, including viruses of bacteria, plants, and animals. With plant viruses, homologous interference initially was used as a test of virus relatedness to define whether two virus isolates were "strains" of the same virus or represented different viruses, and subsequently purposeful infection with a mild isolate was implemented as a protective measure against isolates of the virus causing severe disease. In this study we examined superinfection exclusion of Citrus tristeza virus (CTV), a positive-sense RNA closterovirus. Thirteen naturally occurring isolates of CTV representing five different virus strains and a set of isolates originated from virus constructs engineered based on an infectious cDNA clone of T36 isolate of CTV, including hybrids containing sequences from different isolates, were examined for their ability to prevent superinfection by another isolate of the virus. We show that superinfection exclusion occurred only between isolates of the same strain and not between isolates of different strains. When isolates of the same strain were used for sequential plant inoculation, the primary infection provided complete exclusion of the challenge isolate, whereas isolates from heterologous strains appeared to have no effect on replication, movement or systemic infection by the challenge virus. Surprisingly, substitution of extended cognate sequences from isolates of the T68 or T30 strains into T36 did not confer the ability of resulting hybrid viruses to exclude superinfection by those donor strains. Overall, these results do not appear to be explained by mechanisms proposed previously for other viruses. Moreover, these observations bring an understanding of some previously unexplained fundamental features of CTV biology and, most importantly, build a foundation for the strategy of selecting mild isolates that would efficiently exclude severe virus isolates as a practical means to control CTV diseases. PMID:19923189

  2. Citrus tristeza virus: Evolution of Complex and Varied Genotypic Groups.

    PubMed

    Harper, S J

    2013-01-01

    Amongst the Closteroviridae, Citrus tristeza virus (CTV) is almost unique in possessing a number of distinct and characterized strains, isolates of which produce a wide range of phenotype combinations among its different hosts. There is little understanding to connect genotypes to phenotypes, and to complicate matters more, these genotypes are found throughout the world as members of mixed populations within a single host plant. There is essentially no understanding of how combinations of genotypes affect symptom expression and disease severity. We know little about the evolution of the genotypes that have been characterized to date, little about the biological role of their diversity and particularly, about the effects of recombination. Additionally, genotype grouping has not been standardized. In this study we utilized an extensive array of CTV genomic information to classify the major genotypes, and to determine the major evolutionary processes that led to their formation and subsequent retention. Our analyses suggest that three major processes act on these genotypes: (1) ancestral diversification of the major CTV lineages, followed by (2) conservation and co-evolution of the major functional domains within, though not between CTV genotypes, and (3) extensive recombination between lineages that have given rise to new genotypes that have subsequently been retained within the global population. The effects of genotype diversity and host-interaction are discussed, as is a proposal for standardizing the classification of existing and novel CTV genotypes. PMID:23630519

  3. Evaluación de Anticuerpos Desarrollados Contra la Proteína Recombinante de la Cápside del Virus Tristeza de los Cítricos

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyclonal antibodies specific for the recombinant coat protein (rCP) p25 gene of (Citrus tristeza virus = CTV), were developed for isolates MX08 and MX14 from México and B227 from India. The reactivity of rCP antibodies was evaluated using healthy and CTV infected tissue. The combination of rCP ant...

  4. Citrus tristeza virus: making an ally from an enemy.

    PubMed

    Dawson, William O; Bar-Joseph, Moshe; Garnsey, Stephen M; Moreno, Pedro

    2015-01-01

    Virus diseases of perennial trees and vines have characteristics not amenable to study using small model annual plants. Unique disease symptoms such as graft incompatibilities and stem pitting cause considerable crop losses. Also, viruses in these long-living plants tend to accumulate complex populations of viruses and strains. Considerable progress has been made in understanding the biology and genetics of Citrus tristeza virus (CTV) and in developing it into a tool for crop protection and improvement. The diseases in tree and vine crops have commonalities for which CTV can be used to develop a baseline. The purpose of this review is to provide a necessary background of systems and reagents developed for CTV that can be used for continued progress in this area and to point out the value of the CTV-citrus system in answering important questions on plant-virus interactions and developing new methods for controlling plant diseases. PMID:25973695

  5. Two distinct evolutionary pathways for Citrus tristeza virus: recombination defines two gene modules and provides for increased genetic diversity in a narrow host range plant virus.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phylogenetic analysis of the full or partial genomic sequences of the Citrus tristeza virus (CTV) isolates T36, T68-1 and NS25 showed phylogenetic incongruities between sequences involved in viral RNA replication and those involved in movement and other viral functions. This incongruity was not fou...

  6. QTL analysis of citrus tristeza virus-citradia interaction.

    PubMed

    Asins, M J; Bernet, G P; Ruiz, C; Cambra, M; Guerri, J; Carbonell, E A

    2004-02-01

    Citrus tristeza virus (CTV) has caused the death of millions of trees grafted on sour orange ( Citrus aurantium). However, this rootstock is very well adapted to the Mediterranean, semi-arid conditions. The aim of the present research is to genetically analyze the accumulation of CTV in a progeny derived from the cross between C. aurantium and Poncirus trifoliata, both resistant to CTV isolate T-346. Graft propagation of 104 hybrids was done on healthy sweet orange as a rootstock. Three months later, each rootstock was graft inoculated with two patches of infected tissue (isolate T-346). One, 2, and sometimes, 3 and 4 years after inoculation, hybrids and infected patches were tested for CTV by tissue-blot immuno-assay. Additionally, CTV multiplication was evaluated every year as the optical density of double-antibody sandwich enzyme-linked immuno-sorbent assay reactions. Linkage maps for P. trifoliata based on 63 markers, and for C. aurantium based on 157 markers, were used. Most molecular markers were microsatellites and IRAP (inter-retrotransposon amplified polymorphisms). Some analogues of resistance and expressed sequences were also included for candidate gene analysis. Resistance against CTV was analyzed as a quantitative trait (CTV accumulation) by QTL (quantitative trait loci) analysis to avoid the assumption of monogenic control. Three major resistance QTLs were detected where the P. trifoliata resistance gene, Ctv-R, had been previously located in other progenies. Up to five minor QTLs were detected ( Ctv-A(1) to Ctv-A(5)). A significant epistatic interaction involving Ctv-R(1) and Ctv-A(1) was also found. An analogue of a resistance gene is a candidate for Ctv-A(3), and two expressed sequences are candidates for Ctv-A(1) and Ctv-A(5). Single-strand conformational polymorphism analysis of CTV genes QTL P20 and P25 (coat protein) in susceptible hybrids, was carried out to test whether or not any QTL accumulation was a defeated resistance gene. Since the same haplotype of the virus was visualized independently on the CTV titer, differences in the amount of virions are not explained through the selection of CTV genotypes by the host, but through differences among citradias in CTV replication and/or movement. PMID:14614564

  7. Citrus tristeza virus: a pathogen that changed the course of the citrus industry.

    PubMed

    Moreno, Pedro; Ambrós, Silvia; Albiach-Martí, Maria R; Guerri, José; Peña, Leandro

    2008-03-01

    Citrus tristeza virus (CTV) (genus Closterovirus, family Closteroviridae) is the causal agent of devastating epidemics that changed the course of the citrus industry. Adapted to replicate in phloem cells of a few species within the family Rutaceae and to transmission by a few aphid species, CTV and citrus probably coevolved for centuries at the site of origin of citrus plants. CTV dispersal to other regions and its interaction with new scion varieties and rootstock combinations resulted in three distinct syndromes named tristeza, stem pitting and seedling yellows. The first, inciting decline of varieties propagated on sour orange, has forced the rebuilding of many citrus industries using tristeza-tolerant rootstocks. The second, inducing stunting, stem pitting and low bearing of some varieties, causes economic losses in an increasing number of countries. The third is usually observed by biological indexing, but rarely in the field. CTV polar virions are composed of two capsid proteins and a single-stranded, positive-sense genomic RNA (gRNA) of approximately 20 kb, containing 12 open reading frames (ORFs) and two untranslated regions (UTRs). ORFs 1a and 1b, encoding proteins of the replicase complex, are directly translated from the gRNA, and together with the 5' and 3'UTRs are the only regions required for RNA replication. The remaining ORFs, expressed via 3'-coterminal subgenomic RNAs, encode proteins required for virion assembly and movement (p6, p65, p61, p27 and p25), asymmetrical accumulation of positive and negative strands during RNA replication (p23), or suppression of post-transcriptional gene silencing (p25, p20 and p23), with the role of proteins p33, p18 and p13 as yet unknown. Analysis of genetic variation in CTV isolates revealed (1) conservation of genomes in distant geographical regions, with a limited repertoire of genotypes, (2) uneven distribution of variation along the gRNA, (3) frequent recombination events and (4) different selection pressures shaping CTV populations. Measures to control CTV damage include quarantine and budwood certification programmes, elimination of infected trees, use of tristeza-tolerant rootstocks, or cross protection with mild isolates, depending on CTV incidence and on the virus strains and host varieties predominant in each region. Incorporating resistance genes into commercial varieties by conventional breeding is presently unfeasible, whereas incorporation of pathogen-derived resistance by plant transformation has yielded variable results, indicating that the CTV-citrus interaction may be more specific and complex than initially thought. A deep understanding of the interactions between viral proteins and host and vector factors will be necessary to develop reliable and sound control measures. PMID:18705856

  8. Distribution, genetic diversity and recombination analysis of Citrus tristeza virus of India.

    PubMed

    Biswas, K K; Tarafdar, A; Diwedi, S; Lee, R F

    2012-08-01

    Citrus tristeza virus (CTV) isolates representing all the citrus-growing geographical zones of India were analyzed for nucleotide sequence of the 5'ORF1a fragments of the partial LProI domain and for the coat protein (CP) gene. The nucleotide sequences were compared with previously reported Indian and CTV genotypes from GenBank. The Indian isolates had 80-99 % sequence identity for the 5'ORF1a and 89-99 % identity for the CP genes. In phylogenetic tree analysis, all the Indian and previously reported isolates segregated into eight clades or groups for the 5'ORF1a region. Indian CTV isolates were clustered in all the clades, four of which, D13, K5, BAN-1, and B165, consisted of only Indian isolates. Phylogenetic tree analysis of the CP genes resulted in seven clades. Indian CTV isolates clustered in six of them, and clades I and VI consisted of only Indian isolates. In the phylogenetic tree the Indian CTV isolates clustered in different groups regardless their geographical origin. Diversities in CTV isolates within individual citrus farms were highlighted. Because incongruent phylogenetic relationships were observed for both of the genomic regions, 5'ORF1a and CP gene, recombination analysis was performed using program RDP3. This analysis detected potential recombination events among the CTV isolates which involved exchange of sequences between divergent CTV variants. The SplitsTree analysis showed evidence of phylogenetic conflicts in evolutionary relationships among CTV isolates. PMID:22562224

  9. Developing an understanding of cross-protection by Citrus tristeza virus.

    PubMed

    Folimonova, Svetlana Y

    2013-01-01

    Citrus tristeza virus (CTV) causes two citrus diseases that have caused devastating losses in global citrus production. The first disease is quick decline of trees propagated on the sour orange rootstock. The second disease is stem pitting, which severely affects a number of economically important citrus varieties regardless of the rootstock used and results in reduced tree growth and vigor as well as in reduced fruit size and quality. Both diseases continue to invade new areas. While quick decline could be effectively managed by the use of resistant and/or tolerant rootstocks, the only means to protect commercial citrus against endemic stem pitting isolates of CTV has been cross-protection with mild isolates of the virus. In some citrus areas cross-protection has been successful and allowed production of certain citrus cultivars despite the presence of severe stem pitting isolates in those regions. However, many other attempts to find isolates that would provide sustained protection against aggressive isolates of the virus had failed. In general, there has been no understanding why some mild isolates were effective and others failed to protect. We have been working on the mechanism of cross-protection by CTV. Recent considerable progress has significantly advanced our understanding of how cross-protection may work in the citrus/CTV pathosystem. As we demonstrated, only isolates that belong to the same strain of the virus cross protect against each other, while isolates from different strains do not. We believe that the results of our research could now make finding protecting isolates relatively straightforward. This review discusses some of the history of CTV cross-protection along with the recent findings and our "recipe" for selection of protecting isolates. PMID:23577008

  10. Developing an understanding of cross-protection by Citrus tristeza virus

    PubMed Central

    Folimonova, Svetlana Y.

    2013-01-01

    Citrus tristeza virus (CTV) causes two citrus diseases that have caused devastating losses in global citrus production. The first disease is quick decline of trees propagated on the sour orange rootstock. The second disease is stem pitting, which severely affects a number of economically important citrus varieties regardless of the rootstock used and results in reduced tree growth and vigor as well as in reduced fruit size and quality. Both diseases continue to invade new areas. While quick decline could be effectively managed by the use of resistant and/or tolerant rootstocks, the only means to protect commercial citrus against endemic stem pitting isolates of CTV has been cross-protection with mild isolates of the virus. In some citrus areas cross-protection has been successful and allowed production of certain citrus cultivars despite the presence of severe stem pitting isolates in those regions. However, many other attempts to find isolates that would provide sustained protection against aggressive isolates of the virus had failed. In general, there has been no understanding why some mild isolates were effective and others failed to protect. We have been working on the mechanism of cross-protection by CTV. Recent considerable progress has significantly advanced our understanding of how cross-protection may work in the citrus/CTV pathosystem. As we demonstrated, only isolates that belong to the same strain of the virus cross protect against each other, while isolates from different strains do not. We believe that the results of our research could now make finding protecting isolates relatively straightforward. This review discusses some of the history of CTV cross-protection along with the recent findings and our “recipe” for selection of protecting isolates. PMID:23577008

  11. Contribution of recombination and selection to molecular evolution of Citrus tristeza virus.

    PubMed

    Martín, Susana; Sambade, Adrián; Rubio, Luis; Vives, María C; Moya, Patricia; Guerri, José; Elena, Santiago F; Moreno, Pedro

    2009-06-01

    The genetic variation of Citrus tristeza virus (CTV) was analysed by comparing the predominant sequence variants in seven genomic regions (p33, p65, p61, p18, p13, p20 and p23) of 18 pathogenically distinct isolates from seven different countries. Analyses of the selective constraints acting on each codon suggest that most regions were under purifying selection. Phylogenetic analysis shows diverse patterns of molecular evolution for different genomic regions. A first clade composed of isolates that are genetically close to the reference mild isolates T385 or T30 was inferred from all genomic regions. A second clade, mostly comprising virulent isolates, was defined from regions p33, p65, p13 and p23. For regions p65, p61, p18, p13 and p23, a third clade that mostly included South American isolates could not be related to any reference genotype. Phylogenetic relationships among isolates did not reflect their geographical origin, suggesting significant gene flow between geographically distant areas. Incongruent phylogenetic trees for different genomic regions suggested recombination events, an extreme that was supported by several recombination-detecting methods. A phylogenetic network incorporating the effect of recombination showed an explosive radiation pattern for the evolution of some isolates and also grouped isolates by virulence. Taken together, the above results suggest that negative selection, gene flow, sequence recombination and virulence may be important factors driving CTV evolution. PMID:19264625

  12. Fifteenth IOCV Conference, 2002--Citrus Tristeza Virus Effects of Chemical Control of Aphid Vectors

    E-print Network

    Ma, Lena

    117 Fifteenth IOCV Conference, 2002--Citrus Tristeza Virus Effects of Chemical Control of Aphid-Rodriguez, R. K. Yokomi, P. A. Stansly, and T. K. Riley ABSTRACT. The recent spread of the brown citrus aphid of CTV where T. citri- cida, and other aphid vectors are present. Plots were established with healthy

  13. Etiology, background, worldwide situation and control of Citrus Tristeza virus and its vectors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Citrus tristeza virus (CTV) is readily graft-transmissible and, in nature, is spread by aphid vectors in a semi-persistent manner. CTV-decline has killed >85 million citrus trees grown on sour orange rootstock worldwide. Citrus in these areas must be grown on CTV-tolerant or resistant rootstocks. ...

  14. Past and future of a century old Citrus tristeza virus collection: a California citrus germplasm tale.

    PubMed

    Wang, Jinbo; Bozan, Orhan; Kwon, Sun-Jung; Dang, Tyler; Rucker, Tavia; Yokomi, Raymond K; Lee, Richard F; Folimonova, Svetlana Y; Krueger, Robert R; Bash, John; Greer, Greg; Diaz, James; Serna, Ramon; Vidalakis, Georgios

    2013-01-01

    Citrus tristeza virus (CTV) isolates collected from citrus germplasm, dooryard and field trees in California from 1914 have been maintained in planta under quarantine in the Citrus Clonal Protection Program (CCPP), Riverside, California. This collection, therefore, represents populations of CTV isolates obtained over time and space in California. To determine CTV genetic diversity in this context, genotypes of CTV isolates from the CCPP collection were characterized using multiple molecular markers (MMM). Genotypes T30, VT, and T36 were found at high frequencies with T30 and T30+VT genotypes being the most abundant. The MMM analysis did not identify T3 and B165/T68 genotypes; however, biological and phylogenetic analysis suggested some relationships of CCPP CTV isolates with these two genotypes. Phylogenetic analysis of the CTV coat protein (CP) gene sequences classified the tested isolates into seven distinct clades. Five clades were in association with the standard CTV genotypes T30, T36, T3, VT, and B165/T68. The remaining two identified clades were not related to any standard CTV genotypes. Spatiotemporal analysis indicated a trend of reduced genotype and phylogenetic diversity as well as virulence from southern California (SC) at early (1907-1957) in comparison to that of central California (CC) isolates collected from later (1957-2009) time periods. CTV biological characterization also indicated a reduced number and less virulent stem pitting (SP) CTV isolates compared to seedling yellows isolates introduced to California. This data provides a historical insight of the introduction, movement, and genetic diversity of CTV in California and provides genetic and biological information useful for CTV quarantine, eradication, and disease management strategies such as CTV-SP cross protection. PMID:24339822

  15. The epitope structure of Citrus tristeza virus coat protein mapped by recombinant proteins and monoclonal antibodies.

    PubMed

    Wu, Guan-Wei; Tang, Min; Wang, Guo-Ping; Wang, Cai-Xia; Liu, Yong; Yang, Fan; Hong, Ni

    2014-01-01

    It has been known that there exists serological differentiation among Citrus tristeza virus (CTV) isolates. The present study reports three linear epitopes (aa 48-63, 97-104, and 114-125) identified by using bacterially expressed truncated coat proteins and ten monoclonal antibodies against the native virions of CTV-S4. Site-directed mutagenesis analysis demonstrated that the mutation D98G within the newly identified epitope (97)DDDSTGIT(104) abolished its reaction to MAbs 1, 4, and 10, and the presence of G98 in HB1-CP also resulted in its failure to recognize the three MAbs. Our results suggest that the conformational differences in the epitope I (48)LGTQQNAALNRDLFLT(63) between the CPs of isolates S4 and HB1 might contribute to the different reactions of two isolates to MAbs 5 and 6. This study provides new information for the antigenic structures of CTV, and will extend the understanding of the processes required for antibody binding and aid the development of epitope-based diagnostic tools. PMID:24314654

  16. Sequence diversity on four ORFs of citrus tristeza virus correlates with pathogenicity.

    PubMed

    Herrera-Isidrón, Lisset; Ochoa-Sánchez, Juan Carlos; Rivera-Bustamante, Rafael; Martínez-Soriano, Juan Pablo

    2009-01-01

    The molecular characterization of isolates of citrus tristeza virus (CTV) from eight locations in Mexico was undertaken by analyzing five regions located at the opposite ends of the virus genome. Two regions have been previously used to study CTV variability (coat protein and p23), while the other three correspond to other genomic segments (p349-B, p349-C and p13). Our comparative nucleotide analyses included CTV sequences from different geographical origins already deposited in the GenBank databases. The largest nucleotide differences were located in two fragments located at the 5' end of the genome (p349-B and p349-C). Phylogenetic analyses on those five regions showed that the degree of nucleotide divergence among strains tended to correlate with their pathogenicity. Two main groups were defined: mild, with almost no noticeable effects on the indicator plants and severe, with drastic symptoms. Mild isolates clustered together in every analyzed ORF sharing a genetic distance below 0.022, in contrast with the severe isolates, which showed a more disperse distribution and a genetic distance of 0.276. Analyses of the p349-B and p349-C regions evidenced two lineages within the severe group: severe common subgroup (most of severe isolates) and severe divergent subgroup (T36-like isolates). This study represents the first attempt to analyze the genetic variability of CTV in Mexico by constructing phylogenetic trees based on new genomic regions that use group-specific nucleotide and amino acid sequences. These results may be useful to implement specific assays for strain discrimination. Moreover, it would be an excellent reference for the CTV situation in México to face the recent arrival of brown citrus aphid. PMID:19642988

  17. Agroinoculation of Citrus tristeza virus causes systemic infection and symptoms in the presumed nonhost Nicotiana benthamiana.

    PubMed

    Ambrós, Silvia; El-Mohtar, Choaa; Ruiz-Ruiz, Susana; Peña, Leandro; Guerri, José; Dawson, William O; Moreno, Pedro

    2011-10-01

    Citrus tristeza virus (CTV) naturally infects only some citrus species and relatives and within these it only invades phloem tissues. Failure to agroinfect citrus plants and the lack of an experimental herbaceous host hindered development of a workable genetic system. A full-genome cDNA of CTV isolate T36 was cloned in binary plasmids and was used to agroinfiltrate Nicotiana benthamiana leaves, with or without coinfiltration with plasmids expressing different silencing-suppressor proteins. A time course analysis in agroinfiltrated leaves indicated that CTV accumulates and moves cell-to-cell for at least three weeks postinoculation (wpi), and then, it moves systemically and infects the upper leaves with symptom expression. Silencing suppressors expedited systemic infection and often increased infectivity. In systemically infected Nicotiana benthamiana plants, CTV invaded first the phloem, but after 7 wpi, it was also found in other tissues and reached a high viral titer in upper leaves, thus allowing efficient transmission to citrus by stem-slash inoculation. Infected citrus plants showed the symptoms, virion morphology, and phloem restriction characteristic of the wild T36 isolate. Therefore, agroinfiltration of Nicotiana benthamiana provided the first experimental herbaceous host for CTV and an easy and efficient genetic system for this closterovirus. PMID:21899435

  18. The evolutionary rate of citrus tristeza virus ranks among the rates of the slowest RNA viruses.

    PubMed

    Silva, Gonçalo; Marques, Natália; Nolasco, Gustavo

    2012-02-01

    Citrus tristeza virus (CTV) has been studied intensively at the molecular level. However, knowledge regarding the dynamics of its evolution is practically non-existent. In the past, diverse authors have referred to CTV as a highly variable virus, implying rapid evolution. Others have, in recent times, referred to CTV as an exceptionally slowly evolving virus. In this work, we used the capsid protein (CP) gene to estimate the rate of evolution. This was obtained from a large set of heterochronous CP gene sequences using a bayesian coalescent approach. The best-fitting evolutionary and population models pointed to an evolutionary rate of 1.58×10(-4) nt per site year(-1) (95?% highest posterior density, 1.73×10(-5)-3.16×10(-4) nt per site year(-1)). For an unbiased comparison with other plant and animal viruses, the evolutionary rate of synonymous substitutions was considered. In a series of 88 synonymous evolutionary rates, ranging from 5.2×10(-6) to 6.2×10(-2) nt per site year(-1), CTV ranks in the 10th percentile, embedded among the slowest animal RNA viruses. At the time of citrus dissemination to Europe and the New World, the major clades that led to the current phylogenetic groups were already defined, which may explain the absence nowadays of geographical speciation. PMID:22071513

  19. Differential tropism in roots and shoots infected by Citrus tristeza virus.

    PubMed

    Harper, S J; Cowell, S J; Robertson, C J; Dawson, W O

    2014-07-01

    Virus tropism is a result of interactions between virus, host and vector species, and determines the fate of an infection. In this study, we examined the infection process of Citrus tristeza virus (CTV) in susceptible and resistant species, and found that the tropism of CTV is not simply phloem limited, but tissue specific. In resistant species, virus infection was not prevented, but mostly restricted to the roots. This phenomenon was also observed after partial replacement of genes of one CTV strain from another, despite both parental strains being capable of systemic infection. Finally, the roots remained susceptible in the absence of viral gene products needed for systemic infection of shoots. Our results suggest that all phloem cells within a plant are not equally susceptible and that changes in host or virus may produce a novel tropism: restriction by the host to a location where further virus spread is prevented. PMID:25010274

  20. Expressed sequence enrichment for candidate gene analysis of citrus tristeza virus resistance.

    PubMed

    Bernet, G P; Bretó, M P; Asins, M J

    2004-02-01

    Several studies have reported markers linked to a putative resistance gene from Poncirus trifoliata ( Ctv-R) located at linkage group 4 that confers resistance against one of the most important citrus pathogens, citrus tristeza virus (CTV). To be successful in both marker-assisted selection and transformation experiments, its accurate mapping is needed. Several factors may affect its localization, among them two are considered here: the definition of resistance and the genetic background of progeny. Two progenies derived from P. trifoliata, by self-pollination and by crossing with sour orange ( Citrus aurantium), a citrus rootstock well-adapted to arid and semi-arid areas, were used for linkage group-4 marker enrichment. Two new methodologies were used to enrich this region with expressed sequences. The enrichment of group 4 resulted in the fusion of several C. aurantium linkage groups. The new one A(7+3+4) is now saturated with 48 markers including expressed sequences. Surprisingly, sour orange was as resistant to the CTV isolate tested as was P. trifoliata, and three hybrids that carry Ctv-R, as deduced from its flanking markers, are susceptible to CTV. The new linkage maps were used to map Ctv-R under the hypothesis of monogenic inheritance. Its position on linkage group 4 of P. trifoliata differs from the location previously reported in other progenies. The genetic analysis of virus-plant interaction in the family derived from C. aurantium after a CTV chronic infection showed the segregation of five types of interaction, which is not compatible with the hypothesis of a single gene controlling resistance. Two major issues are discussed: another type of genetic analysis of CTV resistance is needed to avoid the assumption of monogenic inheritance, and transferring Ctv-R from P. trifoliata to sour orange might not avoid the CTV decline of sweet orange trees. PMID:14624336

  1. Citrus tristeza virus p23: a unique protein mediating key virus-host interactions.

    PubMed

    Flores, Ricardo; Ruiz-Ruiz, Susana; Soler, Nuria; Sánchez-Navarro, Jesús; Fagoaga, Carmen; López, Carmelo; Navarro, Luis; Moreno, Pedro; Peña, Leandro

    2013-01-01

    The large RNA genome of Citrus tristeza virus (CTV; ca. 20 kb) contains 12 open reading frames, with the 3'-terminal one corresponding to a protein of 209 amino acids (p23) that is expressed from an abundant subgenomic RNA. p23, an RNA-binding protein with a putative zinc-finger domain and some basic motifs, is unique to CTV because no homologs have been found in other closteroviruses, including the type species of the genus Beet yellows virus (despite both viruses having many homologous genes). Consequently, p23 might have evolved for the specific interaction of CTV with its citrus hosts. From a functional perspective p23 has been involved in many roles: (i) regulation of the asymmetrical accumulation of CTV RNA strands, (ii) induction of the seedling yellows syndrome in sour orange and grapefruit, (iii) intracellular suppression of RNA silencing, (iv) elicitation of CTV-like symptoms when expressed ectopically as a transgene in several Citrus spp., and (v) enhancement of systemic infection (and virus accumulation) in sour orange and CTV release from the phloem in p23-expressing transgenic sweet and sour orange. Moreover, transformation of Mexican lime with intron-hairpin constructs designed for the co-inactivation of p23 and the two other CTV silencing suppressors results in complete resistance against the homologous virus. From a cellular point of view, recent data indicate that p23 accumulates preferentially in the nucleolus, being the first closterovirus protein with such a subcellular localization, as well as in plasmodesmata. These major accumulation sites most likely determine some of the functional roles of p23. PMID:23653624

  2. Citrus tristeza virus (CTV) Causing Proteomic and Enzymatic Changes in Sweet Orange Variety "Westin".

    PubMed

    Dória, Milena Santos; Sousa, Aurizângela Oliveira de; Barbosa, Cristiane de Jesus; Costa, Márcio Gilberto Cardoso; Gesteira, Abelmon da Silva; Souza, Regina Martins; Freitas, Ana Camila Oliveira; Pirovani, Carlos Priminho

    2015-01-01

    Citrus Tristeza disease, caused by CTV (Citrus tristeza virus), committs citrus plantations around the world and specifically attacks phloem tissues of the plant. The virus exists as a mixture of more or less severe variants, which may or may not cause symptoms of Tristeza. The objective of this study was to analyze the changes caused by CTV in the proteome of stems of sweet orange, as well as in the activity and gene expression of antioxidant enzymes. The CTV-infected sweet orange displayed mild symptoms, which were characterized by the presence of sparse stem pitting throughout their stems. The presence of virus was confirmed by RT-PCR. Proteomic analysis by 2DE-PAGE-MS / MS revealed the identity of 40 proteins differentially expressed between CTV- infected and -non-infected samples. Of these, 33 were up-regulated and 7 were down-regulated in CTV-infected samples. Among the proteins identified stands out a specific from the virus, the coat protein. Other proteins identified are involved with oxidative stress and for this their enzymatic activity was measured. The activity of superoxide dismutase (SOD) was higher in CTV-infected samples, as catalase (CAT) showed higher activity in uninfected samples. The activity of guaiacol peroxidase (GPX) did not vary significantly between samples. However, ascorbate peroxidase (APX) was more active in the infected samples. The relative expression of the genes encoding CAT, SOD, APX and GPX was analyzed by quantitative real time PCR (RT-qPCR). The CTV-infected samples showed greater accumulation of transcripts, except for the CAT gene. This gene showed higher expression in the uninfected samples. Taken together, it can be concluded that the CTV affects the protein profile and activity and gene expression of antioxidant enzymes in plants infected by this virus. PMID:26207751

  3. Citrus tristeza virus (CTV) Causing Proteomic and Enzymatic Changes in Sweet Orange Variety “Westin”

    PubMed Central

    Dória, Milena Santos; de Sousa, Aurizângela Oliveira; Barbosa, Cristiane de Jesus; Costa, Márcio Gilberto Cardoso; Gesteira, Abelmon da Silva; Souza, Regina Martins; Freitas, Ana Camila Oliveira; Pirovani, Carlos Priminho

    2015-01-01

    Citrus Tristeza disease, caused by CTV (Citrus tristeza virus), committs citrus plantations around the world and specifically attacks phloem tissues of the plant. The virus exists as a mixture of more or less severe variants, which may or may not cause symptoms of Tristeza. The objective of this study was to analyze the changes caused by CTV in the proteome of stems of sweet orange, as well as in the activity and gene expression of antioxidant enzymes. The CTV-infected sweet orange displayed mild symptoms, which were characterized by the presence of sparse stem pitting throughout their stems. The presence of virus was confirmed by RT-PCR. Proteomic analysis by 2DE-PAGE-MS / MS revealed the identity of 40 proteins differentially expressed between CTV- infected and -non-infected samples. Of these, 33 were up-regulated and 7 were down-regulated in CTV-infected samples. Among the proteins identified stands out a specific from the virus, the coat protein. Other proteins identified are involved with oxidative stress and for this their enzymatic activity was measured. The activity of superoxide dismutase (SOD) was higher in CTV-infected samples, as catalase (CAT) showed higher activity in uninfected samples. The activity of guaiacol peroxidase (GPX) did not vary significantly between samples. However, ascorbate peroxidase (APX) was more active in the infected samples. The relative expression of the genes encoding CAT, SOD, APX and GPX was analyzed by quantitative real time PCR (RT-qPCR). The CTV-infected samples showed greater accumulation of transcripts, except for the CAT gene. This gene showed higher expression in the uninfected samples. Taken together, it can be concluded that the CTV affects the protein profile and activity and gene expression of antioxidant enzymes in plants infected by this virus. PMID:26207751

  4. Superinfection exclusion by Citrus tristeza virus does not correlate with the production of viral small RNAs.

    PubMed

    Folimonova, Svetlana Y; Harper, Scott J; Leonard, Michael T; Triplett, Eric W; Shilts, Turksen

    2014-11-01

    Superinfection exclusion (SIE), a phenomenon in which a preexisting viral infection prevents a secondary infection with the same or closely related virus, has been described for different viruses, including important pathogens of humans, animals, and plants. Several mechanisms acting at various stages of the viral life cycle have been proposed to explain SIE. Most cases of SIE in plant virus systems were attributed to induction of RNA silencing, a host defense mechanism that is mediated by small RNAs. Here we show that SIE by Citrus tristeza virus (CTV) does not correlate with the production of viral small interfering RNAs (siRNAs). CTV variants, which differed in the SIE ability, had similar siRNAs profiles. Along with our previous observations that the exclusion phenomenon requires a specific viral protein, p33, the new data suggest that SIE by CTV is highly complex and appears to use different mechanisms than those proposed for other viruses. PMID:25248160

  5. Transgenic expression in citrus of single-chain antibody fragments specific to Citrus tristeza virus confers virus resistance.

    PubMed

    Cervera, Magdalena; Esteban, Olga; Gil, Maite; Gorris, M Teresa; Martínez, M Carmen; Peña, Leandro; Cambra, Mariano

    2010-12-01

    Citrus tristeza virus (CTV) causes one of the most destructive viral diseases of citrus worldwide. Generation of resistant citrus genotypes through genetic engineering could be a good alternative to control CTV. To study whether production of single-chain variable fragment (scFv) antibodies in citrus could interfere and immunomodulate CTV infection, transgenic Mexican lime plants expressing two different scFv constructs, separately and simultaneously, were generated. These constructs derived from the well-referenced monoclonal antibodies 3DF1 and 3CA5, specific against CTV p25 major coat protein, whose mixture is able to detect all CTV isolates characterized so far. ScFv accumulation levels were low and could be readily detected just in four transgenic lines. Twelve homogeneous and vigorous lines were propagated and CTV-challenged by graft inoculation with an aggressive CTV strain. A clear protective effect was observed in most transgenic lines, which showed resistance in up to 40-60% of propagations. Besides, both a delay in symptom appearance and attenuation of symptom intensity were observed in infected transgenic plants compared with control plants. This effect was more evident in lines carrying the 3DF1scFv transgene, being probably related to the biological functions of the epitope recognized by this antibody. This is the first report describing successful protection against a pathogen in woody transgenic plants by ectopic expression of scFv recombinant antibodies. PMID:20204695

  6. Exploring the limits of vector construction based on Citrus tristeza virus.

    PubMed

    El-Mohtar, Choaa; Dawson, William O

    2014-01-01

    We examined the limits of manipulation of the Citrus tristeza virus (CTV) genome for expressing foreign genes in plants. We previously created a vector with a foreign gene cassette inserted between the major and minor coat protein genes, which is position 6 from the 3' terminus. Yet, this virus has 10 3'-genes with several other potential locations for expression of foreign genes. Since genes positioned closer to the 3' terminus tend to be expressed in greater amounts, there were opportunities for producing greater amounts of foreign protein. We found that the virus tolerated insertions of an extra gene in most positions within the 3' region of the genome with substantially increased levels of gene product produced throughout citrus trees. CTV was amazingly tolerant to manipulation resulting in a suite of stable transient expression vectors, each with advantages for specific uses and sizes of foreign genes in citrus trees. PMID:24314658

  7. The conundrum of a unique protein encoded by citrus tristeza virus that is dispensable for infection of most hosts yet shows characteristics of a viral movement protein.

    PubMed

    Bak, Aurélie; Folimonova, Svetlana Y

    2015-11-01

    Citrus tristeza virus (CTV), one of the most economically important viruses, produces a unique protein, p33, which is encoded only in the genomes of isolates of CTV. Recently, we demonstrated that membrane association of the p33 protein confers virus ability to extend its host range. In this work we show that p33 shares characteristics of viral movement proteins. Upon expression in a host cell, the protein localizes to plasmodesmata and displays the ability to form extracellular tubules. Furthermore, p33 appears to traffic via the cellular secretory pathway and the actin network to plasmodesmata locations and is likely being recycled through the endocytic pathway. Finally, our study reveals that p33 colocalizes with a putative movement protein of CTV, the p6 protein. These results suggest a potential role of p33 as a noncanonical viral movement protein, which mediates virus translocation in the specific hosts. PMID:26210077

  8. Elevated Background in double antibody sandwich-indirect enzyme-linked immunosorbent assay for the detection of Citrus tristeza virus in mandarin cultivars

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Healthy tissue extracts from mandarin cultivars induced non-specific reaction in double antibody sandwich-indirect (DASI-) enzyme-linked immunosorbent assay (ELISA) for the detection of Citrus tristeza virus (CTV) by the Central California Tristeza Eradication Agency (CCTEA), Tulare, CA. This probl...

  9. LATENCY OF SYSTEMIC INFECTION IN YOUNG FIELD-GROWN SWEET ORANGE TREES FOLLOWING GRAFT-INOCULATION WITH CITRUS TRISTEZA VIRUS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Experiments were conducted to determine the time required for Citrus tristeza virus (CTV) to begin migration from the site of inoculation, and the subsequent incubation period required for systemic infection to occur. Young CTV-free sweet orange trees propagated on Citrus macrophylla rootstocks wer...

  10. Accumulation of a 5’ proximal subgenomic RNA of Citrus tristeza virus is correlated with encapsidation by the minor coat protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    During replication, Citrus tristeza virus (CTV) produces large amounts of two unusual subgenomic (sg) RNAs that are positive-stranded and 5' -coterminal. Although these RNAs are produced in similar amounts and are similar in size, with LMT1 (~750 nt) only slightly larger than LMT2 (~650), we found ...

  11. Development of an asymmetric PCR-ELISA typing method for citrus tristeza virus based on the coat protein gene.

    PubMed

    Nolasco, G; Santos, C; Silva, G; Fonseca, F

    2009-02-01

    The coat protein gene of isolates of citrus tristeza virus (CTV) from 20 citrus-producing regions around the world was amplified by RT-PCR, TA cloned, and characterized by SSCP. Haplotypes that produced different patterns within each geographic region were sequenced and a database of 153 accessions of CTV was assembled. Phylogenetic analysis revealed the existence of seven well-defined clusters (Coefficient of differentiation 0.78). An asymmetric PCR-ELISA typing (APET) assay was developed in the frame of this clustering pattern using a set of eight hybridisation probes. The membership of any unknown haplotype is determined by comparing its pattern of reaction against the whole set of probes and not, as previously done in hybridisation assays, in an all-or-nothing basis. Interpretation of the results is objective and done through a visual basic application that compares the rates of hydrolysis of the ELISA substrate of an assayed isolate to a matrix of rates of hydrolysis obtained from standard haplotypes. This assay was validated and showed a better ability to resolve haplotypes than other assays to which it was compared experimentally. It may be automated to the same extent as any ELISA. PMID:18992281

  12. Differential diagnosis of Brazilian strains of Citrus tristeza virus by epitope mapping of coat protein using monoclonal antibodies.

    PubMed

    Peroni, Luís Antonio; Lorencini, Márcio; dos Reis, José Raimundo Ribeiro; Machado, Marcos Antonio; Stach-Machado, Dagmar Ruth

    2009-10-01

    Citrus tristeza virus (CTV) is one of the most important citrus pathogen, and among Brazilian CTV strains, the genotype Capão Bonito (CB) is the most harmful. Therefore, the coat protein (CP) gene were cloned and expressed as recombinant protein and used to develop four specific monoclonal antibodies (MAbs). Our previously data had showed these MAbs could recognize different strains of CTV and the present goal is to identify the epitopes of the recombinant CP by ELISA screening of overlapping recombinant peptides and to determine the binding specificity of CTV isolates in light of their antigenic domains onto CB strains. Three MAbs, 30.G.02, 37.G.11 and 39.07 recognized linear and no identical epitopes, but the fourth MAb, IC.04-12, probably had a conformational epitope, since it could not be identified by ELISA screening. Our previous data revealed MAb IC.04-12 do not recognize CP under denaturing conditions, but can identify weak CTV strains in ELISA involving crop samples. MAb 30.G.02 recognized an extremely conserved sequence and can be classified as "universal" antibody, and, interestingly, the epitope turned out by MAb 39.07 corresponded to severe CTV isolates. So, these MAbs can be applied in a differential screening by ELISA. PMID:19540276

  13. Enhancement or attenuation of disease by deletion of genes from Citrus tristeza virus.

    PubMed

    Tatineni, Satyanarayana; Dawson, William O

    2012-08-01

    Stem pitting is a common virus-induced disease of perennial woody plants induced by a range of different viruses. The phenotype results from sporadic areas of the stem in which normal xylem and phloem development is prevented during growth of stems. These alterations interfere with carbohydrate transport, resulting in reduced plant growth and yield. Citrus tristeza virus (CTV), a phloem-limited closterovirus, induces economically important stem-pitting diseases of citrus. CTV has three nonconserved genes (p33, p18, and p13) that are not related to genes of other viruses and that are not required for systemic infection of some species of citrus, which allowed us to examine the effect of deletions of these genes on symptom phenotypes. In the most susceptible experimental host, Citrus macrophylla, the full-length virus causes only very mild stem-pitting symptoms. Surprisingly, we found that certain deletion combinations (p33 and p18 and/or p13) induced greatly increased stem-pitting symptoms, while other combinations (p13 or p13 plus p18) resulted in reduced stem pitting. These results suggest that the stem-pitting phenotype, which is one of more economically important disease phenotypes, can result not from a specific sequence or protein but from a balance between the expression of different viral genes. Unexpectedly, using green fluorescent protein-tagged full-length virus and deletion mutants (CTV9?p33 and CTV9?p33?p18?p13), the virus was found at pitted areas in abnormal locations outside the normal ring of phloem. Thus, increased stem pitting was associated not only with a prevention of xylem production but also with a proliferation of cells that supported viral replication, suggesting that at random areas of stems the virus can elicit changes in cellular differentiation and development. PMID:22593155

  14. Enhancement or Attenuation of Disease by Deletion of Genes from Citrus Tristeza Virus

    PubMed Central

    Tatineni, Satyanarayana

    2012-01-01

    Stem pitting is a common virus-induced disease of perennial woody plants induced by a range of different viruses. The phenotype results from sporadic areas of the stem in which normal xylem and phloem development is prevented during growth of stems. These alterations interfere with carbohydrate transport, resulting in reduced plant growth and yield. Citrus tristeza virus (CTV), a phloem-limited closterovirus, induces economically important stem-pitting diseases of citrus. CTV has three nonconserved genes (p33, p18, and p13) that are not related to genes of other viruses and that are not required for systemic infection of some species of citrus, which allowed us to examine the effect of deletions of these genes on symptom phenotypes. In the most susceptible experimental host, Citrus macrophylla, the full-length virus causes only very mild stem-pitting symptoms. Surprisingly, we found that certain deletion combinations (p33 and p18 and/or p13) induced greatly increased stem-pitting symptoms, while other combinations (p13 or p13 plus p18) resulted in reduced stem pitting. These results suggest that the stem-pitting phenotype, which is one of more economically important disease phenotypes, can result not from a specific sequence or protein but from a balance between the expression of different viral genes. Unexpectedly, using green fluorescent protein-tagged full-length virus and deletion mutants (CTV9?p33 and CTV9?p33?p18?p13), the virus was found at pitted areas in abnormal locations outside the normal ring of phloem. Thus, increased stem pitting was associated not only with a prevention of xylem production but also with a proliferation of cells that supported viral replication, suggesting that at random areas of stems the virus can elicit changes in cellular differentiation and development. PMID:22593155

  15. Prevalence of Citrus tristeza virus in Mandarin of Sikkim Himalayan Region.

    PubMed

    Kishore, Kundan; Rahman, H; Kalita, H; Pandey, Brijesh; Monika, N

    2010-10-01

    The assessment of Citrus tristeza virus incidence in mandarin of Sikkim, involving sampling techniques, was estimated by DAS-ELISA. Mandarin orchards had high CTV incidence (46.32%), however, differential prevalence with regard to age of plant and location was observed. The CTV prevalence was relatively high in older orchards (51.01%) than that of younger ones (40.80%). Under all the plant age groups, south district had the highest CTV incidence (52.50%) and east district had the lowest (37.71%). The spatial distribution of CTV in plants indicates high concentration in twig followed by leaf tissue, however, stem had relatively less concentration. High aphid infestation was observed in all mandarin growing groves with the maximum in south district and minimum in east district. Taxoptera citricida was the predominating aphid species followed by T. aurantii, however, Aphis spp population was significantly less. Aphid infestation and CTV prevalence were positively and significantly correlated. PMID:23637493

  16. Virus-viroid interactions: Citrus Tristeza Virus enhances the accumulation of Citrus Dwarfing Viroid in Mexican lime via virus-encoded silencing suppressors.

    PubMed

    Serra, Pedro; Bani Hashemian, Seyed M; Fagoaga, Carmen; Romero, Juan; Ruiz-Ruiz, Susana; Gorris, Maria T; Bertolini, Edson; Duran-Vila, Núria

    2014-01-01

    An assay to identify interactions between Citrus Dwarfing Viroid (CDVd) and Citrus Tristeza Virus (CTV) showed that viroid titer was enhanced by the coinfecting CTV in Mexican lime but not in etrog citron. Since CTV encodes three RNA silencing suppressors (RSSs), p23, p20 and p25, an assay using transgenic Mexican limes expressing each RSS revealed that p23 and, to a lesser extent, p25 recapitulated the effect observed with coinfections of CTV and CDVd. PMID:24227850

  17. Citrus tristeza virus: survival at the edge of the movement continuum.

    PubMed

    Folimonova, Svetlana Y; Folimonov, Alexey S; Tatineni, Satyanarayana; Dawson, William O

    2008-07-01

    Systemic invasion of plants by viruses is thought to involve two processes: cell-to-cell movement between adjacent cells and long-distance movement that allows the virus to rapidly move through sieve elements and unload at the growing parts of the plant. There is a continuum of proportions of these processes that determines the degrees of systemic infection of different plants by different viruses. We examined the systemic distribution of Citrus tristeza virus (CTV) in citrus species with a range of susceptibilities. By using a "pure" culture of CTV from a cDNA clone and green fluorescent protein-labeled virus we show that both cell-to-cell and long-distance movement are unusually limited, and the degree of limitation varies depending on the citrus host. In the more-susceptible hosts CTV infected only a small portion of phloem-associated cells, and moreover, the number of infection sites in less-susceptible citrus species was substantially decreased further, indicating that long-distance movement was reduced in those hosts. Analysis of infection foci in the two most differential citrus species, Citrus macrophylla and sour orange, revealed that in the more-susceptible host the infection foci were composed of a cluster of multiple cells, while in the less-susceptible host infection foci were usually single cells, suggesting that essentially no cell-to-cell movement occurred in the latter host. Thus, CTV in sour orange represents a pattern of systemic infection in which the virus appears to function with only the long-distance movement mechanism, yet is able to survive in nature. PMID:18434397

  18. The RNA-dependent RNA polymerase of Citrus tristeza virus forms oligomers.

    PubMed

    Cevik, Bayram

    2013-12-01

    The RNA-dependent RNA polymerases (RdRp) from Citrus tristeza virus (CTV) were tagged with HA and FLAG epitopes. Differentially tagged proteins were expressed either individually or concomitantly in Escherichia coli. Immunoprecipitation of the expressed proteins with anti-FLAG antibody followed by Western blot with anti-HA antibody demonstrated that molecules of RdRp from CTV interact to form oligomers. Yeast two-hybrid assays showed that molecules of RdRp interact in eukaryotic cells. Co-immunoprecipitation with anti-FLAG antibody of truncated HA-tagged RdRps (RdRp?1-166-HA, RdRp?1-390-HA, RdRp1-169-HA) co-expressed with full-length RdRp-FLAG showed that only RdRp1-169-HA interacted with the full-length FLAG-RdRp. Yeast two-hybrid assays with truncated RdRp constructs confirmed that the oligomerization site resides in the N-terminal region and that the first 169 aa of CTV RdRp are necessary and sufficient for oligomerization both in bacterial and yeast cells. Development of control strategies targeting viral RdRp oligomer formation may inhibit virus replication and prove useful in control of CTV. PMID:24210106

  19. Genetic diversity and evidence for recent modular recombination in Hawaiian Citrus tristeza virus.

    PubMed

    Melzer, Michael J; Borth, Wayne B; Sether, Diane M; Ferreira, Stephen; Gonsalves, Dennis; Hu, John S

    2010-02-01

    The Hawaiian Islands are home to a widespread and diverse population of Citrus tristeza virus (CTV), an economically important pathogen of citrus. In this study, we quantified the genetic diversity of two CTV genes and determined the complete genomic sequence for two strains of Hawaiian CTV. The nucleotide diversity was estimated to be 0.0565 + or - 0.0022 for the coat protein (CP) gene (n = 137) and 0.0822 + or - 0.0033 for the p23 gene (n = 30). The genome size and organization of CTV strains HA18-9 and HA16-5 were similar to other fully sequenced strains of CTV. The 3'-terminal halves of their genomes were nearly identical (98.5% nucleotide identity), whereas the 5'-terminal halves were more distantly related (72.3% nucleotide identity), suggesting a possible recombination event. Closer examination of strain HA16-5 indicated that it arose through recent recombination between the movement module of an HA18-9 genotype, and the replication module of an undescribed CTV genotype. PMID:19834797

  20. Characterization of Citrus tristeza virus strains from southern China based on analysis of restriction patterns and sequences of their coat protein genes.

    PubMed

    Jiang, Bo; Hong, Ni; Wang, Guo-Ping; Hu, John; Zhang, Jian-Kun; Wang, Cai-Xia; Liu, Yong; Fan, Xu-Dong

    2008-10-01

    Citrus tristeza virus (CTV) isolates collected from southern China were characterized by biological indexing on citrus indicators, restriction fragment length polymorphism (RFLP), and bi-directional reverse transcription-polymerase chain reaction (BD-PCR) analysis of their coat protein (CP) genes. Of the 30 isolates, only two isolates, N3 and N4, did not induce visible symptoms. Twenty-eight other isolates induced stem pitting and vein clearing, plant stunting, and leaf yellowing symptoms on Mexican lime, Duncan grapefruit, and sour orange seedlings. In BD-PCR analysis, a 392-bp fragment specific for the mild strains was amplified from isolates N3 and N4, and a 320-bp fragment specific for the severe strains was produced from the other 28 isolates. The RFLP analysis for RT-PCR products of the CP gene with restriction enzyme HinfI identified seven groups representing groups I-VI and a new group, which was not involved in the seven groups defined by Gillings (J Virol Methods 44:305-317, 1993). The sequences of the CP genes from 12 Chinese CTV isolates showed a high divergence, with 91.5-99.7% identities at the nucleotide level and 94.2-99.6% identities at the amino acid level. Our results suggest that the composition of CTV populations from China has a high genetic diversity in the CP gene. PMID:18626763

  1. Development and validation of a multiplex reverse transcription quantitative PCR (RT-qPCR) assay for the rapid detection of Citrus tristeza virus, Citrus psorosis virus, and Citrus leaf blotch virus.

    PubMed

    Osman, Fatima; Hodzic, Emir; Kwon, Sun-Jung; Wang, Jinbo; Vidalakis, Georgios

    2015-08-01

    A single real-time multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay for the simultaneous detection of Citrus tristeza virus (CTV), Citrus psorosis virus (CPsV), and Citrus leaf blotch virus (CLBV) was developed and validated using three different fluorescently labeled minor groove binding qPCR probes. To increase the detection reliability, coat protein (CP) genes from large number of different isolates of CTV, CPsV and CLBV were sequenced and a multiple sequence alignment was generated with corresponding CP sequences from the GenBank and a robust multiplex RT-qPCR assay was designed. The capacity of the multiplex RT-qPCR assay in detecting the viruses was compared to singleplex RT-qPCR designed specifically for each virus and was assessed using multiple virus isolates from diverse geographical regions and citrus species as well as graft-inoculated citrus plants infected with various combination of the three viruses. No significant difference in detection limits was found and specificity was not affected by the inclusion of the three assays in a multiplex RT-qPCR reaction. Comparison of the viral load for each virus using singleplex and multiplex RT-qPCR assays, revealed no significant differences between the two assays in virus detection. No significant difference in Cq values was detected when using one-step and two-step multiplex RT-qPCR detection formats. Optimizing the RNA extraction technique for citrus tissues and testing the quality of the extracted RNA using RT-qPCR targeting the cytochrome oxidase citrus gene as an RNA specific internal control proved to generate better diagnostic assays. Results showed that the developed multiplex RT-qPCR can streamline viruses testing of citrus nursery stock by replacing three separate singleplex assays, thus reducing time and labor while retaining the same sensitivity and specificity. The three targeted RNA viruses are regulated pathogens for California's mandatory "Section 3701: Citrus Nursery Stock Pest Cleanliness Program". Adopting a compatible multiplex RT-qPCR testing protocol for these viruses as well as other RNA and DNA regulated pathogens will provide a valuable alternative tool for virus detection and efficient program implementation. PMID:25907469

  2. A real-time RT-PCR assay for detection and absolute quantitation of Citrus tristeza virus in different plant tissues.

    PubMed

    Ruiz-Ruiz, Susana; Moreno, Pedro; Guerri, José; Ambrós, Silvia

    2007-11-01

    A real-time RT-PCR assay using SYBR Green was developed for specific and reliable quantitative detection of Citrus tristeza virus (CTV) in infected plants. A general primer set designed from conserved sequences in ORFs 1b and 2 enabled amplification of the genomic RNA (gRNA) while excluding most subgenomic and defective RNAs. Single RT-PCR products of 204 bp (isolate T36) or 186 bp (other isolates) were obtained with no primer-dimer or non-specific amplifications detected. Melting curve analysis revealed distinct melting temperature peaks (T(m)) for severe and mild isolates. External standard curves using RNA transcripts of the selected target allowed a reproducible quantitative assay, with a wide dynamic range of detection starting with 10(2) gRNA copies and with very low variation coefficient values. This protocol enabled reliable assessments of CTV accumulation in different tissues and from different citrus species, grown in the greenhouse or under field conditions, and infected with CTV isolates differing in their pathogenicity. CTV accumulation was higher in bark and fruits than in roots or leaves and showed minimal differences among several susceptible citrus species, but it was significantly lower in sour orange. This quantitative detection assay will be a valuable tool for diagnosis and molecular studies on CTV biology. PMID:17573130

  3. Rapid differentiation and identification of potential severe strains of Citrus tristeza virus by real-time reverse transcription-polymerase chain reaction assays.

    PubMed

    Yokomi, R K; Saponari, M; Sieburth, P J

    2010-04-01

    A multiplex Taqman-based real-time reverse transcription (RT) polymerase chain reaction (PCR) assay was developed to identify potential severe strains of Citrus tristeza virus (CTV) and separate genotypes that react with the monoclonal antibody MCA13. Three strain-specific probes were developed using intergene sequences between the major and minor coat protein genes (CPi) in a multiplex reaction. Probe CPi-VT3 was designed for VT and T3 genotypes; probe CPi-T36 for T36 genotypes; and probe CPi-T36-NS to identify isolates in an outgroup clade of T36-like genotypes mild in California. Total nucleic acids extracted by chromatography on silica particles, sodium dodecyl sulfate-potassium acetate, and CTV virion immunocapture all yielded high quality templates for real-time PCR detection of CTV. These assays successfully differentiated CTV isolates from California, Florida, and a large panel of CTV isolates from an international collection maintained in Beltsville, MD. The utility of the assay was validated using field isolates collected in California and Florida. PMID:20205535

  4. Heterologous Minor Coat Proteins of Citrus Tristeza Virus Strains Affect Encapsidation, but the Coexpression of HSP70h and p61 Restores Encapsidation to Wild-Type Levels

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The long flexuous bipolar virions of Citrus tristeza virus (CTV), a Closterovirus, are encapsidated with two capsid proteins at opposite ends: the minor coat protein (CPm) encapsidates the 5’ 630 nts of the genomic RNA and the major coat protein encapsidates the remainder of the genome. In this stud...

  5. Profiling of the small RNA populations derived from sour orange seedlings cross-protected against seedling yellows strains of Citrus tristeza virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Control of Citrus tristeza virus (CTV) in central California changed in 2009 from removal of all CTV-infected trees to only those which react positive in tests with selective probes for potentially severe CTV strains. Therefore, new strategies for CTV control are needed. Greenhouse tests have show...

  6. Genetic diversity of citrus tristeza virus from cross-protected and unprotected citrus trees after 20 years of natural challenge in Peru

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Quick decline, caused by Citrus tristeza virus (CTV), devastated Peruvian citrus on sour orange rootstock between 1950 and 1965. A second epidemic occurred between 1965 to 1985 due to spread of CTV strains causing severe stem pitting (SP) on branches and trunks, regardless of rootstock. SP weakens t...

  7. Ectopic expression of the p23 silencing suppressor of Citrus tristeza virus differentially modifies viral accumulation and tropism in two transgenic woody hosts.

    PubMed

    Fagoaga, Carmen; Pensabene-Bellavia, Giovanni; Moreno, Pedro; Navarro, Luís; Flores, Ricardo; Peña, Leandro

    2011-12-01

    Citrus tristeza virus (CTV), a phloem-restricted closterovirus infecting citrus, encodes three different silencing suppressors (p25, p20 and p23), one of which (p23) is a pathogenicity determinant that induces aberrations resembling CTV symptoms when expressed ectopically in transgenic citrus hosts. In this article, the effect of p23 ectopic expression on virus infection was examined in sweet orange (SwO), a highly susceptible host, and sour orange (SO), which severely restricts CTV cell-to-cell movement. Transgenic plants of both species ectopically expressing p23, or transformed with an empty vector, were graft inoculated with the mild CTV isolate T385 or with CTV-BC1/GFP, a clonal strain derived from the severe isolate T36 carrying the gene for the green fluorescent protein (GFP). CTV distribution in infected tissues was assessed by direct tissue blot immunoassay and fluorescence emission, and virus accumulation was estimated by quantitative real-time reverse transcriptase-polymerase chain reaction. CTV accumulation in p23-expressing and control SwO plants was similar, whereas the viral load in transgenic SO expressing p23 was 10-10(5) times higher than in the cognate control plants. Although few infection foci composed of a single cell were observed in the phloem of CTV-infected control SO, the number of foci in p23-expressing plants was higher and usually comprised two to six cells, indicating viral cell-to-cell movement. CTV was detected in mesophyll protoplasts and cells from infected SO and SwO expressing p23, but not in similar protoplasts and cells from infected control plants. Our results show that the ectopic expression of p23 enables CTV to escape from the phloem and, in addition, facilitates systemic infection of the resistant SO host. This is the first report of a viral-encoded protein that enhances virus accumulation and distribution in woody hosts. PMID:21726389

  8. A sensitive and reliable RT-nested PCR assay for detection of Citrus tristeza virus from naturally infected citrus plants.

    PubMed

    Adkar-Purushothama, Charith Raj; Maheshwar, P K; Sano, Teruo; Janardhana, G R

    2011-05-01

    A specific and sensitive reverse transcriptase-nested polymerase chain reaction assay (RT-nPCR) was developed for the detection of Citrus tristeza virus (CTV) from naturally infected citrus samples. Two sets of primer pairs were designed by alignment of nucleotide sequences available in GenBank database for different genotypes of CTV. RT-nPCR reaction components and thermal cycling parameters were optimized and reaction conditions were standardized. Sequencing of the PCR products from direct and nested-PCR reactions confirmed the specificity of both primer pairs. Presence of CTV specific amplicons in asymptomatic samples which were collected from diseased orchards indicated the sensitivity of the test. As RT-nPCR technique, developed in the present study, is specific and efficient in detecting CTV, this could be envisioned for diagnostic applications and surveillance. PMID:21298268

  9. The pathogenicity determinant of Citrus tristeza virus causing the seedling yellows syndrome maps at the 3'-terminal region of the viral genome.

    PubMed

    Albiach-Marti, Maria R; Robertson, Cecile; Gowda, Siddarame; Tatineni, Satyanarayana; Belliure, Belén; Garnsey, Stephen M; Folimonova, Svetlana Y; Moreno, Pedro; Dawson, William O

    2010-01-01

    Citrus tristeza virus (CTV) (genus Closterovirus, family Closteroviridae) causes some of the more important viral diseases of citrus worldwide. The ability to map disease-inducing determinants of CTV is needed to develop better diagnostic and disease control procedures. A distinctive phenotype of some isolates of CTV is the ability to induce seedling yellows (SY) in sour orange, lemon and grapefruit seedlings. In Florida, the decline isolate of CTV, T36, induces SY, whereas a widely distributed mild isolate, T30, does not. To delimit the viral sequences associated with the SY syndrome, we created a number of T36/T30 hybrids by substituting T30 sequences into different regions of the 3' half of the genome of an infectious cDNA of T36. Eleven T36/T30 hybrids replicated in Nicotiana benthamiana protoplasts. Five of these hybrids formed viable virions that were mechanically transmitted to Citrus macrophylla, a permissive host for CTV. All induced systemic infections, similar to that of the parental T36 clone. Tissues from these C. macrophylla source plants were then used to graft inoculate sour orange and grapefruit seedlings. Inoculation with three of the T30/T36 hybrid constructs induced SY symptoms identical to those of T36; however, two hybrids with T30 substitutions in the p23-3' nontranslated region (NTR) (nucleotides 18 394-19 296) failed to induce SY. Sour orange seedlings infected with a recombinant non-SY p23-3' NTR hybrid also remained symptomless when challenged with the parental virus (T36), demonstrating the potential feasibility of using engineered constructs of CTV to mitigate disease. PMID:20078776

  10. Three genes of Citrus tristeza virus are dispensable for infection and movement throughout some varieties of citrus trees.

    PubMed

    Tatineni, Satyanarayana; Robertson, Cecile J; Garnsey, Stephen M; Bar-Joseph, Moshe; Gowda, Siddarame; Dawson, William O

    2008-07-01

    Citrus tristeza virus (CTV), a member of the Closteroviridae, possesses a 19.3-kb positive-stranded RNA genome that is organized into twelve open reading frames (ORFs). The CTV genome contains two sets of conserved genes, which are characteristic of this virus group, the replication gene block (ORF 1a and 1b) and the quintuple gene block (p6, HSP70 h, p61, CPm, and CP). With the exception of the p6 gene, they are required for replication and virion assembly. CTV contains five additional genes, p33, p18, p13, p20 and p23, in the 3' half of the genome, some of which (p33, p18 and p13) are not conserved among other members of this virus group, and have been proposed to have evolved for specific interactions with the citrus host. In the present study, the requirements for systemic infection of citrus trees of p33, p6, p18, p13 and p20 were examined. Viral mutants with a deletion in the p6 or the p20 ORF failed to infect citrus plants systemically, suggesting their possible roles in virus translocation/systemic infection. However, we found that deletions within the p33, p18 or p13 ORF individually resulted in no significant loss of ability of the virus to infect, multiply, and spread throughout citrus trees. Furthermore, deletions in the p33, p18 and p13 genes in all possible combinations including deletions in all three genes allowed the virus to systemically invade citrus trees. Green fluorescent protein-tagged CTV variants with deletions in the p33 ORF or the p33, p18 and p13 ORFs demonstrated that the movement and distribution of these deletion mutants were similar to that of the wild-type virus. PMID:18456299

  11. Citrus tristeza virus p23: determinants for nucleolar localization and their influence on suppression of RNA silencing and pathogenesis.

    PubMed

    Ruiz-Ruiz, Susana; Soler, Nuria; Sánchez-Navarro, Jesús; Fagoaga, Carmen; López, Carmelo; Navarro, Luis; Moreno, Pedro; Peña, Leandro; Flores, Ricardo

    2013-03-01

    Citrus tristeza virus (CTV) encodes a singular protein (p23, 209 amino acids) with multiple functions, including RNA silencing suppression (RSS). Confocal laser-scanning microscopy of green fluorescent protein (GFP)-p23 agroexpressed in Nicotiana benthamiana revealed its accumulation in the nucleolus, Cajal bodies, and plasmodesmata. To dissect the nucleolar localization signal (NoLS) typically associated with basic motifs, seven truncated and 10 point-mutated versions of p23 were assayed. Deletion mutants showed that regions 50 to 86 and 100 to 157 (excluding fragment 106 to 114), both with basic motifs and the first with a zinc-finger, contain the (bipartite) NoLS. Alanine substitutions delimited this signal to three cysteines of the Zn-finger and some basic amino acids. RSS activity of p23 in N. benthamiana was abolished by essentially all mutants, indicating that it involves most p23 regions. The necrotic-inducing ability of p23 when launched in N. benthamiana from Potato virus X was only retained by deletion mutant 158-209 and one substitution mutant, showing that the Zn-finger and flanking basic motifs form part of the pathogenic determinant. Ectopic expression of p23 and some deletion mutants in transgenic Mexican lime demarcated a similar determinant, suggesting that p23 affects related pathways in citrus and N. benthamiana. Both RSS activity and pathogenicity of p23 appear related to its nucleolar localization. PMID:23387469

  12. Symptoms induced by transgenic expression of p23 from Citrus tristeza virus in phloem-associated cells of Mexican lime mimic virus infection without the aberrations accompanying constitutive expression.

    PubMed

    Soler, Nuria; Fagoaga, Carmen; López, Carmelo; Moreno, Pedro; Navarro, Luis; Flores, Ricardo; Peña, Leandro

    2015-05-01

    Citrus tristeza virus (CTV) is phloem restricted in natural citrus hosts. The 23-kDa protein (p23) encoded by the virus is an RNA silencing suppressor and a pathogenicity determinant. The expression of p23, or its N-terminal 157-amino-acid fragment comprising the zinc finger and flanking basic motifs, driven by the constitutive 35S promoter of cauliflower mosaic virus, induces CTV-like symptoms and other aberrations in transgenic citrus. To better define the role of p23 in CTV pathogenesis, we compared the phenotypes of Mexican lime transformed with p23-derived transgenes from the severe T36 and mild T317 CTV isolates under the control of the phloem-specific promoter from Commelina yellow mottle virus (CoYMV) or the 35S promoter. Expression of the constructs restricted to the phloem induced a phenotype resembling CTV-specific symptoms (vein clearing and necrosis, and stem pitting), but not the non-specific aberrations (such as mature leaf epinasty and yellow pinpoints, growth cessation and apical necrosis) observed when p23 was ectopically expressed. Furthermore, vein necrosis and stem pitting in Mexican lime appeared to be specifically associated with p23 from T36. Phloem-specific accumulation of the p23?158-209(T36) fragment was sufficient to induce the same anomalies, indicating that the region comprising the N-terminal 157 amino acids of p23 is responsible (at least in part) for the vein clearing, stem pitting and, possibly, vein corking in this host. PMID:25171669

  13. A genetic system for Citrus Tristeza Virus using the non-natural host Nicotiana benthamiana: an update.

    PubMed

    Ambrós, Silvia; Ruiz-Ruiz, Susana; Peña, Leandro; Moreno, Pedro

    2013-01-01

    In nature Citrus tristeza virus (CTV), genus Closterovirus, infects only the phloem cells of species of Citrus and related genera. Finding that the CTV T36 strain replicated in Nicotiana benthamiana (NB) protoplasts and produced normal virions allowed development of the first genetic system based on protoplast transfection with RNA transcribed from a full-genome cDNA clone, a laborious and uncertain system requiring several months for each experiment. We developed a more efficient system based on agroinfiltration of NB leaves with CTV-T36-based binary plasmids, which caused systemic infection in this non-natural host within a few weeks yielding in the upper leaves enough CTV virions to readily infect citrus by slash inoculation. Stem agroinoculation of citrus and NB plants with oncogenic strains of Agrobacterium tumefaciens carrying a CTV-T36 binary vector with a GUS marker, induced GUS positive galls in both species. However, while most NB tumors were CTV positive and many plants became systemically infected, no coat protein or viral RNA was detected in citrus tumors, even though CTV cDNA was readily detected by PCR in the same galls. This finding suggests (1) strong silencing or CTV RNA processing in transformed cells impairing infection progress, and (2) the need for using NB as an intermediate host in the genetic system. To maintain CTV-T36 in NB or assay other CTV genotypes in this host, we also tried to graft-transmit the virus from infected to healthy NB, or to mechanically inoculate NB leaves with virion extracts. While these trials were mostly unsuccessful on non-treated NB plants, agroinfiltration with silencing suppressors enabled for the first time infecting NB plants by side-grafting and by mechanical inoculation with virions, indicating that previous failure to infect NB was likely due to virus silencing in early infection steps. Using NB as a CTV host provides new possibilities to study virus-host interactions with a simple and reliable system. PMID:23847598

  14. Accumulation of a 5' proximal subgenomic RNA of Citrus tristeza virus is correlated with encapsidation by the minor coat protein.

    PubMed

    Gowda, Siddarame; Tatineni, Satyanarayana; Folimonova, Svetlana Y; Hilf, Mark E; Dawson, William O

    2009-06-20

    During replication, Citrus tristeza virus (CTV) produces large amounts of two unusual subgenomic (sg) RNAs that are positive-stranded and 5' coterminal. Although these RNAs are produced in similar amounts and are similar in size, with LMT1 ( approximately 750 nt) only slightly larger than LMT2 ( approximately 650), we found that the similar sgRNAs are produced differently. We previously showed that the LMT1 RNA is produced by premature termination during genomic RNA synthesis. However, LMT2 production was found to correlate with virion assembly instead of RNA replication. The time course of accumulation of the LMT2 RNA occurred late, coinciding with virion accumulation. The long flexuous virions of CTV contain two coat proteins that encapsidate the virions in a polar manner. The major coat protein encapsidates approximately 97% of the virion, while the minor capsid protein encapsidates the remainder of the genome beginning in the 5' non-translated region with the transition zone at approximately 630 nucleotides from the 5' end. The section of the virion RNA that was encapsidated by CPm was identical in size to the LMT2 RNA, suggesting that the LMT2 RNA represented a portion of the viral RNA protected by CPm encapsidation. Mutations that abrogated encapsidation by CPm also abolished the accumulation of LMT2 RNA. Thus, these two unusual but similar RNAs are produced via different pathways, one from RNA replication and one processed by the virion assembly process. To our knowledge, this represents the first evidence of a viral RNA processed by the assembly mechanism. PMID:19446304

  15. Volatile Organic Compound (VOC) profiling of Citrus tristeza virus (CTV) infection in sweet orange citrus varietals using thermal desorption gas chromatography time of flight mass spectrometry (TD-GC/TOF-MS)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Citrus tristeza virus (CTV) is a plant pathogen which predominately infects economically important citrus crops such as sweet orange, clementine, lime and grapefruit varietals. Within the last 70 years, an estimated 100 million citrus trees on sour orange rootstock have been destroyed due to CTV inf...

  16. 2006 University Citrus Pest Management Guide: Tristeza

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Citrus tristeza virus (CTV) is a major cause of the decline and eventual death of trees on sour orange rootstocks. Initially, affected trees have small leaves and twig dieback. Diseased trees often produce a crop of very small fruit. Eventually, large limbs die back and the tree gradually declines...

  17. Virus isolation and propagation in embryonating eggs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The embryonating egg is one of the most versatile, easy to work with, and widely used host systems for the isolation and propagation of avian viruses. The embryonating chicken egg (ECE) is the most commonly available system that is both specific pathogen free and supports the replication of viruses...

  18. Accumulation of transgene-derived siRNAs is not sufficient for RNAi-mediated protection against Citrus tristeza virus in transgenic Mexican lime.

    PubMed

    López, Carmelo; Cervera, Magdalena; Fagoaga, Carmen; Moreno, Pedro; Navarro, Luis; Flores, Ricardo; Peña, Leandro

    2010-01-01

    Mexican lime plants transformed with the 3'-terminal 549 nucleotides of the Citrus tristeza virus (CTV) genome in sense, antisense and intron-hairpin formats were analysed for transgene-derived transcript and short interfering RNA (siRNA) accumulation, and for CTV resistance. Propagations from all sense, antisense and empty-vector transgenic lines were susceptible to CTV, except for a single sense-line plant with a complex transgene integration pattern that showed transgene-derived siRNAs in association with low levels of the transgene-derived transcript. In contrast, nine of 30 intron-hairpin lines showed CTV resistance, with 9%-56% of bud-propagated plants, depending on the line, remaining uninfected on graft inoculation, and the others being susceptible. Although resistance was always associated with the presence of transgene-derived siRNAs, their level in different sense and intron-hairpin transformants was variable irrespective of the response to CTV infection. In intron-hairpin lines with single transgene integration, CTV resistance was correlated with low accumulation of the transgene-derived transcript rather than with high accumulation of transgene-derived siRNAs. PMID:20078774

  19. Citrus tristeza virus-based RNAi in citrus plants induces gene silencing in Diaphorina citri, a phloem-sap sucking insect vector of citrus greening disease (Huanglongbing).

    PubMed

    Hajeri, Subhas; Killiny, Nabil; El-Mohtar, Choaa; Dawson, William O; Gowda, Siddarame

    2014-04-20

    A transient expression vector based on Citrus tristeza virus (CTV) is unusually stable. Because of its stability it is being considered for use in the field to control Huanglongbing (HLB), which is caused by Candidatus Liberibacter asiaticus (CLas) and vectored by Asian citrus psyllid, Diaphorina citri. In the absence of effective control strategies for CLas, emphasis has been on control of D. citri. Coincident cohabitation in phloem tissue by CLas, D. citri and CTV was exploited to develop a novel method to mitigate HLB through RNA interference (RNAi). Since CTV has three RNA silencing suppressors, it was not known if CTV-based vector could induce RNAi in citrus. Yet, expression of sequences targeting citrus phytoene desaturase gene by CTV-RNAi resulted in photo-bleaching phenotype. CTV-RNAi vector, engineered with truncated abnormal wing disc (Awd) gene of D. citri, induced altered Awd expression when silencing triggers ingested by feeding D. citri nymphs. Decreased Awd in nymphs resulted in malformed-wing phenotype in adults and increased adult mortality. This impaired ability of D. citri to fly would potentially limit the successful vectoring of CLas bacteria between citrus trees in the grove. CTV-RNAi vector would be relevant for fast-track screening of candidate sequences for RNAi-mediated pest control. PMID:24572372

  20. Comparison of Immunohistochemistry and Virus Isolation for Diagnosis of West Nile Virus

    PubMed Central

    Ellis, Angela E.; Mead, Daniel G.; Allison, Andrew B.; Gibbs, Samantha E. J.; Gottdenker, Nicole L.; Stallknecht, David E.; Howerth, Elizabeth W.

    2005-01-01

    Immunohistochemistry and virus isolation were performed on 1,057 birds. Immunohistochemistry, virus isolation, or both found 325 birds to be West Nile virus positive. Of these, 271 were positive by both methods. These results indicate that virus isolation and immunohistochemistry are approximately equal in their ability to detect West Nile virus. PMID:15956415

  1. Heterologous minor coat proteins of Citrus tristeza virus strains affect encapsidation, but the coexpression of HSP70h and p61 restores encapsidation to wild-type levels.

    PubMed

    Tatineni, Satyanarayana; Gowda, Siddarame; Dawson, William O

    2010-07-01

    The long flexuous bipolar virions of Citrus tristeza virus (CTV), a Closterovirus, are encapsidated with two capsid proteins at opposite ends: the minor coat protein (CPm) encapsidates the 5' 630 nts of the genomic RNA and the major coat protein encapsidates the remainder of the genome. In this study, we found encapsidation of CTV CPm in the absence of other assembly-related proteins is highly specific in contrast to most plant viruses that allow virion assembly by a range of heterologous coat proteins. Heterologous CPms with 95-96% amino acid identity from related strains in CTV-CPm, a replicon with CPm as the only assembly-related ORF, either failed to initiate encapsidation or reduced encapsidation substantially. Substitution of subsets of amino acids revealed that the amino acids that differ between positions 121 and 180 of the VT strain, and 61 and 120 of the T3 strain were involved in specific encapsidation. We further mapped the specific encapsidation to a single amino acid: mutation of methionine(165) to threonine (VT type) or serine(105) to proline (T3 type) in CTV-CPm failed to form nucleocapsids. However, the heterologous CPm in combination with both HSP70h and p61 proteins, but not HSP70h or p61 alone, encapsidated at wild-type levels, suggesting that specific encapsidation by CPm was mitigated by the combination of HSP70h and p61. Thus, in addition to the previously described functions of HSP70h and p61 of greatly enhanced virion formation and restriction of CPm encapsidation to the 5' 630 nts of the genomic RNA, these proteins facilitate encapsidation by heterologous CPms. PMID:20399478

  2. Characterization of Recently Introduced HLB and CTV Isolates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Severe stem-pitting isolates of Citrus tristeza virus (CTV) were found in Florida citrus 5 years ago, followed by the discovery 2 years later of the citrus greening disease (huanglongbing: HLB) caused by the bacterium Candidatus Liberibacter asiaticus. The new CTV isolates are members of the VT grou...

  3. Isolation of Usutu Virus in Germany

    PubMed Central

    Jöst, Hanna; Bialonski, Alexandra; Maus, Deborah; Sambri, Vittorio; Eiden, Martin; Groschup, Martin H.; Günther, Stephan; Becker, Norbert; Schmidt-Chanasit, Jonas

    2011-01-01

    Usutu virus (USUV) is a mosquito-borne flavivirus that emerged 2001 in Austria and caused deaths in wild birds. In Germany, 70,378 female mosquitoes were captured in 2009 and 2010 and assayed for USUV. Virus was isolated in cell culture from one pool of Culex pipiens pipiens mosquitoes trapped exclusively in August 2010 in Weinheim, Germany. Subsequent phylogenetic analysis demonstrated a close relationship between the isolated USUV strain from Germany and a USUV strain from Austria, which was detected in a dead blackbird in 2004. PMID:21896821

  4. Calculation of diagnostic parameters of advanced serological and molecular tissue-print methods for detection of Citrus tristeza virus: a model for other plant pathogens.

    PubMed

    Vidal, E; Yokomi, R K; Moreno, A; Bertolini, E; Cambra, M

    2012-01-01

    Citrus tristeza virus (CTV) is one of the most important virus diseases that affect citrus. Control of CTV is achieved by grafting selected virus-free citrus scions onto CTV-tolerant or -resistant rootstocks. Quarantine and certification programs are essential for avoiding the entry and propagation of severe strains of CTV. Citrus nurseries in Spain and central California (United States) maintain zero-tolerance policies for CTV that require sensitive, specific, and reliable pathogen-detection methods. Tissue-print (TP) real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assay was compared with the validated TP enzyme-linked immunosorbent assay (ELISA), using the CTV-specific monoclonal antibodies 3DF1 and 3CA5, for CTV detection. In total, 1,395 samples from healthy and CTV-infected nursery and mature tree plants were analyzed with both methods. The total agreement between both detection methods was substantial (Cohen's kappa index of 0.77 ± 0.03). The diagnostic parameters of each technique (i.e., the sensitivity, specificity, and likelihood ratios) were evaluated in a second test involving 658 Citrus macrophylla nursery plants. Mexican lime indexing was used to evaluate samples with discrepant results in the analysis. For TP-ELISA, a sensitivity of 0.8015, a specificity of 0.9963, and a positive and negative likelihood ratio of 216.42 and 0.199, respectively, were estimated. For TP real-time RT-PCR, a sensitivity of 0.9820, a specificity of 0.8519, and a positive and negative likelihood ratio of 6.63 and 0.021, respectively, were estimated. These diagnostic parameters show that TP real-time RT-PCR was the most sensitive technique, whereas TP-ELISA showed the highest specificity, validating the use of the molecular technique for routine CTV-detection purposes. In addition, our results show that the combination of both techniques can accurately substitute for the conventional biological Mexican lime index for the detection of CTV. The calculation of diagnostic parameters is discussed, as a necessary tool, to validate detection or diagnostic methods in plant pathology. Furthermore, assessment of the post-test probability of disease after a diagnostic result and CTV prevalence allows selection of the best method for accurate and reliable diagnosis. PMID:21879789

  5. Viral-like symptoms induced by the ectopic expression of the p23 gene of Citrus tristeza virus are citrus specific and do not correlate with the pathogenicity of the virus strain.

    PubMed

    Fagoaga, Carmen; López, Carmelo; Moreno, Pedro; Navarro, Luis; Flores, Ricardo; Peña, Leandro

    2005-05-01

    Ectopic expression of the p23 gene from a severe (T36) strain of Citrus tristeza virus (CTV) induces viral-like symptoms in Mexican lime. Here, we report that expressing the same gene from a mild strain induced similar symptoms that correlated with accumulation of p23 protein irrespective of the source strain. CTV inoculation of transgenic limes showing CTV-like leaf symptoms and high p23 accumulation did not modify symptoms initially, with the virus titer being as in inoculated nontransgenic controls; however, at later stages, symptoms became attenuated. Transformation with p23-T36 of CTV-susceptible sweet and sour orange and CTV-resistant trifoliate orange also led to CTV-like leaf symptoms that did not develop when plants were transformed with a truncated p23 version. In transgenic citrus species and relatives other than Mexican lime, p23 was barely detectable, although symptom intensity correlated with levels of p23 transcripts. The lower accumulation of p23 in sweet and sour orange compared with Mexican lime also was observed in nontransgenic plants inoculated with CTV, suggesting that minimal p23 levels cause deleterious effects in the first two species. Conversely, transgenic expression of p23 in CTV nonhost Nicotiana spp. led to accumulation of p23 without phenotypic aberrations, indicating that p23 interferes with plant development only in citrus species and relatives. PMID:15915642

  6. Transformation of Mexican lime with an intron-hairpin construct expressing untranslatable versions of the genes coding for the three silencing suppressors of Citrus tristeza virus confers complete resistance to the virus.

    PubMed

    Soler, Nuria; Plomer, Montserrat; Fagoaga, Carmen; Moreno, Pedro; Navarro, Luis; Flores, Ricardo; Peña, Leandro

    2012-06-01

    Citrus tristeza virus (CTV), the causal agent of the most devastating viral disease of citrus, has evolved three silencing suppressor proteins acting at intra- (p23 and p20) and/or intercellular level (p20 and p25) to overcome host antiviral defence. Previously, we showed that Mexican lime transformed with an intron-hairpin construct including part of the gene p23 and the adjacent 3' untranslated region displays partial resistance to CTV, with a fraction of the propagations from some transgenic lines remaining uninfected. Here, we transformed Mexican lime with an intron-hairpin vector carrying full-length, untranslatable versions of the genes p25, p20 and p23 from CTV strain T36 to silence the expression of these critical genes in CTV-infected cells. Three transgenic lines presented complete resistance to viral infection, with all their propagations remaining symptomless and virus-free after graft inoculation with CTV-T36, either in the nontransgenic rootstock or in the transgenic scion. Accumulation of transgene-derived siRNAs was necessary but not sufficient for CTV resistance. Inoculation with a divergent CTV strain led to partially breaking the resistance, thus showing the role of sequence identity in the underlying mechanism. Our results are a step forward to developing transgenic resistance to CTV and also show that targeting simultaneously by RNA interference (RNAi) the three viral silencing suppressors appears critical for this purpose, although the involvement of concurrent RNAi mechanisms cannot be excluded. PMID:22405601

  7. Recombination defines two gene modules and provides for increased genetic diversity in a narrow host range plant virus.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Citrus tristeza virus (CTV) isolates T36, T68-1 and NS25 showed phylogenetic incongruities between sequences involved in viral RNA replication and those involved in movement and other viral functions. This incongruity was not found in the sequences of isolates T3, T30, T385, VT and T318A. Distance...

  8. Isolation of pigeon herpes encephalomyelitis virus in Saudi Arabia.

    PubMed

    Shalaby, M A; el-Sisi, M A; Ismail, O E; Afaleque, A I

    1985-07-01

    A virus was isolated from the brains of pigeons suffering from nervous disorders in different localities of the Eastern Province, Kingdom of Saudi Arabia. The new isolate caused a high morbidity, ranging from 33% to 50%, and a mortality rate which reached 40%. The virus produced pinpoint greyish pock lesions on the chorioallantoic membrane of embryonated hens' eggs and induced syncytial formation followed by rounding and lysis of the cells in chicken embryo fibroblast cultures. Virus infectivity was significantly reduced following treatment by 20% ether or chloroform. The isolated virus was identified as pigeon herpes encephalomyelitis virus by serum-neutralization, agar gel diffusion and fluorescent antibody staining techniques. PMID:2994283

  9. Analysis of Iranian Potato virus S isolates.

    PubMed

    Salari, Khadijeh; Massumi, Hossein; Heydarnejad, Jahangir; Hosseini Pour, Akbar; Varsani, Arvind

    2011-10-01

    Two hundred forty potato samples with one or more symptoms of leaf mosaic, distortion, mottling and yellowing were collected between 2005 and 2008 from seven Iranian provinces. Forty-four of these samples tested positive with double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA) using a Potato virus S (PVS) polyclonal antibody. Of these 12 isolates of PVS were selected based on the geographical location for biological and molecular characterization. The full coat protein (CP) and 11K genes from 12 PVS isolates were PCR amplified, cloned and sequenced. All 12 PVS isolates showed mosaic symptoms on Nicotiana debneyii and N. tabacum cv. Whiteburly and local lesion on Chenopodium amaranticolor, C. quinoa and C. album. The Iranian isolates share between 93 and 100% pairwise nucleotide identity with other PVS(O) isolates. Based on maximum likelihood phylogenetic analysis coupled with pairwise identity analysis, we propose 15 genotypes for the PVS(O) strain and 3 genotypes for the PVS(A) strain. PMID:21567245

  10. Mayaro virus isolated from a Trinidadian mosquito, Mansonia venezuelensis.

    PubMed

    AITKEN, T H; DOWNS, W G; ANDERSON, C R; SPENCE, L; CASALS, J

    1960-04-01

    A strain of Mayaro virus has been isolated in Trinidad from the mosquito Mansonia venezuelensis. This is the first record of isolation of this agent from naturally infected mosquitoes, caught in the wild. PMID:13792204

  11. Genetic study of Japanese encephalitis viruses isolated in Malaysia.

    PubMed

    Tsuchie, H; Oda, K; Vythilingam, I; Thayan, R; Vijayamalar, B; Sinniah, M; Hossain, M M; Kurimura, T; Igarashi, A

    1994-04-01

    Two hundred and forty nucleotides from the pre-M gene region of 10 Japanese encephalitis (JE) virus strains isolated in Malaysia in 1992 were sequenced and compared with the other JE virus strains from different geographic areas in Asia. Our JE virus strains belong to the largest genotypic group that includes strains isolated in temperate regions such as Japan, China, and Taiwan. Our Malaysian JE virus strains differed in 32 nucleotides (13.3%) from WTP/70/22 strain isolated from Malaysia in 1970, which belonged to another distinct genotypic group. PMID:7853748

  12. Tobacco streak virus isolated from lettuce.

    PubMed

    Abtahi, F S; Khodai Motlagh, M

    2009-05-01

    Tobacco streak virus (TSV) is an ilarvirus with a worldwide distribution. This virus infects many plants and causes significant yield losses. In this study, 300 samples of lettuce were collected from lettuce fields in Tehran Province. Infected plants show symptoms such as: mosaic, vein clearing, vein necrosis, yellowing and leaf distortion. DAS-ELISA (Double Antibody Sandwich-ELISA) was used with a polyclonal antiserum against TSV. Five isolates (T1, T2, T3, T4 and T5), which are collected, respectively from Mohammad Abad (Karaj), Malek Abad (Karaj), Hashtgerd (Karaj), Tarand Balla (Varamin) and Deh mah sin (Pishva) were inoculated on 29 species of Cucurbitaceae, Amaranthaceae, Solanacea, Compositae, Leguminosae and Chenopodiacea. Chenopodium quinoa 6 days after inoculation showed necrotic local lesions. Gomphrena globosa 10 days after inoculation developed chlorotic local lesions. Systemic symptoms were produced in Datura stramonium. Phaseolus vulgaris cv. Red Kidney 5 days after inoculation developed necrotic local lesions. Nicotiana tabacum 7 days after inoculation showed necrotic and chlorotic local lesions. Nicotiana clevelandii 15 days after inoculation developed leaf distortion and vein necrosis. Lactuca sativa 10-15 days after inoculation developed leaf istortion and mosaic. Reverse Transcription Polymerase Chain Reaction (RT-PCR) was performed using one primer pairs designed by DSMZ. An approximately 710 bp fragment was amplified with a specific primer. PMID:19634475

  13. Tanay virus, a new species of virus isolated from mosquitoes in the Philippines.

    PubMed

    Nabeshima, Takeshi; Inoue, Shingo; Okamoto, Kenta; Posadas-Herrera, Guillermo; Yu, Fuxun; Uchida, Leo; Ichinose, Akitoyo; Sakaguchi, Miako; Sunahara, Toshihiko; Buerano, Corazon C; Tadena, Florencio P; Orbita, Ildefonso B; Natividad, Filipinas F; Morita, Kouichi

    2014-06-01

    In 2005, we isolated a new species of virus from mosquitoes in the Philippines. The virion was elliptical in shape and had a short single projection. The virus was named Tanay virus (TANAV) after the locality in which it was found. TANAV genomic RNA was a 9562 nt+poly-A positive strand, and polycistronic. The longest ORF contained putative RNA-dependent RNA polymerase (RdRP); however, conserved short motifs in the RdRP were permuted. TANAV was phylogenetically close to Negevirus, a recently proposed taxon of viruses isolated from haemophagic insects, and to some plant viruses, such as citrus leprosis virus C, hibiscus green spot virus and blueberry necrotic ring blotch virus. In this paper, we describe TANAV and the permuted structure of its RdRP, and discuss its phylogeny together with those of plant viruses and negevirus. PMID:24646751

  14. [Isolation of the virus of Syr-Darya Valley fever].

    PubMed

    L'vov, D K; Karimov, S K; Kiriushchenko, T V; Chun-Siun, F; Skvortsova, T M

    1984-01-01

    In the course of studies on the ecological structure of acute febrile diseases in the season of activity of blood-sucking arthropods strains of a virus antigenically related to Sikhote-Alyñ virus were isolated from the blood of a patient and from Ixodid ticks. This paper presents the results of the study on the causative agent and the clinical picture of the disease caused by this virus. The virus was found to be a new one for science; its appurtenance to the family Picornaviridae, genus Cardiovirus, the antigenic group of encephalomyocarditis has been determined. The virus has been designated "Syr-Darya Valley fever virus" by the area of its isolation. PMID:6097042

  15. Mild strain cross protection of tristeza: A review of research to protect against decline on sour orange in Florida and a look at the future

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tristeza, caused by Citrus tristeza virus (CTV), has long been present in Florida, but outbreaks of decline on sour orange rootstock were occasional events until the late 1970s. Sour orange rootstock was valued for the high quality of fruit produced. Research was directed towards the selection and...

  16. Variability in alternanthera mosaic virus isolates from different hosts

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have determined the complete genome sequences of Alternanthera mosaic virus phlox isolate PA (AltMV-PA) and four infectious clone variants derived from AltMV-SP, as well as partial sequences of other isolates from various types of phlox, and from portulaca, nandina, and cineraria. Phylogenetic co...

  17. Citrus tristeza virus infection induces the accumulation of viral small RNAs (21-24-nt) mapping preferentially at the 3'-terminal region of the genomic RNA and affects the host small RNA profile.

    PubMed

    Ruiz-Ruiz, Susana; Navarro, Beatriz; Gisel, Andreas; Peña, Leandro; Navarro, Luis; Moreno, Pedro; Di Serio, Francesco; Flores, Ricardo

    2011-04-01

    To get an insight into the host RNA silencing defense induced by Citrus tristeza virus (CTV) and into the counter defensive reaction mediated by its three silencing suppressors (p25, p20 and p23), we have examined by deep sequencing (Solexa-Illumina) the small RNAs (sRNAs) in three virus-host combinations. Our data show that CTV sRNAs: (i) represent more than 50% of the total sRNAs in Mexican lime and sweet orange (where CTV reaches relatively high titers), but only 3.5% in sour orange (where the CTV titer is significantly lower), (ii) are predominantly of 21-22-nt, with a biased distribution of their 5' nucleotide and with those of (+) polarity accumulating in a moderate excess, and (iii) derive from essentially all the CTV genome (ca. 20 kb), as revealed by its complete reconstruction from viral sRNA contigs, but adopt an asymmetric distribution with a prominent hotspot covering approximately the 3'-terminal 2,500 nt. These results suggest that the citrus homologues of Dicer-like (DCL) 4 and 2 most likely mediate the genesis of the 21 and 22 nt CTV sRNAs, respectively, and show that both ribonucleases act not only on the genomic RNA but also on the 3' co-terminal subgenomic RNAs and, particularly, on their double-stranded forms. The plant sRNA profile, very similar and dominated by the 24-nt sRNAs in the three mock-inoculated controls, was minimally affected by CTV infection in sour orange, but exhibited a significant reduction of the 24-nt sRNAs in Mexican lime and sweet orange. We have also identified novel citrus miRNAs and determined how CTV influences their accumulation. PMID:21327514

  18. Variation in the intracellular polypeptide profiles from different isolates of bovine virus diarrhoea virus.

    PubMed

    Pocock, D H; Howard, C J; Clarke, M C; Brownlie, J

    1987-01-01

    Variation of the intracellular polypeptides induced in calf testis cells by 5 cloned isolates of bovine virus diarrhoea virus (BVDV) was examined. Three of the isolates were cytopathic (NADL, C 2415 and Pe 515 c) and two were non-cytopathic (C 1226 and Pe 515 nc) in these cells. The isolates Pe 515 c and Pe 515 nc were both isolated from an animal with clinical signs of mucosal disease. In cells infected with NADL, 8 virus specific proteins (vp 1 to vp 8) with molecular weights ranging from 120,000 (vp 1) to 23,000 (vp 8) were detected. Isolates C 2415 and Pe 515 c gave a similar array of polypeptides to NADL, but the 3 cytopathic isolates could be distinguished by the variation in the molecular weights of some of the proteins. The non-cytopathic isolates could also be distinguished from each other by this type of molecular variation; however, one feature that characterised these strains, when compared to the cytopathic isolates, was the absence of vp 2. Comparison of the polypeptides induced by Pe 515 c and Pe 515 nc showed that apart from the lack of vp 2 in the Pe 515 nc virus profile, the molecular weights of the other viral proteins were similar. This supports serological evidence that for mucosal disease to occur the pair of cytopathic and non-cytopathic viruses must be closely related. Four of the polypeptides induced by Pe 515 c were shown to be glycoproteins. PMID:3034204

  19. Triticum Mosaic Virus: A New Virus Isolated From Wheat in Kansas

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In 2006 a mechanically-transmissible and previously uncharacterized virus was isolated in Kansas from wheat with mosaic symptoms. The physio-chemical properties of the virus were examined by purification on cesium chloride density gradients, electron microscopy, sodium dodecyl sulfate polyacrylalmid...

  20. Comparison of cowpox-like viruses isolated from European zoos. Brief report.

    PubMed

    Baxby, D; Shackleton, W B; Wheeler, J; Turner, A

    1979-01-01

    Poxviruses isolated from captive carnivores in Russia (Moscow virus) and elephants in Germany (elephant virus) were very closely-related to cowpox virus. Immunological analysis with absorbed sera separated elephant virus but not cowpox and Moscow virus, whereas polypeptide analysis separated cowpox but not elephant and Moscow virus. A combination of biological tests separated all three. The epidemiological implications are briefly reviewed. PMID:229799

  1. Vaccine efficacy against challenge with HPAI H5N1 virus isolates from Vietnam

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Multiple isolates of H5N1 were isolated from northern Vietnam in December of 2005. All the viruses characterized were clade 2 viruses, but phylogenetically they formed two separate sub-lineages. The isolation of clade 2 viruses was unexpected, since previous isolations from both northern and south...

  2. Isolated acute dysphagia due to varicella-zoster virus.

    PubMed

    Mantero, Vittorio; Rigamonti, Andrea; Valentini, Sergio; Fiumani, Anna; Piamarta, Francesca; Bonfanti, Paolo; Salmaggi, Andrea

    2014-04-01

    We present a case of zoster sine herpete causing isolated acute dysphagia in an immunocompetent patient. The interest of this paper is the atypical presentation of varicella-zoster virus reactivation. A 77-year-old woman presented with a 3-day history of fever and worsening dysphagia for both liquid and solid foods. Cerebrospinal fluid examination revealed lymphocytic pleocytosis and PCR amplified varicella-zoster virus DNA with high antibody titers in both serum and cerebrospinal fluid. The panel was suggestive of a cranial neuritis due to varicella-zoster virus, involved cranial nerves, even in the absence of a cutaneous and mucosal rash. Varicella-zoster virus reactivation should be included in the differential diagnosis of isolated or multiple cranial nerve palsies, with or without zosteriform skin lesions. A prompt etiologic diagnosis can lead to early administration of antiviral therapy. PMID:24529416

  3. Herpes simplex virus: isolation, cytopathological characterization and antiviral sensitivity*

    PubMed Central

    Nozawa, Carlos; Hattori, Lilian Yumi; Galhardi, Ligia Carla Faccin; Lopes, Nayara; Bomfim, Wesley Andrade; de Cândido, Ligyana Korki; de Azevedo, Elbens Marcos Minoreli; Gon, Airton dos Santos; Linhares, Rosa Elisa Carvalho

    2014-01-01

    BACKGROUND Herpes simplex virus (HSV) infection is an endemic disease and it is estimated that 6095% of the adult population are infected with symptoms that are usually self-limiting, though they can be serious, extensive and prolonged in immunocompromised individuals, highlighted by the emergence of drug-resistant strains. The study of the wild-type HSV strains based on the cytopathogenic features and its antiviral sensitivity are important in the establishment of an antivirogram for controlling the infection. OBJECTIVE This study sought to isolate and examine the cytopathological characteristics of circulating strains of the Herpes simplex virus, from clinical specimens and their sensitivity to commercially available antiherpesvirus drugs, acyclovir, phosphonophormic acid and trifluridine. METHODS Herpes simplex virus isolation, cytopathological features and antiviral sensitivity assays were performed in cell culture by tissue culture infectious dose or plaque forming unit assay. RESULTS From twenty-two clinical specimens, we isolated and adapted nine strains. Overall, the cytopathic effect was detected 24 h post-infection (p.i.) and the presence of syncytia was remarkable 48 h p.i., observed after cell staining. Out of eight isolates, four developed plaques of varying sizes. All the isolates were sensitive to acyclovir, phosphonophormic and trifluridine, with the percentage of virus inhibition (%VI) ranging from 49.7-100%. CONCLUSIONS The methodology for HSV isolation and characterization is a straightforward approach, but the drug sensitivity test, regarded as being of great practical importance, needs to be better understood. PMID:24937819

  4. A Circo-Like Virus Isolated from Penaeus monodon Shrimps

    PubMed Central

    Pham, Hanh T.; Yu, Qian; Boisvert, Maude; Van, Hanh T.; Bergoin, Max

    2014-01-01

    A virus with a circular Rep-encoding single-stranded DNA (ssDNA) (CRESS-DNA) genome (PmCV-1) was isolated from Penaeus monodon shrimps in Vietnam. The gene structure of the 1,777-nucleotide (nt) genome was similar to that of circoviruses and cycloviruses, but the nucleic acid and protein sequence identities to these viruses were very low. PMID:24435870

  5. Mild strain cross protection of tristeza: a review of research to protect against decline on sour orange in Florida.

    PubMed

    Lee, Richard F; Keremane, Manjunath L

    2013-01-01

    Tristeza, caused by Citrus tristeza virus (CTV), has long been present in Florida but outbreaks of decline on sour orange rootstock were occasional events until the late 1970s. Sour orange rootstock was valued for the high quality of fruit produced and was widely used because of its tolerance of citrus blight, a disease of unknown etiology. Research was directed towards the selection and screening of mild strains of CTV which could protect against sour orange decline strains. Following the introduction of Toxoptera citricida (also known as the brown citrus aphid) in 1995 there was a greater concern for maintaining production of existing blocks of citrus on sour orange rootstock. Availability of the CTV genome sequence around the same time as well as molecular characterization of in planta CTV populations led to the selection of mild CTV isolates which when inoculated into existing field trees, extended the productive life of the groves and enabled a more graduate replanting of trees on CTV-tolerant rootstocks. The history of CTV in Florida and the methods developed to select mild isolates for use for mild strain cross protection will be reviewed. PMID:24046764

  6. Mild strain cross protection of tristeza: a review of research to protect against decline on sour orange in Florida

    PubMed Central

    Lee, Richard F.; Keremane, Manjunath L.

    2013-01-01

    Tristeza, caused by Citrus tristeza virus (CTV), has long been present in Florida but outbreaks of decline on sour orange rootstock were occasional events until the late 1970s. Sour orange rootstock was valued for the high quality of fruit produced and was widely used because of its tolerance of citrus blight, a disease of unknown etiology. Research was directed towards the selection and screening of mild strains of CTV which could protect against sour orange decline strains. Following the introduction of Toxoptera citricida (also known as the brown citrus aphid) in 1995 there was a greater concern for maintaining production of existing blocks of citrus on sour orange rootstock. Availability of the CTV genome sequence around the same time as well as molecular characterization of in planta CTV populations led to the selection of mild CTV isolates which when inoculated into existing field trees, extended the productive life of the groves and enabled a more graduate replanting of trees on CTV-tolerant rootstocks. The history of CTV in Florida and the methods developed to select mild isolates for use for mild strain cross protection will be reviewed. PMID:24046764

  7. Aphid Transmission of the Ontario Isolate of Plum Pox Virus.

    PubMed

    Lowery, D Thomas; Vickers, Patricia M; Bittner, Lori A; Stobbs, Lorne W; Foottit, Robert G

    2015-10-01

    Utilization of timed virus acquisition access probes in studies of plum pox virus (PPV) transmission by aphids demonstrated that endemic species transmitted the virus readily from plum, Prunus domestica (L.) Batsch; peach, P. persica (L.); or dwarf flowering almond, P. glandulosa Thunberg., to peach seedlings. The green peach aphid, Myzus persicae (Sulzer), was shown to be the most efficient vector. Acquisition of virus by green peach aphids from infected peach leaves resulted in 18-28% infected peach seedlings, while aphids previously fed on infected leaves of plum transferred virus to 36% of peach seedlings. Although the spirea aphid, Aphis spiraecola (Patch), was a less efficient vector than M. persicae it is perhaps more important for the spread of PPV due to its greater abundance and occurrence earlier in the season when peach trees are thought to be more susceptible to infection. Virus transmission rates varied depending on the virus source and healthy test plant species. In contrast to many previous studies, aphid inoculation of the experimental host Nicotiana benthamiana Domin occurred at a low rate, never exceeding 4%. Acquisition of PPV by M. persicae from infected peach fruit was greatly reduced compared with acquisition from leaves. The results of this research indicate that the Ontario isolate of PPV-D is readily transmissible by aphids to peach and natural spread of the virus needs to be considered in future management or eradication programs. PMID:26453705

  8. Isolation of Newcastle disease virus from birds of prey.

    PubMed

    Chu, H P; Trow, E W; Greenwood, A G; Jennings, A R; Keymer, I F

    1976-01-01

    In the 4 year period 1971-74 11 isolations of Newcastle Disease Virus (NDV) were made from 44 birds of prey that died in captivity. Three species of Falconiformes were involved, including one red-headed falcon (Falco chicquera), 5 European kestrels (F. tinnunculus), and 2 secretary birds (Sagittarius serpentarius), also 2 species of Strigiformes, comprising 2 barn owls (Tyto alba) and one little owl (Athene noctua). All NDV isolates were of the velogenic type. PMID:18777349

  9. Characterization and phylogenic analysis of Mexican Newcastle disease virus isolates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Newcastle disease virus (NDV) was isolated in Mexico for the first time in 1946 and the last report of a field outbreak caused by a highly virulent strain dates from year 2000, when 13.6 million birds were slaughtered and 93 farms quarantined. Mean Death Time test resulted in velogenic classificati...

  10. Isolation of Frog Virus 3 from Pallid Sturgeon (Scaphirhynchus albus)

    E-print Network

    Gray, Matthew

    Isolation of Frog Virus 3 from Pallid Sturgeon (Scaphirhynchus albus) Suggests an Interclass Host #12;· Pallid Sturgeon Conservation within the Missouri River Basin ­ History of the decline · Significance & Future Directions Topics Covered #12;Decline of Pallid Sturgeon within the Missouri River Basin

  11. Methods for the isolation of viruses from environmental samples.

    PubMed

    Wommack, K Eric; Williamson, Kurt E; Helton, Rebekah R; Bench, Shellie R; Winget, Danielle M

    2009-01-01

    Viruses are omnipresent and extraordinarily abundant in the microbial ecosystems of water, soil, and sediment. In nearly every reported case for aquatic and porous media environments (soils and sediments) viral abundance exceeds that of co-occurring host populations by 10-100-fold. If current estimates based on metagenome DNA sequence data are correct, then viruses represent the largest reservoir of unknown genetic diversity on Earth. Microscopy and molecular genetic tools have been critical in demonstrating that viruses are a dynamic component of microbial ecosystems capable of significantly influencing the productivity and population biology of their host communities. Moreover, these approaches have begun to describe and constrain the immense genetic diversity of viral communities. A critical first step in the application of many cultivation-independent approaches to virus ecology is obtaining a concentrate of viruses from an environmental sample. Culture-dependent methods also rely on viruses being present at a high enough abundance to detect. Here, methodological details for the isolation and concentration of viruses from water, soil, and aquatic sediment samples are covered in detail. PMID:19066805

  12. Infectivity of the DNA from four isolates of JC virus.

    PubMed Central

    Frisque, R J; Martin, J D; Padgett, B L; Walker, D L

    1979-01-01

    The infectivity of JC virus DNA was demonstrated in its most permissive cell culture, primary human fetal glial cells. The amount of infectivity observed in these heterogeneous cultures varied considerably between batches of cells. Contrary to results obtained with the papovaviruses simian virus 40 and BK virus, the calcium technique (F. L. Graham and A. J. van der Eb, Virology 52:456--467, 1973) was found to be more efficient at promoting JC virus DNA infectivity than the DEAE-dextran method (J. H. McCutchan and J. S. Pagano, J. Natl. Cancer Inst. 41:351--357, 1968): maximum infectivity titers of 4 x 10-(4) and 6 x 10(3) fluorescent cell units per microgram of DNA, respectively. These values represent an approximate recovery of infectivity from virus of between 0.02 and 0.14%. Comparisons of infectivity of DNAs obtained from four isolates of JC virus and which differed in their degrees of heterogeneity did not reveal significant differences. The JC virus DNA was not infectious in primary human fetal lung and kidney cells. Images PMID:228071

  13. Complete genome sequence of mandarin decline Citrus tristeza virus of the Northeastern Himalayan hill region of India: comparative analyses determine recombinant.

    PubMed

    Biswas, Kajal K; Tarafdar, Avijit; Sharma, Susheel K

    2012-03-01

    The complete genome sequence of a mandarin (Citrus reticulata) decline CTV isolate, Kpg3, of the Darjeeling hills of the Northeastern Himalayan region of India is reported for the first time. The complete Kpg3 genome has 19253 nt, and its nucleotide sequence identity ranged from 79% with the Florida CTV isolate T36 to 94% with the Israel isolate VT, whereas its identity to B165, the other Indian isolate, was 89%. Phylogenetic analysis indicated that the Kpg3 genome is closely related to isolate VT and distantly to T36 and B165. Recombination analysis indicated that Kpg3 is recombinant and originated through multiple recombination events in which parts of the genome were exchanged between divergent CTV sequences. PMID:22160128

  14. Mayaro virus fever in French Guiana: isolation, identification, and seroprevalence.

    PubMed

    Talarmin, A; Chandler, L J; Kazanji, M; de Thoisy, B; Debon, P; Lelarge, J; Labeau, B; Bourreau, E; Vié, J C; Shope, R E; Sarthou, J L

    1998-09-01

    This paper reports the first isolation of Mayaro (MAY) virus from a patient infected in French Guiana. The identification was initially performed using immunofluorescent antibody testing with specific mouse antibody, and confirmed by plaque-reduction neutralization testing and reverse transcription-polymerase chain reaction. To determine if MAY virus infection is widespread in French Guiana, a serosurvey was performed to determine the prevalence of antibody to this virus in various ethnic groups and areas of French Guiana. Human sera (n = 1,962) were screened using the hemagglutination inhibition (HI) test. To determine whether MAY virus circulates in the rain forest, a serosurvey in monkey populations was performed. Monkey sera (n = 150) were also screened for antibody to MAY virus using HI testing. Of the human sera tested, 6.3% were positive for anti-MAY virus antibodies. Significant differences in MAY virus seroprevalence between different age groups were observed. Seroprevalence rates increased with age, with a large increase in people 10-19 years of age in comparison with those less than 10 years of age. After adjustment for age, significant differences were also found between places of residence. The prevalence of anti-MAY virus antibody was higher in people living in contact with the forest, especially in the Haut Oyapock area (odds ratio [OR] = 97.7, 95% confidence interval [CI] = 48.2-197.9) and along the Maroni River (OR = 39.7, 95% CI = 20.6-76.6). The ethnic differences observed in this study were probably due to differences in residence. Among monkeys, higher seroprevalence rates were found in Alouatta seniculus (66.0%) than in Saguinus midas (18.2%). Among Alouatta, the seroprevalence increased significantly with weight (and therefore with age). This study indicates that MAY virus is present in French Guiana, and human infections occur in areas where people live near the tropical rain forest. PMID:9749643

  15. Isolation and molecular characterization of Newcastle disease viruses from raptors.

    PubMed

    Jindal, Naresh; Chander, Yogesh; Primus, Alexander; Redig, Patrick T; Goyal, Sagar M

    2010-12-01

    The present study was undertaken to detect and characterize Newcastle disease virus (NDV) in raptors. Cloacal and oropharyngeal swab samples were collected from 60 casualty raptors during January to March 2009 in Minnesota. Inoculation of all these samples (n=120) in 9-day-old embryonated hens' eggs resulted in isolation of haemagglutinating viruses in three samples from two bald eagles and one great horned owl. These three haemagglutinating viruses were confirmed as NDV by reverse transcription-polymerase chain reaction (RT-PCR) using fusion gene-specific primers, and were negative for avian influenza virus by RT-PCR. Further characterization revealed that all three possessed (112)GKQGRL(117) at the fusion gene cleavage site, indicating that they were lentogenic strains. Phylogenetic analysis revealed that all three isolates clustered with published class II genotype II NDVs. The nucleotide sequence homology of the three NDV isolates among themselves was 98.4 to 99.6% and the sequence homology with lentogenic strains from wild birds used for comparison varied between 94.5 and 100%. Detection of NDV strains from raptors merits further epidemiological studies to determine the prevalence of different NDV strains in raptors and their impact in relation to transmission to domestic poultry. PMID:21154052

  16. Suppression of influenza virus infection by the orf virus isolated in Taiwan

    PubMed Central

    LIN, Fong-Yuan; TSENG, Yeu-Yang; CHAN, Kun-Wei; KUO, Shu-Ting; YANG, Cheng-Hsiung; WANG, Chi-Young; TAKASU, Masaki; HSU, Wei-Li; WONG, Min-Liang

    2015-01-01

    Orf virus (ORFV), a member of parapoxvirus, is an enveloped virus with genome of double-stranded DNA. ORFV causes contagious pustular dermatitis or contagious ecthyma in sheep and goats worldwide. In general, detection of viral DNA and observing ORFV virion in tissues of afflicted animals are two methods commonly used for diagnosis of orf infection; however, isolation of the ORFV in cell culture using virus-containing tissue as inoculum is known to be difficult. In this work, the ORFV (Hoping strain) isolated in central Taiwan was successfully grown in cell culture. We further examined the biochemical characteristic of our isolate, including viral genotyping, viral mRNA and protein expression. By electron microscopy, one unique form of viral particle from ORFV infected cellular lysate was demonstrated in the negative-stained field. Moreover, immunomodulating and anti-influenza virus properties of this ORFV were investigated. ORFV stimulated human monocytes (THP-1) secreting proinflammatory cytokines IL-8 and TNF-?. And, pre-treatment of ORFV-infected cell medium prevents A549 cells from subsequent type A influenza virus (IAV) infection. Similarly, mice infected with ORFV via both intramuscular and subcutaneous routes at two days prior to IAV infection significantly decreased the replication of IAV. In summary, the results of a current study indicated our Hoping strain harbors the immune modulator property; with such a bio-adjuvanticity, we further proved that pre-exposure of ORFV protects animals from subsequent IAV infection. PMID:25855509

  17. Isolation and phylogenetic analysis of caprine Orf virus in Malaysia.

    PubMed

    Abdullah, Ashwaq Ahmed; Ismail, Muhammad Farid Bin; Balakrishnan, Krishnan Nair; Bala, Jamilu Abubakar; Hani, Homayoun; Abba, Yusuf; Awang Isa, Mohd Kamaruddin; Abdullah, Faez Firdaus Jesse; Arshad, Siti Suri; Nazariah, Zeenatul Allaudin; Abdullah, Rasedee; Mustapha, Noordin Mohamed; Mohd-Lila, Mohd-Azmi

    2015-12-01

    Orf virus is a DNA virus that causes contiguous ecthyma in goat and sheep. Infection of animals with this virus cause high mortality in young animals resulting in huge economic losses. In this study, we investigated an outbreak of Orf in a goat farm in Malaysia. Samples were collected from infected animals and viral isolation was done using both LT and MDCK cell lines. Molecular detection was done by conventional PCR for specific primers; B2L and F1L genes and phylogenetic analysis was done on the sequence data obtained. Cytopathic effects (CPE) were observed in both cell lines after 3 days of inoculation and were 50 % by the sixth day. PCR showed positive bands for both B2L and F1L genes and phylogenetic analysis showed that the Malaysian strain had close homology to the Chinese and Indian Orf virus isolates. This study gives more insight into the existing Orf viral strains in Malaysia and their relationship with other strains globally. PMID:26645035

  18. A virus related to soybean mosaic virus from Pinellia ternata in China and its comparison with local soybean SMV isolates.

    PubMed

    Chen, J; Zheng, H-Y; Lin, L; Adams, M J; Antoniw, J F; Zhao, M-F; Shang, Y-F; Chen, J-P

    2004-02-01

    A potyvirus isolated from Pinellia ternata in China was characterised and shown to be related to Soybean mosaic virus (SMV). The virus was pathogenic on P. ternata and some soybean cultivars, whereas the local soybean SMV isolate HH5 did not infect P. ternata. Western blot experiments demonstrated a serological relationship between the virus from Pinellia, SMV and Watermelon mosaic virus (WMV). The complete nucleotide sequences of the Pinellia virus (isolate P-1, 9735 nt) and of the Chinese soybean SMV isolates HH5 (9585 nt) and HZ (9588 nt) were determined. A 1733 nt sequence at the 3'-terminus of a second isolate from Pinellia (isolate P-2) was also determined. The predicted polyprotein of isolate P-1 has 83% amino acid (aa) identity with those of published SMV sequences. In many parts of the genome, aa identity was about 90% but it was much lower in the P1 protein region (24-29%), where it more closely resembled Dasheen mosaic virus (62%). The partial sequence of isolate P-2 had 91% nt identity to P-1 and both isolates resembled a recent sequence in the public databases (AF469171) wrongly named Zantedeschia mosaic virus. The two complete SMV soybean sequences had 93-95% nt identity with those of the previously sequenced isolates and >97% amino acid identity. Phylogenetic analysis and comparisons of coat proteins suggest that the Pinellia, WMV and SMV potyviruses should probably be treated as strains of the same species. PMID:14745600

  19. Complete Genome Sequence of Tomato Mosaic Virus Isolated from Jasmine in the United States

    PubMed Central

    Fillmer, Kornelia; Adkins, Scott; Pongam, Patchara

    2015-01-01

    Tomato mosaic virus was reported from jasmine in Florida. We present the first complete genome sequence of a tomato mosaic virus isolate from this woody perennial plant in the United States. PMID:26159525

  20. Isolation and genetic characterization of a tembusu virus strain isolated from mosquitoes in Shandong, China.

    PubMed

    Tang, Y; Diao, Y; Chen, H; Ou, Q; Liu, X; Gao, X; Yu, C; Wang, L

    2015-04-01

    Tembusu virus (TMUV) is a flavivirus, presumed to be a mosquito-borne flavivirus of the Ntaya virus subgroup. To date, however, there have been no reports indicating that mosquitoes are involved in the spread of TMUV. In this study, we report the first isolation of TMUV from Culex mosquitoes. We describe the isolation and characterization of a field strain of TMUV from mosquitoes collected in Shandong Province, China. The virus isolate, named TMUV-SDMS, grows well in mosquito cell line C6/36, in Vero and duck embryo fibroblast (DEF) cell lines, and causes significant cytopathic effects in these cell cultures. The TMUV-SDMS genome is a single-stranded RNA, 10 989 nt in length, consisting of a single open reading frame encoding a polyprotein of 3410 amino acids, with 5' and 3' untranslated regions of 142 and 617 nt, respectively. Phylogenetic analysis of the E and NS5 genes revealed that the TMUV-SDMS is closely related to the TMUV YY5 and BYD strains which cause severe egg-drop in ducks. The 3'NTR of TMUV-SDMS contains two pairs of tandem repeat CS and one non-duplicate CS, which have sequence similarities to the same repeats in the YY5 and BYD strains. Our findings indicate that mosquitoes carrying the TMUV may play an important role in the spread of this virus and in disease outbreak. PMID:23711093

  1. Isolation, Genetic Characterization, and Seroprevalence of Adana Virus, a Novel Phlebovirus Belonging to the Salehabad Virus Complex, in Turkey

    PubMed Central

    Alkan, Cigdem; Alwassouf, Sulaf; Piorkowski, Géraldine; Bichaud, Laurence; Tezcan, Seda; Dincer, Ender; Ergunay, Koray; Ozbel, Yusuf; Alten, Bulent; de Lamballerie, Xavier

    2015-01-01

    ABSTRACT A new phlebovirus, Adana virus, was isolated from a pool of Phlebotomus spp. (Diptera; Psychodidae) in the province of Adana, in the Mediterranean region of Turkey. Genetic analysis based on complete coding of genomic sequences indicated that Adana virus belongs to the Salehabad virus species of the genus Phlebovirus in the family Bunyaviridae. Adana virus is the third virus of the Salehabad virus species for which the complete sequence has been determined. To understand the epidemiology of Adana virus, a seroprevalence study using microneutralization assay was performed to detect the presence of specific antibodies in human and domestic animal sera collected in Adana as well as Mersin province, located 147 km west of Adana. The results demonstrate that the virus is present in both provinces. High seroprevalence rates in goats, sheep, and dogs support intensive exposure to Adana virus in the region, which has not been previously reported for any virus included in the Salehabad serocomplex; however, low seroprevalence rates in humans suggest that Adana virus is not likely to constitute an important public health problem in exposed human populations, but this deserves further studies. IMPORTANCE Until recently, in the genus Phlebovirus, the Salehabad virus species consisted of two viruses: Salehabad virus, isolated from sand flies in Iran, and Arbia virus, isolated from sand flies in Italy. Here we present the isolation and complete genome characterization of the Adana virus, which we propose to be included in the Salehabad virus species. To our knowledge, this is the first report of the isolation and complete genome characterization, from sand flies in Turkey, of a Salehabad virus-related phlebovirus with supporting seropositivity in the Mediterranean, Aegean, and Central Anatolia regions, where phleboviruses have been circulating and causing outbreaks. Salehabad species viruses have generally been considered to be a group of viruses with little medical or veterinary interest. This view deserves to be revisited according to our results, which indicate a high animal infection rate of Adana virus and recent evidence of human infection with Adria virus in Greece. PMID:25653443

  2. Application of the "best fit" pathotyping assay for evaluation of Russian isolates of Marek's disease virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to investigate the validity of the "best fit" pathotyping assay for testing of field isolates of Marek's disease (MD) virus. Twenty serotype 1 MD viruses were isolated from breeder and commercial flocks in 8 regions of the Russian Federation. These isolates were pat...

  3. Genomic characterization of pseudorabies virus strains isolated in Italy.

    PubMed

    Sozzi, E; Moreno, A; Lelli, D; Cinotti, S; Alborali, G L; Nigrelli, A; Luppi, A; Bresaola, M; Catella, A; Cordioli, P

    2014-08-01

    In this study, we undertook the genomic characterization of 54 pseudorabies virus (PRV) strains isolated in Italy during 1984-2010. The characterization was based on partial sequencing of the UL44 (gC) and US8 (gE) genes; 44 strains (38 for gene gE and 36 for gC) were isolated on pig farms; 9 originated from dogs and 1 from cattle. These porcine PRV strains, which were closely related to those isolated in Europe and America in the last 20 years, and the bovine strain bovine/It/2441/1992 belong to cluster B in both phylogenetic trees. Six porcine strains that do not belong to cluster B are related in both gE and gC phylogenetic trees to the 'old' porcine PRV strains isolated in the 1970s and 1980s. In the last two decades, the presence of these strains in domestic pig populations has been reduced drastically, whereas they are prevalent in wild boar. The two remaining strains have an interesting genomic profile, characterized by the gC gene being closely related to the old porcine PRV strains, and the gE gene being similar to that of recently isolated strains. Three strains originating from working dogs on pig farms are located in cluster B in both phylogenetic trees. Five strains isolated from hunting dogs have a high degree of correlation with PRV strains circulating in wild boar. The last isolate has a gC gene similar to that in the two porcine strains mentioned previously, and the gE gene is correlated with the strains isolated from hunting dogs. These results provide interesting insight into the genomic characterization of PRV strains and reveal a clear differentiation between the strains isolated from hunting dogs that are related to the wild boar strains and those originating from domestic pigs. PMID:23331342

  4. Genotyping of newly isolated infectious bronchitis virus isolates from northeastern Georgia.

    PubMed

    Kulkarni, Arun B; Resurreccion, Reynaldo S

    2010-12-01

    Sixteen infectious bronchitis virus (IBV) field isolates obtained from vaccinated commercial broiler chickens showing clinical respiratory disease were characterized by reverse transcriptase-polymerase chain reaction and sequence analysis of the hypervariable region of the S1 spike glycoprotein gene. The genetic relationship among these variants and reference strains was determined by phylogenetic analysis and use of the basic local alignment search tool. All the isolates formed a distinct phylogenetic group with very short branched distances, suggesting that isolates had a similar origin. All the isolates showed 85% amino acid identity with recently described Australian isolates, particularly N1-62. Given that little was known about this new emergent IBV we have characterized five field isolates by sequencing the entire S1 gene. Multiple sequence alignment of deduced amino acid sequences with commonly used vaccine strains revealed that most substitutions occurred in the 53-148 amino acid region. A possible recombination site with N1-62 isolate was identified between amino acid residues 115-121. All the field isolates shared four or five out of seven amino acid residues with N1-62 in this region as opposed to Ark-DPI and Mass 41 reference strains, which shared only two residues. Results indicate that IBV isolates reported here can be considered as new IBV genotype. PMID:21313832

  5. Isolation of saint louis encephalitis virus from a horse with neurological disease in Brazil.

    PubMed

    Rosa, Roberta; Costa, Erica Azevedo; Marques, Rafael Elias; Oliveira, Taismara Simas; Furtini, Ronaldo; Bomfim, Maria Rosa Quaresma; Teixeira, Mauro Martins; Paixão, Tatiane Alves; Santos, Renato Lima

    2013-11-01

    St. Louis encephalitis virus (SLEV) is a causative agent of encephalitis in humans in the Western hemisphere. SLEV is a positive-sense RNA virus that belongs to the Flavivirus genus, which includes West Nile encephalitis virus, Japanese encephalitis virus, Dengue virus and other medically important viruses. Recently, we isolated a SLEV strain from the brain of a horse with neurological signs in the countryside of Minas Gerais, Brazil. The SLEV isolation was confirmed by reverse-transcription RT-PCR and sequencing of the E protein gene. Virus identity was also confirmed by indirect immunofluorescence using commercial antibodies against SLEV. To characterize this newly isolated strain in vivo, serial passages in newborn mice were performed and led to hemorrhagic manifestations associated with recruitment of inflammatory cells into the central nervous system of newborns. In summary this is the first isolation of SLEV from a horse with neurological signs in Brazil. PMID:24278489

  6. Isolation, identification, and complete genome sequence of an avian reticuloendotheliosis virus isolated from geese.

    PubMed

    Lin, Chia-Yao; Chen, Chiou-Lin; Wang, Chao-Cheng; Wang, Ching-Ho

    2009-05-12

    Naturally occurring lymphoreticular tumors in geese have been found from time to time in Taiwan, but their etiology has not been determined except through morphological descriptions. This study observed a reticuloendotheliosis virus (REV) infection occurring in a white Roman goose (Anser anser) farm in Yunlin, Taiwan in 2006. These geese showed growth-retarded and nodular lymphoma-like tumors in the liver, lung, kidney, and pancreas. Thirty blood samples were taken for REV detection and 21 (70%) of them contained REV genetic sequences using polymerase chain reaction (PCR). Virus isolation was attempted from 11 blood samples by inoculating the buffy coat onto DF1 cells. Nine (81%) REVs were isolated after three blind passages. The complete proviral sequence from one isolate was determined for phylogenetic analysis by direct sequencing using overlapping PCR products. The length of the provial genome is 8284 nucleotides. By comparing with other published REV complete sequences, the nucleotide percent identity ranged from 93.5% to 99.8% with most LTR varieties, ranging from 74.9% to 99.8%. The present isolated goose REV is most close to REV APC-566, a REV isolated from Attwater's Prairie chickens. PMID:19131189

  7. First Isolation of a Giant Virus from Wild Hirudo medicinalis Leech: Mimiviridae isolation in Hirudo medicinalis

    PubMed Central

    Boughalmi, Mondher; Pagnier, Isabelle; Aherfi, Sarah; Colson, Philippe; Raoult, Didier; La Scola, Bernard

    2013-01-01

    Giant viruses and amoebae are common in freshwater, where they can coexist with other living multicellular organisms. We screened leeches from the species Hirudo medicinalis for giant viruses. We analyzed five H. medicinalis obtained from Tunisia (3) and France (2). The leeches were decontaminated and then dissected to remove internal parts for co-culture with Acanthamoeba polyphaga. The genomes of isolated viruses were sequenced on a 454 Roche instrument, and a comparative genomics analysis was performed. One Mimivirus was isolated and the strain was named Hirudovirus. The genome assembly generated two scaffolds, which were 1,155,382 and 25,660 base pairs in length. Functional annotations were identified for 47% of the genes, which corresponds to 466 proteins. The presence of Mimividae in the same ecological niche as wild Hirudo may explain the presence of the mimivirus in the digestive tract of the leech, and several studies have already shown that viruses can persist in the digestive tracts of leeches fed contaminated blood. As leeches can be used medically and Mimiviruses have the potential to be an infectious agent in humans, patients treated with leeches should be surveyed to investigate a possible connection. PMID:24287596

  8. Characterisation of potato virus Y isolates from Iran.

    PubMed

    Hosseini, Atefe; Massumi, Hossein; Heydarnejad, Jahangir; Hosseini Pour, Akbar; Varsani, Arvind

    2011-02-01

    A survey of Potato virus Y (PVY) was conducted in cultivated fields in six Iranian provinces between January 2005 to July 2007. Two hundred samples from potato and tomato were collected and analyzed by enzyme-linked immunosorbent assay (ELISA) for potyviruses. Almost one fourth of the samples were found to be infected by PVY. Analysis of these PVY-positive samples using three monoclonal antibodies (MAbs) facilitating the simultaneous detection of three main strains namely the ordinary (PVY(O)), strain (PVY(N)) and C (PVY(C)) strains. However, the fourth strain (PVY(NTN)) and some others recombinant isolates were also identified by molecular methods. Host range and symptoms analysis using sap inoculation of four different strains of PVY onto a range of plants revealed that the four strains showed biological properties that seemed to be consistent with their molecular grouping. Fourteen isolates of PVY were chosen based on the host and geographical location, primer specificity and serology for further biological and molecular characterisation. The coat protein (CP) and P1 genes and 3'-non-translated region (3'NTR) from 14 representative isolates were sequenced and analysed with the sequences available in GenBank. Composite analysis of the P1, CP and 3'-UTR sequences with all full genome sequences of PVY revealed that there are three potential strains of PVY in Iran, PVY(O), PVY(N)-W and PVY(NTN). Isolate KER.SA(N) was the most divergent of all the 14 isolates reacted with PVY(N) specific MAbs but grouped with PVY(O) strains in maximum likelihood phylogentic analysis. The PVY(NTN) isolates from Iran more closely related to the European than North American PVY(NTN) isolates. PMID:21082231

  9. Isolation and partial characterisation of a new strain of Ebola We have isolated a new strain of Ebola virus from a non-

    E-print Network

    1271 Isolation and partial characterisation of a new strain of Ebola virus Summary We have isolated a new strain of Ebola virus from a non- fatal human case infected during the autopsy of a wild about the natural reservoir of the Ebola virus. Lancet 1995; 345: 1271-74 Introduction Ebola virus

  10. Characterization of reticuloendotheliosis virus isolates obtained from chickens, turkeys and prairie chickens in the United States

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Twelve reticuloendotheliosis virus (REV) isolates obtained from chickens, turkeys and prairie chickens in the United States were characterized using ploymerase chain reaction (PCR) and indirect immunofluoresence (IFA) assays. This study included five REV isolates from Prairie chickens in Texas, two ...

  11. Genotyping of Korean isolates of infectious hematopoietic necrosis virus (IHNV) based on the glycoprotein gene

    USGS Publications Warehouse

    Kim, W.-S.; Oh, M.-J.; Nishizawa, T.; Park, J.-W.; Kurath, G.; Yoshimizu, M.

    2007-01-01

    Glycoprotein (G) gene nucleotide sequences of four Korean isolates of infectious hematopoietic necrosis virus (IHNV) were analyzed to evaluate their genetic relatedness to worldwide isolates. All Korean isolates were closely related to Japanese isolates of genogroup JRt rather than to those of North American and European genogroups. It is believed that Korean IHNV has been most likely introduced from Japan to Korea by the movement of contaminated fish eggs. Among the Korean isolates, phylogenetically distinct virus types were obtained from sites north and south of a large mountain range, suggesting the possibility of more than one introduction of virus from Japan. ?? 2007 Springer-Verlag.

  12. 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

  13. Phylogenetic analysis of Bunyamwera and Ngari viruses (family Bunyaviridae, genus Orthobunyavirus) isolated in Kenya.

    PubMed

    Odhiambo, C; Venter, M; Lwande, O; Swanepoel, R; Sang, R

    2016-01-01

    Orthobunyaviruses, tri-segmented, negative-sense RNA viruses, have long been associated with mild to severe human disease in Africa, but not haemorrhagic fever. However, during a Rift Valley fever outbreak in East Africa in 1997-1998, Ngari virus was isolated from two patients and antibody detected in several others with haemorrhagic fever. The isolates were used to identify Ngari virus as a natural Orthobunyavirus reassortant. Despite their potential to reassort and cause severe human disease, characterization of orthobunyaviruses is hampered by paucity of genetic sequences. Our objective was to obtain complete gene sequences of two Bunyamwera virus and three Ngari virus isolates from recent surveys in Kenya and to determine their phylogenetic positioning within the Bunyamwera serogroup. Newly sequenced Kenyan Bunyamwera virus isolates clustered closest to a Bunyamwera virus isolate from the same locality and a Central African Republic isolate indicating that similar strains may be circulating regionally. Recent Kenyan Ngari isolates were closest to the Ngari isolates associated with the 1997-1998 haemorrhagic fever outbreak. We observed a temporal/geographical relationship among Ngari isolates in all three gene segments suggesting a geographical/temporal association with genetic diversity. These sequences in addition to earlier sequences can be used for future analyses of this neglected but potentially deadly group of viruses. PMID:26118981

  14. First isolation of reticuloendotheliosis virus from mallards in China.

    PubMed

    Jiang, Lili; Deng, Xiaoyun; Gao, Yulong; Li, Kai; Chai, Hongliang; Fan, Zhaobin; Ren, Xiangang; Wang, Qi; Zhang, Lizhou; Yun, Bingling; Yin, Chunhong; Chen, Yuming; Qin, Liting; Gao, Honglei; Wang, Yongqiang; Hua, Yuping; Wang, Xiaomei

    2014-08-01

    Reticuloendotheliosis virus (REV) causes an oncogenic, immunosuppressive and runting syndrome in many avian hosts worldwide. REV infection has never been reported in mallard ducks, however. To identify REV infection in mallards, we collected 40 mallard duck samples from Jilin Province of China. In this study, the REV strain, DBYR1102, was first isolated from a mallard in China and identified by PCR, indirect immunofluorescence assay and electron microscopy. The gp90 gene and complete LTR of DBYR1102 were amplified and sequenced. Phylogenetic analysis based on gp90 genes of REV indicated that the REV strain DBYR1102 is closely related to strain HLJR0901 from northeastern China, the prairie chicken isolate APC-566, and REV subtype III, represented by chick syncytial virus. This new strain is distantly related to two other subtypes of REV, 170A and SNV. Phylogenetic analysis based on the LTR yielded information similar to that obtained with the gp90 genes. The results of this study not only expand our epidemiological understanding of REV in the wild birds of China but also demonstrate the potential role of wild waterfowl in REV transmission. PMID:24643331

  15. Isolation of a Novel Swine Influenza Virus from Oklahoma in 2011 Which Is Distantly Related to Human Influenza C Viruses

    PubMed Central

    Hause, Ben M.; Ducatez, Mariette; Collin, Emily A.; Ran, Zhiguang; Liu, Runxia; Sheng, Zizhang; Armien, Anibal; Kaplan, Bryan; Chakravarty, Suvobrata; Hoppe, Adam D.; Webby, Richard J.; Simonson, Randy R.; Li, Feng

    2013-01-01

    Of the Orthomyxoviridae family of viruses, only influenza A viruses are thought to exist as multiple subtypes and has non-human maintenance hosts. In April 2011, nasal swabs were collected for virus isolation from pigs exhibiting influenza-like illness. Subsequent electron microscopic, biochemical, and genetic studies identified an orthomyxovirus with seven RNA segments exhibiting approximately 50% overall amino acid identity to human influenza C virus. Based on its genetic organizational similarities to influenza C viruses this virus has been provisionally designated C/Oklahoma/1334/2011 (C/OK). Phylogenetic analysis of the predicted viral proteins found that the divergence between C/OK and human influenza C viruses was similar to that observed between influenza A and B viruses. No cross reactivity was observed between C/OK and human influenza C viruses using hemagglutination inhibition (HI) assays. Additionally, screening of pig and human serum samples found that 9.5% and 1.3%, respectively, of individuals had measurable HI antibody titers to C/OK virus. C/OK virus was able to infect both ferrets and pigs and transmit to naive animals by direct contact. Cell culture studies showed that C/OK virus displayed a broader cellular tropism than a human influenza C virus. The observed difference in cellular tropism was further supported by structural analysis showing that hemagglutinin esterase (HE) proteins between two viruses have conserved enzymatic but divergent receptor-binding sites. These results suggest that C/OK virus represents a new subtype of influenza C viruses that currently circulates in pigs that has not been recognized previously. The presence of multiple subtypes of co-circulating influenza C viruses raises the possibility of reassortment and antigenic shift as mechanisms of influenza C virus evolution. PMID:23408893

  16. New Genome Sequences of Gamboa Viruses (Family Bunyaviridae, Genus Orthobunyavirus) Isolated in Panama and Argentina

    PubMed Central

    de Lima, Clayton P. S.; Martins, Lívia C.; Aragão Dias, Amarílis; Cardoso, Jedson F.; Silva, Sandro P.; Da Silva, Daisy E. A.; Oliveira, Layanna F.; Vasconcelos, Janaina M.; Ferreira, João Paulo C.; Travassos da Rosa, Amelia P. A.; Guzman, Hilda; Tesh, Robert B.; Vasconcelos, Pedro F. C.

    2014-01-01

    We describe here the nearly complete open reading frame (ORF) of five Gamboa virus strains isolated in Panama and Argentina. The viruses with complete ORF showed the regular genome organization observed in other orthobunyaviruses with exception to the presence of NSs protein. All predicted proteins showed homology with viruses belonging to members of the family Bunyaviridae. PMID:25414487

  17. Genetic relationships between southern African SAT-2 isolates of foot-and-mouth-disease virus.

    PubMed Central

    Vosloo, W.; Knowles, N. J.; Thomson, G. R.

    1992-01-01

    Sequencing of part of the 1D gene of foot-and-mouth disease virus was used to determine the relationships between SAT-2 viruses isolated from outbreaks which occurred in cattle in Zimbabwe and Namibia and in impala in South Africa between 1979 and 1989. The results demonstrated that the outbreaks in different countries were unrelated. Surprisingly close relationships were shown between all SAT-2 viruses isolated from cattle in Zimbabwe since 1983 but the two major epizootics which occurred in 1989 were caused by viruses which were clearly different. Conversely, two apparently unrelated outbreaks in impala in South Africa were caused by viruses which could not be distinguished. PMID:1334842

  18. Complete Genome Sequence of Papaya Ringspot Virus Isolated from Genetically Modified Papaya in Hainan Island, China

    PubMed Central

    Zhao, Guangyuan; Shen, Wentao; Tuo, Decai; Li, Xiaoying

    2015-01-01

    The complete genome sequence (10,326 nucleotides) of a papaya ringspot virus isolate infecting genetically modified papaya in Hainan Island of China was determined through reverse transcription (RT)-PCR. The virus shares 92% nucleotide sequence identity with the isolate that is unable to infect PRSV-resistant transgenic papaya. PMID:26358610

  19. Genetic characterization of epizootic hemorrhagic disease virus strains isolated from cattle in Israel

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Epizootic hemorrhagic disease virus (EHDV), an Orbivirus not previously reported in Israel, was isolated from Israeli cattle during a “bluetongue like” disease outbreak in 2006. To ascertain the origin of this new virus, three isolates from the outbreak were fully sequenced and compared with availab...

  20. Molecular-genetic analysis of field isolates of Avian Leucosis Viruses in the Russian Federation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Commercial poultry farms in 14 regions of Russian Federation were monitored for avian leukosis virus (ALV) infection using virus isolation tests and serology. Results indicated the presence of two subgroups of ALV in farms located in 11 of 14 regions. Analysis of the genomes of 12 field isolates of...

  1. Genetic Transformation of Citrus Paradisi with Antisense and untranslatable RNA-dependent RNA Polymerase Genes of Citrus Tristeza Closterovirus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Expression of the RNA-dependent RNA polymerase (RdRp) of Citrus tristeza virus (CTV) was studied in vivo and in vitro using a polyclonal antiserum raised against the recombinant CTV-RdRp protein. Although 56 kDa CTV-RdRp is thought to be expressed by a +1 translational frameshift at the carboxyl te...

  2. Matrix Gene of Influenza A Viruses Isolated from Wild Aquatic Birds: Ecology and Emergence of Influenza A Viruses

    PubMed Central

    Widjaja, Linda; Krauss, Scott L.; Webby, Richard J.; Xie, Tao; Webster, Robert G.

    2004-01-01

    Wild aquatic birds are the primary reservoir of influenza A viruses, but little is known about the viruses' gene pool in wild birds. Therefore, we investigated the ecology and emergence of influenza viruses by conducting phylogenetic analysis of 70 matrix (M) genes of influenza viruses isolated from shorebirds and gulls in the Delaware Bay region and from ducks in Alberta, Canada, during >18 years of surveillance. In our analysis, we included 61 published M genes of isolates from various hosts. We showed that M genes of Canadian duck viruses and those of shorebird and gull viruses in the Delaware Bay shared ancestors with the M genes of North American poultry viruses. We found that North American and Eurasian avian-like lineages are divided into sublineages, indicating that multiple branches of virus evolution may be maintained in wild aquatic birds. The presence of non-H13 gull viruses in the gull-like lineage and of H13 gull viruses in other avian lineages suggested that gulls' M genes do not preferentially associate with the H13 subtype or segregate into a distinct lineage. Some North American avian influenza viruses contained M genes closely related to those of Eurasian avian viruses. Therefore, there may be interregional mixing of the two clades. Reassortment of shorebird M and HA genes was evident, but there was no correlation among the HA or NA subtype, M gene sequence, and isolation time. Overall, these results support the hypothesis that influenza viruses in wild waterfowl contain distinguishable lineages of M genes. PMID:15280485

  3. Seroepidemiology of infection with hepatitis A and B viruses in an isolated Pacific population.

    PubMed

    Gust, I D; Lehmann, N I; Dimitrakakis, M; Zimmet, P

    1979-05-01

    To determine the prevalence of infection with hepatitis A virus and hepatitis B virus in an isolated population, samples of serum were collected from 574 healthy subjects living on the remote Pacific Island of Funafuti. Each specimen was tested for antibody to hepatitis A virus, hepatitis B surface antigen, and antibody to hepatitis B surface antigen by solid-phase radioimmunoassay. Overall, 79.8% of the population showed evidence of previous infection with hepatitis A virus, and 72.5% with hepatitis B virus; the high prevalence of antibody to both viruses in young adults suggested that the majority of infections were acquired in the first decade of life. Although it is known that hepatitis B virus maintains itself in isolated populations through a reservoir of chronic carriers, the reason for the persistently high rate of infection with hepatitis A virus is unknown. PMID:438552

  4. Isolation of new Brazilian giant viruses from environmental samples using a panel of protozoa

    PubMed Central

    Dornas, Fábio P.; Khalil, Jacques Y. B.; Pagnier, Isabelle; Raoult, Didier; Abrahão, Jônatas; La Scola, Bernard

    2015-01-01

    The Megavirales are a newly described order capable of infecting different types of eukaryotic hosts. For the most part, the natural host is unknown. Several methods have been used to detect these viruses, with large discrepancies between molecular methods and co-cultures. To isolate giant viruses, we propose the use of different species of amoeba as a cellular support. The aim of this work was to isolate new Brazilian giant viruses by comparing the protozoa Acanthamoeba castellanii, A. polyphaga, A. griffini, and Vermamoeba vermiformis (VV) as a platform for cellular isolation using environmental samples. One hundred samples were collected from 3 different areas in September 2014 in the Pampulha lagoon of Belo Horizonte city, Minas Gerais, Brazil. PCR was used to identify the isolated viruses, along with hemacolor staining, labelling fluorescence and electron microscopy. A total of 69 viruses were isolated. The highest ratio of isolation was found in A. polyphaga (46.38%) and the lowest in VV (0%). Mimiviruses were the most frequently isolated. One Marseillevirus and one Pandoravirus were also isolated. With Brazilian environmental samples, we demonstrated the high rate of lineage A mimiviruses. This work demonstrates how these viruses survive and circulate in nature as well the differences between protozoa as a platform for cellular isolation. PMID:26500630

  5. Isolation of Equine rhinitis A virus from a horse semen sample.

    PubMed

    Johnson, Donna J; Ostlund, Eileen N; Palmer, Tiffany J; Fett, Kathryn L; Schmitt, Beverly J

    2012-07-01

    Semen from an apparently healthy 4-year-old American Quarter Horse was submitted to the National Veterinary Services Laboratories for Equine arteritis virus isolation. Visual inspection of the semen sample upon arrival noted it was unusually yellow in color. The semen sample was inoculated onto cell monolayers, and cytopathic effect was observed 5 days postinoculation. The resultant isolate tested negative for Equine arteritis virus, and was subsequently identified as Equine rhinitis A virus. Equine rhinitis A virus has been isolated from horse urine, but has not been described in stallion semen. The present study documents the isolation of Equine rhinitis A virus from stallion semen that was likely contaminated with urine at the time of collection. PMID:22621949

  6. Adaptive evolution of simian immunodeficiency viruses isolated from two conventional progressor macaques with neuroaids

    SciTech Connect

    Foley, Brian T; Korber, Bette T

    2008-01-01

    Simian immunodeficiency virus infection of macaques may result in neuroAIDS, a feature more commonly observed in macaques with rapid progressive disease than in those with conventional disease. This is the first report of two conventional progressors (H631 and H636) with encephalitis in rhesus macaques inoculated with a derivative of SIVsmES43-3. Phylogenetic analyses of viruses isolated from the cerebral spinal fluid (CSF) and plasma from both animals demonstrated tissue compartmentalization. Additionally, virus from the central nervous system (CNS) was able to infect primary macaque monocyte-derived macrophages more efficiently than virus from plasma. Conversely, virus isolated from plasma was able to replicate better in peripheral blood mononuclear cells than virus from CNS. We speculate that these viruses were under different selective pressures in their separate compartments. Furthermore, these viruses appear to have undergone adaptive evolution to preferentially replicate in their respective cell targets. Analysis of the number of potential N-linked glycosylation sites (PNGS) in gp160 showed that there was a statistically significant loss of PNGS in viruses isolated from CNS in both macaques compared to SIVsmE543-3. Moreover, virus isolated from the brain in H631, had statistically significant loss of PNGS compared to virus isolated from CSF and plasma of the same animal. It is possible that the brain isolate may have adapted to decrease the number of PNGS given that humoral immune selection pressure is less likely to be encountered in the brain. These viruses provide a relevant model to study the adaptations required for SIV to induce encephalitis.

  7. 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...

  8. PATHOGENESIS OF CHICKEN-PASSAGED NEWCASTLE DISEASE VIRUSES ISOLATED FROM CHICKENS, WILD, AND EXOTIC BIRDS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The pathogenesis of six Newcastle disease virus (NDV) isolates recovered from chickens and wild (anhinga) and exotic (yellow nape parrot, pheasant, and dove isolate) birds was examined after four passages of the isolates in domestic chickens. Groups of four-week-old specific-pathogen-free White Legh...

  9. Isolation of West Nile Virus from Urine Samples of Patients with Acute Infection

    PubMed Central

    Pacenti, Monia; Franchin, Elisa; Squarzon, Laura; Sinigaglia, Alessandro; Ulbert, Sebastian; Cusinato, Riccardo; Palù, Giorgio

    2014-01-01

    This study demonstrated that West Nile virus (WNV) excreted in the urine of patients with acute infection can be isolated in cell cultures. In addition, the protocols for WNV isolation from urine samples were standardized, and factors that may affect the efficiency of WNV isolation were identified. PMID:24951801

  10. Avian influenza virus isolation, propagation and titration in embryonated chicken eggs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Avian influenza (AI) virus is usually isolated, propagated, and titrated in embryonated chickens eggs (ECE). Most any sample type can be accommodated for culture with appropriate processing. Isolation may also be accomplished in cell culture particularly if mammalian lineage isolates are suspected, ...

  11. Comparison of methods for isolation and titration of Crimean-Congo hemorrhagic fever virus.

    PubMed Central

    Shepherd, A J; Swanepoel, R; Leman, P A; Shepherd, S P

    1986-01-01

    The fluorescence focus assay and the plaque assay in CER cells were compared with mouse inoculation for the isolation and titration of Crimean-Congo hemorrhagic fever virus. The fluorescence focus assay and the plaque assay were of similar sensitivity, but both produced 10- to 100-fold lower titers than did mouse inoculation. For specimens from 26 Crimean-Congo hemorrhagic fever patients in South Africa, virus was isolated from 20 by mouse inoculation and from only 11 by cell culturing. Although cell cultures were less sensitive for the isolation of virus from clinical specimens, they produced diagnostic results much more rapidly. PMID:3095367

  12. Opium poppy mosaic virus, a new umbravirus isolated from Papaver somniferum in New Zealand.

    PubMed

    Tang, Joe; Lebas, Bénédicte; Liefting, Lia; Veerakone, Stella; Wei, Ting; Ward, Lisa

    2016-01-01

    A novel virus, tentatively named "opium poppy mosaic virus" (OPMV), was isolated from Papaver somniferum (opium poppy) with leaf mosaic and mottling symptoms in Auckland, New Zealand, in 2006. The virus was mechanically transmitted to herbaceous plants of several species, in which it induced local and/or systemic symptoms. No virus particles were observed by electron microscopy in the diseased P. somniferum or any of the symptomatic herbaceous plants. The complete genomic sequence of 4230 nucleotides contains four open reading frames (ORF) and is most closely related (59.3 %) to tobacco bushy top virus, a member of the genus Umbravirus. These data suggest that OPMV is a new umbravirus. PMID:26514844

  13. VIRULENCE OF HETEROGENEOUS-ORIGIN NEWCASTLE DISEASE VIRUS ISOLATES BEFORE AND AFTER SEQUENTIAL PASSAGES IN DOMESTIC CHICKENS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Four serial passages of six Newcastle disease virus (NDV) isolates were performed in 2-week-old White Leghorns. The viruses were recovered from chickens (Ckn-Live Bird Market and Ckn-Australia isolates), exotic (Yellow Nape [YN] Parrot, Pheasant, and Dove isolates) and wild birds (Anhinga isolate). ...

  14. Isolation of Tacaribe Virus, a Caribbean Arenavirus, from Host-Seeking Amblyomma americanum Ticks in Florida

    PubMed Central

    Sayler, Katherine A.; Barbet, Anthony F.; Chamberlain, Casey; Clapp, William L.; Alleman, Rick; Loeb, Julia C.; Lednicky, John A.

    2014-01-01

    Arenaviridae are a family of single stranded RNA viruses of mammals and boid snakes. Twenty-nine distinct mammalian arenaviruses have been identified, many of which cause severe hemorrhagic disease in humans, particularly in parts of sub-Saharan Africa, and in Central and South America. Humans typically become infected with an arenavirus through contact with excreta from infected rodents. Tacaribe virus (TCRV) is an arenavirus that was first isolated from bats and mosquitoes during a rabies surveillance survey conducted in Trinidad from 1956 to 1958. Tacaribe virus is unusual because it has never been associated with a rodent host and since that one time isolation, the virus has not been isolated from any vertebrate or invertebrate hosts. We report the re-isolation of the virus from a pool of 100 host-seeking Amblyomma americanum (lone star ticks) collected in a Florida state park in 2012. TCRV was isolated in two cell lines and its complete genome was sequenced. The tick-derived isolate is nearly identical to the only remaining isolate from Trinidad (TRVL-11573), with 99.6% nucleotide identity across the genome. A quantitative RT-PCR assay was developed to test for viral RNA in host-seeking ticks collected from 3 Florida state parks. Virus RNA was detected in 56/500 (11.2%) of surveyed ticks. As this virus was isolated from ticks that parasitize humans, the ability of the tick to transmit the virus to people should be evaluated. Furthermore, reservoir hosts for the virus need to be identified in order to develop risk assessment models of human infection. PMID:25536075

  15. Isolation, identification, and whole genome sequencing of reticuloendotheliosis virus from a vaccine against Marek's disease.

    PubMed

    Li, Junping; Dong, Xuan; Yang, Chenghuai; Li, Qihong; Cui, Zhizhong; Chang, Shuang; Zhao, Peng; Yu, Kangzhen; Yang, Hanchun

    2015-04-01

    According to the requirements of the Ministry of Agriculture of China, all vaccines must be screened for exogenous virus contamination before commercialization. A freeze-dried vaccine against Marek's disease was used to inoculate specific pathogen-free chickens, from which serum samples were collected after 42 days. The results were positive for reticuloendotheliosis virus antibody, which was indicative of reticuloendotheliosis virus contamination. After neutralization with serum positive for Marek's disease virus, chicken embryo fibroblasts were inoculated with the vaccine. Afterward, viral isolation and identification were performed. One reticuloendotheliosis virus strain (MD-2) was isolated and verified using an immunofluorescence assay. Polymerase chain reaction amplification of the provirus MD-2 genome was performed using seven overlapping fragments as primers. The amplified products were sequenced and spliced to obtain the whole MD-2 genome sequence. The full genome length of MD-2 was 8,284 bp, which had an identity greater than 99% with the prairie chicken isolate APC-566 from the US, the goose-derived isolate 3410/06 from Taiwan, and the chicken-derived reticuloendotheliosis virus isolate HLJR0901 from Heilongjiang Province, China. The MD-2 was phylogenetically close to these isolates. The identity with REV isolate HA9901 from Jiangsu Province of China was 96.7%. The MD-2 had the lowest identity with duck-derived Sin Nombre virus from the United States, with the value of only 93.5%. The main difference lay in the U3 region of the long terminal repeat. The present research indicated that some vaccines produced during specific periods in China might be contaminated by reticuloendotheliosis virus. The reticuloendotheliosis virus strain isolated from the vaccine was phylogenetically close to the prevalent strain, with only minor variations. PMID:25725074

  16. [Phylogenetic analysis of rabies viruses isolated from animals in Tokyo in the 1950s].

    PubMed

    Hatakeyama, Kaoru; Sadamasu, Kenji; Kai, Akemi

    2011-05-01

    Molecular epidemiological analysis of 96 rabies viruses isolated from animals in Tokyo in the 1950s involves Japanese fixed virus, Komatsugawa, Takamen, and Nishigahara strains. Strains isolated in Tokyo were divided into Tokyo 1 and Tokyo 2, and grouped into a worldwide distribution cluster differing from Takamen and Nishigahara. Tokyo 1 was grouped into the same cluster as viruses isolated from United States west coast dogs in the 1930s and 1940s. Tokyo 2 was grouped into the same cluster as the Komatsugawa strain, also known as a cluster of viruses from the Khabarovsk raccoon dog, and the Lake Baikal stepped fox in Russia. These findings suggest that 1950s Tokyo rabies viruses were related to those in Russia and the USA. PMID:21706842

  17. Dengue Virus Serotype 2 from a Sylvatic Lineage Isolated from a Patient with Dengue Hemorrhagic Fever

    PubMed Central

    Cardosa, Jane; Ooi, Mong How; Tio, Phaik Hooi; Perera, David; Holmes, Edward C.; Bibi, Khatijar; Abdul Manap, Zahara

    2009-01-01

    Dengue viruses circulate in both human and sylvatic cycles. Although dengue viruses (DENV) infecting humans can cause major epidemics and severe disease, relatively little is known about the epidemiology and etiology of sylvatic dengue viruses. A 20-year-old male developed dengue hemorrhagic fever (DHF) with thrombocytopenia (12,000/ul) and a raised hematocrit (29.5% above baseline) in January 2008 in Malaysia. Dengue virus serotype 2 was isolated from his blood on day 4 of fever. A phylogenetic analysis of the complete genome sequence revealed that this virus was a member of a sylvatic lineage of DENV-2 and most closely related to a virus isolated from a sentinel monkey in Malaysia in 1970. This is the first identification of a sylvatic DENV circulating in Asia since 1975. PMID:19399166

  18. Characterization of H5N2 influenza A viruses isolated from chickens in Japan.

    PubMed

    Okamatsu, Masatoshi; Saito, Takehiko; Mase, Masaji; Tsukamoto, Kenji; Yamaguchi, Shigeo

    2007-03-01

    A low pathogenic avian influenza virus of the H5N2 subtype was isolated for the first time from layer chickens in Japan in 2005. Surveillance in trading restriction zones and epidemiologically related farms revealed 41 seropositive farms, and 16 H5N2 viruses were isolated and characterized from nine of these farms. That these viruses were genetically and antigenically similar to each other suggested that these isolates were derived from a common origin. Complete genomic characterization of all eight gene segments showed that these H5N2 isolates in Japan had high homology to the H5N2 strains prevalent in Central America since 1994. The virus was reisolated from tracheal and cloacal swabs of experimentally inoculated chickens and efficiently transmitted to sentinel chickens in adjacent cages. PMID:17494611

  19. Neurological lesions in chickens experimentally infected with virulent Newcastle disease virus isolates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Neuropil reaction was evaluated in chickens inoculated with four different Newcastle disease virus (NDV) isolates, including Texas GB, Turkey North Dakota, Nevada Cormorant (velogenic neurotropic) and Anhinga (mesogenic). Tissues for this study included archived formalin-fixed, paraffin embedded br...

  20. Complete Genome Sequence of Reticuloendotheliosis Virus Strain MD-2, Isolated from a Contaminated Turkey Herpesvirus Vaccine

    PubMed Central

    Li, Junping; Yang, Chenghuai; Li, Qihong; Li, Huijiao; Xia, Yecai; Liu, Dan

    2013-01-01

    Here, we present the complete genomic sequence of a reticuloendotheliosis virus (REV) isolated from a contaminated turkey herpesvirus (HVT) vaccine. This report will be helpful for epidemiological studies on REV infection in avian flocks. PMID:24092783

  1. Characterization of a U.S. isolate of beet black scorch virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The first reported U.S. isolate of beet black scorch necrovirus (BBSV) was obtained and characterized. Host range of the virus for localized, and occasionally systemic, infection included the Chenopodiaceae and Tetragonia expansa; Nicotianiana benthamiana supported systemic, symptomless infection ...

  2. Virus isolation, genetic characterization and seroprevalence of Toscana virus in Algeria.

    PubMed

    Alkan, C; Allal-Ikhlef, A B; Alwassouf, S; Baklouti, A; Piorkowski, G; de Lamballerie, X; Izri, A; Charrel, R N

    2015-11-01

    Toscana virus (TOSV; Bunyaviridae, Phlebovirus) is transmitted by sandflies of the genus Phlebotomus in the Mediterranean area. One strain of TOSV was isolated from a total of almost 23?000 sandflies collected in Kabylia, Algeria. The complete genome was sequenced, and phylogenetic studies indicated that it was most closely related with TOSV strain from Tunisia within lineage A, which also includes Italian, French and Turkish strains. A seroprevalence study performed on 370 sera collected from people living in the same area showed that almost 50% possessed neutralizing antibodies against TOSV, a rate much higher than that observed in Southern Europe. Sandfly species distribution in the study area suggests that the vector of TOSV in this region belongs to the subgenus Larroussius. These data support the rapid implementation of the diagnosis of TOSV in clinical microbiology laboratories to estimate the burden in patients presenting with neuroinvasive infections and febrile illness. PMID:26235198

  3. Squash vein yellowing virus, a novel ipomovirus, isolated from squash and watermelon in Florida

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A novel whitefly-transmitted member of the family Potyviridae was isolated from a squash plant (Cucurbita pepo) with vein yellowing symptoms in Florida. The virus, for which the name Squash vein yellowing virus (SqVYV) is proposed, has flexuous rod-shaped particles of ~840 nm in length. SqVYV was ...

  4. Full-Genome Sequence of a Novel Varicella-Zoster Virus Clade Isolated in Mexico

    PubMed Central

    Rodríguez-Castillo, Araceli; Ortiz-Alcántara, Joanna María; Gonzalez-Durán, Elizabeth; Segura-Candelas, José Miguel; Pérez-Agüeros, Sandra Ivette; Escobar-Escamilla, Noé; Méndez-Tenorio, Alfonso; Diaz-Quiñonez, José Alberto

    2015-01-01

    Varicella-zoster virus (VZV) is a member of the Herpesviridae family, which causes varicella (chicken pox) and herpes zoster (shingles) in humans. Here, we report the complete genome sequence of varicella-zoster virus, isolated from a vesicular fluid sample, revealing the circulation of VZV clade VIII in Mexico. PMID:26159533

  5. Complete Genome Sequence of a Hepatitis D Virus Genotype 1 Strain Isolated in Guangdong, China.

    PubMed

    Liao, Baolin; Chen, Weilie; Ping, Qu; Shi, Haiyan; He, Haolan; Liu, Huiyuan; Tan, Yizhou; Lei, Chunliang; Cai, Weiping

    2015-01-01

    Here, we report the first complete genome sequence of a hepatitis D virus genotype 1 strain, GZ37, isolated in Guangzhou, Guangdong Province, China, in 2014. The sequence information provided here will help us understand the molecular epidemiology of hepatitis D virus and contribute to disease control in mainland China. PMID:26701082

  6. Characterization of low pathogenicity avian influenza viruses isolated from wild birds in Mongolia 2005 through 2007

    Technology Transfer Automated Retrieval System (TEKTRAN)

    During 2005, 2006 and 2007 2,139 specimens representing 4,077 individual birds of 45 species were tested for avian influenza virus (AIV) as part of a wild bird AIV monitoring program conducted in Mongolia. Samples collected in 2005 were tested by virus isolation directly, samples from 2006 and 2007...

  7. Complete genome sequence of a Tomato mottle mosaic virus isolate from the United States

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tomato mottle mosaic virus (ToMMV) was first identified in the U.S. in tomatoes in Florida in 2010. This report provides the first full genome sequence of a U.S. ToMMV isolate from 2010. The full genome sequence of this emerging virus will enable research scientists to develop additional specific ...

  8. First Complete Genome Sequence of an Emerging Cucumber Green Mottle Mosaic Virus Isolate in North America

    PubMed Central

    Li, Rugang; Zheng, Yi; Fei, Zhangjun

    2015-01-01

    The complete genome sequence (6,423 nucleotides [nt]) of an emerging cucumber green mottle mosaic virus (CGMMV) isolate on cucumber in North America was determined through deep sequencing of small (sRNA) and rapid amplification of cDNA ends. The virus shares 99% nucleotide sequence identity with the Asian genotype but only 90% with the European genotype. PMID:25953166

  9. Characterization of the Taura syndrome virus isolate originating from the 2004 Texas Epizootic in cultured shrimp

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Taura syndrome virus (TSV) is a major viral pathogen of penaeid shrimp worldwide. A comprehensive investigation of the Texas isolate of TSV that caused epizootics in shrimp farms in Texas in 2004 (Us04Pv1) revealed that the virus was highly virulent in laboratory bioassays causing severe symptom dev...

  10. West Nile virus isolated from a Virginia opossum (Didelphis virginiana) in northwestern Missouri, USA, 2012.

    PubMed

    Bosco-Lauth, Angela; Harmon, Jessica R; Lash, R Ryan; Weiss, Sonja; Langevin, Stanley; Savage, Harry M; Godsey, Marvin S; Burkhalter, Kristen; Root, J Jeffrey; Gidlewski, Thomas; Nicholson, William L; Brault, Aaron C; Komar, Nicholas

    2014-10-01

    We describe the isolation of West Nile virus (WNV; Flaviviridae, Flavivirus) from blood of a Virginia opossum (Didelphis virginiana) collected in northwestern Missouri, USA in August 2012. Sequencing determined that the virus was related to lineage 1a WNV02 strains. We discuss the role of wildlife in WNV disease epidemiology. PMID:25098303

  11. Comparison of biological characteristics of H9N2 avian influenza viruses isolated from different hosts.

    PubMed

    Zhu, Yinbiao; Yang, Yang; Liu, Wei; Liu, Xin; Yang, Da; Sun, Zhihao; Ju, Yong; Chen, Sujuan; Peng, Daxin; Liu, Xiufan

    2015-04-01

    The pathogenicity and transmissibility of H9N2 influenza viruses has been widely investigated; however, few studies comparing the biological characteristics of H9N2 viruses isolated from different hosts have been performed. In this study, eight H9N2 viruses, isolated from chickens (Ck/F98, Ck/AH and Ck/TX), pigeons (Pg/XZ), quail/(Ql/A39), ducks (Dk/Y33) and swine (Sw/YZ and Sw/TZ) were selected, and their biological characteristics were determined. The results showed that all H9N2 viruses maintained a preference for both the avian- and human-type receptors, except for Sw/TZ, which had exclusive preference for the human-type receptor. The viruses replicated well in DF-1 and MDCK cells, whereas only three isolates, Ck/F98, Ck/TX and Sw/TZ, could replicate in A549 cells and also replicated in mouse lungs, resulting in body weight loss in mice. All H9N2 viruses were nonpathogenic to chickens and were detected in the trachea and lung tissues. The viruses were shed primarily by the oropharynx and were transmitted efficiently to naïve contact chickens. Our findings suggest that all H9N2 viruses from different hosts exhibit efficient replication and contact-transmission among chickens, and chickens serve as a good reservoir for the persistence and interspecies transmission of H9N2 influenza viruses. PMID:25616845

  12. Characterization of an H4N2 avian influenza virus isolated from domestic duck in Dongting Lake wetland in 2009.

    PubMed

    Zhang, Hongbo; Chen, Quanjiao; Chen, Ze

    2012-02-01

    In January 2009, an H4N2 subtype of avian influenza virus [A/duck/Hunan/8-19/2009 (H4N2)] was isolated from domestic ducks in Dongting Lake wetland. The whole genome of the virus was sequenced and the results indicated that multiple gene segments of the virus had a high homology with viruses isolated from wild waterfowl, which indicated that the virus was probably transmitted from wild waterfowl to domestic ducks. Phylogenetic analysis revealed that the each gene belonged to the Eurasian lineage of avian influenza viruses, but genetic reassortment occurs between viruses of different subtypes. PMID:21853331

  13. Genetic and antigenic relatedness of H3 subtype influenza A viruses isolated from avian and mammalian species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Turkeys are susceptible to some swine influenza viruses based on natural and experimental transmissions of H1 and H3 subtype viruses from swine to turkeys. In 2004, we isolated triple reassortant H3N2 viruses from turkey breeder hens in Ohio and Illinois. These H3N2 viruses are currently the domin...

  14. Complete Genome Sequence of a Fish Nervous Necrosis Virus Isolated from Sea Perch (Lateolabrax japonicus) in China

    PubMed Central

    Jia, Peng

    2015-01-01

    We sequenced and analyzed the complete genome of a fish nervous necrosis virus isolated from diseased sea perch (Lateolabrax japonicus) in Guangdong Province, China. The virus genome contains RNA1 (3,103 bp) and RNA2 (1,433 bp). Phylogenetic analysis shows that the virus belongs to the redspotted grouper nervous necrosis virus genotype of betanodavirus. PMID:26044411

  15. Complete Genome Sequence of a Fish Nervous Necrosis Virus Isolated from Sea Perch (Lateolabrax japonicus) in China.

    PubMed

    Jia, Peng; Jia, Kun-Tong; Yi, Mei-Sheng

    2015-01-01

    We sequenced and analyzed the complete genome of a fish nervous necrosis virus isolated from diseased sea perch (Lateolabrax japonicus) in Guangdong Province, China. The virus genome contains RNA1 (3,103 bp) and RNA2 (1,433 bp). Phylogenetic analysis shows that the virus belongs to the redspotted grouper nervous necrosis virus genotype of betanodavirus. PMID:26044411

  16. Genetic diversity of fusion gene (ORF 117), an analogue of vaccinia virus A27L gene of capripox virus isolates.

    PubMed

    Dashprakash, M; Venkatesan, Gnanavel; Ramakrishnan, Muthannan Andavar; Muthuchelvan, Dhanavelu; Sankar, Muthu; Pandey, Awadh Bihari; Mondal, Bimelendu

    2015-04-01

    The fusion gene (ORF 117) sequences of twelve (n = 12) capripox virus isolates namely sheeppox (SPPV) and goatpox (GTPV) viruses from India were demonstrated for their genetic and phylogenetic relationship among them. All the isolates were confirmed for their identity by routine PCR before targeting ORF 117 gene for sequence analysis. The designed primers specifically amplified ORF 117 gene as 447 bp fragment from total genomic DNA extracted from all the isolates. Sequence analysis revealed a significant percentage of identity among GTPV, SPPV and between them at both nucleotide and amino acid levels. The topology of the phylogenetic tree revealed that three distinct clusters corresponding to SPPV, GTPV and lumpy skin disease virus was formed. However, SPPV Pune/08 and SPPV Roumanian Fanar isolates were clustered into GTPV group as these two isolates showed a 100 and 99.3 % identity with GTPV isolates of India at nt and aa levels, respectively. Protein secondary structure and 3D view was predicted and found that it has high antigenic index and surface probability with low hydrophobicity, and it can be targeted for expression and its evaluation to explore its diagnostic potential in epidemiological investigation in future. PMID:25663144

  17. Characterisation of an isolate of Narcissus degeneration virus from Chinese narcissus (Narcissus tazetta var. chinensis).

    PubMed

    Chen, J; Shi, Y-H; Adams, M J; Zheng, H-Y; Qin, B-X; Chen, J-P

    2007-02-01

    A potyvirus from Chinese narcissus was transmitted mechanically to three species of Narcissus and to Lycoris radiata but not to 22 other test species. In western blot, the coat protein reacted strongly with Narcissus degeneration virus (UK isolate) antiserum. Antiserum raised to the Chinese virus did not react with eighteen other potyviruses. The complete nucleotide sequence (9816 nt) had the typical genome organisation for a member of the genus Potyvirus. Sequence comparisons and phylogenetic analysis showed that the Chinese virus was different from all previously sequenced potyviruses but distantly related to onion yellow dwarf and shallot yellow stripe viruses. PMID:16932980

  18. Characterization of a Tembusu virus isolated from naturally infected house sparrows (Passer domesticus) in Northern China.

    PubMed

    Tang, Y; Diao, Y; Yu, C; Gao, X; Ju, X; Xue, C; Liu, X; Ge, P; Qu, J; Zhang, D

    2013-04-01

    The house sparrow (Passer domesticus) is one of the most widely distributed wild birds in China. Tembusu virus (TMUV) strain, TMUV-SDHS, was isolated from house sparrows living around the poultry farms in Shandong Province, Northern China. Genetic analysis of E and NS5 genes showed that it had a close relationship with that of the YY5 strain, which can cause severe egg drop in ducks. Pathogenicity studies showed that the virus is highly virulent when experimentally inoculated into the ducks. These findings show that house sparrows carrying the Tembusu virus may play an important role in transmitting the virus among other species. PMID:22515847

  19. Archaeal Viruses of the Sulfolobales: Isolation, Infection, and CRISPR Spacer Acquisition.

    PubMed

    Erdmann, Susanne; Garrett, Roger A

    2015-01-01

    Infection of archaea with phylogenetically diverse single viruses, performed in different laboratories, has failed to activate spacer acquisition into host CRISPR loci. The first successful uptake of archaeal de novo spacers was observed on infection of Sulfolobus solfataricus P2 with an environmental virus mixture isolated from Yellowstone National Park (Erdmann and Garrett, Mol Microbiol 85:1044-1056, 2012). Experimental studies of isolated genetic elements from this mixture revealed that SMV1 (S ulfolobus Monocauda Virus 1), a tailed spindle-shaped virus, can induce spacer acquisition in CRISPR loci of Sulfolobus species from a second coinfecting conjugative plasmid or virus (Erdmann and Garrett, Mol Microbiol 85:1044-1056, 2012; Erdmann et al. Mol Microbiol 91:900-917, 2014). Here we describe, firstly, the isolation of archaeal virus mixtures from terrestrial hot springs and the techniques used both to infect laboratory strains with these virus mixtures and to obtain purified virus particles. Secondly, we present the experimental conditions required for activating SMV1-induced spacer acquisition in two different Sulfolobus species. PMID:25981476

  20. Nhumirim virus, a novel flavivirus isolated from mosquitoes from the Pantanal, Brazil.

    PubMed

    Pauvolid-Corrêa, Alex; Solberg, Owen; Couto-Lima, Dinair; Kenney, Joan; Serra-Freire, Nicolau; Brault, Aaron; Nogueira, Rita; Langevin, Stanley; Komar, Nicholas

    2015-01-01

    We describe the isolation of a novel flavivirus, isolated from a pool of mosquitoes identified as Culex (Culex) chidesteri collected in 2010 in the Pantanal region of west-central Brazil. The virus is herein designated Nhumirim virus (NHUV) after the name of the ranch from which the mosquito pool was collected. Flavivirus RNA was detected by real-time RT-PCR of homogenized mosquitoes and from the corresponding C6/36 culture supernatant. Based on full-genome sequencing, the virus isolate was genetically distinct from but most closely related to Barkedji virus (BJV), a newly described flavivirus from Senegal. Phylogenetic analysis demonstrated that NHUV grouped with mosquito-borne flaviviruses forming a clade with BJV. This clade may be genetically intermediate between the Culex-borne flaviviruses amplified by birds and the insect-only flaviviruses. PMID:25252815

  1. Synchrony of Sylvatic Dengue Isolations: A Multi-Host, Multi-Vector SIR Model of Dengue Virus Transmission in

    E-print Network

    Hanley, Kathryn A.

    Synchrony of Sylvatic Dengue Isolations: A Multi-Host, Multi-Vector SIR Model of Dengue Virus, United States of America Abstract Isolations of sylvatic dengue-2 virus from mosquitoes, humans and non of dengue at different periodicities when observed as isolated systems, and that coupling

  2. Complete Genome Sequence of a Tomato Isolate of Parietaria Mottle Virus from Italy

    PubMed Central

    Martínez, Carolina; Aramburu, José; Rubio, Luis

    2015-01-01

    We report here the complete genome sequence of isolate T32 of parietaria mottle virus (PMoV) infecting tomato plants in Turin, Italy, obtained by Sanger sequencing. T32 shares 90.48 to 96.69% nucleotide identity with other two PoMV isolates, CR8 and Pe1, respectively, whose complete genome sequences are available. PMID:26679580

  3. Population Structure of Blueberry Mosaic Associated Virus: Evidence of Genetic Exchange in Geographically Distinct Isolates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The population structure of blueberry mosaic associated virus (BlMaV), a putative member of the family Ophioviridae, was examined using 59 isolates collected from North America and Slovenia. The studied isolates displayed low genetic diversity in the movement and nucleoprotein regions and low ratios...

  4. Biological and molecular characterization of a reticuloendotheliosis virus isolated from turkeys with lymphomas

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two reticuloendotheliosis virus (REV) isolates termed AF-140-11 and AF-140-12 were obtained from turkeys with increased mortality, disseminated lymphoblastoid neoplasia, and decreased egg production. The REV isolates were propagated and titrated in chicken-embryo fibroblasts (CEFs) obtained from a s...

  5. Complete nucleotide sequence of a maize chlorotic mottle virus isolate from Nebraska

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The complete genome of a maize chlorotic mottle virus isolate from Nebraska (MCMV-NE) was cloned and sequenced. The MCMV-NE genome consists of 4,436 nucleotides and shares 99.5% nucleotide sequence identity with an MCMV isolate from Kansas (MCMV-KS). Of 22 polymorphic sites, most resulted from t...

  6. Respiratory transmission of an avian H3N8 influenza virus isolated from a harbour seal

    USGS Publications Warehouse

    Karlsson, Erik A.; Ip, Hon S.; Hall, Jeffrey S.; Yoon, Sun W.; Johnson, Jordan; Beck, Melinda A.; Webby, Richard J.; Schultz-Cherry, Stacey

    2014-01-01

    The ongoing human H7N9 influenza infections highlight the threat of emerging avian influenza viruses. In 2011, an avian H3N8 influenza virus isolated from moribund New England harbour seals was shown to have naturally acquired mutations known to increase the transmissibility of highly pathogenic H5N1 influenza viruses. To elucidate the potential human health threat, here we evaluate a panel of avian H3N8 viruses and find that the harbour seal virus displays increased affinity for mammalian receptors, transmits via respiratory droplets in ferrets and replicates in human lung cells. Analysis of a panel of human sera for H3N8 neutralizing antibodies suggests that there is no population-wide immunity to these viruses. The prevalence of H3N8 viruses in birds and multiple mammalian species including recent isolations from pigs and evidence that it was a past human pandemic virus make the need for surveillance and risk analysis of these viruses of public health importance.

  7. Complete Genome and Clinicopathological Characterization of a Virulent Newcastle Disease Virus Isolate from South America

    PubMed Central

    Diel, Diego G.; Susta, Leonardo; Cardenas Garcia, Stivalis; Killian, Mary L.; Brown, Corrie C.; Afonso, Claudio L.

    2012-01-01

    Newcastle disease (ND) is one of the most important diseases of poultry, negatively affecting poultry production worldwide. The disease is caused by Newcastle disease virus (NDV) or avian paramyxovirus type 1 (APMV-1), a negative-sense single-stranded RNA virus of the genus Avulavirus, family Paramyxoviridae. Although all NDV isolates characterized to date belong to a single serotype of APMV-1, significant genetic diversity has been described between different NDV isolates. Here we present the complete genome sequence and the clinicopathological characterization of a virulent Newcastle disease virus isolate (NDV-Peru/08) obtained from poultry during an outbreak of ND in Peru in 2008. Phylogenetic reconstruction and analysis of the evolutionary distances between NDV-Peru/08 and other isolates representing established NDV genotypes revealed the existence of large genomic and amino differences that clearly distinguish this isolate from viruses of typical NDV genotypes. Although NDV-Peru/08 is a genetically distinct virus, pathogenesis studies conducted with chickens revealed that NDV-Peru/08 infection results in clinical signs characteristic of velogenic viscerotropic NDV strains. Additionally, vaccination studies have shown that an inactivated NDV-LaSota/46 vaccine conferred full protection from NDV-Peru/08-induced clinical disease and mortality. This represents the first complete characterization of a virulent NDV isolate from South America. PMID:22135263

  8. Differentiation of BHV-1 isolates from vaccine virus by high-resolution melting analysis.

    PubMed

    Ostertag-Hill, Claire; Fang, Liang; Izume, Satoko; Lee, Megan; Reed, Aimee; Jin, Ling

    2015-02-16

    An efficacious bovine herpesvirus type-1 (BHV-1) vaccine has been used for many years. However, in the past few years, abortion and respiratory diseases have occurred after administration of the modified live vaccine. To investigate whether BHV-1 isolates from disease outbreaks are identical to those of the vaccines used, selected regions of the BHV-1 genome were investigated by high-resolution melting (HRM) analysis and PCR-DNA sequencing. When a target region within the thymidine kinase (TK) gene was examined by HRM analysis, 6 out of the 11 isolates from abortion cases and 22 out of the 25 isolates from bovine respiratory disease (BRD) cases had different melting curves compared to the vaccine virus. Surprisingly, when a conserved region within the US6 gene that encodes glycoprotein D (gD) was examined by HRM analysis, 5 out of the 11 abortion isolates and 18 out of the 23 BRD isolates had different melting curves from the vaccine virus. To determine whether SNPs within the coding regions of glycoprotein E (gE) and TK genes can be used to differentiate the isolates from the vaccine virus, PCR-DNA sequencing was used to examine these SNPs in all the isolates. This revealed that only 1 out of 11 of the abortion isolates and 4 out of 24 of the BRD isolates are different in the target region of gE from the vaccine virus, while 5 out of 11 abortion isolates and 4 out of 22 BRD isolates are different in the target region of TK from the vaccine virus. No DNA sequence differences were observed in glycoprotein G (gG) region between disease and vaccine isolates. Our study demonstrated that many disease isolates had genetic differences from the vaccine virus in regions examined by HRM and PCR-DNA sequencing analysis. In addition, many isolates contained more than one type of mutation and were composed of mixed variants. Our study suggests that a mixture of variants were present in isolates collected post-vaccination. HRM is a rapid diagnostic method that can be used for rapid differentiation of clinical isolates from vaccine strains. PMID:25556125

  9. Characterization of West Nile viruses isolated from captive American Flamingoes (Phoenicopterus ruber) in Medellin, Colombia.

    PubMed

    Osorio, Jorge E; Ciuoderis, Karl A; Lopera, Juan G; Piedrahita, Leidy D; Murphy, Darby; Levasseur, James; Carrillo, Lina; Ocampo, Martha C; Hofmeister, Erik

    2012-09-01

    Serum samples from a total of 71 healthy captive birds belonging to 18 species were collected in July of 2008 in Medellin (Colombia) and tested for flaviviruses. Eighteen of 29 samples from American Flamingoes (Phoenicopterus ruber) were positive for West Nile virus (WNV) by reverse transcription-polymerase chain reaction. Selected positive samples were serially passaged and WNV was confirmed by immunofluorescence. Two isolates (524/08, 9835/08) were characterized in vitro and in vivo. Sequence analysis revealed WNV with 16 nucleotide substitutions resulting in six amino acid changes when compared with the NY99 strain. Colombian (COL) viruses were more closely related to Louisiana isolates from 2001. When compared with attenuated strains isolated from Texas, COL isolates differed in their plaque size and temperature sensitivity phenotype. The COL viruses were pathogenic in embryonated chicken eggs and Balb/c mice. PMID:22802436

  10. Characterization of West Nile Viruses Isolated from Captive American Flamingoes (Phoenicopterus ruber) in Medellin, Colombia

    PubMed Central

    Osorio, Jorge E.; Ciuoderis, Karl A.; Lopera, Juan G.; Piedrahita, Leidy D.; Murphy, Darby; LeVasseur, James; Carrillo, Lina; Ocampo, Martha C.; Hofmeister, Erik

    2012-01-01

    Serum samples from a total of 71 healthy captive birds belonging to 18 species were collected in July of 2008 in Medellin (Colombia) and tested for flaviviruses. Eighteen of 29 samples from American Flamingoes (Phoenicopterus ruber) were positive for West Nile virus (WNV) by reverse transcription-polymerase chain reaction. Selected positive samples were serially passaged and WNV was confirmed by immunofluorescence. Two isolates (524/08, 9835/08) were characterized in vitro and in vivo. Sequence analysis revealed WNV with 16 nucleotide substitutions resulting in six amino acid changes when compared with the NY99 strain. Colombian (COL) viruses were more closely related to Louisiana isolates from 2001. When compared with attenuated strains isolated from Texas, COL isolates differed in their plaque size and temperature sensitivity phenotype. The COL viruses were pathogenic in embryonated chicken eggs and Balb/c mice. PMID:22802436

  11. Isolation and characterisation of an Aujeszky's disease virus naturally infecting a wild boar (Sus scrofa).

    PubMed

    Capua, I; Fico, R; Banks, M; Tamba, M; Calzetta, G

    1997-04-01

    Isolation of Aujeszky's disease virus (ADV) from an injured, female wild boar (Sus scrofa), shot dead by hunters, in an area adjacent to the Abruzzo National Park is reported. The brain was submitted for attempted virus isolation following episodes of mortality in several dogs and cats fed with meat from the wild boar. Virus was isolated on first passage from the brain of the wild boar. The restriction fragment length polymorphism profile of the isolate was assessed as a type I. The role of stress in reactivating latent ADV in wild boars, the possibility of transmitting infection to endangered species such as bears (Ursus arctos), wolves (Canis lupus), wild cats (Felis silvestris) and lynx (Lynx lynx), present in the Abruzzo National Park and the possible role of wild boars as reservoirs for ADV is discussed. PMID:9220606

  12. Isolation of the Thogoto virus from a Haemaphysalis longicornis in Kyoto City, Japan.

    PubMed

    Yoshii, Kentaro; Okamoto, Natsumi; Nakao, Ryo; Klaus Hofstetter, Robert; Yabu, Tomoko; Masumoto, Hiroki; Someya, Azusa; Kariwa, Hiroaki; Maeda, Akihiko

    2015-08-01

    Ticks transmit viruses responsible for severe emerging and re-emerging infectious diseases, some of which have a significant impact on public health. In Japan, little is known about the distribution of tick-borne viruses. In this study, we collected and tested ticks to investigate the distribution of tick-borne arboviruses in Kyoto, Japan, and isolated the first Thogoto virus (THOV) to our knowledge from Haemaphysalis longicornis in far-eastern Asia. The Japanese isolate was genetically distinct from a cluster of other isolates from Africa, Europe and the Middle East. Various cell lines derived from mammals and ticks were susceptible to the isolate, but it was not pathogenic in mice. These results advance understanding of the distribution and ecology of THOV. PMID:25957096

  13. Characterization of West Nile viruses isolated form captive American flamingoes (Phoenicopterus ruber) in Medellin, Colombia.

    USGS Publications Warehouse

    Osorio, Jorge E.; Ciuoderis, Karl A.; Lopera, Juan G.; Piedrahita, Leidy D.; Murphy, Darby; LeVasseur, James; Carrillo, Lina; Ocampo, Martha C.; Hofmeister, Erik

    2012-01-01

    Serum samples from a total of 71 healthy captive birds belonging to 18 species were collected in July of 2008 in Medellin (Colombia) and tested for flaviviruses. Eighteen of 29 samples from American Flamingoes (Phoenicopterus ruber) were positive for West Nile virus (WNV) by reverse transcription-polymerase chain reaction. Selected positive samples were serially passaged and WNV was confirmed by immunofluorescence. Two isolates (524/08, 9835/08) were characterized in vitro and in vivo. Sequence analysis revealed WNV with 16 nucleotide substitutions resulting in six amino acid changes when compared with the NY99 strain. Colombian (COL) viruses were more closely related to Louisiana isolates from 2001. When compared with attenuated strains isolated from Texas, COL isolates differed in their plaque size and temperature sensitivity phenotype. The COL viruses were pathogenic in embryonated chicken eggs and Balb/c mice.

  14. Complete Genome Sequences of Three Clinical Isolates of Dengue Virus Serotype 1 from South Korean Travelers

    PubMed Central

    Jung, Eunhye; Jeong, Young Eui; Balasuriya, Udeni B. R.

    2015-01-01

    In this study, we report the complete genome sequences of three clinical isolates of dengue virus serotype 1 isolated from South Korean travelers returning from different countries in Southeast Asia. The nucleotide sequence identities ranged from 91.5 to 92.2%, while the amino acid sequence identities ranged from 97.5 to 97.9% among the three clinical isolates. PMID:26607895

  15. Complete Genome Sequences of Three Clinical Isolates of Dengue Virus Serotype 1 from South Korean Travelers.

    PubMed

    Go, Yun Young; Jung, Eunhye; Jeong, Young Eui; Balasuriya, Udeni B R

    2015-01-01

    In this study, we report the complete genome sequences of three clinical isolates of dengue virus serotype 1 isolated from South Korean travelers returning from different countries in Southeast Asia. The nucleotide sequence identities ranged from 91.5 to 92.2%, while the amino acid sequence identities ranged from 97.5 to 97.9% among the three clinical isolates. PMID:26607895

  16. Genetic characterization of dengue virus type 1 isolated in Brunei in 2005-2006.

    PubMed

    Osman, Osmali; Fong, Mun Yik; Sekaran, Shamala Devi

    2009-03-01

    The full-length genomes of two DENV-1 viruses isolated during the 2005-2006 dengue incidents in Brunei were sequenced. Twenty five primer sets were designed to amplify contiguous overlapping fragments of approximately 500-600 base pairs spanning the entire sequence of the genome. The amplified PCR products were sent to a commercial laboratory for sequencing and the nucleotides and the deduced amino acids were determined. Sequence analysis of the envelope gene at the nucleotide and amino acid levels between the two isolates showed 92 and 96 % identity, respectively. Comparison of the envelope gene sequences with 68 other DENV-1 viruses of known genotypes placed the two isolates into two different genotypic groups. Isolate DS06/210505 belongs to genotype V together with some of the recent isolates from India (2003) and older isolates from Singapore (1990) and Burma (1976), while isolate DS212/110306 was clustered in genotype IV with the prototype Nauru strain (1974) and with some of the recent isolates from Indonesia (2004) and the Philippines (2002, 2001). In the full-length genome analysis at the nucleotide level, isolate DS06/210505 showed 94 % identity to the French Guyana strain (1989) in genotype V while isolate DS212/110306 had 96 % identity to the Nauru Island strain (1974) in genotype IV. This work constitutes the first complete genetic characterization of not only Brunei DENV-1 virus isolates, but also the first strain from Borneo Island. This study was the first to report the isolation of dengue virus in the country. PMID:19218214

  17. Superinfection exclusion is an active virus-controlled function that requires a specific viral protein.

    PubMed

    Folimonova, Svetlana Y

    2012-05-01

    Superinfection exclusion, a phenomenon in which a preexisting viral infection prevents a secondary infection with the same or a closely related virus, has been described for various viruses, including important pathogens of humans, animals, and plants. The phenomenon was initially used to test the relatedness of plant viruses. Subsequently, purposeful infection with a mild isolate has been implemented as a protective measure against virus isolates that cause severe disease. In the medical and veterinary fields, superinfection exclusion was found to interfere with repeated applications of virus-based vaccines to individuals with persistent infections and with the introduction of multicomponent vaccines. In spite of its significance, our understanding of this phenomenon is surprisingly incomplete. Recently, it was demonstrated that superinfection exclusion of Citrus tristeza virus (CTV), a positive-sense RNA closterovirus, occurs only between isolates of the same strain, but not between isolates of different strains of the virus. In this study, I show that superinfection exclusion by CTV requires production of a specific viral protein, the p33 protein. Lack of the functional p33 protein completely eliminated the ability of the virus to exclude superinfection by the same or a closely related virus. Remarkably, the protein appeared to function only in a homology-dependent manner. A cognate protein from a heterologous strain failed to confer the exclusion, suggesting the existence of precise interactions of the p33 protein with other factors involved in this complex phenomenon. PMID:22398285

  18. Genetic characterization and pathogenicity assessment of Newcastle disease virus isolated from wild peacock.

    PubMed

    Khulape, Sagar A; Gaikwad, Satish S; Chellappa, Madhan Mohan; Mishra, Bishnu Prasad; Dey, Sohini

    2014-12-01

    The continued spread and occurrence of Newcastle disease virus (NDV) has posed potential threat to domestic poultry industry around the globe. Mainly, wild avian species has always been implicated for the natural reservoir for virus and spread of the disease. In the present study, we report the isolation of Newcastle disease virus (NDV/Peacock/India/2012) in necropsy brain tissue sample of wild peacock from North India. Complete genome of the virus was found to be 15,186 nucleotides (nts) with six genes in order of 3'-N-P-M-F-HN-L-5', which was limited by 55-nts leader region at the 3' end and a 114-nts trailer sequence at 5' end. Sequence analysis of fusion protein revealed the dibasic amino acid cleavage site (112)R-R-Q-K-R-F(117), a characteristic motif of virulent virus. Phylogenetic analysis placed the isolate in genotype II of Newcastle disease virus showing the lowest mean percent divergence (6 %) with other genotype II counterparts. The isolate was characterized as mesogenic (intermediate pathotype) based on the mean death time (63 h) in embryonated chicken eggs and the intra-cerebral pathogenicity index (1.40) in day-old chicks. The report emphasizes the dynamic ecology of NDV strains circulating in a wild avian host during the outbreak of 2012 in North India. Further the genotypic and pathotypical characterizations of the isolate could help in development of homologous vaccine against NDV strain circulating in avian population. PMID:25260553

  19. Analysis of new aphid lethal paralysis virus (ALPV) isolates suggests evolution of two ALPV species.

    PubMed

    Liu, Sijun; Vijayendran, Diveena; Carrillo-Tripp, Jimena; Miller, W Allen; Bonning, Bryony C

    2014-12-01

    Aphid lethal paralysis virus (ALPV; family Dicistroviridae) was first isolated from the bird cherry-oat aphid, Rhopalosiphum padi. ALPV-like virus sequences have been reported from many insects and insect predators. We identified a new isolate of ALPV (ALPV-AP) from the pea aphid, Acyrthosiphon pisum, and a new isolate (ALPV-DvV) from western corn rootworm, Diabrotica virgifera virgifera. ALPV-AP has an ssRNA genome of 9940 nt. Based on phylogenetic analysis, ALPV-AP was closely related to ALPV-AM, an ALPV isolate from honeybees, Apis mellifera, in Spain and Brookings, SD, USA. The distinct evolutionary branches suggested the existence of two lineages of the ALPV virus. One consisted of ALPV-AP and ALPV-AM, whilst all other isolates of ALPV grouped into the other lineage. The similarity of ALPV-AP and ALPV-AM was up to 88?% at the RNA level, compared with 78-79?% between ALPV-AP and other ALPV isolates. The sequence identity of proteins between ALPV-AP and ALPV-AM was 98-99?% for both ORF1 and ORF2, whilst only 85-87?% for ORF1 and 91-92?% for ORF2 between ALPV-AP and other ALPV isolates. Sequencing of RACE (rapid amplification of cDNA ends) products and cDNA clones of the virus genome revealed sequence variation in the 5' UTRs and in ORF1, indicating that ALPV may be under strong selection pressure, which could have important biological implications for ALPV host range and infectivity. Our results indicated that ALPV-like viruses infect insects in the order Coleoptera, in addition to the orders Hemiptera and Hymenoptera, and we propose that ALPV isolates be classified as two separate viral species. PMID:25170050

  20. The preparation of chicken tracheal organ cultures for virus isolation, propagation, and titration.

    PubMed

    Hennion, Ruth M

    2015-01-01

    Chicken tracheal organ cultures (TOCs), comprising transverse sections of chick embryo trachea with beating cilia, have proved useful in the isolation of several respiratory viruses and as a viral assay system, using ciliostasis as the criterion for infection. A simple technique for the preparation of chicken tracheal organ cultures in glass test tubes, in which virus growth and ciliostasis can be readily observed, is described. PMID:25720470

  1. Isolation and characterization of a Korean porcine epidemic diarrhea virus strain KNU-141112.

    PubMed

    Lee, Sunhee; Kim, Youngnam; Lee, Changhee

    2015-10-01

    Severe outbreaks of porcine epidemic diarrhea virus (PEDV) have re-emerged in Korea and rapidly swept across the country, causing tremendous economic losses to producers and customers. Despite the availability of PEDV vaccines in the domestic market, the disease continues to plague the Korean pork industry, raising issues regarding their protective efficacy and new vaccine development. Therefore, PEDV isolation in cell culture is urgently needed to develop efficacious vaccines and diagnostic assays and to conduct further studies on the virus biology. In the present study, one Korean PEDV strain, KOR/KNU-141112/2014, was successfully isolated and serially propagated in Vero cells for over 30 passages. The in vitro and in vivo characteristics of the Korean PEDV isolate were investigated. Virus production in cell culture was confirmed by cytopathology, immunofluorescence, and real-time RT-PCR. The infectious virus titers of the viruses during the first 30 passages ranged from 10(5.1) to 10(8.2) TCID50 per ml. The inactivated KNU-141112 virus was found to mediate potent neutralizing antibody responses in immunized guinea pigs. Animal studies showed that KNU-141112 virus causes severe diarrhea and vomiting, fecal shedding, and acute atrophic enteritis, indicating that strain KNU-141112 is highly enteropathogenic in the natural host. In addition, the entire genomes or complete S genes of KNU-141112 viruses at selected cell culture passages were sequenced to assess the genetic stability and relatedness. Our genomic analyses indicated that the Korean isolate KNU-141112 is genetically stable during the first 30 passages in cell culture and is grouped within subgroup G2b together with the recent re-emergent Korean strains. PMID:26196056

  2. Isolation of an exotic serotype of bluetongue virus from imported cattle in quarantine.

    PubMed Central

    Groocock, C M; Campbell, C H

    1982-01-01

    In 1980, 60 zebu cattle from Brazil were admitted into quarantine in Florida for 150 days. During the 30 days between their last test in Brazil and their first test in Florida, four animals developed antibody to bluetongue virus detectable by agar gel immunodiffusion test. Within 62 days after arrival in Florida, three more seroconverted and one more was positive by the 86th day. Virus neutralizing titers of serums from the first four cattle were highest against bluetongue virus serotype 4 and 20; both of these serotypes are exotic to the United States. A bluetongue virus serotype 4 was isolated from one of these animals. The eight positive reactors were slaughtered; the other 52 cattle, which did not develop detectable antibody titers to bluetongue virus, were released into the United States. PMID:6284326

  3. Induction of brain tumors by a newly isolated JC virus (Tokyo-1 strain).

    PubMed Central

    Nagashima, K.; Yasui, K.; Kimura, J.; Washizu, M.; Yamaguchi, K.; Mori, W.

    1984-01-01

    A newly isolated virus from a patient with progressive multifocal leukoencephalopathy (PML) (Tokyo-1 strain) was found serologically identical to JC virus (Mad-1 strain) and showed high neurooncogenicity in hamsters. Twenty-one animals inoculated intracerebrally with the virus developed brain tumors during a period that averaged 5 months. The tumors were cerebellar medulloblastoma (n = 20); plexus tumor (n = 2) occurred in 1 animal as a single tumor and in another in combination with a medulloblastoma. Thalamic gliomatosis was also present in 6 animals with medulloblastoma. Five mock-infected animals did not develop tumors. Medulloblastoma cells were shown to contain papovavirus T-antigen. In 20 animals examined the medulloblastoma showed a close resemblance to the human medulloblastoma in its histologic, immunocytochemical, and ultrastructural features. Examination of the incipient tumors indicated that the hamster medulloblastoma originated in cells in the neonatal external granular layer. Following infection the cells apparently migrated into the internal granular layer, carrying integrated virus genes and expressing phenotypical transformation. These findings confirm previous reports on the oncogenicity of virus isolates from PML (ZuRhein and Varakis, 1979), but are novel in that with this new isolate tumors could be induced with comparatively low levels of virus inocula. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 PMID:6089567

  4. Genome characterisation of two Ljungan virus isolates from wild bank voles (Myodes glareolus) in Sweden.

    PubMed

    Pounder, Kieran C; Watts, Phillip C; Niklasson, Bo; Kallio, Eva R K; Marston, Denise A; Fooks, Anthony R; Begon, Michael; McElhinney, Lorraine M

    2015-12-01

    Ljungan virus (LV) (family Picornaviridae, genus Parechovirus) is a suspected zoonotic pathogen with associations to human disease in Sweden. LV is a single-stranded RNA virus with a positive sense genome. There are five published Ljungan virus strains, three isolated from Sweden and two from America, and are classified into four genotypes. A further two strains described here were isolated from wild bank voles (Myodes glareolus) caught in Västmanlands county, Sweden in 1994. These strains were sequenced using next generation pyrosequencing technology on the GS454flx platform. Genetic and phylogenetic analysis of the obtained genomes confirms isolates LV340 and LV342 as two new putative members of genotype 2 along with LV145SL, with 92% and 99% nucleotide identities respectively. Only two codon sites throughout the entire genome were identified as undergoing positive selection, both situated within the VP3 structural region, in or near to major antigenic sites. Whilst these two strains do not constitute new genotypes they provide evidence, though weakly supported, which suggests the evolution of Ljungan viruses to be relatively slow, a characteristic unlike other picornaviruses. Additional genomic sequences are urgently required for Ljungan virus strains, particularly from different locations or hosts, to fully understand the evolutionary and epidemiological properties of this potentially zoonotic virus. PMID:26375731

  5. Sequence analysis and genetic diversity of five new Indian isolates of cucumber mosaic virus.

    PubMed

    Kumar, S; Gautam, K K; Raj, S K

    2015-01-01

    Cucumber mosaic virus (CMV) is an important virus since it causes severe losses to many economically important crops worldwide. Five new isolates of CMV were isolated from naturally infected Hippeastrum hybridum, Dahlia pinnata, Hemerocallis fulva, Acorus calamus and Typhonium trilobatum plants, all exhibiting severe leaf mosaic symptoms. For molecular identification and sequence analyses, the complete coat protein (CP) gene of these isolates was amplified by RT-PCR. The resulting amplicons were cloned and sequenced and isolates were designated as HH (KP698590), DP (JF682239), HF (KP698589), AC (KP698588) and TT (JX570732). For study of genetic diversity among these isolates, the sequence data were analysed by BLASTn, multiple alignment and generating phylogenetic trees along with the respective sequences of other CMV isolates available in GenBank Database were done. The isolates under study showed 82-99% sequence diversity among them at nucleotide and amino acid levels; however they showed close relationships with CMV isolates of subgroup IB. In alignment analysis of amino acid sequences of HH and AC isolates, we have found fifteen and twelve unique substitutions, compared to HF, DP and TT isolates, suggesting the cause of high genetic diversity. PMID:26666188

  6. Genomic and phylogenetic characterization of Leanyer virus, a novel orthobunyavirus isolated in northern Australia

    PubMed Central

    Savji, Nazir; Travassos da Rosa, Amelia; Hutchison, Stephen; Celone, Christopher; Hui, Jeffrey; Briese, Thomas; Calisher, Charles H.; Lipkin, W. Ian

    2011-01-01

    Leanyer virus (LEAV), currently classified as a member of the genus Orthobunyavirus, in the family Bunyaviridae, was originally isolated from a pool of Anopheles meraukensis mosquitoes, collected at Leanyer, Northern Territory, Australia in 1974. When it failed to react in serological tests with antisera from other known viruses, full-length genomic sequencing was pursued to determine the relationship of LEAV to other orthobunyavirus species. Genetic and serological characterization confirmed its antigenic distance from other orthobunyaviruses, including to its closest genetic neighbours, the Simbu group viruses, suggesting that it may represent a new antigenic complex. PMID:21402599

  7. Molecular characterization and phylogenetic analysis of human parainfluenza virus type 3 isolated from Saudi Arabia.

    PubMed

    Almajhdi, Fahad N; Alshaman, Mohamed S; Amer, Haitham M

    2012-08-01

    Human parainfluenza virus 3 (HPIV-3) is a leading cause of respiratory disease in children worldwide. Previous sequence analyses of the entire virus genome, among different HPIV-3 strains, demonstrated that HN is the most variable gene. There is a dearth of data on HPIV-3 strains circulating in Saudi Arabia. In this report, HPIV-3 was screened in nasopharyngeal aspirates collected from hospitalized children with acute respiratory disease during two successive seasons (2007/08 and 2008/09) using nested RT-PCR. Out of 73 samples collected during 2007/08, seven (9.59%) were positive; while 3 out of 107 samples collected during 2008/09 (2.8%) were positive. Virus isolation in cell culture was successful using HEp2, but not Vero cells. The identity of the isolated viruses was confirmed using immunofluorescence and neutralization assays. To elucidate the genetic characteristics and phylogeny of Saudi HPIV-3 strains, the complete HN gene sequence of two selected Saudi strains was analyzed in comparison to 20 strains isolated by others from different countries worldwide. Both strains showed the highest degree of sequence homology with Indian strains, followed by Chinese and most Japanese strains. Phylogenetic analysis confirmed that these strains fell into a distinct Asian lineage. This study is the first in Saudi Arabia to recover HPIV-3 isolates of confirmed identity, and to generate sequence data that may help in understanding virus diversity and evolution. PMID:22711360

  8. Rapid identification of dengue virus isolates by using monoclonal antibodies in an indirect immunofluorescence assay.

    PubMed

    Henchal, E A; McCown, J M; Seguin, M C; Gentry, M K; Brandt, W E

    1983-01-01

    Type-specific monoclonal antibodies prepared against the four dengue (DEN) virus serotypes were evaluated for their ability to identify low-passage human and mosquito isolates from Jamaica and West Africa by an indirect immunofluorescence assay. Serotyped human isolates from Jamaican dengue fever patients included 12 DEN-1, two DEN-2, and five DEN-4 viruses. Viruses from West Africa included 84 DEN-2 mosquito strains as well as two DEN-1 and one DEN-2 from humans. Results obtained using the immunofluorescence assay were consistent with virus identifications obtained using the more classical but costly and time-consuming plaque-reduction neutralization test. More viral isolates and higher virus yields were obtained using the C6/36 clone of Aedes albopictus cells rather than LLC-MK2 (monkey kidney) cells. Dengue type-specific monoclonal antibodies detected prototype viral antigens 24-48 hours postinfection in C6/36 cells. This is the first time that monoclonal antibodies have been used to serotype low-passage flavivirus isolates. PMID:6401944

  9. Isolation and Characterization of Influenza C Viruses in the Philippines and Japan

    PubMed Central

    Odagiri, Takashi; Matsuzaki, Yoko; Okamoto, Michiko; Suzuki, Akira; Saito, Mariko; Tamaki, Raita; Lupisan, Socorro P.; Sombrero, Lydia T.; Hongo, Seiji

    2014-01-01

    From November 2009 to December 2013 in the Philippines, 15 influenza C viruses were isolated, using MDCK cells, from specimens obtained from children with severe pneumonia and influenza-like illness (ILI). This is the first report of influenza C virus isolation in the Philippines. In addition, from January 2008 to December 2013, 7 influenza C viruses were isolated from specimens that were obtained from children with acute respiratory illness (ARI) in Sendai city, Japan. Antigenic analysis with monoclonal antibodies to the hemagglutinin-esterase (HE) glycoprotein showed that 19 strains (12 from the Philippines and 7 from Japan) were similar to the influenza C virus reference strain C/Sao Paulo/378/82 (SP82). Phylogenetic analysis of the HE gene showed that the strains from the Philippines and Japan formed distinct clusters within an SP82-related lineage. The clusters that included the Philippine and Japanese strains were shown to have diverged from a common ancestor around 1993. In addition, phylogenetic analysis of the internal genes showed that all strains isolated in the Philippines and Japan had emerged through reassortment events. The composition of the internal genes of the Philippine strains was different from that of the Japanese strains, although all strains were classified into an SP82-related lineage by HE gene sequence analysis. These observations suggest that the influenza C viruses analyzed here had emerged through different reassortment events; however, the time and place at which the reassortment events occurred were not determined. PMID:25552361

  10. Molecular Characteristics of H6N6 Influenza Virus Isolated from Pigeons in Guangxi, Southern China.

    PubMed

    Li, Meng; Xie, Zhixun; Xie, Zhiqin; Luo, Sisi; Xie, Liji; Huang, Li; Deng, Xianwen; Huang, Jiaoling; Zhang, Yanfang; Zeng, Tingting; Wang, Sheng

    2015-01-01

    Here, we report the complete genome sequence of an H6N6 avian influenza virus (AIV) isolated from a pigeon in Guangxi, southern China, in 2014. The eight RNA segment genes shared a high nucleotide identity (97 to 99%) with H6N6 subtypes of AIV isolated from ducks in the regions around Guangxi Province. The finding of this study will help us understand the ecology and molecular characteristics of H6 avian influenza virus in wild birds in southern China. PMID:26634763

  11. A respiratory syncytial virus isolate enables the testing of virucidal products.

    PubMed

    Thevenin, Thomas; Lobert, P E; Dewilde, A; Hober, D

    2012-04-01

    The respiratory syncytial virus (RSV) is known as a major cause of respiratory infections and nosocomial diseases. Testing this virus is rather difficult due to the problems encountered in producing it at a high titer without using any purification method. A RSV isolate which replicates to high level on a Hep-2 cell line with an infectious titer of at least 10(7)TCID(50)mL(-1) in culture supernatant fluids has been identified. Thanks to this isolate, the virucidal effects of two products, a hand rub solution and a surface disinfectant, were conveniently tested according to the EN 14476:2007-02 procedure. PMID:22079427

  12. Characterisation of the welsh onion isolate of Shallot yellow stripe virus from China.

    PubMed

    Chen, J; Wei, C-B; Zheng, H-Y; Shi, Y-H; Adams, M J; Lin, L; Zhang, Q-Y; Wang, S-J; Chen, J-P

    2005-10-01

    The host range and nucleotide sequence of shallot yellow stripe virus (SYSV) from welsh onion in Shandong province, China is described. Of the plants tested, only shallot and welsh onion became infected but most shallot plants were symptomless. The complete sequence of one isolate (10429 nt) and the 3'-terminal 3540 nts of a second isolate were determined. They had c. 90% nt identity to one another and to published (partial) sequences of SYSV. SYSV was most closely related to onion yellow dwarf virus (OYDV) and resembled it in having a much larger P3 protein than other species in the genus. PMID:15968472

  13. Molecular Characteristics of H6N6 Influenza Virus Isolated from Pigeons in Guangxi, Southern China

    PubMed Central

    Li, Meng; Xie, Zhiqin; Luo, Sisi; Xie, Liji; Huang, Li; Deng, Xianwen; Huang, Jiaoling; Zhang, Yanfang; Zeng, Tingting; Wang, Sheng

    2015-01-01

    Here, we report the complete genome sequence of an H6N6 avian influenza virus (AIV) isolated from a pigeon in Guangxi, southern China, in 2014. The eight RNA segment genes shared a high nucleotide identity (97 to 99%) with H6N6 subtypes of AIV isolated from ducks in the regions around Guangxi Province. The finding of this study will help us understand the ecology and molecular characteristics of H6 avian influenza virus in wild birds in southern China. PMID:26634763

  14. Receptor specificity of subtype H1 influenza A viruses isolated from swine and humans in the United States

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The evolution of receptor specificity of classical swine influenza viruses leading to the 2009 H1N1 pandemic virus was analyzed in glycan microarrays. Classical influenza viruses from the alpha, beta, and gamma antigenic clusters isolated between 1945 and 2009 revealed a binding profile very simila...

  15. Infectious hematopoietic necrosis virus: Monophyletic origin of European isolates from North American Genogroup M

    USGS Publications Warehouse

    Enzmann, P.-J.; Kurath, G.; Fichtner, D.; Bergmann, S.M.

    2005-01-01

    Infectious hematopoietic necrosis virus (IHNV) was first detected in Europe in 1987 in France and Italy, and later, in 1992, in Germany. The source of the virus and the route of introduction are unknown. The present study investigates the molecular epidemiology of IHNV outbreaks in Germany since its first introduction. The complete nucleotide sequences of the glycoprotein (G) and non-virion (NV) genes from 9 IHNV isolates from Germany have been determined, and this has allowed the identification of characteristic differences between these isolates. Phylogenetic analysis of partial G gene sequences (mid-G, 303 nucleotides) from North American IHNV isolates (Kurath et al. 2003) has revealed 3 major genogroups, designated U, M and L. Using this gene region with 2 different North American IHNV data sets, it was possible to group the European IHNV strains within the M genogroup, but not in any previously defined subgroup. Analysis of the full length G gene sequences indicated that an independent evolution of IHN viruses had occurred in Europe. IHN viruses in Europe seem to be of a monophyletic origin, again most closely related to North American isolates in the M genogroup. Analysis of the NV gene sequences also showed the European isolates to be monophyletic, but resolution of the 3 genogroups was poor with this gene region. As a result of comparative sequence analyses, several different genotypes have been identified circulating in Europe. ?? Inter-Research 2005.

  16. Complete Genome Sequence of Chikungunya Virus Isolated from an Aedes aegypti Mosquito during an Outbreak in Yemen, 2011.

    PubMed

    Fahmy, Nermeen T; Klena, John D; Mohamed, Amr S; Zayed, Alia; Villinski, Jeffrey T

    2015-01-01

    Chikungunya virus is recognized as a serious public health problem. The complete genome was sequenced for a chikungunya virus isolated from the mosquito Aedes aegypti during a 2011 outbreak in Al Hodayda, Yemen, which resulted in significant human fatalities. Phylogenetic analysis demonstrated that this Yemeni isolate is most closely related to Indian Ocean strains of the east/central/south African genotype. PMID:26184944

  17. Isolation and characterization of bovine parainfluenza virus type 3 from water buffaloes (Bubalus bubalis) in Argentina

    PubMed Central

    2012-01-01

    Background Parainfluenza virus type 3 (PIV3) was isolated from dairy buffaloes (Bubalus bubalis) naturally affected with respiratory and reproductive clinical conditions. Results Examination of nasal and vaginal swabs collected from 12 diseased buffaloes led to the isolation of three paramyxovirus isolates from two animals. Antigenic, morphological and biological characteristics of these three isolates were essentially similar to those of members of the Paramyxoviridae family. Antigenic analysis by direct immunofluorescence and cross neutralization test placed these isolates together with bovine parainfluenza virus type 3 (BPIV3). Nucleotide and amino acid phylogenetic analysis of partial matrix gene sequences of the buffalo isolates and six field BPIV3 isolates from bovines in Argentina were studied. Buffalo isolates were similar to genotype B (BPIV3b) while the six BPIV3 isolates were similar to genotypes A (BPIV3a) and C (BPIV3c). Conclusions This is the first characterization of BPIV3 in water buffalo. According to the samples analyzed, in Argentina, the genotype B was found in buffalo and the genotypes A and C were found in cattle. PMID:22716217

  18. 'Kwanzan Stunting' syndrome: detection and molecular characterization of an Italian isolate of Little cherry virus 1.

    PubMed

    Matic, Slavica; Minafra, Angelantonio; Sánchez-Navarro, Jesús A; Pallás, Vicente; Myrta, Arben; Martelli, Giovanni P

    2009-07-01

    Evident stunting was observed for the first time on Prunus serrulata 'Kwanzan' indicator trees in Southern Italy during the indexing of two sour cherry accessions from cultivars 'Marasca di Verona' and 'Spanska'. Bud break and shooting were delayed and the developing leaves remained small. During the third year many Kwanzan plants died, regardless of the indexed cultivar. Electrophoretic analysis showed the presence of dsRNA pattern in extracts of stunted Kwanzan with a similar size to that of viruses of the family Closteroviridae. An identical pattern of more abundant dsRNA bands was obtained from GF305 seedlings grafted with the same sour cherry accessions. Observations by electron microscopy revealed the presence of long flexuous virus particles in both indicators (Kwanzan and GF305), characteristic of closteroviruses. Subsequent cloning work, starting from the dsRNA extracts of cultivar Marasca di Verona grafted on GF305 indicator, yielded 7 different clones, all showing high identity to the Little cherry virus 1 genome. Full sequencing of this virus isolate (ITMAR) was then done resulting in a complete genome composed of 16,936nt. Primers designed on the obtained sequences for RT-PCR detection confirmed the presence of Little cherry virus 1 in Kwanzan and GF305 trees, inoculated with both sour cherry cultivars. Phylogenetic analysis of the minor coat protein grouped virus isolates into two clusters: one including Italian isolates of sweet cherry, Japanese plum, peach and almond, together with German sweet cherry UW1 isolate, and a second one containing the Italian isolates of sour cherry (ITMAR and ITSPA), that were found associated with strong symptoms of 'Kwanzan Stunting'. PMID:19463722

  19. Pathogenesis and Transmission of Feral Swine Pseudorabies Virus Isolates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Introduction. Aujesky’s Disease or pseudorabies, is one of the oldest recognized swine diseases. It is caused by pseudorabies virus (PRV), an alpha-herpesvirus that can induce respiratory disease, reproductive failure, and affect the central nervous system. PRV vaccines, in conjunction with serologi...

  20. Specific Insect-Virus Interactions Are Responsible for Variation in Competency of Different Thrips tabaci Isolines to Transmit Different Tomato Spotted Wilt Virus Isolates

    PubMed Central

    Jacobson, Alana L.; Kennedy, George G.

    2013-01-01

    Local adaptation between sympatric host and parasite populations driven by vector genetics appears to be a factor that influences dynamics of disease epidemics and evolution of insect-vectored viruses. Although T. tabaci is the primary vector of Tomato spotted wilt virus (TSWV) in some areas of the world, it is not an important vector of this economically important plant virus in many areas where it occurs. Previous studies suggest that genetic variation of thrips populations, virus isolates, or both are important factors underlying the localized importance of this species as a vector of TSWV. This study was undertaken to quantify variation in transmissibility of TSWV isolates by T. tabaci, in the ability of T. tabaci to transmit isolates of TSWV, and to examine the possibility that genetic interactions and local adaptation contribute to the localized nature of this species as a vector of TSWV. Isofemale lines of Thrips tabaci from multiple locations were tested for their ability to transmit multiple TSWV isolates collected at the same and different locations as the thrips. Results revealed that the probability of an isofemale line transmitting TSWV varied among virus isolates, and the probability of an isolate being transmitted varied among isofemale lines. These results indicate that the interaction of T. tabaci and TSWV isolate genetic determinants underlie successful transmission of TSWV by T. tabaci. Further analysis revealed sympatric vector-virus pairing resulted in higher transmission than allopatric pairing, which suggests that local adaptation is occurring between T. tabaci and TSWV isolates. PMID:23358707

  1. Isolation and Genetic Characterization of Mangshi Virus: A Newly Discovered Seadornavirus of the Reoviridae Family Found in Yunnan Province, China

    PubMed Central

    Wang, Jinglin; Li, Huachun; He, Yuwen; Zhou, Yang; Meng, Jingxing; Zhu, Wuyang; Chen, Hongyu; Liao, Defang; Man, Yunping

    2015-01-01

    Background Seadornavirus is a genus of viruses in the family Reoviridae, which consists of Banna virus, Kadipiro virus, and Liao ning virus. Banna virus is considered a potential pathogen for zoonotic diseases. Here, we describe a newly discovered Seadornavirus isolated from mosquitos (Culex tritaeniorhynchus) in Yunnan Province, China, which is related to Banna virus, and referred to as Mangshi virus. Methods and Results The Mangshi virus was isolated by cell culture in Aedes albopictus C6/36 cells, in which it replicated and caused cytopathic effects, but not in mammalian BHK-21 or Vero cells. Polyacrylamide gel analysis revealed a genome consisting of 12 segments of double-stranded RNA, with a “6–4–2” pattern in which the migrating bands were different from those of the Banna virus. Complete genome sequencing was performed by full-length amplification of cDNAs. Sequence analysis showed that seven highly conserved nucleotides and three highly conserved nucleotides were present at the ends of the 5?- and 3?-UTRs in each of 12 genome segments. The amino acid identities of Mangshi virus shared with Balaton virus varied from 27.3% (VP11) to 72.3% (VP1) with Banna virus varying from 18.0% (VP11) to 63.9% (VP1). Phylogenetic analysis based on amino acid sequences demonstrated that Mangshi virus is a member of the genus Seadornavirus and is most closely related to, but distinct from, Balaton virus and Banna virus in the genus Seadornavirus of the family Reoviridae. Conclusion Mangshi virus isolated from mosquitoes (C. tritaeniorhynchus) was identified as a newly discovered virus in the genus Seadornavirus and is phylogenetically close to Banna virus, suggesting that there is genetic diversity of seadornaviruses in tropical and subtropical areas of Southeast Asia. PMID:26630378

  2. Complete genome sequence of nine isolates of canna yellow streak virus reveals its relationship to the sugarcane mosaic virus (SCMV) subgroup of potyviruses.

    PubMed

    Chauhan, Ravendra P; Rajakaruna, Punsasi; Verchot, Jeanmarie

    2015-03-01

    Complete genome sequences were obtained from nine isolates of canna yellow streak virus (CaYSV). CaYSV belongs to the sugarcane mosaic virus (SCMV) subgroup of potyviruses with johnsongrass mosaic virus (JGMV) as its closest relative. Multiple sequence alignments showed a pattern of amino acid substitutions in the CP sequences, which enabled us to relate these isolates to South East Asian or European isolates. Biological characterization of CaYSV identified Nicotiana benthamiana, Chenopodium quinoa and Phaseolus vulgaris as experimental hosts. Given the popularity and global trade of cannas, a clear picture of the genetic diversity of CaYSV is critical to disease management. PMID:25567205

  3. Syrian hamsters (Mesocricetus auratus) oronasally inoculated with a Nipah virus isolate from Bangladesh or Malaysia develop similar respiratory tract lesions.

    PubMed

    Baseler, L; de Wit, E; Scott, D P; Munster, V J; Feldmann, H

    2015-01-01

    Nipah virus is a paramyxovirus in the genus Henipavirus, which has caused outbreaks in humans in Malaysia, India, Singapore, and Bangladesh. Whereas the human cases in Malaysia were characterized mainly by neurological symptoms and a case fatality rate of ?40%, cases in Bangladesh also exhibited respiratory disease and had a case fatality rate of ?70%. Here, we compared the histopathologic changes in the respiratory tract of Syrian hamsters, a well-established small animal disease model for Nipah virus, inoculated oronasally with Nipah virus isolates from human cases in Malaysia and Bangladesh. The Nipah virus isolate from Bangladesh caused slightly more severe rhinitis and bronchointerstitial pneumonia 2 days after inoculation in Syrian hamsters. By day 4, differences in lesion severity could no longer be detected. Immunohistochemistry demonstrated Nipah virus antigen in the nasal cavity and pulmonary lesions; the amount of Nipah virus antigen present correlated with lesion severity. Immunohistochemistry indicated that both Nipah virus isolates exhibited endotheliotropism in small- and medium-caliber arteries and arterioles, but not in veins, in the lung. This correlated with the location of ephrin B2, the main receptor for Nipah virus, in the vasculature. In conclusion, Nipah virus isolates from outbreaks in Malaysia and Bangladesh caused a similar type and severity of respiratory tract lesions in Syrian hamsters, suggesting that the differences in human disease reported in the outbreaks in Malaysia and Bangladesh are unlikely to have been caused by intrinsic differences in these 2 virus isolates. PMID:25352203

  4. Isolation of mixed subtypes of influenza A virus from a bald eagle (Haliaeetus leucocephalus)

    PubMed Central

    2010-01-01

    From April 2007 to March 2008, cloacal swabs were obtained from 246 casualty raptors recovered by various wildlife rehabilitation centers in the United States. The swabs were placed in a virus transport medium and transported to the laboratory on ice packs. At the laboratory, the samples were pooled with each pool consisting of five samples. All pools (n = 50) were screened for the presence of avian influenza virus (AIV) using a real time reverse transcription-polymerase chain reaction (rRT-PCR); one of the pools was found positive. All five samples in this pool were tested individually by rRT-PCR; one sample from a bald eagle was found positive. This sample was inoculated in embryonated chicken eggs for virus isolation and a hemagglutinating virus was isolated. Complete genome sequencing of the isolate revealed a mixed infection with H1N1 and H2N1 subtypes. Further analysis revealed that the PB1-F2 gene sequence of H1N1 virus had the N66S virulence-associated substitution. Further studies on ecology and epidemiology of AIV in raptors are needed to help understand their role in the maintenance and evolution of AIV. PMID:20667110

  5. Isolation of mixed subtypes of influenza A virus from a bald eagle (Haliaeetus leucocephalus).

    PubMed

    Goyal, Sagar M; Jindal, Naresh; Chander, Yogesh; Ramakrishnan, Muthanan A; Redig, Patrick T; Sreevatsan, Srinand

    2010-01-01

    From April 2007 to March 2008, cloacal swabs were obtained from 246 casualty raptors recovered by various wildlife rehabilitation centers in the United States. The swabs were placed in a virus transport medium and transported to the laboratory on ice packs. At the laboratory, the samples were pooled with each pool consisting of five samples. All pools (n = 50) were screened for the presence of avian influenza virus (AIV) using a real time reverse transcription-polymerase chain reaction (rRT-PCR); one of the pools was found positive. All five samples in this pool were tested individually by rRT-PCR; one sample from a bald eagle was found positive. This sample was inoculated in embryonated chicken eggs for virus isolation and a hemagglutinating virus was isolated. Complete genome sequencing of the isolate revealed a mixed infection with H1N1 and H2N1 subtypes. Further analysis revealed that the PB1-F2 gene sequence of H1N1 virus had the N66S virulence-associated substitution. Further studies on ecology and epidemiology of AIV in raptors are needed to help understand their role in the maintenance and evolution of AIV. PMID:20667110

  6. Avian influenza virus with Hemagglutinin-Neuraminidase combination H8N8, isolated in Russia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study reports the genome sequence of an avian influenza virus (AIV) subtype H8N8 isolated in Russia. The genome analysis shows that all genes belong to AIV Eurasian lineages. The PB2 gene was similar to a Mongolian low pathogenic (LP) AIV H7N1 and a Chinese high pathogenic (HP) AIV H5N2....

  7. Molecular detection and characterization of Chinese Yam mild mosaic virus isolates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An improved RT-PCR was developed and validated to be sensitive and reliable for the detection of Yam mild mosaic virus (YMMV). Sequences of coat protein core region of 19 Chinese isolates were obtained, and analysis indicated the presence of different genetic variants. Phylogenetic analyses showed t...

  8. Neurological lesions in chickens experimentally infected with virulent Newcastle disease virus isolates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Distribution, character, and severity of lesions were evaluated in tissues from the central nervous system of chickens inoculated with 10 different Newcastle disease virus (NDV) isolates: CA 1083, Korea 97-147, Australia (all velogenic viscerotropic); Texas GB and Turkey North Dakota (both velogenic...

  9. SIMILARITIES IN SEED AND APHID TRANSMISSION OF SOYBEAN MOSAIC VIRUS ISOLATES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The strain specificity of transmission of Soybean mosaic virus (SMV) through seed and SMV-induced seed-coat mottling were investigated in field experiments. Six soybean plant introductions (PIs) were inoculated with eight SMV isolates. Transmission of SMV through seed ranged from 0% to 42.6% in see...

  10. Complete Genome Sequence of a Bovine Viral Diarrhea Virus Strain Isolated in Southern China

    PubMed Central

    Fan, Qing; Xie, Zhiqin; Liu, Jiabo; Pang, Yaoshan; Deng, Xianwen; Xie, Liji; Luo, Sisi

    2014-01-01

    We report here the full-length RNA genomic sequence of the bovine viral diarrhea virus (BVDV) strain GX4, isolated from a cow in southern China. Studies indicate that BVDV GX4 belongs to the BVDV-1b subtype. This report will help in understanding the epidemiology and molecular characteristics of BVDV in southern China cattle. PMID:24948756

  11. Induction of type I interferons by a novel porcine reproductive and respiratory syndrome virus isolate

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Porcine reproductive and respiratory syndrome virus (PRRSV) inhibits synthesis of type I interferons (IFNs) in infected pigs and in cultured cells. Here we report that one PRRSV mutant A2MC2 induces type I IFNs in cultured cells and has no effect on IFN downstream signaling. The mutant isolate was p...

  12. Complete Genome Sequences of Two Japanese Eel Endothelial Cell-Infecting Virus Strains Isolated in Japan.

    PubMed

    Naoi, Yuki; Okazaki, Sachiko; Katayama, Yukie; Omatsu, Tsutomu; Ono, Shin-Ichi; Mizutani, Tetsuya

    2015-01-01

    Japanese eel endothelial cell-infecting virus (JEECV) causes viral endothelial cell necrosis of eel (VECNE), resulting in severe economic losses in eel aquaculture in Japan. Here, we report the complete genome sequences of two new JEECV strains isolated from farmed Japanese eels. PMID:26564031

  13. Complete genome sequences of new emerging Newcastle disease virus strains isolated from China

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Five virulent Newcastle disease virus (NDV) strains were isolated from geese in China during 2010 to 2011. The complete sequences of two NDV strains and the sequences of the envelop glyprotein genes (F and HN) of three other strains were determined. Phylogenetic analysis classified then into a new g...

  14. First complete genome sequence of an emerging cucumber green mottle mosaic virus isolate in North America

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The complete genome sequence (6,423 nt) of an emerging Cucumber green mottle mosaic virus (CGMMV) isolate on cucumber in North America was determined through deep sequencing of sRNA and rapid amplification of cDNA ends. It shares 99% nucleotide sequence identity to the Asian genotype, but only 90% t...

  15. Complete Genome Sequence of a Newcastle Disease Virus Isolated from Wild Peacock (Pavo cristatus) in India

    PubMed Central

    Khulape, Sagar A.; Gaikwad, Satish S.; Chellappa, Madhan Mohan; Mishra, Bishnu Prasad

    2014-01-01

    We report here the complete genome sequence of a Newcastle disease virus (NDV) isolated from a wild peacock. Phylogenetic analysis showed that it belongs to genotype II, class II of NDV strains. This study helps to understand the ecology of NDV strains circulating in a wild avian host of this geographical region during the outbreak of 2012 in northwest India. PMID:24903868

  16. Molecular analysis of complete genomic sequences of four isolates of Gooseberry vein banding associated virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Presence of Gooseberry vein banding associated virus (GVBaV), a badnavirus in the family Caulimoviridae, is strongly correlated with gooseberry vein banding disease in Ribes spp. In this study, full-length genomic sequences of four GVBaV isolates from different hosts and geographic regions were det...

  17. Molecular Diversity of Grapevine leafroll-associated virus-2 Isolates in Pacific Northwest Vineyards

    Technology Transfer Automated Retrieval System (TEKTRAN)

    One hundred and five isolates of Grapevine leafroll-associated virus-2 (GLRaV-2) collected from vineyard blocks located in different geographical regions of Washington and Oregon states were characterized based on coat protein (CP) and heat-shock protein 70 homologue (HSP-70h) gene sequences. The re...

  18. Ilheus virus isolation in the Pantanal, west-central Brazil.

    PubMed

    Pauvolid-Corrêa, Alex; Kenney, Joan L; Couto-Lima, Dinair; Campos, Zilca M S; Schatzmayr, Hermann G; Nogueira, Rita M R; Brault, Aaron C; Komar, Nicholas

    2013-01-01

    The wetlands of the Brazilian Pantanal host large concentrations of diverse wildlife species and hematophagous arthropods, conditions that favor the circulation of zoonotic arboviruses. A recent study from the Nhecolândia sub-region of Pantanal reported serological evidence of various flaviviruses, including West Nile virus and Ilheus virus (ILHV). According to the age of seropositive horses, at least three flaviviruses, including ILHV, circulated in the Brazilian Pantanal between 2005 and 2009. To extend this study, we collected 3,234 adult mosquitoes of 16 species during 2009 and 2010 in the same sub-region. Mosquito pool homogenates were assayed for infectious virus on C6/36 and Vero cell monolayers and also tested for flaviviral RNA by a group-specific real-time RT-PCR. One pool containing 50 non-engorged female specimens of Aedes scapularis tested positive for ILHV by culture and for ILHV RNA by real-time RT-PCR, indicating a minimum infection rate of 2.5 per 1000. Full-length genomic sequence exhibited 95% identity to the only full genome sequence available for ILHV. The present data confirm the circulation of ILHV in the Brazilian Pantanal. PMID:23875051

  19. Phylogeography of rabies virus isolated from herbivores and bats in the Espírito Santo State, Brazil.

    PubMed

    Vieira, Luiz Fernando Pereira; Pereira, Sílvia Regina Ferreira Gonçalves; Carnieli, Pedro; Tavares, Luiz Carlos Barbosa; Kotait, Ivanete

    2013-04-01

    Rabies is enzootic in the State of Espírito Santo, Brazil. Every year, cattle and horses die from rabies that is transmitted by the vampire bat Desmodus rotundus. This paper describes the spread of the rabies virus by the continuous diffusion model using relaxed random walks with BEAST software. Forty-one (41) sequences of gene G from the rabies virus that was isolated from bats and domestic herbivores from several areas of the state between 2006 and 2010 were analyzed. The phylogenetic tree showed three main clusters as well as two sub-clusters under cluster 2. A spatial analysis showed that three strains of the rabies virus spread independently. In general, central Espírito Santo, which is mountainous, was the area where separation of the virus strains occurred. This physical barrier, however, was overcome at some point in time, as samples from different lineages were found in the same microarea. PMID:23264105

  20. Comparison of biological and molecular characterization of Iranian lettuce mosaic virus isolates.

    PubMed

    Ormaz, B; Winter, S; Koohi-Habibi, M; Mosahebi, Gh; Izadpanah, K

    2006-01-01

    Lettuce mosaic virus (LMV) is one of the most damaging viruses in lettuce and endive cultivating regions. In order to review the characteristics of different LMV isolates of Iran during 2004-2005 samples were collected from lettuce fields in Esfahan, Ghom, Khorasan, Khuzestan and Tehran provinces. All of the isolates were detected by LMV polyclonal antiserum (AS-0155, DSMZ Germany) in ELISA and TIPA tests. Biological purification was done for the LMV isolates and then they were maintained and propagated on Chenopodium quinoa. A range of plant species such as C. amaranticolor, C. album, Carthamus tinctorius, Gazania sp., Gomphrena globosa, Pisum sativum, Spinacia oleracea were inoculated with these isolates using potassium phosphate buffer (0/05M). Molecular weight of coat protein was determined by Polyacrylamid gel electrophoresis (PAGE). Immunocapture reverse transcription polymerase chain reaction (IC-RT-PCR) was performed using LMV polyclonal antiserum and specific primer pairs of LMV as described by Zerbini et al. (1995). The amplified fragments were included the whole CP and 3'UTR regions and the nucleotide sequences of them determined. All isolates induced chlorotic local lesions on C. amaranticolor and chlorotic local lesions with symptoms of systemic infection (vein clearing) on C. album. Tehran isolate in addition, caused local lesions on Gomphrena globosa with red border and white centre. This isolate infected Pisum sativum without any symptoms. Back inoculation on C. quinoa and DAS-ELISA confirmed the latent infection. None of these isolates infected Carthamus tinctorius, Gazania sp. and Spinacia oleracea. The molecular weight of coat protein was determined 30.33 kDa. Western-blot proved this band as the coat protein of the virus. IC-RT-PCR amplification of LMV isolates produced the expected size IC-RT-PCR product of 1300 bps. The comparison of nucleotide sequences showed that there were 98% identities. PMID:17390892

  1. Isolation and Characterization of a Single-Stranded DNA Virus Infecting Chaetoceros lorenzianus Grunow?

    PubMed Central

    Tomaru, Yuji; Takao, Yoshitake; Suzuki, Hidekazu; Nagumo, Tamotsu; Koike, Kanae; Nagasaki, Keizo

    2011-01-01

    Diatoms are one of the most significant primary producers in the ocean, and the importance of viruses as a potential source of mortality for diatoms has recently been recognized. Thus far, eight different diatom viruses infecting the genera Rhizosolenia and Chaetoceros have been isolated and characterized to different extents. We report the isolation of a novel diatom virus (ClorDNAV), which causes the lysis of the bloom-forming species Chaetoceros lorenzianus, and show its physiological, morphological, and genomic characteristics. The free virion was estimated to be ?34 nm in diameter. The arrangement of virus particles appearing in cross-section was basically a random aggregation in the nucleus. Occasionally, distinctive formations such as a ring-like array composed of 9 or 10 spherical virions or a centipede-like array composed of rod-shaped particles were also observed. The latent period and the burst size were estimated to be <48 h and 2.2 × 104 infectious units per host cell, respectively. ClorDNAV harbors a covalently closed circular single-stranded DNA (ssDNA) genome (5,813 nucleotides [nt]) that includes a partially double-stranded DNA region (979 nt). At least three major open reading frames were identified; one showed a high similarity to putative replicase-related proteins of the other ssDNA diatom viruses, Chaetoceros salsugineum DNA virus (previously reported as CsNIV) and Chaetoceros tenuissimus DNA virus. ClorDNAV is the third member of the closed circular ssDNA diatom virus group, the genus Bacilladnavirus. PMID:21666026

  2. Population structure of blueberry mosaic associated virus: Evidence of reassortment in geographically distinct isolates.

    PubMed

    Thekke-Veetil, Thanuja; Polashock, James J; Marn, Mojca V; Plesko, Irena M; Schilder, Annemiek C; Keller, Karen E; Martin, Robert R; Tzanetakis, Ioannis E

    2015-04-01

    The population structure of blueberry mosaic associated virus (BlMaV), a putative member of the family Ophioviridae, was examined using 61 isolates collected from North America and Slovenia. The studied isolates displayed low diversity in the movement and nucleocapsid proteins and low ratios of non-synonymous to synonymous nucleotide substitutions, indicative of strong purifying selection. Phylogenetic analyses revealed grouping primarily based on geography with some isolates deviating from this rule. Phylogenetic incongruence in the two regions, coupled with detection of reassortment events, indicated the possible role of genetic exchange in the evolution of BlMaV. PMID:25733053

  3. Differentiation of Aujeszky's disease virus strains isolated in Poland using DNA biotinylated probes.

    PubMed

    Kochan, G; Lipowski, A; Fici?ska, J; Szewczyk, B

    1994-01-01

    The aim of this study was to compare 17 different Aujeszky's disease virus (ADV) isolates from clinical outbreaks of AD by using DNA biotinylated probes. All isolates were collected in Poland between 1984 and 1991. The restriction fragment pattern (RFP) analysis done by hybridization to NIA-3 DNA biotinylated probe indicated that all Polish ADV field strains can be classified as type I of Suid herpesvirus 1. Hybridization with BamHI fragment 7 and gI gene biotinylated probes revealed an unusual heterogeneity of BamHI fragment 7 in almost 50% of strains isolated in Poland. The nature of the molecular changes in this fragment will be discussed. PMID:7810432

  4. Genetic variation in potato virus M isolates infecting pepino (Solanum muricatum) in China.

    PubMed

    Ge, Beibei; He, Zhen; Zhang, Zhixiang; Wang, Hongqing; Li, Shifang

    2014-12-01

    Potato virus M (PVM, genus Carlavirus, family Betaflexviridae) is considered to be one of most economically important pathogens of pepino in China. However, the details and the mechanisms underlying PVM evolution are unknown. In this study, we determined and analyzed 40 TGB 1 gene sequences, 67 TGB 2 and TGB 3 gene sequences, and 88 CP and NABP gene sequences from viruses isolated from 19 samples of pepino (Solanum muricatum) and one sample of tomato (S. lycopersicum) collected from different areas of China. Recombination analysis identified only one clear recombinant in the TGB2-TGB3-CP region, but no recombinants were detected for each of the five individual genes. Phylogenetic analysis showed that all PVM isolates could be divided into at least two lineages in trees derived from the TGB 2, CP, and NABP gene sequences, and the lineages seemed to reflect geographical origin. The five PVM genes in this study were found to be under strong negative selection pressure. The PVM isolates examined showed frequent gene flow between the Chinese and European populations, and also within the Chinese population. Clear star phylogenies and the neutral equilibrium model test showed that pepino isolates of PVM appear to be experiencing a new expansion after a recent introduction into China, and these isolates display low levels of genetic diversity. To our knowledge, this study is the first report describing genetic structure, recombination, and gene flow in PVM populations, and it provides strong evolutionary evidence for the virus populations from different geographic regions of China. PMID:25233939

  5. Genotypic and pathotypic characterization of Newcastle disease virus isolated from racing pigeons in China.

    PubMed

    Liu, Mengda; Qu, Yajin; Wang, Fangkun; Liu, Sidang; Sun, Honglei

    2015-07-01

    A Newcastle disease virus (NDV) isolated from an outbreak in racing pigeons in China was characterized in this study. Complete gene of the NDV isolate was sequenced and phylogenetic analysis. Pathogenicity experiment was carried out in pigeons, chickens, and ducks. Phylogenetic analysis revealed that the strain clustered with the Class II viruses, has highly phylogenetically similar to NDV strains isolated from pigeons in China, but was distant from the viruses prevalence in chickens and vaccine strains used in China. The deduced amino acid sequence of the cleavage site of the fusion (F) protein confirmed that the isolate contained the virulent motif (112)RRQKRF(117) at the cleavage site, but it caused no appearance disease in chickens and ducks. However, the isolate had virulence in pigeons, resulting in severe nervous signs and highly mortality. Pigeons were considered as a potential source of NDV infection and disease for commercial poultry flocks. Therefore, new vaccines to prevent the NDV infection in the pigeon flocks should be developed as soon as possible, and strict biosecurity measures should be taken to reduce the risk of pigeon Newcastle disease outbreaks. PMID:25877412

  6. First Report of Cucumber mosaic virus Isolated from Wild Vigna angularis var. nipponensis in Korea

    PubMed Central

    Kim, Mi-Kyeong; Jeong, Rae-Dong; Kwak, Hae-Ryun; Lee, Su-Heon; Kim, Jeong-Soo; Kim, Kook-Hyung; Cha, Byeongjin; Choi, Hong-Soo

    2014-01-01

    A viral disease causing severe mosaic, necrotic, and yellow symptoms on Vigna angularis var. nipponensis was prevalent around Suwon area in Korea. The causal virus was characterized as Cucumber mosaic virus (CMV) on the basis of biological and nucleotide sequence properties of RNAs 1, 2 and 3 and named as CMV-wVa. CMV-wVa isolate caused mosaic symptoms on indicator plants, Nicotiana tabacum cv. Xanthi-nc, Petunia hybrida, and Cucumis sativus. Strikingly, CMV-wVa induced severe mosaic and malformation on Cucurbita pepo, and Solanum lycopersicum. Moreover, it caused necrotic or mosaic symptoms on V. angularis and V. radiate of Fabaceae. Symptoms of necrotic local or pin point were observed on inoculated leaves of V. unguiculata, Vicia fava, Pisum sativum and Phaseolus vulgaris. However, CMV-wVa isolate failed to infect in Glycine max cvs. ‘Sorok’, ‘Sodam’ and ‘Somyeong’. To assess genetic variation between CMV-wVa and the other known CMV isolates, phylogenetic analysis using 16 complete nucleotide sequences of CMV RNA1, RNA2, and RNA3 including CMV-wVa was performed. CMV-wVa was more closely related to CMV isolates belonging to CMV subgroup I showing about 85.1–100% nucleotide sequences identity to those of subgroup I isolates. This is the first report of CMV as the causal virus infecting wild Vigna angularis var. nipponensis in Korea. PMID:25289004

  7. Epitope-specific antibody responses to virulent and avirulent feline infectious peritonitis virus isolates.

    PubMed Central

    Fiscus, S A; Rivoire, B L; Teramoto, Y A

    1987-01-01

    Feline infectious peritonitis virus (FIPV) has been isolated several times from infected cats. Some of these isolates vary markedly in their ability to cause disease. Specific-pathogen-free cats were inoculated with the avirulent FIPV-UCD-2 isolate or the extremely virulent FIPV-79-1146 isolate or both. After 1 month, cats which had received FIPV-79-1146 were either dead or showed clinical signs of FIP. All cats which received only FIPV-UCD-2 remained healthy up to 6 months after inoculation. Antibody-mediated immune enhancement of disease was not observed in cats which received FIPV-UCD-2 before inoculation with FIPV-79-1146. Monoclonal antibodies which recognized type-specific epitopes on each of the structural polypeptides of these two viruses were used in competitive-inhibition enzyme-linked immunosorbent assays to analyze the humoral immune responses of the cats. All cats produced antibodies to epitopes found on the homologous virus. In addition, cats inoculated with FIPV-79-1146 also produced antibodies which inhibited the binding of the anti-FIPV-UCD-2 E1 monoclonal antibody. One cat inoculated twice with FIPV-UCD-2 produced antibodies which inhibited the binding of the anti-FIPV-79-1146 N- and E1-specific monoclonal antibodies. Competitive enzyme-linked immunosorbent assays may prove useful in distinguishing cats which are infected with virulent FIPV isolates from cats infected with avirulent feline coronaviruses. PMID:2442191

  8. Molecular characterization of Dasheen mosaic virus isolates infecting edible aroids in India.

    PubMed

    Babu, B; Hegde, V

    2014-01-01

    Dasheen mosaic virus (DsMV) infecting three major edible aroids namely Amorphophallus paeoniifolius, Colocasia esculenta, and Xanthosoma sagittifolium cultivated in India was characterized. Infected plants showing typical DsMV symptoms were subjected to reverse transcription-polymerase chain reaction, and an amplification of a 963 bp fragment which encoded the coat protein (CP) gene was obtained. BLAST analysis of the cloned DNA amplicon revealed the identity of the virus to be that of DsMV. Sequence identity matrix of the nucleotide sequences among the three isolates showed that the DsMV isolate infecting A. paeoniifolius and C. esculenta shared an identity as high as 93%, while the DsMV isolate from X. sagittifolium shared an identity of only 73% and 76% with the DsMV isolates from A. paeoniifolius and C. esculenta, respectively. Comparative analysis of the coat protein of the three DsMV isolates showed the presence of DVG motif (A. paeoniifolius and C. esculenta) and DTG motif in X. sagittifolium and several varying potential threonine and asparagine rich N-glycosylation motifs. Single amino acid substitution of the several conserved motifs occurs in all the three DsMV isolates. This is the first characterization of DsMV isolates infecting A. paeoniifolius, C. esculenta, and X. sagittifolium plants in India. PMID:24717027

  9. A comparative study of rabies virus isolates from hematophagous bats in Brazil.

    PubMed

    Castilho, Juliana G; Carnieli, Pedro; Oliveira, Rafael N; Fahl, Willian O; Cavalcante, Rosangela; Santana, Antonio A; Rosa, Wellington L G A; Carrieri, Maria L; Kotait, Ivanete

    2010-10-01

    The Brazilian chiropteran fauna consists of 167 species; of which, three are hematophagous: the common vampire bat (Desmodus rotundus), the white-winged vampire bat (Diaemus youngi), and the hairy-legged vampire bat (Diphylla ecaudata). The aim of this study was to describe the isolation of Rabies virus from common and hairy-legged vampire bats and to report the first comparative antigenic and genetic studies of isolates from these bats. Antigenic and genetic typing of both isolates identified them as antigenic variant 3 (AgV3), the variant frequently isolated from common vampire bats. Phylogenetic analysis showed 99.3% identity between the isolates. This is the first time since 1934 that Rabies virus has been isolated from hairy-legged vampire bats in Brazil. Our analysis provides evidence that the existence of rabies-positive isolates from hairy-legged vampire bats may be the result of an interspecific rabies transmission event from common vampire bats and suggests that roost cohabitation may occur. PMID:20966291

  10. Virus Isolates during Acute and Chronic Human Immunodeficiency Virus Type 1 Infection Show Distinct Patterns of Sensitivity to Entry Inhibitors

    PubMed Central

    Rusert, Peter; Kuster, Herbert; Joos, Beda; Misselwitz, Benjamin; Gujer, Cornelia; Leemann, Christine; Fischer, Marek; Stiegler, Gabriela; Katinger, Hermann; Olson, William C.; Weber, Rainer; Aceto, Leonardo; Günthard, Huldrych F.; Trkola, Alexandra

    2005-01-01

    We studied the effect of entry inhibitors on 58 virus isolates derived during acute and chronic infection to validate these inhibitors in vitro and to probe whether viruses at early and chronic disease stages exhibit general differences in the interaction with entry receptors. We included members of all types of inhibitors currently identified: (i) agents that block gp120 binding to CD4 (CD4-IgG2 and monoclonal antibody [MAb] IgG1b12), (ii) compounds that block the interaction with CCR5 (the chemokine RANTES/CCL5, the small-molecule inhibitor AD101, and the anti-CCR5 antibody PRO 140), (iii) the fusion inhibitor enfuvirtide (T-20), and (iv) neutralizing antibodies directed against gp120 (MAb 2G12) and gp41 (MAbs 2F5 and 4E10). No differences between viruses from acute and chronic infections in the susceptibility to inhibitors targeting the CD4 binding site, CCR5, or fusion or to MAb 2G12 were apparent, rendering treatment with entry inhibitors feasible across disease stages. The notable exceptions were antibodies 2F5 and 4E10, which were more potent in inhibiting viruses from acute infection (P = 0.0088 and 0.0005, respectively), although epitopes of these MAbs were equally well preserved in both groups. Activities of these MAbs correlated significantly with each other, suggesting that common features of the viral envelope modulate their potencies. PMID:15956589

  11. Viral Replication, Persistence in Water and Genetic Characterization of Two Influenza A Viruses Isolated from Surface Lake Water

    PubMed Central

    Lebarbenchon, Camille; Yang, My; Keeler, Shamus P.; Ramakrishnan, Muthannan A.; Brown, Justin D.; Stallknecht, David E.; Sreevatsan, Srinand

    2011-01-01

    Water-borne transmission has been suggested as an important transmission mechanism for Influenza A (IA) viruses in wild duck populations; however, relatively few studies have attempted to detect IA viruses from aquatic habitats. Water-isolated viruses have rarely been genetically characterized and evaluation for persistence in water and infectivity in natural hosts has never been documented. In this study, we focused on two IA viruses (H3N8 and H4N6 subtypes) isolated from surface lake water in Minnesota, USA. We investigated the relative prevalence of the two virus subtypes in wild duck populations at the sampling site and their genetic relatedness to IA viruses isolated in wild waterbirds in North America. Viral persistence under different laboratory conditions (temperature and pH) and replication in experimentally infected Mallards (Anas platyrhynchos) were also characterized. Both viruses were the most prevalent subtype one year following their isolation in lake water. The viruses persisted in water for an extended time period at constant temperature (several weeks) but infectivity rapidly reduced under multiple freeze-thaw cycles. Furthermore, the two isolates efficiently replicated in Mallards. The complete genome characterization supported that these isolates originated from genetic reassortments with other IA viruses circulating in wild duck populations during the year of sampling. Based on phylogenetic analyses, we couldn't identify genetically similar viruses in duck populations in the years following their isolation from lake water. Our study supports the role for water-borne transmission for IA viruses but also highlights that additional field and experimental studies are required to support inter-annual persistence in aquatic habitats. PMID:22028909

  12. Genetic diversity of African swine fever virus isolates from soft ticks (Ornithodoros moubata) inhabiting warthog burrows in Zambia.

    PubMed

    Dixon, L K; Wilkinson, P J

    1988-12-01

    The genomes of African swine fever virus isolates collected from soft ticks (Ornithodoros moubata) inhabiting warthog burrows in four areas of Zambia were compared by restriction enzyme site mapping. Isolates from different areas showed considerable diversity. The regions of genomes that differed between isolates were distributed throughout the virus genome, although some more conserved regions were identified, such as the right-hand third of the genome. The genomes of seven isolates from neighbouring warthog burrows within Livingstone Game Park in southern Zambia were more similar to each other than those from different areas. However, a number of differences were observed even between the genomes of isolates from the same warthog burrow. The variation between these latter isolates probably resulted from point mutations located at various positions along the genome, in addition to small additions or deletions at both terminal regions. Restriction enzyme site mapping indicated that one isolate may have originated by earlier recombination between two distinguishable viruses. PMID:3199101

  13. Properties of a virus isolated from Vernonia amygdalina Del. in Lagos, Nigeria.

    PubMed

    Taiwo, M A; Dijkstra, J

    2004-01-01

    A previously uncharacterized virus tentatively named Vernonia green vein-banding virus (VGVBV) was isolated from Vernonia amygdalina Del. ("bitterleaf") from Lagos, Nigeria. The virus was mechanically transmissible but had a narrow host range restricted to Nicotiana benthamiana, Chenopodium quinoa and C. amaranticolor. It was also transmissible in a non-persistent manner by Myzus persicae. The virus was purified from N. benthamiana and about 750 nm long flexuous rod-shaped particles were observed in purified preparations as well as in leaf-dips of Vernonia sp. Inclusion bodies in the form of pinwheels and scrolls were observed in ultrathin sections of Vernonia leaves by electron microscopy. M(r), of the viral coat protein was estimated to be about 34 K. In indirect ELISA, all 20 samples from naturally infected Vernonia sp. reacted positively with a potyvirus-specific monoclonal antibody (MAb) as well as with an antiserum raised against VGVBV. Apart from the homologous antigen, the VGVBV antiserum reacted only with Plum poxvirus (PPV). The VGVBV reacted strongly with the antisera to Bean yellow mosaic virus (BYMV), Bean common mosaic virus (BCMV) and Amaranthus leaf mottle virus (AmLMV) but weakly with antisera to PPV and Cowpea aphid-borne mosaic virus (CABMV) (all members of the family Potyviridae, the genus Potyvirus) in at least one of the assays used (indirect ELISA, dot-blot immunoassay and Western blot analysis). The results of our host range, cytopathological and serological studies and the available literature indicate that a hitherto difficult to transmit VGVBV has only been reported from Nigeria. We consider VGVBV a candidate for a new potyvirus. This virus should be further investigated to collect sufficient data for a qualified proposal of VGVBV as a new potyvirus. PMID:15745049

  14. A new subgenotype 2.1d isolates of classical swine fever virus in China, 2014.

    PubMed

    Zhang, Hongliang; Leng, Chaoliang; Feng, Liping; Zhai, Hongyue; Chen, Jiazeng; Liu, Chunxiao; Bai, Yun; Ye, Chao; Peng, Jinmei; An, Tongqing; Kan, Yunchao; Cai, Xuehui; Tian, Zhijun; Tong, Guangzhi

    2015-08-01

    The lapinized attenuated vaccine against classical swine fever (CSF) has been used in China for over half a century and has generally prevented large-scale outbreaks in recent years. However, since late 2014, a large number of new cases of CSF were detected in many immunized pig farms in China. Several of these CSV viruses were isolated and characterized. Phylogenetic and genomic sequence analyses indicate that these new isolates, as well as some reference isolates, form a new subgenotype named 2.1d, and share several consistent molecular characteristics. Since these new isolates emerged in disparate geographic regions within 5 months, this suggests that these isolates may be widespread. Given that current vaccines do not appear to provide effective protection against this new subgenotype, further investigation of these strains is urgently needed. PMID:26031602

  15. Molecular characterization of the Tobacco rattle virus RNA2 genome isolated from Gladiolus.

    PubMed

    Kim, Yoon J; Lim, Mi S; Kim, Su M; Ryu, Ki H; Choi, Sun H

    2015-06-01

    Tobacco rattle virus (TRV-K) was first identified in a symptomatic Gladiolus plant cultivated in Korea. We analyzed the TRV-K genome and compared its phylogeny with other TRV isolates. After constructing of a full-length genomic RNA2 strand clone, a complete sequence was generated from several overlapping clones. The cloned genome was 3261 bases in length, identical to TRV-K, and had three open reading frames. TRV-K had the highest sequence identity with the American isolate TRV-ORY. Sequence analysis of the RNA2 genome showed that TRV-K contains an intact 2a, 2b, and 2c coding sequence and an RNA1-related 3' terminus, which is typical of TRV RNA2. Phylogenetic analysis revealed that TRV-K is in the same cluster as the American isolates and another Korean isolate, TRV-SK; however, it was in a different cluster than the European isolates. PMID:26081277

  16. Monoclonal antibodies specific to transforming polyproteins encoded by independent isolates of feline sarcoma virus.

    PubMed Central

    Veronese, F; Kelloff, G J; Reynolds, F H; Hill, R W; Stephenson, J R

    1982-01-01

    Hybridomas secreting monoclonal antibodies directed against polyprotein gene products of the Gardner, Snyder-Theilen, and McDonough strain of feline sarcoma virus have been isolated. Antibody produced by one hybridoma recognizes immunological determinants localized within a feline leukemia virus gag gene structural component (p15) common to polyproteins encoded by each feline sarcoma virus isolate while antibody produced by a second is specific for p30 determinants unique to P170gag-fms. Additional hybridomas secrete antibody directed against v-fes specific determinants common to the Gardner and Snyder-Theilen feline sarcoma virus-encoded polyproteins and to v-fms determinants unique to P170gas-fms polyprotein. GA P110gas-fes and ST P85gas-fes immunoprecipitated by antibody directed against p15 exhibit readily detectable levels of protein kinase activity but lack such activity when precipitated by antibody specific for their acquired sequence (v-fes) components. P170gas-fms immunoprecipitated by monoclonal antibody to either p15 or p30 lacks detectable levels of autophosphorylation but represents a substrate for the GA P110gag-fes and ST P85gag-fes enzymatic activities. These findings argue that the v-fes-associated protein kinase represents an intrinsic property of the v-fes gene product and recognizes tyrosine acceptor sites within polyprotein gene products of all three strains of feline sarcoma virus. Images PMID:6183442

  17. Isolation and full-genome sequence of two reticuloendotheliosis virus strains from mixed infections with Marek's disease virus in China.

    PubMed

    Bao, Ke-yan; Zhang, Yan-ping; Zheng, Hui-wen; Lv, Hong-chao; Gao, Yu-long; Wang, Jing-fei; Gao, Hong-lei; Qi, Xiao-le; Cui, Hong-yu; Wang, Yong-qiang; Ren, Xian-gang; Wang, Xiao-mei; Liu, Chang-jun

    2015-06-01

    Reticuloendotheliosis virus (REV), classified as a gammaretrovirus, has a variety of hosts, including chickens, ducks, geese, turkeys, and wild birds. REV causes a series of pathological syndromes, especially the immunosuppression of the host, which may lead to an increased susceptibility to other pathogens, thus greatly damaging the poultry industry. Mixed infections of REV and Marek's disease virus (MDV) have been reported in many countries, including China. Previous reports revealed that MDV vaccines were not efficacious, and even less-virulent MDV strains would cause some losses due to mixed infections with REV. Additionally, contaminants in the MDV vaccine might be the main source of REV. In this study, two clinical samples were collected from two flocks of chickens that were diagnosed with MDV. Subsequently, two REV isolates were obtained from the clinical samples. The isolates, named CY1111 and SY1209, were further confirmed through an indirect immunofluorescence assay and electron microscopy. Complete genome sequences of the two REV strains were determined to test the relationship between them and other REV strains. Phylogenetic trees showed that the two REV strains were closely related to most REV strains that were isolated from a variety of hosts. Therefore, REVs might spread freely among these hosts under natural conditions. Additionally, most REV strains in China were in the same clade. The present work offers some information regarding REV in China. PMID:25850423

  18. Phylogenetic analysis of Chinese sheeppox and goatpox virus isolates

    PubMed Central

    2012-01-01

    Background Sheeppox virus (SPPV) and goatpox virus (GTPV), members of the Capripoxvirus genus of the Poxviridae family are causative agents of sheep pox and goat pox respectively, which are important contagious diseases and endemic in central and northern Africa, the Middle and Far East, and the Indian sub-continent. Both sheep pox and goat pox can cause wool and hide damage, and reduce the production of mutton and milk, which may result in significant economic losses and threaten the stockbreeding. In this study, three SPPVs and two GTPVs were collected from China in 2009 and 2011. We described the sequence features and phylogenetic analysis of the P32 gene, GPCR gene and RPO30 gene of the SPPVs and GTPVs to reveal their genetic relatedness. Results Sequence and phylogenetic analysis showed that there was a close relationship among SPPV/GanS/2/2011/China, SPPV/GanS/1/2011/China and SPPV/NingX/2009/China. They were clustered on the same SPPV clade. GTPV/HuB/2009/China and GS-V1 belonged to the GTPV lineage. GS-V1 was closely related to other GTPV vaccine strains. GTPV/HuB/2009/China and GS-V1 were clustered with GTPVs from China and some southern Asian countries. Conclusion This study may expand the datum for spread trend research of Chinese SPPVs and GTPVs, meanwhile provide theoretical references to improve the preventive and control strategy. PMID:22264255

  19. Phospholipase A2 Isolated from the Venom of Crotalus durissus terrificus Inactivates Dengue virus and Other Enveloped Viruses by Disrupting the Viral Envelope

    PubMed Central

    Muller, Vanessa Danielle; Soares, Ricardo Oliveira; dos Santos-Junior, Nilton Nascimento; Trabuco, Amanda Cristina; Cintra, Adelia Cristina; Figueiredo, Luiz Tadeu; Caliri, Antonio; Sampaio, Suely Vilela; Aquino, Victor Hugo

    2014-01-01

    The Flaviviridae family includes several virus pathogens associated with human diseases worldwide. Within this family, Dengue virus is the most serious threat to public health, especially in tropical and sub-tropical regions of the world. Currently, there are no vaccines or specific antiviral drugs against Dengue virus or against most of the viruses of this family. Therefore, the development of vaccines and the discovery of therapeutic compounds against the medically most important flaviviruses remain a global public health priority. We previously showed that phospholipase A2 isolated from the venom of Crotalus durissus terrificus was able to inhibit Dengue virus and Yellow fever virus infection in Vero cells. Here, we present evidence that phospholipase A2 has a direct effect on Dengue virus particles, inducing a partial exposure of genomic RNA, which strongly suggests inhibition via the cleavage of glycerophospholipids at the virus lipid bilayer envelope. This cleavage might induce a disruption of the lipid bilayer that causes a destabilization of the E proteins on the virus surface, resulting in inactivation. We show by computational analysis that phospholipase A2 might gain access to the Dengue virus lipid bilayer through the pores found on each of the twenty 3-fold vertices of the E protein shell on the virus surface. In addition, phospholipase A2 is able to inactivate other enveloped viruses, highlighting its potential as a natural product lead for developing broad-spectrum antiviral drugs. PMID:25383618

  20. Genetic diversity of the coat protein of olive latent virus 1 isolates.

    PubMed

    Varanda, C M R; Nolasco, G; Clara, M I; Félix, M R

    2014-06-01

    The CP gene variability among 21 olive latent virus 1 (OLV-1) isolates obtained from different hosts and locations and at different times was assessed. Amplicons obtained by RT-PCR were cloned, and at least 10 sequences from each isolate were analyzed and compared. OLV-1 sequences available in GenBank were included. The encoded CPs consisted of 270 amino acids, except those of isolates G1S and C7 (269 aa) and G6 (271 aa). Comparison of CP genomic sequences of the isolates under study showed very low values of nucleotide diversity, 0.02, and maximum nucleotide distances between (0.087) or within isolates (0.001). Although very few nucleotide sequence differences were observed among the isolates, olive isolates exhibited lower diversity (0.012). In addition, at position 158 (157 in C7 and G1S and 159 in G6) of the deduced aa sequences, an alanine residue was found to be conserved among the olive isolates. In citrus and tulip isolates, a threonine residue was present at position 158, whereas a valine was present at this same position in tomato isolates. Phylogenetic analysis indicated that OLV-1 isolates clustered in five groups according to original host. However, G6, originally recovered from olive but repeatedly inoculated and maintained in N. benthamiana plants for 8 years in our laboratory, was separated from other isolates. This may be attributable to adaptation to the experimental host over time. There was no correlation of phylogenetic grouping of isolates based on geographical location or year of collection. Strong negative selection may have contributed to the low diversity among the OLV-1 CP isolates. PMID:24352437

  1. CHARACTERIZATION OF VARIOUS ISOLATES OF A NATURALLY OCCURRING RECOMBINANT AVIAN LEUKOSIS VIRUS USING BIOLOGICAL ASSAYS AND POLYMERASE CHAIN REACTION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recently, we have isolated a naturally occurring recombinant avian leukosis virus (ALV) containing the envelope of ALV-B and LTR of ALV-J from commercial layer flocks affected with myeloid leukosis. Seven new isolates of the recombinant ALV, isolated from the same flock, were characterized using bio...

  2. NS-gene based phylogenetic analysis of equine influenza viruses isolated in Poland.

    PubMed

    Kwasnik, Malgorzata; Gora, Ilona M; Rola, Jerzy; Zmudzinski, Jan F; Rozek, Wojciech

    2016-01-15

    The phylogenetic analysis of influenza virus is based mainly on the variable hemagglutinin or neuraminidase genes. However, some discrete evolutionary trends might be revealed when more conservative genes are considered. We compared all available in GenBank database full length NS sequences of equine influenza virus including Polish isolates. Four nucleotides at positions A202, A237, T672 and A714 and three amino acids at positions H59, K71 and S216 which are also present in A/eq/Pulawy/2006 and A/eq/Pulawy/2008 may be discriminating for the Florida sublineage. Threonine at position 83 seems to be characteristic for EIV strains of Florida 2 isolated after 2007. There are nine common substitutions in the NS sequences of A/eq/Pulawy/2005, A/eq/Aboyne/1/2005 and A/eq/Lincolnshire/1/2006 in relation to the reference strain A/eq/Miami/63, resulting in four amino acid changes in NS1 protein (I56, E76, K140, E179) and one in NEP (R22). We grouped these strains as "Aboyne-like". Some of the listed changes were also observed in H7N7 strains isolated between 1956 and 1966, in A/eq/Jilin/89 or in pre-divergent H3N8 strains. Two hypotheses regarding the origin of this group were postulated: three independent transfers of avian influenza viruses into the equine population or reassortation between H7N7 and H3N8 EIV. Similarities of the NS sequences of "Aboyne like" viruses to viruses isolated in the fifties or seventies can reflect a phenomenon of "frozen evolution". PMID:26711034

  3. Hepatitis delta virus: Making the point from virus isolation up to 2014

    PubMed Central

    Romeo, Raffaella; Perbellini, Riccardo

    2015-01-01

    Chronic infection with hepatitis delta virus (HDV) has lately regained clinical importance because of the recent evidence of increasing prevalence in several European countries, due to immigration from highly endemic areas. HDV requires the mandatory presence of hepatitis B virus (HBV) for propagation to hepatocytes. It is transmitted by the same routes of HBV and it can be acquired either by co-infection (simultaneous transmission of the two viruses) or super-infection (acquisition of HDV by an already chronic carrier of HBV). As a consequence, every HBV carrier is potentially at risk for HDV superinfection. Since the clinical course of super-infection can be severe, early diagnosis of HDV infection is necessary. PMID:26464754

  4. Hepatitis delta virus: Making the point from virus isolation up to 2014.

    PubMed

    Romeo, Raffaella; Perbellini, Riccardo

    2015-10-01

    Chronic infection with hepatitis delta virus (HDV) has lately regained clinical importance because of the recent evidence of increasing prevalence in several European countries, due to immigration from highly endemic areas. HDV requires the mandatory presence of hepatitis B virus (HBV) for propagation to hepatocytes. It is transmitted by the same routes of HBV and it can be acquired either by co-infection (simultaneous transmission of the two viruses) or super-infection (acquisition of HDV by an already chronic carrier of HBV). As a consequence, every HBV carrier is potentially at risk for HDV superinfection. Since the clinical course of super-infection can be severe, early diagnosis of HDV infection is necessary. PMID:26464754

  5. Enzyme-linked immunosorbent assay typing of California serogroup viruses isolated in Canada.

    PubMed Central

    Artsob, H; Spence, L P; Th'ng, C

    1984-01-01

    A procedure was developed to type California serogroup viruses by an antibody-capture, enzyme-linked immunosorbent assay. Seven California serogroup members from North America were distinguished, including snowshoe hare, La Crosse, California encephalitis, San Angelo, Jamestown Canyon, Keystone, and trivittatus. Extensive cross-reactions were observed between Jamestown Canyon and the closely related South River strain. The enzyme-linked immunosorbent assay method was successfully applied to the typing of 77 California serogroup viruses isolated in Canada, including 61 snowshoe hare, 13 Jamestown Canyon, and 3 trivittatus topotypes. PMID:6149231

  6. Evolutionary and phenotypic analysis of live virus isolates suggests arthropod origin of a pathogenic RNA virus family.

    PubMed

    Marklewitz, Marco; Zirkel, Florian; Kurth, Andreas; Drosten, Christian; Junglen, Sandra

    2015-06-16

    The evolutionary origins of arboviruses are unknown because their typical dual host tropism is paraphyletic within viral families. Here we studied one of the most diversified and medically relevant RNA virus families, the Bunyaviridae, in which four of five established genera are transmitted by arthropods. We define two cardinally novel bunyavirus groups based on live isolation of 26 viral strains from mosquitoes (Jonchet virus [JONV], eight strains; Ferak virus [FERV], 18 strains). Both viruses were incapable of replicating at vertebrate-typical temperatures but replicated efficiently in insect cells. Replication involved formation of virion-sense RNA (vRNA) and mRNA, including cap-snatching activity. SDS/PAGE, mass spectrometry, and Edman degradation identified translation products corresponding to virion-associated RNA-dependent RNA polymerase protein (RdRp), glycoprotein precursor protein, glycoproteins Gn and Gc, as well as putative nonstructural proteins NSs and NSm. Distinct virion morphologies suggested ancient evolutionary divergence, with bunyavirus-typical morphology for FERV (spheres of 60-120 nm) as opposed to an unusual bimorphology for JONV (tubular virions of 60 × 600 nm and spheres of 80 nm). Both viruses were genetically equidistant from all other bunyaviruses, showing <15% amino acid identity in the RdRp palm domain. Both had different and unique conserved genome termini, as in separate bunyavirus genera. JONV and FERV define two novel sister taxons to the superclade of orthobunyaviruses, tospoviruses, and hantaviruses. Phylogenetic ancestral state reconstruction with probabilistic hypothesis testing suggested ancestral associations with arthropods at deep nodes throughout the bunyavirus tree. Our findings suggest an arthropod origin of bunyaviruses. PMID:26038576

  7. Evolutionary and phenotypic analysis of live virus isolates suggests arthropod origin of a pathogenic RNA virus family

    PubMed Central

    Marklewitz, Marco; Zirkel, Florian; Kurth, Andreas; Drosten, Christian; Junglen, Sandra

    2015-01-01

    The evolutionary origins of arboviruses are unknown because their typical dual host tropism is paraphyletic within viral families. Here we studied one of the most diversified and medically relevant RNA virus families, the Bunyaviridae, in which four of five established genera are transmitted by arthropods. We define two cardinally novel bunyavirus groups based on live isolation of 26 viral strains from mosquitoes (Jonchet virus [JONV], eight strains; Ferak virus [FERV], 18 strains). Both viruses were incapable of replicating at vertebrate-typical temperatures but replicated efficiently in insect cells. Replication involved formation of virion-sense RNA (vRNA) and mRNA, including cap-snatching activity. SDS/PAGE, mass spectrometry, and Edman degradation identified translation products corresponding to virion-associated RNA-dependent RNA polymerase protein (RdRp), glycoprotein precursor protein, glycoproteins Gn and Gc, as well as putative nonstructural proteins NSs and NSm. Distinct virion morphologies suggested ancient evolutionary divergence, with bunyavirus-typical morphology for FERV (spheres of 60–120 nm) as opposed to an unusual bimorphology for JONV (tubular virions of 60 × 600 nm and spheres of 80 nm). Both viruses were genetically equidistant from all other bunyaviruses, showing <15% amino acid identity in the RdRp palm domain. Both had different and unique conserved genome termini, as in separate bunyavirus genera. JONV and FERV define two novel sister taxons to the superclade of orthobunyaviruses, tospoviruses, and hantaviruses. Phylogenetic ancestral state reconstruction with probabilistic hypothesis testing suggested ancestral associations with arthropods at deep nodes throughout the bunyavirus tree. Our findings suggest an arthropod origin of bunyaviruses. PMID:26038576

  8. Genetic variation in swine influenza virus A isolate associated with proliferative and necrotizing pneumonia in pigs.

    PubMed Central

    Rekik, M R; Arora, D J; Dea, S

    1994-01-01

    A new antigenic variant of H1N1 swine influenza virus A (Sw/QC/5393/91 [QC/91]) has been found to be associated with porcine proliferative and necrotizing pneumonia. Analysis of its genomic RNA by T1 oligonucleotide mapping revealed that considerable genomic divergence exists between QC/91 and the swine influenza viruses currently circulating in North American swine herds. Analysis of the nucleotide sequence of the HA1 region of the hemagglutinin RNA of QC/91, in comparison with those of most common H1N1 human and swine influenza A viruses, showed the presence of multiple point mutations. Two amino acid substitutions appeared to be located in antigenic sites Sb and Ca. This correlates with antigenic variations demonstrated between A/NJ/8/76, A/Sw/WI/49/76, and Québec isolate A/Sw/QC/5393/91 of swine influenza virus A. Another mutation was responsible for the loss of a glycosylation site, which may have also affected the antigenicity. The other mutations seem to have been accumulated progressively over time. This significant constancy in the fixation of mutations with time suggests that genetic diversity of these viruses may best be interpreted as the result of drifts in the population of circulating swine influenza viruses in Québec. Images PMID:7545918

  9. Structure of a Venezuelan equine encephalitis virus assembly intermediate isolated from infected cells

    SciTech Connect

    Lamb, Kristen; Lokesh, G.L.; Sherman, Michael; Watowich, Stanley

    2010-10-25

    Venezuelan equine encephalitis virus (VEEV) is a prototypical enveloped ssRNA virus of the family Togaviridae. To better understand alphavirus assembly, we analyzed newly formed nucleocapsid particles (termed pre-viral nucleocapsids) isolated from infected cells. These particles were intermediates along the virus assembly pathway, and ultimately bind membrane-associated viral glycoproteins to bud as mature infectious virus. Purified pre-viral nucleocapsids were spherical with a unimodal diameter distribution. The structure of one class of pre-viral nucleocapsids was determined with single particle reconstruction of cryo-electron microscopy images. These studies showed that pre-viral nucleocapsids assembled into an icosahedral structure with a capsid stoichiometry similar to the mature nucleocapsid. However, the individual capsomers were organized significantly differently within the pre-viral and mature nucleocapsids. The pre-viral nucleocapsid structure implies that nucleocapsids are highly plastic and undergo glycoprotein and/or lipid-driven rearrangements during virus self-assembly. This mechanism of self-assembly may be general for other enveloped viruses.

  10. Pinhal Virus, a New Arenavirus Isolated from Calomys tener in Brazil.

    PubMed

    Bisordi, Ivani; Levis, Silvana; Maeda, Adriana Y; Suzuki, Akemi; Nagasse-Sugahara, Teresa K; de Souza, Renato P; Pereira, Luiz E; Garcia, Jorge B; Cerroni, Matheus de P; E Silva, Franko de A; Dos Santos, Cecília L S; da Fonseca, Benedito A L

    2015-11-01

    Arenavirus Sabiá was originally isolated from a fatal human infection in Brazil, and after the occurrence of the second fatal human case in São Paulo state, epidemiologic and virologic studies were performed in the area where the patient lived, aiming at the identification of the Sabiá natural rodent reservoir. A broadly cross-reactive enzyme-linked immunosorbent assay (ELISA) was used to screen for antibody-positive samples. Antibodies to arenavirus were detected in two of the 55 samples of Calomys tener, and from these results, samples of rodents were analyzed by a broad RT-PCR assay. RT-PCR amplification detected arenavirus sequences in five of the 55 C. tener samples, and sequencing showed that this virus is a distinct form of Sabiá virus. Thus, we describe here the evidence for the circulation of a new arenavirus in Brazil (proposed name Pinhal virus) and its genetic characterization compared to other arenaviruses. This study also suggests C. tener as a probable rodent reservoir for this virus and associates this new virus with the lineage C of New World arenaviruses. Although we have defined some characteristics of this virus, so far, there is no evidence of its involvement in human disease. PMID:26501215

  11. Respiratory infectivity of a recently isolated Egyptian strain of Rift Valley fever virus.

    PubMed Central

    Brown, J L; Dominik, J W; Morrissey, R L

    1981-01-01

    The respiratory infectivity of a strain of Rift Valley fever virus isolated in Egypt (strain ZH-501) was compared with that of one isolate from Uganda (Entebbe strain) and two isolates from South Africa (strains SA-51 and SA-75). Studies were performed with ICR mice which were infected by exposure to infectious aerosols composed of particles with a mass median diameter of 0.96 micrometer. The respiratory median lethal doses for ZH-501, Entebbe, SA-51, and SA-75 were 2.2, 1.9, 2.6, and 1.9 log10 plaque-forming units, respectively. Although these values are statistically different, the biological implications of such differences seem unimportant. In an additional study of pathogenesis, a single group of mice was infected with 3.1 log10 plaque-forming units of ZH-501, and tissues were assayed sequentially through 96 h postinfection. Between 6 and 30 h, demonstration of an increasing virus concentration only in the lungs indicated that initial replication occurred there; however, determination of histopathological changes did not reveal evidence of pneumonia. Virus was isolated from the liver by 48 h, and the ultimate outcome of infection was a fulminating and fatal hepatic necrosis. PMID:7287187

  12. Genome sequence analysis of dengue virus 1 isolated in Key West, Florida.

    PubMed

    Shin, Dongyoung; Richards, Stephanie L; Alto, Barry W; Bettinardi, David J; Smartt, Chelsea T

    2013-01-01

    Dengue virus (DENV) is transmitted to humans through the bite of mosquitoes. In November 2010, a dengue outbreak was reported in Monroe County in southern Florida (FL), including greater than 20 confirmed human cases. The virus collected from the human cases was verified as DENV serotype 1 (DENV-1) and one isolate was provided for sequence analysis. RNA was extracted from the DENV-1 isolate and was used in reverse transcription polymerase chain reaction (RT-PCR) to amplify PCR fragments to sequence. Nucleic acid primers were designed to generate overlapping PCR fragments that covered the entire genome. The DENV-1 isolate found in Key West (KW), FL was sequenced for whole genome characterization. Sequence assembly, Genbank searches, and recombination analyses were performed to verify the identity of the genome sequences and to determine percent similarity to known DENV-1 sequences. We show that the KW DENV-1 strain is 99% identical to Nicaraguan and Mexican DENV-1 strains. Phylogenetic and recombination analyses suggest that the DENV-1 isolated in KW originated from Nicaragua (NI) and the KW strain may circulate in KW. Also, recombination analysis results detected recombination events in the KW strain compared to DENV-1 strains from Puerto Rico. We evaluate the relative growth of KW strain of DENV-1 compared to other dengue viruses to determine whether the underlying genetics of the strain is associated with a replicative advantage, an important consideration since local transmission of DENV may result because domestic tourism can spread DENVs. PMID:24098658

  13. Phylogenetic analysis of Indian rabies virus isolates targeting the complete glycoprotein gene.

    PubMed

    Cherian, Susan; Singh, Rajendra; Singh, K P; Manjunatha Reddy, G B; Anjaneya; Ravi Kumar, G V P P S; Sumithra, T G; Singh, R P

    2015-12-01

    Rabies a fatal viral zoonosis is endemic in India. There is no report on phylogenetic study of Indian rabies virus isolates based on the complete G gene. In the present study, a total of 25 rabies positive brain samples collected during 2001-2014 from North India (UP, MP, Delhi, Rajasthan), South India (Kerala and Karnataka) and Gujarat states belonging to six different host species were subjected to G gene amplification by RT-PCR as three overlapping fragments of 881bp, 991bp and 618bp. Phylogenetic analysis revealed that all Indian rabies virus isolates are genetically closely related with Arctic-like 1a lineage viruses. However, two distinct clusters were identified namely, India South and India North. All the Indian rabies isolates had 95.5-100% homology related to geography, but not to host species. Deduced amino acids on comparison revealed two amino acid changes, aa 356 in ECTO; N?K and aa 458; M?I, which were found to distinguish between the India South and India North isolates. PMID:26427850

  14. A survey of fish viruses isolated from wild marine fishes from the coastal waters of southern Korea.

    PubMed

    Kim, Wi-Sik; Choi, Shin-Young; Kim, Do-Hyung; Oh, Myung-Joo

    2013-11-01

    A survey was conducted to investigate viral infection in 253 wild marine fishes harvested in the southern coastal area of Korea from 2010 to 2012. The fish that were captured by local anglers were randomly bought and sampled for virus examination. The samples were tested for presence of virus by virus isolation with FHM, FSP, and BF-2 cells and molecular methods (polymerase chain reaction and sequencing). Of the 253 fish sampled, 9 fish were infected with virus. Aquabirnaviruses (ABVs), Viral hemorrhagic septicemia virus (VHSV), and Red seabream iridovirus (RSIV) were detected in 7, 1, and 1 fish, respectively. Molecular phylogenies demonstrated the detected viruses (ABV, VHSV, and RSIV) were more closely related to viruses reported of the same type from Korea and Japan than from other countries, suggesting these viruses may be indigenous to Korean and Japanese coastal waters. PMID:24081931

  15. Isolation and identification of highly pathogenic avian influenza virus subtype H5N1 in peafowl (Pavo cristatus).

    PubMed

    Ismail, Mahmoud Moussa; Khan, Owais Ahmed; Cattoli, Giovanni; Lu, Huaguang

    2010-03-01

    An outbreak of highly pathogenic avian influenza (HPAI) virus subtype H5N1 was first diagnosed in a "backyard" flock of peafowl (Pavo cristatus) raised on palace premises in the Kingdom of Saudi Arabia in December 3, 2007. The flock consisted of 40 peafowl, and their ages ranged from 3 to 5 years old. Affected birds suffered from depression, anorexia, and white diarrhea. Four dead birds were submitted for HPAI diagnosis at the Central Veterinary Diagnostic Laboratory in Riyadh. Brain and liver tissues and tracheal and cloacal swabs were taken from the dead birds and processed for a real-time reverse transcriptase (RT)-PCR test and virus isolation in specific-pathogen-free embryonating chicken eggs. The H5N1 subtype of avian influenza virus was isolated from the four dead birds and identified by a real-time RT-PCR before and after egg inoculation. The virus isolates were characterized as HPAI H5N1 virus by sequencing analysis. Phylogenetic comparisons revealed that the H5N1 viruses isolated from peafowl belong to the genetic clade 2.2 according to the World Health Organization nomenclature. The peafowl H5N1 virus falls into 2.2.2 sublineage II and clusters with the H5N1 viruses isolated from poultry in Saudi Arabia in 2007-08. PMID:20521659

  16. Genetics, Receptor Binding, and Virulence in Mice of H10N8 Influenza Viruses Isolated from Ducks and Chickens in Live Poultry Markets in China.

    PubMed

    Deng, Guohua; Shi, Jianzhong; Wang, Jing; Kong, Huihui; Cui, Pengfei; Zhang, Fang; Tan, Dan; Suzuki, Yasuo; Liu, Liling; Jiang, Yongping; Guan, Yuntao; Chen, Hualan

    2015-06-01

    We analyzed eight H10N8 viruses isolated from ducks and chickens in live poultry markets from 2009 to 2013 in China. These viruses showed distinct genetic diversity and formed five genotypes: the four duck isolates formed four different genotypes, whereas the four chicken viruses belong to a single genotype. The viruses bound to both human- and avian-type receptors, and four of the viruses caused 12.7% to 22.5% body weight loss in mice. PMID:25855738

  17. Genetics, Receptor Binding, and Virulence in Mice of H10N8 Influenza Viruses Isolated from Ducks and Chickens in Live Poultry Markets in China

    PubMed Central

    Deng, Guohua; Shi, Jianzhong; Wang, Jing; Kong, Huihui; Cui, Pengfei; Zhang, Fang; Tan, Dan; Suzuki, Yasuo; Liu, Liling; Jiang, Yongping; Guan, Yuntao

    2015-01-01

    We analyzed eight H10N8 viruses isolated from ducks and chickens in live poultry markets from 2009 to 2013 in China. These viruses showed distinct genetic diversity and formed five genotypes: the four duck isolates formed four different genotypes, whereas the four chicken viruses belong to a single genotype. The viruses bound to both human- and avian-type receptors, and four of the viruses caused 12.7% to 22.5% body weight loss in mice. PMID:25855738

  18. Genetic and serological typing of European infectious haematopoietic necrosis virus (IHNV) isolates

    USGS Publications Warehouse

    Johansson, T.; Einer-Jensen, K.; Batts, W.; Ahrens, P.; Bjorkblom, C.; Kurath, G.; Bjorklund, H.; Lorenzen, N.

    2009-01-01

    Infectious haematopoietic necrosis virus (IHNV) causes the lethal disease infectious haematopoietic necrosis (IHN) in juvenile salmon and trout. The nucleocapsid (N) protein gene and partial glycoprotein (G) gene (nucleotides 457 to 1061) of the European isolates IT-217A, FR-32/87, DE-DF 13/98 11621, DE-DF 4/99-8/99, AU-9695338 and RU-FR1 were sequenced and compared with IHNV isolates from the North American genogroups U, M and L. In phylogenetic studies the N gene of the Italian, French, German and Austrian isolates clustered in the M genogroup, though in a different subgroup than the isolates from the USA. Analyses of the partial G gene of these European isolates clustered them in the M genogroup close to the root while the Russian isolate clustered in the U genogroup. The European isolates together with US-WRAC and US-Col-80 were also tested in an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies (MAbs) against the N protein. MAbs 136-1 and 136-3 reacted equally at all concentrations with the isolates tested, indicating that these antibodies identify a common epitope. MAb 34D3 separated the M and L genogroup isolates from the U genogroup isolate. MAb 1DW14D divided the European isolates into 2 groups. MAb 1DW14D reacted more strongly with DE-DF 13/98 11621 and RU-FR1 than with IT-217A, FR- 32/87, DE-DF 4/99-8/99 and AU-9695338. In the phylogenetic studies, the Italian, French, German and Austrian isolates clustered in the M genogroup, whereas in the serological studies using MAbs, the European M genogroup isolates could not be placed in the same specific group. These results indicate that genotypic and serotypic classification do not correlate. ?? 2009 Inter-Research.

  19. VIRUSES THAT INTERACT SYNGERISTICALLY WITH SWEET POTATO CHLOROTIC STUNT VIRUS-NIGERIAN ISOLATE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Early studies of sweet potato virus disease (SPVD)in Nigeria showed that it was caused by co-infection of Ipomoea batatas with an aphid-transmitted and a whitefly-transmitted agent. The agents were later found to be Sweet potato feathery mottle (SPFMV, genus Potyvirus)and Sweet potato chlorotic stu...

  20. ISOLATION AND CHARACTERIZATION OF AN ADVENTITIOUS AVIAN LEUKOSIS VIRUS ISOLATED FROM COMMERCIAL MAREK'S DISEASE VACCINES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Commercial Marek's disease (MD) vaccines produced by two manufacturers were tested for possible contamination with avian leukosis virus (ALV). Samples of MD vaccines manufactured by two companies (A & B) were received from a breeder company; samples were also received directly from vaccine company B...

  1. Complete genome sequences of two biologically distinct isolates of Asparagus virus 1.

    PubMed

    Blockus, S; Lesker, T; Maiss, E

    2015-02-01

    The complete genome sequences of two asparagus virus 1 (AV-1) isolates differing in their ability to cause systemic infection in Nicotiana benthamiana were determined. Their genomes had 9,741 nucleotides excluding the 3'-terminal poly(A) tail, encoded a polyprotein of 3,112 amino acids, and shared 99.6 % nucleotide sequence identity. They differed at 37 nucleotide and 15 amino acid sequence positions (99.5 % identity) scattered over the polyprotein. The closest relatives of AV-1 in amino acid sequence identity were plum pox virus (54 %) and turnip mosaic virus (53 %), corroborating the classification of AV-1 as a member of a distinct species in the genus Potyvirus. PMID:25216774

  2. Gouléako Virus Isolated from West African Mosquitoes Constitutes a Proposed Novel Genus in the Family Bunyaviridae?

    PubMed Central

    Marklewitz, M.; Handrick, S.; Grasse, W.; Kurth, A.; Lukashev, A.; Drosten, C.; Ellerbrok, H.; Leendertz, F. H.; Pauli, G.; Junglen, S.

    2011-01-01

    The family Bunyaviridae is the most diversified family of RNA viruses. We describe a novel prototypic bunyavirus, tentatively named Gouléako virus, isolated from various mosquito species trapped in Côte d'Ivoire. The S segment comprised 1,087 nucleotides (nt), the M segment 3,188 nt, and the L segment 6,358 nt, constituting the shortest bunyavirus genome known so far. The virus had shorter genome termini than phleboviruses and showed no evidence of encoded NSs and NSm proteins. An uncharacterized 105-amino-acid (aa) putative open reading frame (ORF) was detected in the S segment. Genetic equidistance to other bunyaviruses (74 to 88% aa identity) and absence of serological cross-reactivity with phleboviruses suggested a proposed novel Bunyaviridae genus. PMID:21715500

  3. First isolation of La Crosse virus from naturally infected Aedes albopictus.

    PubMed Central

    Gerhardt, R. R.; Gottfried, K. L.; Apperson, C. S.; Davis, B. S.; Erwin, P. C.; Smith, A. B.; Panella, N. A.; Powell, E. E.; Nasci, R. S.

    2001-01-01

    La Crosse (LAC) virus, a California serogroup bunyavirus, is the leading cause of pediatric arboviral encephalitis in the United States and an emerging disease in Tennessee, West Virginia, and North Carolina. Human cases of LAC encephalitis in Tennessee and North Carolina have increased above endemic levels during 1997 to 1999 and may represent an expansion of a new southeastern endemic focus. This report describes the isolation of LAC virus from the exotic mosquito Aedes albopictus. The discovery of LAC virus in wild populations of Ae. albopictus coupled with its expanding distribution in the southeastern United States, suggests that this mosquito may become an important accessory vector, potentially increasing the number of human cases in endemic foci or expanding the range of the disease. PMID:11747692

  4. Isolation and Physiological Characterization of a Novel Algicidal Virus Infecting the Marine Diatom Skeletonema costatum

    PubMed Central

    Kim, JinJoo; Kim, Chang-Hoon; Youn, Seok-Hyun; Choi, Tae-Jin

    2015-01-01

    Diatoms are a major component of the biological community, serving as the principal primary producers in the food web and sustaining oxygen levels in aquatic environments. Among marine planktonic diatoms, the cosmopolitan Skeletonema costatum is one of the most abundant and widespread species in the world’s oceans. Here, we report the basic characteristics of a new diatom-infecting S. costatum virus (ScosV) isolated from Jaran Bay, Korea, in June 2008. ScosV is a polyhedral virus (45–50 nm in diameter) that propagates in the cytoplasm of host cells and causes lysis of S. costatum cultures. The infectivity of ScosV was determined to be strain- rather than species-specific, similar to other algal viruses. The burst size and latent period were roughly estimated at 90–250 infectious units/cell and <48 h, respectively. PMID:26060438

  5. Cymbidium chlorotic mosaic virus, a new sobemovirus isolated from a spring orchid (Cymbidium goeringii) in Japan.

    PubMed

    Kondo, Hideki; Takemoto, Shogo; Maruyama, Kazuyuki; Chiba, Sotaro; Andika, Ida Bagus; Suzuki, Nobuhiro

    2015-08-01

    Cymbidium chlorotic mosaic virus (CyCMV), isolated from a spring orchid (Cymbidium goeringii), was characterized molecularly. CyCMV isometric virions comprise a single, positive-strand RNA genome of 4,083 nucleotides and 30-kDa coat protein. The virus genome contains five overlapping open reading frames with a genomic organization similar to that of sobemoviruses. BLAST searches and phylogenetic analysis revealed that CyCMV is most closely related to papaya lethal yellowing virus, a proposed dicot-infecting sobemovirus (58.8 % nucleotide sequence identity), but has a relatively distant relationship to monocot-infecting sobemoviruses, with only modest sequence identities. This suggests that CyCMV is a new monocot-infecting member of the floating genus Sobemovirus. PMID:26025156

  6. First genome analysis and molecular characterization of Chickpea chlorotic dwarf virus Egyptian isolate infecting squash.

    PubMed

    Fahmy, Inas Farouk; Taha, Omnia; El-Ashry, Abdel Nasser

    2015-06-01

    This study aims to identifying and characterizing some molecular properties of geminiviruses co-infection in squash field crop cultivated in Egypt. Squash crops observed to be heavily infected with several insect vectors, also severe chlorosis and stunting was observed. Electron microscopic analysis has revealed geminate capsid particles which indicate the infection of Geminiviruses, especially SqLCV which represent an economic problem to squash filed crop in Egypt. We have investigated possible mixed infections with different plant viruses associated with chlorotic stunt diseases and or other genus groups of geminiviruses. The main objective of this study is to investigate the recombination events, possible recombinants and variants among these genera in the same family differing in vector transmission. This is the first report of the molecular characterization, phylogenetic analysis and putative recombination events of the full length genome of the Chickpea Chlorotic Dwarf Mastrevirus in Egypt. And the first report of co-infection with another begomovirus infecting squash plants. A full length clone of both viruses were isolated and characterized at the molecular level. The complete nucleotide sequence of DNA-A was determined (2,572 bp) and submitted to the genbank under accession no. KF692356. The isolate from Egypt has about 97.8 % homology with the Chickpea chlorotic dwarf virus (CpCDV) isolate from Syria DNA-A isolate FR687959, a 83.2 % homology with the Sudan isolate AM933134 and a 82.7 % homology with Pakistan isolate FR687960. To best of our knowledge this is the first report of complete genome of CpCDV that infect squash plants in Egypt and worldwide. PMID:26436119

  7. Genetic variability of blueberry scorch virus isolates from highbush blueberry in New York State.

    PubMed

    Kalinowska, El?bieta; Marsella-Herrick, Patricia; Fuchs, Marc

    2015-06-01

    The genetic variability of blueberry scorch virus (BlScV) isolates from New York was determined within a portion of the RNA-dependent RNA polymerase gene and the triple gene block and coat protein (CP) genes. Phylogenetic analysis of 19 New York isolates and other isolates for which sequence information is available in GenBank revealed two distinct clades, regardless of the coding region analyzed, and limited variability within (0.029 ± 0.007) and between (0.183 ± 0.032) phylogroups. Recombination events were identified in the CP gene of three New York isolates, and codons of the five BlScV genes characterized were found to be under neutral or negative selective pressure. PMID:25809019

  8. Detection and partial molecular characterization of atypical plum pox virus isolates from naturally infected sour cherry.

    PubMed

    Chirkov, Sergei; Ivanov, Peter; Sheveleva, Anna

    2013-06-01

    Atypical isolates of plum pox virus (PPV) were discovered in naturally infected sour cherry in urban ornamental plantings in Moscow, Russia. The isolates were detected by polyclonal double antibody sandwich ELISA and RT-PCR using universal primers specific for the 3'-non-coding and coat protein (CP) regions of the genome but failed to be recognized by triple antibody sandwich ELISA with the universal monoclonal antibody 5B and by RT-PCR using primers specific to for PPV strains D, M, C and W. Sequence analysis of the CP genes of nine isolates revealed 99.2-100 % within-group identity and 62-85 % identity to conventional PPV strains. Phylogenetic analysis showed that the atypical isolates represent a group that is distinct from the known PPV strains. Alignment of the N-terminal amino acid sequences of CP demonstrated their close similarity to those of a new tentative PPV strain, CR. PMID:23404462

  9. Molecular characterizations of two grapevine rupestris stem pitting-associated virus isolates from China.

    PubMed

    Hu, Guo-Jun; Dong, Ya-Feng; Zhu, Hong-Juan; Zhang, Zun-Ping; Fan, Xu-Dong; Ren, Fang; Zhou, Jun

    2015-10-01

    The complete nucleotide sequences of two isolates of grapevine rupestris stem pitting-associated virus (LSL and JF) collected from grapevine of Xingcheng in Liaoning Province, China, were determined. The genomes of both LSL and JF were found to contain five open reading frames (ORFs). Sequence alignments showed that the genomic sequences of JF were 76.1 %-83.5 % identical to the other ten GRSPaV isolates that have been reported previously and that the nucleotide sequence identity of isolate LSL to other isolates was no more than 78 %. Phylogenetic analysis based on the complete genome sequence indicated that JF belongs to group III and that LSL belongs to a new group (group IV). The average genetic distances of the new genetic lineage from groups I, II and III were 0.34, 0.32 and 0.33, respectively. PMID:26215445

  10. Genome sequence of a pathogenic isolate of monkey B virus (species Macacine herpesvirus 1).

    PubMed

    Ohsawa, Kazutaka; Black, Darla; Ohsawa, Makiko; Eberle, R

    2014-10-01

    The only genome sequence for monkey B virus (BV; species Macacine herpesvirus 1) is that of an attenuated vaccine strain originally isolated from a rhesus monkey (BVrh). Here we report the genome sequence of a virulent BV strain isolated from a cynomolgus macaque (BVcy). The overall genome organization is the same, although sequence differences exist. The greatest sequence divergence is located in non-coding areas of the long and short repeat regions. Like BVrh, BVcy has duplicated Ori elements and lacks an ORF corresponding to the ?34.5 gene of herpes simplex virus. Nine of ten miRNAs and the majority of ORFs are conserved between BVrh and BVcy. The most divergent genes are several membrane-associated proteins and those encoding immediate early proteins. PMID:24903602

  11. Genome sequence of a pathogenic isolate of monkey B virus (species Macacine herpesvirus 1)

    PubMed Central

    Ohsawa, Kazutaka; Black, Darla; Ohsawa, Makiko; Eberle, R.

    2014-01-01

    The only genome sequence for monkey B virus (BV; species Macacine herpesvirus 1) is that of an attenuated vaccine strain originally isolated from a rhesus monkey (BVrh). Here we report the genome sequence of a virulent BV strain isolated from a cynomolgus macaque (BVcy). The overall genome organization is the same, although sequence differences exist. The greatest sequence divergence is located in non-coding areas of the long and short repeat regions. Like BVrh, BVcy has duplicated Ori elements and lacks an ORF corresponding to the ?34.5 gene of herpes simplex virus. Nine of ten miRNAs and the majority of ORFs are conserved between BVrh and BVcy. The most divergent genes are several membrane-associated proteins and those encoding immediate early proteins. PMID:24903602

  12. Identify, isolate, inform: Background and considerations for Ebola virus disease preparedness in U.S. ambulatory care settings.

    PubMed

    Chea, Nora; Perz, Joseph F; Srinivasan, Arjun; Laufer, Alison S; Pollack, Lori A

    2015-11-01

    Public health activities to identify and monitor persons at risk for Ebola virus disease in the United States include directing persons at risk to assessment facilities that are prepared to safely evaluate for Ebola virus disease. Although it is unlikely that a person with Ebola virus disease will unexpectedly present to a nonemergency ambulatory care facility, the Centers for Disease Control and Prevention have provided guidance for this setting that can be summarized as identify, isolate, and inform. PMID:26277570

  13. Isolation and Characterization of H4N6 Avian Influenza Viruses from Pigs with Pneumonia in Canada

    PubMed Central

    Karasin, Alexander I.; Brown, Ian H.; Carman, Suzanne; Olsen, Christopher W.

    2000-01-01

    In October 1999, H4N6 influenza A viruses were isolated from pigs with pneumonia on a commercial swine farm in Canada. Phylogenetic analyses of the sequences of all eight viral RNA segments demonstrated that these are wholly avian influenza viruses of the North American lineage. To our knowledge, this is the first report of interspecies transmission of an avian H4 influenza virus to domestic pigs under natural conditions. PMID:10982381

  14. Screening, isolation and optimization of anti–white spot syndrome virus drug derived from terrestrial plants

    PubMed Central

    Ghosh, Upasana; Chakraborty, Somnath; Balasubramanian, Thangavel; Das, Punyabrata

    2014-01-01

    Objective To screen, isolate and optimize anti-white spot syndrome virus (WSSV) drug derived from various terrestrial plants and to evaluate the efficacy of the same in host–pathogen interaction model. Methods Thirty plants were subjected to Soxhlet extraction using water, ethanol, methanol and hexane as solvents. The 120 plant isolates thus obtained were screened for their in vivo anti–WSSV property in Litopenaeus vannamei. The best anti–WSSV plant isolate, TP22C was isolated and further analyzed. The drug was optimized at various concentrations. Viral and immune genes were analysed using reverse transcriptase PCR to confirm the potency of the drug. Results Seven plant isolates exhibited significant survivability in host. The drug TP22C thus formulated showed 86% survivability in host. The surviving shrimps were nested PCR negative at the end of the 15 d experimentation. The lowest concentration of TP22C required intramuscularly for virucidal property was 10 mg/mL. The oral dosage of 750 mg/kg body weight/day survived at the rate of 86%. Neither VP28 nor ie 1 was expressed in the test samples at 42nd hour and 84th hour post viral infection. Conclusions The drug TP22C derived from Momordica charantia is a potent anti-white spot syndrome virus drug. PMID:25183066

  15. Isolation and phylogenetic analysis of Mucambo virus (Venezuelan equine encephalitis complex subtype IIIA) in Trinidad.

    PubMed

    Auguste, Albert J; Volk, Sara M; Arrigo, Nicole C; Martinez, Raymond; Ramkissoon, Vernie; Adams, A Paige; Thompson, Nadin N; Adesiyun, Abiodun A; Chadee, Dave D; Foster, Jerome E; Travassos Da Rosa, Amelia P A; Tesh, Robert B; Weaver, Scott C; Carrington, Christine V F

    2009-09-15

    In the 1950s and 1960s, alphaviruses in the Venezuelan equine encephalitis (VEE) antigenic complex were the most frequently isolated arboviruses in Trinidad. Since then, there has been very little research performed with these viruses. Herein, we report on the isolation, sequencing, and phylogenetic analyses of Mucambo virus (MUCV; VEE complex subtype IIIA), including 6 recently isolated from Culex (Melanoconion) portesi mosquitoes and 11 previously isolated in Trinidad and Brazil. Results show that nucleotide and amino acid identities across the complete structural polyprotein for the MUCV isolates were 96.6-100% and 98.7-100%, respectively, and the phylogenetic tree inferred for MUCV was highly geographically- and temporally-structured. Bayesian analyses suggest that the sampled MUCV lineages have a recent common ancestry of approximately 198 years (with a 95% highest posterior density (HPD) interval of 63-448 years) prior to 2007, and an overall rate of evolution of 1.28 x 10(-4) substitutions/site/yr. PMID:19631956

  16. Sequence determination of a quadripartite dsRNA virus isolated from Aspergillus foetidus.

    PubMed

    Kozlakidis, Zisis; Herrero, Noemi; Ozkan, Selin; Kanhayuwa, Lakkhana; Jamal, Atif; Bhatti, Muhammad F; Coutts, Robert H A

    2013-01-01

    Virus infection of Aspergillus foetidus was documented over 40 years ago and was one of the first mycovirus infections described in a filamentous fungus. The virus, named Aspergillus foetidus virus (AfV), contains at least two types of icosahedral particles, called AfV-fast (-F) and AfV-slow (-S) virions, based on their relative electrophoretic mobilities. Here, we report the complete nucleotide sequence of the AfV-F genome isolated from virions purified from the prototype isolate of the fungus. The AfV-F double-stranded (ds) RNA genome is tetra-segmented, and the plus strands of each of the four segments, but not the minus strands, are polyadenylated. The organisation and sequences of the four AfV-F dsRNAs are similar to those described for Alternaria alternata virus 1, which we propose is a member of an emerging mycovirus genus ("Alternavirus") and family ("Alternaviridae"), which also includes AfV-F. PMID:22760661

  17. Characterization and pathogenicity for pigs of a hog cholera virus strain isolated from wild boars.

    PubMed

    Leforban, Y; Cariolet, R

    1992-01-01

    One hog cholera virus strain isolated from an outbreak of the disease in a wild boar breeding herd in Brittany (France) in 1990 has been characterized with a panel of monoclonal antibodies to hog cholera virus and ruminant pestiviruses: the strain was found to be indistinguishable from that of other domestic pig isolates. The pathogenicity of the strain to domestic pigs was evaluated by infecting intranasally, intramuscularly and by contact 17 specific pathogen-free 6-week- and 12-week-old pigs. Sixteen of the 17 pigs showed symptoms of hog cholera. The virus was detected in the blood of the 16 pigs during all phases of hyperthermia which persisted up to death or the terminal phase, ie between 16 and 29 days post-infection. One animal recovered after presenting a mild form of the disease. This pig was the only one which raised antibodies to the virus. Typical hog cholera lesions were observed in 2 pigs only; the other animal showed very few pathological changes. No relationship between intensity or duration of the disease and pathological changes could be established. PMID:1387299

  18. Isolation and characterization of tick-borne encephalitis virus from Ixodes persulcatus in Mongolia in 2012.

    PubMed

    Muto, Memi; Bazartseren, Boldbaatar; Tsevel, Bazartseren; Dashzevge, Erdenechimeg; Yoshii, Kentaro; Kariwa, Hiroaki

    2015-07-01

    Tick-borne encephalitis virus (TBEV) is a zoonotic virus belonging to the genus Flavivirus, in the family Flaviviridae. The virus, which is endemic in Europe and northern parts of Asia, causes severe encephalitis. Tick-borne encephalitis (TBE) has been reported in Mongolia since the 1980s, but details about the biological characteristics of the endemic virus are lacking. In this study, 680 ticks (Ixodes persulcatus) were collected in Selenge aimag, northern Mongolia, in 2012. Nine Mongolian TBEV strains were isolated from tick homogenates. A sequence analysis of the envelope protein gene revealed that all isolates belonged to the Siberian subtype of TBEV. Two strains showed similar growth properties in cultured cells, but their virulence in mice differed. Whole genome sequencing revealed only thirteen amino acid differences between these Mongolian TBEV strains. Our results suggest that these naturally occurring amino acid mutations affected the pathogenicity of Mongolian TBEV. Our results may be an important platform for monitoring TBEV to evaluate the epidemiological risk in TBE endemic areas of Mongolia. PMID:26025267

  19. Genetic Structure and Molecular Variability of Cucumber mosaic virus Isolates in the United States

    PubMed Central

    Nouri, Shahideh; Arevalo, Rafael; Falk, Bryce W.; Groves, Russell L.

    2014-01-01

    Cucumber mosaic virus (CMV) has a worldwide distribution and the widest host range of any known plant virus. From 2000 to 2012, epidemics of CMV severely affected the production of snap bean (Phaseulos vulgaris L.) in the Midwest and Northeastern United States. Virus diversity leading to emergence of new strains is often considered a significant factor in virus epidemics. In addition to epidemics, new disease phenotypes arising from genetic exchanges or mutation can compromise effectiveness of plant disease management strategies. Here, we captured a snapshot of genetic variation of 32 CMV isolates collected from different regions of the U.S including new field as well as historic isolates. Nucleotide diversity (?) was low for U.S. CMV isolates. Sequence and phylogenetic analyses revealed that CMV subgroup I is predominant in the US and further showed that the CMV population is a mixture of subgroups IA and IB. Furthermore, phylogenetic analysis suggests likely reassortment between subgroups IA and IB within five CMV isolates. Based on phylogenetic and computational analysis, recombination between subgroups I and II as well as IA and IB in RNA 3 was detected. This is the first report of recombination between CMV subgroups I and II. Neutrality tests illustrated that negative selection was the major force operating upon the CMV genome, although some positively selected sites were detected for all encoded proteins. Together, these data suggest that different regions of the CMV genome are under different evolutionary constraints. These results also delineate composition of the CMV population in the US, and further suggest that recombination and reassortment among strain subgroups does occur but at a low frequency, and point towards CMV genomic regions that differ in types of selection pressure. PMID:24801880

  20. Influenza A(H5N8) virus isolation in Russia, 2014.

    PubMed

    Marchenko, Vasiliy Y; Susloparov, Ivan M; Kolosova, Nataliya P; Goncharova, Nataliya I; Shipovalov, Andrey V; Durymanov, Alexander G; Ilyicheva, Tatyana N; Budatsirenova, Lubov V; Ivanova, Valentina K; Ignatyev, Georgy A; Ershova, Svetlana N; Tulyahova, Valeriya S; Mikheev, Valeriy N; Ryzhikov, Alexander B

    2015-11-01

    In this study, we report the isolation of influenza A(H5N8) virus from a Eurasian wigeon (Anas penelope) in Sakha Republic of the Russian Far East. The strain A/wigeon/Sakha/1/2014 (H5N8) has been shown to be pathogenic for mammals. It is similar to the strains that caused outbreaks in wild birds and poultry in Southeast Asia and Europe in 2014. PMID:26306756

  1. Complete Genome Sequence of Nervous Necrosis Virus Isolated from Sevenband Grouper (Epinephelus septemfasciatus) in South Korea

    PubMed Central

    Kim, Jong-Oh; Kim, Wi-Sik; Cho, Jae-Kwon; Kim, Kyong-Min; Son, Maeng-Hyun

    2014-01-01

    The draft genome sequence of the nervous necrosis virus (NNV) SGYeosu08, isolated from sevenband grouper (Epinephelus septemfasciatus) in Yeosu, South Korea, was cloned and analyzed. The full-length RNA1 was a 3,103-nucleotide-encoding region of RNA-dependent RNA polymerase, and the RNA2 encoding a coat protein was 1,433 nucleotides in length. This genome sequence might be useful in the development of an accurate diagnostic tool. PMID:25502666

  2. Molecular and antigenic characteristics of Newcastle disease virus isolates from domestic ducks in China.

    PubMed

    Wu, Wei; Liu, Huairan; Zhang, Tingting; Han, Zongxi; Jiang, Yanyu; Xu, Qianqian; Shao, Yuhao; Li, Huixin; Kong, Xiangang; Chen, Hongyan; Liu, Shengwang

    2015-06-01

    Newcastle disease (ND) is one of the most devastating diseases to the poultry industry. The causative agents of ND are virulent strains of Newcastle disease virus (NDV), which are members of the genus Avulavirus within the family Paramyxoviridae. Waterfowl, such as ducks and geese, are generally considered potential reservoirs of NDV and may show few or no clinical signs when infected with viruses that are obviously virulent in chickens. However, ND outbreaks in domestic waterfowl have been frequently reported in many countries in the past decade. In this study, 18 NDV strains isolated from domestic ducks in southern and eastern China, between 2005 and 2013, were genetically and phylogenetically characterized. The complete genomes of these strains were sequenced, and they exhibited genome sizes of 15,186 nucleotides (nt), 15,192 nt, and 15,198 nt, which follow the "rule of six" that is required for the replication of NDV strains. Based on the cleavage site of the F protein and pathogenicity tests in chickens, 17 of our NDV isolates were categorized as lentogenic viruses, and one was characterized as a velogenic virus. Phylogenetic analysis based on the partial sequences of the F gene and the complete genome sequences showed that there are at least four genotypes of NDV circulating in domestic ducks; GD1, AH224, and AH209 belong to genotypes VIId, Ib, and II of class II NDVs, respectively, and the remaining 15 isolates belong to genotype 1b of class I NDVs. Cross-reactive hemagglutination inhibition tests demonstrated that the antigenic relatedness between NDV strains may be associated with their genotypes, rather than their hosts. These results suggest that though those NDV isolates were from duck, they still don't form a phylogenetic group because they came from the same species; however, they may play an important role in promoting the evolution of NDVs. PMID:25725159

  3. Isolation of avian influenza virus (H10N7) from an emu (Dromaius novaehollandiae) with conjunctivitis and respiratory disease.

    PubMed

    Woolcock, P R; Shivaprasad, H L; De Rosa, M

    2000-01-01

    Avian influenza virus was isolated from the conjunctiva of a male emu chick. Clinical observations included ocular discharge, dyspnea, and mild respiratory signs. Lesions included conjunctivitis, tracheitis, bronchopneumonia, and airsacculitis. Escherichia coli was isolated from the conjunctiva and the sinus, and Staphylococcus sp. was isolated from the conjunctiva. Influenza A viral nucleoprotein was detected immunohistochemically in epithelial cells of the bronchi, lung parenchyma and tracheal mucosa, and mononuclear inflammatory cells within the exudate of the bronchial lumen; conjunctiva, air sacs, kidney, intestine, and liver were negative for the viral nucleoprotein. The isolated influenza virus was typed as H10N7 and was determined to be nonpathogenic for chickens. PMID:11007030

  4. Phylogenetic and biological characterization of virulent Newcastle disease viruses isolated in wild birds during 2002-2007

    Technology Transfer Automated Retrieval System (TEKTRAN)

    As part of a West Nile virus surveillance program in the Houston Metropolitan Area and in Rhode Island, extracts from brain from 5608 dead birds representing 21 avian orders, were cultured in Vero cells. Sixteen Newcastle disease virus isolates were recovered from birds of the order Columbiformes. ...

  5. Characaterization of H5N1 highly pathogenic avian influenza viruses isolated from poultry in Pakistan 2006-2008

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nine avian influenza viruses (AIV), H5N1 subtype, were isolated from dead poultry in the Karachi region of Pakistan from 2006-2008. The intravenous pathogenicity indices and HA protein cleavage sites of all nine viruses were consistent with highly pathogenic AIV. Based on phylogenetic analysis of ...

  6. Isolation of exotic Newcastle disease virus (ENDV) from field collected flies and experimental ENDV infections of three arthropod species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    During the 2002 Exotic Newcastle Disease (END) outbreak in California arthropods were collected from two quarantined backyard poultry premises after removal of END virus infected birds. The END virus (ENDV) isolated from field collected pools of three fly species was found to have >98% homology by ...

  7. Evaluation and optimization of avian embryos and cell culture methods for efficient isolation and propagation of avian influenza viruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Surveillance of wild bird populations for avian influenza viruses (AIV) contributes to our understanding of AIV evolution and ecology. Both real-time reverse transcriptase polymerase chain reaction (RRT-PCR) and virus isolation in embryonating chicken eggs (ECE) are standard methods for detecting A...

  8. Broad-spectrum detection and quantitation methods of Soil-borne cereal mosaic virus isolates.

    PubMed

    Vaïanopoulos, Céline; Legrève, Anne; Moreau, Virginie; Bragard, Claude

    2009-08-01

    A broad-spectrum reverse transcription-polymerase chain reaction (RT-PCR) protocol was developed for detecting Soil-borne cereal mosaic virus (SBCMV) isolates, responsible for mosaic diseases in Europe, using primers targeting the highly conserved 3'-untranslated region of RNA-1 and RNA-2 of SBCMV. The 3'-end region is a privileged target for the detection of a wide range of isolates, because of sequence conservation, of the tRNA-like structure, the major role in viral replication and the signal amplification due to the presence of numerous genomic and subgenomic RNAs. The primers were also designed for virus quantitation using real-time RT-PCR with SYBR-Green chemistry. No cross-reaction with Wheat spindle streak mosaic virus, frequently associated with SBCMV, was observed. The use of RT-PCR and real-time quantitative RT-PCR allowed a more sensitive detection and quantitation of SBCMV to be made than was the case with ELISA. The methods enabled European isolates of SBCMV from Belgium, France, Germany, Italy and the UK to be detected and quantified. Real-time RT-PCR represents a new tool for comparing soil inoculum potential as well as cultivar resistance to SBCMV. PMID:19490978

  9. Human isolates of dengue type 1 virus induce apoptosis in mouse neuroblastoma cells.

    PubMed Central

    Desprès, P; Flamand, M; Ceccaldi, P E; Deubel, V

    1996-01-01

    Human isolates of dengue (DEN) type 1 viruses FGA/89 and BR/90 differ in their membrane fusion properties in mosquito cell lines (P. Desprès et al., Virology 196:209-216, 1993). FGA/89 and BR/90 were assayed for their neurovirulence in newborn mice, and neurons were the major target cells for both DEN-1 virus strains within the central nervous system. To study the susceptibility of neurons to DEN virus infection, DEN virus replication was analyzed in the murine neuroblastoma cell line Neuro 2a. Infection of Neuro 2a cells with FGA/89 or BR/90 induced apoptotic DNA degradation after 25 h of infection. Studies of DEN protein synthesis revealed that accumulation of viral proteins leads to apoptotic cell death. The apoptotic process progressed more rapidly following BR/90 infection than it did after FGA/89 infection. The higher cytotoxicity of BR/90 for Neuro 2a cells was linked to an incomplete maturation of the envelope proteins, resulting in abortive virus assembly. Accumulation of viral proteins in the endoplasmic reticulum may induce stress and thereby activate the apoptotic pathway in mouse neuroblastoma cells. PMID:8648748

  10. Efficacy of rabies vaccines against Duvenhage virus isolated from European house bats (Eptesicus serotinus), classic rabies and rabies-related viruses.

    PubMed

    Fekadu, M; Shaddock, J H; Sanderlin, D W; Smith, J S

    1988-12-01

    Isolates of rabies from separate enzootics can be distinguished by their reactions with panels of monoclonal antibodies (mAbs) directed to different sites on the nucleocapsid and glycoproteins of the virus. Estimates of antigenic relatedness can be made by comparing similarities among groups. In this manner it can be shown that while classic strains of rabies react with most of the mAbs, the rabies related Lyssaviruses (Mokola, Lagos and Duvenhage) react with only a few of the mAbs and isolates of rabies from Eptesicus serotinus bats in Europe are intermediate between the two groups. Mice immunized intraperitoneally with human diploid vaccine (HDCV) or animal vaccines (Rabisin and Rabiffa) were protected against a challenge with DBV, DUV-1 and most classic rabies strains. HDCV gave only partial protection against human virus isolates from Finland and Saudi Arabia. The HDCV did not protect mice against challenges with Lagos bat or Mokola virus (rabies-like viruses). The animal vaccines, however, did protect mice against Lagos bat virus, but not against Mokola. Dogs immunized with Rabisin were protected against an intracerebral challenge with DBV. Dogs developed rabies-neutralizing antibody titres after intramuscular or intravenous inoculation with live DBV or DUV-1 virus; these dogs were protected against an intramuscular canine street rabies virus challenge. We conclude that the rabies vaccines tested protect against DBV/DUV-1 and classic street rabies strains, but not Mokola. PMID:3245296

  11. Genomic Characterization of a Novel Virus of the Family Tymoviridae Isolated from Mosquitoes

    PubMed Central

    Fu, Shihong; Wang, David; Rayner, Simon; Tang, Qing; Liang, Guodong

    2012-01-01

    Background The family Tymoviridae comprises three plant virus genera, including Tymovirus, Marafivirus, and Maculavirus, which are found in most parts of the world and cause severe agricultural losses. We describe a putatively novel member of the family Tymoviridae, which is isolated from mosquitoes (Culex spp.), referred to as CuTLV. Methods and Results The CuTLV was isolated by cell culture, which replicates and causes cytopathic effects in Aedes albopictus C6/36 cells, but not in mammalian BHK-21 or Vero cells. The complete 6471 nucleotide sequence of CuTLV was determined. The genome of CuTLV is predicted to contain three open reading frames (ORFs). The largest ORF1 is 5307 nucleotides (nt) in length and encodes a putative polypeptide of 1769 amino acids (aa), which contains the conserved motifs for the methyltransferase (MTR), Tymovirus endopeptidase (PRO), helicase (HEL), and RNA-dependent RNA polymerase (RdRp) of the replication-associated proteins (RPs) of positive-stranded RNA viruses. In contrast, the ORF1 sequence does not contain the so-called “tymobox” or “marafibox”, the conserved subgenomic RNA promoter present in all tymoviruses or marafiviruses, respectively. ORF2 and ORF3 putatively encode a 248-aa coat protein (CP) and a proline-rich 149-aa polypeptide. The whole genomic nucleotide identity of CuTLV with other members of family Tymoviridae ranged from 46.2% (ChiYMV) to 52.4% (GFkV). Phylogenetic analysis based on the putative RP and CP genes of CuTLV demonstrated that the virus is most closely related to viruses in the genus Maculavirus. Conclusions The CuTLV is a novel virus related to members of the family Tymoviridae, with molecular characters that are distinct from those of tymoviruses, marafiviruses, and other maculaviruses or macula-like viruses. This is the first report of the isolation of a Tymoviridae-like virus from mosquitoes. Further investigations are required to clarify the origin, replication strategy, and the public health or agricultural importance of the CuTLV. PMID:22848363

  12. Genetic and biological characterization of Muko virus, a new distinct member of the species Great Island virus (genus Orbivirus, family Reoviridae), isolated from ixodid ticks in Japan.

    PubMed

    Ejiri, Hiroko; Lim, Chang-Kweng; Isawa, Haruhiko; Kuwata, Ryusei; Kobayashi, Daisuke; Yamaguchi, Yukie; Takayama-Ito, Mutsuyo; Kinoshita, Hitomi; Kakiuchi, Satsuki; Horiya, Madoka; Kotaki, Akira; Takasaki, Tomohiko; Maeda, Ken; Hayashi, Toshihiko; Sasaki, Toshinori; Kobayashi, Mutsuo; Saijo, Masayuki; Sawabe, Kyoko

    2015-12-01

    Among the tick-borne orbiviruses (genus Orbivirus, family Reoviridae), 36 serotypes are currently classified within a single virus species, Great Island virus. In this study, we report the first characterization of a tick-borne orbivirus isolated from the tick Ixodes turdus in Japan, which we identified as a new member of the species Great Island virus. The virus isolate, designated Muko virus (MUV), replicated and induced cytopathic effects in BHK-21, Vero E6, and CCL-141 cells and caused high mortality in suckling mice after intracerebral inoculation. Full genome sequence analysis showed that MUV shared the greatest phylogenetic similarity with Tribe? virus in terms of the amino acid sequences of all viral proteins except for outer capsid protein 1 (OC1; VP4 of MUV). Analysis of genome segment 9 in MUV detected an uninterrupted open reading frame that overlaps with VP6 (Hel), which putatively encodes a molecular and functional equivalent of NS4 from Great Island virus. Our study provides new insights into the geographic distribution, genetic diversity, and evolutionary history of the members of the species Great Island virus. PMID:26350980

  13. [Isolation, identification and full-length genome sequence analysis of encephalomyocarditis virus from local aardvarks].

    PubMed

    Chang, Hong-Tao; Liu, Hui-Min; He, Xiu-Yuan; Zhao, Jun; Chen, Lu; Wang, Xin-Wei; Yang, Xia; Yao, Hui-Xia; Wang, Chuan-Qing

    2014-07-01

    Encephalomyocarditis virus (EMCV) is a natural epidemic zoonotic pathogen. However, no reports have been published regarding the isolation, identification and full-length genome of EMCV from a local aardvark population. In present study, an EMCV isolate HNXX13 was isolated from aardvarks named Huainan-pig in Henan Province. The systematic identification, full-length genome sequencing and molecular characteristic analysis of the isolate HNXX13 were conducted. The result showed that the isolate was spherical with a diameter of 24-30 nm, neither heat- nor acid-resistant, sensitive to trypsin, insensitive to chloroform, not protected by bivalent cationic, and the specific fluorescence was observed in the cytoplasm of BHK-21 cells infected with the isolate by using indirect fluorescence assay. The full-length genome of EMCV HNXX13 generated a 7 725bp sequence (GenBank: F771002), with 81.0%-99.9% nucleotide identity to reference strains from different animals, and 99.5% with a Chinese reference strain isolated earlier from a commercial pig herd. The phylogenetic tree based on the full-length genome and ORF sequences identified that all EMCV strains were divided into three groups G1, G2 and G3, and strain HNXX13 belonging to the G1 group with other Chinese reference strains. The result also identified that this EMCV infection could cause severe clinical signs in a local aardvark population, and enriches the molecular epidemiological data of EMCV in China. Regional differences exist in EMCV genome and transmission is limited within a certain area. However, the cross-infection and transmission of EMCV between aardvark and mice appears most likely. Mutations have occurred in some amino acids of EMCV strain HNXX13 during the transmission in local aardvark herd and these mutations might make the virus easier to infect the aardvark. PMID:25272589

  14. Genotypes of Japanese encephalitis virus isolated in three states in Malaysia.

    PubMed

    Tsuchie, H; Oda, K; Vythilingam, I; Thayan, R; Vijayamalar, B; Sinniah, M; Singh, J; Wada, T; Tanaka, H; Kurimura, T; Igarashi, A

    1997-02-01

    Two hundred forty nucleotides from the pre-membrane gene region of 12 Japanese encephalitis virus (JEV) strains isolated from three different regions of Malaysia from 1993 to 1994 were sequenced and compared with each other and with the JEV strains from different geographic areas in Asia. These 12 Malaysian isolates were classified into two genotypes. The four JEV strains isolated from Sarawak in 1994 and the four JEV strains isolated from Sepang, Selangor in 1993 were classified into one genotype that included earlier isolated strains from Malaysia (JE-827 from Sarawak in 1968 and WTP/70/22 from Kuala Lumpur in 1970). The four JEV strains from Ipoh, Perak in 1994 were classified into another genotype that included JEV strains isolated from northern Thailand and Cambodia. In an earlier report, 10 JEV strains from Sabak Bernam, Selangor in 1992 were classified into the largest genotype that included strains isolated in temperate regions such as Japan, China, and Taiwan. The data indicate that at least three genotypes of JEV have been circulating in Malaysia. PMID:9080873

  15. Lassa virus isolates from Mali and the Ivory Coast represent an emerging fifth lineage

    PubMed Central

    Manning, John T.; Forrester, Naomi; Paessler, Slobodan

    2015-01-01

    Previous imported cases of Lassa fever (LF) into the United Kingdom from the Ivory Coast and Mali, as well as the detection of Lassa virus (LASV) among the Mastomys natalensis population within Mali has led to the suggestion that the endemic area for LF is expanding. Initial phylogenetic analyses arrange isolates from Mali and the Ivory Coast separately from the classical lineage IV isolates taken from Sierra Leone, Guinea, and Liberia. The availability of full genome sequences continues to increase, allowing for a more complete phylogenetic comparison of the isolates from Mali and the Ivory Coast to the other existing isolates. In this study, we utilized a Bayesian approach to infer the demographic histories of each LASV isolate for which the full sequence was available. Our results indicate that the isolates from Mali and the Ivory Coast group separately from the isolates of lineage IV, comprising a distinct fifth lineage. The split between lineages IV and V is estimated to have occurred around 200–300 years ago, which coincides with the colonial period of West Africa. PMID:26483768

  16. Biochemical analysis of murine leukemia viruses isolated from radiation-induced leukemias of strain BALB/c

    SciTech Connect

    Ellis, R.W.; Hopkins, N.; Fleissner, E.

    1980-02-01

    Murine leukemia viruses isolated from radiation-induced BALB/c leukemias were characterized with respect to viral proteins and RNA. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the viral structural proteins revealed that for p12, p15, p30, and gp70, three to four electrophoretic variants of each could be detected. There was no correlation found between any of these mobilities and N- or B-tropism of the viruses. Proteins of all xenotropic viral isolates were identical in their gel electrophoretic profiles. The similar phenotypes of multiple viral clones from individual leukemias and of isolates grown in different cells suggest that the polymorphism of ecotropic viruses was generated in vivo rather than during in vitro virus growth. By two-dimensional fingerprinting of RNase T1-resistant oligonucleotides from 70S viral RNA, the previously reported association of N- and B-tropism with two distinct oligonucleotides was confirmed. The presence of two other oligonucleotides was correlated with positive and negative phenotypes of the virus-coded G/sub IX/ cell surface antigen. The RNAs of two B-tropic isolates with distinctive p15 and p12 phenotypes differed from the RNA of a prototype N-tropic virus by the absence of three oligonucleotides mapping in the 5' portion (gag region) of the prototype RNA. In addition, one small-plaque B-tropic virus displayed extensive changes in the RNA sequences associated with the env region of the prototype.

  17. Isolation and characterization of Mayaro virus from a human in Acre, Brazil.

    PubMed

    Terzian, Ana Carolina B; Auguste, Albert J; Vedovello, Danila; Ferreira, Marcelo U; da Silva-Nunes, Mônica; Sperança, Márcia A; Suzuki, Rodrigo B; Juncansen, Camila; Araújo, João P; Weaver, Scott C; Nogueira, Maurício L

    2015-02-01

    Mayaro virus (MAYV) is widely distributed throughout South America and is the etiologic agent of Mayaro fever, an acute febrile illness often presenting with arthralgic manifestations. The true incidence of MAYV infection is likely grossly underestimated because the symptomatic presentation is very similar to that of dengue fever and other acute febrile tropical diseases. We report the complete genome sequence of a MAYV isolate detected from an Acrelândia patient presenting with fever, chills, and sweating, but with no arthralgia. Results show that this isolate belongs to genotype D and is closely related to Bolivian strains. Our results suggest that the Acre/Mayaro strain is closely related to the progenitor of these Bolivian strains that were isolated between 2002 and 2006. PMID:25510721

  18. Single Strain Isolation Method for Cell Culture-Adapted Hepatitis C Virus by End-Point Dilution and Infection

    PubMed Central

    Sugiyama, Nao; Murayama, Asako; Suzuki, Ryosuke; Watanabe, Noriyuki; Shiina, Masaaki; Liang, T. Jake; Wakita, Takaji; Kato, Takanobu

    2014-01-01

    The hepatitis C virus (HCV) culture system has enabled us to clarify the HCV life cycle and essential host factors for propagation. However, the virus production level of wild-type JFH-1 (JFH-1/wt) is limited, and this leads to difficulties in performing experiments that require higher viral concentrations. As the cell culture-adapted JFH-1 has been reported to have robust virus production, some mutations in the viral genome may play a role in the efficiency of virus production. In this study, we obtained cell culture-adapted virus by passage of full-length JFH-1 RNA-transfected Huh-7.5.1 cells. The obtained virus produced 3 log-fold more progeny viruses as compared with JFH-1/wt. Several mutations were identified as being responsible for robust virus production, but, on reverse-genetics analysis, the production levels of JFH-1 with these mutations did not reach the level of cell culture-adapted virus. By using the single strain isolation method by end-point dilution and infection, we isolated two strains with additional mutations, and found that these strains have the ability to produce more progeny viruses. On reverse-genetics analysis, the strains with these additional mutations were able to produce robust progeny viruses at comparable levels as cell culture-adapted JFH-1 virus. The strategy used in this study will be useful for identifying strains with unique characteristics, such as robust virus production, from a diverse population, and for determining the responsible mutations for these characteristics. PMID:24848954

  19. Complete Genome Sequence of Genotype Ib Newcastle Disease Virus Isolated from a Mallard (Anas platyrhynchos) in Russia

    PubMed Central

    Sobolev, Ivan A.; Glushchenko, Alexandra V.; Shestopalov, Alexander M.

    2015-01-01

    We report here the complete genome sequence of a Newcastle disease virus isolate, NDV/Yakutiya/mallard/852/2011, isolated from a mallard in Russia. On the basis of phylogenetic analysis, this strain was clustered into class II genotype Ib. PMID:26634762

  20. THE COMPLETE NUCLEOTIDE SEQUENCE OF BEAN YELLOW MOSAIC VIRUS ISOLATE BYMV-GDD, AND COMPARISON TO OTHER POTYVIRUSES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The complete nucleotide sequence of Bean yellow mosaic virus (BYMV) gladiolus isolate GDD was determined and compared to broad bean isolates BYMV-MB4 and BYMV-S. The BYMV-GDD genome (9528nt) was more similar to BYMV-MB4 (9532nt) than to BYMV-S (9547nt), which has atypical symptom expression and host...

  1. Leek yellow stripe virus isolates from Brazil form a distant clade based on the P1 gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The complete genomic sequence of a garlic isolate of Leek yellow stripe virus from Brazil (LYSV-MG) has been determined, and phylogenetic comparisons made to LYSV isolates from other parts of the world. In addition, the nucleotide sequence of the 5'UTR and part of the P1 gene of multiple LYSV isolat...

  2. Median infectious dose (ID??) of porcine reproductive and respiratory syndrome virus isolate MN-184 via aerosol exposure.

    PubMed

    Cutler, Timothy D; Wang, Chong; Hoff, Steven J; Kittawornrat, Apisit; Zimmerman, Jeffrey J

    2011-08-01

    The median infectious dose (ID(50)) of porcine reproductive and respiratory syndrome (PRRS) virus isolate MN-184 was determined for aerosol exposure. In 7 replicates, 3-week-old pigs (n=58) respired 10l of airborne PRRS virus from a dynamic aerosol toroid (DAT) maintained at -4°C. Thereafter, pigs were housed in isolation and monitored for evidence of infection. Infection occurred at virus concentrations too low to quantify by microinfectivity assays. Therefore, exposure dose was determined using two indirect methods ("calculated" and "theoretical"). "Calculated" virus dose was derived from the concentration of rhodamine B monitored over the exposure sequence. "Theoretical" virus dose was based on the continuous stirred-tank reactor model. The ID(50) estimate was modeled on the proportion of pigs that became infected using the probit and logit link functions for both "calculated" and "theoretical" exposure doses. Based on "calculated" doses, the probit and logit ID(50) estimates were 1 × 10(-0.13)TCID(50) and 1 × 10(-0.14)TCID(50), respectively. Based on "theoretical" doses, the probit and logit ID(50) were 1 × 10(0.26)TCID(50) and 1 × 10(0.24)TCID(50), respectively. For each point estimate, the 95% confidence interval included the other three point estimates. The results indicated that MN-184 was far more infectious than PRRS virus isolate VR-2332, the only other PRRS virus isolate for which ID(50) has been estimated for airborne exposure. Since aerosol ID(50) estimates are available for only these two isolates, it is uncertain whether one or both of these isolates represent the normal range of PRRS virus infectivity by this route. PMID:21474258

  3. A novel quadripartite dsRNA virus isolated from a phytopathogenic filamentous fungus, Rosellinia necatrix.

    PubMed

    Lin, Yu-Hsin; Chiba, Sotaro; Tani, Akio; Kondo, Hideki; Sasaki, Atsuko; Kanematsu, Satoko; Suzuki, Nobuhiro

    2012-04-25

    Here we report the biological and molecular attributes of a novel dsRNA virus isolated from Rosellinia necatrix, a filamentous phytopathogenic fungus. The virus, termed Rosellinia necatrix quadrivirus 1 (RnQV1), forms rigid spherical particles approximately 45 nm in diameter in infected mycelia. The particles contain 4 dsRNA segments, dsRNA1 to dsRNA4, with a size range of 4.9 to 3.7 kbp, each possessing a single large ORF. A comparison of the virus-infected and -cured isogenic fungal strains suggested that RnQV1 infection has no appreciable phenotypic effects. Phylogenetic analysis using the dsRNA3-encoded RdRp sequence revealed that RnQV1 is more distantly related to quadripartite chrysoviruses than to monopartite totiviruses, and is placed in a distinct group from other mycoviruses. No significant sequence similarities were evident between known proteins and RnQV1 structural proteins shown to be encoded by dsRNA2 or dsRNA4. These suggest that RnQV1 is a novel latent virus, belonging to a new family. PMID:22321722

  4. Agnogene Deletion in a Novel Pathogenic JC Virus Isolate Impairs VP1 Expression and Virion Production

    PubMed Central

    Ellis, Laura C.; Norton, Elizabeth; Dang, Xin; Koralnik, Igor J.

    2013-01-01

    Infection of glial cells by the human polyomavirus JC (JCV) causes progressive multifocal leukoencephalopathy (PML). JCV Encephalopathy (JCVE) is a newly identified disease characterized by JCV infection of cortical pyramidal neurons. The virus JCVCPN associated with JCVE contains a unique 143 base pair deletion in the agnogene. Contrary to most JCV brain isolates, JCVCPN has an archetype-like regulatory region (RR) usually found in kidney strains. This provided us with the unique opportunity to determine for the first time how each of these regions contributed to the phenotype of JCVCPN. We characterized the replication of JCVCPN compared to the prototype virus JCVMad-1 in kidney, glial and neuronal cell lines. We found that JCVCPN is capable of replicating viral DNA in all cell lines tested, but is unable to establish persistent infection seen with JCVMad-1. JCVCPN does not have an increased ability to replicate in the neuronal cell line tested. To determine whether this phenotype results from the archetype-like RR or the agnogene deletion, we generated chimeric viruses between JCVCPN of JCVMad-1. We found that the deletion in the agnogene is the predominant cause of the inability of the virus to maintain a persistent infection, with the introduction of a full length agnogene, either with or without agnoprotein expression, rescues the replication of JCVCPN. Studying this naturally occurring pathogenic variant of JCV provides a valuable tool for understanding the functions of the agnogene and RR form in JCV replication. PMID:24265839

  5. Molecular characterization of an influenza A virus (H4N2) isolated from waterfowl habitats in the State of Mexico.

    PubMed

    Ornelas-Eusebio, Erika; Obregón-Ascencio, Alejandro; Chávez-Maya, Fernando; García-Espinosa, Gary

    2015-03-01

    Wild waterfowl and their habitats are the main reservoirs of influenza A virus (IAV) mainly during the breeding season and prior to migration. This study describes the molecular characterization of an IAV isolated from 240 water samples of a small wetland during non-breeding season of migratory wild ducks in the State of Mexico, Mexico. The results showed that the virus belongs to the H4N2 subtype and each of its eight segments of the viral genome has similarity to IAV isolated from ducks in North America. This study suggests that IAV can be isolated from small wetland during non-breeding season of migrating waterfowl. PMID:25482497

  6. Molecular characterization of an influenza A virus (H4N2) isolated from waterfowl habitats in the State of Mexico

    PubMed Central

    ORNELAS-EUSEBIO, Erika; OBREGÓN-ASCENCIO, Alejandro; CHÁVEZ-MAYA, Fernando; GARCÍA-ESPINOSA, Gary

    2014-01-01

    Wild waterfowl and their habitats are the main reservoirs of influenza A virus (IAV) mainly during the breeding season and prior to migration. This study describes the molecular characterization of an IAV isolated from 240 water samples of a small wetland during non-breeding season of migratory wild ducks in the State of Mexico, Mexico. The results showed that the virus belongs to the H4N2 subtype and each of its eight segments of the viral genome has similarity to IAV isolated from ducks in North America. This study suggests that IAV can be isolated from small wetland during non-breeding season of migrating waterfowl. PMID:25482497

  7. A novel highly pathogenic H5N8 avian influenza virus isolated from a wild duck in China.

    PubMed

    Fan, Shengtao; Zhou, Lichen; Wu, Di; Gao, Xiaolong; Pei, Enle; Wang, Tianhou; Gao, Yuwei; Xia, Xianzhu

    2014-11-01

    Migrating wild birds are considered natural reservoirs of influenza viruses and serve as a potential source of novel influenza strains in humans and livestock. During routine avian influenza surveillance conducted in eastern China, a novel H5N8 (SH-9) reassortant influenza virus was isolated from a mallard duck in China. blast analysis revealed that the HA, NA, PB1, PA, NP, and M segments of SH-9 were most closely related to the corresponding segments of A/duck/Jiangsu/k1203/2010 (H5N8). The SH-9 virus preferentially recognized avian-like influenza virus receptors and was highly pathogenic in mice. Our results suggest that wild birds could acquire the H5N8 virus from breeding ducks and spread the virus via migratory bird flyways. PMID:25363159

  8. First isolation of a rabies-related virus from a Daubenton's bat in the United Kingdom.

    PubMed

    Whitby, J E; Heaton, P R; Black, E M; Wooldridge, M; McElhinney, L M; Johnstone, P

    2000-09-30

    On May 30, 1996, a sick Daubenton's bat (Myotis daubentonii) was recovered from the cellar of a public house in Newhaven, East Sussex. Its condition deteriorated rapidly, and it was euthanased and examined. Positive results, establishing the presence of a rabies or rabies-related virus in its brain, were obtained from the fluorescent antibody test, the rabies tissue culture isolation test, and a hemi-nested reverse-transcription PCR. The complete sequence of the nucleoprotein gene was determined and a phylogenetic analysis, based on the 470 nucleotide bases of the amino terminus of the nucleoprotein, established the genotype of the virus as European bat lyssavirus 2. Bat rabies had not previously been recorded in the UK but does occur in mainland Europe. A study of the back-trajectories of the wind on May 29 and 30, established that the infected bat possibly came from near the Franco-Swiss border. PMID:11073000

  9. Evolutionary and Ecological Characterization of Mayaro Virus Strains Isolated during an Outbreak, Venezuela, 2010.

    PubMed

    Auguste, Albert J; Liria, Jonathan; Forrester, Naomi L; Giambalvo, Dileyvic; Moncada, Maria; Long, Kanya C; Morón, Dulce; de Manzione, Nuris; Tesh, Robert B; Halsey, Eric S; Kochel, Tadeusz J; Hernandez, Rosa; Navarro, Juan-Carlos; Weaver, Scott C

    2015-10-01

    In 2010, an outbreak of febrile illness with arthralgic manifestations was detected at La Estación village, Portuguesa State, Venezuela. The etiologic agent was determined to be Mayaro virus (MAYV), a reemerging South American alphavirus. A total of 77 cases was reported and 19 were confirmed as seropositive. MAYV was isolated from acute-phase serum samples from 6 symptomatic patients. We sequenced 27 complete genomes representing the full spectrum of MAYV genetic diversity, which facilitated detection of a new genotype, designated N. Phylogenetic analysis of genomic sequences indicated that etiologic strains from Venezuela belong to genotype D. Results indicate that MAYV is highly conserved genetically, showing ?17% nucleotide divergence across all 3 genotypes and 4% among genotype D strains in the most variable genes. Coalescent analyses suggested genotypes D and L diverged ?150 years ago and genotype diverged N ?250 years ago. This virus commonly infects persons residing near enzootic transmission foci because of anthropogenic incursions. PMID:26401714

  10. Evolutionary and Ecological Characterization of Mayaro Virus Strains Isolated during an Outbreak, Venezuela, 2010

    PubMed Central

    Auguste, Albert J.; Liria, Jonathan; Forrester, Naomi L.; Giambalvo, Dileyvic; Moncada, Maria; Long, Kanya C.; Morón, Dulce; de Manzione, Nuris; Tesh, Robert B.; Halsey, Eric S.; Kochel, Tadeusz J.; Hernandez, Rosa; Navarro, Juan-Carlos

    2015-01-01

    In 2010, an outbreak of febrile illness with arthralgic manifestations was detected at La Estación village, Portuguesa State, Venezuela. The etiologic agent was determined to be Mayaro virus (MAYV), a reemerging South American alphavirus. A total of 77 cases was reported and 19 were confirmed as seropositive. MAYV was isolated from acute-phase serum samples from 6 symptomatic patients. We sequenced 27 complete genomes representing the full spectrum of MAYV genetic diversity, which facilitated detection of a new genotype, designated N. Phylogenetic analysis of genomic sequences indicated that etiologic strains from Venezuela belong to genotype D. Results indicate that MAYV is highly conserved genetically, showing ?17% nucleotide divergence across all 3 genotypes and 4% among genotype D strains in the most variable genes. Coalescent analyses suggested genotypes D and L diverged ?150 years ago and genotype diverged N ?250 years ago. This virus commonly infects persons residing near enzootic transmission foci because of anthropogenic incursions. PMID:26401714

  11. Research note: the isolation of a herpes virus from captive cranes with an inclusion body disease

    USGS Publications Warehouse

    Docherty, D.E.; Henning, D.J.

    1980-01-01

    A viral agent, identified as a herpesvirus and tentatively called 'inclusion body disease of cranes' (IBDC), was isolated from captive cranes involved in a die-off at the International Crane Foundation near Baraboo, Wisconsin. Preliminary animal susceptibility tests, based on experimental infections, suggested that White Pekin ducklings up to 17 days old and adult coots were susceptible to the IBDC virus whereas 16-day-old White Leghorn chicks and 64-day-old Muscovy ducks were not. No serum antibody to IBDC virus was detected in 95 wild sandhill cranes collected in Wisconsin or Indiana in 1976 and 1977. However, 9 of 11 captive cranes in the affected area at the ICF had antibody to this agent.

  12. A comparison of virulence of influenza A virus isolates from mallards in experimentally inoculated turkeys.

    PubMed

    Mondal, Shankar; Xing, Zheng; Cardona, Carol

    2013-12-01

    Low pathogenic avian influenza viruses (LPAIV) from wild waterfowl can and do cross species barriers, infecting and sometimes becoming established in domestic poultry. Turkeys are naturally highly susceptible to LPAIV infections, especially with viruses from ducks. In this study, we describe clinical signs and lesions in experimentally inoculated commercial turkeys produced by a LPAIV, A/mallard/MN/1714/09 (H7N1), isolated from a mallard duck. Our results demonstrate that this H7N1 isolate produced clinical signs, including severe edema of the head and face because of an early inflammatory response in both inoculated and contact turkeys. In comparison, an isolate, A/mallard/MN/2749/09 (H6N8) from the same mallard population, infected and was transmitted between naive turkeys but did not cause clinical disease or lesions. Our data indicate that proinflammatory (IL-1beta, TNF-alpha, and IL-6) and antiviral (IFN-gamma and IL-2) cytokines are expressed at different levels in H7N1- and H6N8-infected turkey peripheral blood mononuclear cells. These differences correlate inversely with clinical lesions, suggesting that differences in host responses result in variances in viral pathogenesis and in virulence of LPAIV in commercial turkeys. Based on these results, we can conclude that turkeys may exhibit variable immunologic responses to infection with different AIV strains. PMID:24597123

  13. Phylogenetic analyses of highly pathogenic avian influenza virus isolates from Germany in 2006 and 2007 suggest at least three separate introductions of H5N1 virus.

    PubMed

    Starick, E; Beer, M; Hoffmann, B; Staubach, C; Werner, O; Globig, A; Strebelow, G; Grund, C; Durban, M; Conraths, F J; Mettenleiter, T; Harder, T

    2008-04-30

    In spring 2006, highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 was detected in Germany in 343 dead wild birds, as well as in a black swan (Cygnus atratus) kept in a zoo, three stray cats, one stone marten (Martes foina), and in a single turkey farm. In 2007 (June-July) the virus reoccurred in 96 wild birds at six geographically separate locations in the Southeast of Germany. In addition, a backyard mixed duck and goose holding was affected. Real-time RT-PCR [Hoffmann, B., Harder, T., Starick, E., Depner, K., Werner, O., Beer, M., 2007. Rapid and highly sensitive pathotyping of avian influenza A H5N1 virus by using real-time reverse transcription-PCR. J. Clin. Microbiol. 45, 600-603] and nucleotide sequencing confirmed that these H5-viruses belonged to the Qinghai lineage of HPAIV H5N1 (clade 2.2). For a more detailed analysis, the hemagglutinin and neuraminidase genes of 27 selected German H5N1 viruses isolated 2006 or 2007 and originating from different regions and animal species were sequenced and analysed phylogenetically. As a result, three closely related but distinguishable H5N1 subclades could be defined: In 2006 a 'Northern type' (subclade 2.2.2), representing virus isolates from the German federal states Mecklenburg-Western Pomerania, Schleswig-Holstein, Brandenburg, and Lower Saxony, and a 'Southern type' (subclade 2.2.1) from Baden-Württemberg and Bavaria were detected. Interestingly, representatives of both types were present in Central Germany and caused the outbreak in turkeys (subclade 2.2.2) and in a case in a tufted duck (Aythya fuligula) (subclade 2.2.1) in Saxony. Furthermore, one isolate from the South of Germany was identified as 2.2.2 and vice versa a 2.2.1-like isolate was found in Northern Germany. H5N1 viruses isolated in 2007 belonged to a third type (subclade 2.2.3) which was not detected in 2006. Our data suggest the introduction of three distinct H5N1 variants into the wild bird population of Germany. The source of these viruses and the exact time of introduction remain obscure. Based on the identification of closely related H5N1 viruses from Southern and Central Russia, a recent introduction via wild birds on winter escape from these regions, early in 2006 constitutes the most likely scenario for the 2006 outbreaks. The viruses detected in 2007 most likely represent another new incursion from an as yet unknown source. PMID:18031958

  14. Isolation and characterization of influenza A viruses from environmental water at an overwintering site of migratory birds in Japan.

    PubMed

    Okuya, Kosuke; Kawabata, Toshiko; Nagano, Kiori; Tsukiyama-Kohara, Kyoko; Kusumoto, Isamu; Takase, Kozo; Ozawa, Makoto

    2015-12-01

    The Izumi plain in Kagoshima prefecture, Japan, is an overwintering site of more than 10,000 cranes. The wet paddy areas are artificially created to provide roosting sites for the cranes every winter. Since wild ducks, known to be a natural reservoir of influenza A viruses, also overwinter in this area, the cranes' roost water likely serves as a source of influenza A virus infection. To assess this potential risk, we collected 126 water samples from the cranes' roost in the 2012/2013 winter season for virus isolation. We isolated six influenza viruses of three subtypes (H3N8, H4N6, and H4N8) from the water samples collected in the months of November and December. Genetic analysis of our isolates indicated that these viruses were genetically similar to the low-pathogenic avian influenza viruses circulating among Eurasian waterfowl. These findings suggest the possibility of the cranes becoming infected with the avian influenza viruses that are present in their roost water. PMID:26392284

  15. Analysis of the complete sequences of two biologically distinct Zucchini yellow mosaic virus isolates further evidences the involvement of a single amino acid in the virus pathogenicity.

    PubMed

    Nováková, S; Svoboda, J; Glasa, M

    2014-01-01

    The complete genome sequences of two Slovak Zucchini yellow mosaic virus isolates (ZYMV-H and ZYMV-SE04T) were determined. These isolates differ significantly in their pathogenicity, producing either severe or very mild symptoms on susceptible cucurbit hosts. The viral genome of both isolates consisted of 9593 nucleotides in size, and contained an open reading frame encoding a single polyprotein of 3080 amino acids. Despite their different biological properties, an extremely high nucleotide identity could be noted (99.8%), resulting in differences of only 5 aa, located in the HC-Pro, P3, and NIb, respectively. In silico analysis including 5 additional fully-sequenced and phylogenetically closely-related isolates known to induce different symptoms in cucurbits was performed. This suggested that the key single mutation responsible for virus pathogenicity is likely located in the N-terminal part of P3, adjacent to the PIPO. PMID:25518719

  16. Mallard or chicken? Comparing the isolation of avian influenza A viruses in embryonated Mallard and chicken eggs

    PubMed Central

    Järhult, Josef D.; Wahlgren, John; Hasan, Badrul; Salaneck, Erik; Lundkvist, Åke

    2015-01-01

    Background To date, the most efficient and robust method for isolating avian influenza A viruses (IAVs) is using embryonated chicken eggs (ECEs). It is known that low-pathogenic avian IAVs undergo rapid genetic changes when introduced to poultry holdings, but the factors driving mutagenesis are not well understood. Despite this, there is limited data on the effects of the standard method of virus isolation of avian-derived viruses, that is, whether isolation in ECEs causes adaptive changes in avian IAVs. Eggs from a homologous species could potentially offer an isolation vessel less prone to induce adaptive changes. Methods We performed eight serial passages of two avian IAVs isolated from fecal samples of wild Mallards in both ECEs and embryonated Mallard eggs, and hemagglutination assay titers and hemagglutinin sequences were compared. Results There was no obvious difference in titers between ECEs and embryonated Mallard eggs. Sequence analyses of the isolates showed no apparent difference in the rate of introduction of amino acid substitutions in the hemagglutinin gene (three substitutions in total in embryonated Mallard eggs and two substitutions in ECEs). Conclusion Embryonated Mallard eggs seem to be good isolation vessels for avian IAVs but carry some practical problems such as limited availability and short egg-laying season of Mallards. Our study finds isolation of Mallard-derived avian IAVs in ECEs non-inferior to isolation in embryonated Mallard eggs, but more research in the area may be warranted as this is a small-scale study. PMID:26356095

  17. Genetic and evolutionary characterization of Venezuelan equine encephalitis virus isolates from Argentina.

    PubMed

    Pisano, María Belén; Torres, Carolina; Ré, Viviana Elizabeth; Farías, Adrián Alejandro; Sánchez Seco, María Paz; Tenorio, Antonio; Campos, Rodolfo; Contigiani, Marta Silvia

    2014-08-01

    Venezuelan equine encephalitis viruses (VEEV) are emerging pathogens of medical and veterinary importance circulating in America. Argentina is a country free from epizootic VEEV activity, with circulation of enzootic strains belonging to Rio Negro virus (RNV; VEEV subtype VI) and Pixuna virus (PIXV, VEEV subtype IV). In this work, we aim to report the sequencing and phylogenetic analyses of all Argentinean VEE viruses, including 7 strains previously isolated from mosquitoes in 1980, 5 sequences obtained from rodents in 1991 and 11 sequences amplified from mosquitoes between 2003 and 2005. Two genomic regions, corresponding to the non-structural protein 4 (nsP4) and the protein E3/E2 (PE2) genes were analyzed, but only 8 samples could be amplified in the last one (longer and more variable fragment of 702 bp). For both genomic fragments, phylogenetic trees showed the absence of lineages within RNV group, and a close genetic relationship between Argentinean strains and the prototype strain BeAr35645 for PIXV clade. The analysis of nsP4 gene opens the possibility to propose a possible geographic clustering of strains within PIXV group (Argentina and Brazil). Coalescent analysis performed on RNV sequences suggested a common ancestor of 58.3 years (with a 95% highest posterior density [HPD] interval of 16.4-345.7) prior to 1991 and inferred a substitution rate of 9.8×10(-5)substitutions/site/year, slightly lower than other enzootic VEE viruses. These results provide, for the first time, information about genetic features and variability of all VEEVs detected in Argentina, creating a database that will be useful for future detections in our country. This is particularly important for RNV, which has indigenous circulation. PMID:24833218

  18. Analysis of the DNAs from seven varicella-zoster virus isolates.

    PubMed Central

    Richards, J C; Hyman, R W; Rapp, F

    1979-01-01

    The 32P-labeled DNAs from seven different clinical isolates of human varicella-zoster virus (VZV) were independently digested with five site-specific restriction endonucleases, EcoRI, HindIII, SmaI, BamHI, and AvaI. The digestion products were analyzed by electrophoresis on 0.5% agarose gels followed by autoradiography of the dried gels. Evaluation of the restriction enzyme cleavage patterns revealed small variations among the VZV DNAs. The VZV DNAs were also compared based on their buoyant densities in CsCl. No significant buoyant density differences were detected among the VZV DNAs. Images PMID:229268

  19. Study of oseltamivir and zanamivir resistance-related mutations in influenza viruses isolated from wild mallards in Sweden.

    PubMed

    Orozovic, Goran; Orozovic, Kanita; Järhult, Josef D; Olsen, Björn

    2014-01-01

    Resistance to neuraminidase inhibitors (NAIs) is a growing problem in battle against influenza A virus. However, little is known about the resistance of viruses isolated from dabbling ducks, the natural reservoir of the influenza virus. To our knowledge, no low-pathogenic avian influenza (LPAI) virus resistant to NAIs has been detected. The aim of this study was to investigate mallard isolates of influenza A virus previously identified to carry oseltamivir carboxylate (OC) or zanamivir (ZA) resistance-related mutations. In this work, 21 viruses belonging to the N1, N3, N6 and N9 subtypes were analyzed using a colorimetric NA inhibition assay. The results of assay showed no NAIs-resistant phenotype for any of the viruses. The R118K mutation was the most recurrent, as it was observed in all subtypes except for N6. IC50 values confirmed the differences in sensitivity to OC or ZA observed in the N1 and N2 groups of NAs. Furthermore, both wild types (WTs) in the N6 and one WT in the N9 subtype were less sensitive to ZA than were genotypically related mutants with R152K and R118K change in the respective subtypes. This may indicate that these and probably even other NAIs resistance-related mutations found in our virus collection were not induced by NAIs residuals in the environment and that the impact of such mutations in an avian influenza could be dependent on subtype, strain and host species. PMID:24558492

  20. Genetic and antigenic characterization of H5 and H7 influenza viruses isolated from migratory water birds in Hokkaido, Japan and Mongolia from 2010 to 2014.

    PubMed

    Hiono, Takahiro; Ohkawara, Ayako; Ogasawara, Kohei; Okamatsu, Masatoshi; Tamura, Tomokazu; Chu, Duc-Huy; Suzuki, Mizuho; Kuribayashi, Saya; Shichinohe, Shintaro; Takada, Ayato; Ogawa, Hirohito; Yoshida, Reiko; Miyamoto, Hiroko; Nao, Naganori; Furuyama, Wakako; Maruyama, Junki; Eguchi, Nao; Ulziibat, Gerelmaa; Enkhbold, Bazarragchaa; Shatar, Munkhduuren; Jargalsaikhan, Tserenjav; Byambadorj, Selenge; Damdinjav, Batchuluun; Sakoda, Yoshihiro; Kida, Hiroshi

    2015-08-01

    Migratory water birds are the natural reservoir of influenza A viruses. H5 and H7 influenza viruses are isolated over the world and also circulate among poultry in Asia. In 2010, two H5N1 highly pathogenic avian influenza viruses (HPAIVs) were isolated from fecal samples of water birds on the flyway of migration from Siberia, Russia to the south in Hokkaido, Japan. H7N9 viruses are sporadically isolated from humans and circulate in poultry in China. To monitor whether these viruses have spread in the wild bird population, we conducted virological surveillance of avian influenza in migratory water birds in autumn from 2010 to 2014. A total of 8103 fecal samples from migratory water birds were collected in Japan and Mongolia, and 350 influenza viruses including 13 H5 and 19 H7 influenza viruses were isolated. A phylogenetic analysis revealed that all isolates are genetically closely related to viruses circulating among wild water birds. The results of the antigenic analysis indicated that the antigenicity of viruses in wild water birds is highly stable despite their nucleotide sequence diversity but is distinct from that of HPAIVs recently isolated in Asia. The present results suggest that HPAIVs and Chinese H7N9 viruses were not predominantly circulating in migratory water birds; however, continued monitoring of H5 and H7 influenza viruses both in domestic and wild birds is recommended for the control of avian influenza. PMID:26036326

  1. Identification and molecular characterization of a novel duck Tembusu virus isolate from Southwest China.

    PubMed

    Zhu, Kesen; Huang, Juan; Jia, Renyong; Zhang, Bin; Wang, Mingshu; Zhu, Dekang; Chen, Shun; Liu, Mafeng; Yin, Zhongqiong; Cheng, Anchun

    2015-11-01

    Tembusu virus (TMUV) has caused significant economic losses in the Chinese duck industry and may have been overlooked regarding its zoonotic transmission potential. A novel TMUV isolate (named CQW1) was separated from the liver tissue of a young duck in Southwest China. The CQW1 isolate proliferated in embryonated duck eggs and led to death within 3-4 days post-inoculation. Furthermore, CQW1 replicated in duck embryo fibroblast (DEF) cells and caused a cytopathic effect (CPE). The disease emerged on a duck farm in Southwest China and was reproduced by animal experiment. We found that CQW1 was detectable by RT-PCR in brain and liver tissues of dead ducklings within 5 days after inoculation. Most importantly, concentrated nuclei, neuronophagia and microglial nodules were observed in the brain tissue of the inoculated ducklings, and additionally, the liver tissue was affected, mainly by disordered lobular architecture, degeneration, necrosis and regenerated hepatocytes. Analysis of the complete genome sequence showed that CQW1 was 10,992 nt in length with two nucleotide insertions and shared 96.8 % to 99.1 % and 98.4 % to 99.6 % identity at nucleotide and amino acid level, respectively, with Chinese isolates. Phylogenetic analysis of the nucleotide sequences demonstrated that the CQW1 isolate was closely related to other members of the genus Flavivirus and formed a new clade together with the GX2013H isolate. Also, the CQW1 isolate demonstrated the highest average pairwise distance value among the Chinese isolates. In the present study, we obtained evidence that TMUV is present in Southwest China. Extensive pathological and epidemiological studies are urgently needed. PMID:26303137

  2. Hepatitis B Virus Subgenotype A1: Evolutionary Relationships between Brazilian, African and Asian Isolates

    PubMed Central

    Lago, Bárbara V.; Mello, Francisco C.; Kramvis, Anna; Niel, Christian; Gomes, Selma A.

    2014-01-01

    Brazil is a country of low hepatitis B virus (HBV) endemicity in which the genotype A of HBV (HBV/A) is the most prevalent. The complete nucleotide sequences of 26 HBV/A isolates, originating from eight Brazilian states, were determined. All were adw2. Twenty-three belonged to subgenotype A1 and three to A2. By phylogenetic analysis, it was shown that all the 23 HBV/A1 isolates clustered together with isolates from Bangladesh, India, Japan, Nepal, the Philippines and United Arab Emirates, but not with those of Congo, Kenya, Malawi, Rwanda, South Africa, Tanzania, Uganda and Zimbabwe. Four amino acid residues in the polymerase (His138 in the terminal protein domain, Pro18 and His90 in the spacer, and Ser109 in the reverse transcriptase), and one (Phe17) in the precore region, predominated in Latin American and Asian HBV/A1 isolates, but were rarely encountered in African isolates, with the exception of those from Somalia. Specific variations of two adjacent amino acids in the C-terminal domain of the HBx protein, namely Ala146 and Pro147, were found in all the Brazilian, but rarely in the other HBV/A1 isolates. By Bayesian analysis, the existence of an ‘Asian-American’ clade within subgenotype A1 was supported by a posterior probability value of 0.996. The close relatedness of the Brazilian, Asian and Somalian isolates suggests that the HBV/A1 strains predominant in Brazil did not originate from the five million slaves who were imported from Central and Western Africa from 1551 to 1840, but rather from the 300–400,000 captives forcibly removed from southeast Africa at the middle of the 19th century. PMID:25122004

  3. Genotyping hepatitis C virus isolates from Spain, Brazil, China, and Macau by a simplified PCR method.

    PubMed Central

    Holland, P V; Barrera, J M; Ercilla, M G; Yoshida, C F; Wang, Y; de Olim, G A; Betlach, B; Kuramoto, K; Okamoto, H

    1996-01-01

    An improved and simplified method of genotyping was developed for classifying hepatitis C virus (HCV) isolates into the five common genotypes, i.e., I/1a, II/1b, III/2a, IV/2b, and V/3a, by PCR with genotype-specific primers deduced from the core gene. Sense and antisense primers, specific for each of the five common genotypes, were designed by comparison of 319 core gene sequences from HCV isolates of various genotypes from genetic groups 1 to 9. In the first round of PCR, a sequence of 433 bp representing nucleotides 319 to 751 was amplified with universal primers. The second round of PCR was performed with respective sense and antisense primers in two separate reactions, one for the amplification of genotypes I/1a and II/1b and the other for the amplification of genotypes III/2a, IV/2b, and V/3a. The specificity of genotyping was confirmed with a panel of 191 serum samples containing HCV isolates whose core gene sequences were known: 110 serum samples infected with HCV of the five common genotypes and 81 serum samples infected with HCV of other genotypes. The use of sense and antisense primers for genotype II/1b (primers 389 and 492) abolished the cross-reaction of the antisense primer for genotype II/1b (primer 133) with some HCV isolates of genotype I/1a found by our original method. The new method was used for genotyping 130 HCV isolates from Spain, 53 from Brazil, 106 from China, and 30 from Macau. A total of 329 bp of the NS5b region (nucleotides 8279 to 8607) of five isolates from Spain and five isolates from Macau which could not be classified as any of the five common HCV genotypes or genotype 2c were sequenced, and the sequences were compared with those of HCV isolates of known genotypes; two isolates from Spain were deduced to be of genotype 4d and one was deduced to be of genotype 1d, while the remaining two isolates from Spain had novel genotypes in genetic group 2; however, all five isolates from Macau were of genotype 6a. PMID:8880482

  4. Capsid Gene Divergence of Black Queen Cell Virus Isolates in Thailand and Japan Honey Bee Species.

    PubMed

    Mookhploy, Wannapha; Kimura, Kiyoshi; Disayathanoowat, Terd; Yoshiyama, Mikio; Hondo, Kai; Chantawannakul, Panuwan

    2015-06-01

    Black queen cell virus (BQCV) has been found in honey bees worldwide. By using the reverse transcription polymerase chain reaction (RT-PCR) technique, BQCV was detected in a non-native species, Apis mellifera L., collected in both Thailand and Japan, and three other honey bee species (Apis cerana indica F., Apis dorsata F., and Apis florae F.) native to Thailand and Apis cerana japonica F. native to Japan. Based on the capsid coding region, the phylogenetic analysis showed that the BQCV strains found in A. cerana indica and A. cerana japonica were similar within the group and closer to BQCV in Asia. It is interesting to note that the genetic variation of the BQCV isolates was more associated with geographic origin than the host bee species from which the isolates were obtained. PMID:26470278

  5. 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

  6. Characterization of an Avian Influenza Virus H9N2 Strain Isolated from a Wild Bird in Southern China

    PubMed Central

    Xu, Qian; Xie, Liji; Xie, Zhiqin; Deng, Xianwen; Liu, Jiabo; Luo, Sisi

    2014-01-01

    We isolated an avian influenza virus H9N2 strain from a wild bird in the Guangxi Province of southern China in 2013 named A/turtledove/Guangxi/49B6/2013(H9N2) (GX49B6). We aimed to understand the genetic characters of the GX49B6 strain by analyzing the complete genome sequence. The results showed that our isolated strain has features of low pathogenic avian influenza viruses and viruses that infect humans. The discovery of the complete genome sequence of the GX49B6 strain may be helpful to further the understanding of the epidemiology and surveillance of avian influenza viruses in the field. PMID:24948768

  7. Characterization of reticuloendotheliosis virus strain T DNA and isolation of a novel variant of reticuloendotheliosis virus strain T by molecular cloning.

    PubMed Central

    Chen, I S; Mak, T W; O'Rear, J J; Temin, H M

    1981-01-01

    Reticuloendotheliosis virus strain T (REV-T) is a highly oncogenic avian retrovirus which causes a rapid neoplastic disease of the lymphoreticular system. Upon infection, this virus gives rise to two species of unintegrated linear viral DNA, which are 8.3 and 5.5 kilobase pairs long and represent the helper virus (REV-A) and the oncogenic component (REV-T), respectively. Restriction endonuclease cleavage maps of these two DNA components indicate that REV-T DNA has a large portion of the genome deleted with respect to REV-A DNA and a substitution about 0.8 to 1.5 kilobase pairs long that is unrelated to REV-A DNA. These additional sequences comprise the putative transforming region of REV-T (rel). A chicken spleen cell line transformed by REV-T produced virus which upon infection gives rise to three species of unintegrated linear viral DNA (8.3, 5.5, and 3,3 kilobase pairs). We isolated the proviruses of the 8.3- and 3.3-kilobase pair species from this cell line by cloning in the phage vector Charon 4A. Restriction enzyme mapping showed that the two proviral clones are proviruses of REV-A and a variant of REV-T, respectively. A subclone of the variant REV-T provirus specific for the rel sequences of REV-T was used as a hybridization probe to demonstrate that the rel sequences are different from the putative transforming sequences of Schmidt-Ruppin Rous sarcoma virus strain A, avain myelocytomatosis virus, avian myeloblastosis virus, avian erythroblastosis virus, Abelson murine leukemia virus, and Friend erythroleukemia virus. In addition, the rel-specific hybridization probe was used to identify a specific set of sequences which are present in uninfected avian DNAs digested with several restriction enzymes. The corresponding cell sequences are not arranged like rel in REV-T. Images PMID:6275117

  8. Infection of Mice, Ferrets, and Rhesus Macaques with a Clinical Mumps Virus Isolate

    PubMed Central

    Xu, Pei; Huang, Zhixiang; Gao, Xiudan; Michel, Frank J.; Hirsch, Gwen; Hogan, Robert J.; Sakamoto, Kaori; Ho, Wenzhe; Wu, Jianguo

    2013-01-01

    In recent years, many mumps outbreaks have occurred in vaccinated populations worldwide. The reasons for these outbreaks are not clear. Animal models are needed to investigate the causes of outbreaks and to understand the pathogenesis of mumps virus (MuV). In this study, we have examined the infection of three animal models with an isolate of mumps virus from a recent outbreak (MuV-IA). We have found that while both ferrets and mice generated humoral and cellular immune responses to MuV-IA infection, no obvious signs of illness were observed in these animals; rhesus macaques were the most susceptible to MuV-IA infection. Infection of rhesus macaques via both intranasal and intratracheal routes with MuV-IA led to the typical clinical signs of mumps 2 weeks to 4 weeks postinfection. However, none of the infected macaques showed any fever or neurologic signs during the experimental period. Mumps viral antigen was detected in parotid glands by immunohistochemistry (IHC). Rhesus macaques represent the best animal model for the study of mumps virus pathogenesis. PMID:23678169

  9. Endemic human T cell lymphotropic virus type II infection among isolated Brazilian Amerindians.

    PubMed

    Maloney, E M; Biggar, R J; Neel, J V; Taylor, M E; Hahn, B H; Shaw, G M; Blattner, W A

    1992-07-01

    Evidence for human T cell lymphotropic viruses (HTLV) was sought in sera and cells collected from adults in 13 isolated South and Central American Indian tribes. Serologic tests identified frequent HTLV-II-like reactivity among the Cayapo and Kraho tribes, who live 330 km apart in Central Brazil. Polymerase chain reaction analyses of viral DNA in cell pellet and plasma fractions confirmed the virus as HTLV-II. Both tribes speak Gé and, at the time of blood collection (1974), subsisted as hunter/gatherers and slash and burn agriculturalists. Further testing of plasma from Cayapo and Kraho of all ages revealed overall HTLV-II prevalence rates of 33.3% and 12.2%, respectively, with increasing prevalence associated with age and female gender. These data reveal for the first time a high prevalence of HTLV-II infection in remote South American Indians with little contact with non-Indians. Thus, HTLV-II is postulated to be an ancient human virus in the New World. PMID:1607683

  10. Isolation of recombinant phage antibodies targeting the hemagglutinin cleavage site of highly pathogenic avian influenza virus.

    PubMed

    Dong, Jinhua; Sakurai, Akira; Nomura, Namiko; Park, Enoch Y; Shibasaki, Futoshi; Ueda, Hiroshi

    2013-01-01

    Highly pathogenic avian influenza (HPAI) H5N1 viruses, which have emerged in poultry and other wildlife worldwide, contain a characteristic multi-basic cleavage site (CS) in the hemagglutinin protein (HA). Because this arginine-rich CS is unique among influenza virus subtypes, antibodies against this site have the potential to specifically diagnose pathogenic H5N1. By immunizing mice with the CS peptide and screening a phage display library, we isolated four antibody Fab fragment clones that specifically bind the antigen peptide and several HPAI H5N1 HA proteins in different clades. The soluble Fab fragments expressed in Escherichia coli bound the CS peptide and the H5N1 HA protein with nanomolar affinity. In an immunofluorescence assay, these Fab fragments stained cells infected with HPAI H5N1 but not those infected with a less virulent strain. Lastly, all the Fab clones could detect the CS peptide and H5N1 HA protein by open sandwich ELISA. Thus, these recombinant Fab fragments will be useful novel reagents for the rapid and specific detection of HPAI H5N1 virus. PMID:23577205

  11. The isolation and characterization of a Norwalk virus-specific cDNA.

    PubMed Central

    Matsui, S M; Kim, J P; Greenberg, H B; Su, W; Sun, Q; Johnson, P C; DuPont, H L; Oshiro, L S; Reyes, G R

    1991-01-01

    Norwalk virus, an important cause of epidemic, acute, nonbacterial gastroenteritis in adults and children, has eluded adaptation to tissue culture, the development of an animal model, and molecular cloning. In this study, a portion of the Norwalk viral genome encoding an immunoreactive region was cloned from very small quantities of infected stool using sequence-independent single primer amplification. Six overlapping complementary DNA (cDNA) clones were isolated by immunologic screening. The expressed recombinant protein from a representative clone reacted with six of seven high titer. Norwalk-specific, postinfection sera but not with corresponding preinfection sera. Nucleic acid sequence for all clones defined a single open reading frame contiguous with the lambda gt11-expressed beta-galactosidase protein. Only oligonucleotide probes specific for the positive strand (defined by the open reading frame) hybridized to an RNaseA-sensitive, DNaseI-resistant nucleic acid sequence extracted from Norwalk-infected stool. Furthermore, RNA extracted from serial postinfection, but not preinfection, stools from three of five volunteers hybridized to a Norwalk virus cDNA probe. Clone-specific oligonucleotide probes hybridized with cesium chloride gradient fractions containing purified Norwalk virion. In conclusion, an antigenic, protein-coding region of the Norwalk virus genome has been identified. This epitope has potential utility in future sero- and molecular epidemiologic studies of Norwalk viral gastroenteritis. Images PMID:2010555

  12. Isolation and genetic characterization of naturally NS-truncated H3N8 equine influenza virus in South Korea.

    PubMed

    Na, W; Kang, B; Kim, H-I; Hong, M; Park, S-J; Jeoung, H-Y; An, D-J; Moon, H; Kim, J-K; Song, D

    2014-04-01

    Equine influenza virus (EIV) causes a highly contagious respiratory disease in equids, with confirmed outbreaks in Europe, America, North Africa, and Asia. Although China, Mongolia, and Japan have reported equine influenza outbreaks, Korea has not. Since 2011, we have conducted a routine surveillance programme to detect EIV at domestic stud farms, and isolated H3N8 EIV from horses showing respiratory disease symptoms. Here, we characterized the genetic and biological properties of this novel Korean H3N8 EIV isolate. This H3N8 EIV isolate belongs to the Florida sublineage clade 1 of the American H3N8 EIV lineage, and surprisingly, possessed a non-structural protein (NS) gene segment, where 23 bases of the NS1-encoding region were naturally truncated. Our preliminary biological data indicated that this truncation did not affect virus replication; its effect on biological and immunological properties of the virus will require further study. PMID:23800580

  13. Detection and isolation of Sindbis virus from mosquitoes captured during an outbreak in Sweden, 2013.

    PubMed

    Bergqvist, Joakim; Forsman, Oscar; Larsson, Pär; Näslund, Jonas; Lilja, Tobias; Engdahl, Cecilia; Lindström, Anders; Gylfe, Åsa; Ahlm, Clas; Evander, Magnus; Bucht, Göran

    2015-02-01

    Mosquito-borne alphaviruses have the potential to cause large outbreaks throughout the world. Here we investigated the causative agent of an unexpected Sindbis virus (SINV) outbreak during August-September, 2013, in a previously nonendemic region of Sweden. Mosquitoes were collected using carbon dioxide-baited CDC traps at locations close to human cases. The mosquitoes were initially screened as large pools by SINV-specific quantitative RT-PCR, and the SINV-positive mosquitoes were species determined by single-nucleotide polymorphism (SNP) analysis, followed by sequencing the barcoding region of the cytochrome oxidase I gene. The proportion of the collected mosquitoes was determined by a metabarcoding strategy. By using novel strategies for PCR screening and genetic typing, a new SINV strain, Lövånger, was isolated from a pool of 1600 mosquitoes composed of Culex, Culiseta, and Aedes mosquitoes as determined by metabarcoding. The SINV-positive mosquito Culiseta morsitans was identified by SNP analysis and sequencing. After whole-genome sequencing and phylogenetic analysis, the SINV Lövånger isolate was shown to be most closely similar to recent Finnish SINV isolates. In conclusion, within a few weeks, we were able to detect and isolate a novel SINV strain and identify the mosquito vector during a sudden SINV outbreak. PMID:25700044

  14. Screening, isolation and optimization of anti–white spot syndrome virus drug derived from marine plants

    PubMed Central

    Chakraborty, Somnath; Ghosh, Upasana; Balasubramanian, Thangavel; Das, Punyabrata

    2014-01-01

    Objective To screen, isolate and optimize anti-white spot syndrome virus (WSSV) drug derived from various marine floral ecosystems and to evaluate the efficacy of the same in host–pathogen interaction model. Methods Thirty species of marine plants were subjected to Soxhlet extraction using water, ethanol, methanol and hexane as solvents. The 120 plant isolates thus obtained were screened for their in vivo anti-WSSV property in Litopenaeus vannamei. By means of chemical processes, the purified anti-WSSV plant isolate, MP07X was derived. The drug was optimized at various concentrations. Viral and immune genes were analysed using reverse transcriptase PCR to confirm the potency of the drug. Results Nine plant isolates exhibited significant survivability in host. The drug MP07X thus formulated showing 85% survivability in host. The surviving shrimps were nested PCR negative at the end of the 15 d experimentation. The lowest concentration of MP07X required intramuscularly for virucidal property was 10 mg/mL. The oral dosage of 1?000 mg/kg body weight/day survived at the rate of 85%. Neither VP28 nor ie 1 was expressed in the test samples at 42nd hour and 84th hour post viral infection. Conclusions The drug MP07X derived from Rhizophora mucronata is a potent anti-WSSV drug. PMID:25183065

  15. The genome sequence of ectromelia virus Naval and Cornell isolates from outbreaks in North America

    PubMed Central

    Mavian, Carla; López-Bueno, Alberto; Bryant, Neil A.; Seeger, Kathy; Quail, Michael A.; Harris, David; Barrell, Bart; Alcami, Antonio

    2014-01-01

    Ectromelia virus (ECTV) is the causative agent of mousepox, a disease of laboratory mouse colonies and an excellent model for human smallpox. We report the genome sequence of two isolates from outbreaks in laboratory mouse colonies in the USA in 1995 and 1999: ECTV-Naval and ECTV-Cornell, respectively. The genome of ECTV-Naval and ECTV-Cornell was sequenced by the 454-Roche technology. The ECTV-Naval genome was also sequenced by the Sanger and Illumina technologies in order to evaluate these technologies for poxvirus genome sequencing. Genomic comparisons revealed that ECTV-Naval and ECTV-Cornell correspond to the same virus isolated from independent outbreaks. Both ECTV-Naval and ECTV-Cornell are extremely virulent in susceptible BALB/c mice, similar to ECTV-Moscow. This is consistent with the ECTV-Naval genome sharing 98.2% DNA sequence identity with that of ECTV-Moscow, and indicates that the genetic differences with ECTV-Moscow do not affect the virulence of ECTV-Naval in the mousepox model of footpad infection. PMID:24999046

  16. Molecular Characterization of Highly Pathogenic H5N1 Avian Influenza A Viruses Isolated from Raccoon Dogs in China

    PubMed Central

    Qi, Xian; Li, Xihan; Rider, Paul; Fan, Weixing; Gu, Hongwei; Xu, Longtao; Yang, Yonghua; Lu, Sangwei; Wang, Hua; Liu, Fenyong

    2009-01-01

    Background The highly pathogenic avian influenza H5N1 virus can infect a variety of animals and continually poses a threat to animal and human health. While many genotypes of H5N1 virus can be found in chicken, few are associated with the infection of mammals. Characterization of the genotypes of viral strains in animal populations is important to understand the distribution of different viral strains in various hosts. This also facilitates the surveillance and detection of possible emergence of highly pathogenic strains of specific genotypes from unknown hosts or hosts that have not been previously reported to carry these genotypes. Methodology/Principal Findings Two H5N1 isolates were obtained from lung samples of two raccoon dogs that had died from respiratory disease in China. Pathogenicity experiments showed that the isolates were highly pathogenic to chicken. To characterize the genotypes of these viruses, their genomic sequences were determined and analyzed. The genetic contents of these isolates are virtually identical and they may come from the same progenitor virus. Phylogenetic analysis indicated that the isolates were genetically closely related to genotype V H5N1 virus, which was first isolated in China in 2003, and were distinct from the dominant virus genotypes (e.g. genotype Z) of recent years. The isolates also contain a multibasic amino acid motif at their HA cleavage sites and have an E residue at position 627 of the PB2 protein similar to the previously-identified avian viruses. Conclusions/Significance This is the first report that genotype V H5N1 virus is found to be associated with a mammalian host. Our results strongly suggest that genotype V H5N1 virus has the ability to cross species barriers to infect mammalian animals. These findings further highlight the risk that avian influenza H5N1 virus poses to mammals and humans, which may be infected by specific genotypes that are not known to infect these hosts. PMID:19270752

  17. Genetic characterization and phylogenetic analysis of host-range genes of Camelpox virus isolates from India.

    PubMed

    Bera, B C; Barua, S; Shanmugasundaram, K; Anand, T; Riyesh, T; Vaid, R K; Virmani, N; Kundu, S; Yadav, N K; Malik, P; Singh, R K

    2015-09-01

    Camelpox virus (CMLV), a close variant of variola virus (VARV) infects camels worldwide. The zoonotic infections reported from India signify the need to study the host-range genes-responsible for host tropism. We report sequence and phylogenetic analysis of five host-range genes: cytokine response modifier B (crmB), chemokine binding protein (ckbp), viral schlafen-like (v-slfn), myxomavirus T4-like (M-T4-like) and b5r of CMLVs isolated from outbreaks in India. Comparative analysis revealed that these genes are conserved among CMLVs and shared 94.5-100 % identity at both nucleotide (nt) and amino acid (aa) levels. All genes showed identity (59.3-98.4 %) with cowpox virus (CPXV) while three genes-crmB, ckbp and b5r showed similarity (92-96.5 %) with VARVs at both nt and aa levels. Interestingly, three consecutive serine residue insertions were observed in CKBP protein of CMLV-Delhi09 isolate which was similar to CPXV-BR and VACVs, besides five point mutations (K53Q, N67I, F84S, A127T and E182G) were also similar to zoonotic OPXVs. Further, few inconsistent point mutation(s) were also observed in other gene(s) among Indian CMLVs. These indicate that different strains of CMLVs are circulating in India and these mutations could play an important role in adaptation of CMLVs in humans. The phylogeny revealed clustering of all CMLVs together except CMLV-Delhi09 which grouped separately due to the presence of specific point mutations. However, the topology of the concatenated phylogeny showed close evolutionary relationship of CMLV with VARV and TATV followed by CPXV-RatGer09/1 from Germany. The availability of this genetic information will be useful in unveiling new strategies to control emerging zoonotic poxvirus infections. PMID:26396982

  18. Characterization of a herpes virus isolated from domestic geese in Australia

    USGS Publications Warehouse

    Gough, R.E.; Hansen, W.R.

    2000-01-01

    A herpesvirus (GHV 552/89) associated with high mortality in a flock of domestic geese in Australia was compared with duck virus enteritis (DVE) herpesvirus by cross-protection studies in domestic geese, Muscovy ducks and commercial Pekin ducks. In DVE-vaccinated geese, Muscovy ducks and Pekin ducks, mortality levels of 100, 50 and 0%, respectively, were recorded following challenge with GHV 552/89. Conversely, in geese, Muscovy ducks and Pekin ducks immunized with inactivated GHV 552/89, 100% mortality was observed in the geese and Muscovy ducks, and 80% in the Pekin ducks following challenge with DVE virus. The isolate was also compared with six other avian herpesviruses using cross-neutralization tests in cell cultures. No detectable cross-neutralization occurred with any of the avian herpesviruses tested. Further characterization of GHV 552/89 was undertaken by comparing its genome with strains of DVE herpesvirus using restriction endonuclease analysis of the viral DNA and a polymerase chain reaction (PCR) test. Following digestion with HindIII, the DNA fragment pattern of GHV 552/89 was found to be completely different from the DVE viruses. Similarities were found between the digestion patterns of a UK and a US DVE isolate, but both were distinguishable from a UK vaccine strain. The results of the PCR analysis and comparison using two DVE-specific primer sets did not produce specific amplification products of expected molecular weights (603 and 446 base pairs) from the GHV 552/89 genome. The PCR products derived from the DVE strains were similar to those derived from the DVE control DNA. From the results of this study, it is concluded that the goose herpesvirus GHV 552/89 is antigenically and genomically distinct from DVE herpesvirus.

  19. Isolation, Identification, and Sequencing of a Goose-Derived Newcastle Disease Virus and Determination of Its Pathogenicity.

    PubMed

    Chen, Xiao-Qing; Li, Zi-Bing; Hu, Gui-Xue; Gu, Song-Zhi; Zhang, Shuang; Ying, Ying; Gao, Feng-Shan

    2015-06-01

    In August 2010, geese in the Meihekou area of Jilin province in China were found to be infected by a pathogen that caused a disease similar to Newcastle disease. To determine the causative agent of the infections, a virus was isolated from liver tissues of infected geese, followed by a pathogenicity determination. The isolated virus was named NDV/White Goose/China/Jilin(Meihekou)/MHK-1/2010. Specific primers were designed to amplify the whole genome of the MHK-1 virus, followed by sequencing and splicing of the entire genome. Sequencing and phylogenetic analysis of MHK-1 showed that the isolate was a virulent strain of Newcastle disease virus. The MHK-1 genome is 15,192 nucleotides long, and it belongs to the class II branch of Newcastle disease viruses, as evidenced by the amino acid sequence (112R-R-Q-K-R-F117) of the F protein. The hemagglutinin titer was 1:128 to 1:512. The chicken embryo mean death time, the intracerebral pathogenicity index, and the median lethal dose of chicken embryos of MHK-1 were 43 hr, 1.63, and 10(9)/ml, respectively, which revealed that the newly isolated MHK-1 strain is strongly pathogenic to geese. PMID:26473673

  20. Molecular characterisation of an isolate of Dasheen mosaic virus from Zantedeschia aethiopica in China and comparisons in the genus Potyvirus.

    PubMed

    Chen, J; Chen, J; Chen, J; Adams, M J

    2001-01-01

    The complete nucleotide sequence of an isolate of Dasheen mosaic virus from Zantedeschia aethiopica in Zhejiang Province, China, was determined. The 9991 nucleotide genome was typical of the genus Potyvirus and phylogenetic analysis showed it to be a member of the Bean common mosaic virus subgroup. The 3'-terminal sequence, including the coat protein region, was determined for three further isolates from China and Japan. Variations in the length and composition of the N-terminus of the coat protein were not related to geographic origin or plant host. An analysis of all potyvirus cleavage sites revealed patterns related to phylogenetic groupings. PMID:11699967

  1. First isolation of Bunyamwera virus (Bunyaviridae family) from horses with neurological disease and an abortion in Argentina.

    PubMed

    Tauro, Laura B; Rivarola, Maria E; Lucca, Eduardo; Mariño, Betina; Mazzini, Rubén; Cardoso, Jedson Ferreira; Barrandeguy, María Edith; Teixeira Nunes, Marcio Roberto; Contigiani, Marta S

    2015-10-01

    Bunyamwera virus (BUNV) is the prototype virus for both the Orthobunyavirus genus and the Bunyaviridae family. Different strains of BUNV have been associated with clinical diseases in domestic animals, mainly ruminants. During 2013, in Argentina's Santa Fe Province, three new isolates of BUNV were recovered from the brain and spleen of two horses with encephalitis, and from the brain of an aborted equine fetus. This isolation of BUNV from domestic animals provided the first association of BUNV infection with disease of the central nervous system and abortion in equines in Argentina. PMID:26183295

  2. Newcastle disease virus isolates from US waterfowl reveal novel genomic subgroups with diverse and evolving genetics and the potential for spillover into US live bird markets

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Distribution of genetic groups (genogroups) of Newcastle disease virus (NDV) isolated during 1986 to 2005 in the U.S. from 209 waterfowl and shorebirds (W&S) and 17 live bird market (LBM) samples was investigated. Waterfowl and shorebirds viruses were distinct from vaccine viruses and from the viru...

  3. Characterization of an Influenza A Virus Isolated from Pigs During an Outbreak of Respiratory Disease in Swine and People at a County Fair in the United States

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In August 2007, pigs and people became clinically affected by an influenza-like illness during attendance at an Ohio county fair. Influenza A virus was identified from pigs and people, and the virus isolates were characterized as swine H1N1 similar to the swine H1N1 viruses currently circulating in...

  4. Genetic Diversity in the Coat Protein Genes of Prune dwarf virus Isolates from Sweet Cherry Growing in Turkey

    PubMed Central

    Öztürk, Yusuf; Çevik, Bayram

    2015-01-01

    Sweet cherry is an important fruit crop with increasing economical value in Turkey and the world. A number of viruses cause diseases and economical losses in sweet cherry. Prune dwarf virus (PDV), is one of the most common viruses of stone fruits including sweet cherry in the world. In this study, PDV was detected from 316 of 521 sweet cherry samples collected from 142 orchards in 10 districts of Isparta province of Turkey by double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA). The presence of PDV in ELISA positive samples was confirmed in 37 isolates by reverse transcription- polymerase chain reaction (RT-PCR) method. A genomic region of 862 bp containing the coat protein (CP) gene of PDV was re-amplified from 21 selected isolates by RT-PCR. Amplified DNA fragments of these isolates were purified and sequenced for molecular characterization and determining genetic diversity of PDV. Sequence comparisons showed 84–99% to 81–100% sequence identity at nucleotide and amino acid level, respectively, of the CP genes of PDV isolates from Isparta and other parts of the world. Phylogenetic analyses of the CP genes of PDV isolates from different geographical origins and diverse hosts revealed that PDV isolates formed different phylogenetic groups. While isolates were not grouped solely based on their geographical origins or hosts, some association between phylogenetic groups and geographical origins or hosts were observed. PMID:25774109

  5. Full-Genome Sequence Analysis of a Natural Reassortant H4N2 Avian Influenza Virus Isolated from a Domestic Duck in Southern China.

    PubMed

    Wu, Aiqiong; Xie, Zhixun; Xie, Liji; Xie, Zhiqin; Luo, Sisi; Deng, Xianwen; Huang, Li; Huang, Jiaoling; Zeng, Tingting

    2014-01-01

    We report here the complete genome sequence of a novel reassortant H4N2 avian influenza virus strain, A/duck/Guangxi/125D17/2012(H4N2) (GX125D17), isolated from a duck in Guangxi Province, China in 2012. We obtained the complete genome sequence of the GX125D17 virus isolation by PCR, cloning, and sequencing. Sequence analysis revealed that this H4N2 virus strain was a novel reassortant avian in?uenza virus (AIV). Information about the complete genome sequence of the GX125D17 virus strain will be useful for epidemiological studies. PMID:25212619

  6. Full-Genome Sequence Analysis of a Natural Reassortant H4N2 Avian Influenza Virus Isolated from a Domestic Duck in Southern China

    PubMed Central

    Wu, Aiqiong; Xie, Liji; Xie, Zhiqin; Luo, Sisi; Deng, Xianwen; Huang, Li; Huang, Jiaoling; Zeng, Tingting

    2014-01-01

    We report here the complete genome sequence of a novel reassortant H4N2 avian influenza virus strain, A/duck/Guangxi/125D17/2012(H4N2) (GX125D17), isolated from a duck in Guangxi Province, China in 2012. We obtained the complete genome sequence of the GX125D17 virus isolation by PCR, cloning, and sequencing. Sequence analysis revealed that this H4N2 virus strain was a novel reassortant avian in?uenza virus (AIV). Information about the complete genome sequence of the GX125D17 virus strain will be useful for epidemiological studies. PMID:25212619

  7. Complete genome sequencing and evolutionary analysis of Indian isolates of Dengue virus type 2

    SciTech Connect

    Dash, Paban Kumar Sharma, Shashi; Soni, Manisha; Agarwal, Ankita; Parida, Manmohan; Rao, P.V.Lakshmana

    2013-07-05

    Highlights: •Complete genome of Indian DENV-2 was deciphered for the first time in this study. •The recent Indian DENV-2 revealed presence of many unique amino acid residues. •Genotype shift (American to Cosmopolitan) characterizes evolution of DENV-2 in India. •Circulation of a unique clade of DENV-2 in South Asia was identified. -- Abstract: Dengue is the most important arboviral infection of global public health significance. It is now endemic in most parts of the South East Asia including India. Though Dengue virus type 2 (DENV-2) is predominantly associated with major outbreaks in India, complete genome information of Indian DENV-2 is not available. In this study, the full-length genome of five DENV-2 isolates (four from 2001 to 2011 and one from 1960), from different parts of India was determined. The complete genome of the Indian DENV-2 was found to be 10,670 bases long with an open reading frame coding for 3391 amino acids. The recent Indian DENV-2 (2001–2011) revealed a nucleotide sequence identity of around 90% and 97% with an older Indian DENV-2 (1960) and closely related Sri Lankan and Chinese DENV-2 respectively. Presence of unique amino acid residues and non-conservative substitutions in critical amino acid residues of major structural and non-structural proteins was observed in recent Indian DENV-2. Selection pressure analysis revealed positive selection in few amino acid sites of the genes encoding for structural and non-structural proteins. The molecular phylogenetic analysis based on comparison of both complete coding region and envelope protein gene with globally diverse DENV-2 viruses classified the recent Indian isolates into a unique South Asian clade within Cosmopolitan genotype. A shift of genotype from American to Cosmopolitan in 1970s characterized the evolution of DENV-2 in India. Present study is the first report on complete genome characterization of emerging DENV-2 isolates from India and highlights the circulation of a unique clade in South Asia.

  8. Molecular Characterization of Subtype H11N9 Avian Influenza Virus Isolated from Shorebirds in Brazil

    PubMed Central

    Hurtado, Renata; Fabrizio, Thomas; Vanstreels, Ralph Eric Thijl; Krauss, Scott; Webby, Richard J.; Webster, Robert G.; Durigon, Edison Luiz

    2015-01-01

    Migratory aquatic birds play an important role in the maintenance and spread of avian influenza viruses (AIV). Many species of aquatic migratory birds tend to use similar migration routes, also known as flyways, which serve as important circuits for the dissemination of AIV. In recent years there has been extensive surveillance of the virus in aquatic birds in the Northern Hemisphere; however in contrast only a few studies have been attempted to detect AIV in wild birds in South America. There are major flyways connecting South America to Central and North America, whereas avian migration routes between South America and the remaining continents are uncommon. As a result, it has been hypothesized that South American AIV strains would be most closely related to the strains from North America than to those from other regions in the world. We characterized the full genome of three AIV subtype H11N9 isolates obtained from ruddy turnstones (Arenaria interpres) on the Amazon coast of Brazil. For all gene segments, all three strains consistently clustered together within evolutionary lineages of AIV that had been previously described from aquatic birds in North America. In particular, the H11N9 isolates were remarkably closely related to AIV strains from shorebirds sampled at the Delaware Bay region, on the Northeastern coast of the USA, more than 5000 km away from where the isolates were retrieved. Additionally, there was also evidence of genetic similarity to AIV strains from ducks and teals from interior USA and Canada. These findings corroborate that migratory flyways of aquatic birds play an important role in determining the genetic structure of AIV in the Western hemisphere, with a strong epidemiological connectivity between North and South America. PMID:26689791

  9. Development of a surveillance scheme for equine influenza in the UK and characterisation of viruses isolated in Europe, Dubai and the USA from 2010-2012.

    PubMed

    Woodward, Alana L; Rash, Adam S; Blinman, Donna; Bowman, Samantha; Chambers, Thomas M; Daly, Janet M; Damiani, Armando; Joseph, Sunitha; Lewis, Nicola; McCauley, John W; Medcalf, Liz; Mumford, Jenny; Newton, J Richard; Tiwari, Ashish; Bryant, Neil A; Elton, Debra M

    2014-03-14

    Equine influenza viruses are a major cause of respiratory disease in horses worldwide and undergo antigenic drift. Several outbreaks of equine influenza occurred worldwide during 2010-2012, including in vaccinated animals, highlighting the importance of surveillance and virus characterisation. Virus isolates were characterised from more than 20 outbreaks over a 3-year period, including strains from the UK, Dubai, Germany and the USA. The haemagglutinin-1 (HA1) sequence of all isolates was determined and compared with OIE-recommended vaccine strains. Viruses from Florida clades 1 and 2 showed continued divergence from each other compared with 2009 isolates. The antigenic inter-relationships among viruses were determined using a haemagglutination-inhibition (HI) assay with ferret antisera and visualised using antigenic cartography. All European isolates belonged to Florida clade 2, all those from the USA belonged to Florida clade 1. Two subpopulations of clade 2 viruses were isolated, with either substitution A144V or I179V. Isolates from Dubai, obtained from horses shipped from Uruguay, belonged to Florida clade 1 and were similar to viruses isolated in the USA the previous year. The neuraminidase (NA) sequence of representative strains from 2007 and 2009 to 2012 was also determined and compared with that of earlier isolates dating back to 1963. Multiple changes were observed at the amino acid level and clear distinctions could be made between viruses belonging to Florida clade 1 and clade 2. PMID:24480583

  10. Identification of amino acid changes in the envelope glycoproteins of bovine viral diarrhea viruses isolated from alpaca that may be involved in host adaptation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bovine viral diarrhea viruses (BVDV) are most commonly associated with infections of cattle. However, BVDV is often isolated from closely related ruminants with a number of BVDV-1b viruses being isolated from alpacas that were both acutely and persistently infected (PI). The complete nucleotide se...

  11. Genetic characterization of a rare H12N3 avian influenza virus isolated from a green-winged teal in Japan.

    PubMed

    Bui, Vuong Nghia; Ogawa, Haruko; Hussein, Islam T M; Hill, Nichola J; Trinh, Dai Quang; AboElkhair, Mohammed; Sultan, Serageldeen; Ma, Eric; Saito, Keisuke; Watanabe, Yukiko; Runstadler, Jonathan A; Imai, Kunitoshi

    2015-04-01

    This study reports on the genetic characterization of an avian influenza virus, subtype H12N3, isolated from an Eurasian green-winged teal (Anas crecca) in Japan in 2009. The entire genome sequence of the isolate was analyzed, and phylogenetic analyses were conducted to characterize the evolutionary history of the isolate. Phylogenetic analysis of the hemagglutinin and neuraminidase genes indicated that the virus belonged to the Eurasian-like avian lineage. Molecular dating indicated that this H12 virus is likely a multiple reassortant influenza A virus. This is the first reported characterization of influenza A virus subtype H12N3 isolated in Japan and these data contribute to the accumulation of knowledge on the genetic diversity and generation of novel influenza A viruses. PMID:25557930

  12. Complete genome sequence of a banana bract mosaic virus isolate infecting the French plantain cv. Nendran in India.

    PubMed

    Balasubramanian, V; Selvarajan, R

    2012-02-01

    The first complete genome sequence of an Indian isolate (TRY) of Banana bract mosaic virus (BBrMV) was determined following virus RNA extraction from the French plantain cv. Nendran (AAB). The complete genome was 9711 nucleotides excluding the poly(A) tail and had a genome organization similar to that of a Philippine (PHI) isolate characterized earlier. When compared to BBrMV-PHI, the complete genome sequence of BBrMV-TRY was 94% identical at the nucleotide level and its ten mature proteins had amino acid sequence identities ranging from 88 to 98%. Phylogenetic analysis suggests that the BBrMV-TRY isolate is closely related to the BBrMV-PHI isolate. PMID:22134527

  13. Biological and Molecular Characterization of a Korean Isolate of Cucurbit aphid-borne yellows virus Infecting Cucumis Species in Korea

    PubMed Central

    Choi, Seung-Kook; Yoon, Ju-Yeon; Choi, Gug-Seoun

    2015-01-01

    Surveys of yellowing viruses in plastic tunnels and in open field crops of melon (Cucumis melo cultivar catalupo), oriental melon (C. melo cultivar oriental melon), and cucumber (C. sativus) were carried out in two melon-growing areas in 2014, Korea. Severe yellowing symptoms on older leaves of melon and chlorotic spots on younger leaves of melon were observed in the plastic tunnels. The symptoms were widespread and included initial chlorotic lesions followed by yellowing of whole leaves and thickening of older leaves. RT-PCR analysis using total RNA extracted from diseased leaves did not show any synthesized products for four cucurbit-infecting viruses; Beet pseudo-yellows virus, Cucumber mosaic virus, Cucurbit yellows stunting disorder virus, and Melon necrotic spot virus. Virus identification using RT-PCR showed Cucurbit aphid-borne yellows Virus (CABYV) was largely distributed in melon, oriental melon and cucumber. This result was verified by aphid (Aphis gossypii) transmission of CABYV. The complete coat protein (CP) gene amplified from melon was cloned and sequenced. The CP gene nucleotide and the deduced amino acid sequence comparisons as well as phylogenetic tree analysis of CABYV CPs showed that the CABYV isolates were undivided into subgroups. Although the low incidence of CABYV in infections to cucurbit crops in this survey, CABYV may become an important treat for cucurbit crops in many different regions in Korea, suggesting that CABYV should be taken into account in disease control of cucurbit crops in Korea. PMID:26673519

  14. Biological and Molecular Characterization of a Korean Isolate of Cucurbit aphid-borne yellows virus Infecting Cucumis Species in Korea.

    PubMed

    Choi, Seung-Kook; Yoon, Ju-Yeon; Choi, Gug-Seoun

    2015-12-01

    Surveys of yellowing viruses in plastic tunnels and in open field crops of melon (Cucumis melo cultivar catalupo), oriental melon (C. melo cultivar oriental melon), and cucumber (C. sativus) were carried out in two melon-growing areas in 2014, Korea. Severe yellowing symptoms on older leaves of melon and chlorotic spots on younger leaves of melon were observed in the plastic tunnels. The symptoms were widespread and included initial chlorotic lesions followed by yellowing of whole leaves and thickening of older leaves. RT-PCR analysis using total RNA extracted from diseased leaves did not show any synthesized products for four cucurbit-infecting viruses; Beet pseudo-yellows virus, Cucumber mosaic virus, Cucurbit yellows stunting disorder virus, and Melon necrotic spot virus. Virus identification using RT-PCR showed Cucurbit aphid-borne yellows Virus (CABYV) was largely distributed in melon, oriental melon and cucumber. This result was verified by aphid (Aphis gossypii) transmission of CABYV. The complete coat protein (CP) gene amplified from melon was cloned and sequenced. The CP gene nucleotide and the deduced amino acid sequence comparisons as well as phylogenetic tree analysis of CABYV CPs showed that the CABYV isolates were undivided into subgroups. Although the low incidence of CABYV in infections to cucurbit crops in this survey, CABYV may become an important treat for cucurbit crops in many different regions in Korea, suggesting that CABYV should be taken into account in disease control of cucurbit crops in Korea. PMID:26673519

  15. Characterization of sour cherry isolates of plum pox virus from the Volga Basin in Russia reveals a new cherry strain of the virus.

    PubMed

    Glasa, Miroslav; Prikhodko, Yuri; Predaj?a, Lukáš; Nagyová, Alžbeta; Shneyder, Yuri; Zhivaeva, Tatiana; Subr, Zdeno; Cambra, Mariano; Candresse, Thierry

    2013-09-01

    Plum pox virus (PPV) is the causal agent of sharka, the most detrimental virus disease of stone fruit trees worldwide. PPV isolates have been assigned into seven distinct strains, of which PPV-C regroups the genetically distinct isolates detected in several European countries on cherry hosts. Here, three complete and several partial genomic sequences of PPV isolates from sour cherry trees in the Volga River basin of Russia have been determined. The comparison of complete genome sequences has shown that the nucleotide identity values with other PPV isolates reached only 77.5 to 83.5%. Phylogenetic analyses clearly assigned the RU-17sc, RU-18sc, and RU-30sc isolates from cherry to a distinct cluster, most closely related to PPV-C and, to a lesser extent, PPV-W. Based on their natural infection of sour cherry trees and genomic characterization, the PPV isolates reported here represent a new strain of PPV, for which the name PPV-CR (Cherry Russia) is proposed. The unique amino acids conserved among PPV-CR and PPV-C cherry-infecting isolates (75 in total) are mostly distributed within the central part of P1, NIa, and the N terminus of the coat protein (CP), making them potential candidates for genetic determinants of the ability to infect cherry species or of adaptation to these hosts. The variability observed within 14 PPV-CR isolates analyzed in this study (0 to 2.6% nucleotide divergence in partial CP sequences) and the identification of these isolates in different localities and cultivation conditions suggest the efficient establishment and competitiveness of the PPV-CR in the environment. A specific primer pair has been developed, allowing the specific reverse-transcription polymerase chain reaction detection of PPV-CR isolates. PMID:23581702

  16. Comparative full length genome sequence analysis of usutu virus isolates from Africa

    PubMed Central

    2013-01-01

    Background Usutu virus (USUV), a flavivirus belonging to the Japanese encephalitis serocomplex, was identified in South Africa in 1959 and reported for the first time in Europe in 2001. To date, full length genome sequences have been available only for the reference strain from South Africa and a single isolate from each of Austria, Hungary, and Italy. Methods We sequenced four USUV isolates from Senegal and the Central African Republic (CAR) between 1974 and 2007 and compared the sequence data to USUV strains from Austria, Hungary, Italy, and South Africa using a Bayesian Markov chain Monte Carlo method. We further clarified the taxonomic status of a USUV strain isolated in CAR in 1969 and proposed earlier as a subtype of USUV due to an asymetric serological cross-reactivity with USUV reference strain. Results A comparison of the four newly obtained USUV sequences with those from SouthAfrica_1959, Vienna_2001, Budapest_2005, and Italy_2009 revealed that they are all 96-99% and 99% similar at the nucleotide and amino acid levels, respectively. The phylogenetic relationships between these sequences indicated that a strain isolated in Senegal in 1993 is most closely related to the USUV strains detected in Europe. Analysis of a strain isolated from a human in CAR in 1981 (CAR_1981) revealed the presence of specific amino acid substitutions and a deletion in the 3? noncoding region. This is the first fully sequenced human USUV isolate. The putative USUV subtype, CAR_1969, was 81% and 94% identical at the nucleotide and amino acid levels, respectively, compared to the other USUV strains. Our phylogenetic analyses support the serological identification of CAR_1969 as a subtype of USUV. Conclusions In this study, we investigate the genetic diversity of USUV in Africa and the phylogenetic relationship of isolates from Africa and Europe for the first time. The results suggest a low genetic diversity within USUV, the existence of a distinct USUV subtype strain, and support the hypothesis that USUV was introduced to Europe from Africa. Further sequencing and analysis of USUV isolates from other African countries would contribute to a better understanding of its genetic diversity and geographic distribution. PMID:23816256

  17. Detection and Characterization of Clade 1 Reassortant H5N1 Viruses Isolated from Human Cases in Vietnam during 2013

    PubMed Central

    Balish, Amanda; Hoang, Anh Nguyen; Gustin, Kortney M.; Nhung, Pham Thi; Jones, Joyce; Thu, Ngoc Nguyen; Davis, William; Ngoc, Thao Nguyen Thi; Jang, Yunho; Sleeman, Katrina; Villanueva, Julie; Kile, James; Gubareva, Larisa V.; Lindstrom, Stephen; Tumpey, Terrence M.; Davis, C. Todd; Long, Nguyen Thanh

    2015-01-01

    Highly pathogenic avian influenza (HPAI) H5N1 is endemic in Vietnamese poultry and has caused sporadic human infection in Vietnam since 2003. Human infections with HPAI H5N1 are of concern due to a high mortality rate and the potential for the emergence of pandemic viruses with sustained human-to-human transmission. Viruses isolated from humans in southern Vietnam have been classified as clade 1 with a single genome constellation (VN3) since their earliest detection in 2003. This is consistent with detection of this clade/genotype in poultry viruses endemic to the Mekong River Delta and surrounding regions. Comparison of H5N1 viruses detected in humans from southern Vietnamese provinces during 2012 and 2013 revealed the emergence of a 2013 reassortant virus with clade 1.1.2 hemagglutinin (HA) and neuraminidase (NA) surface protein genes but internal genes derived from clade 2.3.2.1a viruses (A/Hubei/1/2010-like; VN12). Closer analysis revealed mutations in multiple genes of this novel genotype (referred to as VN49) previously associated with increased virulence in animal models and other markers of adaptation to mammalian hosts. Despite the changes identified between the 2012 and 2013 genotypes analyzed, their virulence in a ferret model was similar. Antigenically, the 2013 viruses were less cross-reactive with ferret antiserum produced to the clade 1 progenitor virus, A/Vietnam/1203/2004, but reacted with antiserum produced against a new clade 1.1.2 WHO candidate vaccine virus (A/Cambodia/W0526301/2012) with comparable hemagglutination inhibition titers as the homologous antigen. Together, these results indicate changes to both surface and internal protein genes of H5N1 viruses circulating in southern Vietnam compared to 2012 and earlier viruses. PMID:26244768

  18. Identification of a Genotype VIId Newcastle Disease Virus Isolated from Sansui Sheldrake Ducks in Guizhou Province, China

    PubMed Central

    Ji, Xinqin; Xu, Houqiang; Zhao, Jiafu; Ruan, Yong; Chen, Jiaqi

    2015-01-01

    In this study, we report the complete genome sequence of a novel Newcastle disease virus (NDV) strain, Sheldrake duck/China/Guizhou/SS1/2014, isolated from Sansui Sheldrake duck flocks in Guizhou Province, southwestern China. The genome of this isolate is 15,192 nucleotides in length, which belongs to NDV genotype VIId in class II. This discovery will help us further study the epidemiology characteristics and molecular pathogenesis of genotype VIId NDV in Sansui Sheldrake ducks. PMID:25858828

  19. Phylogenetic analysis of a swine influenza A(H3N2) virus isolated in Korea in 2012.

    PubMed

    Kim, Jin Il; Lee, Ilseob; Park, Sehee; Lee, Sangmoo; Hwang, Min-Woong; Bae, Joon-Yong; Heo, Jun; Kim, Donghwan; Jang, Seok-Il; Kim, Kabsu; Park, Man-Seong

    2014-01-01

    Influenza A virus (IAV) can infect avian and mammalian species, including humans. The genome nature of IAVs may contribute to viral adaptation in different animal hosts, resulting in gene reassortment and the reproduction of variants with optimal fitness. As seen again in the 2009 swine-origin influenza A H1N1 pandemic, pigs are known to be susceptible to swine, avian, and human IAVs and can serve as a 'mixing vessel' for the generation of novel IAV variants. To this end, the emergence of swine influenza viruses must be kept under close surveillance. Herein, we report the isolation and phylogenetic study of a swine IAV, A/swine/Korea/PL01/2012 (swPL01, H3N2 subtype). After screening nasopharyngeal samples from pigs in the Gyeongsangnam-do region of Korea from December 2011 to May 2012, we isolated the swPL01 virus and sequenced its all of 8 genome segments (polymerase basic 2, PB2; polymerase basic 1, PB1; polymerase acidic, PA; hemagglutinin, HA; nucleocapsid protein, NP; neuraminidase, NA; matrix protein, M; and nonstructural protein, NS). The phylogenetic study, analyzed with reference strains registered in the National Center for Biotechnology Information (NCBI) database, indicated that the swPL01 virus was similar to the North American triple-reassortant swine strains and that the HA gene of the swPL01 virus was categorized into swine H3 cluster IV. The swPL01 virus had the M gene of the triple-reassortant swine H3N2 viruses, whereas that of other contemporary strains in Korea was transferred from the 2009 pandemic H1N1 virus. These data suggest the possibility that various swine H3N2 viruses may co-circulate in Korea, which underlines the importance of a sustained surveillance system against swine IAVs. PMID:24523938

  20. ISOLATE-SPECIFIC SYNERGY IN DEISEASE SYMPTOMS BETWEEN CAULIFLOWER MOSAIC AND TURNIP-CLEARING VIRUSES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Simultaneous infection of a plant by two viruses can cause more severe disease than is caused by infection with either virus alone. Such synergy may be due to effects on the replication of one virus by the second virus or to other causes. The tobamovirus turnip vein-clearing virus (TVCV), itself cau...

  1. Molecular characterization of highly pathogenic H5N1 avian influenza viruses isolated in Sweden in 2006

    PubMed Central

    Kiss, István; Gyarmati, Péter; Zohari, Siamak; Ramsay, Karin Wilbe; Metreveli, Giorgi; Weiss, Elisabeth; Brytting, Maria; Stivers, Marielle; Lindström, Sofia; Lundkvist, Ake; Nemirov, Kirill; Thorén, Peter; Berg, Mikael; Czifra, György; Belák, Sándor

    2008-01-01

    Background The analysis of the nonstructural (NS) gene of the highly pathogenic (HP) H5N1 avian influenza viruses (AIV) isolated in Sweden early 2006 indicated the co-circulation of two sub-lineages of these viruses at that time. In order to complete the information on their genetic features and relation to other HP H5N1 AIVs the seven additional genes of twelve Swedish isolates were amplified in full length, sequenced, and characterized. Results The presence of two sub-lineages of HP H5N1 AIVs in Sweden in 2006 was further confirmed by the phylogenetic analysis of approximately the 95% of the genome of twelve isolates that were selected on the base of differences in geographic location, timing and animal species of origin. Ten of the analyzed viruses belonged to sub-clade 2.2.2. and grouped together with German and Danish isolates, while two 2.2.1. sub-clade viruses formed a cluster with isolates of Egyptian, Italian, Slovenian, and Nigerian origin. The revealed amino acid differences between the two sub-groups of Swedish viruses affected the predicted antigenicity of the surface glycoproteins, haemagglutinin and neuraminidase, rather than the nucleoprotein, polymerase basic protein 2, and polymerase acidic protein, the main targets of the cellular immune responses. The distinctive characteristics between members of the two subgroups were identified and described. Conclusion The comprehensive genetic characterization of HP H5N1 AIVs isolated in Sweden during the spring of 2006 is reported. Our data support previous findings on the coincidental spread of multiple sub-lineage H5N1 HPAIVs via migrating aquatic birds to large distance from their origin. The detection of 2.2.1. sub-clade viruses in Sweden adds further data regarding their spread in the North of Europe in 2006. The close genetic relationship of Swedish isolates sub-clade 2.2.2. to the contemporary German and Danish isolates supports the proposition of the introduction and spread of a single variant of 2.2.2. sub-clade H5N1 avian influenza viruses in the Baltic region. The presented findings underline the importance of whole genome analysis. PMID:18837987

  2. Genotyping of Cucumber mosaic virus isolates in western New York State during epidemic years: Characterization of an emergent plant virus population.

    PubMed

    Thompson, Jeremy R; Langenhan, Jamie L; Fuchs, Marc; Perry, Keith L

    2015-12-01

    In the early 2000s an epidemic of cucumber mosaic virus (CMV) spread within the Midwestern and Eastern US affecting snap and dry bean (Phaseolus vulgaris L.) cultivation. Fifty one CMV isolates from this period were partially characterized from varied hosts by sequencing a section from each of the three genomic RNAs. Aside from one subgroup II strain from pepper, all isolates, including those from snap bean, fell within the IA subgroup. The nucleotide sequence diversity of virus populations sampled at multiple sites and at different years was significantly higher than that of a population from single site in a single year, although in general the number of polymorphisms was low (<11%). Complementary DNA (cDNA) clones of Bn57, a representative isolate from snap bean, were engineered for the production of infectious in vitro RNA transcripts initiated from a T7 promoter. Infections from these cDNAs resulted in symptoms consistent with those of the original field isolate, indicating that a satellite RNA is not involved in symptom expression in snap bean. These infectious clones were used to assess symptom determinants and the effects of virus infection on plant growth. Inoculations with pseudorecombinants derived from Bn57 and the non-bean infecting strain Fny confirmed RNA2 as a specific determinant for snap bean infection. Bn57, along with almost all isolates identified in this study contained the Y631 locus in the 2a protein, a determinant for systemic infection in bean. The presence of this locus extended to all non-bean hosts except two pepper infecting isolates. Infection by Bn57 in snap bean had a significant effect on pod number and mass with a 55 and 41 percent reduction in greenhouse assays, respectively. To our knowledge Bn57 is the first CMV strain isolated from P. vulgaris to be fully sequenced and cloned, providing a useful tool for analyses of CMV-host interactions. PMID:26254084

  3. Avian Influenza Virus with Hemagglutinin-Neuraminidase Combination H3N6, Isolated from a Domestic Pigeon in Guangxi, Southern China

    PubMed Central

    Liu, Tingting; Song, Degui; Huang, Li; Xie, Zhiqing; Deng, Xianweng; Luo, Sisi; Huang, Jiaoling; Zeng, Tingting

    2015-01-01

    The H3 subtype of avian influenza virus can provide genes for human influenza virus through gene reassortment, which has raised great concerns about its potential threat to human health. An H3N6 subtype of avian influenza virus was isolated from Guangxi Province, China, in 2009. All eight gene segments of the strain were sequenced. The sequence analysis indicated that this H3N6 virus was a nature reassortant virus. The genome sequences now can be used to understand the epidemiological and molecular characteristics of the H3N6 influenza virus in southern China. PMID:25657287

  4. Molecular characterization and phylogenetics of a reassortant H13N8 influenza virus isolated from gulls in Mongolia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A double reassortant H13N8 influenza A virus was isolated from gulls in Mongolia. The basic virological characteristics were studied. Complete genome sequence analysis indicated the complicated evolutionary history. The PA gene belongs to classical Avian-like lineage and more likely originated fro...

  5. Complete Genome Sequence of an Emerging Melon Necrotic Spot Virus Isolate Infecting Greenhouse Cucumber in North America

    PubMed Central

    Li, Rugang; Zheng, Yi; Fei, Zhangjun

    2015-01-01

    The complete genome sequence (4,267 nt) of a Melon necrotic spot virus (MNSV) isolate (ABCA13-01) infecting greenhouse cucumber in Canada was determined through deep sequencing of small RNAs. Its genome sequence was most closely related to MNSV-N (97%) but lacked a 55-nucleotide insertion at the 3? untranslated region for resistance breaking. PMID:26184937

  6. Characterization and Sequencing of a Genotype VIId Newcastle Disease Virus Isolated from Laying Ducks in Jiangsu, China.

    PubMed

    Liu, Mei; Shen, Xinyue; Cheng, Xu; Li, Jianmei; Dai, Yabin

    2015-01-01

    We report here the complete genome sequence and biological characterization of a virulent Newcastle disease virus (NDV) strain, NDV/duck/Jiangsu/JSD0812/2008, isolated from laying ducks in Jiangsu Province, China. The genome is 15,192 nucleotides in length and is classified in subgenotype VIId of genotype VII, class II. PMID:26634760

  7. Isolation of a recent Korean epizootic strain of Newcastle disease virus from Eurasian Scops Owls affected with severe diarrhea.

    PubMed

    Choi, Kang-Seuk; Lee, Eun-Kyoung; Jeon, Woo-Jin; Nah, Jin-Ju; Kim, Young-Jun; Lee, Mu-Yeong; Lee, Hang; Kwon, Jun-Hun

    2008-01-01

    Velogenic Newcastle disease virus (NDV) was recovered from two dead Eurasian Scops Owls (Otus scops) from a wildlife rescue center in Korea during 2005. Phylogenetic analysis based on the sequence of the partial fusion (F) protein revealed that the isolates had the highest level of homology to recent Korean NDV strains from poultry. PMID:18263840

  8. Complete genome sequence of an Indian field isolate of classical Swine Fever virus belonging to subgenotype 1.1.

    PubMed

    Kamboj, Aman; Patel, Chhabi L; Chaturvedi, V K; Saini, Mohini; Gupta, Praveen K

    2014-01-01

    We report the complete genome sequence of an Indian field isolate of classical swine fever virus (CSFV) belonging to predominant subgenotype 1.1 prevalent in India. This report will help in understanding the molecular diversity of CSFV strains circulating worldwide and to select and develop a suitable vaccine candidate for classical swine fever (CSF) control in India. PMID:25278522

  9. Characterization and Sequencing of a Genotype VIId Newcastle Disease Virus Isolated from Laying Ducks in Jiangsu, China

    PubMed Central

    Liu, Mei; Shen, Xinyue; Cheng, Xu; Li, Jianmei

    2015-01-01

    We report here the complete genome sequence and biological characterization of a virulent Newcastle disease virus (NDV) strain, NDV/duck/Jiangsu/JSD0812/2008, isolated from laying ducks in Jiangsu Province, China. The genome is 15,192 nucleotides in length and is classified in subgenotype VIId of genotype VII, class II. PMID:26634760

  10. Characterization of rabies virus isolated from a colony of Eptesicus furinalis bats in Brazil.

    PubMed

    Almeida, Marilene Fernandes de; Favoretto, Silvana R; Martorelli, Luzia F Alves; Trezza-Netto, José; Campos, Angélica Cristine de Almeida; Ozahata, Carlos H; Sodré, Miriam Martos; Kataoka, Ana Paula A G; Sacramento, Débora R Veiga; Durigon, Edison L

    2011-01-01

    Some bat species have adapted to the expanding human population by acquiring the ability to roost in urban buildings, increasing the exposure risk for people and domestic animals, and consequently, the likelihood of transmitting rabies. Three dead bats were found in the yard of a house in an urban area of Jundiaí city in the state of São Paulo in southeast Brazil. Two of the three bats tested positive for rabies, using Fluorescent Antibody and Mouse Inoculation techniques. A large colony of Eptesicus furinalis was found in the house's attic, and of the 119 bats captured, four more tested positive for rabies. The objectives of this study were to report the rabies diagnosis, characterize the isolated virus antigenically and genetically, and study the epidemiology of the colony. PMID:21412617

  11. Broome virus, a new fusogenic Orthoreovirus species isolated from an Australian fruit bat

    SciTech Connect

    Thalmann, Claudia M.; Cummins, David Michael; Yu Meng; Lunt, Ross; Pritchard, Lindsay Ian; Hansson, Eric; Crameri, Sandra; Hyatt, Alex; Wang Linfa

    2010-06-20

    This report describes the discovery and characterization of a new fusogenic orthoreovirus, Broome virus (BroV), isolated from a little red flying-fox (Pteropus scapulatus). The BroV genome consists of 10 dsRNA segments, each having a 3' terminal pentanucleotide sequence conserved amongst all members of the genus Orthoreovirus, and a unique 5' terminal pentanucleotide sequence. The smallest genome segment is bicistronic and encodes two small nonstructural proteins, one of which is a novel fusion associated small transmembrane (FAST) protein responsible for syncytium formation, but no cell attachment protein. The low amino acid sequence identity between BroV proteins and those of other orthoreoviruses (13-50%), combined with phylogenetic analyses of structural and nonstructural proteins provide evidence to support the classification of BroV in a new sixth species group within the genus Orthoreovirus.

  12. Genomic sequence and virulence of clonal isolates of vaccinia virus Tiantan, the Chinese smallpox vaccine strain.

    PubMed

    Zhang, Qicheng; Tian, Meijuan; Feng, Yi; Zhao, Kai; Xu, Jing; Liu, Ying; Shao, Yiming

    2013-01-01

    Despite the worldwide eradication of smallpox in 1979, the potential bioterrorism threat from variola virus and the ongoing use of vaccinia virus (VACV) as a vector for vaccine development argue for continued research on VACV. In China, the VACV Tiantan strain (TT) was used in the smallpox eradication campaign. Its progeny strain is currently being used to develop a human immunodeficiency virus (HIV) vaccine. Here we sequenced the full genomes of five TT clones isolated by plaque purification from the TT (752-1) viral stock. Phylogenetic analysis with other commonly used VACV strains showed that TT (752-1) and its clones clustered and exhibited higher sequence diversity than that found in Dryvax clones. The ?190 kbp genomes of TT appeared to encode 273 open reading frames (ORFs). ORFs located in the middle of the genome were more conserved than those located at the two termini, where many virulence and immunomodulation associated genes reside. Several patterns of nucleotide changes including point mutations, insertions and deletions were identified. The polymorphisms in seven virulence-associated proteins and six immunomodulation-related proteins were analyzed. We also investigated the neuro- and skin- virulence of TT clones in mice and rabbits, respectively. The TT clones exhibited significantly less virulence than the New York City Board of Health (NYCBH) strain, as evidenced by less extensive weight loss and morbidity in mice as well as produced smaller skin lesions and lower incidence of putrescence in rabbits. The complete genome sequences, ORF annotations, and phenotypic diversity yielded from this study aid our understanding of the Chinese historic TT strain and are useful for HIV vaccine projects employing TT as a vector. PMID:23593246

  13. Identification and characterization of a novel lymphocystis disease virus isolate from cultured grouper in China.

    PubMed

    Huang, X; Huang, Y; Xu, L; Wei, S; Ouyang, Z; Feng, J; Qin, Q

    2015-04-01

    Grouper Epinephelus spp. is one of the most important mariculture fish species in China and South-East Asian countries. The emerging viral diseases, evoked by iridovirus which belongs to genus Megalocytivirus and Ranavirus, have been well characterized in recent years. To date, few data on lymphocystis disease in grouper which caused by lymphocystis disease virus (LCDV) were described. Here, a novel LCDV isolate was identified and characterized. Based on the sequence of LCDV major capsid protein (MCP) and DNA polymerase gene, we found that the causative agents from different species of diseased groupers were the same one and herein were uniformly defined as grouper LCDV (GLCDV). Furthermore, H&E staining revealed that the nodules on the skin were composed of giant cells that contained inclusion bodies in the cytoplasm. Numerous virus particles with >210 nm in diameter and with hexagonal profiles were observed in the cytoplasm. In addition, phylogenetic analysis based on four iridovirus core genes, MCP, DNA polymerase, myristoylated membrane protein (MMP) and ribonucleotide reductase (RNR), consistently showed that GLCDV was mostly related to LCDV-C, followed by LCDV-1. Taken together, our data firstly provided the molecular evidence that GLCDV was a novel emerging iridovirus pathogen in grouper culture. PMID:24720572

  14. [Molecular identification and sequence analysis of broad bean wilt virus 2 isolates from atractylodes macrocephala Koidz].

    PubMed

    Niu, Yanbing; Shi, Xiaoli; Zhang, Ximei; Zhao, Huiqi; Zhao, Baojia

    2015-01-01

    To identity the pathogen that causes the mosaic and yellowing symptoms on Atractylodes macrocephala Koidz in Jiangxian, Shanxi province, biological inoculation, sequence-independent amplification (SIA),RT-PCR and other identification methods were used. The results showed that the chlorotic and necrosis symptoms occurred in the indicator plant Chenopodium quinoa after it was infected with the pathogen,and the same symptoms appeared after the reinoculation of healthy Atractylodes macrocephala Koidz; this reflected that the disease was likely to be caused by a virus. The results of SIA and sequencing showed that Broad bean wilt virus 2 (BBWV2) was present in severely mosaic Atractylodes macrocephala Koidz leaves. To further characterize the BBWV2 isolate from Atractylodes macrocephala (BBWV2-Am), the polyprotein partial gene encoded by BBWV2-Am RNA2 was cloned and sequenced. Sequence alignments showed that the nucleotide sequence identity of BBWV2-Am SCP and LCP genes ranged from 79.3% to 87.2% and from 80.1% to 89.2% compared to other BBWV2 strains,respectively; the deduced amino acid sequence similarities of the two gene products ranged from 91.2% to 95.7% and from 89.44 to 95.5%, respectively,compared to those of other BBWV2 strains. Phylogenetic comparisons showed that BBWV2-Am was most likely to be related to BBWV2-Rg,but formed an independent branch. This is the first report of BBWV2 in Atractylodes macrocephala Koidz. PMID:25997332

  15. Molecular characterization of pig epidemic diarrhoea viruses isolated in Japan from 2013 to 2014.

    PubMed

    Suzuki, Tohru; Murakami, Satoshi; Takahashi, Osamu; Kodera, Aya; Masuda, Tsuneyuki; Itoh, Sakie; Miyazaki, Ayako; Ohashi, Seiichi; Tsutsui, Toshiyuki

    2015-12-01

    Since October 2013, approximately 1000 outbreaks of porcine epidemic diarrhoea (PED) have occurred, spanning almost all prefectures of Japan, after a period of seven years without a reported case. In order to consider occurrence factor of PED outbreaks, we determined the whole-genome sequences of 38 PED virus (PEDV) strains from diarrheal samples collected at swine farms in 18 prefectures between 2013 and 2014 using next-generation sequencing technology. Using these data, we investigated genetic variation among the recent Japanese PEDV strains and the genetic relationships between these strains and global PEDV strains isolated recently from multiple swine-industrial countries. Eleven out of 38 PEDV strains were isolated successfully on Vero cells with trypsin treatment and subjected to genome sequence analysis. In a comparative genome analysis, we detected two novel PEDV variants, TTR-2/JPN/2014 and MYG-1/JPN/2014, with large deletions in the spike and ORF3 genes, respectively. A phylogenetic analysis based on the spike gene showed that the 38 Japanese PEDV strains were classified into two PEDV types: the North American type with high virulence (n=34) and the INDEL type (n=4). In addition, the recent Japanese PEDV isolates had a close relationship to global PEDV strains isolated in recent years than to the classical PEDV strains detected in Japan the past decades ago. Moreover, the phylogenetic dendrogram of the complete genomes also indicated that the 38 Japanese PEDV strains, including the two novel PEDV variants discovered in this study, are closely related to the PEDV strains that were widespread in the United States and Korea in 2013-2014. These findings suggest that the re-emergence of PED outbreaks since the last reported case in 2006 was caused by the introduction of recent PEDV strains to Japan from overseas. PMID:26477934

  16. Two different genotypes of H1N2 swine influenza virus isolated in northern China and their pathogenicity in animals.

    PubMed

    Yang, Huanliang; Chen, Yan; Qiao, Chuanling; Xu, Chuantian; Yan, Minghua; Xin, Xiaoguang; Bu, Zhigao; Chen, Hualan

    2015-02-25

    During 2006 and 2007, two swine-origin triple-reassortant influenza A (H1N2) viruses were isolated from pigs in northern China, and the antigenic characteristics of the hemagglutinin protein of the viruses were examined. Genotyping and phylogenetic analyses demonstrated different emergence patterns for the two H1N2 viruses, Sw/Hebei/10/06 and Sw/Tianjin/1/07. Sequences for the other genes encoding the internal proteins were compared with the existing data to determine their origins and establish the likely mechanisms of genetic reassortment. Sw/Hebei/10/06 is an Sw/Indiana/9K035/99-like virus, whereas Sw/Tianjin/1/07 represents a new H1N2 genotype with surface genes of classic swine and human origin and internal genes originating from the Eurasian avian-like swine H1N1 virus. Six-week-old female BALB/c mice infected with the Sw/HeB/10/06 and Sw/TJ/1/07 viruses showed an average weight loss of 12.8% and 8.1%, respectively. Healthy six-week-old pigs were inoculated intranasally with either the Sw/HeB/10/06 or Sw/TJ/1/07 virus. No considerable changes in the clinical presentation were observed post-inoculation in any of the virus-inoculated groups, and the viruses effectively replicated in the nasal cavity and lung tissue. Based on the results, it is possible that the new genotype of the swine H1N2 virus that emerged in China may become widespread in the swine population and pose a potential threat to public health. PMID:25542286

  17. Natural compounds isolated from Brazilian plants are potent inhibitors of hepatitis C virus replication in vitro

    PubMed Central

    Jardim, A.C.G.; Igloi, Z.; Shimizu, J.F.; Santos, V.A.F.F.M.; Felippe, L.G.; Mazzeu, B.F.; Amako, Y.; Furlan, M.; Harris, M.; Rahal, P.

    2015-01-01

    Compounds extracted from plants can provide an alternative approach to new therapies. They present characteristics such as high chemical diversity, lower cost of production and milder or inexistent side effects compared with conventional treatment. The Brazilian flora represents a vast, largely untapped, resource of potential antiviral compounds. In this study, we investigate the antiviral effects of a panel of natural compounds isolated from Brazilian plants species on hepatitis C virus (HCV) genome replication. To do this we used firefly luciferase-based HCV sub-genomic replicons of genotypes 2a (JFH-1), 1b and 3a and the compounds were assessed for their effects on both HCV replication and cellular toxicity. Initial screening of compounds was performed using the maximum non-toxic concentration and 4 compounds that exhibited a useful therapeutic index (favourable ratio of cytotoxicity to antiviral potency) were selected for extra analysis. The compounds APS (EC50 = 2.3 ?M), a natural alkaloid isolated from Maytrenus ilicifolia, and the lignans 3?43 (EC50 = 4.0 ?M), 3?20 (EC50 = 8.2 ?M) and 5?362 (EC50 = 38.9 ?M) from Peperomia blanda dramatically inhibited HCV replication as judged by reductions in luciferase activity and HCV protein expression in both the subgenomic and infectious systems. We further show that these compounds are active against a daclatasvir resistance mutant subgenomic replicon. Consistent with inhibition of genome replication, production of infectious JFH-1 virus was significantly reduced by all 4 compounds. These data are the first description of Brazilian natural compounds possessing anti-HCV activity and further analyses are being performed in order to investigate the mode of action of those compounds. PMID:25557602

  18. A novel single-stranded RNA virus isolated from a phytopathogenic filamentous fungus, Rosellinia necatrix, with similarity to hypo-like viruses.

    PubMed

    Zhang, Rui; Liu, Shengxue; Chiba, Sotaro; Kondo, Hideki; Kanematsu, Satoko; Suzuki, Nobuhiro

    2014-01-01

    Here we report a biological and molecular characterization of a novel positive-sense RNA virus isolated from a field isolate (NW10) of a filamentous phytopathogenic fungus, the white root rot fungus that is designated as Rosellinia necatrix fusarivirus 1 (RnFV1). A recently developed technology using zinc ions allowed us to transfer RnFV1 to two mycelially incompatible Rosellinia necatrix strains. A biological comparison of the virus-free and -recipient isogenic fungal strains suggested that RnFV1 infects latently and thus has no potential as a virocontrol agent. The virus has an undivided positive-sense RNA genome of 6286 nucleotides excluding a poly (A) tail. The genome possesses two non-overlapping open reading frames (ORFs): a large ORF1 that encodes polypeptides with RNA replication functions and a smaller ORF2 that encodes polypeptides of unknown function. A lack of coat protein genes was suggested by the failure of virus particles from infected mycelia. No evidence was obtained by Northern analysis or classical 5'-RACE for the presence of subgenomic RNA for the downstream ORF. Sequence similarities were found in amino-acid sequence between RnFV1 putative proteins and counterparts of a previously reported mycovirus, Fusarium graminearum virus 1 (FgV1). Interestingly, several related sequences were detected by BLAST searches of independent transcriptome assembly databases one of which probably represents an entire virus genome. Phylogenetic analysis based on the conserved RNA-dependent RNA polymerase showed that RnFV1, FgV1, and these similar sequences are grouped in a cluster distinct from distantly related hypoviruses. It is proposed that a new taxonomic family termed Fusariviridae be created to include RnFV1 and FgV1. PMID:25101066

  19. A novel single-stranded RNA virus isolated from a phytopathogenic filamentous fungus, Rosellinia necatrix, with similarity to hypo-like viruses

    PubMed Central

    Zhang, Rui; Liu, Shengxue; Chiba, Sotaro; Kondo, Hideki; Kanematsu, Satoko; Suzuki, Nobuhiro

    2014-01-01

    Here we report a biological and molecular characterization of a novel positive-sense RNA virus isolated from a field isolate (NW10) of a filamentous phytopathogenic fungus, the white root rot fungus that is designated as Rosellinia necatrix fusarivirus 1 (RnFV1). A recently developed technology using zinc ions allowed us to transfer RnFV1 to two mycelially incompatible Rosellinia necatrix strains. A biological comparison of the virus-free and -recipient isogenic fungal strains suggested that RnFV1 infects latently and thus has no potential as a virocontrol agent. The virus has an undivided positive-sense RNA genome of 6286 nucleotides excluding a poly (A) tail. The genome possesses two non-overlapping open reading frames (ORFs): a large ORF1 that encodes polypeptides with RNA replication functions and a smaller ORF2 that encodes polypeptides of unknown function. A lack of coat protein genes was suggested by the failure of virus particles from infected mycelia. No evidence was obtained by Northern analysis or classical 5?-RACE for the presence of subgenomic RNA for the downstream ORF. Sequence similarities were found in amino-acid sequence between RnFV1 putative proteins and counterparts of a previously reported mycovirus, Fusarium graminearum virus 1 (FgV1). Interestingly, several related sequences were detected by BLAST searches of independent transcriptome assembly databases one of which probably represents an entire virus genome. Phylogenetic analysis based on the conserved RNA-dependent RNA polymerase showed that RnFV1, FgV1, and these similar sequences are grouped in a cluster distinct from distantly related hypoviruses. It is proposed that a new taxonomic family termed Fusariviridae be created to include RnFV1 and FgV1. PMID:25101066

  20. Phylogenetic and pathogenic analysis of a novel H6N2 avian influenza virus isolated from a green peafowl in a wildlife park.

    PubMed

    Fan, Zhaobin; Ci, Yanpeng; Ma, Yixin; Liu, Liling; Ma, Jianzhang; Li, D Yanbing; Chen, Hualan

    2014-12-01

    H6 subtype avian influenza virus, which has been circulating among different species, causes considerable concern for both veterinary medicine and public health. We isolated a strain of H6N2 avian influenza virus from healthy green peafowl (Pavo muticus) in Qinghuangdao Wildlife Park in Hebei Province, China, in 2012. A phylogenetic analysis indicated that the isolated H6N2 strain had the same gene constellation as southern China strains, which were predominantly isolated from waterfowl distributed in Shantou, Guangxi, and Hunan in 2001-2010. The isolate showed no and low pathogenicity in chickens and ducks, respectively. However, it replicated efficiently in the lungs and turbinate of infected mice, resulting in thickened alveolar septa and moderate interstitial pneumonia. This finding raises concerns that the H6N2 subtype maybe evolve into a novel endemic avian influenza virus. Therefore, periodical surveillance of avian influenza viruses must be undertaken to monitor the advent of novel viruses. PMID:25619010

  1. Characterization of Synthetic Chikungunya Viruses Based on the Consensus Sequence of Recent E1-226V Isolates

    PubMed Central

    Scholte, Florine E. M.; Tas, Ali; Martina, Byron E. E.; Cordioli, Paolo; Narayanan, Krishna; Makino, Shinji; Snijder, Eric J.; van Hemert, Martijn J.

    2013-01-01

    Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that re-emerged in 2004 and has caused massive outbreaks in recent years. The lack of a licensed vaccine or treatment options emphasize the need to obtain more insight into the viral life cycle and CHIKV-host interactions. Infectious cDNA clones are important tools for such studies, and for mechanism of action studies on antiviral compounds. Existing CHIKV cDNA clones are based on a single genome from an individual clinical isolate, which is expected to have evolved specific characteristics in response to the host environment, and possibly also during subsequent cell culture passaging. To obtain a virus expected to have the general characteristics of the recent E1-226V CHIKV isolates, we have constructed a new CHIKV full-length cDNA clone, CHIKV LS3, based on the consensus sequence of their aligned genomes. Here we report the characterization of this synthetic virus and a green fluorescent protein-expressing variant (CHIKV LS3-GFP). Their characteristics were compared to those of natural strain ITA07-RA1, which was isolated during the 2007 outbreak in Italy. In cell culture the synthetic viruses displayed phenotypes comparable to the natural isolate, and in a mouse model they caused lethal infections that were indistinguishable from infections with a natural strain. Compared to ITA07-RA1 and clinical isolate NL10/152, the synthetic viruses displayed similar sensitivities to several antiviral compounds. 3-deaza-adenosine was identified as a new inhibitor of CHIKV replication. Cyclosporin A had no effect on CHIKV replication, suggesting that cyclophilins -opposite to what was found for other +RNA viruses- do not play an essential role in CHIKV replication. The characterization of the consensus sequence-based synthetic viruses and their comparison to natural isolates demonstrated that CHIKV LS3 and LS3-GFP are suitable and representative tools to study CHIKV-host interactions, screen for antiviral compounds and unravel their mode of action. PMID:23936484

  2. Characterization of synthetic Chikungunya viruses based on the consensus sequence of recent E1-226V isolates.

    PubMed

    Scholte, Florine E M; Tas, Ali; Martina, Byron E E; Cordioli, Paolo; Narayanan, Krishna; Makino, Shinji; Snijder, Eric J; van Hemert, Martijn J

    2013-01-01

    Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that re-emerged in 2004 and has caused massive outbreaks in recent years. The lack of a licensed vaccine or treatment options emphasize the need to obtain more insight into the viral life cycle and CHIKV-host interactions. Infectious cDNA clones are important tools for such studies, and for mechanism of action studies on antiviral compounds. Existing CHIKV cDNA clones are based on a single genome from an individual clinical isolate, which is expected to have evolved specific characteristics in response to the host environment, and possibly also during subsequent cell culture passaging. To obtain a virus expected to have the general characteristics of the recent E1-226V CHIKV isolates, we have constructed a new CHIKV full-length cDNA clone, CHIKV LS3, based on the consensus sequence of their aligned genomes. Here we report the characterization of this synthetic virus and a green fluorescent protein-expressing variant (CHIKV LS3-GFP). Their characteristics were compared to those of natural strain ITA07-RA1, which was isolated during the 2007 outbreak in Italy. In cell culture the synthetic viruses displayed phenotypes comparable to the natural isolate, and in a mouse model they caused lethal infections that were indistinguishable from infections with a natural strain. Compared to ITA07-RA1 and clinical isolate NL10/152, the synthetic viruses displayed similar sensitivities to several antiviral compounds. 3-deaza-adenosine was identified as a new inhibitor of CHIKV replication. Cyclosporin A had no effect on CHIKV replication, suggesting that cyclophilins -opposite to what was found for other +RNA viruses- do not play an essential role in CHIKV replication. The characterization of the consensus sequence-based synthetic viruses and their comparison to natural isolates demonstrated that CHIKV LS3 and LS3-GFP are suitable and representative tools to study CHIKV-host interactions, screen for antiviral compounds and unravel their mode of action. PMID:23936484

  3. Suppression of Resistance-breaking Beet Necrotic Yellow Vein Virus Isolates by Beet Oak-leaf Virus in Sugar Beet.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rhizomania, a very serious disease of sugar beet (Beta vulgaris L.), is caused by Beet necrotic yellow vein virus (BNYVV). Resistance allele Rz1 has been widely incorporated into commercial cultivars. Recently, resistance-breaking strains of Beet necrotic yellow vein virus (RB-BNYVV) were identified...

  4. Complete Coding Sequences of Eastern Equine Encephalitis Virus and Venezuelan Equine Encephalitis Virus Strains Isolated from Human Cases

    PubMed Central

    Yu, Guo-Yun; Wiley, Michael R.; Kugelman, Jeffrey R.; Ladner, Jason T.; Beitzel, Brett F.; Eccleston, Lori T.; Morazzani, Elaine M.; Glass, Pamela J.

    2015-01-01

    We obtained the complete coding genome of an eastern equine encephalitis virus (EEEV) strain, EEEV V105-00210, and the complete genome of a Venezuelan equine encephalitis virus (VEEV) strain, VEEV INH-9813. They were obtained from human cases and are proposed as reference challenge strains for vaccine and therapeutic development in animal models. PMID:25908124

  5. Genetic and antigenic characterization of H5N1 viruses of clade 2.3.2.1 isolated in India.

    PubMed

    Bhat, Sushant; Bhatia, Sandeep; Pillai, Aravind S; Sood, Richa; Singh, Vikas Kumar; Shrivas, Om Prakash; Mishra, Suchitra K; Mawale, Namrata

    2015-11-01

    The recurrent circulation of highly pathogenic avian influenza (HPAI) H5N1 in Indian poultry since 2006 resulted in emergence of the viruses of distinct antigenic clades of haemagglutinin (HA) with the majority of the H5N1 outbreaks since 2011 belonging to clade 2.3.2.1. The present study was aimed to characterize the antigenic profile of a collection of H5N1 HPAI viruses of clade 2.3.2.1 isolated in India by applying antigenic cartography, serological data and phylogenetic analysis. Eleven H5N1 viruses (2 of clade 2.2 and 9 of clade 2.3.2.1) were selected based on genetic analysis and were further characterized by antigenic cartography analysis based on cross HI (hemagglutination inhibition) data. This study highlights the intercladal antigenic differences between clades 2.3.2.1 and 2.2 and the intracladal antigenic divergence among the clade 2.3.2.1 viruses. Five viruses of clade 2.3.2.1 were also studied for analysis of glycosylation pattern of Hemagglutinin (HA) gene and the growth kinetics analysis in MDCK cells in which the viruses CL03485/H5N1 and 03CL488/H5N1 showed better replication kinetics than other viruses. The study presents a baseline data of antigenicity and other factors that can be used in the selection of suitable H5 vaccine strains or HA donor viruses to develop H5 vaccine strains by reverse genetics or other methods for control of currently circulating H5N1 viruses in Indian region. PMID:26299902

  6. Analysis of genome comparison of two Indian isolates of Cowpea aphid-borne mosaic virus from India.

    PubMed

    Mishra, Ritesh; Verma, Rakesh Kumar; Gaur, Rajarshi Kumar

    2015-10-01

    The complete sequence of two Cowpea aphid-borne mosaic virus (CABMV) isolates (RR3 and RR4) from India was determined. Phylogenetic analysis showed that both isolates showed different closeness with other isolates of CABMV. CABMV-RR3 showed maximum identity of 99 % with CABMV-BR1 from Brazil at nucleotide and protein levels, whereas CABMV-RR4 showed identity of 73 and 95 % with CABMV-Z isolate from Zimbabwe at nucleotide and protein levels respectively. Similarity identity matrix revealed 69 % identity at nucleotide level and 91 % at protein level with each other. Recombination breakpoint detection showed that CABMV-MG-Avr from Brazil and CABMV-Z from Zimbabwe act as major parents in our isolates RR3 and RR4, respectively. PMID:26184969

  7. Molecular Characterization and Variation of the Broad bean wilt virus 2 Isolates Based on Analyses of Complete Genome Sequences

    PubMed Central

    Kwak, Hae-Ryun; Kim, Mi-Kyeong; Lee, Ye-Ji; Seo, Jang-Kyun; Kim, Jeong-Soo; Kim, Kook-Hyung; Cha, Byeongjin; Choi, Hong-Soo

    2013-01-01

    The full-genome sequences of fourteen isolates of Broad bean wilt virus 2 (BBWV2), collected from broad bean, pea, spinach, bell pepper and paprika plants in Korea during the years 2006–2012, were determined and analyzed comparatively along with fifteen previously reported BBWV2 genome sequences. Sequence analyses showed that RNA-1 and RNA-2 sequences of BBWV2 Korean isolates consisted of 5950–5956 and 3568–3604 nucleotides, respectively. Full-length genome sequence-based phylogenetic analyses revealed that the BBWV2 Korean isolates could be divided into three major groups comprising GS-I (isolates BB2 and RP7) along with isolate IP, GS-II (isolates BB5, P2, P3 and RP3) along with isolate B935, and GS-III including 16 BBWV2 Korean isolates. Interestingly, GS-III appears to be newly emerged and predominant in Korea. Recombination analyses identified two recombination events in the analyzed BBWV2 population: one in the RNA-1 of isolate K and another one in the RNA-2 of isolate XJ14-3. However, no recombination events were detected in the other 21 Korean isolates. On the other hand, out of 29 BBWV2 isolates, 16 isolates were found to be reassortants, of which each RNA segment (i.e. RNA1 and RNA2) was originated from different parental isolates. Our findings suggested that reassortment rather than recombination is a major evolutionary force in the genetic diversification of BBWV population in Korea. PMID:25288968

  8. Molecular Characterization and Variation of the Broad bean wilt virus 2 Isolates Based on Analyses of Complete Genome Sequences.

    PubMed

    Kwak, Hae-Ryun; Kim, Mi-Kyeong; Lee, Ye-Ji; Seo, Jang-Kyun; Kim, Jeong-Soo; Kim, Kook-Hyung; Cha, Byeongjin; Choi, Hong-Soo

    2013-12-01

    The full-genome sequences of fourteen isolates of Broad bean wilt virus 2 (BBWV2), collected from broad bean, pea, spinach, bell pepper and paprika plants in Korea during the years 2006-2012, were determined and analyzed comparatively along with fifteen previously reported BBWV2 genome sequences. Sequence analyses showed that RNA-1 and RNA-2 sequences of BBWV2 Korean isolates consisted of 5950-5956 and 3568-3604 nucleotides, respectively. Full-length genome sequence-based phylogenetic analyses revealed that the BBWV2 Korean isolates could be divided into three major groups comprising GS-I (isolates BB2 and RP7) along with isolate IP, GS-II (isolates BB5, P2, P3 and RP3) along with isolate B935, and GS-III including 16 BBWV2 Korean isolates. Interestingly, GS-III appears to be newly emerged and predominant in Korea. Recombination analyses identified two recombination events in the analyzed BBWV2 population: one in the RNA-1 of isolate K and another one in the RNA-2 of isolate XJ14-3. However, no recombination events were detected in the other 21 Korean isolates. On the other hand, out of 29 BBWV2 isolates, 16 isolates were found to be reassortants, of which each RNA segment (i.e. RNA1 and RNA2) was originated from different parental isolates. Our findings suggested that reassortment rather than recombination is a major evolutionary force in the genetic diversification of BBWV population in Korea. PMID:25288968

  9. Pandemic Swine-Origin H1N1 Influenza A Virus Isolates Show Heterogeneous Virulence in Macaques ? ‡

    PubMed Central

    Safronetz, David; Rockx, Barry; Feldmann, Friederike; Belisle, Sarah E.; Palermo, Robert E.; Brining, Douglas; Gardner, Don; Proll, Sean C.; Marzi, Andrea; Tsuda, Yoshimi; LaCasse, Rachel A.; Kercher, Lisa; York, Anthony; Korth, Marcus J.; Long, Dan; Rosenke, Rebecca; Shupert, W. Lesley; Aranda, Celia Alpuche; Mattoon, John S.; Kobasa, Darwyn; Kobinger, Gary; Li, Yan; Taubenberger, Jeffery K.; Richt, Jürgen A.; Parnell, Michael; Ebihara, Hideki; Kawaoka, Yoshihiro; Katze, Michael G.; Feldmann, Heinz

    2011-01-01

    The first influenza pandemic of the new millennium was caused by a newly emerged swine-origin influenza virus (SOIV) (H1N1). This new virus is characterized by a previously unknown constellation of gene segments derived from North American and Eurasian swine lineages and the absence of common markers predictive of human adaptation. Overall, human infections appeared to be mild, but an alarming number of young individuals presented with symptoms atypical for seasonal influenza. The new SOIV also showed a sustained human-to-human transmissibility and higher reproduction ratio than common seasonal viruses, altogether indicating a higher pathogenic potential for this newly emerged virus. To study the virulence of the SOIV, we used a recently established cynomolgus macaque model and compared parameters of clinical disease, virology, host responses, and pathology/histopathology with a current seasonal H1N1 virus. We here show that infection of macaques with two genetically similar but clinically distinct SOIV isolates from the early stage of the pandemic (A/Mexico/4108/2009 and A/Mexico/InDRE4487/2009) resulted in upper and lower respiratory tract infections and clinical disease ranging from mild to severe pneumonia that was clearly advanced over the mild infection caused by A/Kawasaki/UTK-4/2009, a current seasonal strain. Unexpectedly, we observed heterogeneity among the two SOIV isolates in virus replication, host transcriptional and cytokine responses, and disease progression, demonstrating a higher pathogenic potential for A/Mexico/InDRE4487/2009. Differences in virulence may explain more severe disease, as was seen with certain individuals infected with the emerged pandemic influenza virus. Thus, the nonhuman primate model closely mimics influenza in humans. PMID:21084481

  10. Characterization of Hungarian isolates of zucchini yellow mosaic virus (ZYMV, potyvirus) transmitted by seeds of Cucurbita pepo var Styriaca.

    PubMed

    Tóbiás, István; Palkovics, László

    2003-04-01

    Zucchini yellow mosaic virus (ZYMV) has emerged as an important pathogen of cucurbits within the last few years in Hungary. The Hungarian isolates show a high biological variability, have specific nucleotide and amino acid sequences in the N-terminal region of coat protein and form a distinct branch in the phylogenetic tree. The virus is spread very efficiently in the field by several aphid species in a non-persistent manner. It can be transmitted by seed in holl-less seeded oil pumpkin (Cucurbita pepo (L) var Styriaca), although at a very low rate. Three isolates from seed transmission assay experiments were chosen and their nucleotide sequences of coat proteins have been compared with the available CP sequences of ZYMV. According to the sequence analysis, the Hungarian isolates belong to the Central European branch in the phylogenetic tree and, together with the ZYMV isolates from Austria and Slovenia, share specific amino acids at positions 16, 17, 27 and 37 which are characteristic only to these isolates. The phylogenetic tree suggests the common origin of distantly distributed isolates which can be attributed to widespread seed transmission. PMID:12701712

  11. Phylogenetic analysis of Newcastle disease viruses isolated from wild birds in the Poyang Lake region of China

    PubMed Central

    FAN, Shengtao; WANG, Tiecheng; GAO, Xiaolong; YING, Ying; LI, Xue; LI, Yongcheng; LI, Yuanguo; MA, Jinzhu; SUN, Heting; CHU, Dong; XU, Yu; YANG, Songtao; LI, Qihan; GAO, Yuwei; XIA, Xianzhu

    2015-01-01

    Newcastle disease virus (NDV) causes a highly contagious viral disease in poultry and wild birds, and it can cause significant economic loss worldwide. Eight viral strains were isolated by inoculating embryonated chicken eggs from the Poyang Lake region of China with swab samples. All eight of the NDV isolates were identified as class I genotype 3 strains, but they diverged notablely from class II viruses. Further analysis revealed that all eight NDV isolates were lentogenic strains containing the 112ERQER?L117 motif at the F protein cleavage site. The strains were highly identical and were more species specific (chicken and waterfowl) than site specific (Nanchang and Duchang regions). The close phylogenetic proximity of these isolates indicates that viral transmission may happen between poultry and wild birds. Our study demonstrates that lentogenic class I NDVs exist in clinically healthy wild waterfowl and poultry within the Poyang Lake region. Active surveillance of these viruses to determine their evolution and origin is one of the most realistic strategies for preventing and controlling NDV outbreaks. PMID:25843743

  12. Full genome sequences and molecular characterization of tick-borne encephalitis virus strains isolated from human patients.

    PubMed

    Formanová, Petra; ?erný, Ji?í; Bolfíková, Barbora ?erná; Valdés, James J; Kozlova, Irina; Dzhioev, Yuri; R?žek, Daniel

    2015-02-01

    Tick-borne encephalitis virus (TBEV) causes tick-borne encephalitis (TBE), one of the most important human neuroinfections across Eurasia. Up to date, only three full genome sequences of human European TBEV isolates are available, mostly due to difficulties with isolation of the virus from human patients. Here we present full genome characterization of an additional five low-passage TBEV strains isolated from human patients with severe forms of TBE. These strains were isolated in 1953 within Central Bohemia in the former Czechoslovakia, and belong to the historically oldest human TBEV isolates in Europe. We demonstrate here that all analyzed isolates are distantly phylogenetically related, indicating that the emergence of TBE in Central Europe was not caused by one predominant strain, but rather a pool of distantly related TBEV strains. Nucleotide identity between individual sequenced TBEV strains ranged from 97.5% to 99.6% and all strains shared large deletions in the 3' non-coding region, which has been recently suggested to be an important determinant of virulence. The number of unique amino acid substitutions varied from 3 to 9 in individual isolates, but no characteristic amino acid substitution typical exclusively for all human TBEV isolates was identified when compared to the isolates from ticks. We did, however, correlate that the exploration of the TBEV envelope glycoprotein by specific antibodies were in close proximity to these unique amino acid substitutions. Taken together, we report here the largest number of patient-derived European TBEV full genome sequences to date and provide a platform for further studies on evolution of TBEV since the first emergence of human TBE in Europe. PMID:25311899

  13. Isolation and Characterization of a Novel Single-Stranded RNA Virus Infecting the Bloom-Forming Diatom Rhizosolenia setigera

    PubMed Central

    Nagasaki, Keizo; Tomaru, Yuji; Katanozaka, Noriaki; Shirai, Yoko; Nishida, Kensho; Itakura, Shigeru; Yamaguchi, Mineo

    2004-01-01

    A novel single-stranded RNA (ssRNA) virus specifically infecting the bloom-forming diatom Rhizosolenia setigera (R. setigera RNA virus [RsRNAV]) was isolated from Ariake Sea, Japan. Viral replication occurred within the cytoplasm, and the virus particle was icosahedral, lacked a tail, and was 32 nm in diameter on average. The major nucleic acid extracted from the RsRNAV particles was an ssRNA molecule 11.2 kb in length, although smaller RNA molecules (0.6, 1.2, and 1.5 kb) were occasionally observed. The major structural proteins of RsRNAV were 41.5, 41.0, and 29.5 kDa. Inter- and intraspecies host specificity tests revealed that RsRNAV is not only species specific but also strain specific and that its intraspecies host specificity is diverse among virus clones. The latent period of RsRNAV was 2 days, and the burst sizes were 3,100 and 1,010 viruses per host cell when viruses were inoculated into the host culture at the exponential and stationary growth phases, respectively, at 15°C under a 12-h-12-h light-dark cycle of ca. 110 ?mol of photons m?2 s?1 with cool white fluorescent illumination. To our knowledge, this is the first report describing the biological properties of a virus infecting a diatom. Further studies on RsRNAV will be helpful in understanding the ecological relationship between diatoms and viruses in nature. PMID:14766545

  14. Isolation of a non-haemadsorbing, non-cytopathic strain of African swine fever virus in Madagascar.

    PubMed Central

    Gonzague, M.; Roger, F.; Bastos, A.; Burger, C.; Randriamparany, T.; Smondack, S.; Cruciere, C.

    2001-01-01

    African swine fever (ASF) suspected clinically in Madagascar (1998-9) was confirmed by polymerase chain reaction (PCR) and nucleotide sequencing, following virus isolation. No haemadsorption or cytopathic effect could be detected following leukocyte inoculation, but viral growth in cells was confirmed by PCR. Detection of ASF virus genome was carried out by amplification of a highly conserved region coding for the p72 protein. Nucleotide sequencing of the amplicon revealed 99.2% nucleotide identity between the recent Malagasy strains and a virus recovered from the 1994 outbreak in Mozambique (SPEC265). A serological survey performed on 449 sera, revealed that only 5.3% of the sera taken from pigs between 1998 and 1999 were positive. PMID:11467803

  15. The complete genome sequences of two isolates of cnidium vein yellowing virus, a tentative new member of the family Secoviridae.

    PubMed

    Yoo, Ran Hee; Zhao, Fumei; Lim, Seungmo; Igori, Davaajargal; Kim, Sang-Mok; An, Tae-Jin; Lee, Su-Heon; Moon, Jae Sun

    2015-11-01

    We determined the complete genome sequences of two isolates of cnidium vein yellowing virus (CnVYV-1 and -2) that co-infected all field samples collected from Cnidium officinale in Korea. Unlike CnVYV-2, however, CnVYV-1 was sap-transmissible to Nicotiana benthamiana. CnVYV-1 and -2 have bipartite genomes of 7,263 and 3,110 nucleotides and 7,278 and 3,112 nucleotides, respectively, excluding the poly(A) tails. Phylogenetic analysis of the CnVYV-1 and -2 sequences indicated close relationships to strawberry latent ringspot virus, an unassigned member of the family Secoviridae. CnVYV-1 and CnVYV-2 are closely related viruses that may represent a tentative new species of the family Secoviridae. PMID:26282235

  16. Ring test evaluation of the detection of influenza A virus in swine oral fluids by real-time, reverse transcription polymerase chain reaction (rRT-PCR) and virus isolation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The probability of detecting influenza A virus (IAV) in oral fluid (OF) specimens was calculated for each of 13 real-time, reverse transcription polymerase chain reaction (rRT-PCR) and 7 virus isolation (VI) assays. To conduct the study, OF was inoculated with H1N1 or H3N2 IAV and serially 10-fold d...

  17. Limited evidence of trans-hemispheric movement of avian influenza viruses among contemporary North American shorebird isolates

    USGS Publications Warehouse

    Pearce, John M.; Ramey, Andrew M.; Ip, Hon S.; Gill, Robert E., Jr.

    2010-01-01

    Migratory routes of gulls, terns, and shorebirds (Charadriiformes) are known to cross hemispheric boundaries and intersect with outbreak areas of highly pathogenic avian influenza (HPAI). Prior assessments of low pathogenic avian influenza (LPAI) among species of this taxonomic order found some evidence for trans-hemispheric movement of virus genes. To specifically clarify the role of shorebird species in the trans-hemispheric movement of influenza viruses, assess the temporal variation of Eurasian lineages observed previously among North American shorebirds, and evaluate the necessity for continued sampling of these birds for HPAI in North America, we conducted a phylogenetic analysis of >700 contemporary sequences isolated between 2000 and 2008. Evidence for trans-hemispheric reassortment among North American shorebird LPAI gene segments was lower (0.88%) than previous assessments and occurred only among eastern North American isolates. Furthermore, half of the reassortment events occurred in just two isolates. Unique phylogenetic placement of these samples suggests secondary infection and or involvement of other migratory species, such as gulls. Eurasian lineages observed in North American shorebirds before 2000 were not detected among contemporary samples, suggesting temporal variation of LPAI lineages. Results suggest that additional bird migration ecology and virus phylogenetics research is needed to determine the exact mechanisms by which shorebirds in eastern North America become infected with LPAI that contain Eurasian lineage genes. Because of the low prevalence of avian influenza in non-eastern North America sites, thousands more shorebirds will need to be sampled to sufficiently examine genetic diversity and trans-hemispheric exchange of LPAI viruses in these areas. Alternatively, other avian taxa with higher virus prevalence could serve as surrogates to shorebirds for optimizing regional surveillance programs for HPAI through the LPAI phylogenetic approach.

  18. Ring test evaluation of the detection of influenza A virus in swine oral fluids by real-time reverse-transcription polymerase chain reaction and virus isolation.

    PubMed

    Goodell, Christa K; Zhang, Jianqiang; Strait, Erin; Harmon, Karen; Patnayak, Devi; Otterson, Tracy; Culhane, Marie; Christopher-Hennings, Jane; Clement, Travis; Leslie-Steen, Pamela; Hesse, Richard; Anderson, Joe; Skarbek, Kevin; Vincent, Amy; Kitikoon, Pravina; Swenson, Sabrina; Jenkins-Moore, Melinda; McGill, Jodi; Rauh, Rolf; Nelson, William; O'Connell, Catherine; Shah, Rohan; Wang, Chong; Main, Rodger; Zimmerman, Jeffrey J

    2016-01-01

    The probability of detecting influenza A virus (IAV) in oral fluid (OF) specimens was calculated for each of 13 assays based on real-time reverse-transcription polymerase chain reaction (rRT-PCR) and 7 assays based on virus isolation (VI). The OF specimens were inoculated with H1N1 or H3N2 IAV and serially diluted 10-fold (10(-1) to 10(-8)). Eight participating laboratories received 180 randomized OF samples (10 replicates × 8 dilutions × 2 IAV subtypes plus 20 IAV-negative samples) and performed the rRT-PCR and VI procedure(s) of their choice. Analysis of the results with a mixed-effect logistic-regression model identified dilution and assay as variables significant (P < 0.0001) for IAV detection in OF by rRT-PCR or VI. Virus subtype was not significant for IAV detection by either rRT-PCR (P = 0.457) or VI (P = 0.101). For rRT-PCR the cycle threshold (Ct) values increased consistently with dilution but varied widely. Therefore, it was not possible to predict VI success on the basis of Ct values. The success of VI was inversely related to the dilution of the sample; the assay was generally unsuccessful at lower virus concentrations. Successful swine health monitoring and disease surveillance require assays with consistent performance, but significant differences in reproducibility were observed among the assays evaluated. PMID:26733728

  19. Complete genome sequence of an isolate of Potato virus X (PVX) infecting Cape gooseberry (Physalis peruviana) in Colombia.

    PubMed

    Gutiérrez, Pablo A; Alzate, Juan F; Montoya, Mauricio Marín

    2015-06-01

    Transcriptome analysis of a Cape gooseberry (Physalis peruviana) plant with leaf symptoms of a mild yellow mosaic typical of a viral disease revealed an infection with Potato virus X (PVX). The genome sequence of the PVX-Physalis isolate comprises 6435 nt and exhibits higher sequence similarity to members of the Eurasian group of PVX (~95 %) than to the American group (~77 %). Genome organization is similar to other PVX isolates with five open reading frames coding for proteins RdRp, TGBp1, TGBp2, TGBp3, and CP. 5' and 3' untranslated regions revealed all regulatory motifs typically found in PVX isolates. The PVX-Physalis genome is the only complete sequence available for a Potexvirus in Colombia and is a new addition to the restricted number of available sequences of PVX isolates infecting plant species different to potato. PMID:25680343

  20. Serological and Molecular Studies of a Novel Virus Isolate Causing Yellow Mosaic of Patchouli [Pogostemon cablin (Blanco) Benth

    PubMed Central

    Zaim, Mohammad; Ali, Ashif; Joseph, Jomon; Khan, Feroz

    2013-01-01

    Here we have identified and characterized a devastating virus capable of inducing yellow mosaic on the leaves of Patchouli [Pogostemon cablin (Blanco) Benth]. The diagnostic tools used were host range, transmission studies, cytopathology, electron microscopy, serology and partial coat protein (CP) gene sequencing. Evidence from biological, serological and sequence data suggested that the causal virus belonged to genus Potyvirus, family Potyviridae. The isolate, designated as Patchouli Yellow Mosaic Virus (PaYMV), was transmitted through grafting, sap and the insect Myzus persicae (Sulz.). Flexuous rod shaped particles with a mean length of 800 nm were consistently observed in leaf-dip preparations from natural as well as alternate hosts, and in purified preparation. Cytoplasmic cylindrical inclusions, pinwheels and laminar aggregates were observed in ultra-thin sections of infected patchouli leaves. The purified capsid protein has a relative mass of 43 kDa. Polyclonal antibodies were raised in rabbits against the coat protein separated on SDS – PAGE; which were used in ELISA and western blotting. Using specific antibodies in ELISA, PaYMV was frequently detected at patchouli plantations at Lucknow and Bengaluru. Potyvirus-specific degenerate primer pair (U335 and D335) had consistently amplified partial CP gene from crude preparations of infected tissues by reverse transcription polymerase chain reaction (RT-PCR). Comparison of the PCR product sequence (290 bp) with the corresponding regions of established potyviruses showed 78–82% and 91–95% sequence similarity at the nucleotide and amino acid levels, respectively. The results clearly established that the virus under study has close homology with watermelon mosaic virus (WMV) in the coat protein region and therefore could share a common ancestor family. Further studies are required to authenticate the identity of PaYMV as a distinct virus or as an isolate of WMV. PMID:24386278

  1. Full genome sequence and some biological properties of reticuloendotheliosis virus strain APC-566 isolated from endangered Attwater's prairie chickens.

    PubMed

    Barbosa, Taylor; Zavala, Guillermo; Cheng, Sunny; Villegas, Pedro

    2007-03-01

    Reticuloendotheliosis virus (REV) causes runting, high mortality, immunosuppression, and chronic neoplasia associated with T and/or B cell lymphomas in a variety of domestic and wild birds, including Attwater's prairie chickens (APC) (Tympanuchus cupido attwateri). The complete proviral sequence of a recent REV isolate from APC (REV APC-566) was determined. This virus was isolated from an APC maintained in captivity in a reproduction program intended to avoid its extinction. REV APC-566 was determined to be oncogenic in Japanese quail (Coturnix coturnix japonica), chickens (Gallus gallus) and turkeys (Meleagris gallopavo). Immune responses against bacteria and viruses were significantly reduced in turkeys infected with REV APC-566. The proviral genome is 8286 nucleotides in length and exhibits a genetic organization characteristic of replication-competent gammaretroviruses. The REV APC-566 provirus contains two identical long terminal repeats (LTR) and a complete set of genes including gag, gag-pol and env. As previously reported, alignments with other REV sequences showed high similarity with sequences found in the gag and pol genes from other REVs. The REV APC-566 env gene showed high nucleotide sequence homology with REV sequences inserted in fowl poxvirus (99.8%), and with spleen necrosis virus (SNV) (95.1%). Sequences coding for a previously reported immunosuppressive peptide contained in the transmembrane region of the env gene are well conserved among all REV sequences analyzed. The LTR was the most divergent region, exhibiting various deletions and insertions. REV APC-566 has a unique insertion of 23 bp in U3 and shares deletions of 19 and 5 bp with chicken syncytial virus and REV inserts in fowlpox virus. PMID:17098316

  2. Porcine reproductive and respiratory syndrome virus: antigenic and molecular diversity of British isolates and implications for diagnosis.

    PubMed

    Frossard, Jean-Pierre; Fearnley, Catherine; Naidu, Brindha; Errington, Jane; Westcott, David G; Drew, Trevor W

    2012-08-17

    Porcine reproductive and respiratory syndrome (PRRS) is an endemic disease of pigs, caused by PRRS virus, a member of the Arteriviridae family. First seen in Britain in 1991, the disease continues to be a significant economic and welfare problem for pig producers. To date, only PRRSV genotype 1 has been found in Britain. At the genetic level, a considerable increase has been reported in the diversity of PRRS viruses isolated in Britain between 2003 and 2007, versus the early 1990 s. In this study, the diversity has been shown to extend to the antigenic level too, with potential consequences for diagnostic methods. Antigenic diversity was assessed using a panel of twelve monoclonal antibodies, only one of which reacted with all isolates tested. Nine diverse viruses were compared as potential antigens in immunoperoxidase monolayer assays, where each one produced quite different results for a common panel of sera. As a single virus is used in each diagnostic assay, results must therefore be interpreted cautiously. For a real-time RT-PCR assay, published oligonucleotide primer and probe sequences were evaluated against available genetic sequences of British and European viruses, and were re-designed where considerable mismatches were found. The multiplex assay incorporating these modified primers to detect genotype 1 and 2 PRRS viruses was then validated for use with diagnostic sera and tissues. As the increasing degree of diversity exhibited by British strains is mirrored in other countries, PRRSV will continue to provide an ongoing challenge to diagnosis at a global, as well as national level. PMID:22472704

  3. Resistance testing of clinical herpes simplex virus type 2 isolates collected over 4 decades.

    PubMed

    Bohn-Wippert, Kathrin; Schmidt, Susanne; Runtze, Anna; Zell, Roland; Sauerbrei, Andreas

    2015-10-01

    There is only little information about the role of mutations of the thymidine kinase (TK) and DNA polymerase (pol) genes of herpes simplex virus type 2 (HSV-2) for the development of antiviral resistance. In this study, the polymorphism of TK and DNA pol genes was examined in 82 clinical isolates collected routinely between 1973 and 2013. If novel, presently unclear or resistance-related mutations were found, the resistance phenotype against acyclovir (ACV) and foscarnet (FOS) was analyzed. The four novel amino acid changes G150D, A157T, R248W, L342W and the hitherto phenotypically unclear substitution T131M within the TK gene were identified as natural polymorphisms. Within the DNA pol gene, 17 novel substitutions and the to-date unclear change R628C were characterized as part of natural gene polymorphism. Two novel DNA pol mutations were linked to resistance (M910T) and weak susceptibility to ACV (684 insertion ED), respectively. In one isolate, the genomic cause of ACV resistance could not be identified. Phylogenetic analysis including sequences of this study and of the GenBank revealed a hierarchy of mutation clusters in TK displaying G39E as first common mutation step, followed by N78D and L140F. In conclusion, the present findings allow a deeper insight in the variability of HSV-2 TK and DNA pol genes. The most common substitution G39E can be excluded as unique cause of HSV-2 resistance. This study supports once more the importance of phenotypic adjustment of genotypic results to enhance the clinical utility of genotypic findings. PMID:26338148

  4. Hepatitis B Virus Genotype D Isolates Circulating in Chapecó, Southern Brazil, Originate from Italy

    PubMed Central

    Gusatti, Carolina Souza; Costi, Cintia; Halon, Maria Laura; Grandi, Tarciana; Medeiros, Arlete Ferrari Rech; Silva, Cláudia Maria Dornelles; Gomes, Selma Andrade; Silva, Marcia Susana Nunes; Niel, Christian; Rossetti, Maria Lucia Rosa

    2015-01-01

    Hepatitis B virus genotype A1 (HBV/A1), of African origin, is the most prevalent genotype in Brazil, while HBV/F predominates in the other South American countries. However, HBV/D is the most common in the three states of southern Brazil, where ‘islands’ of elevated prevalence, as Chapecó and other cities, have been described. In this study, 202 HBV chronic carriers attending in 2013 the viral hepatitis ambulatory of Chapecó, were investigated. In comparison with previous studies performed in the same ambulatory, a rapid aging of the HBV infected population was observed (mean age of the newly diagnosed patients increasing from 29.9 ± 10.3 years in 1996 to 44.4 ± 13.3 years in 2013), probably due to a singular vaccination schedule at Chapecó that included not only children but also adolescents. Phylogenetic and BLAST analyses (S region) classified 91 HBV isolates into genotypes A (n = 3) and D (n = 88). The majority of HBV/D isolates were closely related to D3 sequences. To understand the reasons for the absence or near absence of genotypes A and F, and how HBV/D was introduced in the south of Brazil, HBV/D infected patients were inquired about their genealogical and geographical origins. Forty-three (52%) patients have their four grandparents of Italian origin, vs. seven (8%) who have their four grandparents of Brazilian origin. At all, 65 out of 83 (78%) patients had at least one grandparent originating from Italy. Taking into consideration the fact that Italy is one of the few countries where subgenotype D3 is predominant, the results strongly suggested that HBV/D was introduced in Brazil through Italian immigration which culminated between 1870 and 1920. PMID:26275046

  5. The First Outbreak of Eastern Equine Encephalitis in Vermont: Outbreak Description and Phylogenetic Relationships of the Virus Isolate

    PubMed Central

    Saxton-Shaw, Kali D.; Ledermann, Jeremy P.; Kenney, Joan L.; Berl, Erica; Graham, Alan C.; Russo, Joel M.; Powers, Ann M.; Mutebi, John-Paul

    2015-01-01

    The first known outbreak of eastern equine encephalitis (EEE) in Vermont occurred on an emu farm in Rutland County in 2011. The first isolation of EEE virus (EEEV) in Vermont (VT11) was during this outbreak. Phylogenetic analysis revealed that VT11 was most closely related to FL01, a strain from Florida isolated in 2001, which is both geographically and temporally distinct from VT11. EEEV RNA was not detected in any of the 3,905 mosquito specimens tested, and the specific vectors associated with this outbreak are undetermined. PMID:26043136

  6. Comparative full genome analysis revealed E1: A226V shift in 2007 Indian Chikungunya virus isolates.

    PubMed

    Santhosh, S R; Dash, P K; Parida, M M; Khan, M; Tiwari, M; Lakshmana Rao, P V

    2008-07-01

    The resurgence of Chikungunya virus (CHIKV) in the form of unprecedented explosive epidemic after a gap of 32 years in India is a point of major public health concern. In 2007 again there was outbreak in Kerala, India, affecting more than 25,000 cases with many reported mortalities. To understand the molecular basis of this high virulence and its implication in large-scale epidemic, a detailed systematic serological, virological and molecular investigation was undertaken with the epidemic samples of Kerala-2007. The comparative analysis of full genome sequence of Chikungunya virus isolate of 2007 with 2006 revealed three unique substitutions in structural and non-structural genes of 2007 isolate [two in E1 region (V14A and A226V) and one in Nsp1 (M184T)]. Our finding further substantiates the association of A226V shift in E1 gene with evolutionary success possibly due to adaptation in the mosquito vector with progression of epidemic, as observed in Reunion Island. This A226V shift which was absent in all 2006 Indian isolates, is found to be present in the four 2007 isolates, analysed in this study. These unique molecular features of the 2007 isolates with the progression of the epidemic from 2005 to 2007 demonstrate their high evolutionary and epidemic potential and thereby suggesting possible implication in higher magnitude and virulence of this outbreak. PMID:18384900

  7. Isolation of ortho- and paramyxoviruses from wild birds in Western Australia, and the characterization of novel influenza A viruses.

    PubMed

    Mackenzie, J S; Edwards, E C; Holmes, R M; Hinshaw, V S

    1984-02-01

    As part of the World Health Organization's international programme on the ecology of influenza, cloacal swabs were collected from 3,736 birds belonging to 67 species over a 3-year period in Western Australia for the isolation of ortho- and paramyxoviruses. A total of 24 influenza A viruses were isolated from various species of ducks, shearwaters , noddies , terns and a coot , and were subtyped as H1N9 , H3N8 , H4N4 , H4N6 , H6N2 , H6N4 , H?N2, H?N6 and H? N9 . The H? haemagglutinins did not react in tests with reference antisera. Whether they represent a novel haemagglutinin subtype or atypical members of an established subtype remains to be determined, although preliminary results indicate that they may be atypical members of the H7 subtype. The H1N9 isolate is the first reported isolate of this particular antigenic combination. A total of 17 Newcastle disease viruses was isolated from ducks, noddies , terns and a black- fronted plover : preliminary results suggest that they are avirulent for domestic chickens. This study indicates that ortho- and paramyxoviruses are present in a variety of wild birds in Australia. PMID:6430260

  8. Genetic variability between isolates of human immunodeficiency virus (HIV) type 2 is comparable to the variability among HIV type 1.

    PubMed Central

    Zagury, J F; Franchini, G; Reitz, M; Collalti, E; Starcich, B; Hall, L; Fargnoli, K; Jagodzinski, L; Guo, H G; Laure, F

    1988-01-01

    The isolation from macaques of retroviruses related to human immunodeficiency virus (HIV) led to the identification of a second group of human retroviruses (termed HIV-2), which are prevalent in West Africa and closely related to the simian immunodeficiency virus (SIV). We have cloned and determined the complete nucleotide sequence of the human West African retrovirus HIV-2NIH-Z and compared it to that of a previously described strain of HIV-2 (HIV-2ROD) as well as to SIV and HIV-1. We have reached the following conclusions: (i) The HIV-2 isolates are (slightly) more closely related to each other than to SIV, compatible with their isolation from different species. (ii) The variability between HIV-2 isolates is similar in degree and kind to that found among HIV-1 isolates. The equivalent degrees of intragroup divergence suggest that HIV-1 and HIV-2 have existed in their present ranges in Africa for approximately equal lengths of time. The fact that acquired immunodeficiency syndrome is widespread in regions where HIV-1 is prevalent but not in regions where HIV-2 is prevalent suggests a substantial difference in the morbidity rates associated with HIV-1 vs. HIV-2 infection. (iii) HIV-2 and SIV are related to each other more closely than they are to HIV-1. PMID:3261862

  9. Complete nucleotide sequence of a South African isolate of Grapevine fanleaf virus and its associated satellite RNA.

    PubMed

    Lamprecht, Renate L; Spaltman, Monique; Stephan, Dirk; Wetzel, Thierry; Burger, Johan T

    2013-07-01

    The complete sequences of RNA1, RNA2 and satellite RNA have been determined for a South African isolate of Grapevine fanleaf virus (GFLV-SACH44). The two RNAs of GFLV-SACH44 are 7,341 nucleotides (nt) and 3,816 nt in length, respectively, and its satellite RNA (satRNA) is 1,104 nt in length, all excluding the poly(A) tail. Multiple sequence alignment of these sequences showed that GFLV-SACH44 RNA1 and RNA2 were the closest to the South African isolate, GFLV-SAPCS3 (98.2% and 98.6% nt identity, respectively), followed by the French isolate, GFLV-F13 (87.3% and 90.1% nt identity, respectively). Interestingly, the GFLV-SACH44 satRNA is more similar to three Arabis mosaic virus satRNAs (85%-87.4% nt identity) than to the satRNA of GFLV-F13 (81.8% nt identity) and was most distantly related to the satRNA of GFLV-R2 (71.0% nt identity). Full-length infectious clones of GFLV-SACH44 satRNA were constructed. The infectivity of the clones was tested with three nepovirus isolates, GFLV-NW, Arabis mosaic virus (ArMV)-NW and GFLV-SAPCS3. The clones were mechanically inoculated in Chenopodium quinoa and were infectious when co-inoculated with the two GFLV helper viruses, but not when co-inoculated with ArMV-NW. PMID:23867805

  10. Complete Nucleotide Sequence of a South African Isolate of Grapevine Fanleaf Virus and Its Associated Satellite RNA

    PubMed Central

    Lamprecht, Renate L.; Spaltman, Monique; Stephan, Dirk; Wetzel, Thierry; Burger, Johan T.

    2013-01-01

    The complete sequences of RNA1, RNA2 and satellite RNA have been determined for a South African isolate of Grapevine fanleaf virus (GFLV-SACH44). The two RNAs of GFLV-SACH44 are 7,341 nucleotides (nt) and 3,816 nt in length, respectively, and its satellite RNA (satRNA) is 1,104 nt in length, all excluding the poly(A) tail. Multiple sequence alignment of these sequences showed that GFLV-SACH44 RNA1 and RNA2 were the closest to the South African isolate, GFLV-SAPCS3 (98.2% and 98.6% nt identity, respectively), followed by the French isolate, GFLV-F13 (87.3% and 90.1% nt identity, respectively). Interestingly, the GFLV-SACH44 satRNA is more similar to three Arabis mosaic virus satRNAs (85%–87.4% nt identity) than to the satRNA of GFLV-F13 (81.8% nt identity) and was most distantly related to the satRNA of GFLV-R2 (71.0% nt identity). Full-length infectious clones of GFLV-SACH44 satRNA were constructed. The infectivity of the clones was tested with three nepovirus isolates, GFLV-NW, Arabis mosaic virus (ArMV)-NW and GFLV-SAPCS3. The clones were mechanically inoculated in Chenopodium quinoa and were infectious when co-inoculated with the two GFLV helper viruses, but not when co-inoculated with ArMV-NW. PMID:23867805

  11. Raspberry latent virus, a New Reovirus Isolated from Crumbly Fruited Red Raspberry Plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A virus induced crumbly fruit disease in 'Meeker' and other red raspberry cultivars has been observed in northern Washington, USA and British Columbia, Canada. Raspberry bushy dwarf virus (RBDV) and Raspberry leaf mottle virus (RLMV) were detected in raspberries with severe crumbly fruit. In additi...

  12. Complete Nucleotide Sequence of an Isolate of Coleus vein necrosis virus from Verbena

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A plant of 'Taylor Town Red' verbena exhibiting mottling, necrosis and low vigor was tested for the presence of viruses by extracting double-stranded RNA which is indicative of infection with an RNA virus. The dsRNA was cloned and sequenced and a novel carlavirus identified. The new virus was dete...

  13. Pandemic H1N1 influenza virus in Chilean commercial turkeys with genetic and serologic comparisons to U.S. H1N1 avian influenza vaccine isolates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Beginning in April 2009, a novel H1N1 influenza virus has caused acute respiratory disease in humans, first in Mexico and then spreading around the world. The resulting pandemic influenza A H1N1 2009 (pH1N1) virus was isolated in swine in Canada in June, 2009, and later in turkey breeders in Chile, ...

  14. Characterization of Class I Newcastle disease virus isolates from Hong Kong bird markets and detection using real-time reverse transcription PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Of twenty-three viral isolates from Hong Kong bird market poultry samples (2003-05) that hemmagglutinated chicken red blood cells and demonstrated Newcastle disease virus (NDV) specific hemagglutination-inhibition activity with polyclonal antiserum, 21 tested negative for avian influenza virus and N...

  15. Coastal Plain Experiment Station, Department of Plant Pathology, University of Georgia, Tifton, GA, USA Phylogenetic Analysis of Iris yellow spot virus Isolates from Onion (Allium cepa)

    E-print Network

    Pappu, Hanu R.

    , USA Phylogenetic Analysis of Iris yellow spot virus Isolates from Onion (Allium cepa) in Georgia (USA observed in sweet onions in Georgia (USA) in 2003 in the Vidalia region. The virus had been reported in the onion- growing regions in western USA several years before being detected in Georgia in the east

  16. Evaluation and attempted optimization of avian embryos and cell culture methods for efficient isolation and propagation of low pathogenicity avian influenza viruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Surveillance of wild bird populations for avian influenza viruses (AIV) contributes to our understanding of AIV evolution and ecology. Both real-time reverse transcriptase polymerase chain reaction (RRT-PCR) and virus isolation in embryonating chicken eggs (ECE) are standard methods for detecting A...

  17. Evaluation of homologous inactivated influenza vaccine for protection of chickens against the H7N9 virus isolated in Anhui, China during 2013

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The recent outbreak of avian influenza (AI) H7N9 in humans in China in 2013 has resulted in approximately 30 % mortality. The genetic composition of these H7N9 viruses appears to be solely of avian origin. Although few isolations of these viruses have been demonstrated on poultry farms, the correlat...

  18. Efficacy of inactivated influenza vaccines for protection of poultry against the H7N9 low pathogenic avian influenza virus isolated in China during 2013

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The recent outbreak in China of avian influenza (AI) H7N9 in birds and humans underscores the interspecies movement of these viruses. Interestingly, the genetic composition of these H7N9 viruses appears to be solely of avian origin and of low pathogenicity in birds. Although few isolations of these ...

  19. Phylogenetic and antigenic characterization of reassortant H9N2 avian influenza viruses isolated from wild waterfowl in the East Dongting Lake wetland in 2011–2012

    PubMed Central

    2014-01-01

    Background Wild waterfowl are recognized as the natural reservoir for influenza A viruses. Two distinct lineages, the American and Eurasian lineages, have been identified in wild birds. Gene flow between the two lineages is limited. The H9N2 virus has become prevalent in poultry throughout Eurasia, and mainly circulates in wild ducks and shorebirds in North America. Methods In this study, 22 H9N2 avian influenza viruses were isolated from wild waterfowl feces in East Dongting Lake Nature Reserve in November 2011 and March 2012. The phylogenetic, molecular, and antigenic characteristics of these viruses were analyzed based on analyses of the whole genome sequence of each isolate. Results Phylogenetic analyses indicated that these H9N2 viruses were generated by reassortment events. The HA, NA, PA, and NS genes were derived from the American gene pool, and the other four genes were derived from the Eurasian gene pool. Antigenic analyses indicated that these viruses were significantly different from the Eurasian lineage viruses. Conclusions This study presents the isolation of novel intercontinental recombinant H9N2 viruses from wild waterfowl in the East Dongting Lake wetland. The novel genotype H9N2 virus has not been detected in poultry in the region yet, and may be transmitted to naïve birds in poultry farms. Therefore, our results highlight the need for ongoing surveillance of wild birds and poultry in this region. PMID:24779444

  20. Sequence diversity of the nucleoprotein gene of iris yellow spot virus (genus Tospovirus, family Bunyaviridae) isolates from the western region of the United States.

    PubMed

    Pappu, H R; du Toit, L J; Schwartz, H F; Mohan, S K

    2006-05-01

    Iris yellow spot virus (IYSV), a tentative virus species in the genus Tospovirus and family Bunyaviridae, is considered a rapidly emerging threat to onion production in the western United States (US). The present study was undertaken to determine the sequence diversity of IYSV isolates from infected onion plants grown in California, Colorado, Idaho, Oregon, Utah and Washington. Using primers derived from the small RNA of IYSV, the complete sequence of the nucleoprotein (NP) gene of each isolate was determined and the sequences compared. In addition, a shallot isolate of IYSV from Washington was included in the study. The US isolates of IYSV shared a high degree of sequence identity (95 to 99%) with one another and to previously reported isolates. Phylogenetic analyses showed that with the exception of one isolate from central Oregon and one isolate from California, all the onion and shallot isolates from the western US clustered together. This cluster also included onion and lisianthus isolates from Japan. A second distinct cluster consisted of isolates from Australia (onion), Brazil (onion), Israel (lisianthus), Japan (alstroemeria), The Netherlands (iris) and Slovenia (leek). The IYSV isolates evaluated in this study appear to represent two distinct groups, one of which largely represents isolates from the western US. Understanding of the population structure of IYSV would potentially provide insights into the molecular epidemiology of this virus. PMID:16320007

  1. Genome analysis of orf virus isolates from goats in the Fujian Province of southern China

    PubMed Central

    Chi, Xuelin; Zeng, Xiancheng; Li, Wei; Hao, Wenbo; Li, Ming; Huang, Xiaohong; Huang, Yifan; Rock, Daniel L.; Luo, Shuhong; Wang, Shihua

    2015-01-01

    Orf virus (ORFV), a species of the genus Parapoxvirus of the family Poxviridae, causes non-systemic, highly contagious, and eruptive disease in sheep, goat, and other wild and domestic ruminants. Our previous work shows orf to be ubiquitous in the Fujian Province of China, a region where there is considerable heterogeneity among ORFVs. In this study, we sequenced full genomes of four Fujian goat ORFV strains (OV-GO, OV-YX, OV-NP, and OV-SJ1). The four strains were 132–139 kb in length, with each containing 124–132 genes and about 64% G+C content. The most notable differences between the four strains were found near the genome termini. OV-NP lacked seven and OV-SJ1 lacked three genes near the right terminus when compared against other ORFVs. We also investigated the skin-virulence of the four Fujian ORFVs in goats. The ORFVs with gene deletions showed low virulence while the ORFVs without gene deletions showed high virulence in goats suggesting gene deletion possibly leads to attenuation of ORFVs. Gene 134 was disrupted in OV-NP genome due to the lack of initial code. The phylogenetic tree based on complete Parapoxviruse genomes showed that sheep originated and goat originated ORFVs formed distinctly separate branches with 100% bootstrap. Based on the single gene phylogenetic tree of 132 genes of ORFVs, 47 genes can be easily distinguished as having originated from sheep or goats. In order to further reveal genetic variation presented in goat ORFVs and sheep ORFVs, we analyzed the deduced amino acid sequences of gene 008, multiple alignment of amino acid sequences of gene 008 from the genome of five goat ORFVs and four sheep ORFVs revealed 33 unique amino acids differentiating it as having sheep or goats as host. The availability of genomic sequences of four Fujian goat ORFVs aids in our understanding of the diversity of orf virus isolates in this region and can assist in distinguishing between orf strains that originate in sheep and goats. PMID:26557108

  2. Different Pathogenesis of CCR5-Using Primary HIV-1 Isolates from Non-Switch and Switch Virus Patients in Human Lymphoid Tissue Ex Vivo

    NASA Technical Reports Server (NTRS)

    Iarlsson, Ingrid; Grivel, Jean-Charles; Chen. Silvia; Karlsson, Anders; Albert, Jan; Fenyol, Eva Maria; Margolis, Leonid B.

    2005-01-01

    CCR5-utilizing HIV-1 variants (R5) typically transmit infection and dominate its early stages, whereas emergence of CXCR4-using (X4 or R5X4) HIV-1 is often associated with disease progression. However, such a switch in co-receptor usage can only be detected in approximately onehalf of HIV-infected patients (switch virus patients), and progression to immunodeficiency may also occur in patients without detectable switch in co-receptor usage (non-switch virus patients). Here, we used a system of ex vivo-infected tonsillar tissue to compare the pathogenesis of sequential primary R5 HIV-1 isolates from the switch and non-switch patients. Inoculation of ex vivo tissue with these R5 isolates resulted in viral replication and CCR5(+)CD4(+) T cell depletion. The levels of such depletion by HIV-1 isolated from non-switch virus patients were significantly higher than those by R5 HIV-1 isolates from switch virus patients. T cell depletion seemed to be controlled by viral factors and did not significantly vary between tissues from different donors. In contrast, viral replication did not correlate with the switch status of the patients; in tissues fiom different donors it varied 30-fold and seemed to be controlled by a combination of viral and tissue factors. Nevertheless, replication-level hierarchy among sequential isolates remained constant in tissues from various donors. Viral load in vivo was higher in switch virus patients compared to non-switch virus patients. The high cytopathogenicity of CCR5(+)CD4(+) T cells by R5 HIV-1 isolates from non-switch virus patients may explain the steady decline of CD4(+) T cells in the absence of CXCR4 using virus; elimination of target cells by these isolates may limit their own replication in vivo.

  3. Relationship Between Ebola Virus Real-Time Quantitative Polymerase Chain Reaction-Based Threshold Cycle Value and Virus Isolation From Human Plasma.

    PubMed

    Spengler, Jessica R; McElroy, Anita K; Harmon, Jessica R; Ströher, Ute; Nichol, Stuart T; Spiropoulou, Christina F

    2015-10-01

    We performed a longitudinal analysis of plasma samples obtained from 4 patients with Ebola virus (EBOV) disease (EVD) to determine the relationship between the real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)-based threshold cycle (Ct) value and the presence of infectious EBOV. EBOV was not isolated from plasma samples with a Ct value of >35.5 or >12 days after onset of symptoms. EBOV was not isolated from plasma samples in which anti-EBOV nucleoprotein immunoglobulin G was detected. These data demonstrate the utility of interpreting qRT-PCR results in the context of the course of EBOV infection and associated serological responses for patient-management decisions. PMID:25941333

  4. African Swine Fever Virus Georgia Isolate Harboring Deletions of MGF360 and MGF505 Genes Is Attenuated in Swine and Confers Protection against Challenge with Virulent Parental Virus

    PubMed Central

    O'Donnell, Vivian; Holinka, Lauren G.; Gladue, Douglas P.; Sanford, Brenton; Krug, Peter W.; Lu, Xiqiang; Arzt, Jonathan; Reese, Bo; Carrillo, Consuelo; Risatti, Guillermo R.

    2015-01-01

    ABSTRACT African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal disease of domestic pigs that has significant economic consequences for the swine industry. The control of African swine fever (ASF) has been hampered by the unavailability of vaccines. Experimental vaccines have been developed using genetically modified live attenuated ASFVs where viral genes involved in virus virulence were removed from the genome. Multigene family 360 (MGF360) and MGF505 represent a group of genes sharing partial sequence and structural identities that have been connected with ASFV host range specificity, blocking of the host innate response, and virus virulence. Here we report the construction of a recombinant virus (ASFV-G-?MGF) derived from the highly virulent ASFV Georgia 2007 isolate (ASFV-G) by specifically deleting six genes belonging to MGF360 or MGF505: MGF505-1R, MGF360-12L, MGF360-13L, MGF360-14L, MGF505-2R, and MGF505-3R. ASFV-G-?MGF replicates as efficiently in primary swine macrophage cell cultures as the parental virus. In vivo, ASFV-G-?MGF is completely attenuated in swine, since pigs inoculated intramuscularly (i.m.) with either 102 or 104 50% hemadsorbing doses (HAD50) remained healthy, without signs of the disease. Importantly, when these animals were subsequently exposed to highly virulent parental ASFV-G, no signs of the disease were observed, although a proportion of these animals harbored the challenge virus. This is the first report demonstrating the role of MGF genes acting as independent determinants of ASFV virulence. Additionally, ASFV-G-?MGF is the first experimental vaccine reported to induce protection in pigs challenged with highly virulent and epidemiologically relevant ASFV-G. IMPORTANCE The main problem for controlling ASF is the lack of vaccines. Studies focusing on understanding ASFV virulence led to the production of genetically modified recombinant viruses that, while attenuated, are able to confer protection in pigs challenged with homologous viruses. Here we have produced an attenuated recombinant ASFV derived from highly virulent ASFV strain Georgia (ASFV-G) lacking only six of the multigene family 360 (MGF360) and MGF505 genes (ASFV-G-?MGF). It is demonstrated, by first time, that deleting specific MGF genes alone can completely attenuate a highly virulent field ASFV isolate. Recombinant virus ASFV-G-?MGF effectively confers protection in pigs against challenge with ASFV-G when delivered once via the intramuscular (i.m.) route. The protection against ASFV-G is highly effective by 28 days postvaccination. This is the first report of an experimental vaccine that induces solid protection against virulent ASFV-G. PMID:25810553

  5. Complete genome sequences of a novel reassortant H4N2 avian influenza virus isolated from a live poultry market in eastern China.

    PubMed

    Teng, Qiaoyang; Ji, Xiwen; Li, Guoxin; Li, Xuesong; Li, Zejun

    2012-11-01

    A/duck/Shanghai/28-1/2009(H4N2) (DK28) was isolated from a live poultry market in Shanghai, China. Using PCR and sequencing analysis, we obtained the complete genome sequences of the DK28 virus. The sequence analysis demonstrated that this H4N2 virus was a novel multiple-gene reassortant avian influenza virus (AIV) whose genes originated from H1N1, H1N3, H3N3, H4N2, and H4N6. Knowledge regarding the complete genome sequences of the DK28 virus will be useful for epidemiological surveillance. PMID:23043180

  6. Complete Genome Sequences of a Novel Reassortant H4N2 Avian Influenza Virus Isolated from a Live Poultry Market in Eastern China

    PubMed Central

    Teng, Qiaoyang; Ji, Xiwen; Li, Guoxin; Li, Xuesong

    2012-01-01

    A/duck/Shanghai/28-1/2009(H4N2) (DK28) was isolated from a live poultry market in Shanghai, China. Using PCR and sequencing analysis, we obtained the complete genome sequences of the DK28 virus. The sequence analysis demonstrated that this H4N2 virus was a novel multiple-gene reassortant avian influenza virus (AIV) whose genes originated from H1N1, H1N3, H3N3, H4N2, and H4N6. Knowledge regarding the complete genome sequences of the DK28 virus will be useful for epidemiological surveillance. PMID:23043180

  7. Full-genome sequences of hepatitis B virus subgenotype D3 isolates from the Brazilian Amazon Region.

    PubMed

    Spitz, Natália; Mello, Francisco C A; Araujo, Natalia Motta

    2015-02-01

    The Brazilian Amazon Region is a highly endemic area for hepatitis B virus (HBV). However, little is known regarding the genetic variability of the strains circulating in this geographical region. Here, we describe the first full-length genomes of HBV isolated in the Brazilian Amazon Region; these genomes are also the first complete HBV subgenotype D3 genomes reported for Brazil. The genomes of the five Brazilian isolates were all 3,182 base pairs in length and the isolates were classified as belonging to subgenotype D3, subtypes ayw2 (n = 3) and ayw3 (n = 2). Phylogenetic analysis suggested that the Brazilian sequences are not likely to be closely related to European D3 sequences. Such results will contribute to further epidemiological and evolutionary studies of HBV. PMID:25742278

  8. Citrus Viruses in Guatemala: Application of Laboratory-Based Assays

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In preparation for a citrus certification in Guatemala, there was an urgent need to determine which graft transmissible citrus pathogens were present. Because of the lack of biological indicator plants, Citrus tristeza virus (CTV) and Xylella fastidiosa, causal agent for citrus variegated chlorosis...

  9. Equine influenza A(H3N8) virus isolated from Bactrian camel, Mongolia.

    PubMed

    Yondon, Myagmarsukh; Zayat, Batsukh; Nelson, Martha I; Heil, Gary L; Anderson, Benjamin D; Lin, Xudong; Halpin, Rebecca A; McKenzie, Pamela P; White, Sarah K; Wentworth, David E; Gray, Gregory C

    2014-12-01

    Because little is known about the ecology of influenza viruses in camels, 460 nasal swab specimens were collected from healthy (no overt illness) Bactrian camels in Mongolia during 2012. One specimen was positive for influenza A virus (A/camel/Mongolia/335/2012[H3N8]), which is phylogenetically related to equine influenza A(H3N8) viruses and probably represents natural horse-to-camel transmission. PMID:25418532

  10. Phylogenetic analysis of a novel H6N6 avian influenza virus isolated from a green peafowl in China and its pathogenic potential in mice.

    PubMed

    Fan, Zhaobin; Ci, Yanpeng; Ma, Yixin; Liu, Liling; Wang, Deli; Ma, Jianzhang; Li, Yanbing; Chen, Hualan

    2014-12-01

    To explore the ecology of the H6 subtype avian influenza viruses in Hebei Province, China, a long-term surveillance was conducted in 2012 among domestic poultry and birds in a wildlife park. In this study, we report the characterization of a novel H6N6 avian influenza virus isolated from a healthy green peafowl in Qinghuangdao Wildlife Park in 2012. A phylogenetic analysis indicated that the isolated H6N6 strain has the same gene constellation as the ST3367-like strains, which are mainly distributed in southern and eastern China. A mouse experiment showed that the isolate replicated efficiently in the lungs and turbinates of infected mice without previous adaptation, resulting in locally thickened alveolar septa and interstitial pneumonia. Further studies of the H6 subtype viruses are required to clarify their evolutionary pattern in north China, which will benefit disease control and pandemic preparedness for novel viruses. PMID:25220620

  11. Lack of detection of host associated differences in Newcastle disease viruses of genotype VIId isolated from chickens and geese

    PubMed Central

    2012-01-01

    Background The goose is usually considered to be resistant even to strains of Newcastle disease virus (NDV) that are markedly virulent for chickens. However, ND outbreaks have been frequently reported in goose flocks in China since the late 1990s with the concurrent emergence of genotype VIId NDV in chickens. Although the NDVs isolated from both chickens and geese in the past 15?years have been predominantly VIId viruses, published data comparing goose- and chicken-originated ND viruses are scarce and controversial. Results In this paper, we compared genotype VIId NDVs originated from geese and chickens genetically and pathologically. Ten entire genomic sequences and 329 complete coding sequences of individual genes from genotype VIId NDVs of both goose- and chicken-origin were analyzed. We then randomly selected two goose-originated and two chicken-originated VIId NDVs and compared their pathobiology in both geese and chickens in vivo and in vitro with genotype IV virus Herts/33 as a reference. The results showed that all the VIId NDVs either from geese or from chickens shared high sequence homology and characteristic amino acid substitutions and clustered together in phylogenetic trees. In addition, geese and chickens infected by goose or chicken VIId viruses manifested very similar pathological features distinct from those of birds infected with Herts/33. Conclusions There is no genetic or phenotypic difference between genotype VIId NDVs originated from geese and chickens. Therefore, no species-preference exists for either goose or chicken viruses and more attention should be paid to the trans-species transmission of VIId NDVs between geese and chickens for the control and eradication of ND. PMID:22971647

  12. Increased sensitivity of RT-PCR for Potato virus Y detection using RNA isolated by a procedure with differential centrifugation.

    PubMed

    Zhang, Jianhua; Nie, Xianzhou; Boquel, Sébastien; Al-Daoud, Fadi; Pelletier, Yvan

    2015-12-01

    The sensitivity of reverse transcription-polymerase chain reaction (RT-PCR) for virus detection is influenced by many factors such as specificity of primers and quality of templates. These factors become extremely important for successful detection when virus concentration is low. Total RNA isolated from Potato virus Y (PVY)-infected potato plants using the sodium sulfite RNA isolation method or RNeasy plant mini kit contains a high proportion of host RNA and may also contain trace amount of phenolic and polysaccharide residues, which may inhibit RT-PCR. The goal of this study was to enhance the sensitivity of PVY detection by reducing host RNA in the extract by differential centrifugation followed by extraction using an RNeasy mini kit (DCR method). One-step RT-PCR had relatively low amplification efficiency for PVY RNA when a high proportion of plant RNA was present. SYBR Green-based real time RT-PCR showed that the RNA isolated by the DCR method had a higher cycle threshold value (Ct) for the elongation factor 1-? mRNA (Ef1?) of potato than the Ct value of the RNA extracted using the RNeasy plant mini kit, indicating that the DCR method significantly reduced the proportion of potato RNA in the extract. The detectable amount of RNA extracted using the DCR method was <0.001ng when plant sap from 10 PVY-infected and PVY-free potato leaflets in a 1.5:100 fresh weight ratio was extracted, compared with 0.01 and 0.02ng of RNA using the RNeasy plant mini kit and sodium sulfite RNA isolation methods, respectively. PMID:26210699

  13. An in vitro study of antifungal drug susceptibility of Candida species isolated from human immunodeficiency virus seropositive and human immunodeficiency virus seronegative individuals in Lucknow population Uttar Pradesh

    PubMed Central

    Dar, Mohammad Shafi; Sreedar, Gadiputi; Shukla, Abhilasha; Gupta, Prashant; Rehan, Ahmad Danish; George, Jiji

    2015-01-01

    Background: Candidiasis is the most common opportunistic infection in human immunodeficiency virus (HIV) seropositive patients, starting from asymptomatic colonization to pathogenic forms and gradual colonization of non-albicans in patients with advanced immunosuppression leads to resistance for azole group of antifungal drugs with high rate of morbidity and mortality. Objectives: To isolate the Candida species and determine of antifungal drug susceptibility against fluconazole, itraconazole, nystatin, amphotericin B, and clotrimazolein HIV seropositive and control individuals, with or without clinical oropharyngeal candidiasis (OPC). Materials and Methods: Includes samples from faucial region of 70 subjects with and without clinical candidiasis in HIV seropositive and controls were aseptically inoculated onto Sabaraud's Dextrose Agar media and yeasts were identified for the specific species by Corn Meal Agar, sugar fermentation and heat tolerance tests. Antifungal drug susceptibility of the isolated species was done against above-mentioned drugs by E-test and disc diffusion method. Results: The commonly isolated species in HIV seropositive and controls were Candida albicans, Candida glabrata and Candida tropicalis Candida guilliermondii and Candida dubliniensis isolated only in HIV seropositive patients. Susceptibility against selected antifungal drugs was observed more in HIV-negative individuals whereas susceptible dose-dependent and resistance were predominant in HIV-positive patients. Conclusion: Resistance is the major problem in the therapy of OPC, especially in HIV seropositive patients due to aggressive and prolonged use of antifungal agents, therefore, our study emphasizes the need for antifungal drug susceptibility testing whenever antifungal treatment is desired, especially in HIV-infected subjects.

  14. Genome of a mononucleosis Epstein-Barr virus contains DNA fragments previously regarded to be unique to Burkitt's lymphoma isolates.

    PubMed

    Fischer, D K; Miller, G; Gradoville, L; Heston, L; Westrate, M W; Maris, W; Wright, J; Brandsma, J; Summers, W C

    1981-05-01

    We wished to learn whether the genomes of strains of EMB isolated from patients with infectious mononucleosis are consistently distinguishable from those of strains from Burkitt's lymphoma. The genome of a new transforming strains (FF41) of EBV isolated from saliva of a patient with uncomplicated infectious mononucleosis was compared with the DNA of B95-8, the only other available virus from mononucleosis. It had been found previously that B95-8 has a deletion of about 8 Md in the region of the physical map represented by the Eco RI C, Hind III D, and Bam HI I fragments. The W91 and HR-1 isolates for Burkitt's lymphoma are not deleted in this region and it had been proposed that additional information was characteristic of EBV isolates from Burkitt's lymphoma. By means of restriction enzyme analysis, blot hybridization experiments and molecular cloning of FF41 DNA we demonstrate that the deletion found in B95-8 is not present in the new mononucleosis isolate. The FF41 genome contains an extra 8 Md of DNA, represented by Bam HI fragments B', W' and I', which are located in a larger Eco RI C fragment. Thus the genome of this salivary isolate contains DNA that had previously been regarded to be unique to strains from Burkitt's lymphoma. It is therefore unlikely that major insertions or deletions in the EBV genome account for differences in disease manifestation following EBV infection. PMID:6263500

  15. Sequence analysis of the NSP2, ORF5, and ORF7 genes of 11 PRRS virus isolates from China.

    PubMed

    Li, Jida; Yin, Yanbo; Guo, Baoqing; Zhou, Shun; Zhang, Yi; Liu, Xingcai; Sun, Tingting

    2012-10-01

    During a tracing investigation of blue ear disease in China conducted from January to November 2008, 11 porcine reproductive and respiratory syndrome virus (PRRSV) isolates were collected from eight provinces including Liaoning, Jilin, Hebei, Shandong, Henan, Shanghai, Zhejiang, and Guangxi. The complete gene sequences of the NSP2, ORF5, and ORF7 genes from these 11 PRRSV isolates were amplified, cloned and detected by RT-PCR and then compared with the published sequences of other strains. The results showed that all of the isolates genotypically belonged to an American strain, but shared high homology with JXA1, the highly pathogenic strain endemic to China. All of the 11 PRRSV isolates demonstrated a 90-nucleotide deletion in the NSP2 gene, suggesting that the main epidemic of PRRSV in 2008 was due to this gene deletion isolate. More consistent mutations were found in specific regions of the ORF5 and ORF7 genes of the 11 PRRSV isolates, such as the signal peptide and transmembrane regions of GP5 and the Pat 7 motif of the N protein. Whether these mutations influence nuclear localization of PRRSV requires confirmation by future studies. PMID:22644762

  16. Differential Viral Fitness Between H1N1 and H3N8 Avian Influenza Viruses Isolated from Mallards (Anas platyrhynchos).

    PubMed

    Ferreira, Helena Lage; Vangeluwe, Didier; Van Borm, Steven; Poncin, Olivier; Dumont, Nathalie; Ozhelvaci, Orkun; Munir, Muhammad; van den Berg, Thierry; Lambrecht, Bénédicte

    2015-12-01

    Homosubtypic and heterosubtypic immunity in mallards (Anas platyrhynchos) play an important role in the avian influenza virus (AIV) diversity. The mechanisms of AIV replication among wild birds and the role of immunity in AIV diversity have thus not been completely clarified. During the monitoring of AI circulation among wild waterfowl in 2007-2008, two viruses (H3N8 and H1N1) were isolated from ducks caught in a funnel trap located in La Hulpe wetland in Belgium. H3N8 viruses were revealed to be more prevalent in the mallard population than was H1N1, which might suggest a better adaptation to this species. In order to investigate this hypothesis, we characterized both isolated viruses biologically by experimental inoculation. Virus excretion and humoral response induced by both isolated viruses were evaluated in mallards after a first infection followed by a homo- or heterosubtypic reinfection under controlled experimental conditions. The H1N1 virus had a delayed peak of excretion of 4 days compared to the H3N8, but the virus shedding was more limited, earlier, and shorter after each reinfection. Moreover, the H3N8 virus could spread to all ducks after homo- or heterosubtypic reinfections and during a longer period. Although the humoral response induced by both viruses after infection and reinfection could be detected efficiently by competitive ELISA, only a minimal H1 antibody response and almost no H3-specific antibodies could be detected by the HI test. Our results suggest that the H3N8 isolate replicates better in mallards under experimental controlled conditions. PMID:26629623

  17. A single-tube nucleotide isolation reagent for the quantitative PCR detection of virus in body fluids.

    PubMed

    Liu, Hong; Wu, Yuansheng; Liu, Cuihua; He, Jinyang

    2014-07-01

    A high-salt reagent composed of guanidinium thiocyanate, guanidine hydrochloride, urea, sodium citrate, and other compounds was designed for the single-tube isolation of viral nucleotides from body fluids. The single-tube reagent was used for the extraction of SIV RNA and HBV DNA from standard virus stock dilutions and virus-positive samples. The sensitivity and reproducibility of the single-tube reagent were analysed via quantitative PCR assays. The results revealed that the single-tube reagent can facilitate quantitative PCR-mediated detection in a reaction system with a 25-?l volume using only 100 ?l of a body fluid sample and reaches a sensitivity of up to 50 copies/ml. The low coefficients of variance of both the HBV and SIV standard stock results indicate the excellent reproducibility of the single-tube reagent. A Bland-Altman analysis of the assay results from the SIV- and HBV-positive samples revealed that the single-tube reagent can precisely extract both RNA and DNA viral nucleotides from virus-positive samples. All of the isolation steps were performed in a single tube and were completed in no more than 35 min. The only major equipment required is a high-speed freezing centrifuge. The single-tube reagent is economical and easy to use and does not require any complex equipment. PMID:24720911

  18. Characterization of antigenic domains and epitopes in the ORF3 protein of a Chinese isolate of avian hepatitis E virus.

    PubMed

    Zhao, Qin; Sun, Ya-ni; Hu, Shou-bin; Wang, Xin-jie; Xiao, Yi-hong; Hsu, Walter H; Xiao, Shu-qi; Wang, Cheng-bao; Mu, Yang; Hiscox, Julian A; Zhou, En-Min

    2013-12-27

    Avian hepatitis E virus (HEV) is an emerging virus associated with the big liver and spleen disease or hepatitis-splenomegaly syndrome in chickens and subclinical infections by the virus are also common. The complete genome of avian HEV contains three open-reading frames (ORFs) in which ORF2 protein is part of virus particles and thus contains primary epitopes. Antigenic epitopes of avian HEV ORF2 protein have been described but those associated with the ORF3 have not. To analyze the antigenic domains and epitopes in the ORF3 protein of a Chinese isolate of avian HEV (CaHEV), we generated a series of antigens comprised of the complete ORF3 and also five truncated overlapping ORF3 peptides. The antibodies used in this study were mouse antisera and monoclonal antibodies against ORF3, positive chicken sera from Specific Pathogen Free chickens experimentally infected with CaHEV and clinical chicken sera. Using these antigens and antibodies, we identified three antigenic domains at amino acids (aa) 1-28, 55-74 and 75-88 in which aa 75-88 was a dominant domain. The dominant domain contained at least two major epitopes since field chickens infected with avian HEV produced antibodies against the domain and epitopes. These results provide useful information for future development of immunoassays for the diagnosis of avian HEV infection. PMID:24021883

  19. Twelve isolations of Zika virus from Aedes (Stegomyia) africanus (Theobald) taken in and above a Uganda forest*

    PubMed Central

    Haddow, A. J.; Williams, M. C.; Woodall, J. P.; Simpson, D. I. H.; Goma, L. K. H.

    1964-01-01

    In continuation of a series of studies of arboreal mosquitos as virus vectors in Uganda, 12 strains of Zika virus and one strain of another Group B arbovirus were isolated between November 1961 and June 1963 from pools of Aedes (Stegomyia) africanus caught on a 120-foot (36.5-m) tower in Zika forest. For five strains it is known at what height the mosquitos were caught: one was from mosquitos taken at ground level, and the other four were from mosquitos taken in or above the upper canopy after sunset. No small mammal trapped in the forest either on the ground or in the trees showed serum antibody for Zika virus. These findings suggest that in Zika forest, A. (S.) africanus becomes infected from a virus reservoir that is probably not among the small animals tested and that infected mosquitos are liable to be spread widely beyond the forest by convection currents above the tree-tops in the first two or three hours after sunset. ImagesFIG. 1 PMID:14230895

  20. TWELVE ISOLATIONS OF ZIKA VIRUS FROM AEDES (STEGOMYIA) AFRICANUS (THEOBALD) TAKEN IN AND ABOVE A UGANDA FOREST.

    PubMed

    HADDOW, A J; WILLIAMS, M C; WOODALL, J P; SIMPSON, D I; GOMA, L K

    1964-01-01

    In continuation of a series of studies of arboreal mosquitos as virus vectors in Uganda, 12 strains of Zika virus and one strain of another Group B arbovirus were isolated between November 1961 and June 1963 from pools of Aedes (Stegomyia) africanus caught on a 120-foot (36.5-m) tower in Zika forest. For five strains it is known at what height the mosquitos were caught: one was from mosquitos taken at ground level, and the other four were from mosquitos taken in or above the upper canopy after sunset. No small mammal trapped in the forest either on the ground or in the trees showed serum antibody for Zika virus.These findings suggest that in Zika forest, A. (S.) africanus becomes infected from a virus reservoir that is probably not among the small animals tested and that infected mosquitos are liable to be spread widely beyond the forest by convection currents above the tree-tops in the first two or three hours after sunset. PMID:14230895

  1. Completion sequence and cloning of the infectious cDNA of a chb isolate of cucumber green mottle mosaic virus.

    PubMed

    Zhong, M; Zhao, X; Liu, Y; Wang, Y; Cao, K

    2015-03-01

    Cucumber green mottle mosaic virus (CGMMV) is an important and widespread seed-borne virus that infects Cucurbitaceous plants. It is a member of the genus Tobamovirus in the family Virgaviridae with a monopartite (+) ssRNA genome. Here we report the complete genome sequence, construction and testing of the infectious clones of a chb isolate of CGMMV. Full-length CGMMV cDNA was cloned into the vector pUC19. The linearized vector containing full-length cDNA was used as template for in vitro transcription, and the synthesized capped transcript was highly infectious in Chenopodium amaranticolor and cucumber (Cucumis sativus). Inoculated plants showed symptoms typical of CGMMV infection. The infectivity was confirmed by mechanical transmission to new plants, RT-PCR and western blot. Progeny virus derived from infectious transcripts had the same biological and biochemical properties as wild-type virus. To our knowledge, this is the first detailed report of a biologically active transcript from CGMMV. PMID:25790051

  2. Isolation of deer tick virus (Powassan virus, lineage II) from Ixodes scapularis and detection of antibody in vertebrate hosts sampled in the Hudson Valley, New York State

    PubMed Central

    2013-01-01

    Background Deer tick virus, DTV, is a genetically and ecologically distinct lineage of Powassan virus (POWV) also known as lineage II POWV. Human incidence of POW encephalitis has increased in the last 15 years potentially due to the emergence of DTV, particularly in the Hudson Valley of New York State. We initiated an extensive sampling campaign to determine whether POWV was extant throughout the Hudson Valley in tick vectors and/or vertebrate hosts. Methods More than 13,000 ticks were collected from hosts or vegetation and tested for the presence of DTV using molecular and virus isolation techniques. Vertebrate hosts of Ixodes scapularis (black-legged tick) were trapped (mammals) or netted (birds) and blood samples analyzed for the presence of neutralizing antibodies to POWV. Maximum likelihood estimates (MLE) were calculated to determine infection rates in ticks at each study site. Results Evidence of DTV was identified each year from 2007 to 2012, in nymphal and adult I. scapularis collected from the Hudson Valley. 58 tick pools were positive for virus and/or RNA. Infection rates were higher in adult ticks collected from areas east of the Hudson River. MLE limits ranged from 0.2-6.0 infected adults per 100 at sites where DTV was detected. Virginia opossums, striped skunks and raccoons were the source of infected nymphal ticks collected as replete larvae. Serologic evidence of POWV infection was detected in woodchucks (4/6), an opossum (1/6), and birds (4/727). Lineage I, prototype POWV, was not detected. Conclusions These data demonstrate widespread enzootic transmission of DTV throughout the Hudson Valley, in particular areas east of the river. High infection rates were detected in counties where recent POW encephalitis cases have been identified, supporting the hypothesis that lineage II POWV, DTV, is responsible for these human infections. PMID:24016533

  3. Genetic structure in natural populations of barley/cereal yellow dwarf virus isolates from Alaska

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The genetic structure of natural populations of Alaskan Barley yellow dwarf virus (BYDV)-PAV, BYDV-PAS, and Cereal yellow dwarf virus (CYDV)-RPV from barley (Hordeum vulgare) and oats (Avena sativa) in Alaska were analyzed between 2001-04. PCR products spanning the viral coat protein gene of 186 iso...

  4. Complete genome and clinicopathological characterization of a virulent Newcastle disease virus isolate from South America

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Newcastle disease (ND) is one of the most important diseases of poultry, negatively affecting trade and poultry production worldwide. The disease is caused by Newcastle disease virus (NDV) or avian paramyxovirus type-1 (APMV-1), a negative sense single-stranded RNA virus of the genus Avulavirus, fam...

  5. Differential acquisition and transmission of Florida Tomato spotted wilt virus isolates by Western flower thrips

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Thrips-vectored Tomato spotted wilt virus (TSWV) is one of the most important insect-vectored plant pathogens globally. The virus host range encompasses many key vegetable, ornamental and agronomic crops. TSWV populations are highly heterogeneous, which has important implications for vector relati...

  6. PHYLOGENY OF BLUETONGUE VIRUS ISOLATES BY SEQUENCE ANALYSIS OF THE VP5 CODING GENE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bluetongue virus (BTV) is an arthropod-borne Orbivirus that infects domestic and wild ruminants. Worldwide, there are at least 24 serotypes and numerous strains within each serotype. Bluetongue virus has 10 double-stranded genome segments that encode the viral structural and non-structural proteins...

  7. Pathogenesis of novel reassortant avian influenza virus A (H5N8) Isolates in the ferret.

    PubMed

    Kim, Heui Man; Kim, Chi-Kyeong; Lee, Nam-Joo; Chu, Hyuk; Kang, Chun; Kim, Kisoon; Lee, Joo-Yeon

    2015-07-01

    Outbreaks of avian influenza virus H5N8 first occurred in 2014, and spread to poultry farms in Korea. Although there was no report of human infection by this subtype, it has the potential to threaten human public health. Therefore, we evaluated the pathogenesis of H5N8 viruses in ferrets. Two representative Korean H5N8 strains did not induce mortality and significant respiratory signs after an intranasal challenge in ferrets. However, ferrets intratracheally infected with A/broiler duck/Korea/Buan2/2014 virus showed dose-dependent mortality. Although the Korean H5N8 strains were classified as the HPAI virus, possessing multiple basic amino acids in the cleavage site of the hemagglutinin sequence, they did not produce pathogenesis in ferrets challenged intranasally, similar to the natural infection route. These results could be useful for public health by providing the pathogenic characterization of H5N8 viruses. PMID:25776760

  8. Genome Analysis of a Tembusu Virus, GX2013H, Isolated from a Cheery Valley Duck in Guangxi, China

    PubMed Central

    Zeng, Tingting; Xie, Liji; Deng, Xianwen; Xie, Zhiqin; Liu, Jiabo; Fan, Qing; Pang, Yaoshan; Luo, Sisi

    2014-01-01

    We report here the complete genome sequence of a duck Tembusu virus (DTMUV) strain, GX2013H, isolated from a duck from Cheery Valley in the Guangxi Province of southern China in 2013. We obtained the strain GX2013H from a Cheery Valley duck with severely decreased egg production and neurological signs. The genome of GX2013H is 10,990 nucleotides (nt) in length and contains a single open reading frame encoding a putative polyprotein of 3,425 amino acids (aa). A comparison of the complete sequence and the deduced amino acid sequence of GX2013H with published sequences of 15 other chicken anemia viruses from China showed that the homologies of the nucleotides are approximately 96.5% to 97.5% and the homologies of the deduced amino acid sequences are approximately 98.9% to 99.3%. This report will help to understand the epidemiology and molecular characteristics of TMUV in Guangxi. PMID:25013132

  9. Pollen transmission of asparagus virus 2 (AV-2) may facilitate mixed infection by two AV-2 isolates in asparagus plants.

    PubMed

    Kawamura, Ryusuke; Shimura, Hanako; Mochizuki, Tomofumi; Ohki, Satoshi T; Masuta, Chikara

    2014-09-01

    Asparagus virus 2 (AV-2) is a member of the genus Ilarvirus and thought to induce the asparagus decline syndrome. AV-2 is known to be transmitted by seed, and the possibility of pollen transmission was proposed 25 years ago but not verified. In AV-2 sequence analyses, we have unexpectedly found mixed infection by two distinct AV-2 isolates in two asparagus plants. Because mixed infections by two related viruses are normally prevented by cross protection, we suspected that pollen transmission of AV-2 is involved in mixed infection. Immunohistochemical analyses and in situ hybridization using AV-2-infected tobacco plants revealed that AV-2 was localized in the meristem and associated with pollen grains. To experimentally produce a mixed infection via pollen transmission, two Nicotiana benthamiana plants that were infected with each of two AV-2 isolates were crossed. Derived cleaved-amplified polymorphic sequence analysis identified each AV-2 isolate in the progeny seedlings, suggesting that pollen transmission could indeed result in a mixed infection, at least in N. benthamiana. PMID:25116643

  10. Molecular characterization and phylogenetics of Fennoscandian cowpox virus isolates based on the p4c and atip genes

    PubMed Central

    2014-01-01

    Background Cowpox virus (CPXV), a rodent-borne Orthopoxvirus (OPV) that is indigenous to Eurasia can infect humans, cattle, felidae and other animals. Molecular characterization of CPXVs isolated from different geographic locations is important for the understanding of their biology, geographic distribution, classification and evolution. Our aim was to characterize CPXVs isolated from Fennoscandia on the basis of A-type inclusion (ATI) phenotype, restriction fragment length polymorphism (RFLP) profiles of atip gene fragment amplicon, and phylogenetic tree topology in conjunction with the patristic and genetic distances based on full length DNA sequence of the atip and p4c genes. Methods ATI phenotypes were determined by transmission electron microcopy and RFLP profiles were obtained by restriction enzyme digestion of the atip gene fragment PCR product. A 6.2 kbp region spanning the entire atip and p4c genes of Fennoscandian CPXV isolates was amplified and sequenced. The phylogenetic affinity of Fennoscandian CPXV isolates to OPVs isolated from other geographic regions was determined on the basis of the atip and p4c genes. Results Fennoscandian CPXV isolates encoded full length atip and p4c genes. They produce wild type V+ ATI except for CPXV-No-H2. CPXVs were resolved into six and seven species clusters based on the phylogeny of the atip and p4c genes respectively. The CPXVs isolated from Fennoscandia were grouped into three distinct clusters that corresponded to isolates from Norway, Sweden and Finland. Conclusion CPXV is a polyphyletic assemblage of six or seven distinct clusters and the current classification in which CPXVs are united as one single species should be re-considered. Our results are of significance to the classification and evolution of OPVs. PMID:24972911

  11. Metadata beyond the sequence enables the phylodynamic inference of bovine viral diarrhea virus type 1a isolates from Western Canada.

    PubMed

    Chernick, Adam; Godson, Dale L; van der Meer, Frank

    2014-12-01

    Bovine viral diarrhea virus (BVDV) has been recognized as an important pathogen of livestock in Canada. The high mutation rate of this virus leads to a great degree of diversity between isolates resulting in the ability to infer precise evolutionary relationships. Many studies have attempted to elucidate the regional and global evolution of BVDV, but so far few have applied Bayesian methods to this end. We aimed to describe the molecular epidemiology and phylodynamics of BVDV 1a isolates in Western Canada using 5'UTR and E1-E2 sequence data, collection dates and locations. Sequences were obtained from isolates submitted to a diagnostic laboratory in Saskatoon, Canada. Path sampling and stepping stone model testing were employed to identify the model that best fit the data. We found that these Western Canadian isolates share a most recent common ancestor dated near 1909. Furthermore, the E1-E2 region shows a median substitution rate about ten times greater than the 5'UTR. It was also noted that caution should be exercised when inferring phylogenetic relationships using the 5'UTR alone, as it becomes difficult to resolve relationships within major clades. Phylogeographic and population size fluctuation estimates require more thorough sampling than was performed here to be reliable. We have found that there are significant gains to be made by utilizing a Bayesian analysis and by incorporating additional types of data beyond the sequence. These include the estimation of most common recent ancestor dates and the precise inference of transmission routes. Future work will expand upon these findings by more thoroughly sampling BVDV isolates spatially and temporally and further refining the Bayesian model employed here. PMID:24424090

  12. Phylogenetic and pathogenic analyses of three H5N1 avian influenza viruses (clade 2.3.2.1) isolated from wild birds in Northeast China.

    PubMed

    Fan, Zhaobin; Ci, Yanpeng; Liu, Liling; Ma, Yixin; Jia, Ying; Wang, Deli; Guan, Yuntao; Tian, Guobin; Ma, Jianzhang; Li, Yanbing; Chen, Hualan

    2015-01-01

    From April to September 2012, periodic surveillance of avian influenza H5N1 viruses from different wild bird species was conducted in Northeast China. Three highly pathogenic avian influenza (HPAI) H5N1 viruses were isolated from a yellow-browed warbler, common shoveler, and mallard. To trace the genetic lineage of the isolates, nucleotide sequences of all eight gene segments were determined and phylogenetically analyzed. The data indicated that three viruses belonged to the same antigenic virus group: clade 2.3.2.1. To investigate the pathogenicity of these three viruses in different hosts, chickens, ducks, and mice were inoculated. The results showed that chickens were susceptible to each of the three HPAI H5N1 viruses, resulting in 100% mortality within 2-6 days after infection, whereas the three isolates exhibited distinctly different virulence in ducks and mice. The results of this study demonstrated that HPAI H5N1 viruses of clade 2.3.2.1 are still circulating in wild birds through overlapping migratory flyways. Therefore, continuous monitoring of H5N1 in both domestic and wild birds is necessary to prevent a potentially wider outbreak. PMID:25461692

  13. Whole genome sequence analysis of circulating Bluetongue virus serotype 11 strains from the United States including two domestic canine isolates.

    PubMed

    Gaudreault, Natasha N; Jasperson, Dane C; Dubovi, Edward J; Johnson, Donna J; Ostlund, Eileen N; Wilson, William C

    2015-07-01

    Bluetongue virus (BTV) is a vector-transmitted pathogen that typically infects and causes disease in domestic and wild ruminants. BTV is also known to infect domestic canines as discovered when dogs were vaccinated with a BTV-contaminated vaccine. Canine BTV infections have been documented through serological surveys, and natural infection by the Culicoides vector has been suggested. The report of isolation of BTV serotype 11 (BTV-11) from 2 separate domestic canine abortion cases in the states of Texas in 2011 and Kansas in 2012, were apparently unrelated to BTV-contaminated vaccination or consumption of BTV-contaminated raw meat as had been previously speculated. To elucidate the origin and relationship of these 2 domestic canine BTV-11 isolates, whole genome sequencing was performed. Six additional BTV-11 field isolates from Texas, Florida, and Washington, submitted for diagnostic investigation during 2011 and 2013, were also fully sequenced and analyzed. The phylogenetic analysis indicates that the BTV-11 domestic canine isolates are virtually identical, and both share high identity with 2 BTV-11 isolates identified from white-tailed deer in Texas in 2011. The results of the current study further support the hypothesis that a BTV-11 strain circulating in the Midwestern states could have been transmitted to the dogs by the infected Culicoides vector. Our study also expands the short list of available BTV-11 sequences, which may aid BTV surveillance and epidemiology. PMID:26069226

  14. Genomic detection and characterization of a Korean isolate of Little cherry virus 1 sampled from a peach tree.

    PubMed

    Lim, Seungmo; Igori, Davaajargal; Yoo, Ran Hee; Zhao, Fumei; Cho, In-Sook; Choi, Gug-Seoun; Lim, Hyoun-Sub; Lee, Su-Heon; Moon, Jae Sun

    2015-10-01

    A peach tree (Prunus persica) showing yellowing and mild mottle symptoms was analyzed using high-throughput RNA sequencing to determine the causal agent. A total of nine contigs similar to Little cherry virus 1 (LChV-1) were produced, and all the contigs showed nucleotide sequence identity (lower than 83 %) and query coverage (higher than 73 %) with LChV-1. The symptomatic peach sample was confirmed to be infected with LChV-1-like virus as a result of reverse transcription-polymerase chain reaction using primers designed based on sequences of the contigs. Occurrence of diseases caused by LChV-1 in Prunus species has been reported. Complete 16,931-nt genome of the peach virus composed of eight open reading frames was determined, and conserved domains including viral methyltransferase, viral helicase 1, RNA-dependent RNA polymerase (RdRp), heat shock protein 70 homologue (HSP70h), HSP90h and closterovirus coat protein (CP) were identified. Phylogenetic trees based on amino acid sequence alignments between the peach virus and members in the family Closteroviridae showed that the virus was most similar to LChV-1. Pairwise comparisons based on amino acid sequence alignments of three genes (RdRp, HSP70h and CP) between the peach virus and LChV-1 isolates showed the highest amino acid sequence identities, with 84.32 % for RdRp, 85.48 % for HSP70h and 80.45 % for CP. These results indicate that this is the first report for the presence of LChV-1 in South Korea and may be one of the first reports of natural infection of peach by LChV-1. Although it is not clear if LChV-1 YD isolate was responsible for specific symptoms observed, detection and characterization of the peach tree-infecting LChV-1 in South Korea would be useful in terms of the epidemiology of LChV-1. PMID:26315329

  15. Rapid detection of genetically diverse tomato black ring virus isolates using reverse transcription loop-mediated isothermal amplification.

    PubMed

    Hasiów-Jaroszewska, Beata; Budzy?ska, Daria; Borodynko, Natasza; Pospieszny, Henryk

    2015-12-01

    A reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) has been developed for detection of tomato black ring virus (TBRV) isolates collected from different hosts. One-step RT-LAMP was performed with a set of four primers, the design of which was based on the coat protein gene. Results of RT-LAMP were visualized by direct staining of products with fluorescent dyes, agarose gel electrophoresis, and analysis of amplification curves. The sensitivity of RT-LAMP was 100-fold greater than that of RT-PCR. The RT-LAMP assay developed here is a useful and practical method for diagnosis of TBRV. PMID:26338092

  16. In vivo evaluation of vaccine efficacy against challenge with a contemporary field isolate from the ? cluster of H1N1 swine influenza virus

    PubMed Central

    Detmer, Susan E.; Gramer, Marie R.; King, Vickie L.; Mathur, Sheerin; Rapp-Gabrielson, Vicki J.

    2013-01-01

    Influenza A virus vaccines currently contain a mixture of isolates that reflect the genetic and antigenic characteristics of the currently circulating strains. This study was conducted to evaluate the efficacy of a trivalent inactivated swine influenza virus vaccine (Flusure XP) in pigs challenged with a contemporary ?-cluster H1N1 field isolate of Canadian swine origin. Pigs were allocated to vaccinated, placebo, and negative-control groups and monitored for respiratory disease for 5 d after challenge. On the challenge day and 5 d after challenge the serum of the vaccinated pigs had reciprocal hemagglutination inhibition antibody titers 40 for all the vaccine viruses but ? 20 for the challenge virus. Gross lesions were present in the lungs of all pigs that had been inoculated with the challenge virus, but the proportion of lung tissue consolidated did not differ significantly between the placebo and vaccinated pigs. However, the amount of virus was significantly reduced in the nasal secretions, lungs, and bronchoalveolar lavage fluid in the vaccinated pigs compared with the placebo pigs. These results indicate that swine vaccinated with Flusure XP were partially protected against experimental challenge with a swine ?-cluster H1N1 virus that is genetically similar to viruses currently circulating in Canadian swine. PMID:23814353

  17. In vivo evaluation of vaccine efficacy against challenge with a contemporary field isolate from the ? cluster of H1N1 swine influenza virus.

    PubMed

    Detmer, Susan E; Gramer, Marie R; King, Vickie L; Mathur, Sheerin; Rapp-Gabrielson, Vicki J

    2013-01-01

    Influenza A virus vaccines currently contain a mixture of isolates that reflect the genetic and antigenic characteristics of the currently circulating strains. This study was conducted to evaluate the efficacy of a trivalent inactivated swine influenza virus vaccine (Flusure XP) in pigs challenged with a contemporary ?-cluster H1N1 field isolate of Canadian swine origin. Pigs were allocated to vaccinated, placebo, and negative-control groups and monitored for respiratory disease for 5 d after challenge. On the challenge day and 5 d after challenge the serum of the vaccinated pigs had reciprocal hemagglutination inhibition antibody titers 40 for all the vaccine viruses but ? 20 for the challenge virus. Gross lesions were present in the lungs of all pigs that had been inoculated with the challenge virus, but the proportion of lung tissue consolidated did not differ significantly between the placebo and vaccinated pigs. However, the amount of virus was significantly reduced in the nasal secretions, lungs, and bronchoalveolar lavage fluid in the vaccinated pigs compared with the placebo pigs. These results indicate that swine vaccinated with Flusure XP were partially protected against experimental challenge with a swine ?-cluster H1N1 virus that is genetically similar to viruses currently circulating in Canadian swine. PMID:23814353

  18. Isolation and phylogenetic analysis of hemagglutinin gene of H9N2 influenza viruses from chickens in South China from 2012 to 2013

    PubMed Central

    Shen, Han-Qin; Yan, Zhuan-Qiang; Zeng, Fan-Gui; Liao, Chang-Tao; Zhou, Qing-Feng; Qin, Jian-Ping; Xie, Qing-Mei; Bi, Ying-Zuo

    2015-01-01

    As part of our ongoing influenza surveillance program in South China, 19 field strains of H9N2 subtype avian influenza viruses (AIVs) were isolated from dead or diseased chicken flocks in Guangdong province, South China, between 2012 and 2013. Hemagglutinin (HA) genes of these strains were sequenced and analyzed and phylogenic analysis showed that 12 of the 19 isolates belonged to the lineage h9.4.2.5, while the other seven belonged to h9.4.2.6. Specifically, we found that all of the viruses isolated in 2013 belonged to lineage h9.4.2.5. The lineage h9.4.2.5 viruses contained a PSRSSR?GLF motif at HA cleavage site, while the lineage h9.4.2.6 viruses contained a PARSSR?GLF at the same position. Most of the isolates in lineage h9.4.2.5 lost one potential glycosylation site at residues 200-202, and had an additional one at residues 295-297 in HA1. Notably, 19 isolates had an amino acid exchange (Q226L) in the receptor binding site, which indicated that the viruses had potential affinity of binding to human like receptor. The present study shows the importance of continuing surveillance of new H9N2 strains to better prepare for the next epidemic or pandemic outbreak of H9N2 AIV infections in chicken flocks. PMID:25643797

  19. Identification of amino acid changes in the envelope glycoproteins of bovine viral diarrhea viruses isolated from alpaca that may be involved in host adaptation.

    PubMed

    Neill, John D; Dubovi, Edward J; Ridpath, Julia F

    2015-09-30

    Bovine viral diarrhea viruses (BVDV) are most commonly associated with infections of cattle. However, BVDV are often isolated from closely related ruminants with a number of BVDV-1b viruses being isolated from alpacas that were both acutely and persistently infected. The complete nucleotide sequence of the open reading frame of eleven alpaca-adapted BVDV isolates and the region encoding the envelope glycoproteins of an additional three isolates were determined. With the exception of one, all alpaca isolates were >99.2% similar at the nucleotide level. The Hercules isolate was more divergent, with 95.7% sequence identity to the other viruses. Sequence similarity of the 14 viruses indicated they were isolates of a single BVDV strain that had adapted to and were circulating through alpaca herds. Hercules was a more distantly related strain that has been isolated only once in Canada and represented a separate adaptation event that possessed the same adaptive changes. Comparison of amino acid sequences of alpaca and bovine-derived BVDV strains revealed three regions with amino acid sequences unique to all alpaca isolates. The first contained two small in-frame deletions near the N-terminus of the E2 glycoprotein. The second was found near the C-terminus of the E2 protein where four altered amino acids were located within a 30 amino acid domain that participates in E2 homodimerization. The third region contained three variable amino acids in the C-terminus of the E(rns) within the amphipathic helix membrane anchor. These changes were found in the polar side of the amphipathic helix and resulted in an increased charge within the polar face. Titration of bovine and alpaca viruses in both bovine and alpaca cells indicated that with increased charge in the amphipathic helix, the ability to infect alpaca cells also increased. PMID:26072370

  20. Complete genome analysis of a frog virus 3 (FV3) isolate and sequence comparison with isolates of differing levels of virulence

    PubMed Central

    2014-01-01

    Background Frog virus 3 (FV3) is the type species of the genus Ranavirus, and in the past few decades, FV3 infections have resulted in considerable morbidity and mortality in a range of wild and cultivated amphibian species in the Americas, Europe, and Asia. The reasons for the pathogenicity of FV3 are not well understood. Findings We investigated three FV3 isolates designated SSME, wt-FV3, and aza-Cr, and reported that our wt-FV3 and aza-Cr strains showed similar levels of virulence, while SSME was the least virulent in an in vivo study with Lithiobates pipiens tadpoles. Using 454 GS-FLX sequencing technology, we sequenced SSME and compared it to the published wt-FV3 genome. SSME had multiple amino acid deletions in ORFs 49/50L, 65L, 66L, and 87L, which may explain its reduced virulence. We also investigated repeat regions and found that repeat copy number differed between isolates, with only one group of 3 isolates and 1 pair of isolates being identical at all 3 locations. Conclusions In this study we have shown that genetic variability is present between closely related FV3 isolates, both in terms of deletions/insertions, and even more so at select repeat locations. These genomic areas with deletions/insertions may represent regions that affect virulence, and therefore require investigation. Furthermore, we have identified repeat regions that may prove useful in future phylogeographical tracking and identification of ranaviral strains across different environmental regions. PMID:24620832