Identification of reassortant pandemic H1N1 influenza virus in Korean pigs.

PubMed

Han, Jae Yeon; Park, Sung Jun; Kim, Hye Kwon; Rho, Semi; Nguyen, Giap Van; Song, Daesub; Kang, Bo Kyu; Moon, Hyung Jun; Yeom, Min Joo; Park, Bong Kyun

2012-05-01

Since the 2009 pandemic human H1N1 influenza A virus emerged in April 2009, novel reassortant strains have been identified throughout the world. This paper describes the detection and isolation of reassortant strains associated with human pandemic influenza H1N1 and swine influenza H1N2 (SIV) viruses in swine populations in South Korea. Two influenza H1N2 reassortants were detected, and subtyped by PCR. The strains were isolated using Madin- Darby canine kidney (MDCK) cells, and genetically characterized by phylogenetic analysis for genetic diversity. They consisted of human, avian, and swine virus genes that were originated from the 2009 pandemic H1N1 virus and a neuraminidase (NA) gene from H1N2 SIV previously isolated in North America. This identification of reassortment events in swine farms raises concern that reassortant strains may continuously circulate within swine populations, calling for the further study and surveillance of pandemic H1N1 among swine.

Temperature sensitivity on growth and/or replication of H1N1, H1N2 and H3N2 influenza A viruses isolated from pigs and birds in mammalian cells.

PubMed

Massin, Pascale; Kuntz-Simon, Gaëlle; Barbezange, Cyril; Deblanc, Céline; Oger, Aurélie; Marquet-Blouin, Estelle; Bougeard, Stéphanie; van der Werf, Sylvie; Jestin, Véronique

2010-05-19

Influenza A viruses have been isolated from a wide range of animal species, aquatic birds being the reservoir for their genetic diversity. Avian influenza viruses can be transmitted to humans, directly or indirectly through an intermediate host like pig. This study aimed to define in vitro conditions that could prove useful to evaluate the potential of influenza viruses to adapt to a different host. Growth of H1N1, H1N2 and H3N2 influenza viruses belonging to different lineages isolated from birds or pigs prior to 2005 was tested on MDCK or NPTr cell lines in the presence or absence of exogenous trypsin. Virus multiplication was compared at 33, 37 and 40 degrees C, the infection site temperatures in human, swine and avian hosts, respectively. Temperature sensitivity of PB2-, NP- and M-RNA replication was also tested by quantitative real-time PCR. Multiplication of avian viruses was cold-sensitive, whatever cell type. By contrast, temperature sensitivity of swine viruses was found to depend on the virus and the host cell: for an H1N1 swine isolate from 1982, multiplication was cold-sensitive on NPTr cells and undetectable at 40 degrees C. From genetic analyses, it appears that temperature sensitivity could involve other residues than PB2 residue 627 and could affect other steps of the replication cycle than replication. Copyright 2009 Elsevier B.V. All rights reserved.

Modified vaccinia virus Ankara expressing the hemagglutinin of pandemic (H1N1) 2009 virus induces cross-protective immunity against Eurasian 'avian-like' H1N1 swine viruses in mice.

PubMed

Castrucci, Maria R; Facchini, Marzia; Di Mario, Giuseppina; Garulli, Bruno; Sciaraffia, Ester; Meola, Monica; Fabiani, Concetta; De Marco, Maria A; Cordioli, Paolo; Siccardi, Antonio; Kawaoka, Yoshihiro; Donatelli, Isabella

2014-05-01

To examine cross-reactivity between hemagglutinin (HA) derived from A/California/7/09 (CA/09) virus and that derived from representative Eurasian "avian-like" (EA) H1N1 swine viruses isolated in Italy between 1999 and 2008 during virological surveillance in pigs. Modified vaccinia virus Ankara (MVA) expressing the HA gene of CA/09 virus (MVA-HA-CA/09) was used as a vaccine to investigate cross-protective immunity against H1N1 swine viruses in mice. Two classical swine H1N1 (CS) viruses and four representative EA-like H1N1 swine viruses previously isolated during outbreaks of respiratory disease in pigs on farms in Northern Italy were used in this study. Female C57BL/6 mice were vaccinated with MVA/HA/CA/09 and then challenged intranasally with H1N1 swine viruses. Cross-reactive antibody responses were determined by hemagglutination- inhibition (HI) and virus microneutralizing (MN) assays of sera from MVA-vaccinated mice. The extent of protective immunity against infection with H1N1 swine viruses was determined by measuring lung viral load on days 2 and 4 post-challenge. Systemic immunization of mice with CA/09-derived HA, vectored by MVA, elicited cross-protective immunity against recent EA-like swine viruses. This immune protection was related to the levels of cross-reactive HI antibodies in the sera of the immunized mice and was dependent on the similarity of the antigenic site Sa of H1 HAs. Our findings suggest that the herd immunity elicited in humans by the pandemic (H1N1) 2009 virus could limit the transmission of recent EA-like swine HA genes into the influenza A virus gene pool in humans. © 2013 The Authors Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.

Novel triple reassortant H1N2 influenza viruses bearing six internal genes of the pandemic 2009/H1N1 influenza virus were detected in pigs in China.

PubMed

Qiao, Chuanling; Liu, Liping; Yang, Huanliang; Chen, Yan; Xu, Huiyang; Chen, Hualan

2014-12-01

The pandemic A/H1N1 influenza viruses emerged in both Mexico and the United States in March 2009, and were transmitted efficiently in the human population. Transmissions of the pandemic 2009/H1N1 virus from humans to poultry and other species of mammals were reported from several continents during the course of the 2009 H1N1 pandemic. Reassortant H1N1, H1N2, and H3N2 viruses containing genes of the pandemic 2009/H1N1 viruses appeared in pigs in some countries. In winter of 2012, a total of 2600 nasal swabs were collected from healthy pigs in slaughterhouses located throughout 10 provinces in China. The isolated viruses were subjected to genetic and antigenic analysis. Two novel triple-reassortant H1N2 influenza viruses were isolated from swine in China in 2012, with the HA gene derived from Eurasian avian-like swine H1N1, the NA gene from North American swine H1N2, and the six internal genes from the pandemic 2009/H1N1 viruses. The two viruses had similar antigenic features and some significant changes in antigenic characteristics emerged when compared to the previously identified isolates. We inferred that the novel reassortant viruses in China may have arisen from the accumulation of the three types of influenza viruses, which further indicates that swine herds serve as "mixing vessels" for influenza viruses. Influenza virus reassortment is an ongoing process, and our findings highlight the urgent need for continued influenza surveillance among swine herds. Copyright © 2014 Elsevier B.V. All rights reserved.

Antigenic variation of H1N1, H1N2 and H3N2 swine influenza viruses in Japan and Vietnam.

PubMed

Takemae, Nobuhiro; Nguyen, Tung; Ngo, Long Thanh; Hiromoto, Yasuaki; Uchida, Yuko; Pham, Vu Phong; Kageyama, Tsutomu; Kasuo, Shizuko; Shimada, Shinichi; Yamashita, Yasutaka; Goto, Kaoru; Kubo, Hideyuki; Le, Vu Tri; Van Vo, Hung; Do, Hoa Thi; Nguyen, Dang Hoang; Hayashi, Tsuyoshi; Matsuu, Aya; Saito, Takehiko

2013-04-01

The antigenicity of the influenza A virus hemagglutinin is responsible for vaccine efficacy in protecting pigs against swine influenza virus (SIV) infection. However, the antigenicity of SIV strains currently circulating in Japan and Vietnam has not been well characterized. We examined the antigenicity of classical H1 SIVs, pandemic A(H1N1)2009 (A(H1N1)pdm09) viruses, and seasonal human-lineage SIVs isolated in Japan and Vietnam. A hemagglutination inhibition (HI) assay was used to determine antigenic differences that differentiate the recent Japanese H1N2 and H3N2 SIVs from the H1N1 and H3N2 domestic vaccine strains. Minor antigenic variation between pig A(H1N1)pdm09 viruses was evident by HI assay using 13 mAbs raised against homologous virus. A Vietnamese H1N2 SIV, whose H1 gene originated from a human strain in the mid-2000s, reacted poorly with post-infection ferret serum against human vaccine strains from 2000-2010. These results provide useful information for selection of optimal strains for SIV vaccine production.

Avian Influenza (H5N1) Viruses Isolated from Humans in Asia in 2004 Exhibit Increased Virulence in Mammals

PubMed Central

Maines, Taronna R.; Lu, Xui Hua; Erb, Steven M.; Edwards, Lindsay; Guarner, Jeannette; Greer, Patricia W.; Nguyen, Doan C.; Szretter, Kristy J.; Chen, Li-Mei; Thawatsupha, Pranee; Chittaganpitch, Malinee; Waicharoen, Sunthareeya; Nguyen, Diep T.; Nguyen, Tung; Nguyen, Hanh H. T.; Kim, Jae-Hong; Hoang, Long T.; Kang, Chun; Phuong, Lien S.; Lim, Wilina; Zaki, Sherif; Donis, Ruben O.; Cox, Nancy J.; Katz, Jacqueline M.; Tumpey, Terrence M.

2005-01-01

The spread of highly pathogenic avian influenza H5N1 viruses across Asia in 2003 and 2004 devastated domestic poultry populations and resulted in the largest and most lethal H5N1 virus outbreak in humans to date. To better understand the potential of H5N1 viruses isolated during this epizootic event to cause disease in mammals, we used the mouse and ferret models to evaluate the relative virulence of selected 2003 and 2004 H5N1 viruses representing multiple genetic and geographical groups and compared them to earlier H5N1 strains isolated from humans. Four of five human isolates tested were highly lethal for both mice and ferrets and exhibited a substantially greater level of virulence in ferrets than other H5N1 viruses isolated from humans since 1997. One human isolate and all four avian isolates tested were found to be of low virulence in either animal. The highly virulent viruses replicated to high titers in the mouse and ferret respiratory tracts and spread to multiple organs, including the brain. Rapid disease progression and high lethality rates in ferrets distinguished the highly virulent 2004 H5N1 viruses from the 1997 H5N1 viruses. A pair of viruses isolated from the same patient differed by eight amino acids, including a Lys/Glu disparity at 627 of PB2, previously identified as an H5N1 virulence factor in mice. The virus possessing Glu at 627 of PB2 exhibited only a modest decrease in virulence in mice and was highly virulent in ferrets, indicating that for this virus pair, the K627E PB2 difference did not have a prevailing effect on virulence in mice or ferrets. Our results demonstrate the general equivalence of mouse and ferret models for assessment of the virulence of 2003 and 2004 H5N1 viruses. However, the apparent enhancement of virulence of these viruses in humans in 2004 was better reflected in the ferret. PMID:16140756

Identification of swine H1N2/pandemic H1N1 reassortant influenza virus in pigs, United States.

PubMed

Ali, Ahmed; Khatri, Mahesh; Wang, Leyi; Saif, Yehia M; Lee, Chang-Won

2012-07-06

In October and November 2010, novel H1N2 reassortant influenza viruses were identified from pigs showing mild respiratory signs that included cough and depression. Sequence and phylogenetic analysis showed that the novel H1N2 reassortants possesses HA and NA genes derived from recent H1N2 swine isolates similar to those isolated from Midwest. Compared to the majority of reported reassortants, both viruses preserved human-like host restrictive and putative antigenic sites in their HA and NA genes. The four internal genes, PB2, PB1, PA, and NS were similar to the contemporary swine triple reassortant viruses' internal genes (TRIG). Interestingly, NP and M genes of the novel reassortants were derived from the 2009 pandemic H1N1. The NP and M proteins of the two isolates demonstrated one (E16G) and four (G34A, D53E, I109T, and V313I) amino acid changes in the M2 and NP proteins, respectively. Similar amino acid changes were also noticed upon incorporation of the 2009 pandemic H1N1 NP in other reassortant viruses reported in the U.S. Thus the role of those amino acids in relation to host adaptation need to be further investigated. The reassortments of pandemic H1N1 with swine influenza viruses and the potential of interspecies transmission of these reassortants from swine to other species including human indicate the importance of systematic surveillance of swine population to determine the origin, the prevalence of similar reassortants in the U.S. and their impact on both swine production and public health. Copyright © 2012 Elsevier B.V. All rights reserved.

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.

Genetic characterization of H1N2 swine influenza virus isolated in China and its pathogenesis and inflammatory responses in mice.

PubMed

Zhang, Yan; Wang, Nan; Cao, Jiyue; Chen, Huanchun; Jin, Meilin; Zhou, Hongbo

2013-09-01

In 2009, two H1N2 influenza viruses were isolated from trachea swabs of pigs in Hubei in China. We compared these sequences with the other 18 complete genome sequences of swine H1N2 isolates from China during 2004 to 2010 and undertook extensive analysis of their evolutionary patterns. Six different genotypes - two reassortants between triple reassortant (TR) H3N2 and classical swine (CS) H1N1 virus, three reassortants between TR H1N2, Eurasian avian-like H1N1 swine virus and H9N2 swine virus, and one reassortant between H1N1, H3N2 human virus and CS H1N1 virus - were observed in these 20 swine H1N2 isolates. The TR H1N2 swine virus is the predominant genotype, and the two Hubei H1N2 isolates were located in this cluster. We also used a mouse model to examine the pathogenesis and inflammatory responses of the two isolates. The isolates replicated efficiently in the lung, and exhibited a strong inflammatory response, serious pathological changes and mortality in infected mice. Given the role that swine can play as putative "genetic mixing vessels" and the observed transmission of TR H1N2 in ferrets, H1N2 influenza surveillance in pigs should be increased to minimize the potential threat to public health.

Characterization of a newly emerged genetic cluster of H1N1 and H1N2 swine influenza virus in the United States.

PubMed

Vincent, Amy L; Ma, Wenjun; Lager, Kelly M; Gramer, Marie R; Richt, Juergen A; Janke, Bruce H

2009-10-01

H1 influenza A viruses that were distinct from the classical swine H1 lineage were identified in pigs in Canada in 2003–2004; antigenic and genetic characterization identified the hemagglutinin (HA) as human H1 lineage. The viruses identified in Canadian pigs were human lineage in entirety or double (human–swine) reassortants. Here, we report the whole genome sequence analysis of four human-like H1 viruses isolated from U.S. swine in 2005 and 2007. All four isolates were characterized as triple reassortants with an internal gene constellation similar to contemporary U.S. swine influenza virus (SIV), with HA and neuraminidase (NA) most similar to human influenza virus lineages. A 2007 human-like H1N1 was evaluated in a pathogenesis and transmission model and compared to a 2004 reassortant H1N1 SIV isolate with swine lineage HA and NA. The 2007 isolate induced disease typical of influenza virus and was transmitted to contact pigs; however, the kinetics and magnitude differed from the 2004 H1N1 SIV. This study indicates that the human-like H1 SIV can efficiently replicate and transmit in the swine host and now co-circulates with contemporary SIVs as a distinct genetic cluster of H1 SIV.

Elicitation of Protective Antibodies against a Broad Panel of H1N1 Viruses in Ferrets Preimmune to Historical H1N1 Influenza Viruses.

PubMed

Carter, Donald M; Darby, Christopher A; Johnson, Scott K; Carlock, Michael A; Kirchenbaum, Greg A; Allen, James D; Vogel, Thorsten U; Delagrave, Simon; DiNapoli, Joshua; Kleanthous, Harold; Ross, Ted M

2017-12-15

Most preclinical animal studies test influenza vaccines in immunologically naive animal models, even though the results of vaccination may not accurately reflect the effectiveness of vaccine candidates in humans that have preexisting immunity to influenza. In this study, novel, broadly reactive influenza vaccine candidates were assessed in preimmune ferrets. These animals were infected with different H1N1 isolates before being vaccinated or infected with another influenza virus. Previously, our group has described the design and characterization of computationally optimized broadly reactive hemagglutinin (HA) antigens (COBRA) for H1N1 isolates. Vaccinating ferrets with virus-like particle (VLP) vaccines expressing COBRA HA proteins elicited antibodies with hemagglutination inhibition (HAI) activity against more H1N1 viruses in the panel than VLP vaccines expressing wild-type HA proteins. Specifically, ferrets infected with the 1986 virus and vaccinated with a single dose of the COBRA HA VLP vaccines elicited antibodies with HAI activity against 11 to 14 of the 15 H1N1 viruses isolated between 1934 and 2013. A subset of ferrets was infected with influenza viruses expressing the COBRA HA antigens. These COBRA preimmune ferrets had superior breadth of HAI activity after vaccination with COBRA HA VLP vaccines than COBRA preimmune ferrets vaccinated with VLP vaccines expressing wild-type HA proteins. Overall, priming naive ferrets with COBRA HA based viruses or using COBRA HA based vaccines to boost preexisting antibodies induced by wild-type H1N1 viruses, COBRA HA antigens elicited sera with the broadest HAI reactivity against multiple antigenic H1N1 viral variants. This is the first report demonstrating the effectiveness of a broadly reactive or universal influenza vaccine in a preimmune ferret model. IMPORTANCE Currently, many groups are testing influenza vaccine candidates to meet the challenge of developing a vaccine that elicits broadly reactive and long

Elicitation of Protective Antibodies against a Broad Panel of H1N1 Viruses in Ferrets Preimmune to Historical H1N1 Influenza Viruses

PubMed Central

Carter, Donald M.; Darby, Christopher A.; Johnson, Scott K.; Carlock, Michael A.; Kirchenbaum, Greg A.; Allen, James D.; Vogel, Thorsten U.; Delagrave, Simon; DiNapoli, Joshua; Kleanthous, Harold

2017-01-01

ABSTRACT Most preclinical animal studies test influenza vaccines in immunologically naive animal models, even though the results of vaccination may not accurately reflect the effectiveness of vaccine candidates in humans that have preexisting immunity to influenza. In this study, novel, broadly reactive influenza vaccine candidates were assessed in preimmune ferrets. These animals were infected with different H1N1 isolates before being vaccinated or infected with another influenza virus. Previously, our group has described the design and characterization of computationally optimized broadly reactive hemagglutinin (HA) antigens (COBRA) for H1N1 isolates. Vaccinating ferrets with virus-like particle (VLP) vaccines expressing COBRA HA proteins elicited antibodies with hemagglutination inhibition (HAI) activity against more H1N1 viruses in the panel than VLP vaccines expressing wild-type HA proteins. Specifically, ferrets infected with the 1986 virus and vaccinated with a single dose of the COBRA HA VLP vaccines elicited antibodies with HAI activity against 11 to 14 of the 15 H1N1 viruses isolated between 1934 and 2013. A subset of ferrets was infected with influenza viruses expressing the COBRA HA antigens. These COBRA preimmune ferrets had superior breadth of HAI activity after vaccination with COBRA HA VLP vaccines than COBRA preimmune ferrets vaccinated with VLP vaccines expressing wild-type HA proteins. Overall, priming naive ferrets with COBRA HA based viruses or using COBRA HA based vaccines to boost preexisting antibodies induced by wild-type H1N1 viruses, COBRA HA antigens elicited sera with the broadest HAI reactivity against multiple antigenic H1N1 viral variants. This is the first report demonstrating the effectiveness of a broadly reactive or universal influenza vaccine in a preimmune ferret model. IMPORTANCE Currently, many groups are testing influenza vaccine candidates to meet the challenge of developing a vaccine that elicits broadly reactive and long

Isolation of avian influenza H5N1 virus from vaccinated commercial layer flock in Egypt

PubMed Central

2012-01-01

Background Uninterrupted transmission of highly pathogenic avian influenza virus (HPAIV) H5N1 of clade 2.2.1 in Egypt since 2006 resulted in establishment of two main genetic clusters. The 2.2.1/C group where all recent human and majority of backyard origin viruses clustered together, meanwhile the majority of viruses derived from vaccinated poultry in commercial farms grouped in 2.2.1.1 clade. Findings In the present investigation, an HPAIV H5N1 was isolated from twenty weeks old layers chickens that were vaccinated with a homologous H5N1 vaccine at 1, 7 and 16 weeks old. At twenty weeks of age, birds showed cyanosis of comb and wattle, decrease in egg production and up to 27% mortality. Examined serum samples showed low antibody titer in HI test (Log2 3.2± 4.2). The hemagglutinin (HA) and neuraminidase (NA) genes of the isolated virus were closely related to viruses in 2.2.1/C group isolated from poultry in live bird market (LBM) and backyards or from infected people. Conspicuous mutations in the HA and NA genes including a deletion within the receptor binding domain in the HA globular head region were observed. Conclusions Despite repeated vaccination of layer chickens using a homologous H5N1 vaccine, infection with HPAIV H5N1 resulted in significant morbidity and mortality. In endemic countries like Egypt, rigorous control measures including enforcement of biosecurity, culling of infected birds and constant update of vaccine virus strains are highly required to prevent circulation of HPAIV H5N1 between backyard birds, commercial poultry, LBM and humans. PMID:23185975

Characterization of H1N1 swine influenza viruses circulating in Canadian pigs in 2009.

PubMed

Nfon, Charles K; Berhane, Yohannes; Hisanaga, Tamiko; Zhang, Shunzhen; Handel, Katherine; Kehler, Helen; Labrecque, Olivia; Lewis, Nicola S; Vincent, Amy L; Copps, John; Alexandersen, Soren; Pasick, John

2011-09-01

The 2009 pandemic H1N1 (pH1N1), of apparent swine origin, may have evolved in pigs unnoticed because of insufficient surveillance. Consequently, the need for surveillance of influenza viruses circulating in pigs has received added attention. In this study we characterized H1N1 viruses isolated from Canadian pigs in 2009. Isolates from May 2009 were comprised of hemagglutinin and neuraminidase (NA) genes of classical SIV origin in combination with the North American triple-reassortant internal gene (TRIG) cassette, here termed contemporary SIV (conSIV) H1N1. These conSIV H1N1 viruses were contiguous with the North American αH1 cluster, which was distinct from the pH1N1 isolates that were antigenically more related to the γH1 cluster. After the initial isolation of pH1N1 from an Alberta pig farm in early May 2009, pH1N1 was found several times in Canadian pigs. These pH1N1 isolates were genetically and antigenically homogeneous. In addition, H1N1 viruses bearing seasonal human H1 and N1 genes together with the TRIG cassette and an NA encoding an oseltamivir-resistance marker were isolated from pigs. The NS gene of one of these seasonal human-like SIV (shSIV) H1N1 isolates was homologous to pH1N1 NS, implicating reassortment between the two strains. Antigenic cross-reactivity was observed between pH1N1 and conSIV but not with shSIV H1N1. In summary, although there was cocirculation of pH1N1 with conSIV and shSIV H1N1 in Canadian pigs after May 2009, there was no evidence supporting the presence of pH1N1 in pigs prior to May 2009. The possibility for further reassortants being generated exists and should be closely monitored.

Multiplex RT-PCR assay for differentiating European swine influenza virus subtypes H1N1, H1N2 and H3N2.

PubMed

Chiapponi, Chiara; Moreno, Ana; Barbieri, Ilaria; Merenda, Marianna; Foni, Emanuela

2012-09-01

In Europe, three major swine influenza viral (SIV) subtypes (H1N1, H1N2 and H3N2) have been isolated in pigs. Developing a test that is able to detect and identify the subtype of the circulating strain rapidly during an outbreak of respiratory disease in the pig population is of essential importance. This study describes two multiplex RT-PCRs which distinguish the haemagglutinin (HA) gene and the neuraminidase (NA) gene of the three major subtypes of SIV circulating in Europe. The HA PCR was able to identify the lineage (avian or human) of the HA of H1 subtypes. The analytical sensitivity of the test, considered to be unique, was assessed using three reference viruses. The detection limit corresponded to 1×10(-1) TCID(50)/200μl for avian-like H1N1, 1×10(0) TCID(50)/200μl for human-like H1N2 and 1×10(1) TCID(50)/200μl for H3N2 SIV. The multiplex RT-PCR was first carried out on a collection of 70 isolated viruses showing 100% specificity and then on clinical samples, from which viruses had previously been isolated, resulting in an 89% positive specificity of the viral subtype. Finally, the test was able to identify the viral subtype correctly in 56% of influenza A positive samples, from which SIV had not been isolated previously. It was also possible to identify mixed viral infections and the circulation of a reassortant strain before performing genomic studies. Copyright © 2012 Elsevier B.V. All rights reserved.

A Historical Perspective of Influenza A(H1N2) Virus

PubMed Central

McVernon, Jodie; Hall, Robert; Leder, Karin

2014-01-01

The emergence and transition to pandemic status of the influenza A(H1N1)A(H1N1)pdm09) virus in 2009 illustrated the potential for previously circulating human viruses to re-emerge in humans and cause a pandemic after decades of circulating among animals. Within a short time of the initial emergence of A(H1N1)pdm09 virus, novel reassortants were isolated from swine. In late 2011, a variant (v) H3N2 subtype was isolated from humans, and by 2012, the number of persons infected began to increase with limited person-to-person transmission. During 2012 in the United States, an A(H1N2)v virus was transmitted to humans from swine. During the same year, Australia recorded its first H1N2 subtype infection among swine. The A(H3N2)v and A(H1N2)v viruses contained the matrix protein from the A(H1N1)pdm09 virus, raising the possibility of increased transmissibility among humans and underscoring the potential for influenza pandemics of novel swine-origin viruses. We report on the differing histories of A(H1N2) viruses among humans and animals. PMID:24377419

A historical perspective of influenza A(H1N2) virus.

PubMed

Komadina, Naomi; McVernon, Jodie; Hall, Robert; Leder, Karin

2014-01-01

The emergence and transition to pandemic status of the influenza A(H1N1)A(H1N1)pdm09) virus in 2009 illustrated the potential for previously circulating human viruses to re-emerge in humans and cause a pandemic after decades of circulating among animals. Within a short time of the initial emergence of A(H1N1)pdm09 virus, novel reassortants were isolated from swine. In late 2011, a variant (v) H3N2 subtype was isolated from humans, and by 2012, the number of persons infected began to increase with limited person-to-person transmission. During 2012 in the United States, an A(H1N2)v virus was transmitted to humans from swine. During the same year, Australia recorded its first H1N2 subtype infection among swine. The A(H3N2)v and A(H1N2)v viruses contained the matrix protein from the A(H1N1)pdm09 virus, raising the possibility of increased transmissibility among humans and underscoring the potential for influenza pandemics of novel swine-origin viruses. We report on the differing histories of A(H1N2) viruses among humans and animals.

Natural co-infection of influenza A/H3N2 and A/H1N1pdm09 viruses resulting in a reassortant A/H3N2 virus.

PubMed

Rith, Sareth; Chin, Savuth; Sar, Borann; Y, Phalla; Horm, Srey Viseth; Ly, Sovann; Buchy, Philippe; Dussart, Philippe; Horwood, Paul F

2015-12-01

Despite annual co-circulation of different subtypes of seasonal influenza, co-infections between different viruses are rarely detected. These co-infections can result in the emergence of reassortant progeny. We document the detection of an influenza co-infection, between influenza A/H3N2 with A/H1N1pdm09 viruses, which occurred in a 3 year old male in Cambodia during April 2014. Both viruses were detected in the patient at relatively high viral loads (as determined by real-time RT-PCR CT values), which is unusual for influenza co-infections. As reassortment can occur between co-infected influenza A strains we isolated plaque purified clonal viral populations from the clinical material of the patient infected with A/H3N2 and A/H1N1pdm09. Complete genome sequences were completed for 7 clonal viruses to determine if any reassorted viruses were generated during the influenza virus co-infection. Although most of the viral sequences were consistent with wild-type A/H3N2 or A/H1N1pdm09, one reassortant A/H3N2 virus was isolated which contained an A/H1N1pdm09 NS1 gene fragment. The reassortant virus was viable and able to infect cells, as judged by successful passage in MDCK cells, achieving a TCID50 of 10(4)/ml at passage number two. There is no evidence that the reassortant virus was transmitted further. The co-infection occurred during a period when co-circulation of A/H3N2 and A/H1N1pdm09 was detected in Cambodia. It is unclear how often influenza co-infections occur, but laboratories should consider influenza co-infections during routine surveillance activities. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Phylogenetic characterization of H5N1 avian influenza viruses isolated in Indonesia from 2003-2007

PubMed Central

Takano, Ryo; Nidom, Chairul A.; Kiso, Maki; Muramoto, Yukiko; Yamada, Shinya; Sakai-Tagawa, Yuko; Macken, Catherine; Kawaoka, Yoshihiro

2010-01-01

The wide distribution of H5N1 highly pathogenic avian influenza viruses is a global threat to human health. Indonesia has had the largest number of human infections and fatalities caused by these viruses. To understand the enzootic conditions of the viruses in Indonesia, twenty-four H5N1 viruses isolated from poultry from 2003 to 2007 were phylogenetically characterized. Although previous studies exclusively classified the Indonesian viruses into clades 2.1.1-2.1.3, our phylogenetic analyses showed a new sub-lineage that did not belong to any of the present clades. In addition, novel reassortant viruses were identified that emerged between this new sub-lineage and other clades in 2005-2006 on Java Island. H5N1 viruses were introduced from Java Island to Sulawesi, Kalimantan, and Sumatra Island on multiple occasions from 2003-2007, causing the geographical expansion of these viruses in Indonesia. These findings identify Java Island as the epicenter of the Indonesian H5N1 virus expansion. PMID:19464724

Isolation and characterization of highly pathogenic avian influenza virus subtype H5N1 from donkeys

PubMed Central

2010-01-01

Background The highly pathogenic H5N1 is a major avian pathogen that crosses species barriers and seriously affects humans as well as some mammals. It mutates in an intensified manner and is considered a potential candidate for the possible next pandemic with all the catastrophic consequences. Methods Nasal swabs were collected from donkeys suffered from respiratory distress. The virus was isolated from the pooled nasal swabs in specific pathogen free embryonated chicken eggs (SPF-ECE). Reverse transcriptase polymerase chain reaction (RT-PCR) and sequencing of both haemagglutingin and neuraminidase were performed. H5 seroconversion was screened using haemagglutination inhibition (HI) assay on 105 donkey serum samples. Results We demonstrated that H5N1 jumped from poultry to another mammalian host; donkeys. Phylogenetic analysis showed that the virus clustered within the lineage of H5N1 from Egypt, closely related to 2009 isolates. It harboured few genetic changes compared to the closely related viruses from avian and humans. The neuraminidase lacks oseltamivir resistant mutations. Interestingly, HI screening for antibodies to H5 haemagglutinins in donkeys revealed high exposure rate. Conclusions These findings extend the host range of the H5N1 influenza virus, possess implications for influenza virus epidemiology and highlight the need for the systematic surveillance of H5N1 in animals in the vicinity of backyard poultry units especially in endemic areas. PMID:20398268

Novel triple-reassortant H1N1 swine influenza viruses in pigs in Tianjin, Northern China.

PubMed

Sun, Ying-Feng; Wang, Xiu-Hui; Li, Xiu-Li; Zhang, Li; Li, Hai-Hua; Lu, Chao; Yang, Chun-Lei; Feng, Jing; Han, Wei; Ren, Wei-Ke; Tian, Xiang-Xue; Tong, Guang-Zhi; Wen, Feng; Li, Ze-Jun; Gong, Xiao-Qian; Liu, Xiao-Min; Ruan, Bao-Yang; Yan, Ming-Hua; Yu, Hai

2016-02-01

Pigs are susceptible to both human and avian influenza viruses and therefore have been proposed to be mixing vessels for the generation of pandemic influenza viruses through reassortment. In this study, for the first time, we report the isolation and genetic analyses of three novel triple-reassortant H1N1 swine influenza viruses from pigs in Tianjin, Northern China. Phylogenetic analysis showed that these novel viruses contained genes from the 2009 pandemic H1N1 (PB2, PB1, PA and NP), Eurasian swine (HA, NA and M) and triple-reassortant swine (NS) lineages. This indicated that the reassortment among the 2009 pandemic H1N1, Eurasian swine and triple-reassortant swine influenza viruses had taken place in pigs in Tianjin and resulted in the generation of new viruses. Furthermore, three human-like H1N1, two classical swine H1N1 and two Eurasian swine H1N1 viruses were also isolated during the swine influenza virus surveillance from 2009 to 2013, which indicated that multiple genetic lineages of swine H1N1 viruses were co-circulating in the swine population in Tianjin, China. The emergence of novel triple-reassortant H1N1 swine influenza viruses may be a potential threat to human health and emphasizes the importance of further continuous surveillance. Copyright © 2015 Elsevier B.V. All rights reserved.

A Simple Restriction Fragment Length Polymorphism-Based Strategy That Can Distinguish the Internal Genes of Human H1N1, H3N2, and H5N1 Influenza A Viruses

PubMed Central

Cooper, Lynn A.; Subbarao, Kanta

2000-01-01

A simple molecular technique for rapid genotyping was developed to monitor the internal gene composition of currently circulating influenza A viruses. Sequence information from recent H1N1, H3N2, and H5N1 human virus isolates was used to identify conserved regions within each internal gene, and gene-specific PCR primers capable of amplifying all three virus subtypes were designed. Subtyping was based on subtype-specific restriction fragment length polymorphism (RFLP) patterns within the amplified regions. The strategy was tested in a blinded fashion using 10 control viruses of each subtype (total, 30) and was found to be very effective. Once standardized, the genotyping method was used to identify the origin of the internal genes of 51 influenza A viruses isolated from humans in Hong Kong during and immediately following the 1997–1998 H5N1 outbreak. No avian-human or H1-H3 reassortants were detected. Less than 2% (6 of 486) of the RFLP analyses were inconclusive; all were due to point mutations within a restriction site. The technique was also used to characterize the internal genes of two avian H9N2 viruses isolated from children in Hong Kong during 1999. PMID:10878047

Full-Genome Sequence of a Reassortant H1N2 Influenza A Virus Isolated from Pigs in Brazil.

PubMed

Schmidt, Candice; Cibulski, Samuel Paulo; Muterle Varela, Ana Paula; Mengue Scheffer, Camila; Wendlant, Adrieli; Quoos Mayer, Fabiana; Lopes de Almeida, Laura; Franco, Ana Cláudia; Roehe, Paulo Michel

2014-12-18

In this study, the full-genome sequence of a reassortant H1N2 swine influenza virus is reported. The isolate has the hemagglutinin (HA) and neuraminidase (NA) genes from human lineage (H1-δ cluster and N2), and the internal genes (polymerase basic 1 [PB1], polymerase basic 2 [PB2], polymerase acidic [PA], nucleoprotein [NP], matrix [M], and nonstructural [NS]) are derived from human 2009 pandemic H1N1 (H1N1pdm09) virus. Copyright © 2014 Schmidt et al.

Protective efficacy of an inactivated Eurasian avian-like H1N1 swine influenza vaccine against homologous H1N1 and heterologous H1N1 and H1N2 viruses in mice.

PubMed

Sui, Jinyu; Yang, Dawei; Qiao, Chuanling; Xu, Huiyang; Xu, Bangfeng; Wu, Yunpu; Yang, Huanliang; Chen, Yan; Chen, Hualan

2016-07-19

Eurasian avian-like H1N1 (EA H1N1) swine influenza viruses are prevalent in pigs in Europe and Asia, but occasionally cause human infection, which raises concern about their pandemic potential. Here, we produced a whole-virus inactivated vaccine with an EA H1N1 strain (A/swine/Guangxi/18/2011, SW/GX/18/11) and evaluated its efficacy against homologous H1N1 and heterologous H1N1 and H1N2 influenza viruses in mice. A strong humoral immune response, which we measured by hemagglutination inhibition (HI) and virus neutralization (VN), was induced in the vaccine-inoculated mice upon challenge. The inactivated SW/GX/18/11 vaccine provided complete protection against challenge with homologous SW/GX/18/11 virus in mice and provided effective protection against challenge with heterologous H1N1 and H1N2 viruses with distinctive genomic combinations. Our findings suggest that this EA H1N1 vaccine can provide protection against both homologous H1N1 and heterologous H1N1 or H1N2 virus infection. As such, it is an excellent vaccine candidate to prevent H1N1 swine influenza. Copyright © 2016 Elsevier Ltd. All rights reserved.

Adaptation of Pandemic H1N1 Influenza Viruses in Mice▿

PubMed Central

Ilyushina, Natalia A.; Khalenkov, Alexey M.; Seiler, Jon P.; Forrest, Heather L.; Bovin, Nicolai V.; Marjuki, Henju; Barman, Subrata; Webster, Robert G.; Webby, Richard J.

2010-01-01

The molecular mechanism by which pandemic 2009 influenza A viruses were able to sufficiently adapt to humans is largely unknown. Subsequent human infections with novel H1N1 influenza viruses prompted an investigation of the molecular determinants of the host range and pathogenicity of pandemic influenza viruses in mammals. To address this problem, we assessed the genetic basis for increased virulence of A/CA/04/09 (H1N1) and A/TN/1-560/09 (H1N1) isolates, which are not lethal for mice, in a new mammalian host by promoting their mouse adaptation. The resulting mouse lung-adapted variants showed significantly enhanced growth characteristics in eggs, extended extrapulmonary tissue tropism, and pathogenicity in mice. All mouse-adapted viruses except A/TN/1-560/09-MA2 grew faster and to higher titers in cells than the original strains. We found that 10 amino acid changes in the ribonucleoprotein (RNP) complex (PB2 E158G/A, PA L295P, NP D101G, and NP H289Y) and hemagglutinin (HA) glycoprotein (K119N, G155E, S183P, R221K, and D222G) controlled enhanced mouse virulence of pandemic isolates. HA mutations acquired during adaptation affected viral receptor specificity by enhancing binding to α2,3 together with decreasing binding to α2,6 sialyl receptors. PB2 E158G/A and PA L295P amino acid substitutions were responsible for the significant enhancement of transcription and replication activity of the mouse-adapted H1N1 variants. Taken together, our findings suggest that changes optimizing receptor specificity and interaction of viral polymerase components with host cellular factors are the major mechanisms that contribute to the optimal competitive advantage of pandemic influenza viruses in mice. These modulators of virulence, therefore, may have been the driving components of early evolution, which paved the way for novel 2009 viruses in mammals. PMID:20592084

Genetic characterization of highly pathogenic avian influenza H5N1 viruses isolated from naturally infected pigeons in Egypt.

PubMed

Elgendy, Emad Mohamed; Watanabe, Yohei; Daidoji, Tomo; Arai, Yasuha; Ikuta, Kazuyoshi; Ibrahim, Madiha Salah; Nakaya, Takaaki

2016-12-01

Avian influenza viruses impose serious public health burdens with significant mortality and morbidity not only in poultry but also in humans. While poultry susceptibility to avian influenza virus infection is well characterized, pigeons have been thought to have low susceptibility to these viruses. However, recent studies reported natural pigeon infections with highly pathogenic avian influenza H5N1 viruses. In Egypt, which is one of the H5N1 endemic areas for birds, pigeons are raised in towers built on farms in backyards and on house roofs, providing a potential risk for virus transmission from pigeons to humans. In this study, we performed genetic analysis of two H5N1 virus strains that were isolated from naturally infected pigeons in Egypt. Genetic and phylogenetic analyses showed that these viruses originated from Egyptian H5N1 viruses that were circulating in chickens or ducks. Several unique mutations, not reported before in any Egyptian isolates, were detected in the internal genes (i.e., polymerase residues PB1-V3D, PB1-K363R, PA-A369V, and PA-V602I; nucleoprotein residue NP-R38K; and nonstructural protein residues NS1-D120N and NS2-F55C). Our findings suggested that pigeons are naturally infected with H5N1 virus and can be a potential reservoir for transmission to humans, and showed the importance of genetic analysis of H5N1 internal genes.

Genomic characterization of H1N2 swine influenza viruses in Italy.

PubMed

Moreno, Ana; Chiapponi, Chiara; Boniotti, Maria Beatrice; Sozzi, Enrica; Foni, Emanuela; Barbieri, Ilaria; Zanoni, Maria Grazia; Faccini, Silvia; Lelli, Davide; Cordioli, Paolo

2012-05-04

Three subtypes (H1N1, H1N2, and H3N2) are currently diffused worldwide in pigs. The H1N2 subtype was detected for the first time in Italian pigs in 1998. To investigate the genetic characteristics and the molecular evolution of this subtype in Italy, we conducted a phylogenetic analysis of whole genome sequences of 26 strains isolated from 1998 to 2010. Phylogenetic analysis of HA and NA genes showed differences between the older (1998-2003) and the more recent strains (2003-2010). The older isolates were closely related to the established European H1N2 lineage, whereas the more recent isolates possessed a different NA deriving from recent human H3N2 viruses. Two other reassortant H1N2 strains have been detected: A/sw/It/22530/02 has the HA gene that is closely related to H1N1 viruses; A/sw/It/58769/10 is an uncommon strain with an HA that is closely related to H1N1 and an NA similar to H3N2 SIVs. Amino acid analysis revealed interesting features: a deletion of two amino acids (146-147) in the HA gene of the recent isolates and two strains isolated in 1998; the presence of the uncommon aa change (N66S), in the PB1-F2 protein in strains isolated from 2009 to 2010, which is said to have contributed to the increased virulence. These results demonstrate the importance of pigs as mixing vessels for animal and human influenza and show the presence and establishment of reassortant strains involving human viruses in pigs in Italy. These findings also highlighted different genomic characteristics of the NA gene the recent Italian strains compared to circulating European viruses. Published by Elsevier B.V.

H7N9 Influenza Virus Is More Virulent in Ferrets than 2009 Pandemic H1N1 Influenza Virus.

PubMed

Yum, Jung; Ku, Keun Bon; Kim, Hyun Soo; Seo, Sang Heui

2015-12-01

The novel H7N9 influenza virus has been infecting humans in China since February 2013 and with a mortality rate of about 40%. This study compared the pathogenicity of the H7N9 and 2009 pandemic H1N1 influenza viruses in a ferret model, which shows similar symptoms to those of humans infected with influenza viruses. The H7N9 influenza virus caused a more severe disease than did the 2009 pandemic H1N1 influenza virus. All of the ferrets infected with the H7N9 influenza virus had died by 6 days after infection, while none of those infected with the 2009 pandemic H1N1 influenza virus died. Ferrets infected with the H7N9 influenza virus had higher viral titers in their lungs than did those infected with the 2009 pandemic H1N1 influenza virus. Histological findings indicated that hemorrhagic pneumonia was caused by infection with the H7N9 influenza virus, but not with the 2009 pandemic H1N1 influenza virus. In addition, the lung tissues of ferrets infected with the H7N9 influenza virus contained higher levels of chemokines than did those of ferrets infected with the 2009 pandemic H1N1 influenza virus. This study suggests that close monitoring is needed to prevent human infection by the lethal H7N9 influenza virus.

Novel reassortant of swine influenza H1N2 virus in Germany.

PubMed

Zell, Roland; Motzke, Susann; Krumbholz, Andi; Wutzler, Peter; Herwig, Volker; Dürrwald, Ralf

2008-01-01

European porcine H1N2 influenza viruses arose after multiple reassortment steps involving a porcine influenza virus with avian-influenza-like internal segments and human H1N1 and H3N2 viruses in 1994. In Germany, H1N2 swine influenza viruses first appeared in 2000. Two German H1N2 swine influenza virus strains isolated from pigs with clinical symptoms of influenza are described. They were characterized by the neutralization test, haemagglutination inhibition (HI) test and complete sequencing of the viral genomes. The data demonstrate that these viruses represent a novel H1N2 reassortant. The viruses showed limited neutralization by sera raised against heterologous A/sw/Bakum/1,832/00-like H1N2 viruses. Sera pools from recovered pigs showed a considerably lower HI reaction, indicative of diagnostic difficulties in using the HI test to detect these viruses with A/sw/Bakum/1,832/00-like H1N2 antigens. Genome sequencing revealed the novel combination of the human-like HAH1 gene of European porcine H1N2 influenza viruses and the NAN2 gene of European porcine H3N2 viruses.

Multiple introductions of highly pathogenic avian influenza H5N1 viruses into Bangladesh

PubMed Central

Marinova-Petkova, Atanaska; Feeroz, Mohammed M; Rabiul Alam, SM; Kamrul Hasan, M; Akhtar, Sharmin; Jones-Engel, Lisa; Walker, David; McClenaghan, Laura; Rubrum, Adam; Franks, John; Seiler, Patrick; Jeevan, Trushar; McKenzie, Pamela; Krauss, Scott; Webby, Richard J; Webster, Robert G

2014-01-01

Highly pathogenic H5N1 and low pathogenic H9N2 influenza viruses are endemic to poultry markets in Bangladesh and have cocirculated since 2008. H9N2 influenza viruses circulated constantly in the poultry markets, whereas highly pathogenic H5N1 viruses occurred sporadically, with peaks of activity in cooler months. Thirty highly pathogenic H5N1 influenza viruses isolated from poultry were characterized by antigenic, molecular, and phylogenetic analyses. Highly pathogenic H5N1 influenza viruses from clades 2.2.2 and 2.3.2.1 were isolated from live bird markets only. Phylogenetic analysis of the 30 H5N1 isolates revealed multiple introductions of H5N1 influenza viruses in Bangladesh. There was no reassortment between the local H9N2 influenza viruses and H5N1 genotype, despite their prolonged cocirculation. However, we detected two reassortant H5N1 viruses, carrying the M gene from the Chinese H9N2 lineage, which briefly circulated in the Bangladesh poultry markets and then disappeared. On the other hand, interclade reassortment occurred within H5N1 lineages and played a role in the genesis of the currently dominant H5N1 viruses in Bangladesh. Few ‘human-like' mutations in H5N1 may account for the limited number of human cases. Antigenically, clade 2.3.2.1 H5N1 viruses in Bangladesh have evolved since their introduction and are currently mainly homogenous, and show evidence of recent antigenic drift. Although reassortants containing H9N2 genes were detected in live poultry markets in Bangladesh, these reassortants failed to supplant the dominant H5N1 lineage. PMID:26038508

Reassortant Eurasian Avian-Like Influenza A(H1N1) Virus from a Severely Ill Child, Hunan Province, China, 2015.

PubMed

Zhu, Wenfei; Zhang, Hong; Xiang, Xingyu; Zhong, Lili; Yang, Lei; Guo, Junfeng; Xie, Yiran; Li, Fangcai; Deng, Zhihong; Feng, Hong; Huang, Yiwei; Hu, Shixiong; Xu, Xin; Zou, Xiaohui; Li, Xiaodan; Bai, Tian; Chen, Yongkun; Li, Zi; Li, Junhua; Shu, Yuelong

2016-11-01

In 2015, a novel influenza A(H1N1) virus was isolated from a boy in China who had severe pneumonia. The virus was a genetic reassortant of Eurasian avian-like influenza A(H1N1) (EA-H1N1) virus. The hemagglutinin, neuraminidase, and matrix genes of the reassortant virus were highly similar to genes in EA-H1N1 swine influenza viruses, the polybasic 1 and 2, polymerase acidic, and nucleoprotein genes originated from influenza A(H1N1)pdm09 virus, and the nonstructural protein gene derived from classical swine influenza A(H1N1) (CS H1N1) virus. In a mouse model, the reassortant virus, termed influenza A/Hunan/42443/2015(H1N1) virus, showed higher infectivity and virulence than another human EA-H1N1 isolate, influenza A/Jiangsu/1/2011(H1N1) virus. In the respiratory tract of mice, virus replication by influenza A/Hunan/42443/2015(H1N1) virus was substantially higher than that by influenza A/Jiangsu/1/2011(H1N1) virus. Human-to-human transmission of influenza A/Hunan/42443/2015(H1N1) virus has not been detected; however, given the circulation of novel EA-H1N1 viruses in pigs, enhanced surveillance should be instituted among swine and humans.

Reassortant Eurasian Avian-Like Influenza A(H1N1) Virus from a Severely Ill Child, Hunan Province, China, 2015

PubMed Central

Zhu, Wenfei; Zhang, Hong; Xiang, Xingyu; Zhong, Lili; Yang, Lei; Guo, Junfeng; Xie, Yiran; Li, Fangcai; Deng, Zhihong; Feng, Hong; Huang, Yiwei; Hu, Shixiong; Xu, Xin; Zou, Xiaohui; Li, Xiaodan; Bai, Tian; Chen, Yongkun; Li, Zi

2016-01-01

In 2015, a novel influenza A(H1N1) virus was isolated from a boy in China who had severe pneumonia. The virus was a genetic reassortant of Eurasian avian-like influenza A(H1N1) (EA-H1N1) virus. The hemagglutinin, neuraminidase, and matrix genes of the reassortant virus were highly similar to genes in EA-H1N1 swine influenza viruses, the polybasic 1 and 2, polymerase acidic, and nucleoprotein genes originated from influenza A(H1N1)pdm09 virus, and the nonstructural protein gene derived from classical swine influenza A(H1N1) (CS H1N1) virus. In a mouse model, the reassortant virus, termed influenza A/Hunan/42443/2015(H1N1) virus, showed higher infectivity and virulence than another human EA-H1N1 isolate, influenza A/Jiangsu/1/2011(H1N1) virus. In the respiratory tract of mice, virus replication by influenza A/Hunan/42443/2015(H1N1) virus was substantially higher than that by influenza A/Jiangsu/1/2011(H1N1) virus. Human-to-human transmission of influenza A/Hunan/42443/2015(H1N1) virus has not been detected; however, given the circulation of novel EA-H1N1 viruses in pigs, enhanced surveillance should be instituted among swine and humans. PMID:27767007

A novel H6N1 virus-like particle vaccine induces long-lasting cross-clade antibody immunity against human and avian H6N1 viruses.

PubMed

Yang, Ji-Rong; Chen, Chih-Yuan; Kuo, Chuan-Yi; Cheng, Chieh-Yu; Lee, Min-Shiuh; Cheng, Ming-Chu; Yang, Yu-Chih; Wu, Chia-Ying; Wu, Ho-Sheng; Liu, Ming-Tsan; Hsiao, Pei-Wen

2016-02-01

Avian influenza A(H6N1) virus is one of the most common viruses isolated from migrating birds and domestic poultry in many countries. The first and only known case of human infection by H6N1 virus in the world was reported in Taiwan in 2013. This led to concern that H6N1 virus may cause a threat to public health. In this study, we engineered a recombinant H6N1 virus-like particle (VLP) and investigated its vaccine effectiveness compared to the traditional egg-based whole inactivated virus (WIV) vaccine. The H6N1-VLPs exhibited similar morphology and functional characteristics to influenza viruses. Prime-boost intramuscular immunization in mice with unadjuvanted H6N1-VLPs were highly immunogenic and induced long-lasting antibody immunity. The functional activity of the VLP-elicited IgG antibodies was proved by in vitro seroprotective hemagglutination inhibition and microneutralization titers against the homologous human H6N1 virus, as well as in vivo viral challenge analyses which showed H6N1-VLP immunization significantly reduced viral load in the lung, and protected against human H6N1 virus infection. Of particular note, the H6N1-VLPs but not the H6N1-WIVs were able to confer cross-reactive humoral immunity; antibodies induced by H6N1-VLP vaccine robustly inhibited the hemagglutination activities and in vitro replication of distantly-related heterologous avian H6N1 viruses. Furthermore, the H6N1-VLPs were found to elicit significantly greater anti-HA2 antibody responses in immunized mice than H6N1-WIVs. Collectively, we demonstrated for the first time a novel H6N1-VLP vaccine that effectively provides broadly protective immunity against both human and avian H6N1 viruses. These results, which uncover the underlying mechanisms for induction of wide-range immunity against influenza viruses, may be useful for future influenza vaccine development. Copyright © 2015 Elsevier B.V. All rights reserved.

Whole genome characterization of human influenza A(H1N1)pdm09 viruses isolated from Kenya during the 2009 pandemic.

PubMed

Gachara, George; Symekher, Samuel; Otieno, Michael; Magana, Japheth; Opot, Benjamin; Bulimo, Wallace

2016-06-01

An influenza pandemic caused by a novel influenza virus A(H1N1)pdm09 spread worldwide in 2009 and is estimated to have caused between 151,700 and 575,400 deaths globally. While whole genome data on new virus enables a deeper insight in the pathogenesis, epidemiology, and drug sensitivities of the circulating viruses, there are relatively limited complete genetic sequences available for this virus from African countries. We describe herein the full genome analysis of influenza A(H1N1)pdm09 viruses isolated in Kenya between June 2009 and August 2010. A total of 40 influenza A(H1N1)pdm09 viruses isolated during the pandemic were selected. The segments from each isolate were amplified and directly sequenced. The resulting sequences of individual gene segments were concatenated and used for subsequent analysis. These were used to infer phylogenetic relationships and also to reconstruct the time of most recent ancestor, time of introduction into the country, rates of substitution and to estimate a time-resolved phylogeny. The Kenyan complete genome sequences clustered with globally distributed clade 2 and clade 7 sequences but local clade 2 viruses did not circulate beyond the introductory foci while clade 7 viruses disseminated country wide. The time of the most recent common ancestor was estimated between April and June 2009, and distinct clusters circulated during the pandemic. The complete genome had an estimated rate of nucleotide substitution of 4.9×10(-3) substitutions/site/year and greater diversity in surface expressed proteins was observed. We show that two clades of influenza A(H1N1)pdm09 virus were introduced into Kenya from the UK and the pandemic was sustained as a result of importations. Several closely related but distinct clusters co-circulated locally during the peak pandemic phase but only one cluster dominated in the late phase of the pandemic suggesting that it possessed greater adaptability. Copyright © 2016 Elsevier B.V. All rights reserved.

The Potential of Avian H1N1 Influenza A Viruses to Replicate and Cause Disease in Mammalian Models

PubMed Central

Koçer, Zeynep A.; Krauss, Scott; Stallknecht, David E.; Rehg, Jerold E.; Webster, Robert G.

2012-01-01

H1N1 viruses in which all gene segments are of avian origin are the most frequent cause of influenza pandemics in humans; therefore, we examined the disease-causing potential of 31 avian H1N1 isolates of American lineage in DBA/2J mice. Thirty of 31 isolates were very virulent, causing respiratory tract infection; 22 of 31 resulted in fecal shedding; and 10 of 31 were as pathogenic as the pandemic 2009 H1N1 viruses. Preliminary studies in BALB/cJ mice and ferrets showed that 1 of 4 isolates tested was more pathogenic than the pandemic 2009 H1N1 viruses in BALB/cJ mice, and 1 of 2 strains transmitted both by direct and respiratory-droplet contact in ferrets. Preliminary studies of other avian subtypes (H2, H3, H4, H6, H10, H12) in DBA/2J mice showed lower pathogenicity than the avian H1N1 viruses. These findings suggest that avian H1N1 influenza viruses are unique among influenza A viruses in their potential to infect mammals. PMID:22848544

Vaccination with virus-like particles containing H5 antigens from three H5N1 clades protects chickens from H5N1 and H5N8 influenza viruses

PubMed Central

Kapczynski, Darrell R.; Tumpey, Terrence M.; Hidajat, Rachmat; Zsak, Aniko; Chrzastek, Klaudia; Tretyakova, Irina; Pushko, Peter

2016-01-01

Highly pathogenic avian influenza (HPAI) viruses, especially H5N1 strains, represent a public health threat and cause widespread morbidity and mortality in domestic poultry. Recombinant virus-like particles (VLPs) represent a promising novel vaccine approach to control avian influenza including HPAI strains. Influenza VLPs contain viral hemagglutinin (HA), which can be expressed in cell culture within highly immunogenic VLPs that morphologically and antigenically resemble influenza virions, except VLPs are non-infectious. Here we describe a recombinant VLP containing HA proteins derived from three distinct clades of H5N1 viruses as an experimental, broadly protective H5 avian influenza vaccine. A baculovirus vector was configured to co-express the H5 genes from recent H5N1 HPAI isolates A/chicken/Germany/2014 (clade 2.3.4.4), A/chicken/West Java/Subang/29/2007 (clade 2.1.3) and A/chicken/Egypt/121/2012 (clade 2.2.1). Co-expression of these genes in Sf9 cells along with influenza neuraminidase (NA) and retrovirus gag genes resulted in production of triple-clade H555 VLPs that exhibited hemagglutination activity and morphologically resembled influenza virions. Vaccination of chickens with these VLPs resulted in induction of serum antibody responses and efficient protection against experimental challenges with three different viruses including the recent U.S. H5N8 HPAI isolate. We conclude that these novel triple-clade VLPs represent a feasible strategy for simultaneously evoking protective antibodies against multiple variants of H5 influenza virus. PMID:26868083

Genetic characterization and diversity of circulating influenza A/H1N1pdm09 viruses isolated in Jeddah, Saudi Arabia between 2014 and 2015.

PubMed

Hashem, Anwar M; Azhar, Esam I; Shalhoub, Sarah; Abujamel, Turki S; Othman, Norah A; Al Zahrani, Abdulwahab B; Abdullah, Hanan M; Al-Alawi, Maha M; Sindi, Anees A

2018-05-01

The emerged influenza A/H1N1pdm09 viruses have replaced the previously circulating seasonal H1N1 viruses. The close antigenic properties of these viruses to the 1918 H1N1 pandemic viruses and their post-pandemic evolution pattern could further enhance their adaptation and pathogenicity in humans representing a major public health threat. Given that data on the dynamics and evolution of these viruses in Saudi Arabia is sparse we investigated the genetic diversity of circulating influenza A/H1N1pdm09 viruses from Jeddah, Saudi Arabia, by analyzing 39 full genomes from isolates obtained between 2014-2015, from patients with varying symptoms. Phylogenetic analysis of all gene segments and concatenated genomes showed similar topologies and co-circulation of clades 6b, 6b.1 and 6b.2, with clade 6b.1 being the most predominate since 2015. Most viruses were more closely related to the vaccine strain (Michigan/45/2015) recommended for the 2017/2018 season, than to the California/07/2009 strain. Low sequence variability was observed in the haemagglutinin protein compared to the neuraminidase protein. Resistance to neuraminidase inhibitors was limited as only one isolate had the H275Y substitution. Interestingly, two isolates had short PA-X proteins of 206 amino acids compared to the 232 amino acid protein found in most influenza A/H1N1pdm09 viruses. Together, the co-circulation of several clades and the predominance of clade 6b.1, despite its low circulation in Asia in 2015, suggests multiple introductions most probably during the mass gathering events of Hajj and Umrah. Jeddah represents the main port of entry to the holy cities of Makkah and Al-Madinah, emphasizing the need for vigilant surveillance in the kingdom.

Divergent genetic evolution of hemagglutinin in influenza A H1N1 and A H1N2 subtypes isolated in the south-France since the winter of 2001-2002.

PubMed

Al Faress, Shaker; Cartet, Gaëlle; Ferraris, Olivier; Norder, Helene; Valette, Martine; Lina, Bruno

2005-07-01

Influenza A viruses are divided into subtypes based on their hemagglutinin (H1 to H15) and neuraminidase (N1 to N9) glycoproteins. Of these, three A subtypes H1N1, H3N2 and H1N2 circulate in the human population. Influenza A viruses display a high antigenic variability called "antigenic drift" which allows the virus to escape antibody neutralization. Evaluate the mutations apparition that might predict a divergent antigenic evolution of hemagglutinin in influenza A H1N1 and A H1N2 viruses. During the three winters of 2001-2002 to 2003-2004, 58 A H1N1 and 23 A H1N2 subtypes have been isolated from patients with influenza-like illness in the south of France. The HA1 region was analyzed by RT-PCR and subsequently sequenced to compare the HA1 genetic evolution of influenza A H1N1 and A H1N2 subtypes. Our results showed that 28 amino acid substitutions have accumulated in the HA1 region since the circulation of A/New Caledonia/20/99-like viruses in France. Of these, fifteen were located in four antigenic sites (B, C, D and E). Six of them were observed only in the A H1N2 isolates, six only in the A H1N1 isolates and three in both subtypes. Furthermore, nine of twenty two A H1N2 isolates from the winter of 2002-2003 shared a T90A amino acid change which has not been observed in any A H1N1 isolate; resulting in the introduction of a new glycosylation site close to the antigenic site E. This might mask some antigenic E determinants and therefore, modify the A H1N2 antigenicity. The divergent genetic evolution of hemagglutinin may ultimately lead to a significant different antigenicity between A H1N1 and A H1N2 subtypes that would require the introduction of a new subtype in the vaccine batches.

Possible basis for the emergence of H1N1 viruses with pandemic potential from avian hosts.

PubMed

Koçer, Zeynep A; Krauss, Scott; Zanin, Mark; Danner, Angela; Gulati, Shelly; Jones, Jeremy C; Friedman, Kimberly; Graham, Allison; Forrest, Heather; Seiler, Jon; Air, Gillian M; Webster, Robert G

2015-07-01

Influenza A viruses of the H1N1 subtype have emerged from the avian influenza gene pool in aquatic birds and caused human pandemics at least twice during the past century. Despite this fact, surprisingly little is known about the H1N1 gene pool in the aquatic bird reservoir. A preliminary study showed that an H1N1 virus from a shorebird of the Charadriiformes order was transmitted between animals through the airborne route of infection, whereas an H1N1 virus from a bird of the Anseriformes order was not. Here we show that two of the three H1N1 viruses isolated from Charadriiformes species in 2009 were transmitted between animals through the airborne route of infection, and five H1N1 isolates from Anseriformes species were not. The one H1N1 virus from a Charadriiformes species that failed to transmit through the airborne route was a reassortant possessing multiple internal gene segments from Anseriformes species. The molecular differences between the airborne-transmissible and non-airborne-transmissible H1N1 viruses were multigenic, involving the selection of virus with human-like receptor-binding specificity (α2-6 sialic acid) and multiple differences in the polymerase complex, mainly in the PB2, PB1-F2, and nonstructural genes.

Possible basis for the emergence of H1N1 viruses with pandemic potential from avian hosts

PubMed Central

Koçer, Zeynep A; Krauss, Scott; Zanin, Mark; Danner, Angela; Gulati, Shelly; Jones, Jeremy C; Friedman, Kimberly; Graham, Allison; Forrest, Heather; Seiler, Jon; Air, Gillian M; Webster, Robert G

2015-01-01

Influenza A viruses of the H1N1 subtype have emerged from the avian influenza gene pool in aquatic birds and caused human pandemics at least twice during the past century. Despite this fact, surprisingly little is known about the H1N1 gene pool in the aquatic bird reservoir. A preliminary study showed that an H1N1 virus from a shorebird of the Charadriiformes order was transmitted between animals through the airborne route of infection, whereas an H1N1 virus from a bird of the Anseriformes order was not. Here we show that two of the three H1N1 viruses isolated from Charadriiformes species in 2009 were transmitted between animals through the airborne route of infection, and five H1N1 isolates from Anseriformes species were not. The one H1N1 virus from a Charadriiformes species that failed to transmit through the airborne route was a reassortant possessing multiple internal gene segments from Anseriformes species. The molecular differences between the airborne-transmissible and non-airborne-transmissible H1N1 viruses were multigenic, involving the selection of virus with human-like receptor-binding specificity (α2-6 sialic acid) and multiple differences in the polymerase complex, mainly in the PB2, PB1-F2, and nonstructural genes. PMID:26251829

Highly pathogenic avian influenza virus (H5N1) isolated from whooper swans, Japan.

PubMed

Uchida, Yuko; Mase, Masaji; Yoneda, Kumiko; Kimura, Atsumu; Obara, Tsuyoshi; Kumagai, Seikou; Saito, Takehiko; Yamamoto, Yu; Nakamura, Kikuyasu; Tsukamoto, Kenji; Yamaguchi, Shigeo

2008-09-01

On April 21, 2008, four whooper swans were found dead at Lake Towada, Akita prefecture, Japan. Highly pathogenic avian influenza virus of the H5N1 subtype was isolated from specimens of the affected birds. The hemagglutinin (HA) gene of the isolate belongs to clade 2.3.2 in the HA phylogenetic tree.

Isolation and Characterization of Avian Influenza Viruses, Including Highly Pathogenic H5N1, from Poultry in Live Bird Markets in Hanoi, Vietnam, in 2001

PubMed Central

Nguyen, Doan C.; Uyeki, Timothy M.; Jadhao, Samadhan; Maines, Taronna; Shaw, Michael; Matsuoka, Yumiko; Smith, Catherine; Rowe, Thomas; Lu, Xiuhua; Hall, Henrietta; Xu, Xiyan; Balish, Amanda; Klimov, Alexander; Tumpey, Terrence M.; Swayne, David E.; Huynh, Lien P. T.; Nghiem, Ha K.; Nguyen, Hanh H. T.; Hoang, Long T.; Cox, Nancy J.; Katz, Jacqueline M.

2005-01-01

Since 1997, outbreaks of highly pathogenic (HP) H5N1 and circulation of H9N2 viruses among domestic poultry in Asia have posed a threat to public health. To better understand the extent of transmission of avian influenza viruses (AIV) to humans in Asia, we conducted a cross-sectional virologic study in live bird markets (LBM) in Hanoi, Vietnam, in October 2001. Specimens from 189 birds and 18 environmental samples were collected at 10 LBM. Four influenza A viruses of the H4N6 (n = 1), H5N2 (n = 1), and H9N3 (n = 2) subtypes were isolated from healthy ducks for an isolation frequency of over 30% from this species. Two H5N1 viruses were isolated from healthy geese. The hemagglutinin (HA) genes of these H5N1 viruses possessed multiple basic amino acid motifs at the cleavage site, were HP for experimentally infected chickens, and were thus characterized as HP AIV. These HA genes shared high amino acid identities with genes of other H5N1 viruses isolated in Asia during this period, but they were genetically distinct from those of H5N1 viruses isolated from poultry and humans in Vietnam during the early 2004 outbreaks. These viruses were not highly virulent for experimentally infected ducks, mice, or ferrets. These results establish that HP H5N1 viruses with properties similar to viruses isolated in Hong Kong and mainland China circulated in Vietnam as early as 2001, suggest a common source for H5N1 viruses circulating in these Asian countries, and provide a framework to better understand the recent widespread emergence of HP H5N1 viruses in Asia. PMID:15767421

Design and Characterization of a Computationally Optimized Broadly Reactive Hemagglutinin Vaccine for H1N1 Influenza Viruses

PubMed Central

Carter, Donald M.; Darby, Christopher A.; Lefoley, Bradford C.; Crevar, Corey J.; Alefantis, Timothy; Oomen, Raymond; Anderson, Stephen F.; Strugnell, Tod; Cortés-Garcia, Guadalupe; Vogel, Thorsten U.; Parrington, Mark; Kleanthous, Harold

2016-01-01

ABSTRACT One of the challenges of developing influenza A vaccines is the diversity of antigenically distinct isolates. Previously, a novel hemagglutinin (HA) for H5N1 influenza was derived from a methodology termed computationally optimized broadly reactive antigen (COBRA). This COBRA HA elicited a broad antibody response against H5N1 isolates from different clades. We now report the development and characterization of a COBRA-based vaccine for both seasonal and pandemic H1N1 influenza virus isolates. Nine prototype H1N1 COBRA HA proteins were developed and tested in mice using a virus-like particle (VLP) format for the elicitation of broadly reactive, functional antibody responses and protection against viral challenge. These candidates were designed to recognize H1N1 viruses isolated within the last 30 years. In addition, several COBRA candidates were designed based on sequences of H1N1 viruses spanning the past 100 years, including modern pandemic H1N1 isolates. Four of the 9 H1N1 COBRA HA proteins (X1, X3, X6, and P1) had the broadest hemagglutination inhibition (HAI) activity against a panel of 17 H1N1 viruses. These vaccines were used in cocktails or prime-boost combinations. The most effective regimens that both elicited the broadest HAI response and protected mice against a pandemic H1N1 challenge were vaccines that contained the P1 COBRA VLP and either the X3 or X6 COBRA VLP vaccine. These mice had little or no detectable viral replication, comparable to that observed with a matched licensed vaccine. This is the first report describing a COBRA-based HA vaccine strategy that elicits a universal, broadly reactive, protective response against seasonal and pandemic H1N1 isolates. IMPORTANCE Universal influenza vaccine approaches have the potential to be paradigm shifting for the influenza vaccine field, with the goal of replacing the current standard of care with broadly cross-protective vaccines. We have used COBRA technology to develop an HA head

H5N1 influenza viruses: outbreaks and biological properties

PubMed Central

Neumann, Gabriele; Chen, Hualan; Gao, George F; Shu, Yuelong; Kawaoka, Yoshihiro

2010-01-01

All known subtypes of influenza A viruses are maintained in wild waterfowl, the natural reservoir of these viruses. Influenza A viruses are isolated from a variety of animal species with varying morbidity and mortality rates. More importantly, influenza A viruses cause respiratory disease in humans with potentially fatal outcome. Local or global outbreaks in humans are typically characterized by excess hospitalizations and deaths. In 1997, highly pathogenic avian influenza viruses of the H5N1 subtype emerged in Hong Kong that transmitted to humans, resulting in the first documented cases of human death by avian influenza virus infection. A new outbreak started in July 2003 in poultry in Vietnam, Indonesia, and Thailand, and highly pathogenic avian H5N1 influenza viruses have since spread throughout Asia and into Europe and Africa. These viruses continue to infect humans with a high mortality rate and cause worldwide concern of a looming pandemic. Moreover, H5N1 virus outbreaks have had devastating effects on the poultry industries throughout Asia. Since H5N1 virus outbreaks appear to originate from Southern China, we here examine H5N1 influenza viruses in China, with an emphasis on their biological properties. PMID:19884910

Sensitization with vaccinia virus encoding H5N1 hemagglutinin restores immune potential against H5N1 influenza virus.

PubMed

Yasui, Fumihiko; Itoh, Yasushi; Ikejiri, Ai; Kitabatake, Masahiro; Sakaguchi, Nobuo; Munekata, Keisuke; Shichinohe, Shintaro; Hayashi, Yukiko; Ishigaki, Hirohito; Nakayama, Misako; Sakoda, Yoshihiro; Kida, Hiroshi; Ogasawara, Kazumasa; Kohara, Michinori

2016-11-28

H5N1 highly pathogenic avian influenza (H5N1 HPAI) virus causes elevated mortality compared with seasonal influenza viruses like H1N1 pandemic influenza (H1N1 pdm) virus. We identified a mechanism associated with the severe symptoms seen with H5N1 HPAI virus infection. H5N1 HPAI virus infection induced a decrease of dendritic cell number in the splenic extrafollicular T-cell zone and impaired formation of the outer layers of B-cell follicles, resulting in insufficient levels of antibody production after infection. However, in animals vaccinated with a live recombinant vaccinia virus expressing the H5 hemagglutinin, infection with H5N1 HPAI virus induced parafollicular dendritic cell accumulation and efficient antibody production. These results indicate that a recombinant vaccinia encoding H5 hemagglutinin gene does not impair dendritic cell recruitment and can be a useful vaccine candidate.

Efficacy of a high-growth reassortant H1N1 influenza virus vaccine against the classical swine H1N1 subtype influenza virus in mice and pigs.

PubMed

Wen, Feng; Yu, Hai; Yang, Fu-Ru; Huang, Meng; Yang, Sheng; Zhou, Yan-Jun; Li, Ze-Jun; Tong, Guang-Zhi

2014-11-01

Swine influenza (SI) is an acute, highly contagious respiratory disease caused by swine influenza A viruses (SwIVs), and it poses a potential global threat to human health. Classical H1N1 (cH1N1) SwIVs are still circulating and remain the predominant subtype in the swine population in China. In this study, a high-growth reassortant virus (GD/PR8) harboring the hemagglutinin (HA) and neuraminidase (NA) genes from a novel cH1N1 isolate in China, A/Swine/Guangdong/1/2011 (GD/11) and six internal genes from the high-growth A/Puerto Rico/8/34(PR8) virus was generated by plasmid-based reverse genetics and tested as a candidate seed virus for the preparation of an inactivated vaccine. The protective efficacy of this vaccine was evaluated in mice and pigs challenged with GD/11 virus. Prime and boost inoculation of GD/PR8 vaccine yielded high-titer serum hemagglutination inhibiting (HI) antibodies and IgG antibodies for GD/11 in both mice and pigs. Complete protection of mice and pigs against cH1N1 SIV challenge was observed, with significantly fewer lung lesions and reduced viral shedding in vaccine-inoculated animals compared with unvaccinated control animals. Our data demonstrated that the GD/PR8 may serve as the seed virus for a promising SwIVs vaccine to protect the swine population.

Effect of Priming with H1N1 Influenza Viruses of Variable Antigenic Distances on Challenge with 2009 Pandemic H1N1 Virus

PubMed Central

O'Donnell, Christopher D.; Wright, Amber; Vogel, Leatrice N.; Wei, Chih-Jen; Nabel, Gary J.

2012-01-01

Compared to seasonal influenza viruses, the 2009 pandemic H1N1 (pH1N1) virus caused greater morbidity and mortality in children and young adults. People over 60 years of age showed a higher prevalence of cross-reactive pH1N1 antibodies, suggesting that they were previously exposed to an influenza virus or vaccine that was antigenically related to the pH1N1 virus. To define the basis for this cross-reactivity, ferrets were infected with H1N1 viruses of variable antigenic distance that circulated during different decades from the 1930s (Alaska/35), 1940s (Fort Monmouth/47), 1950s (Fort Warren/50), and 1990s (New Caledonia/99) and challenged with 2009 pH1N1 virus 6 weeks later. Ferrets primed with the homologous CA/09 or New Jersey/76 (NJ/76) virus served as a positive control, while the negative control was an influenza B virus that should not cross-protect against influenza A virus infection. Significant protection against challenge virus replication in the respiratory tract was observed in ferrets primed with AK/35, FM/47, and NJ/76; FW/50-primed ferrets showed reduced protection, and NC/99-primed ferrets were not protected. The hemagglutinins (HAs) of AK/35, FM/47, and FW/50 differ in the presence of glycosylation sites. We found that the loss of protective efficacy observed with FW/50 was associated with the presence of a specific glycosylation site. Our results suggest that changes in the HA occurred between 1947 and 1950, such that prior infection could no longer protect against 2009 pH1N1 infection. This provides a mechanistic understanding of the nature of serological cross-protection observed in people over 60 years of age during the 2009 H1N1 pandemic. PMID:22674976

Effect of priming with H1N1 influenza viruses of variable antigenic distances on challenge with 2009 pandemic H1N1 virus.

PubMed

O'Donnell, Christopher D; Wright, Amber; Vogel, Leatrice N; Wei, Chih-Jen; Nabel, Gary J; Subbarao, Kanta

2012-08-01

Compared to seasonal influenza viruses, the 2009 pandemic H1N1 (pH1N1) virus caused greater morbidity and mortality in children and young adults. People over 60 years of age showed a higher prevalence of cross-reactive pH1N1 antibodies, suggesting that they were previously exposed to an influenza virus or vaccine that was antigenically related to the pH1N1 virus. To define the basis for this cross-reactivity, ferrets were infected with H1N1 viruses of variable antigenic distance that circulated during different decades from the 1930s (Alaska/35), 1940s (Fort Monmouth/47), 1950s (Fort Warren/50), and 1990s (New Caledonia/99) and challenged with 2009 pH1N1 virus 6 weeks later. Ferrets primed with the homologous CA/09 or New Jersey/76 (NJ/76) virus served as a positive control, while the negative control was an influenza B virus that should not cross-protect against influenza A virus infection. Significant protection against challenge virus replication in the respiratory tract was observed in ferrets primed with AK/35, FM/47, and NJ/76; FW/50-primed ferrets showed reduced protection, and NC/99-primed ferrets were not protected. The hemagglutinins (HAs) of AK/35, FM/47, and FW/50 differ in the presence of glycosylation sites. We found that the loss of protective efficacy observed with FW/50 was associated with the presence of a specific glycosylation site. Our results suggest that changes in the HA occurred between 1947 and 1950, such that prior infection could no longer protect against 2009 pH1N1 infection. This provides a mechanistic understanding of the nature of serological cross-protection observed in people over 60 years of age during the 2009 H1N1 pandemic.

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

Microevolution of Highly Pathogenic Avian Influenza A(H5N1) Viruses Isolated from Humans, Egypt, 2007–2011

PubMed Central

Younan, Mary; Poh, Mee Kian; Elassal, Emad; Davis, Todd; Rivailler, Pierre; Balish, Amanda L.; Simpson, Natosha; Jones, Joyce; Deyde, Varough; Loughlin, Rosette; Perry, Ije; Gubareva, Larisa; ElBadry, Maha A.; Truelove, Shaun; Gaynor, Anne M.; Mohareb, Emad; Amin, Magdy; Cornelius, Claire; Pimentel, Guillermo; Earhart, Kenneth; Naguib, Amel; Abdelghani, Ahmed S.; Refaey, Samir; Klimov, Alexander I.; Kandeel, Amr

2013-01-01

We analyzed highly pathogenic avian influenza A(H5N1) viruses isolated from humans infected in Egypt during 2007–2011. All analyzed viruses evolved from the lineage of subtype H5N1 viruses introduced into Egypt in 2006; we found minimal evidence of reassortment and no exotic introductions. The hemagglutinin genes of the viruses from 2011 formed a monophyletic group within clade 2.2.1 that also included human viruses from 2009 and 2010 and contemporary viruses from poultry; this finding is consistent with zoonotic transmission. Although molecular markers suggestive of decreased susceptibility to antiviral drugs were detected sporadically in the neuraminidase and matrix 2 proteins, functional neuraminidase inhibition assays did not identify resistant viruses. No other mutations suggesting a change in the threat to public health were detected in the viral proteomes. However, a comparison of representative subtype H5N1 viruses from 2011 with older subtype H5N1 viruses from Egypt revealed substantial antigenic drift. PMID:23260983

Sensitivity of influenza rapid diagnostic tests to H5N1 and 2009 pandemic H1N1 viruses.

PubMed

Sakai-Tagawa, Yuko; Ozawa, Makoto; Tamura, Daisuke; Le, Mai thi Quynh; Nidom, Chairul A; Sugaya, Norio; Kawaoka, Yoshihiro

2010-08-01

Simple and rapid diagnosis of influenza is useful for making treatment decisions in the clinical setting. Although many influenza rapid diagnostic tests (IRDTs) are available for the detection of seasonal influenza virus infections, their sensitivity for other viruses, such as H5N1 viruses and the recently emerged swine origin pandemic (H1N1) 2009 virus, remains largely unknown. Here, we examined the sensitivity of 20 IRDTs to various influenza virus strains, including H5N1 and 2009 pandemic H1N1 viruses. Our results indicate that the detection sensitivity to swine origin H1N1 viruses varies widely among IRDTs, with some tests lacking sufficient sensitivity to detect the early stages of infection when the virus load is low.

Pathogenicity and Transmissibility of Novel Reassortant H3N2 Influenza Viruses with 2009 Pandemic H1N1 Genes in Pigs

PubMed Central

Ma, Jingjiao; Shen, Huigang; Liu, Qinfang; Bawa, Bhupinder; Qi, Wenbao; Duff, Michael; Lang, Yuekun; Lee, Jinhwa; Yu, Hai; Bai, Jianfa; Tong, Guangzhi; Hesse, Richard A.; Richt, Jürgen A.

2014-01-01

viruses with 3 or 5 genes from A(H1N1)pdm09 isolated from diseased pigs are pathogenic and transmissible in pigs, but the reassortant H3N2 virus with 5 A(H1N1)pdm09 genes displayed less efficient transmissibility than the endemic and reassortant H3N2 viruses with 3 A(H1N1)pdm09 genes. Further studies revealed that an avian-like glycine at the HA 228 receptor binding site of the reassortant H3N2 virus with 5 A(H1N1)pdm09 genes is responsible for less efficient transmissibility in pigs. Our results provide insights into viral pathogenesis and the transmission of novel reassortant H3N2 viruses that are circulating in U.S. swine herds and warrant future surveillance. PMID:25540372

Sequential Infection in Ferrets with Antigenically Distinct Seasonal H1N1 Influenza Viruses Boosts Hemagglutinin Stalk-Specific Antibodies

PubMed Central

Kirchenbaum, Greg A.; Carter, Donald M.

2015-01-01

ABSTRACT Broadly reactive antibodies targeting the conserved hemagglutinin (HA) stalk region are elicited following sequential infection or vaccination with influenza viruses belonging to divergent subtypes and/or expressing antigenically distinct HA globular head domains. Here, we demonstrate, through the use of novel chimeric HA proteins and competitive binding assays, that sequential infection of ferrets with antigenically distinct seasonal H1N1 (sH1N1) influenza virus isolates induced an HA stalk-specific antibody response. Additionally, stalk-specific antibody titers were boosted following sequential infection with antigenically distinct sH1N1 isolates in spite of preexisting, cross-reactive, HA-specific antibody titers. Despite a decline in stalk-specific serum antibody titers, sequential sH1N1 influenza virus-infected ferrets were protected from challenge with a novel H1N1 influenza virus (A/California/07/2009), and these ferrets poorly transmitted the virus to naive contacts. Collectively, these findings indicate that HA stalk-specific antibodies are commonly elicited in ferrets following sequential infection with antigenically distinct sH1N1 influenza virus isolates lacking HA receptor-binding site cross-reactivity and can protect ferrets against a pathogenic novel H1N1 virus. IMPORTANCE The influenza virus hemagglutinin (HA) is a major target of the humoral immune response following infection and/or seasonal vaccination. While antibodies targeting the receptor-binding pocket of HA possess strong neutralization capacities, these antibodies are largely strain specific and do not confer protection against antigenic drift variant or novel HA subtype-expressing viruses. In contrast, antibodies targeting the conserved stalk region of HA exhibit broader reactivity among viruses within and among influenza virus subtypes. Here, we show that sequential infection of ferrets with antigenically distinct seasonal H1N1 influenza viruses boosts the antibody responses

Experimental infection with H1N1 European swine influenza virus protects pigs from an infection with the 2009 pandemic H1N1 human influenza virus.

PubMed

Busquets, Núria; Segalés, Joaquim; Córdoba, Lorena; Mussá, Tufaria; Crisci, Elisa; Martín-Valls, Gerard E; Simon-Grifé, Meritxell; Pérez-Simó, Marta; Pérez-Maíllo, Monica; Núñez, Jose I; Abad, Francesc X; Fraile, Lorenzo; Pina, Sonia; Majó, Natalia; Bensaid, Albert; Domingo, Mariano; Montoya, María

2010-01-01

The recent pandemic caused by human influenza virus A(H1N1) 2009 contains ancestral gene segments from North American and Eurasian swine lineages as well as from avian and human influenza lineages. The emergence of this A(H1N1) 2009 poses a potential global threat for human health and the fact that it can infect other species, like pigs, favours a possible encounter with other influenza viruses circulating in swine herds. In Europe, H1N1, H1N2 and H3N2 subtypes of swine influenza virus currently have a high prevalence in commercial farms. To better assess the risk posed by the A(H1N1) 2009 in the actual situation of swine farms, we sought to analyze whether a previous infection with a circulating European avian-like swine A/Swine/Spain/53207/2004 (H1N1) influenza virus (hereafter referred to as SwH1N1) generated or not cross-protective immunity against a subsequent infection with the new human pandemic A/Catalonia/63/2009 (H1N1) influenza virus (hereafter referred to as pH1N1) 21 days apart. Pigs infected only with pH1N1 had mild to moderate pathological findings, consisting on broncho-interstitial pneumonia. However, pigs inoculated with SwH1N1 virus and subsequently infected with pH1N1 had very mild lung lesions, apparently attributed to the remaining lesions caused by SwH1N1 infection. These later pigs also exhibited boosted levels of specific antibodies. Finally, animals firstly infected with SwH1N1 virus and latter infected with pH1N1 exhibited undetectable viral RNA load in nasal swabs and lungs after challenge with pH1N1, indicating a cross-protective effect between both strains. © INRA, EDP Sciences, 2010.

Enhanced virulence of clade 2.3.2.1 highly pathogenic avian influenza A H5N1 viruses in ferrets.

PubMed

Pearce, Melissa B; Pappas, Claudia; Gustin, Kortney M; Davis, C Todd; Pantin-Jackwood, Mary J; Swayne, David E; Maines, Taronna R; Belser, Jessica A; Tumpey, Terrence M

2017-02-01

Sporadic avian to human transmission of highly pathogenic avian influenza (HPAI) A(H5N1) viruses necessitates the analysis of currently circulating and evolving clades to assess their potential risk. Following the spread and sustained circulation of clade 2 viruses across multiple continents, numerous subclades and genotypes have been described. To better understand the pathogenesis associated with the continued diversification of clade 2A(H5N1) influenza viruses, we investigated the relative virulence of eleven human and poultry isolates collected from 2006 to 2013 by determining their ability to cause disease in the ferret model. Numerous clade 2 viruses, including a clade 2.2 avian isolate, a 2.2.2.1 human isolate, and two 2.2.1 human isolates, were found to be of low virulence in the ferret model, though lethality was detected following infection with one 2.2.1 human isolate. In contrast, three of six clade 2.3.2.1 avian isolates tested led to severe disease and death among infected ferrets. Clade 2.3.2.1b and 2.3.2.1c isolates, but not 2.3.2.1a isolates, were associated with ferret lethality. All A(H5N1) viruses replicated efficiently in the respiratory tract of ferrets regardless of their virulence and lethality. However, lethal isolates were characterized by systemic viral dissemination, including detection in the brain and enhanced histopathology in lung tissues. The finding of disparate virulence phenotypes between clade 2A(H5N1) viruses, notably differences between subclades of 2.3.2.1 viruses, suggests there are distinct molecular determinants present within the established subclades, the identification of which will assist in molecular-based surveillance and public health efforts against A(H5N1) viruses. Published by Elsevier Inc.

Enhanced virulence of clade 2.3.2.1 highly pathogenic avian influenza A H5N1 viruses in ferrets

PubMed Central

Pearce, Melissa B.; Pappas, Claudia; Gustin, Kortney M.; Davis, C. Todd; Pantin-Jackwood, Mary J.; Swayne, David E.; Maines, Taronna R.; Belser, Jessica A.; Tumpey, Terrence M.

2017-01-01

Sporadic avian to human transmission of highly pathogenic avian influenza (HPAI) A(H5N1) viruses necessitates the analysis of currently circulating and evolving clades to assess their potential risk. Following the spread and sustained circulation of clade 2 viruses across multiple continents, numerous subclades and genotypes have been described. To better understand the pathogenesis associated with the continued diversification of clade 2 A(H5N1) influenza viruses, we investigated the relative virulence of eleven human and poultry isolates collected from 2006 to 2013 by determining their ability to cause disease in the ferret model. Numerous clade 2 viruses, including a clade 2.2 avian isolate, a 2.2.2.1 human isolate, and two 2.2.1 human isolates, were found to be of low virulence in the ferret model, though lethality was detected following infection with one 2.2.1 human isolate. In contrast, three of six clade 2.3.2.1 avian isolates tested led to severe disease and death among infected ferrets. Clade 2.3.2.1b and 2.3.2.1c isolates, but not 2.3.2.1a isolates, were associated with ferret lethality. All A(H5N1) viruses replicated efficiently in the respiratory tract of ferrets regardless of their virulence and lethality. However, lethal isolates were characterized by systemic viral dissemination, including detection in the brain and enhanced histopathology in lung tissues. The finding of disparate virulence phenotypes between clade 2 A(H5N1) viruses, notably differences between subclades of 2.3.2.1 viruses, suggests there are distinct molecular determinants present within the established subclades, the identification of which will assist in molecular-based surveillance and public health efforts against A(H5N1) viruses. PMID:28038412

[Phylogenetic analysis of human/swine/avian gene reassortant H1N2 influenza A virus isolated from a pig in China].

PubMed

Chen, Yixiang; Meng, Xueqiong; Liu, Qi; Huang, Xia; Huang, Shengbin; Liu, Cuiquan; Shi, Kaichuang; Guo, Jiangang; Chen, Fangfang; Hu, Liping

2008-04-01

Our aim in this study was to determine the genetic characterization and probable origin of the H1N2 swine influenza virus (A/Swine/Guangxi/13/2006) (Sw/GX/13/06) from lung tissue of a pig in Guangxi province, China. Eight genes of Sw/GX/13/06 were cloned and genetically analyzed. The hemagglutinin (HA), nucleoprotein (NP), matrix (M) and non-structural (NS) genes of Sw/GX/13/06 were most closely related to genes from the classical swine H1N1 influenza virus lineage. The neuraminidase (NA) and PB1 genes were most closely related to the corresponding genes from the human influenza H3N2 virus lineage. The remaining two genes PA and PB2 polymerase genes were most closely related to the genes from avian influenza virus lineage. Phylogenetic analyses revealed that Sw/GX/13/06 was a human/swine/avian H1N2 virus, and closely related to H1N2 viruses isolated from pigs in United States (1999-2001) and Korea (2002). To our knowledge, Sw/GX/13/06 was the first triple-reassortant H1N2 influenza A virus isolated from a pig in China. Whether the Sw/GX/13/06 has a potential threat to breeding farm and human health remains to be further investigated.

Influenza A(H1N1)pdm09 virus infection in giant pandas, China.

PubMed

Li, Desheng; Zhu, Ling; Cui, Hengmin; Ling, Shanshan; Fan, Shengtao; Yu, Zhijun; Zhou, Yuancheng; Wang, Tiecheng; Qian, Jun; Xia, Xianzhu; Xu, Zhiwen; Gao, Yuwei; Wang, Chengdong

2014-03-01

We confirmed infection with influenza A(H1N1)pdm09 in giant pandas in China during 2009 by using virus isolation and serologic analysis methods. This finding extends the host range of influenza viruses and indicates a need for increased surveillance for and control of influenza viruses among giant pandas.

Two genotypes of H1N2 swine influenza viruses appeared among pigs in China.

PubMed

Xu, Chuantian; Zhu, Qiyun; Yang, Huanliang; Zhang, Xiumei; Qiao, Chuanling; Chen, Yan; Xin, Xiaoguang; Chen, Hualan

2009-10-01

H1N2 is one of the main subtypes of influenza, which circulates in swine all over the world. To investigate the prevalence and genetic of H1N2 in swine of China. Two H1N2 swine influenza viruses were isolated from Tianjin and Guangdong province of China in 2004 and 2006, respectively. The molecular evolution of eight gene segments was analyzed. A/Swine/Tianjin/1/2004 has low identity with A/Swine/Guangdong/2006; in the phylogenetic tree of PA gene, A/Swine/Guangdong/1/2006 and A/Swine/Guangxi/1/2006 along with the H1N2 swine isolates of North America formed a cluster; and A/Swine/Tianjin/2004 and A/Swine/Zhejiang/2004, along with the classical H1N1 swine isolates formed another cluster; except that NA gene of A/Swine/Tianjin/1/2004 fell into the cluster of the H3N2 human influenza virus, indicating the reassortment between H3N2 human and H1N1 swine influenza viruses. Two different genotypes of H1N2 appeared among pigs in China. A/swine/Guangdong/1/06 was probably from H1N2 swine influenza viruses of North America; while A/swine/Tianjin/1/04 maybe come from reassortments of classical H1N1 swine and H3N2 human viruses prevalent in North America.

The Continuing Evolution of H5N1 and H9N2 Influenza Viruses in Bangladesh

PubMed Central

Marinova-Petkova, Atanaska; Shanmuganatham, Karthik; Feeroz, Mohammed M.; Jones-Engel, Lisa; Hassan, M. Kamrul; Akhtar, Sharmin; Turner, Jasmine; Walker, David; Seiler, Patrick; Franks, John; McKenzie, Pamela; Krauss, Scott; Webby, Richard J.; Webster, Robert G.

2017-01-01

Summary In 2011, avian influenza surveillance at the Bangladesh live bird markets (LBMs) showed complete replacement of the highly pathogenic avian influenza (HPAI) H5N1 virus of clade 2.2.2 (Qinghai-like H5N1 lineage) by the HPAI H5N1 clade 2.3.2.1. This clade, which continues to circulate in Bangladesh and neighboring countries, is an intra- and inter-clade reassortant; its HA, PB1, PA and NS genes come from subclade 2.3.2.1a; PB2 from subclade 2.3.2.1c; and NA, NP, and M from clade 2.3.4.2. The H9N2 influenza viruses co-circulating in the Bangladesh LBMs are also reassortants, possessing five genes (NS, M, NP, PA, and PB1) from a HPAI H7N3 virus previously isolated in Pakistan. Despite frequent co-infection of chickens and ducks, reassortment between these H5N1 and H9N2 viruses has been rare. However, all such reassortants detected in 2011 through 2013 have carried 7 genes from HPAI H5N1 clade 2.3.2.1a and the PB1 gene from the Bangladeshi H9N2 clade G1 Mideast, itself derived from HPAI H7N3 virus. Although, the live birds which we sampled in Bangladesh showed no clinical signs of morbidity, the emergence of this reassortant HPAI H5N1 lineage further complicates endemic circulation of H5N1 viruses in Bangladesh, posing a threat to both poultry and humans. PMID:27309046

Genetic and phylogenetic analysis of multi-continent human influenza A(H1N2) reassortant viruses isolated in 2001 through 2003.

PubMed

Chen, M-J; La, T; Zhao, P; Tam, J S; Rappaport, R; Cheng, S-M

2006-12-01

Genetic analyses were performed on 228 influenza A(H1) viruses derived from clinical subjects participating in an experimental vaccine trial conducted in 20 countries on four continents between 2001 and 2003. HA1 phylogenetic analysis of these viruses showed multiple clades circulated around the world with regional prevalence patterns. Sixty-five of the A(H1) viruses were identified as A(H1N2), 40 of which were isolated from South Africa. The A(H1) sequences of these viruses cluster with published H1N2 viruses phylogenetically and share with them diagnostic signature V169A and A193T changes. The results also showed for the first time that H1N2 viruses were prominent in South Africa during the 2001-2002 influenza season, accounting for over 90% of the A(H1) cases in our study, and infecting both children (29/31) and the elderly (11/13). Phylogenetic analysis of the 65 H1N2 viruses we identified, in conjunction with the 56 recent H1N2 viruses currently available in the database, provided a comprehensive view of the circulation and evolution of distinct clades of H1N2 viruses in a temporal manner between early 2001 and mid-2003, shortly after the appearance of these recent reassortant viruses in or near year 2000.

Prevention of influenza virus shedding and protection from lethal H1N1 challenge using a consensus 2009 H1N1 HA and NA adenovirus vector vaccine

PubMed Central

Jones, Frank R.; Gabitzsch, Elizabeth S.; Xu, Younong; Balint, Joseph P.; Borisevich, Viktoriya; Smith, Jennifer; Smith, Jeanon; Peng, Bi-Hung; Walker, Aida; Salazar, Magda; Paessler, Slobodan

2013-01-01

Vaccines against emerging pathogens such as the 2009 H1N1 pandemic virus can benefit from current technologies such as rapid genomic sequencing to construct the most biologically relevant vaccine. A novel platform (Ad5 [E1-, E2b-]) has been utilized to induce immune responses to various antigenic targets. We employed this vector platform to express hemagglutinin (HA) and neuraminidase (NA) genes from 2009 H1N1 pandemic viruses. Inserts were consensuses sequences designed from viral isolate sequences and the vaccine was rapidly constructed and produced. Vaccination induced H1N1 immune responses in mice, which afforded protection from lethal virus challenge. In ferrets, vaccination protected from disease development and significantly reduced viral titers in nasal washes. H1N1 cell mediated immunity as well as antibody induction correlated with the prevention of disease symptoms and reduction of virus replication. The Ad5 [E1-, E2b-] should be evaluated for the rapid development of effective vaccines against infectious diseases. PMID:21821082

Fatal influenza A (H5N1) virus Infection in zoo-housed Tigers in Yunnan Province, China

PubMed Central

Hu, Tingsong; Zhao, Huanyun; Zhang, Yan; Zhang, Wendong; Kong, Qiang; Zhang, Zhixiao; Cui, Qinghua; Qiu, Wei; Deng, Bo; Fan, Quanshui; Zhang, Fuqiang

2016-01-01

From 2014 to 2015, three cases of highly pathogenic avian influenza infection occurred in zoo-housed north-east China tigers (Panthera tigris ssp.altaica) and four tigers died of respiratory distress in succession in Yunnan Province, China. We isolated and characterized three highly pathogenic avian influenza A(H5N1) viruses from these tigers. Phylogenetic analysis indicated that A/tiger /Yunnan /tig1404 /2014(H5N1) belongs to the provisional subclade 2.3.4.4e which were novel reassortant influenza A (H5N1) viruses with six internal genes from avian influenza A (H5N2) viruses. The HA gene of the isolated A/tiger /Yunnan /tig1412 /2014(H5N1) virus belongs to the subclade 2.3.2.1b. The isolated A/tiger /Yunnan /tig1508/2015 (H5N1) virus was a novel reassortant influenza A (H5N1) virus with three internal genes (PB2, PB1 and M) from H9N2 virus and belongs to the subclade 2.3.2.1c. PMID:27162026

Fatal influenza A (H5N1) virus Infection in zoo-housed Tigers in Yunnan Province, China.

PubMed

Hu, Tingsong; Zhao, Huanyun; Zhang, Yan; Zhang, Wendong; Kong, Qiang; Zhang, Zhixiao; Cui, Qinghua; Qiu, Wei; Deng, Bo; Fan, Quanshui; Zhang, Fuqiang

2016-05-10

From 2014 to 2015, three cases of highly pathogenic avian influenza infection occurred in zoo-housed north-east China tigers (Panthera tigris ssp.altaica) and four tigers died of respiratory distress in succession in Yunnan Province, China. We isolated and characterized three highly pathogenic avian influenza A(H5N1) viruses from these tigers. Phylogenetic analysis indicated that A/tiger /Yunnan /tig1404 /2014(H5N1) belongs to the provisional subclade 2.3.4.4e which were novel reassortant influenza A (H5N1) viruses with six internal genes from avian influenza A (H5N2) viruses. The HA gene of the isolated A/tiger /Yunnan /tig1412 /2014(H5N1) virus belongs to the subclade 2.3.2.1b. The isolated A/tiger /Yunnan /tig1508/2015 (H5N1) virus was a novel reassortant influenza A (H5N1) virus with three internal genes (PB2, PB1 and M) from H9N2 virus and belongs to the subclade 2.3.2.1c.

Genetic characterization of H1N2 influenza a virus isolated from sick pigs in Southern China in 2010.

PubMed

Kong, Wei Li; Huang, Liang Zong; Qi, Hai Tao; Cao, Nan; Zhang, Liang Quan; Wang, Heng; Guan, Shang Song; Qi, Wen Bao; Jiao, Pei Rong; Liao, Ming; Zhang, Gui Hong

2011-10-13

In China H3N2 and H1N1 swine influenza viruses have been circulating for many years. In January 2010, before swine were infected with foot and mouth disease in Guangdong, some pigs have shown flu-like symptoms: cough, sneeze, runny nose and fever. We collected the nasopharyngeal swab of all sick pigs as much as possible. One subtype H1N2 influenza viruses were isolated from the pig population. The complete genome of one isolate, designated A/swine/Guangdong/1/2010(H1N2), was sequenced and compared with sequences available in GenBank. The nucleotide sequences of all eight viral RNA segments were determined, and then phylogenetic analysis was performed using the neighbor-joining method. HA, NP, M and NS were shown to be closely to swine origin. PB2 and PA were close to avian origin, but NA and PB1were close to human origin. It is a result of a multiple reassortment event. In conclusion, our finding provides further evidence about the interspecies transmission of avian influenza viruses to pigs and emphasizes the importance of reinforcing swine influenza virus (SIV) surveillance, especially before the emergence of highly pathogenic FMDs in pigs in Guangdong.

North American triple reassortant and Eurasian H1N1 swine influenza viruses do not readily reassort to generate a 2009 pandemic H1N1-like virus.

PubMed

Ma, Wenjun; Liu, Qinfang; Qiao, Chuanling; del Real, Gustavo; García-Sastre, Adolfo; Webby, Richard J; Richt, Jürgen A

2014-03-11

The 2009 pandemic H1N1 virus (pH1N1) was derived through reassortment of North American triple reassortant and Eurasian avian-like swine influenza viruses (SIVs). To date, when, how and where the pH1N1 arose is not understood. To investigate viral reassortment, we coinfected cell cultures and a group of pigs with or without preexisting immunity with a Eurasian H1N1 virus, A/Swine/Spain/53207/2004 (SP04), and a North American triple reassortant H1N1 virus, A/Swine/Kansas/77778/2007 (KS07). The infected pigs were cohoused with one or two groups of contact animals to investigate viral transmission. In coinfected MDCK or PK15 continuous cell lines with KS07 and SP04 viruses, more than 20 different reassortant viruses were found. In pigs without or with preexisting immunity (immunized with commercial inactivated swine influenza vaccines) and coinfected with both viruses, six or seven reassortant viruses, as well as the parental viruses, were identified in bronchoalveolar lavage fluid samples from the lungs. Interestingly, only one or two viruses transmitted to and were detected in contact animals. No reassortant containing a gene constellation similar to that of pH1N1 virus was found in either coinfected cells or pigs, indicating that the reassortment event that resulted in the generation of this virus is a rare event that likely involved specific viral strains and/or a favorable, not-yet-understood environment. IMPORTANCE The 2009 pandemic-like H1N1 virus could not be reproduced either in cell cultures or in pigs coinfected with North American triple reassortant H1N1 and Eurasian H1N1 swine influenza viruses. This finding suggests that the generation of the 2009 pandemic H1N1 virus by reassortment was a rare event that likely involved specific viral strains and unknown factors. Different reassortant viruses were detected in coinfected pigs with and without preexisting immunity, indicating that host immunity plays a relevant role in driving viral reassortment of

Phenotypic Differences in Virulence and Immune Response in Closely Related Clinical Isolates of Influenza A 2009 H1N1 Pandemic Viruses in Mice

PubMed Central

Camp, Jeremy V.; Chu, Yong-Kyu; Chung, Dong-Hoon; McAllister, Ryan C.; Adcock, Robert S.; Gerlach, Rachael L.; Wiemken, Timothy L.; Peyrani, Paula; Ramirez, Julio A.; Summersgill, James T.; Jonsson, Colleen B.

2013-01-01

To capture the possible genotypic and phenotypic differences of the 2009 influenza A virus H1N1 pandemic (H1N1pdm) strains circulating in adult hospitalized patients, we isolated and sequenced nine H1N1pdm viruses from patients hospitalized during 2009–2010 with severe influenza pneumonia in Kentucky. Each viral isolate was characterized in mice along with two additional H1N1 pandemic strains and one seasonal strain to assess replication and virulence. All isolates showed similar levels of replication in nasal turbinates and lung, but varied in their ability to cause morbidity. Further differences were identified in cytokine and chemokine responses. IL-6 and KC were expressed early in mice infected with strains associated with higher virulence. Strains that showed lower pathogenicity in mice had greater IFNγ, MIG, and IL-10 responses. A principal component analysis (PCA) of the cytokine and chemokine profiles revealed 4 immune response phenotypes that correlated with the severity of disease. A/KY/180/10, which showed the greatest virulence with a rapid onset of disease progression, was compared in additional studies with A/KY/136/09, which showed low virulence in mice. Analyses comparing a low (KY/136) versus a high (KY/180) virulent isolate showed a significant difference in the kinetics of infection within the lower respiratory tract and immune responses. Notably by 4 DPI, virus titers within the lung, bronchoalveolar lavage fluid (BALf), and cells within the BAL (BALc) revealed that the KY/136 replicated in BALc, while KY/180 replication persisted in lungs and BALc. In summary, our studies suggest four phenotypic groups based on immune responses that result in different virulence outcomes in H1N1pdm isolates with a high degree of genetic similarity. In vitro studies with two of these isolates suggested that the more virulent isolate, KY/180, replicates productively in macrophages and this may be a key determinant in tipping the response toward a more severe

Higher titers of some H5N1 and recent human H1N1 and H3N2 influenza viruses in Mv1 Lu vs. MDCK cells

PubMed Central

2011-01-01

Background The infectivity of influenza A viruses can differ among the various primary cells and continuous cell lines used for such measurements. Over many years, we observed that all things equal, the cytopathic effects caused by influenza A subtype H1N1, H3N2, and H5N1 viruses were often detected earlier in a mink lung epithelial cell line (Mv1 Lu) than in MDCK cells. We asked whether virus yields as measured by the 50% tissue culture infectious dose and plaque forming titer also differed in MDCK and Mv1 Lu cells infected by the same influenza virus subtypes. Results The 50% tissue culture infectious dose and plaque forming titer of many influenza A subtype H1N1, H3N2, and H5N1 viruses was higher in Mv1 Lu than in MDCK cells. Conclusions The yields of influenza subtype H1N1, H3N2, and H5N1 viruses can be higher in Mv1 Lu cells than in MDCK cells. PMID:21314955

Characterization of influenza A(H1N1)pdm09 viruses isolated from Nepalese and Indian outbreak patients in early 2015.

PubMed

Nakamura, Kazuya; Shirakura, Masayuki; Fujisaki, Seiichiro; Kishida, Noriko; Burke, David F; Smith, Derek J; Kuwahara, Tomoko; Takashita, Emi; Takayama, Ikuyo; Nakauchi, Mina; Chadha, Mandeep; Potdar, Varsha; Bhushan, Arvind; Upadhyay, Bishnu Prasad; Shakya, Geeta; Odagiri, Takato; Kageyama, Tsutomu; Watanabe, Shinji

2017-09-01

We characterized influenza A(H1N1)pdm09 isolates from large-scale outbreaks that occurred in Nepal and India in early 2015. Although no specific viral features, which may have caused the outbreaks, were identified, an S84N substitution in hemagglutinin was frequently observed. Chronological phylogenetic analysis revealed that these Nepalese and Indian viruses possessing the S84N substitution constitute potential ancestors of the novel genetic subclade 6B.1 virus that spread globally in the following (2015/16) influenza season. Thus, active surveillance of circulating influenza viruses in the Southern Asia region, including Nepal and India, would be beneficial for detecting novel variant viruses prior to their worldwide spread. © 2017 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.

[Differences in oligomerization of nucleocapsid protein of epidemic human influenza A(H1N1), A(H1N2) and B viruses].

PubMed

Prokudina, E N; Semenova, N P; Chumakov, V M; Burtseva, E I; Slepushkin, A N

2003-01-01

A comparative analysis of involving the nucleocapsid protein (NP) into shaping-up of SDS-resistant oligomers was carried out presently in circulating epidemic strains of human influenza, viruses A and B. The study results of viral isolates obtained from clinical samples and recent standard strains revealed that the involvement of NP in the SDS-resistant oligomers, which are different in various subtypes of influenza A viruses. According to this sign, the human viruses A(9H3N2) are close to the avian ones, in which, as proved by us previously, virtually the entire NP transforms itself into the oligomers resistant to SDS. About 10-20% of NP are involved in shaping-up the virus influenza A(H1N1) of SDS-resistant oligomers. No SDS-resistant NP-oligomers were detected in influenza of type B. It is suggested that the prevalence of human viruses A(H3N2) in NP-oligomers are the peculiarities of NP structure and of the presence of the PB1 protein from avian influenza virus.

Comparative virulence of wild-type H1N1pdm09 influenza A isolates in swine.

PubMed

Henningson, Jamie N; Rajao, Daniela S; Kitikoon, Pravina; Lorusso, Alessio; Culhane, Marie R; Lewis, Nicola S; Anderson, Tavis K; Vincent, Amy L

2015-03-23

In 2009, a novel swine-origin H1N1 (H1N1pdm09) influenza A virus (IAV) reached pandemic status and was soon after detected in pigs worldwide. The objective of this study was to evaluate whether differences in the HA protein can affect pathogenicity and antigenicity of H1N1pdm09 in swine. We compared lung pathology, viral replication and shedding and the antigenic relationships of four wild-type H1N1pdm09 viruses in pigs: one human (CA/09) and three isolated in swine after the pandemic (IL/09, IL/10, and MN/10). The swine strains were selected based upon unique amino acid substitutions in the HA protein. All selected viruses resulted in mild disease and viral shedding through nasal and oral fluids, however, viral replication and the degree of pathology varied between the isolates. A/Swine/IL/5265/2010 (IL/10), with substitutions I120M, S146G, S186P, V252M, had lower viral titers in the lungs and nasal secretions and fewer lung lesions. The other two swine viruses caused respiratory pathology and replicated to titers similar to the human CA/09, although MN/10 (with mutations D45Y, K304E, A425S) had lower nasal shedding. Swine-adapted H1N1pdm09 have zoonotic potential, and have reassorted with other co-circulating swine viruses, influencing the evolution of IAV in swine globally. Further, our results suggest that amino acid changes in the HA gene have the potential to alter the virulence of H1N1pdm09 in swine. Importantly, the limited clinical signs in pigs could result in continued circulation of these viruses with other endemic swine IAVs providing opportunities for reassortment. Published by Elsevier B.V.

Development of a real-time RT-PCR assay for a novel influenza A (H1N1) virus.

PubMed

Jiang, Tao; Kang, Xiaoping; Deng, Yongqiang; Zhao, Hui; Li, Xiaofeng; Yu, Xuedong; Yu, Man; Qin, Ede; Zhu, Qingyu; Yang, Yinhui; Qin, Chengfeng

2010-02-01

A pandemic caused by a novel influenza A virus (H1N1) poses a serious public health threat. In this study, a real-time reverse transcriptase PCR (RT-PCR) assay based on the hemagglutinin gene was developed that discriminates the novel H1N1 from swine influenza virus, seasonal H1N1/H3N2 virus and the highly pathogenic H5N1 avian influenza virus. The sensitivity of this assay was 0.2 50% tissue culture infective dose of virus and 200 copies of in vitro-transcribed target RNA. Three hundred and forty-eight clinical specimens from suspected H1N1 patients were tested using this assay, and forty-two (12.07%) were found to be positive. Tests using the real-time PCR assay recommended by WHO and virus isolation gave identical results. This sensitive and specific real-time RT-PCR assay will contribute to the early diagnosis and control of the emerging H1N1 influenza pandemic. 2009 Elsevier B.V. All rights reserved.

Genetic characterization of influenza A virus subtype H12N1 isolated from a watercock and lesser whistling ducks in Thailand.

PubMed

Wongphatcharachai, Manoosak; Wisedchanwet, Trong; Lapkuntod, Jiradej; Nonthabenjawan, Nutthawan; Jairak, Waleemas; Amonsin, Alongkorn

2012-06-01

Monitoring of influenza A virus (IAV) was conducted in wild bird species in central Thailand. Four IAV subtype H12N1 strains were isolated from a watercock (order Gruiformes, family Rallidae) (n = 1) and lesser whistling ducks (order Anseriformes, family Anatidae) (n = 3). All H12N1 viruses were characterized by whole-genome sequencing. Phylogenetic analysis of all eight genes of the Thai H12N1 viruses indicated that they are most closely related to the Eurasian strains. Analysis of the HA gene revealed the strains to be of low pathogenicity. This study is the first to report the circulation of IAV subtype H12N1 in Thailand and to describe the genetic characteristics of H12N1 in Eurasia. Moreover, the genetic information obtained on H12N1 has contributed a new Eurasian strain of H12N1 to the GenBank database.

Pathogenicity of an H5N1 avian influenza virus isolated in Vietnam in 2012 and reliability of conjunctival samples for diagnosis of infection

PubMed Central

Bui, Vuong N.; Dao, Tung D.; Nguyen, Tham T. H.; Nguyen, Lien T.; Bui, Anh N.; Trinh, Dai Q.; Pham, Nga T.; Inui, Kenjiro; Runstadler, Jonathan; Ogawa, Haruko; Nguyen, Khong V.; Imai, Kunitoshi

2013-01-01

The continued spread of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 among poultry in Vietnam poses a potential threat to animals and public health. To evaluate the pathogenicity of a 2012 H5N1 HPAIV isolate and to assess the utility of conjunctival swabs for viral detection and isolation in surveillance, an experimental infection with HPAIV subtype H5N1 was carried out in domestic ducks. Ducks were infected with 107.2 TCID50 of A/duck/Vietnam/QB1207/2012 (H5N1), which was isolated from a moribund domestic duck. In the infected ducks, clinical signs of disease, including neurological disorder, were observed. Ducks started to die at 3 days-post-infection (dpi), and the study mortality reached 67%. Viruses were recovered from oropharyngeal and conjunctival swabs until 7 dpi and from cloacal swabs until 4 dpi. In the ducks that died or were sacrificed on 3, 5, or 6 dpi, viruses were recovered from lung, brain, heart, pancreas and intestine, among which the highest virus titers were in the lung, brain or heart. Results of virus titration were confirmed by real-time RT-PCR. Genetic and phylogenetic analysis of the HA gene revealed that the isolate belongs to clade 2.3.2.1 similarly to the H5N1 viruses isolated in Vietnam in 2012. The present study demonstrated that this recent HPAI H5N1 virus of clade 2.3.2.1 could replicate efficiently in the systemic organs, including the brain, and cause severe disease with neurological symptoms in domestic ducks. Therefore, this HPAI H5N1 virus seems to retain the neurotrophic feature and has further developed properties of shedding virus from the oropharynx and conjunctiva in addition to the cloaca, potentially posing a higher risk of virus spread through cross-contact and/or environmental transmission. Continued surveillance and diagnostic programs using conjuntcival swabs in the field would further verify the apparent reliability of conjunctival samples for the detection of AIV. PMID:24211664

Functional Evolution of Influenza Virus NS1 Protein in Currently Circulating Human 2009 Pandemic H1N1 Viruses.

PubMed

Clark, Amelia M; Nogales, Aitor; Martinez-Sobrido, Luis; Topham, David J; DeDiego, Marta L

2017-09-01

In 2009, a novel H1N1 influenza virus emerged in humans, causing a global pandemic. It was previously shown that the NS1 protein from this human 2009 pandemic H1N1 (pH1N1) virus was an effective interferon (IFN) antagonist but could not inhibit general host gene expression, unlike other NS1 proteins from seasonal human H1N1 and H3N2 viruses. Here we show that the NS1 protein from currently circulating pH1N1 viruses has evolved to encode 6 amino acid changes (E55K, L90I, I123V, E125D, K131E, and N205S) with respect to the original protein. Notably, these 6 residue changes restore the ability of pH1N1 NS1 to inhibit general host gene expression, mainly by their ability to restore binding to the cellular factor CPSF30. This is the first report describing the ability of the pH1N1 NS1 protein to naturally acquire mutations that restore this function. Importantly, a recombinant pH1N1 virus containing these 6 amino acid changes in the NS1 protein (pH1N1/NSs-6mut) inhibited host IFN and proinflammatory responses to a greater extent than that with the parental virus (pH1N1/NS1-wt), yet virus titers were not significantly increased in cell cultures or in mouse lungs, and the disease was partially attenuated. The pH1N1/NSs-6mut virus grew similarly to pH1N1/NSs-wt in mouse lungs, but infection with pH1N1/NSs-6mut induced lower levels of proinflammatory cytokines, likely due to a general inhibition of gene expression mediated by the mutated NS1 protein. This lower level of inflammation induced by the pH1N1/NSs-6mut virus likely accounts for the attenuated disease phenotype and may represent a host-virus adaptation affecting influenza virus pathogenesis. IMPORTANCE Seasonal influenza A viruses (IAVs) are among the most common causes of respiratory infections in humans. In addition, occasional pandemics are caused when IAVs circulating in other species emerge in the human population. In 2009, a swine-origin H1N1 IAV (pH1N1) was transmitted to humans, infecting people then and up

Functional Evolution of Influenza Virus NS1 Protein in Currently Circulating Human 2009 Pandemic H1N1 Viruses

PubMed Central

Clark, Amelia M.; Nogales, Aitor; Martinez-Sobrido, Luis

2017-01-01

ABSTRACT In 2009, a novel H1N1 influenza virus emerged in humans, causing a global pandemic. It was previously shown that the NS1 protein from this human 2009 pandemic H1N1 (pH1N1) virus was an effective interferon (IFN) antagonist but could not inhibit general host gene expression, unlike other NS1 proteins from seasonal human H1N1 and H3N2 viruses. Here we show that the NS1 protein from currently circulating pH1N1 viruses has evolved to encode 6 amino acid changes (E55K, L90I, I123V, E125D, K131E, and N205S) with respect to the original protein. Notably, these 6 residue changes restore the ability of pH1N1 NS1 to inhibit general host gene expression, mainly by their ability to restore binding to the cellular factor CPSF30. This is the first report describing the ability of the pH1N1 NS1 protein to naturally acquire mutations that restore this function. Importantly, a recombinant pH1N1 virus containing these 6 amino acid changes in the NS1 protein (pH1N1/NSs-6mut) inhibited host IFN and proinflammatory responses to a greater extent than that with the parental virus (pH1N1/NS1-wt), yet virus titers were not significantly increased in cell cultures or in mouse lungs, and the disease was partially attenuated. The pH1N1/NSs-6mut virus grew similarly to pH1N1/NSs-wt in mouse lungs, but infection with pH1N1/NSs-6mut induced lower levels of proinflammatory cytokines, likely due to a general inhibition of gene expression mediated by the mutated NS1 protein. This lower level of inflammation induced by the pH1N1/NSs-6mut virus likely accounts for the attenuated disease phenotype and may represent a host-virus adaptation affecting influenza virus pathogenesis. IMPORTANCE Seasonal influenza A viruses (IAVs) are among the most common causes of respiratory infections in humans. In addition, occasional pandemics are caused when IAVs circulating in other species emerge in the human population. In 2009, a swine-origin H1N1 IAV (pH1N1) was transmitted to humans, infecting people

Comparisons of Highly Virulent H5N1 Influenza A Viruses Isolated from Humans and Chickens from Hong Kong

PubMed Central

Suarez, David L.; Perdue, Michael L.; Cox, Nancy; Rowe, Thomas; Bender, Catherine; Huang, Jing; Swayne, David E.

1998-01-01

Genes of an influenza A (H5N1) virus from a human in Hong Kong isolated in May 1997 were sequenced and found to be all avian-like (K. Subbarao et al., Science 279:393–395, 1998). Gene sequences of this human isolate were compared to those of a highly pathogenic chicken H5N1 influenza virus isolated from Hong Kong in April 1997. Sequence comparisons of all eight RNA segments from the two viruses show greater than 99% sequence identity between them. However, neither isolate’s gene sequence was closely (>95% sequence identity) related to any other gene sequences found in the GenBank database. Phylogenetic analysis demonstrated that the nucleotide sequences of at least four of the eight RNA segments clustered with Eurasian origin avian influenza viruses. The hemagglutinin gene phylogenetic analysis also included the sequences from an additional three human and two chicken H5N1 virus isolates from Hong Kong, and the isolates separated into two closely related groups. However, no single amino acid change separated the chicken origin and human origin isolates, but they all contained multiple basic amino acids at the hemagglutinin cleavage site, which is associated with a highly pathogenic phenotype in poultry. In experimental intravenous inoculation studies with chickens, all seven viruses were highly pathogenic, killing most birds within 24 h. All infected chickens had virtually identical pathologic lesions, including moderate to severe diffuse edema and interstitial pneumonitis. Viral nucleoprotein was most frequently demonstrated in vascular endothelium, macrophages, heterophils, and cardiac myocytes. Asphyxiation from pulmonary edema and generalized cardiovascular collapse were the most likely pathogenic mechanisms responsible for illness and death. In summary, a small number of changes in hemagglutinin gene sequences defined two closely related subgroups, with both subgroups having human and chicken members, among the seven viruses examined from Hong Kong, and

Influenza A(H6N1) Virus in Dogs, Taiwan

PubMed Central

Lin, Hui-Ting; Wang, Ching-Ho; Chueh, Ling-Ling; Su, Bi-Ling

2015-01-01

We determined the prevalence of influenza A virus in dogs in Taiwan and isolated A/canine/Taiwan/E01/2014. Molecular analysis indicated that this isolate was closely related to influenza A(H6N1) viruses circulating in Taiwan and harbored the E627K substitution in the polymerase basic 2 protein, which indicated its ability to replicate in mammalian species. PMID:26583707

H1N1 influenza viruses varying widely in hemagglutinin stability transmit efficiently from swine to swine and to ferrets.

PubMed

Russier, Marion; Yang, Guohua; Marinova-Petkova, Atanaska; Vogel, Peter; Kaplan, Bryan S; Webby, Richard J; Russell, Charles J

2017-03-01

A pandemic-capable influenza virus requires a hemagglutinin (HA) surface glycoprotein that is immunologically unseen by most people and is capable of supporting replication and transmission in humans. HA stabilization has been linked to 2009 pH1N1 pandemic potential in humans and H5N1 airborne transmissibility in the ferret model. Swine have served as an intermediate host for zoonotic influenza viruses, yet the evolutionary pressure exerted by this host on HA stability was unknown. For over 70 contemporary swine H1 and H3 isolates, we measured HA activation pH to range from pH 5.1 to 5.9 for H1 viruses and pH 5.3 to 5.8 for H3 viruses. Thus, contemporary swine isolates vary widely in HA stability, having values favored by both avian (pH >5.5) and human and ferret (pH ≤5.5) species. Using an early 2009 pandemic H1N1 (pH1N1) virus backbone, we generated three viruses differing by one HA residue that only altered HA stability: WT (pH 5.5), HA1-Y17H (pH 6.0), and HA2-R106K (pH 5.3). All three replicated in pigs and transmitted from pig-to-pig and pig-to-ferret. WT and R106 viruses maintained HA genotype and phenotype after transmission. Y17H (pH 6.0) acquired HA mutations that stabilized the HA protein to pH 5.8 after transmission to pigs and 5.5 after transmission to ferrets. Overall, we found swine support a broad range of HA activation pH for contact transmission and many recent swine H1N1 and H3N2 isolates have stabilized (human-like) HA proteins. This constitutes a heightened pandemic risk and underscores the importance of ongoing surveillance and control efforts for swine viruses.

H1N1 influenza viruses varying widely in hemagglutinin stability transmit efficiently from swine to swine and to ferrets

PubMed Central

Yang, Guohua; Marinova-Petkova, Atanaska; Kaplan, Bryan S.; Webby, Richard J.

2017-01-01

A pandemic-capable influenza virus requires a hemagglutinin (HA) surface glycoprotein that is immunologically unseen by most people and is capable of supporting replication and transmission in humans. HA stabilization has been linked to 2009 pH1N1 pandemic potential in humans and H5N1 airborne transmissibility in the ferret model. Swine have served as an intermediate host for zoonotic influenza viruses, yet the evolutionary pressure exerted by this host on HA stability was unknown. For over 70 contemporary swine H1 and H3 isolates, we measured HA activation pH to range from pH 5.1 to 5.9 for H1 viruses and pH 5.3 to 5.8 for H3 viruses. Thus, contemporary swine isolates vary widely in HA stability, having values favored by both avian (pH >5.5) and human and ferret (pH ≤5.5) species. Using an early 2009 pandemic H1N1 (pH1N1) virus backbone, we generated three viruses differing by one HA residue that only altered HA stability: WT (pH 5.5), HA1-Y17H (pH 6.0), and HA2-R106K (pH 5.3). All three replicated in pigs and transmitted from pig-to-pig and pig-to-ferret. WT and R106 viruses maintained HA genotype and phenotype after transmission. Y17H (pH 6.0) acquired HA mutations that stabilized the HA protein to pH 5.8 after transmission to pigs and 5.5 after transmission to ferrets. Overall, we found swine support a broad range of HA activation pH for contact transmission and many recent swine H1N1 and H3N2 isolates have stabilized (human-like) HA proteins. This constitutes a heightened pandemic risk and underscores the importance of ongoing surveillance and control efforts for swine viruses. PMID:28282440

Possible Increased Pathogenicity of Pandemic (H1N1) 2009 Influenza Virus upon Reassortment

PubMed Central

Schrauwen, Eefje J.A.; Herfst, Sander; Chutinimitkul, Salin; Bestebroer, Theo M.; Rimmelzwaan, Guus F.; Osterhaus, Albert D.M.E.; Kuiken, Thijs

2011-01-01

Since emergence of the pandemic (H1N1) 2009 virus in April 2009, three influenza A viruses—seasonal (H3N2), seasonal (H1N1), and pandemic (H1N1) 2009—have circulated in humans. Genetic reassortment between these viruses could result in enhanced pathogenicity. We compared 4 reassortant viruses with favorable in vitro replication properties with the wild-type pandemic (H1N1) 2009 virus with respect to replication kinetics in vitro and pathogenicity and transmission in ferrets. Pandemic (H1N1) 2009 viruses containing basic polymerase 2 alone or in combination with acidic polymerase of seasonal (H1N1) virus were attenuated in ferrets. In contrast, pandemic (H1N1) 2009 with neuraminidase of seasonal (H3N2) virus resulted in increased virus replication and more severe pulmonary lesions. The data show that pandemic (H1N1) 2009 virus has the potential to reassort with seasonal influenza viruses, which may result in increased pathogenicity while it maintains the capacity of transmission through aerosols or respiratory droplets. PMID:21291589

Evolutionary genetics of highly pathogenic H5N1 avian influenza viruses isolated from whooper swans in northern Japan in 2008.

PubMed

Usui, Tatsufumi; Yamaguchi, Tsuyoshi; Ito, Hiroshi; Ozaki, Hiroichi; Murase, Toshiyuki; Ito, Toshihiro

2009-12-01

In April and May 2008, highly pathogenic avian influenza viruses subtype H5N1 were isolated from dead or moribund whooper swans in Aomori, Akita and Hokkaido prefectures in northern Japan. To trace the genetic lineage of the isolates, the nucleotide sequences of all eight genes were determined and phylogenetically analyzed. The Japanese strains were nearly identical to chicken viruses isolated in Russia in April 2008 and closely related to viruses isolated from dead wild birds in Hong Kong in 2007-2008. Their HA genes clustered in clade 2.3.2. On the other hand, NA and the other internal genes were closely related to those of clade 2.3.4 viruses (genotype V) whose NP genes originated from an HA clade 2.3.2 virus. In conclusion, the H5N1 viruses isolated in Japan, Russia and Hong Kong were derived from a common ancestor virus belonging to genotype V that was generated from genetic reassortment events between viruses of HA clades 2.3.2 and 2.3.4.

Virological and pathological characterization of an avian H1N1 influenza A virus.

PubMed

Koo, Bon-Sang; Kim, Hye Kwon; Song, Daesub; Na, Woonsung; Song, Min-Suk; Kwon, Jin Jung; Wong, Sook-San; Noh, Ji Yeong; Ahn, Min-Ju; Kim, Doo-Jin; Webby, Richard J; Yoon, Sun-Woo; Jeong, Dae Gwin

2018-05-01

Gene segments from avian H1N1 influenza A viruses have reassorted with other influenza viruses to generate pandemic strains over the past century. Nevertheless, little effort has been invested in understanding the characteristics of avian H1N1 influenza viruses. Here, we present the genome sequence and a molecular and virological characterization of an avian influenza A virus, A/wild bird/Korea/SK14/2014 (A/SK14, H1N1), isolated from migratory birds in South Korea during the winter season of 2014-2015. Full-genome sequencing and phylogenetic analysis revealed that the virus belongs to the Eurasian avian lineage. Although it retained avian-receptor binding preference, A/SK14 virus also exhibited detectable human-like receptor binding and was able to replicate in differentiated primary normal human bronchial epithelial cells. In animal models, A/SK14 virus was moderately pathogenic in mice, and virus was detected in nasal washes from inoculated guinea pigs, but not in direct-contact guinea pigs. Although A/SK14 showed moderate pathogenicity and no evidence of transmission in a mammalian model, our results suggest that the dual receptor specificity of A/SK14-like virus might allow for a more rapid adaptation to mammals, emphasizing the importance of further continuous surveillance and risk-assessment activities.

A duplex real-time RT-PCR assay for detecting H5N1 avian influenza virus and pandemic H1N1 influenza virus.

PubMed

Kang, Xiao-ping; Jiang, Tao; Li, Yong-qiang; Lin, Fang; Liu, Hong; Chang, Guo-hui; Zhu, Qing-yu; Qin, E-de; Qin, Cheng-feng; Yang, Yin-hui

2010-06-02

A duplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay was improved for simultaneous detection of highly pathogenic H5N1 avian influenza virus and pandemic H1N1 (2009) influenza virus, which is suitable for early diagnosis of influenza-like patients and for epidemiological surveillance. The sensitivity of this duplex real-time RT-PCR assay was 0.02 TCID50 (50% tissue culture infective dose) for H5N1 and 0.2 TCID50 for the pandemic H1N1, which was the same as that of each single-target RT-PCR for pandemic H1N1 and even more sensitive for H5N1 with the same primers and probes. No cross reactivity of detecting other subtype influenza viruses or respiratory tract viruses was observed. Two hundred and thirty-six clinical specimens were tested by comparing with single real-time RT-PCR and result from the duplex assay was 100% consistent with the results of single real-time RT-PCR and sequence analysis.

Swine Influenza Virus (H1N2) Characterization and Transmission in Ferrets, Chile.

PubMed

Bravo-Vasquez, Nicolás; Karlsson, Erik A; Jimenez-Bluhm, Pedro; Meliopoulos, Victoria; Kaplan, Bryan; Marvin, Shauna; Cortez, Valerie; Freiden, Pamela; Beck, Melinda A; Hamilton-West, Christopher; Schultz-Cherry, Stacey

2017-02-01

Phylogenetic analysis of the influenza hemagglutinin gene (HA) has suggested that commercial pigs in Chile harbor unique human seasonal H1-like influenza viruses, but further information, including characterization of these viruses, was unavailable. We isolated influenza virus (H1N2) from a swine in a backyard production farm in Central Chile and demonstrated that the HA gene was identical to that in a previous report. Its HA and neuraminidase genes were most similar to human H1 and N2 viruses from the early 1990s and internal segments were similar to influenza A(H1N1)pdm09 virus. The virus replicated efficiently in vitro and in vivo and transmitted in ferrets by respiratory droplet. Antigenically, it was distinct from other swine viruses. Hemagglutination inhibition analysis suggested that antibody titers to the swine Chilean H1N2 virus were decreased in persons born after 1990. Further studies are needed to characterize the potential risk to humans, as well as the ecology of influenza in swine in South America.

Swine Influenza Virus (H1N2) Characterization and Transmission in Ferrets, Chile

PubMed Central

Bravo-Vasquez, Nicolás; Karlsson, Erik A.; Jimenez-Bluhm, Pedro; Meliopoulos, Victoria; Kaplan, Bryan; Marvin, Shauna; Cortez, Valerie; Freiden, Pamela; Beck, Melinda A.

2017-01-01

Phylogenetic analysis of the influenza hemagglutinin gene (HA) has suggested that commercial pigs in Chile harbor unique human seasonal H1-like influenza viruses, but further information, including characterization of these viruses, was unavailable. We isolated influenza virus (H1N2) from a swine in a backyard production farm in Central Chile and demonstrated that the HA gene was identical to that in a previous report. Its HA and neuraminidase genes were most similar to human H1 and N2 viruses from the early 1990s and internal segments were similar to influenza A(H1N1)pdm09 virus. The virus replicated efficiently in vitro and in vivo and transmitted in ferrets by respiratory droplet. Antigenically, it was distinct from other swine viruses. Hemagglutination inhibition analysis suggested that antibody titers to the swine Chilean H1N2 virus were decreased in persons born after 1990. Further studies are needed to characterize the potential risk to humans, as well as the ecology of influenza in swine in South America. PMID:28098524

Partial heterologous protection by low pathogenic H9N2 virus against natural H9N2-PB1 gene reassortant highly pathogenic H5N1 virus in chickens.

PubMed

Dash, Sandeep Kumar; Kumar, Manoj; Kataria, Jag Mohan; Nagarajan, Shanmugasundaram; Tosh, Chakradhar; Murugkar, Harshad V; Kulkarni, Diwakar D

2016-06-01

Low pathogenic avian influenza H9N2 and highly pathogenic avian influenza H5N1 viruses continue to co-circulate in chickens. Prior infection with low pathogenic avian influenza can modulate the outcome of H5N1 infection. In India, low pathogenic H9N2 and highly pathogenic H5N1 avian influenza viruses are co-circulating in poultry. Herein, by using chickens with prior infection of A/chicken/India/04TI05/2012 (H9N2) virus we explored the outcome of infection with H5N1 virus A/turkey/India/10CA03/2012 natural PB1 gene reassortant from H9N2. Four groups (E1-E4) of SPF chickens (n = 6) prior inoculated with 10(6) EID50 of H9N2 virus were challenged with 10(6) EID50 of H5N1 natural reassortant (PB1-H9N2) virus at days 1 (group E1); 3 (group E2); 7 (group E3) and 14 (group E4) post H9N2 inoculation. The survival percentage in groups E1-E4 was 0, 100, 66.6 and 50%, respectively. Virus shedding periods for groups E1-E4 were 3, 4, 7 and 9 days, respectively post H5N1 challenge. Birds of group E1 and E2 were shedding both H9N2 and H5N1 viruses and mean viral RNA copy number was higher in oropharyngeal swabs than cloacal swabs. In group, E3 and E4 birds excreted only H5N1 virus and mean viral RNA copy number was higher in most cloacal swabs than oral swabs. These results indicate that prior infection with H9N2 virus could protect from lethal challenge of reassortant H5N1 virus as early as with three days prior H9N2 inoculation and protection decreased in groups E3 and E4 as time elapsed. However, prior infection with H9N2 did not prevent infection with H5N1 virus and birds continue to excrete virus in oropharyngeal and cloacal swabs. Amino acid substitution K368E was found in HA gene of excreted H5N1 virus of group E3. Hence, concurrent infection can also cause emergence of viruses with mutations leading to virus evolution. The results of this study are important for the surveillance and epidemiological data analysis where both H9N2 and H5N1 viruses are co

Comparative pathology of pigs infected with Korean H1N1, H1N2, or H3N2 swine influenza A viruses.

PubMed

Lyoo, Kwang-Soo; Kim, Jeong-Ki; Jung, Kwonil; Kang, Bo-Kyu; Song, Daesub

2014-09-24

The predominant subtypes of swine influenza A virus (SIV) in Korea swine population are H1N1, H1N2, and H3N2. The viruses are genetically close to the classical U.S. H1N1 and triple-reassortant H1N2 and H3N2 viruses, respectively. Comparative pathogenesis caused by Korean H1N1, H1N2, and H3N2 SIV was evaluated in this study. The H3N2 infected pigs had severe scores of gross and histopathological lesions at post-inoculation days (PID) 2, and this then progressively decreased. Both the H1N1 and H1N2 infected pigs lacked gross lesions at PID 2, but they showed moderate to severe pneumonia on PID 4, 7 and 14. The pigs infected with H1N1 had significant scores of gross and histopathological lesions when compared with the other pigs infected with H1N2, H3N2, and mock at PID 14. Mean SIV antigen-positive scores were rarely detected for pigs infected with H1N2 and H3N2 from PID 7, whereas a significantly increased amount of viral antigens were found in the bronchioles and alveolar epithelium of the H1N1infected pigs at PID 14. We demonstrated that Korean SIV subtypes had different pulmonary pathologic patterns. The Korean H3N2 rapidly induced acute lung lesions such as broncho-interstitial pneumonia, while the Korean H1N1 showed longer course of infection as compared to other strains.

Pathogenicity of an H5N1 avian influenza virus isolated in Vietnam in 2012 and reliability of conjunctival samples for diagnosis of infection.

PubMed

Bui, Vuong N; Dao, Tung D; Nguyen, Tham T H; Nguyen, Lien T; Bui, Anh N; Trinh, Dai Q; Pham, Nga T; Inui, Kenjiro; Runstadler, Jonathan; Ogawa, Haruko; Nguyen, Khong V; Imai, Kunitoshi

2014-01-22

The continued spread of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 among poultry in Vietnam poses a potential threat to animals and public health. To evaluate the pathogenicity of a 2012 H5N1 HPAIV isolate and to assess the utility of conjunctival swabs for viral detection and isolation in surveillance, an experimental infection with HPAIV subtype H5N1 was carried out in domestic ducks. Ducks were infected with 10(7.2) TCID50 of A/duck/Vietnam/QB1207/2012 (H5N1), which was isolated from a moribund domestic duck. In the infected ducks, clinical signs of disease, including neurological disorder, were observed. Ducks started to die at 3 days-post-infection (dpi), and the study mortality reached 67%. Viruses were recovered from oropharyngeal and conjunctival swabs until 7 dpi and from cloacal swabs until 4 dpi. In the ducks that died or were sacrificed on 3, 5, or 6 dpi, viruses were recovered from lung, brain, heart, pancreas and intestine, among which the highest virus titers were in the lung, brain or heart. Results of virus titration were confirmed by real-time RT-PCR. Genetic and phylogenetic analysis of the HA gene revealed that the isolate belongs to clade 2.3.2.1 similarly to the H5N1 viruses isolated in Vietnam in 2012. The present study demonstrated that this recent HPAI H5N1 virus of clade 2.3.2.1 could replicate efficiently in the systemic organs, including the brain, and cause severe disease with neurological symptoms in domestic ducks. Therefore, this HPAI H5N1 virus seems to retain the neurotrophic feature and has further developed properties of shedding virus from the oropharynx and conjunctiva in addition to the cloaca, potentially posing a higher risk of virus spread through cross-contact and/or environmental transmission. Continued surveillance and diagnostic programs using conjunctival swabs in the field would further verify the apparent reliability of conjunctival samples for the detection of AIV. Copyright © 2013 Elsevier B

Novel reassortant H9N2 viruses in pigeons and evidence for antigenic diversity of H9N2 viruses isolated from quails in Egypt.

PubMed

Kandeil, Ahmed; El-Shesheny, Rabeh; Maatouq, Asmaa; Moatasim, Yassmin; Cai, Zhipeng; McKenzie, Pamela; Webby, Richard; Kayali, Ghazi; Ali, Mohamed A

2017-04-01

The endemicity of avian influenza viruses (AIVs) among Egyptian poultry represents a public health risk. Co-circulation of low pathogenic AIV H9N2 subtype with highly pathogenic AIV H5N1 subtype in Egyptian farms provides a possibility to generate novel reassortant viruses. Here, the genetic characteristics of surface glycoproteins of 59 Egyptian H9N2 viruses, isolated between 2013 and 2015, were analysed. To elucidate the potential of genetic reassortment, 10 H9N2 isolates were selected based on different avian hosts (chickens, ducks, pigeons and quails) and phylogenetic analyses of their full genome sequences were conducted. Additionally, we performed antigenic analysis to further investigate the antigenic evolution of H9N2 viruses isolated during 2011-2015. Different viral characteristics including receptor-binding affinity and drug resistance of representative Egyptian H9N2 viruses were further investigated. The surface glycoproteins of current Egyptian H9N2 viruses were closely related to viruses of the G1-like lineage isolated from Egypt. Several genetic markers that enhance virulence in poultry and transmission to humans were detected. Analysis of the full genome of 10 H9N2 isolates indicated that two pigeon isolates inherited five internal genes from Eurasian AIVs circulating in wild birds. Antigenic conservation of different Egyptian H9N2 isolates from chickens, pigeons and ducks was observed, whereas quail isolates showed antigenic drift. The Egyptian H9N2 viruses preferentially bound to the human-like receptor rather than to the avian-like receptor. Our results suggest that the endemic H9N2 viruses in Egypt contain elements that may favour avian-to-human transmission and thus represent a public health risk.

Novel reassortant H9N2 viruses in pigeons and evidence for antigenic diversity of H9N2 viruses isolated from quails in Egypt

PubMed Central

Kandeil, Ahmed; El-Shesheny, Rabeh; Maatouq, Asmaa; Moatasim, Yassmin; Cai, Zhipeng; McKenzie, Pamela; Webby, Richard

2017-01-01

The endemicity of avian influenza viruses (AIVs) among Egyptian poultry represents a public health risk. Co-circulation of low pathogenic AIV H9N2 subtype with highly pathogenic AIV H5N1 subtype in Egyptian farms provides a possibility to generate novel reassortant viruses. Here, the genetic characteristics of surface glycoproteins of 59 Egyptian H9N2 viruses, isolated between 2013 and 2015, were analysed. To elucidate the potential of genetic reassortment, 10 H9N2 isolates were selected based on different avian hosts (chickens, ducks, pigeons and quails) and phylogenetic analyses of their full genome sequences were conducted. Additionally, we performed antigenic analysis to further investigate the antigenic evolution of H9N2 viruses isolated during 2011–2015. Different viral characteristics including receptor-binding affinity and drug resistance of representative Egyptian H9N2 viruses were further investigated. The surface glycoproteins of current Egyptian H9N2 viruses were closely related to viruses of the G1-like lineage isolated from Egypt. Several genetic markers that enhance virulence in poultry and transmission to humans were detected. Analysis of the full genome of 10 H9N2 isolates indicated that two pigeon isolates inherited five internal genes from Eurasian AIVs circulating in wild birds. Antigenic conservation of different Egyptian H9N2 isolates from chickens, pigeons and ducks was observed, whereas quail isolates showed antigenic drift. The Egyptian H9N2 viruses preferentially bound to the human-like receptor rather than to the avian-like receptor. Our results suggest that the endemic H9N2 viruses in Egypt contain elements that may favour avian-to-human transmission and thus represent a public health risk. PMID:27902350

Characaterization of H5N1 highly pathogenic avian influenza viruses isolated from poultry in Pakistan 2006-2008

USDA-ARS?s Scientific Manuscript database

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 ...

Phylogenetic and nucleotide sequence analysis of influenza A (H1N1) HA and NA genes of strains isolated from Saudi Arabia.

PubMed

Al-Qahtani, Ahmed Ali; Mubin, Muhammad; Dela Cruz, Damian M; Althawadi, Sahar Isa; Ul Rehman, Muhammad Shah Nawaz; Bohol, Marie Fe F; Al-Ahdal, Mohammed N

2017-01-30

In early 2009, a novel influenza A (H1N1) virus appeared in Mexico and rapidly disseminated worldwide. Little is known about the phylogeny and evolutionary dynamics of the H1N1 strain found in Saudi Arabia. Nucleotide sequencing and bioinformatics analyses were used to study molecular variation between the virus isolates. In this report, 72 hemagglutinin (HA) and 45 neuraminidase (NA) H1N1 virus gene sequences, isolated in 2009 from various regions of Saudi Arabia, were analyzed. Genetic characterization indicated that viruses from two different clades, 6 and 7, were circulating in the region, with clade 7, the most widely circulating H1N1 clade globally in 2009, being predominant. Sequence analysis of the HA and NA genes revealed a high degree of sequence identity with the corresponding genes from viruses circulating in the South East Asia region and with the A/California/7/2009 strain. New mutations in the HA gene of pandemic H1N1 (pH1N1) viruses, that could alter viral fitness, were identified. Relaxed-clock and Bayesian Skyline Plot analyses, based on the isolates used in this study and closely related globally representative strains, indicated marginally higher substitution rates than the type strain (5.14×10-3 and 4.18×10-3 substitutions/nucleotide/year in the HA and NA genes, respectively). The Saudi isolates were antigenically homogeneous and closely related to the prototype vaccine strain A/California/7/2009. The antigenic site of the HA gene had acquired novel mutations in some isolates, making continued monitoring of these viruses vital for the identification of potentially highly virulent and drug resistant variants.

Influenza virus A(H1N1)2009 antibody-dependent cellular cytotoxicity in young children prior to the H1N1 pandemic.

PubMed

Mesman, Annelies W; Westerhuis, Brenda M; Ten Hulscher, Hinke I; Jacobi, Ronald H; de Bruin, Erwin; van Beek, Josine; Buisman, Annemarie M; Koopmans, Marion P; van Binnendijk, Robert S

2016-09-01

Pre-existing immunity played a significant role in protection during the latest influenza A virus H1N1 pandemic, especially in older age groups. Structural similarities were found between A(H1N1)2009 and older H1N1 virus strains to which humans had already been exposed. Broadly cross-reactive antibodies capable of neutralizing the A(H1N1)2009 virus have been implicated in this immune protection in adults. We investigated the serological profile of a group of young children aged 9 years (n=55), from whom paired blood samples were available, just prior to the pandemic wave (March 2009) and shortly thereafter (March 2010). On the basis of A(H1N1)2009 seroconversion, 27 of the 55 children (49 %) were confirmed to be infected between these two time points. Within the non-infected group of 28 children (51 %), high levels of seasonal antibodies to H1 and H3 HA1 antigens were detected prior to pandemic exposure, reflecting past infection with H1N1 and H3N2, both of which had circulated in The Netherlands prior to the pandemic. In some children, this reactivity coincided with specific antibody reactivity against A(H1N1)2009. While these antibodies were not able to neutralize the A(H1N1)2009 virus, they were able to mediate antibody-dependent cellular cytotoxicity (ADCC) in vitro upon interaction with the A(H1N1)2009 virus. This finding suggests that cross-reactive antibodies could contribute to immune protection in children via ADCC.

Pathogenic analysis of the pandemic 2009 H1N1 influenza A viruses in ferrets.

PubMed

Tsuda, Yoshimi; Weisend, Carla; Martellaro, Cynthia; Feldmann, Friederike; Haddock, Elaine

2017-08-18

The pandemic 2009 H1N1 influenza A virus emerged in humans and caused the first influenza pandemic of the 21st century. Mexican isolates, A/Mexico/4108/2009 (H1N1) (Mex4108) and A/Mexico/InDRE4478/2009 (H1N1) (Mex4487) derived from a mild case and from a cluster of severe cases, showed heterogeneity in virulence in a cynomolgus macaque model. To compare the more pathogenic differences, we generated recombinant viruses and compared their virulence in ferrets. Ferrets infected with recombinant Mex4487 displayed a slightly higher rate of viral replication and severe pneumonia in the early stage of infection. In contrast, prolonged lower virus shedding of recombinant Mex4108 than that of recombinant Mex4487 was detected in throat swabs. Thus, Mex4487 induces severe pneumonia in infected individuals, whereas Mex4108 might have wide-spreading potential with mild disease.

Pathobiological Characterization of a Novel Reassortant Highly Pathogenic H5N1 Virus Isolated in British Columbia, Canada, 2015

PubMed Central

Berhane, Yohannes; Kobasa, Darwyn; Embury-Hyatt, Carissa; Pickering, Brad; Babiuk, Shawn; Joseph, Tomy; Bowes, Victoria; Suderman, Mathew; Leung, Anders; Cottam-Birt, Colleen; Hisanaga, Tamiko; Pasick, John

2016-01-01

In the current study, we describe the pathobiologic characteristics of a novel reassortant virus - A/chicken/BC/FAV-002/2015 (H5N1) belonging to clade 2.3.4.4 that was isolated from backyard chickens in British Columbia, Canada. Sequence analyses demonstrate PB1, PA, NA and NS gene segments were of North American lineage while PB2, HA, NP and M were derived from a Eurasian lineage H5N8 virus. This novel virus had a 19 amino acid deletion in the neuraminidase stalk. We evaluated the pathogenic potential of this isolate in various animal models. The virus was highly pathogenic to mice with a LD50 of 10 plaque forming units (PFU), but had limited tissue tropism. It caused only subclinical infection in pigs which did result in seroconversion. This virus was highly pathogenic to chickens, turkeys, juvenile Muscovy ducks (Cairnia moschata foma domestica) and adult Chinese geese (Anser cynoides domesticus) causing a systemic infection in all species. The virus was also efficiently transmitted and resulted in mortality in naïve contact ducks, geese and chickens. Our findings indicate that this novel H5N1 virus has a wide host range and enhanced surveillance of migratory waterfowl may be necessary in order to determine its potential to establish itself in the wild bird reservoir. PMID:26988892

Humans and Ferrets with Prior H1N1 Influenza Virus Infections Do Not Exhibit Evidence of Original Antigenic Sin after Infection or Vaccination with the 2009 Pandemic H1N1 Influenza Virus

PubMed Central

O'Donnell, Christopher D.; Wright, Amber; Vogel, Leatrice; Boonnak, Kobporn; Treanor, John J.

2014-01-01

The hypothesis of original antigenic sin (OAS) states that the imprint established by an individual's first influenza virus infection governs the antibody response thereafter. Subsequent influenza virus infection results in an antibody response against the original infecting virus and an impaired immune response against the newer influenza virus. The purpose of our study was to seek evidence of OAS after infection or vaccination with the 2009 pandemic H1N1 (2009 pH1N1) virus in ferrets and humans previously infected with H1N1 viruses with various antigenic distances from the 2009 pH1N1 virus, including viruses from 1935 through 1999. In ferrets, seasonal H1N1 priming did not diminish the antibody response to infection or vaccination with the 2009 pH1N1 virus, nor did it diminish the T-cell response, indicating the absence of OAS in seasonal H1N1 virus-primed ferrets. Analysis of paired samples of human serum taken before and after vaccination with a monovalent inactivated 2009 pH1N1 vaccine showed a significantly greater-fold rise in the titer of antibody against the 2009 pH1N1 virus than against H1N1 viruses that circulated during the childhood of each subject. Thus, prior experience with H1N1 viruses did not result in an impairment of the antibody response against the 2009 pH1N1 vaccine. Our data from ferrets and humans suggest that prior exposure to H1N1 viruses did not impair the immune response against the 2009 pH1N1 virus. PMID:24648486

Humans and ferrets with prior H1N1 influenza virus infections do not exhibit evidence of original antigenic sin after infection or vaccination with the 2009 pandemic H1N1 influenza virus.

PubMed

O'Donnell, Christopher D; Wright, Amber; Vogel, Leatrice; Boonnak, Kobporn; Treanor, John J; Subbarao, Kanta

2014-05-01

The hypothesis of original antigenic sin (OAS) states that the imprint established by an individual's first influenza virus infection governs the antibody response thereafter. Subsequent influenza virus infection results in an antibody response against the original infecting virus and an impaired immune response against the newer influenza virus. The purpose of our study was to seek evidence of OAS after infection or vaccination with the 2009 pandemic H1N1 (2009 pH1N1) virus in ferrets and humans previously infected with H1N1 viruses with various antigenic distances from the 2009 pH1N1 virus, including viruses from 1935 through 1999. In ferrets, seasonal H1N1 priming did not diminish the antibody response to infection or vaccination with the 2009 pH1N1 virus, nor did it diminish the T-cell response, indicating the absence of OAS in seasonal H1N1 virus-primed ferrets. Analysis of paired samples of human serum taken before and after vaccination with a monovalent inactivated 2009 pH1N1 vaccine showed a significantly greater-fold rise in the titer of antibody against the 2009 pH1N1 virus than against H1N1 viruses that circulated during the childhood of each subject. Thus, prior experience with H1N1 viruses did not result in an impairment of the antibody response against the 2009 pH1N1 vaccine. Our data from ferrets and humans suggest that prior exposure to H1N1 viruses did not impair the immune response against the 2009 pH1N1 virus.

Canine susceptibility to human influenza viruses (A/pdm 09H1N1, A/H3N2 and B).

PubMed

Song, Daesub; Kim, Hyekwon; Na, Woonsung; Hong, Minki; Park, Seong-Jun; Moon, Hyoungjoon; Kang, Bokyu; Lyoo, Kwang-Soo; Yeom, Minjoo; Jeong, Dae Gwin; An, Dong-Jun; Kim, Jeong-Ki

2015-02-01

We investigated the infectivity and transmissibility of the human seasonal H3N2, pandemic (pdm) H1N1 (2009) and B influenza viruses in dogs. Dogs inoculated with human seasonal H3N2 and pdm H1N1 influenza viruses exhibited nasal shedding and were seroconverted against the viruses; this did not occur in the influenza B virus-inoculated dogs. Transmission of human H3N2 virus between dogs was demonstrated by observing nasal shedding and seroconversion in naïve dogs after contact with inoculated dogs. The seroprevalence study offered evidence of human H3N2 infection occurring in dogs since 2008. Furthermore, serological evidence of pdm H1N1 influenza virus infection alone and in combination with canine H3N2 virus was found in the serum samples collected from field dogs during 2010 and 2011. Our results suggest that dogs may be hosts for human seasonal H3N2 and pdm H1N1 influenza viruses. © 2015 The Authors.

Genetic makeup of amantadine-resistant and oseltamivir-resistant human influenza A/H1N1 viruses.

PubMed

Zaraket, Hassan; Saito, Reiko; Suzuki, Yasushi; Baranovich, Tatiana; Dapat, Clyde; Caperig-Dapat, Isolde; Suzuki, Hiroshi

2010-04-01

The emergence and widespread occurrence of antiviral drug-resistant seasonal human influenza A viruses, especially oseltamivir-resistant A/H1N1 virus, are major concerns. To understand the genetic background of antiviral drug-resistant A/H1N1 viruses, we performed full genome sequencing of prepandemic A/H1N1 strains. Seasonal influenza A/H1N1 viruses, including antiviral-susceptible viruses, amantadine-resistant viruses, and oseltamivir-resistant viruses, obtained from several areas in Japan during the 2007-2008 and 2008-2009 influenza seasons were analyzed. Sequencing of the full genomes of these viruses was performed, and the phylogenetic relationships among the sequences of each individual genome segment were inferred. Reference genome sequences from the Influenza Virus Resource database were included to determine the closest ancestor for each segment. Phylogenetic analysis revealed that the oseltamivir-resistant strain evolved from a reassortant oseltamivir-susceptible strain (clade 2B) which circulated in the 2007-2008 season by acquiring the H275Y resistance-conferring mutation in the NA gene. The oseltamivir-resistant lineage (corresponding to the Northern European resistant lineage) represented 100% of the H1N1 isolates from the 2008-2009 season and further acquired at least one mutation in each of the polymerase basic protein 2 (PB2), polymerase basic protein 1 (PB1), hemagglutinin (HA), and neuraminidase (NA) genes. Therefore, a reassortment event involving two distinct oseltamivir-susceptible lineages, followed by the H275Y substitution in the NA gene and other mutations elsewhere in the genome, contributed to the emergence of the oseltamivir-resistant lineage. In contrast, amantadine-resistant viruses from the 2007-2008 season distinctly clustered in clade 2C and were characterized by extensive amino acid substitutions across their genomes, suggesting that a fitness gap among its genetic components might have driven these mutations to maintain it in the

Molecular analysis of hemagglutinin-1 fragment of avian influenza H5N1 viruses isolated from chicken farms in Indonesia from 2008 to 2010.

PubMed

Mahardika, Gusti N; Jonas, Melina; Murwijati, Theresia; Fitria, Nur; Suartha, I Nyoman; Suartini, I Gusti A A; Wibawan, I Wayan Teguh

2016-04-15

Highly pathogenic avian influenza virus of subtype H5N1 (AIV-H5N1) has been circulating in Indonesia since 2003. To understand the genetic diversity of these viruses, and to predict vaccine efficacy, the hemaglutinin-1 (HA-1) fragment of viruses isolated from chicken farms in Indonesia from 2008 to 2010 was sequenced and analyzed. The effects of these molecular changes were investigated in challenge experiments and HI assays of homologous and heterologous strains. Molecular analysis showed that these AIV-H5N1 isolates had evolved into three distinct sub-lineages from an ancestor circulating since 2003. Although no significant positive selection of residues was detected, 12 negatively selected sites were identified (p<0.05). Moreover, four sites showed evidence of significant episodic diversifying selection. The findings indicated complete protectivity and high HI titers with homologous strains, compared with protectivity ranging from 40 to 100% and lower HI titers with heterologous strains resulting from polymorphisms at antigenic sites. Our findings provide valuable insight into the molecular evolution of AIV and have important implications for vaccine efficacy and future vaccination strategies. Copyright © 2016 Elsevier B.V. All rights reserved.

H1N1 seasonal influenza virus evolutionary rate changed over time.

PubMed

Suptawiwat, Ornpreya; Kongchanagul, Alita; Boonarkart, Chompunuch; Auewarakul, Prasert

2018-05-02

It was previously shown that the seasonal H1N1 influenza virus antigenic drift occurred at a slower rate than the seasonal H3N2 virus during the first decade of the 21th century. It was hypothesized that the slower antigenic evolution led to a decrease in average ages of infection, which in turn resulted in lower level of global viral circulation. It is unclear what caused the difference between the two viruses, but a plausible explanation may be related to the fact that the H1N1 virus had been in human population for much longer than the H3N2 virus. This would suggest that H1N1 antigenic drift in an earlier period may have been different from a more recent period. To test this hypothesis, we analyzed seasonal H1N1 influenza sequences during various time periods. In comparison to more recent H1N1 virus, the older H1N1 virus during the first half of the 20th century showed evidences of higher nonsynnonymous/synonymous ration (dN/dS) in its hemagglutinin (HA) gene. We compared amino acid sequence changes in the HA epitopes for each outbreak season and found that there were less changes in later years. Amino acid sequence diversity in the epitopes as measured by sequence entropy became smaller for each passing decade. These suggest that there might be some limit to the antigenic drift. The longer an influenza virus has drifted in human population, the less flexibility it may become. With less flexibility to adapt and escape the host immunity, the virus may have to rely more on younger naïve population. Copyright © 2018 Elsevier B.V. All rights reserved.

Pathogenicity and Transmission in Pigs of the Novel A(H3N2)v Influenza Virus Isolated from Humans and Characterization of Swine H3N2 Viruses Isolated in 2010-2011

PubMed Central

Kitikoon, Pravina; Gauger, Phillip C.; Schlink, Sarah N.; Bayles, Darrell O.; Gramer, Marie R.; Darnell, Daniel; Webby, Richard J.; Lager, Kelly M.; Swenson, Sabrina L.; Klimov, Alexander

2012-01-01

Swine influenza virus (SIV) H3N2 with triple reassorted internal genes (TRIG) has been enzootic in Unites States since 1998. Transmission of the 2009 pandemic H1N1 (pH1N1) virus to pigs in the United States was followed by reassortment with endemic SIV, resulting in reassorted viruses that include novel H3N2 genotypes (rH3N2p). Between July and December 2011, 12 cases of human infections with swine-lineage H3N2 viruses containing the pandemic matrix (pM) gene [A(H3N2)v] were detected. Whole-genome analysis of H3N2 viruses isolated from pigs from 2009 to 2011 sequenced in this study and other available H3N2 sequences showed six different rH3N2p genotypes present in the U.S. swine population since 2009. The presence of the pM gene was a common feature among all rH3N2p genotypes, but no specific genotype appeared to predominate in the swine population. We compared the pathogenic, transmission, genetic, and antigenic properties of a human A(H3N2)v isolate and two swine H3N2 isolates, H3N2-TRIG and rH3N2p. Our in vivo study detected no increased virulence in A(H3N2)v or rH3N2p viruses compared to endemic H3N2-TRIG virus. Antibodies to cluster IV H3N2-TRIG and rH3N2p viruses had reduced cross-reactivity to A(H3N2)v compared to other cluster IV H3N2-TRIG and rH3N2p viruses. Genetic analysis of the hemagglutinin gene indicated that although rH3N2p and A(H3N2)v are related to cluster IV of H3N2-TRIG, some recent rH3N2p isolates appeared to be forming a separate cluster along with the human isolates of A(H3N2)v. Continued monitoring of these H3N2 viruses is necessary to evaluate the evolution and potential loss of population immunity in swine and humans. PMID:22491461

Pathogenicity and transmission in pigs of the novel A(H3N2)v influenza virus isolated from humans and characterization of swine H3N2 viruses isolated in 2010-2011.

PubMed

Kitikoon, Pravina; Vincent, Amy L; Gauger, Phillip C; Schlink, Sarah N; Bayles, Darrell O; Gramer, Marie R; Darnell, Daniel; Webby, Richard J; Lager, Kelly M; Swenson, Sabrina L; Klimov, Alexander

2012-06-01

Swine influenza virus (SIV) H3N2 with triple reassorted internal genes (TRIG) has been enzootic in Unites States since 1998. Transmission of the 2009 pandemic H1N1 (pH1N1) virus to pigs in the United States was followed by reassortment with endemic SIV, resulting in reassorted viruses that include novel H3N2 genotypes (rH3N2p). Between July and December 2011, 12 cases of human infections with swine-lineage H3N2 viruses containing the pandemic matrix (pM) gene [A(H3N2)v] were detected. Whole-genome analysis of H3N2 viruses isolated from pigs from 2009 to 2011 sequenced in this study and other available H3N2 sequences showed six different rH3N2p genotypes present in the U.S. swine population since 2009. The presence of the pM gene was a common feature among all rH3N2p genotypes, but no specific genotype appeared to predominate in the swine population. We compared the pathogenic, transmission, genetic, and antigenic properties of a human A(H3N2)v isolate and two swine H3N2 isolates, H3N2-TRIG and rH3N2p. Our in vivo study detected no increased virulence in A(H3N2)v or rH3N2p viruses compared to endemic H3N2-TRIG virus. Antibodies to cluster IV H3N2-TRIG and rH3N2p viruses had reduced cross-reactivity to A(H3N2)v compared to other cluster IV H3N2-TRIG and rH3N2p viruses. Genetic analysis of the hemagglutinin gene indicated that although rH3N2p and A(H3N2)v are related to cluster IV of H3N2-TRIG, some recent rH3N2p isolates appeared to be forming a separate cluster along with the human isolates of A(H3N2)v. Continued monitoring of these H3N2 viruses is necessary to evaluate the evolution and potential loss of population immunity in swine and humans.

Characterization of neuraminidase inhibitor-resistant influenza A(H1N1)pdm09 viruses isolated in four seasons during pandemic and post-pandemic periods in Japan.

PubMed

Takashita, Emi; Fujisaki, Seiichiro; Kishida, Noriko; Xu, Hong; Imai, Masaki; Tashiro, Masato; Odagiri, Takato

2013-11-01

Japan has the highest frequency of neuraminidase (NA) inhibitor use against influenza in the world. Therefore, Japan could be at high risk of the emergence and spread of NA inhibitor-resistant viruses. The aim of this study was to monitor the emergence of NA inhibitor-resistant viruses and the possibility of human-to-human transmission during four influenza seasons in Japan. To monitor antiviral-resistant A(H1N1)pdm09 viruses, we examined viruses isolated in four seasons from the 2008-2009 season through the 2011-2012 season in Japan by allelic discrimination, NA gene sequencing, and NA inhibitor susceptibility. We found that 157 (1·3%) of 12 026 A(H1N1)pdm09 isolates possessed an H275Y substitution in the NA protein that confers about 400- and 140-fold decreased susceptibility to oseltamivir and peramivir, respectively, compared with 275H wild-type viruses. The detection rate of resistant viruses increased from 1·0% during the pandemic period to 2·0% during the post-pandemic period. The highest detection rate of the resistant viruses was found in patients who were 0-9 years old. Furthermore, among the cases with resistant viruses, the percentage of no known exposure to antiviral drugs increased from 16% during the pandemic period to 44% during the post-pandemic period, implying that suspected human-to-human transmission of the resistant viruses gradually increased in the post-pandemic period. A(H1N1)pdm09 viruses resistant to oseltamivir and peramivir were sporadically detected in Japan, but they did not spread throughout the community. No viruses resistant to zanamivir and laninamivir were detected. © 2013 John Wiley & Sons Ltd.

Isolation of Highly Pathogenic Avian Influenza H5N1 Virus from Saker Falcons (Falco cherrug) in the Middle East

PubMed Central

Marjuki, Henju; Wernery, Ulrich; Yen, Hui-Ling; Franks, John; Seiler, Patrick; Walker, David; Krauss, Scott; Webster, Robert G.

2009-01-01

There is accumulating evidence that birds of prey are susceptible to fatal infection with highly pathogenic avian influenza (HPAI) virus. We studied the antigenic, molecular, phylogenetic, and pathogenic properties of 2 HPAI H5N1 viruses isolated from dead falcons in Saudi Arabia and Kuwait in 2005 and 2007, respectively. Phylogenetic and antigenic analyses grouped both isolates in clade 2.2 (Qinghai-like viruses). However, the viruses appeared to have spread westward via different flyways. It remains unknown how these viruses spread so rapidly from Qinghai after the 2005 outbreak and how they were introduced into falcons in these two countries. The H5N1 outbreaks in the Middle East are believed by some to be mediated by wild migratory birds. However, sporting falcons may be at additional risk from the illegal import of live quail to feed them. PMID:20148178

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. Copyright © 2014 Elsevier B.V. All rights reserved.

Influenza A (H5N1) Viruses from Pigs, Indonesia

PubMed Central

Nidom, Chairul A.; Takano, Ryo; Yamada, Shinya; Sakai-Tagawa, Yuko; Daulay, Syafril; Aswadi, Didi; Suzuki, Takashi; Suzuki, Yasuo; Shinya, Kyoko; Iwatsuki-Horimoto, Kiyoko; Muramoto, Yukiko

2010-01-01

Pigs have long been considered potential intermediate hosts in which avian influenza viruses can adapt to humans. To determine whether this potential exists for pigs in Indonesia, we conducted surveillance during 2005–2009. We found that 52 pigs in 4 provinces were infected during 2005–2007 but not 2008–2009. Phylogenetic analysis showed that the viruses had been introduced into the pig population in Indonesia on at least 3 occasions. One isolate had acquired the ability to recognize a human-type receptor. No infected pig had influenza-like symptoms, indicating that influenza A (H5N1) viruses can replicate undetected for prolonged periods, facilitating avian virus adaptation to mammalian hosts. Our data suggest that pigs are at risk for infection during outbreaks of influenza virus A (H5N1) and can serve as intermediate hosts in which this avian virus can adapt to mammals. PMID:20875275

Antigenicity of the 2015-2016 seasonal H1N1 human influenza virus HA and NA proteins.

PubMed

Clark, Amelia M; DeDiego, Marta L; Anderson, Christopher S; Wang, Jiong; Yang, Hongmei; Nogales, Aitor; Martinez-Sobrido, Luis; Zand, Martin S; Sangster, Mark Y; Topham, David J

2017-01-01

Antigenic drift of the hemagglutinin (HA) and neuraminidase (NA) influenza virus proteins contributes to reduced vaccine efficacy. To analyze antigenic drift in human seasonal H1N1 viruses derived from the 2009 pandemic H1N1 virus (pH1N1-like viruses) accounts for the limited effectiveness (around 40%) of vaccination against pH1N1-like viruses during the 2015-2016 season, nasal washes/swabs collected from adult subjects in the Rochester, NY area, were used to sequence and isolate the circulating viruses. The HA and NA proteins from viruses circulating during the 2015-2016 season encoded eighteen and fourteen amino acid differences, respectively, when compared to A/California/04/2009, a strain circulating at the origin of the 2009 pandemic. The circulating strains belonged to subclade 6B.1, defined by HA amino acid substitutions S101N, S179N, and I233T. Hemagglutination-inhibiting (HAI) and HA-specific neutralizing serum antibody (Ab) titers from around 50% of pH1N1-like virus-infected subjects and immune ferrets were 2-4 fold lower for the 2015-2016 circulating strains compared to the vaccine strain. In addition, using a luminex-based mPlex HA assay, the binding of human sera from subjects infected with pH1N1-like viruses to the HA proteins from circulating and vaccine strains was not identical, strongly suggesting antigenic differences in the HA protein. Additionally, NA inhibition (NAI) Ab titers in human sera from pH1N1-like virus-infected subjects increased after the infection and there were measurable antigenic differences between the NA protein of circulating strains and the vaccine strain using both ferret and human antisera. Despite having been vaccinated, infected subjects exhibited low HAI Ab titers against the vaccine and circulating strains. This suggests that poor responses to the H1N1 component of the vaccine as well as antigenic differences in the HA and NA proteins of currently circulating pH1N1-like viruses could be contributing to risk of

Oseltamivir-resistant pandemic influenza a (H1N1) 2009 viruses in Spain.

PubMed

Ledesma, Juan; Vicente, Diego; Pozo, Francisco; Cilla, Gustavo; Castro, Sonia Pérez; Fernández, Jonathan Suárez; Ruiz, Mercedes Pérez; Navarro, José María; Galán, Juan Carlos; Fernández, Mirian; Reina, Jordi; Larrauri, Amparo; Cuevas, María Teresa; Casas, Inmaculada; Breña, Pilar Pérez

2011-07-01

Pandemic influenza A (H1N1) 2009 virus appeared in Spain on April 25, 2009 for the first time. This new virus was adamantane-resistant but it was sensitive to neuraminidase (NA) inhibitors oseltamivir and zanamivir. To detect oseltamivir-resistant pandemic influenza A (H1N1) 2009 viruses by the Spanish Influenza Surveillance System (SISS) and a possible spread of oseltamivir-resistant viruses in Spain since starting of the pandemic situation. A total of 1229 respiratory samples taken from 413 severe and 766 non-severe patients with confirmed viral detection of pandemic influenza A (H1N1) 2009 viruses from different Spanish regions were analyzed for the specific detection of the H275Y mutation in NA between April 2009 and May 2010. H275Y NA substitution was found in 8 patients infected with pandemic influenza A (H1N1) 2009 viruses collected in November and December 2009 and in January 2010. All oseltamivir-resistant viruses were detected in severe patients (8/413, 1.93%) who previously received treatment with oseltamivir. Six of these patients were immunocompromised. In Spain, the number of oseltamivir-resistant pandemic influenza A (H1N1) 2009 viruses is until now very low. No evidence for any spread of oseltamivir-resistant H1N1 viruses is achieved in our Country. Copyright © 2011 Elsevier B.V. All rights reserved.

Development of a dual-protective live attenuated vaccine against H5N1 and H9N2 avian influenza viruses by modifying the NS1 gene.

PubMed

Choi, Eun-hye; Song, Min-Suk; Park, Su-Jin; Pascua, Philippe Noriel Q; Baek, Yun Hee; Kwon, Hyeok-il; Kim, Eun-Ha; Kim, Semi; Jang, Hyung-Kwan; Poo, Haryoung; Kim, Chul-Joong; Choi, Young Ki

2015-07-01

An increasing number of outbreaks of avian influenza H5N1 and H9N2 viruses in poultry have caused serious economic losses and raised concerns for human health due to the risk of zoonotic transmission. However, licensed H5N1 and H9N2 vaccines for animals and humans have not been developed. Thus, to develop a dual H5N1 and H9N2 live-attenuated influenza vaccine (LAIV), the HA and NA genes from a virulent mouse-adapted avian H5N2 (A/WB/Korea/ma81/06) virus and a recently isolated chicken H9N2 (A/CK/Korea/116/06) virus, respectively, were introduced into the A/Puerto Rico/8/34 backbone expressing truncated NS1 proteins (NS1-73, NS1-86, NS1-101, NS1-122) but still possessing a full-length NS gene. Two H5N2/NS1-LAIV viruses (H5N2/NS1-86 and H5N2/NS1-101) were highly attenuated compared with the full-length and remaining H5N2/NS-LAIV viruses in a mouse model. Furthermore, viruses containing NS1 modifications were found to induce more IFN-β activation than viruses with full-length NS1 proteins and were correspondingly attenuated in mice. Intranasal vaccination with a single dose (10(4.0) PFU/ml) of these viruses completely protected mice from a lethal challenge with the homologous A/WB/Korea/ma81/06 (H5N2), heterologous highly pathogenic A/EM/Korea/W149/06 (H5N1), and heterosubtypic highly virulent mouse-adapted H9N2 viruses. This study clearly demonstrates that the modified H5N2/NS1-LAIV viruses attenuated through the introduction of mutations in the NS1 coding region display characteristics that are desirable for live attenuated vaccines and hold potential as vaccine candidates for mammalian hosts.

Isolation and genetic characterization of a novel 2.2.1.2a H5N1 virus from a vaccinated meat-turkeys flock in Egypt.

PubMed

Salaheldin, Ahmed H; Veits, Jutta; Abd El-Hamid, Hatem S; Harder, Timm C; Devrishov, Davud; Mettenleiter, Thomas C; Hafez, Hafez M; Abdelwhab, Elsayed M

2017-03-09

Vaccination of poultry to control highly pathogenic avian influenza virus (HPAIV) H5N1 is used in several countries. HPAIV H5N1 of clade 2.2.1 which is endemic in Egypt has diversified into two genetic clades. Clade 2.2.1.1 represents antigenic drift variants in vaccinated commercial poultry while clade 2.2.1.2 variants are detected in humans and backyard poultry. Little is known about H5N1 infection in vaccinated turkeys under field conditions. Here, we describe an HPAI H5N1 outbreak in a vaccinated meat-turkey flock in Egypt. Birds were vaccinated with inactivated H5N2 and H5N1 vaccines at 8 and 34 days of age, respectively. At 72 nd day of age (38 days post last vaccination), turkeys exhibited mild respiratory signs, cyanosis of snood and severe congestion of the internal organs. Survivors had a reduction in feed consumption and body gain. A mortality of ~29% cumulated within 10 days after the onset of clinical signs. Laboratory diagnosis using RT-qPCRs revealed presence of H5N1 but was negative for H7 and H9 subtypes. A substantial antigenic drift against different serum samples from clade 2.2.1.1 and clade 2.3.4.4 was observed. Based on full genome sequence analysis the virus belonged to clade 2.2.1.2 but clustered with recent H5N1 viruses from 2015 in poultry in Israel, Gaza and Egypt in a novel subclade designated here 2.2.1.2a which is distinct from 2014/2015 2.2.1.2 viruses. These viruses possess 2.2.1.2 clade-specific genetic signatures and also mutations in the HA similar to those in clade 2.2.1.1 that enabled evasion from humoral immune response. Taken together, this manuscript describes a recent HPAI H5N1 outbreak in vaccinated meat-turkeys in Egypt after infection with a virus representing novel distinct 2.2.1.2a subclade. Infection with HPAIV H5N1 in commercial turkeys resulted in significant morbidity and mortality despite of vaccination using H5 vaccines. The isolated virus showed antigenic drift and clustered in a novel cluster designated here

Novel antigenic shift in HA sequences of H1N1 viruses detected by big data analysis.

PubMed

Zhang, Ruiying; Xu, Chongfeng; Duan, Ziyuan

2017-07-01

The influenza virus H1N1 has been prevalent all over the world for nearly a century. Many studies on its evolutionary history, substitution rate and antigenicity-associated sites have been done with small datasets. To have a complete view, we analysed 3171 full-length HA sequences from human H1N1 viruses sampled from 1918 to 2016, and discovered a new clade has formed with sequences isolated in Iran. Based on genetic distance calculations, we revealed an uneven evolutionary rate among sequences isolated in different years. We also found that the HA1 fragment of the new clade is like that of viruses that existed in the 1930s, while the HA2 fragment is closely associated with strains isolated after the 2009 pandemic. This new, "mixed" HA sequence indicates a cryptic antigenic shift event occurred, and it should draw more attention to the new clade identified from sequences from Iran. Copyright © 2017. Published by Elsevier B.V.

Multiple introductions of a reassortant H5N1 avian influenza virus of clade 2.3.2.1c with PB2 gene of H9N2 subtype into Indian poultry.

PubMed

Tosh, Chakradhar; Nagarajan, Shanmugasundaram; Kumar, Manoj; Murugkar, Harshad V; Venkatesh, Govindarajulu; Shukla, Shweta; Mishra, Amit; Mishra, Pranav; Agarwal, Sonam; Singh, Bharati; Dubey, Prashant; Tripathi, Sushil; Kulkarni, Diwakar D

2016-09-01

Highly pathogenic avian influenza (HPAI) H5N1 viruses are a threat to poultry in Asia, Europe, Africa and North America. Here, we report isolation and characterization of H5N1 viruses isolated from ducks and turkeys in Kerala, Chandigarh and Uttar Pradesh, India between November 2014 and March 2015. Genetic and phylogenetic analyses of haemagglutinin gene identified that the virus belonged to a new clade 2.3.2.1c which has not been detected earlier in Indian poultry. The virus possessed molecular signature for high pathogenicity to chickens, which was corroborated by intravenous pathogenicity index of 2.96. The virus was a reassortant which derives its PB2 gene from H9N2 virus isolated in China during 2007-2013. However, the neuraminidase and internal genes are of H5N1 subtype. Phylogenetic and network analysis revealed that after detection in China in 2013/2014, the virus moved to Europe, West Africa and other Asian countries including India. The analyses further indicated multiple introductions of H5N1 virus in Indian poultry and internal spread in Kerala. One of the outbreaks in ducks in Kerala is linked to the H5N1 virus isolated from wild birds in Dubai suggesting movement of virus probably through migration of wild birds. However, the outbreaks in ducks in Chandigarh and Uttar Pradesh were from an unknown source in Asia which also contributed gene pools to the outbreaks in Europe and West Africa. The widespread incidence of the novel H5N1 HPAI is similar to the spread of clade 2.2 ("Qinghai-like") virus in 2005, and should be monitored to avoid threat to animal and public health. Copyright © 2016 Elsevier B.V. All rights reserved.

Signal Immune Reactions of Macrophages Differentiated from THP-1 Monocytes to Infection with Pandemic H1N1PDM09 Virus and H5N2 and H9N2 Avian Influenza A Virus.

PubMed

Sokolova, T M; Poloskov, V V; Shuvalov, A N; Rudneva, I A; Timofeeva, T A

2018-03-01

In culture of THP-1 cells differentiated into macrophages with PMA (THP-PMA macrophages) infected with influenza viruses of subtypes H1, H5 and H9, we measured the expression of TLR7 and RIG1 receptor genes, sensors of viral RNA and ribonucleoprotein, and the levels of production of inflammatory cytokines IL-1β, TNFα, IL-10, and IFNα. The sensitivity and inflammatory response of THP-PMA macrophages to pandemic influenza A virus H1N1pdm09 and avian influenza H5N2 and H9N2 viruses correlate with the intracellular level of their viral RNA and activation of the RIG1 gene. Abortive infection is accompanied by intensive macrophage secretion of TNFα, IL-1β, and toxic factors inducing cell death. Activity of endosomal TLR7 receptor gene changed insignificantly in 24 h after infection and significantly decreased in 48 and 72 h under the action of H5N2 and H9N2, which correlated with manifestation of the cytopathogenic effect of these viruses. H5N2 and H9N2 avian viruses in THP-PMA macrophages are strong activators of the expression of the gene of the cytoplasmic RIG1 receptor 24 and 48 h after infection, and the pandemic virus H1N1pdm09 is a weak stimulator of RIG1 gene. Avian influenza H5N2 and H9N2 viruses are released by rapid induction of the inflammatory response in macrophages. At the late stages of infection, we observed a minor increase in IL-10 secretion in macrophages and, probably, the polarization of a part of the population in type M2. The studied influenza A viruses are weak inductors of IFN in THP-PMA macrophages. In the culture medium of THP-PMA macrophages infected with H9N2 and H5N2 viruses, MTT test revealed high levels of toxic factors causing the death of Caco-2 cells. In contrast to avian viruses, pandemic virus H1N1pdm09 did not induce production of toxic factors.

Antigenic Drift of the Pandemic 2009 A(H1N1) Influenza Virus in a Ferret Model

PubMed Central

Guarnaccia, Teagan; Carolan, Louise A.; Maurer-Stroh, Sebastian; Lee, Raphael T. C.; Job, Emma; Reading, Patrick C.; Petrie, Stephen; McCaw, James M.; McVernon, Jodie; Hurt, Aeron C.; Kelso, Anne; Mosse, Jennifer; Barr, Ian G.; Laurie, Karen L.

2013-01-01

Surveillance data indicate that most circulating A(H1N1)pdm09 influenza viruses have remained antigenically similar since they emerged in humans in 2009. However, antigenic drift is likely to occur in the future in response to increasing population immunity induced by infection or vaccination. In this study, sequential passaging of A(H1N1)pdm09 virus by contact transmission through two independent series of suboptimally vaccinated ferrets resulted in selection of variant viruses with an amino acid substitution (N156K, H1 numbering without signal peptide; N159K, H3 numbering without signal peptide; N173K, H1 numbering from first methionine) in a known antigenic site of the viral HA. The N156K HA variant replicated and transmitted efficiently between naïve ferrets and outgrew wildtype virus in vivo in ferrets in the presence and absence of immune pressure. In vitro, in a range of cell culture systems, the N156K variant rapidly adapted, acquiring additional mutations in the viral HA that also potentially affected antigenic properties. The N156K escape mutant was antigenically distinct from wildtype virus as shown by binding of HA-specific antibodies. Glycan binding assays demonstrated the N156K escape mutant had altered receptor binding preferences compared to wildtype virus, which was supported by computational modeling predictions. The N156K substitution, and culture adaptations, have been detected in human A(H1N1)pdm09 viruses with N156K preferentially reported in sequences from original clinical samples rather than cultured isolates. This study demonstrates the ability of the A(H1N1)pdm09 virus to undergo rapid antigenic change to evade a low level vaccine response, while remaining fit in a ferret transmission model of immunization and infection. Furthermore, the potential changes in receptor binding properties that accompany antigenic changes highlight the importance of routine characterization of clinical samples in human A(H1N1)pdm09 influenza surveillance

Diversifying Selection Analysis Predicts Antigenic Evolution of 2009 Pandemic H1N1 Influenza A Virus in Humans.

PubMed

Lee, Alexandra J; Das, Suman R; Wang, Wei; Fitzgerald, Theresa; Pickett, Brett E; Aevermann, Brian D; Topham, David J; Falsey, Ann R; Scheuermann, Richard H

2015-05-01

Although a large number of immune epitopes have been identified in the influenza A virus (IAV) hemagglutinin (HA) protein using various experimental systems, it is unclear which are involved in protective immunity to natural infection in humans. We developed a data mining approach analyzing natural H1N1 human isolates to identify HA protein regions that may be targeted by the human immune system and can predict the evolution of IAV. We identified 16 amino acid sites experiencing diversifying selection during the evolution of prepandemic seasonal H1N1 strains and found that 11 sites were located in experimentally determined B-cell/antibody (Ab) epitopes, including three distinct neutralizing Caton epitopes: Sa, Sb, and Ca2 [A. J. Caton, G. G. Brownlee, J. W. Yewdell, and W. Gerhard, Cell 31:417-427, 1982, http://dx.doi.org/10.1016/0092-8674(82)90135-0]. We predicted that these diversified epitope regions would be the targets of mutation as the 2009 H1N1 pandemic (pH1N1) lineage evolves in response to the development of population-level protective immunity in humans. Using a chi-squared goodness-of-fit test, we identified 10 amino acid sites that significantly differed between the pH1N1 isolates and isolates from the recent 2012-2013 and 2013-2014 influenza seasons. Three of these sites were located in the same diversified B-cell/Ab epitope regions as identified in the analysis of prepandemic sequences, including Sa and Sb. As predicted, hemagglutination inhibition (HI) assays using human sera from subjects vaccinated with the initial pH1N1 isolate demonstrated reduced reactivity against 2013-2014 isolates. Taken together, these results suggest that diversifying selection analysis can identify key immune epitopes responsible for protective immunity to influenza virus in humans and thereby predict virus evolution. The WHO estimates that approximately 5 to 10% of adults and 20 to 30% of children in the world are infected by influenza virus each year. While an adaptive

Prevalence of Influenza A(H1N1)pdm09 Virus Resistant to Oseltamivir in Shiraz, Iran, During 2012 - 2013.

PubMed

Khodadad, Nastaran; Moattari, Afagh; Shamsi Shahr Abadi, Mahmoud; Kadivar, Mohammad Rahim; Sarvari, Jamal; Tavakoli, Forough; Pirbonyeh, Neda; Emami, Amir

2015-08-01

Oseltamivir has been used as a drug of choice for the prophylaxis and treatment of human influenza A(H1N1)pdm09 infection across the world. However, the most frequently identified oseltamivir resistant virus, influenza A(H1N1)pdm09, exhibit the H275Y substitution in NA gene. This study aimed to determine the prevalence and phylogenetic relationships of oseltamivir resistance in influenza A(H1N1)pdm09 viruses isolated in Shiraz, Iran. Throat swab samples were collected from 200 patients with influenza-like disease from December 2012 until February 2013. A total of 77 influenza A(H1N1)pdm09 positive strains were identified by real-time polymerase chain reaction (PCR). Oseltamivir resistance was detected using quantal assay and nested-PCR method. The NA gene sequencing was conducted to detect oseltamivir-resistant mutants and establish the phylogeny of the prevalent influenza variants. Our results revealed that A(H1N1)pdm09 viruses present in these samples were susceptible to oseltamivir, and contained 5 site specific mutations (V13G, V106I, V241I, N248D, and N369K) in NA gene. These mutations correlated with increasing expression and enzymatic activity of NA protein in the influenza A(H1N1)pdm09 viruses, which were closely related to a main influenza A(H1N1)pdm09 cluster isolated around the world. A(H1N1)pdm09 viruses, identified in this study in Shiraz, Iran, contained 5 site specific mutations and were susceptible to oseltamivir.

Association of swine influenza H1N1 pandemic virus (SIV-H1N1p) with porcine respiratory disease complex in sows from commercial pig farms in Colombia.

PubMed

Jiménez, Luisa Fernanda Mancipe; Ramírez Nieto, Gloria; Alfonso, Victor Vera; Correa, Jairo Jaime

2014-08-01

Porcine respiratory disease complex (PRDC) is a serious health problem that mainly affects growing and finishing pigs. PRDC is caused by a combination of viral and bacterial agents, such as porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), Mycoplasma hyopneumoniae (Myh), Actinobacillus pleuropneumoniae (APP), Pasteurella multocida and Porcine circovirus 2 (PCV2). To characterize the specific role of swine influenza virus in PRDC presentation in Colombia, 11 farms from three major production regions in Colombia were examined in this study. Nasal swabs, bronchial lavage and lung tissue samples were obtained from animals displaying symptoms compatible with SIV. Isolation of SIV was performed in 9-day embryonated chicken eggs or Madin-Darby Canine Kidney (MDCK) cells. Positive isolates, identified via the hemagglutination inhibition test, were further analyzed using PCR. Overall, 7 of the 11 farms were positive for SIV. Notably, sequencing of the gene encoding the hemagglutinin (HA) protein led to grouping of strains into circulating viruses identified during the human outbreak of 2009, classified as pandemic H1N1-2009. Serum samples from 198 gilts and multiparous sows between 2008 and 2009 were obtained to determine antibody presence of APP, Myh, PCV2 and PRRSV in both SIV-H1N1p-negative and -positive farms, but higher levels were recorded for SIV-H1N1p-positive farms. Odds ratio (OR) and P values revealed statistically significant differences (p<0.05) in PRDC presentation in gilts and multiparous sows of farms positive for SIV-H1N1p. Our findings indicate that positive farms have increased risk of PRDC presentation, in particular, PCV2, APP and Myh.

Virological and serological study of human infection with swine influenza A H1N1 virus in China.

PubMed

Zu, Rongqiang; Dong, Libo; Qi, Xian; Wang, Dayan; Zou, Shumei; Bai, Tian; Li, Ming; Li, Xiaodan; Zhao, Xiang; Xu, Cuiling; Huo, Xiang; Xiang, Nijuan; Yang, Shuai; Li, Zi; Xu, Zhen; Wang, Hua; Shu, Yuelong

2013-11-01

Pigs are considered to be "mixing vessels" for the emergence of influenza viruses with pandemic potential. 2009 Pandemic Influenza H1N1 further proved this hypothesis, and raised the needs for risk assessment of human cases caused by swine influenza virus. A field investigation was conducted after a case identified with infection of European avian-like swine influenza H1N1 virus. The diagnosis was confirmed by real-time PCR, virus isolation, whole genome sequencing and serological assays. Samples from local pigs and close contacts were tested to identify the source of infection and route of transmission. The virus from the index case was similar to viruses circulating in the local pigs. The case's grandfather was asymptomatic with sero-conversion. A total of 42.8% of swine sera were positive for European avian-like swine H1N1. This study highlighted the importance of performing surveillance on swine influenza to monitor new virus emergence in humans. © 2013 Elsevier Inc. All rights reserved.