Identification of four genotypes of H3N2 swine influenza virus in pigs from southern China

In 2011, four H3N2 swine influenza viruses (SIVs) were isolated from nasal swabs of four pigs (800 nasal swabs were collected from pigs showing influenza-like symptoms) in Guangdong province, China. Four different genotypes of H3N2 appeared among pigs in southern China, including wholly human-like H3N2 viruses, intermediate (1975) double-reassortant human H3N2 viruses (resulting from reassortment between an early human lineage and a recent human lineage), recent double-reassortant human H3N2 viruses, and avian-like H3N2 viruses. Because pigs can support the reassortment of human and avian influenza viruses, our surveillance should be enhanced as a part of an overall pandemic preparedness plan.     

Comparison of the pathogenesis of two genetically different H3N2 influenza A viruses in pigs

In 1997 and 1998, H3N2 influenza A viruses emerged among pigs in North America. Genetic analyses of the H3N2 isolates demonstrated that they had distinctly different genotypes. The most commonly isolated viruses in the United States have a triple-reassortant genotype, with the hemagglutinin, neuraminidase, and PB1 polymerase genes being of human influenza virus origin, the nucleoprotein, matrix, and nonstructural genes being of classical swine influenza virus origin, and the PA and PB2 polymerase genes being of avian influenza virus origin. In contrast, a wholly human H3N2 virus was isolated from a single baby pig in Ontario, Canada, in 1997, but it did not spread within the swine population. Genetic differences between this wholly human virus and the triple-reassortant viruses may affect their replication efficiencies in pigs. In the present study we compared the pathogenicities and replication kinetics of the wholly human virus and a triple-reassortant virus in 7-week-old pigs that were infected intranasally with 2 x 10(3) to 2 x 10(6) 50% tissue culture infective doses of virus. Our results demonstrate that the wholly human virus replicated to significantly lower titers and that the onset of virus shedding was delayed compared to the replication titers and the time of onset of virus shedding in triple-reassortant viruses. In addition, infection with the triple-reassortant virus was associated with moderate to severe gross pathological and histological pulmonary lesions, while infection with the wholly human virus induced only mild pulmonary changes.     

Phylogenetic analysis of H1N2 isolates of influenza A virus from pigs in the United States

Twenty-four H1N2 influenza A viruses were newly isolated from pigs in the United States. These isolates originated from 19 farms in 9 different swine producing states between 1999 and 2001. All farms had clinical histories of respiratory problem and/or abortion. The viral isolates were characterized genetically to determine the origin of all eight gene segments. The results showed that all H1N2 isolates were reassortants of classical swine H1N1 and triple reassortant H3N2 viruses. The neuraminidase (NA) and PB1 genes of the H1N2 isolates were of human origin, while the hemagglutinin (HA), nucleoprotein (NP), matrix (M), non-structural (NS), PA and PB2 polymerase genes were of avian or swine origin. Fifteen of the 24 H1N2 isolates were shown to have a close phylogenic relationship and high amino acid homology with the first US isolate of H1N2 (A/SW/IN/9K035/99). The remaining nine isolates had a close phylogenic relationship with classical swine influenza H1N1 in the HA gene. All other genes including NA, M, NP, NS, PA, PB1 and PB2 showed a close phylogenic relationship with the H1N2 (A/SW/IN/9K035/99) strain and triple reassortant H3N2 viruses. However, PB1 genes of two isolates (A/SW/KS/13481-S/00, A/SW/KS/13481-T/00) were originated from avian influenza A virus lineage. These results suggest that although there are some variations in the HA genes, the H1N2 viruses prevalent in the US swine population are of a similar genetic lineage.     

Emergence of a new swine H3N2 and pandemic (H1N1) 2009 influenza A virus reassortant in two Canadian animal populations, mink and swine

A swine H3N2 (swH3N2) and pandemic (H1N1) 2009 (pH1N1) influenza A virus reassortant (swH3N2/pH1N1) was detected in Canadian swine at the end of 2010. Simultaneously, a similar virus was also detected in Canadian mink based on partial viral genome sequencing. The origin of the new swH3N2/pH1N1 viral genes was related to the North American swH3N2 triple-reassortant cluster IV (for hemagglutinin [HA] and neuraminidase [NA] genes) and to pH1N1 for all the other genes (M, NP, NS, PB1, PB2, and PA). Data indicate that the swH3N2/pH1N1 virus can be found in several pigs that are housed at different locations.     

Isolation and phylogenetic analysis of H1N1 swine influenza virus isolated in Korea

A swine influenza H1N1 virus was isolated from a pig during a severe outbreak of respiratory disease in Korea. All genes of the H1N1 isolate, including hemagglutinin (HA), neuraminidase (NA), matrix (M), nucleoprotein (NP), non-structural (NS), PA, PB1 and PB2, were of swine origin. Also, all these genes showed a close phylogenic relationship with those of H1N1 viruses previously isolated from pigs in the United States. These results suggest that North American swine influenza virus has actually been transmitted to pigs in Korea.     

Restricted infectivity of a human-Lineage H3N2 influenza A virus in pigs is hemagglutinin and neuraminidase gene dependent

Influenza A viruses cause pandemics at sporadic intervals. Pandemic viruses can potentially be introduced into the human population through in toto transfer of an avian influenza virus or through reassortment between avian and human strains. Pigs are believed to play a central role in the creation of pandemic viruses through reassortment because of their susceptibility to infection with both avian and human influenza viruses. However, we recently found that a human-lineage H3N2 influenza virus was highly restricted in its ability to infect pigs after intranasal inoculation. We hypothesized that this restricted infectivity phenotype was controlled by the hemagglutinin (HA) and neuraminidase (NA). To test this, we infected pigs with reverse genetics-created HA plus NA reassortant viruses. Specifically, introduction of the HA and NA genes of a contemporary H3N2 swine virus into the genetic background of the wholly human virus resulted in a significant increase in virus shedding and pathogenicity. These data indicate that the HA/NA can play important roles in controlling human influenza virus infectivity in pigs. The results further support the premise that a barrier exists to human influenza virus infection in pigs, which may limit the role of pigs in pandemic virus creation through reassortment of human and avian influenza viruses.     

Genetic characterization of H3N2 influenza viruses isolated from pigs in North America, 1977-1999: evidence for wholly human and reassortant virus genotypes

Since 1998, H3N2 viruses have caused epizootics of respiratory disease in pigs throughout the major swine production regions of the U.S. These outbreaks are remarkable because swine influenza in North America had previously been caused almost exclusively by H1N1 viruses. We sequenced the full-length protein coding regions of all eight RNA segments from four H3N2 viruses that we isolated from pigs in the Midwestern U.S. between March 1998 and March 1999, as well as from H3N2 viruses recovered from a piglet in Canada in January 1997 and from a pig in Colorado in 1977. Phylogenetic analyses demonstrated that the 1977 Colorado and 1997 Ontario isolates are wholly human influenza viruses. However, the viruses isolated since 1998 from pigs in the Midwestern U.S. are reassortant viruses containing hemagglutinin, neuraminidase and PB1 polymerase genes from human influenza viruses, matrix, non-structural and nucleoprotein genes from classical swine viruses, and PA and PB2 polymerase genes from avian viruses. The HA proteins of the Midwestern reassortant swine viruses can be differentiated from those of the 1995 lineage of human H3 viruses by 12 amino acid mutations in HA1. In contrast, the Sw/ONT/97 virus, which did not spread from pig-to-pig, lacks 11 of these changes.     

Pathogenic and antigenic properties of phylogenetically distinct reassortant H3N2 swine influenza viruses cocirculating in the United States

Swine influenza is an acute respiratory disease caused by type A influenza viruses. Before 1998, swine influenza virus isolates in the United States were mainly of the classical H1N1 lineage. Since then, phylogenetically distinct reassortant H3N2 viruses have been identified as respiratory pathogens in pigs on U.S. farms. The H3N2 viruses presently circulating in the U.S. swine population are triple reassortants containing avian-like (PA and PB2), swine-like (M, NP, and NS), and human-like (HA, NA, and PB1) gene segments. Recent sequence data show that the triple reassortants have acquired at least three distinct H3 molecules from human influenza viruses and thus form three distinct phylogenetic clusters (I to III). In this study we analyzed the antigenic and pathogenic properties of viruses belonging to each of these clusters. Hemagglutination inhibition and neutralization assays that used hyperimmune sera obtained from caesarian-derived, colostrum-deprived pigs revealed that H3N2 cluster I and cluster III viruses share common epitopes, whereas a cluster II virus showed only limited cross-reactivity. H3N2 viruses from each of the three clusters were able to induce clinical signs of disease and associated lesions upon intratracheal inoculation into seronegative pigs. There were, however, differences in the severity of lesions between individual strains even within one antigenic cluster. A correlation between the severity of disease and pig age was observed. These data highlight the increased diversity of swine influenza viruses in the United States and would indicate that surveillance should be intensified to determine the most suitable vaccine components.     

猪型(H1N1)流感病毒血凝素和神经氨酸酶基因来源的研究

目的 研究2002年我国内地从猪群中分离的猪型(H1N1)毒株HA和NA基因来源。及其使猪致病的原因。方法 用PCR扩增目的基因，用P^GEM-T Easy Vector，4℃过夜连接，重组质粒转入DH-10B细菌，筛选阳性菌落，酶切鉴定，送六合通公司自动测序，并作进化树分析。结果 3株猪型(H1N1)病毒的HA和NA基因属猪型(H1N1)流感病毒，而不同于其他禽或人的H1N1亚型流感病毒。2002年猪型毒株由1991年猪型毒株演变而来。近来我国内地猪群中猪型毒株活动增强，其对猪能致病是由于病毒粒HA和NA蛋白抗原性发生变异所造成。结论 3株猪型病毒的HA和NA基因来源于猪型(H1N1)毒株。近来猪型毒株对猪具有致病性和活动增强是由于其HA和NA蛋白分子上氨基酸序列发生替换所造成。     

Phylogenetic analysis of an H1N2 influenza A virus isolated from a pig in Korea

Summary. An influenza H1N2 virus was isolated from a pig during an severe outbreak of respiratory disease in a Korean herd. The neuraminidase (NA) and PB1 genes of the H1N2 isolate were of human origin, while the hemagglutinin (HA), matrix (M), nucleoprotein (NP), and non-structural (NS) genes were of swine origin and PA and PB2 gene were of avain origin. Phylogenetic results indicate that the Korean H1N2 isolate was closely related to H1N2 viruses isolated recently from pigs in the United States.     

Further evidence for infection of pigs with human-like H1N1 influenza viruses in China

Classical swine and avian-like H1N1 influenza viruses were reported widely in swine population worldwide, but human-like H1N1 swine viruses were reported occasionally. In 2006, a human-like H1N1 swine virus (A/swine/Guangdong/96/06) was isolated from pigs in Guangdong province, which was reported in China for the first time. To get further evidence for infection of pigs with human-like H1N1 influenza viruses, we analyzed eight gene segments of three human-like swine H1N1 viruses (A/swine/Guangdong/96/06, A/swine/Tianjin/01/04 and A/swine/Henan/01/06) isolated in China. All the eight genes of the three viruses are highly homologous to recent (about 2000) and early (1980s) human H1N1 influenza viruses, respectively. Phylogenetic analyses revealed that A/Swine/Guangdong/96/06 was directly derived from about 2000 human H1N1 influenza viruses, while A/swine/Tianjin/01/04 and A/swine/Henan/01/06 seemed to be descendants of human H1N1 viruses circulating in 1980s. Seroprevalence of our isolate (A/swine/Guangdong/96/06) confirmed the presence of human-like H1N1 virus in pigs in China. Existence of these influenza viruses, especially older viruses (A/swine/Tianjin/01/04 and A/swine/Henan/01/06), indicates that human-like H1N1 influenza viruses may remain invariant for long periods in pigs and provides the evidence that pigs serve as reservoirs of older influenza viruses for human pandemics.     

Identification of human H1N2 and human-swine reassortant H1N2 and H1N1 influenza A viruses among pigs in Ontario, Canada (2003 to 2005)

Since 2003, three novel genotypes of H1 influenza viruses have been recovered from Canadian pigs, including a wholly human H1N2 virus and human-swine reassortants. These isolates demonstrate that human-lineage H1N2 viruses are infectious for pigs and that viruses with a human PB1/swine PA/swine PB2 polymerase complex can replicate in pigs.     

Parotitis in a Child Infected with Triple-Reassortant Influenza A Virus in Canada in 2007

Swine H3N2 influenza virus designated A/Ontario/1252/2007 was isolated from a child with parotitis. Diagnosis was confirmed by viral isolation and serological assays. A/Ontario/1252/2007 was related to H3N2 triple reassortants that emerged in swine in the United States in 1998. Three of five tested household members were also seropositive for A/Ontario/1252/2007.Influenza A viruses infect a broad range of species, with avian and swine strains presenting the most potential for interspecies transmission. Since 1998, H3N2 triple reassortants with genes derived from human (HA, NA, and PB1), swine (M, NS, and NP), and avian (PA and PB2) viruses have caused outbreaks of respiratory disease in pigs throughout the United States, and in 2005, such infections spread across Canada (6, 9). Zoonotic infections of humans with H3N2 swine influenza viruses (SIV) have been reported (5). Here, we report a case of acute parotitis due to the transmission of an H3N2 triple reassortant from pigs to a child. This is the third human H3N2 SIV infection identified in Canada since 2005. These results indicate that SIV infection in humans may be less rare than previously thought and that atypical presentation of the disease may be occurring.     

Swine infection with H9N2 influenza viruses in China in 2004

In 2004, H9N2 influenza A viruses were isolated from pigs with respiratory syndrome in commercial swine farms in Henan province, China. Antigenic and genetic characterization were performed for seven swine H9N2 influenza viruses. The hemagglutinin antigenicity of swine H9N2 viruses was similar to those of avian H9N2 viruses of A/duck/Hong Kong/Y280/1997 (Dk/HK/Y280/97)-like sublineage prevalent in China. It is noteworthy that the neuraminidase of these isolates had no deletions in the stalk, which was seldom observed in those viruses of Dk/HK/Y280/97-like sublineage. Genetic analysis revealed that all seven isolates had an -R-S-S-R- motif at the HA cleavage site, which was the same as those of Dk/HK/Y280/97-like viruses established in avian population in China. Phylogenetic analyses showed that the seven swine H9N2 viruses were completely derived from avian influenza viruses of Dk/HK/Y280/97-like sublineage. The present results indicated that avian-to-pig interspecies transmission of H9N2 viruses continued to exist in China through 2004; therefore, surveillance of swine influenza should be given a high priority. Yan-Long Cong, Chun-Feng Wang and Chun-Mei Yan have contributed equally to this work. (i) All the authors have agreed to its submission and are responsible for its contents and (ii) all the authors have agreed that Yanlong Cong may act on their behalf regarding any subsequent processing of the paper.     

Genetic analysis of H9N2 avian influenza viruses isolated from India

H9N2 avian influenza viruses are endemic in domestic poultry in Asia and are grouped into three major sublineages represented by their prototype strains A/Duck/Hong Kong/Y280/97 (Y280-like), A/Quail/Hong Kong/G1/97 (G1-like) and A/Chicken/Korea/38349-p96323/96 (Korean-like). To understand the genetic relationship of Indian viruses, we determined the partial nucleotide sequence of five H9N2 avian influenza viruses isolated from chicken in India during 2003-2004 and compared them with H9N2 sequences available in GenBank. Deduced amino acid sequence analysis revealed that four isolates shared an R-S-S-R/G motif at the cleavage site of HA, representing low pathogenicity in chickens, while one virus harbors an R-S-N-R/G motif at the same position. All the viruses maintained the human-like motif 226Lysine (H3 numbering) at the HA receptor binding site. Phylogenetic analysis showed that 50% of the genes (HA, NA, NP and M) were similar to G1-like viruses, whereas the remaining genes of the Indian isolates formed a separate, not yet defined, sublineage in the Eurasian lineage. Our finding provides evidence of a novel reassortant H9N2 genotype of G1-like viruses circulating in India.     

Characterization of a new avian-like influenza A virus from horses in China.

In March 1989 a severe outbreak of respiratory disease occurred in horses in the Jilin and Heilongjiang provinces of Northeast China that caused up to 20% mortality in some herds. An influenza virus of the H3N8 subtype was isolated from the infected animals and was antigenically and molecularly distinguishable from the equine 2 (H3N8) viruses currently circulating in the world. The reference strain A/Equine/Jilin/1/89 (H3N8) was most closely related to avian H3N8 influenza viruses. Sequence comparisons of the entire hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix (M), and NS genes along with partial sequences of the three polymerase (PB1, PB2, PA) genes suggest that six of the eight gene segments (PA, HA, NP, NA, M, NS) are closely related to avian influenza viruses. Since direct sequence analysis can only provide a crude measure of relationship, phylogenetic analysis was done on the sequence information. Phylogenetic analyses of the entire HA, NP, M, and NS genes and of partial sequences of PB1, PB2, and PA indicated that these genes are of recent avian origin. The NP gene segment is closely related to the gene segment found in the newly described H14 subtype isolated from ducks in the USSR. The A/Equine/Jilin/1/89 (H3N8) influenza virus failed to replicate in ducks, but did replicate and cause disease in mice on initial inoculation and on subsequent passaging caused 100% mortality. In ferrets, the virus caused severe influenza symptoms. A second outbreak of influenza in horses in Northeast China occurred in April 1990 in the Heilongjiang province with 48% morbidity and no mortality. The viruses isolated from this outbreak were antigenically indistinguishable from those in the 1989 outbreak and it is probable that the reduced mortality was due to the immune status of of the horses in the region. No influenza was detected in horses in Northern China in the spring, summer, or fall of 1991 and no influenza has been detected in horses in adjacent areas. Our analysis suggests that this new equine influenza virus in horses in Northeast China is the latest influenza virus in mammals to emerge from the avian gene pool in nature and that it may have spread to horses without reassortment. The appearance of this new equine virus in China emphasizes the potential for whole avian influenza viruses to successfully enter mammalian hosts and serves as a model and a warning for the appearance of new pandemic influenza viruses in humans.(ABSTRACT TRUNCATED AT 250 WORDS)     

我国猪群中H9N2亚型毒株HA和NA基因特性的研究

目的 了解我国内地从猪中分离到H9N2亚型毒株HA和NA基因来源及它们使猪致病的原因。方法 用PCR扩增目的基因，与P^GEM-T Easy Vector4℃过夜连接，重组质粒转化DH-10β细菌，筛选阳性菌落，酶切鉴定，测序。然后，进行进化树分析。结果 两株猪H9N2毒株HA蛋白分子上第226位上氨基酸为L，这与从人和猪所分离出的H9N2毒株相同，其连接肽属对禽致病的毒株，但它们的序列为R-L-S-R，而不是R-S-S-R；其NA蛋白茎区第62～64位存在掉失，这与A／Shaoguarn/408／98，A／Swine／Hong Kong／9／98及A／Duck／Hong Kong／y280／97(H9N2)毒株相同；HA与NA基因进化树分析表明，两株猪H9N2毒株的HA基因接近于A／Chicken／Hong Kong／G23／97和A／Chicken／Hong Kong／G9／97．而NA基因接近于A／Shaoguan／408／98毒株。结论 两株猪H9N2亚型毒株的HA和NA基因可能性最大来自禽H9N2毒株。由于其HA蛋白分子上连接肽氨基酸序列发生替换，可能造成了它们对猪具有致病性。禽H9N2毒株NA蛋白茎区氨基酸掉失，造成了它们能直接感染猪。     

Characterization of An Avian Influenza Virus of Subtype H7N2 Isolated from Chickens in Northern China

An H7N2 avian influenza virus was isolated from chickens during routine surveillance in northern China in 2002. To understand the origin of this virus, we completely sequenced its genome. The PB1, PA, HA, and M genes of this virus were highly homologous with those of the wild bird virus A/Africa starling/Eng-Q/983/79 (H7N1). The NP and NS genes were closely related to those of two other wild bird viruses isolated 30 years ago. The closest relatives of the PB2 and NA genes of the virus were those of the A/swine/Germany/2/81 (H1NI) and A/Leningrad/134/57 (H2N2), respectively. Animal inoculation tests showed that the virus cannot replicate efficiently in chickens. However, after intranasal inoculation, the virus induced 20% weight loss and replicated well in the lungs of mice. The virus was also recovered from the hearts and brains of the mice. These results suggest that the influenza virus isolated in chickens in northern China in 2002 originated in wild birds and may pose a threat for both avian species and mammalian hosts.     

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

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.     

Human influenza a viral genes responsible for the restriction of its replication in duck intestine

Although influenza A viruses are occasionally transmitted from one animal species to another, their host range tends to be restricted. Currently circulating human influenza A viruses are thought to have originated from avian viruses, yet none of these strains replicate in duck intestine, a major site of avian virus replication. Although the hemagglutinin (HA) and neuraminidase (NA) genes are known to restrict human virus replication in ducks, the contribution of the other viral genes remains unknown. To determine the genetic basis for host range restriction of the replication of human influenza A virus in duck intestine, we first established a reverse genetics system for generating A/Memphis/8/88 (H3N2) (Mem/88) and A/mallard/New York/6750/78 (H2N2) (Mal/NY) viruses from cloned cDNAs. Using this system, we then attempted to generate reassortant viruses with various combinations of candidate genes. We were able to generate single-gene reassortants, which possessed PB2, NP, M, or NS from Mem/88, with the remainder from Mal/NY. Despite unsuccessful production of other single-gene reassortants from Mem/88, we did generate reassortant viruses comprised of both the HA and the NA, all three polymerase genes (PB2, PB1, and PA), or all polymerase genes and the NP gene from Mem/88, with the rest derived from Mal/NY. Among these reassortants, only those possessing the M or NS gene from Mem/88 and the remainder from Mal/NY replicated in duck intestine. These results indicate incompatibility between the genes of avian and human influenza A viruses and indicate that all genes other than the M and NS restrict replication of human influenza A virus in duck intestine.