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.     

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.     

Genetic evolution of swine influenza A (H3N2) viruses in China from 1970 to 2006

Pigs are susceptible to both human and avian influenza viruses and have been proposed to be intermediate hosts, or mixing vessels, for the generation of pandemic influenza viruses through reassortment or adaptation to the mammalian host. In this study, we summarize and report for the first time the coexistence of wholly human-like H3N2 viruses, double-reassortant H3N2 viruses, and triple-reassortant H3N2 viruses in pigs in China by analyzing the eight genes of swine influenza A (H3N2) viruses found in China from 1970 to 2006. In 1970, the first wholly human-like H3N2 (Hong Kong/68-like) viruses were isolated from pigs in Taiwan, and then in the next years Victoria/75-like, Sydney/97-like, New York/99-like, and Moscow/99-like swine H3N2 viruses were regularly isolated in China. In the 1980s, two triple-reassortant viruses were isolated from pigs. Recently, the double-reassortant viruses containing genes from the human (HA and NA) and avian (PB2, PB1, PA, NP, M, and NS) lineages and the triple-reassortant viruses containing genes from the human (HA and NA), classical swine (NP), and avian (PB2, PB1, PA, M, and NS) lineages emerged in pigs in China. The coexistence of wholly human-like and reassortant viruses provides further evidence that pigs serve as intermediate hosts, or mixing vessels, and emphasizes the importance of reinforcing swine influenza virus surveillance in China.     

Identification and characterization of a highly virulent triple reassortant H1N1 swine influenza virus in the United States

A highly virulent H1N1 influenza A virus, A/Swine/Kansas/77778/2007 (KS07), which caused approximately 10% mortality in finishing pigs, was isolated from herds in the Midwestern United States. Molecular and phylogenic analysis revealed this swine isolate was a triple reassortant virus, similar to an H1N1 virus that infected humans and pigs at an Ohio county fair in August 2007. A pig challenge model was developed to evaluate the pathogenicity and transmission capacity of the KS07 virus. The results confirmed that the KS07 virus is highly virulent in pigs and easily transmitted to sentinel animals. The KS07 virus failed to cross-react with a panel of H1-specific swine sera. Interestingly, the KS07 virus shed for a prolonged period up to 7 days in infected pigs, indicating that this virus can spread efficiently between animals. The highly virulent H1N1 swine influenza virus is further evidence of reassortment among avian, human and swine influenza viruses and justifies the need for continued surveillance of influenza viruses in swine.     

Amplification of four genes of influenza A viruses using a degenerate primer set in a one step RT-PCR method

We designed a degenerate primer set that yielded full-length amplification of hemagglutinin (HA), neuraminidase (NA), matrix (M), and non-structural protein (NSP) genes of influenza A viruses in a single reaction mixture. These four genes were amplified from 15 HA (1–15) and 9 NA (1–9) subtypes of influenza A viruses of avian (n = 16) origin. In addition, 272 field isolates of avian origin were tested by this method. Full-length amplification of HA, NA, M, and NSP genes was obtained in 242 (88.9%), 254 (93.4%), 268 (98.5%), and 268 (98.5%) isolates, respectively. No gene was amplified in four isolates. Of these four isolates, two were subtyped as H4N6, one as H7N7, and one as H10N7. Amplification was successful for all 4 genes of H1N1, H2N3, and H3N2 isolates of swine influenza. Also, all four genes were amplified in one equine influenza (H3N8) isolate and seven isolates of human origin (H1N1 and H3N2). This appears to be the first study using degenerate primer set for full-length amplification of four genes of influenza A viruses in a single reaction. Further studies are needed to determine if this primer set can be used for subtyping of influenza virus isolates.     

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.     

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.     

Genetic characterization of novel reassortant H1N2 influenza A viruses isolated from pigs in southeastern China

Summary. In December 2004, three influenza H1N2 viruses were isolated from lung samples of pigs that had died from respiratory disease on a farm in southeastern China. To determine the genetic characterization and probable origin, one of the three isolates, A/Swine/Zhejiang/1/2004 (Sw/ZJ/1/2004), was genetically analyzed. Sw/ZJ/1/2004 was a reassortant with an NA gene most closely related to the corresponding gene from a human-like H3N2 virus circulating in 1995. The remaining seven genes were most closely related to those from the classical swine H1N1 virus. Sw/ZJ/1/2004 appeared to be a novel reassortant H1N2 virus that was genetically distinguishable from other H1N2 viruses found in pigs worldwide. The isolation of Sw/ZJ/1/2004 provided further evidence for pigs serving as a “mixing vessel” for the generation of new reassortant genotypes of influenza viruses and emphasizes the importance of reinforcing influenza virus surveillance in pigs in China.     

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.     

Genetic characterization of Thai swine influenza viruses after the introduction of pandemic H1N1 2009

Pandemic H1N1 2009 (pH1N1), influenza virus containing triple reassortant internal genes (TRIG) from avian, human, and swine influenza viruses emerged in 2009 as a highly infectious virus that was able to be transmitted from humans to pigs. During June 2010–May 2012, influenza virus surveillance was conducted in Thai pig population. Twenty-three samples (1.75 %) were successfully isolated from total of 1,335 samples. Interestingly, pH1N1 (7 isolates, 30.34 %), reassortant pH1N1 (rH1N1) (1 isolate, 4.35 %), Thai endemic H1N1 (enH1N1) (3 isolates, 13.04 %), reassortant H3N2 with pH1N1 internal genes (rH3N2) (9 isolates, 39.13 %), and reassortant H1N2 with pH1N1 internal genes (rH1N2) (3 isolates, 13.04 %) were found. It should be noted that rH1N1, rH1N2, and rH3N2 viruses contained the internal genes of pH1N1 virus having a TRIG cassette descendant from the North American swine lineage. Although all isolates in this study were obtained from mild clinically sick pigs, the viruses were still highly infective and possibly may play an important role in human–animal interfacing transmission. In addition, the TRIG cassette may have an influence on antigenic shift resulting in emergence of novel viruses, as seen in this study. Continuing surveillance of influenza A natural hosts, particularly in pigs is necessary.     

Genetic characterization of H1N1 swine influenza A viruses isolated in eastern China

Three influenza H1N1 viruses were isolated in 2005 from pigs with respiratory disease on a farm in eastern China. The three isolates were characterized to determine their probable origin. Each of the eight genes of the isolates was most closely related to the corresponding gene from the classical swine H1N1 virus. Also, phylogenetic analysis further confirmed that each of the eight genes of the isolates was closely related to the classical swine H1N1 viruses, especially those isolated in China. The HA1 proteins of the three isolates were identical to that of A/Swine/Guangdong/1/01, a virus isolated in 2001 in China, even though three nucleotide differences were observed. These results further support the concept that swine can serve as a reservoir of genetically stable influenza viruses.     

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。     

Differences in transmissibility and pathogenicity of reassortants between H9N2 and 2009 pandemic H1N1 influenza A viruses from humans and swine

Both H9N2 subtype avian influenza and 2009 pandemic H1N1 viruses (pH1N1) can infect humans and pigs, which provides the opportunity for virus reassortment, leading to the genesis of new strains with potential pandemic risk. In this study, we generated six reassortant H9 viruses in the background of three pH1N1 strains from different hosts (A/California/04/2009 [CA04], A/Swine/Jiangsu/48/2010 [JS48] and A/Swine/Jiangsu/285/2010 [JS285]) by replacing either the HA (H9N1-pH1N1) or both the HA and NA genes (H9N2-pH1N1) from an h9.4.2.5-lineage H9N2 subtype influenza virus, A/Swine/Taizhou/5/08 (TZ5). The reassortant H9 viruses replicated to higher titers in vitro and in vivo and gained both efficient transmissibility in guinea pigs and increased pathogenicity in mice compared with the parental H9N2 virus. In addition, differences in transmissibility and pathogenicity were observed among these reassortant H9 viruses. The H9N2-pH1N1viruses were transmitted more efficiently than the corresponding H9N1-pH1N1 viruses but showed significantly decreased pathogenicity. One of the reassortant H9 viruses that were generated, H9N-JS48, showed the highest virulence in mice and acquired respiratory droplet transmissibility between guinea pigs. These results indicate that coinfection of swine with H9N2 and pH1N1viruses may pose a threat for humans if reassortment occurs, emphasizing the importance of surveillance of these viruses in their natural hosts.     

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.     

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 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.     

Origin of the 2009 Mexico influenza virus: a comparative phylogenetic analysis of the principal external antigens and matrix protein

Triple-reassortant swine influenza A (H1) viruses, containing genes from avian, human, and swine influenza viruses, emerged and became an outbreak among humans worldwide. Over a 1,000 cases were identified within the first month, chiefly in Mexico and the United States. Here, the phylogenetic analysis of haemagglutin (HA), neuraminidase (NA), and matrix protein (MP) was carried out. The analysis showed that the H1 of this reassortant originated from American pigs, while NA and MP were more likely from European pigs. All of the 2009 isolates appear homogeneous and cluster together, although they are distinct from classical human A (H1N1) viruses. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

两株京科猪H1N1亚型流感病毒内部基因来源的研究

目的通过内部基因研究了解两株猪(H1N1)亚型流感病毒内部基因是否含有禽流感病毒基因节段及是否与猪群中H9N2亚型毒株发生了基因重配。方法病毒在鸡胚中传代,从收获的尿囊液中提取RNA,通过逆转录合成cDNA,cDNA用PCR扩增。PCR产物用纯化试剂盒纯化,接着进行核苷酸序列测定,然后用MegAlign(Version1.03)和Editseq(Version3.69)软件进行基因进化树分析。结果两株京科猪H1N1病毒内部基因除PB2基因节段有所不同外,其余5个基因节段均相同,但与猪H1N1流感病毒内部基因相近,然而,与古典型猪H1N1毒株有差异。结论两株京科猪H1N1病毒不是基因重配株,它们的内部基因均属猪H1N1流感病毒基因系。     

Neuraminidase inhibitor susceptibility of swine influenza A viruses isolated in Germany between 1981 and 2008

European swine influenza A viruses donated the matrix protein 2 as well as the neuraminidase (NA) gene to pandemic influenza A (H1N1) viruses that emerged in 2009. As a result, the latter became amantadine resistant and neuraminidase inhibitor (NAI) susceptible. These recent developments reflecting the close connection between influenza A virus infection chains in humans and pigs urge an antiviral surveillance within swine influenza A viruses. Here, NAI susceptibility of 204 serologically typed swine influenza A viruses of subtypes H1N1, H1N2, and H3N2 circulating in Germany between 1981 and 2008 was analyzed in chemiluminescence-based NA inhibition assays. Mean 50% inhibitory concentrations of oseltamivir and zanamivir indicate a good drug susceptibility of tested viruses. As found for human isolates, the oseltamivir and zanamivir susceptibility was subtype-specific. So, swine influenza A (H1N1) viruses were just as susceptible to oseltamivir as to zanamivir. In contrast, swine H1N2 and H3N2 influenza A viruses were more sensitive to oseltamivir than to zanamivir. Furthermore, reduction in plaque size and virus spread by both drugs was tested with selected H1N1 and H1N2 isolates in MDCK cells expressing similar amounts of α2.3- and α2.6-linked sialic acid receptors. Data obtained in cell culture-based assays for H1N1 isolates correlated with that from enzyme inhibition assays. But, H1N2 isolates that are additionally glycosylated at Asn158 and Asn163 near the receptor-binding site of hemagglutinin (HA) were resistant to both NAI in MDCK cells. Possibly, these additional HA glycosylations cause a misbalance between HA and NA function that hampers or abolishes NAI activity in cells.     

Identification of amino acids in the HA of H3 influenza viruses that determine infectivity levels in primary swine respiratory epithelial cells

In the late 1990s, triple reassortant H3N2 influenza A viruses emerged and spread widely within the swine population of the United States. We have shown previously that an isolate representative of this lineage of viruses, A/Swine/Minnesota/593/99 (Sw/MN), has higher infectivity and accelerated replication kinetics in pigs, compared to a human-lineage H3N2 virus isolated from a pig during the same time period, A/Swine/Ontario/00130/97 (Sw/ONT [Landolt, G.A., Karasin, A.I., Phillips, L., Olsen, C.W., 2003. Comparison of the pathogenesis of two genetically different H3N2 influenza A viruses in pigs. J. Clin. Microbiol. 41, 1936-1941]). Additional in vivo experiments using reverse genetics-generated reassortant viruses demonstrated that these phenotypes are dependent upon the HA and/or NA genes (Landolt, G.A., Karasin, A.I., Schutten, M.M., Olsen, C.W., 2006. Restricted infectivity of a human-lineage H3N2 influenza A virus in pigs is hemagglutinin and neuraminidase gene dependent. J. Clin. Microbiol. 44, 297-301). To further study the infectivity of influenza viruses for pigs, we developed a primary swine respiratory epithelial cell (SREC) culture model. In SRECs, Sw/MN infects a significantly higher number of cells compared to Sw/ONT. Using reverse genetics-generated Sw/MN x Sw/ONT reassortant viruses we demonstrate that the infectivity phenotypes of these viruses in SRECs are strongly dependent upon the HA gene. Using chimeras and point directed mutations within the HA genes, we have identified amino acids that, either alone or in combination with other amino acids, impact infectivity. In particular, amino acid 138 is the dominant factor in determining infectivity levels in SRECs.