Antigenic and genetic characterization of swine influenza A (H1N1) viruses isolated from pneumonia patients in The Netherlands

It is generally believed that pigs can serve as an intermediate host for the transmission of avian influenza viruses to humans or as mixing vessels for the generation of avian-human reassortant viruses. Here we describe the antigenic and genetic characterization of two influenza A (H1N1) viruses, which were isolated in The Netherlands from two patients who suffered from pneumonia. Both viruses proved to be antigenically and genetically similar to avian-like swine influenza A (H1N1) viruses which currently circulate in European pigs. It is concluded that European swine H1N1 viruses can infect humans directly, causing serious disease without the need for any reassortment event.     

Genetic characterization of H1N1, H1N2 and H3N2 swine influenza virus in Thailand

Swine have been known to be a suitable host for influenza A virus. In Thailand, phylogenetic analysis on swine influenza virus (SIV) has as yet not been attempted. The present report presents molecular and phylogenetic analysis performed on SIV in Thailand. In this study, 12 SIV isolates from the central and eastern part of Thailand were subtyped and the molecular genetics of hemagglutinin and neuraminidase were elucidated. Three subtypes, H1N1, H1N2 and H3N2, are described. Phylogenetic analysis of the SIV hemagglutinin and neuraminidase genes shows individual clusters with swine, human or avian influenza virus at various global locations. Furthermore, amino acid substitutions were detected either at the receptor binding site or the antigenic sites of the hemagglutinin gene.     

2004-2005年中国A(H1N1)亚型流感病毒抗原性及基因特性研究

目的 阐明2004-2005年中国流行的A（H1N1）亚型流感病毒血凝素抗原性及其基因变异情况.方法 对2004-2005年分离的A（H1N1）亚型毒株先进行单向血凝抑制试验;在此基础上选取不同时间、地点的A（H1N1）亚型流感毒株进行血凝素基因HA1区核苷酸序列测定并推导出其氨基酸序列,然后进行基因进化特性分析.结果 单向血凝抑制实验结果表明,2004年A（H1N1）亚型病毒株对鉴定血清的血凝抑制效价与A/Shanghai/1/1999（H1N1）毒株没有4倍差异;2005年分离的A（H1H1）亚型毒株中有62株（占6.2%）病毒与A/Shanghai/1/1999（H1N1）毒株本身的血凝抑制效价相比有4倍差异.HA1区核苷酸序列和氨基酸序列分析表明,我国2005年分离到的A（H1N1）亚型流感病毒株有以下位点发生变异,54 K＞R、90T＞K、101Y＞H、149R＞K、169V＞A、190D＞N、212R＞K、219K＞R、245W＞R、246Y＞F、258T＞N、318V＞A,其中54、190位氨基酸位于抗原决定簇.结论 我国2005年分离的A（H1N1）亚型流感毒株基因特性和抗原性已开始发生变异.     

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.     

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

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.     

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

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.     

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.     

Isolation and characterization of two H5N1 influenza viruses from swine in Jiangsu Province of China

Pigs are susceptible to infection with both human and avian influenza A viruses and are considered intermediate hosts that facilitate virus reassortment. Although H5N1 virus has spread to a wide range of avian and mammalian species, data about swine H5N1 isolates are scarce. To determine whether Asian H5N1 influenza viruses had been transmitted to pigs, a total of 1,107 nasal swab samples from healthy swine were collected from 2008 to 2009 in Jiangsu province of eastern China. In this survey, two H5N1 viruses A/swine/Jiangsu/1/2008 (JS/08) and A/swine/Jiangsu/2/2009 (JS/09) were isolated and identified. Phylogenetic analysis showed that JS/08 and JS/09 belonged to clade 7 and clade 2.3.4, respectively, and shared over 99.0 % sequence identity with poultry H5N1 isolates of the same clade in China. Receptor specificity analysis also showed that both of the swine H5N1 isolates bound preferentially to avian-type receptors. However, experiments in mammals indicated that JS/09 was moderately pathogenic to mice without prior adaption, whereas JS/08 had limited ability to replicate. Our findings suggest that pigs are naturally infected with avian H5N1 virus and highlight the potential threat to public health due to adaption or reassortment of H5N1 virus in this species.     

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

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.     

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.     

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

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.     

Antigenic and structural characterization of multiple subpopulations of H3N2 influenza virus from an individual

Influenza viruses grown in embryonated chicken eggs frequently possess antigenically distinguishable hemagglutinin (HA) compared to virus from the same source grown in mammalian cell culture. To further investigate the extent of variation among viruses from an individual, viruses were isolated from throat washes collected over a 48-hr period during infection with influenza virus designated A/Mem/6/86 (H3N2). Viruses were isolated from limit dilutions in eggs and mammalian Madin-Darby canine kidney (MDCK) cells and the antigenic, structural, and receptor-binding properties of these viruses were determined. Viruses which could be isolated in MDCK cells were present at 10- to 100-fold higher frequency in the original sample than viruses which could be isolated in eggs. The HA of virus clones isolated in MDCK cells were antigenically and structurally identical. In contrast, viruses from the same source, selected at limit dilution in eggs, could be divided into three distinct subpopulations based on the distinguishable antigenic and structural characteristics of their HA molecules. The three groups of egg-grown viruses could be distinguished from each other, and from MDCK cell-grown viruses, not only by a panel of anti-HA monoclonal antibodies, but also by immune ferret sera raised to H3N2 virus strains of recent years and sera raised to the different egg-grown clones themselves. Of these groups, group 1 and group 2 egg-grown viruses each represented a minor subpopulation of viruses which could be isolated in eggs, while viruses of the third antigenic phenotype were the most frequently isolated in eggs. Amino acid substitutions in the HA of egg-grown viruses occurred in antigenic and receptor-binding sites of the molecule. Group 1 viruses each possessed two amino acid substitutions in their HA molecules at residues 193 and 229 in HA1. Group 3 viruses, which displayed altered receptor specificities compared to MDCK cell-grown viruses and other egg-grown viruses, possessed a single amino acid substitution at residue 145 in HA1. The HA of the group 2 egg-grown viruses appeared structurally identical, yet displayed marked differences in antigenic and receptor-binding properties, compared to viruses isolated in MDCK cells. These results demonstrate that multiple, distinct subpopulations of virus can be isolated from a single patient during an infection with influenza and highlights the potential problems in selecting the most appropriate virus for epidemiological and vaccine purposes since selection could result in the use of viruses that are not representative of those which predominate in a human population.     

Classical swine H1N1 influenza viruses confer cross protection from swine-origin 2009 pandemic H1N1 influenza virus infection in mice and ferrets

The hemagglutinin of the 2009 pandemic H1N1 influenza virus is a derivative of and is antigenically related to classical swine but not to seasonal human H1N1 viruses. We compared the A/California/7/2009 (CA/7/09) virus recommended by the WHO as the reference virus for vaccine development, with two classical swine influenza viruses A/swine/Iowa/31 (sw/IA/31) and A/New Jersey/8/1976 (NJ/76) to establish the extent of immunologic cross-reactivity and cross-protection in animal models. Primary infection with 2009 pandemic or NJ/76 viruses elicited antibodies against the CA/7/09 virus and provided complete protection from challenge with this virus in ferrets; the response in mice was variable and conferred partial protection. Although ferrets infected with sw/IA/31 virus developed low titers of cross-neutralizing antibody, they were protected from pulmonary replication of the CA/7/09 virus. The data suggest that prior exposure to antigenically related H1N1 viruses of swine-origin provide some protective immunity against the 2009 pandemic H1N1 virus.     

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)     

Characterization of a highly pathogenic H5N1 avian influenza virus isolated from ducks in Eastern China in 2011

This study describes the characterization of seven H5N1 avian influenza viruses from domestic ducks in Eastern China in 2011. Phylogenetic analysis showed these viruses were closely related to an H5N1 virus circulating in wild birds in Hong Kong. Some characteristics of these viruses were similar to those of an H5N1 strain that circulated in China and Vietnam (2003-2004). The virulence of three isolates was examined in chickens and mice, and they were found to be highly pathogenic in chickens but showed low pathogenicity in mice. These results suggest that continued H5N1 surveillance in poultry should be used as an early warning system for avian influenza outbreaks.     

Antigenic comparison of swine influenza virus isolates

Summary Cross hemagglutination inhibition (HI) and neuraminidase inhibition (NI) tests demonstrated strong antigenic relationship between A/Swine/Wisconsin/1/73 (SW/73) and A/Swine/Shope/15/31 (SW/31) influenza viruses. An eightyone fold purification of virus was achieved by adsorption and elution followed by differential ultracentrifugation and sedimentation through linear sucrose gradient. Radio-immunoassay using purified¹²⁵I labeled viral antigens revealed antigenic variation between the two virus isolates. Neuraminidase of both viruses had pH optima between 6.5 and 7.0, and SW/31 enzyme was relatively more heat stable than SW/73.With 4 FiguresContribution No. 183, Department of Infectious Diseases, and Contribution No. 1239, Division of Biology, Kansas Agricultural Experiment Station, Manhattan, Kansas 66506.     

Antigenic structure of hemagglutinins of influenza H1N1 (Hsw1N1) virus isolated from humans and ducks

The method of specific adsorption followed by the use of antisera in HI test and competitive enzyme immunoassay was used to study the antigenic composition of hemagglutinins (HA) Hsw1 in influenza viruses isolated in 1982 from humans in Bulgaria and in 1976 in Canada from ducks as well as their antigenic relationships with HA of Hsw1 variant isolated from swine and man. Hemagglutinins of Hsw1 strains isolated from man in Bulgaria and Alma-Ata were found to be similar to HA of A/New Jersey/8/76 virus in two determinants and with hemagglutinin of the classic virus of swine in three determinants. The HA of A/duck/Alberta/35/76 virus was similar in three determinants to HA of A/New Jersey/8/76 virus and in two determinants with other Hsw1 variants. The similarities and differences in antigenic determinants of HA in Hsw1 viruses isolated from man and animals attest to their common origin and different modes of variability.