Genetic diversity of H9N2 influenza viruses from pigs in China: a potential threat to human health?

Pandemic strains of influenza A virus might arise by genetic reassortment between viruses from different hosts. 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 10 (A-J) genotypes in pigs in China by analyzing the eight genes of 28 swine H9N2 viruses isolated in China from 1998 to 2007. Swine H9N2 viruses in genotype A and B were completely derived from Y280-like and Shanghai/F/98-like viruses, respectively, which indicated avian-to-pig interspecies transmission of H9N2 viruses did exist in China. The other eight genotype (C-J) viruses might be double-reassortant viruses, in which six genotype (E-J) viruses possessed 1-4 H5-like gene segments indicating they were reassortants of H9 and H5 viruses. In conclusion, genetic diversity of H9N2 influenza viruses from pigs in China provides further evidence that avian to pig interspecies transmission of H9N2 viruses did occur and might result in the generation of new reassortant viruses by genetic reassortment with swine H1N1, H1N2 and H3N2 influenza viruses, therefore, these swine H9N2 influenza viruses might be a potential threat to human health and continuing to carry out swine influenza virus surveillance in China is of great significance.     

Two different genotypes of H1N2 swine influenza virus isolated in northern China and their pathogenicity in animals

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.     

类人源猪H3N2亚型流感病毒HA,NA遗传进化分析

2007-2008年从吉林省某猪场采集疑似流感发病猪的鼻咽拭子,经病毒分离鉴定获得3株H3N2亚型流感病毒,分别命名为A/swine/Jilin/5/2007（Sw/Jilin/5/07）、A/swine/Jilin/19/2007（Sw/Jilin/19/07）、A/swine/Jilin/37/2008（Sw/Jilin/37/08）。HA进化树分析结果表明：3株H3N2亚型流感病毒属于近代人源病毒谱系;但是在NA进化树中,Sw/Jilin/37/08株与早期人源和近代人源关系密切,暗示它可能是早期人源和近代人源H3N2亚型猪流感病毒之间的过渡毒株,进一步证实猪在流感病毒种间传播过程中充当＂中间宿主＂作用。     

3株H9N2亚型禽流感病毒全基因组序列进化分析

为明确3株不同源性的H9N2亚型禽流感病毒(AIV)A/Chicken/Jilin/22/13(简称JL22)、A/Chicken/Jilin/24/13(简称JL24)、A/Duck/Jilin/37/13(简称JL37)基因组的遗传变异情况,本试验采用RT-PCR技术,分别扩增出3株AIV的8个基因片段,克隆后进行序列测定。结果显示,3株H9N2亚型AIV的主要致病基因均属于经典的欧亚种系。氨基酸比对发现JL22的HA氨基酸序列与A/Chicken/Hong Kong/G9/97和A/Duck/Hong Kong/Y280/97的HA氨基酸序列相比,在551位多了1个潜在糖基化位点。JL22、JL24、JL37的HA序列,在226位的氨基酸残基均为Leu,具有同哺乳动物唾液酸受体结合的特性,说明对人的感染性增强。M基因在31位上均发生了Asn取代Ser的现象,说明这些病毒对金刚烷胺产生了耐药性。由系统进化树可知3株毒株亲缘关系较远,各个基因所属分支也不具有统一性,且部分基因分别与鸡源、鸭源和猪源3种源性流感病毒株高度同源,推测这3株毒株是不同动物不同毒株经过长时间进化而发生自然重排的产物。     

Genetic characterization of H7N2 influenza virus isolated from pigs

Because pigs have respiratory epitheliums which express both α2-3 and α2-6 linked sialic acid as receptors to influenza A viruses, they are regarded as mixing vessel for the generation of pandemic influenza viruses through genetic reassortment. A H7N2 influenza virus (A/swine/KU/16/2001) was isolated from pig lungs collected from the slaughterhouse. All eight genes of the influenza virus were sequenced and phylogenetic analysis indicated that A/swine/KU/16/2001 originated in Hong Kong and genetic reassortment had occurred between the avian H7N2 and H5N3 influenza viruses. The first isolation of H7 influenza virus in pigs provides the opportunity for genetic reassortment of influenza viruses with pandemic potential and emphasizes the importance of surveillance for atypical swine influenza viruses.     

Development of vaccine strains of H5 and H7 influenza viruses

To establish vaccine strains of H5 and H7 influenza viruses, A/duck/Hokkaido/Vac-1/04 (H5N1) [Vac-1/04 (H5N1)], A/duck/Hokkaido/Vac-3/07 (H5N1) [Vac-3/07 (H5N1)], and A/duck/Hokkaido/ Vac-2/04 (H7N7) [Vac-2/04 (H7N7)] were generated from non-pathogenic avian influenza viruses isolated from migratory ducks. Vac-1/04 (H5N1) and Vac-3/07 (H5N1) were generated by genetic reassortment between H5N2 or H5N3 virus as an HA gene provider and H7N1 or H6N1 viruses as an NA gene provider. Vac-2/04 (H7N7) was a genetic reassortant obtained using H7N7 and H9 N2 viruses to give high growth character of the H9N2 virus in chicken embryonated eggs. The results of sequence analyses and experimental infections revealed that these H5N1 and H7N7 reassortant viruses were non-pathogenic in chickens and embryos, and had good growth potential in embryonated eggs. These viruses should be useful to develop vaccines against H5 and H7 highly pathogenic avian influenza viruses.     

Avian and swine influenza viruses: our current understanding of the zoonotic risk

The introduction of swine or avian influenza (AI) viruses in the human population can set the stage for a pandemic, and many fear that the Asian H5N1 AI virus will become the next pandemic virus. This article first compares the pathogenesis of avian, swine and human influenza viruses in their natural hosts. The major aim was to evaluate the zoonotic potential of swine and avian viruses, and the possible role of pigs in the transmission of AI viruses to humans. Cross-species transfers of swine and avian influenza to humans have been documented on several occasions, but all these viruses lacked the critical capacity to spread from human-to-human. The extreme virulence of H5N1 in humans has been associated with excessive virus replication in the lungs and a prolonged overproduction of cytokines by the host, but there remain many questions about the exact viral cell and tissue tropism. Though pigs are susceptible to several AI subtypes, including H5N1, there is clearly a serious barrier to infection of pigs with such viruses. AI viruses frequently undergo reassortment in pigs, but there is no proof for a role of pigs in the generation of the 1957 or 1968 pandemic reassortants, or in the transmission of H5N1 or other wholly avian viruses to humans. The major conclusion is that cross-species transmission of influenza viruses per se is insufficient to start a human influenza pandemic and that animal influenza viruses must undergo dramatic but largely unknown genetic changes to become established in the human population.     

Detection of Novel Reassortant Influenza A (H3N2) and H1N1 2009 Pandemic Viruses in Swine in Hanoi,Vietnam

From May to September 2013, monthly samples were collected from swine in a Vietnamese slaughterhouse for influenza virus isolation and serological testing. A(H1N1)pdm09 viruses and a novel H3N2 originating from reassortment between A(H1N1)pdm09 and novel viruses of the North American triple reassortant lineage were isolated. Serological results showed low seroprevalence for the novel H3N2 virus and higher seroprevalence for A(H1N1)pdm09 viruses. In addition, serology suggested that other swine influenza viruses are also circulating in Vietnamese swine.     

Emergence of H3N2 reassortant influenza A viruses in North American pigs

In late summer through early winter of 1998, there were several outbreaks of respiratory disease in the swine herds of North Carolina, Texas, Minnesota and Iowa. Four viral isolates from outbreaks in different states were analyzed, both antigenically and genetically. All of the isolates were identified as H3N2 influenza viruses with antigenic profiles similar to those of recent human H3 strains. Genotyping and phylogenetic analysis demonstrated that the four swine viruses had emerged through two different pathways. The North Carolina isolate is the product of genetic reassortment between human and swine influenza viruses, while the others arose from reassortment of human, swine and avian viral genes. The hemagglutinin genes of the four isolates were all derived from the human H3N2 virus circulating in 1995. It remains to be determined if either of these recently emerged viruses will become established in the pigs in North America and whether they will become an economic burden.     

Simultaneous Infection of Pigs and People with Triple‐Reassortant Swine Influenza Virus H1N1 at a U.S. County Fair

Influenza‐like illness was noted in people and pigs in attendance at an Ohio county fair in August 2007. The morbidity rate in swine approached 100% within 1–2 days of initial clinical signs being recognized, and approximately two dozen people developed influenza‐like illness. Triple‐reassortant swine H1N1 influenza viruses were identified in both pigs and people at the fair. The identified viruses (A/Sw/OH/511445/2007, A/Ohio/01/2007, and A/Ohio/02/2007) were similar to H1N1 swine influenza viruses currently found in the U.S. swine population. This case illustrates the possibility of transmission of swine influenza in settings where there is close human/swine interaction.     

Serological and virological surveillance of swine H1N1 and H3N2 influenza virus infection in two farms located in Hubei province, central China

Swine influenza viruses H1N1 and H3N2 have been reported in the swine population worldwide. From June 2008 to June 2009, we carried out serological and virological surveillance of swine influenza in the Hubei province in central China. The serological results indicated that antibodies to H1N1 swine influenza virus in the swine population were high with a 42.5% (204/480) positive rate, whereas antibodies to H3N2 swine influenza virus were low with a 7.9% (38/480) positive rate. Virological surveillance showed that only one sample from weanling pigs was positive by RT-PCR. Phylogenetic analysis of the hemagglutinin and neuraminidase genes revealed that the A/Sw/HB/S1/2009 isolate was closely related to avian-like H1N1 viruses and seemed to be derived from the European swine H1N1 viruses. In conclusion, H1N1 influenza viruses were more dominant in the pig population than H3N2 influenza viruses in central China, and infection with avian-like H1N1 viruses persistently emerged in the swine population in the area.     

Novel H1N2 swine influenza reassortant strain in pigs derived from the pandemic H1N1/2009 virus

Swine influenza monitoring programs have been in place in Italy since the 1990 s and from 2009 testing for the pandemic H1N1/2009 virus (H1N1pdm) was also performed on all the swine samples positive for type A influenza. This paper reports the isolation and genomic characterization of a novel H1N2 swine influenza reassortant strain from pigs in Italy that was derived from the H1N1pdm virus. In May 2010, mild respiratory symptoms were observed in around 10% of the pigs raised on a fattening farm in Italy. Lung homogenate taken from one pig showing respiratory distress was tested for influenza type A and H1N1pdm by two real time RT-PCR assays. Virus isolation was achieved by inoculation of lung homogenate into specific pathogen free chicken embryonated eggs (SPF CEE) and applied onto Caco-2 cells and then the complete genome sequencing and phylogenetic analysis was performed from the CEE isolate. The lung homogenate proved to be positive for both influenza type A (gene M) and H1N1pdm real time RT-PCRs. Virus isolation (A/Sw/It/116114/2010) was obtained from both SPF CEE and Caco-2 cells. Phylogenetic analysis showed that all of the genes of A/Sw/It/116114/2010, with the exception of neuraminidase (NA), belonged to the H1N1pdm cluster. The NA was closely related to two H1N2 double reassortant swine influenza viruses (SIVs), previously isolated in Sweden and Italy. NA sequences for these three strains were clustering with H3N2 SIVs. The emergence of a novel reassortant H1N2 strain derived from H1N1pdm in swine in Italy raises further concerns about whether these viruses will become established in pigs. The new reassortant not only represents a pandemic (zoonotic) threat but also has unknown livestock implications for the European swine industry.     

Identification and characterization of H2N3 avian influenza virus from backyard poultry and comparison with novel H2N3 swine influenza virus

In early 2007, H2N3 influenza virus was isolated from a duck and a chicken in two separate poultry flocks in Ohio. Since the same subtype influenza virus with hemagglutinin (H) and neuraminidase (N) genes of avian lineage was also identified in a swine herd in Missouri in 2006, the objective of this study was to characterize and compare the genetic, antigenic, and biologic properties of the avian and swine isolates. Avian isolates were low pathogenic by in vivo chicken pathogenicity testing. Sequencing and phylogenetic analyses revealed that all genes of the avian isolates were comprised of avian lineages, whereas the swine isolates contained contemporary swine internal gene segments, demonstrating that the avian H2N3 viruses were not directly derived from the swine virus. Sequence comparisons for the H and N genes demonstrated that the avian isolates were similar but not identical to the swine isolates. Accordingly, the avian and swine isolates were also antigenically related as determined by hemagglutination-inhibition (HI) and virus neutralization assays, suggesting that both avian and swine isolates originated from the same group of H2N3 avian influenza viruses. Although serological surveys using the HI assay on poultry flocks and swine herds in Ohio did not reveal further spread of H2 virus from the index flocks, surveillance is important to ensure the virus is not reintroduced to domestic swine or poultry. Contemporary H2N3 avian influenza viruses appear to be easily adaptable to unnatural hosts such as poultry and swine, raising concern regarding the potential for interspecies transmission of avian viruses to humans.     

The epidemiology and evolution of influenza viruses in pigs

Pigs serve as major reservoirs of H1N1 and H3N2 influenza viruses which are endemic in pig populations world-wide and are responsible for one of the most prevalent respiratory diseases in pigs. The maintenance of these viruses in pigs and the frequent exchange of viruses between pigs and other species is facilitated directly by swine husbandry practices, which provide for a continual supply of susceptible pigs and regular contact with other species, particularly humans. The pig has been a contender for the role of intermediate host for reassortment of influenza A viruses of avian and human origin since it is the only domesticated mammalian species which is reared in abundance and is susceptible to, and allows productive replication, of avian and human influenza viruses. This can lead to the generation of new strains of influenza, some of which may be transmitted to other species including humans. This concept is supported by the detection of human-avian reassortant viruses in European pigs with some evidence for subsequent transmission to the human population. Following interspecies transmission to pigs, some influenza viruses may be extremely unstable genetically, giving rise to variants which could be conducive to the species barrier being breached a second time. Eventually, a stable lineage derived from the dominant variant may become established in pigs. Genetic drift occurs particularly in the genes encoding the external glycoproteins, but does not usually result in the same antigenic variability that occurs in the prevailing strains in the human population. Adaptation of a 'newly' transmitted influenza virus to pigs can take many years. Both human H3N2 and avian H1N1 were detected in pigs many years before they acquired the ability to spread rapidly and become associated with disease epidemics in pigs.     

禽流感病毒宿主特异性与致病性的分子基础研究进展

禽流感病毒不断重排和变异导致新型流感病毒不断出现,其中有些毒株已经获得了感染哺乳动物的能力,严重危害人类公共卫生安全。近年来,对于禽流感病毒致宿主特异性和致病性的研究取得了一定进展。病毒蛋白某些氨基酸位点的突变就能够改变病毒的宿主特异性,使病毒能够跨宿主传播。而且,病毒的RNA聚合酶、NS1非结构蛋白和几种新发现的病毒蛋白都与病毒的致病性密切相关。论文阐述了禽流感病毒宿主特异性与致病性的分子基础,为禽流感跨物种传播机制研究及防控工作提供参考。     

欧亚类禽H1N1与古典H1N1亚型猪流感病毒NA基因遗传进化分析

本研究对2011年分离自吉林省猪群的3株流感病毒进行了遗传进化分析。结果表明欧亚类禽H1N1猪流感病毒和古典H1N1猪流感病毒在吉林省猪群中共同流行,因此加强猪流感流行病学调查具有重要意义。     

福建省鸭禽流感病毒分离株（A/Duck/Fujian/FQ107/2007〔H9N2〕）全基因测序及遗传进化分析

为了解一株可引起产蛋鸭产蛋异常的H9N2亚型禽流感病毒A/Duck/Fujian/FQ107/2007（H9N2）（以下简称Dk/FQ107/07）分离株的分子特性及其遗传进化地位,运用RT-PCR方法对其基因组进行扩增,克隆至pMD18-T载体后测序。结果显示,Dk/FQ107/07病毒株的血凝素（haemagglutini,HA）蛋白裂解位点的氨基酸组成为-PARSSR↓GLF-,其静脉接种指数（intravenous pathogenicity index,IVPI）为0.04,符合低致病性禽流感病毒特征;Dk/FQ107/07株HA基因与A/chicken/Shantou/5269/2005（H9N2）同源性最高,为98.9%,和我国首次哺乳动物流感病毒分离株A/Swine/HongKong/9/98（H9N2）有较近的遗传进化关系,三者均属于经典的H9N2/Y280群系;神经氨酸酶（neuraminidase,NA）基因与中国大陆首次分离株A/chicken/Beijing/1/1994（H9N2）相比,在63、64、65位点上缺失3个氨基酸（T、E、I）;核蛋白（nucleoprotein,NP）基因与高致病性鸭源禽流感分离株A/Duck/Fujian/1734/05（H5N1）和A/Duck/Fujian/9713/2005（H5N1）在同一遗传进化分支上,而从聚合酶（polymerase PA,PA）基因的遗传进化分析发现其基因属于H9N2/Y439群系。由此可见,Dk/FQ107/07可能是由不同禽流感病毒基因亚群间发生自然重排的产物。     

The NS segment of H5N1 avian influenza viruses (AIV) enhances the virulence of an H7N1 AIV in chickens

Some outbreaks involving highly pathogenic avian influenza viruses (HPAIV) of subtypes H5 and H7 were caused by avian-to-human transmissions. In nature, different influenza A viruses can reassort leading to new viruses with new characteristics. We decided to investigate the impact that the NS-segment of H5 HPAIV would have on viral pathogenicity of a classical avian H7 HPAIV in poultry, a natural host. We focussed this study based on our previous work that demonstrated that single reassortment of the NS-segment from an H5 HPAIV into an H7 HPAIV changes the ability of the virus to replicate in mammalian hosts. Our present data show that two different H7-viruses containing an NS-segment from H5–types (FPV NS GD or FPV NS VN) show an overall highly pathogenic phenotype compared with the wild type H7–virus (FPV), as characterized by higher viral shedding and earlier manifestation of clinical signs. Correlating with the latter, higher amounts of IFN-β mRNA were detected in the blood of NS-reassortant infected birds, 48 h post-infection (pi). Although lymphopenia was detected in chickens from all AIV-infected groups, also 48 h pi those animals challenged with NS-reassortant viruses showed an increase of peripheral monocyte/macrophage-like cells expressing high levels of IL-1β, as determined by flow cytometry. Taken together, these findings highlight the importance of the NS-segment in viral pathogenicity which is directly involved in triggering antiviral and pro-inflammatory cytokines found during HPAIV pathogenesis in chickens.     

A low pathogenic H5N2 influenza virus isolated in Taiwan acquired high pathogenicity by consecutive passages in chickens

H5N2 viruses were isolated from cloacal swab samples of apparently healthy chickens in Taiwan in 2003 and 2008 during surveillance of avian influenza. Each of the viruses was eradicated by stamping out. The official diagnosis report indicated that the Intravenous Pathogenicity Indexes (IVPIs) of the isolates were 0.00 and 0.89, respectively, indicating that these were low pathogenic strains, although the hemagglutinin of the strain isolated in 2008 (Taiwan08) had multibasic amino acid residues at the cleavage site (PQRKKR/G). In the present study, these H5N2 viruses were assessed for their intravenous and intranasal pathogenicity for chickens. It was examined whether Taiwan08 acquires pathogenicity through consecutive passages in chickens. Intravenous pathogenicity of Taiwan08 depended upon the age of the chickens used for the IVPI test; all of the eight-week-old chickens intravenously inoculated with Taiwan08 showed clinical signs but survived for ten days post inoculation (IVPI=0.68), whereas all the six-week-old chickens died (IVPI=1.86). Taiwan08-P8, which were passaged in chickens for eight times, killed all the eight-week-old chickens (IVPI=2.36). The four-week-old chickens died after intranasal inoculation of Taiwan08-P8, indicating that Taiwan08 must have become highly pathogenic during circulation in chicken flocks. These results emphasize the importance of a stamping out policy for avian influenza even if the IVPI of the causal virus is low.     

Molecular characterization of low pathogenic avian influenza viruses, isolated from food products imported into Singapore

We have completed the genetic characterization of all eight gene segments for four low pathogenic avian influenza (LPAI) viruses. The objective of this study was to detect the presence of novel signatures that may serve as early warning indicators of the conversion of LPAI viruses to high pathogenic avian influenza (HPAI) viruses. This study included three H5N2 and one H5N3 viruses that were isolated from live poultry imported into Singapore as part of the national avian influenza virus (AIV) surveillance program. Based on the molecular criterion of the World Organisation for Animal Health (OIE), sequence analysis with the translated amino acid (aa) sequence of the hemagglutinin (HA) gene revealed the absence of multibasic aa at the HA cleavage site, identifying all four virus isolates as LPAI. Detailed phylogenetic tree analyses using the HA and neuraminidase (NA) genes clustered these isolates in the Eurasian H5 lineage, but away from the HPAI H5 subtypes. This analysis further revealed that the internal genes clustered to different avian and swine subtypes, suggesting that the four isolates may possibly share their ancestry with these different influenza subtypes. Our results suggest that the four LPAI isolates in this study contained mainly avian signatures, and the phylogenetic tree for the internal genes further suggests the potential for reassortment with other different circulating avian subtypes. This is the first comprehensive report on the genetic characterization of LPAI H5N2/3 viruses isolated in South-East Asia.