Yanbo Yin

Evolution of the H9N2 influenza genotype that facilitated the genesis of the novel H7N9 virus

Significance The emergence of human infection with a novel H7N9 avian influenza reassortant in China raises a pandemic concern. However, it is not fully understood how these H9N2 chicken viruses facilitated the genesis of the novel H7N9 viruses. Here we show that a “fittest” genotype (G57) emerged with changed antigenicity and improved adaptability in chickens. It became predominant in vaccinated farm chickens and caused widespread outbreaks before the H7N9 virus emergence, increasing reassortment between H9N2 and other subtype viruses and finally providing all of their internal genes to the novel H7N9 viruses. The prevalence and variation of H9N2 influenza virus in farmed poultry could provide an important early warning of the emergence of novel reassortants with pandemic potential. The emergence of human infection with a novel H7N9 influenza virus in China raises a pandemic concern. Chicken H9N2 viruses provided all six of the novel reassortant’s internal genes. However, it is not fully understood how the prevalence and evolution of these H9N2 chicken viruses facilitated the genesis of the novel H7N9 viruses. Here we show that over more than 10 y of cocirculation of multiple H9N2 genotypes, a genotype (G57) emerged that had changed antigenicity and improved adaptability in chickens. It became predominant in vaccinated farm chickens in China, caused widespread outbreaks in 2010–2013 before the H7N9 viruses emerged in humans, and finally provided all of their internal genes to the novel H7N9 viruses. The prevalence and variation of H9N2 influenza virus in farmed poultry could provide an important early warning of the emergence of novel reassortants with pandemic potential.

Amino Acid 316 of Hemagglutinin and the Neuraminidase Stalk Length Influence Virulence of H9N2 Influenza Virus in Chickens and Mice

ABSTRACT H9N2 influenza viruses with an A316S substitution in hemagglutinin (HA) and a shorter neuraminidase (NA) stalk have become predominant in China. The A316S was shown to increase HA cleavage efficiency when combined with short stalk NA, and the short stalk NA improved NA enzyme activity and release of virus from erythrocytes. Single mutations or combinations of these mutations strengthened the virulence of H9N2 virus in chickens and mice.

Novel genetic reassortants in H9N2 influenza A viruses and their diverse pathogenicity to mice

BackgroundH9N2 influenza A viruses have undergone extensive reassortments in different host species, and could lead to the epidemics or pandemics with the potential emergence of novel viruses.MethodsTo understand the genetic and pathogenic features of early and current circulating H9N2 viruses, 15 representative H9N2 viruses isolated from diseased chickens in northern China between 1998 and 2010 were characterized and compared with all Chinese H9N2 viruses available in the NCBI database. Then, the representative viruses of different genotypes were selected to study the pathogenicity in mice with the aim to investigate the adaptation and the potential pathogenicity of the novel H9N2 reassortants to mammals.ResultsOur results demonstrated that most of the 15 isolates were reassortants and generated four novel genotypes (B62-B65), which incorporated the gene segments from Eurasian H9N2 lineage, North American H9N2 branch, and H5N1 viruses. It was noteworthy that the newly identified genotype B65 has been prevalent in China since 2007, and more importantly, different H9N2 influenza viruses displayed a diverse pathogenicity to mice. The isolates of the 2008-2010 epidemic (genotypes B55 and B65) were lowly infectious, while two representative viruses of genotypes B0 and G2 isolated from the late 1990s were highly pathogenic to mice. In addition, Ck/SD/LY-1/08 (genotype 63, containing H5N1-like NP and PA genes) was able to replicate well in mouse lungs with high virus titers but caused mild clinical signs.ConclusionSeveral lines of evidence indicated that the H9N2 influenza viruses constantly change their genetics and pathogenicity. Thus, the genetic evolution of H9N2 viruses and their pathogenicity to mammals should be closely monitored to prevent the emergence of novel pandemic viruses.

Molecular and antigenic characterization of H9N2 avian influenza virus isolates from chicken flocks between 1998 and 2007 in China.

Despite extensive vaccination, H9N2 subtype influenza A viruses (IAVs) have prevailed in chicken populations in China. H9N2 IAVs have been a major cause of respiratory disease and reduced egg production, resulting in great economic losses to the Chinese poultry industry. In attempt to find reasons for lack of adequate protection by commercial vaccines, 41 H9N2 viruses isolated from chicken flocks in various regions of China through surveillance between 1998 and 2007 were systemically analyzed using molecular and serological methods in comparison to IAV Ck/Shandong/6/96 and Ck/Shanghai/F/98 that have been used in a majority of commercial vaccines for H9N2 in China since 1998. The analyses showed that the field isolates were predominantly of Beijing/94 lineage and underwent rapid genetic and antigenic changes, forming several antigenic groups. Comparisons between the field isolates and vaccine strains revealed that a majority of the field isolates examined were antigenically distinct from the vaccine strains to some extent. Therefore, the rapid antigenic evolution of H9N2 IAV and resulting antigenic difference from the earlier vaccine strains appears to be a key factor for suboptimal control of H9N2 IAV in China, emphasizing that the vaccine strain should be updated in a timely manner through surveillance and accompanying laboratory evaluation of contemporary viruses for antigenic similarity with existing vaccine strains.

Highly Pathogenic Avian Influenza A(H5N1) Virus Struck Migratory Birds in China in 2015

Approximately 100 migratory birds, including whooper swans and pochards, were found dead in the Sanmenxia Reservoir Area of China during January 2015. The causative agent behind this outbreak was identified as H5N1 highly pathogenic avian influenza virus (HPAIV). Genetic and phylogenetic analyses revealed that this Sanmenxia H5N1 virus was a novel reassortant, possessing a Clade 2.3.2.1c HA gene and a H9N2-derived PB2 gene. Sanmenxia Clade 2.3.2.1c-like H5N1 viruses possess the closest genetic identity to A/Alberta/01/2014 (H5N1), which recently caused a fatal respiratory infection in Canada with signs of meningoencephalitis, a highly unusual symptom with influenza infections in humans. Furthermore, this virus was shown to be highly pathogenic to both birds and mammals, and demonstrate tropism for the nervous system. Due to the geographical location of Sanmenxia, these novel H5N1 viruses also have the potential to be imported to other regions through the migration of wild birds, similar to the H5N1 outbreak amongst migratory birds in Qinghai Lake during 2005. Therefore, further investigation and monitoring is required to prevent this novel reassortant virus from becoming a new threat to public health.

Molecular characterization of Newcastle disease viruses in Ostriches (Struthio camelus L.): Further evidences of recombination within avian paramyxovirus type 1

Newcastle disease virus (NDV) strains isolated from ostriches have been genotyped for the first time by partial sequencing of the F gene to determine the epidemiologic role that this species can play within ND outbreaks. Fifteen additional NDV strains, mostly isolated from chickens but also from pigeons and penguins, were also included in the study to determine genetic relationships with ostriches NDV isolates. High genetic diversity was demonstrated in ostrich NDV isolates, as the 10 isolates were grouped in four distinct NDV genotypes. In agreement with the results obtained when chicken isolates have been molecularly characterized, the predominant genotype in ostriches was the genotype VII. More interestingly, evidences of recombination between genotype II and VII were observed in one ostrich isolate and in two further chicken isolates. Therefore, it seems that ostriches may play a relevant role in the ecology and epidemiology of ND particularly in those regions where they have an increasing farming importance as minor poultry species.

Influenza A Virus Acquires Enhanced Pathogenicity and Transmissibility after Serial Passages in Swine

ABSTRACT Genetic and phylogenetic analyses suggest that the pandemic H1N1/2009 virus was derived from well-established swine influenza lineages; however, there is no convincing evidence that the pandemic virus was generated from a direct precursor in pigs. Furthermore, the evolutionary dynamics of influenza virus in pigs have not been well documented. Here, we subjected a recombinant virus (rH1N1) with the same constellation makeup as the pandemic H1N1/2009 virus to nine serial passages in pigs. The severity of infection sequentially increased with each passage. Deep sequencing of viral quasispecies from the ninth passage found five consensus amino acid mutations: PB1 A469T, PA 1129T, NA N329D, NS1 N205K, and NEP T48N. Mutations in the hemagglutinin (HA) protein, however, differed greatly between the upper and lower respiratory tracts. Three representative viral clones with the five consensus mutations were selected for functional evaluation. Relative to the parental virus, the three viral clones showed enhanced replication and polymerase activity in vitro and enhanced replication, pathogenicity, and transmissibility in pigs, guinea pigs, and ferrets in vivo. Specifically, two mutants of rH1N1 (PB1 A469T and a combination of NS1 N205K and NEP T48N) were identified as determinants of transmissibility in guinea pigs. Crucially, one mutant viral clone with the five consensus mutations, which also carried D187E, K211E, and S289N mutations in its HA, additionally was able to infect ferrets by airborne transmission as effectively as the pandemic virus. Our findings demonstrate that influenza virus can acquire viral characteristics that are similar to those of the pandemic virus after limited serial passages in pigs. IMPORTANCE We demonstrate here that an engineered reassortant swine influenza virus, with the same gene constellation pattern as the pandemic H1N1/2009 virus and subjected to only nine serial passages in pigs, acquired greatly enhanced virulence and transmissibility. In particular, one representative pathogenic passaged virus clone, which carried three mutations in the HA gene and five consensus mutations in PB1, PA, NA, NS1, and NEP genes, additionally was able to confer respiratory droplet transmission as effectively as the pandemic H1N1/2009 virus. Our findings suggest that pigs can readily induce adaptive mutational changes to a precursor pandemic-like virus to transform it into a highly virulent and infectious form akin to that of the pandemic H1N1/2009 virus, which underlines the potential direct role of pigs in promoting influenza A virus pathogenicity and transmissibility.

Assessment of the pathogenesis of Streptococcus suis type 2 infection in piglets for understanding streptococcal toxic shock-like syndrome, meningitis, and sequelae.

Streptococcus suis type 2 (SS2) is an zoonotic pathogen that had caused outbreaks in 1998 and 2005 in China. It is still not very clear how the disease progresses into the streptococcal toxic shock-like syndrome (STSLS) or meningitis, as well as the sequelae from the survivals. The present study used piglets as infection model to systematically investigate the pathogenesis of the infection caused by the SS2 strain 05ZYH33. The infected piglets showed joint swelling, lameness, and crouch at beginning, then developed into septic-like shock syndrome (SLSS) or prostration syndrome, at last the survivals showed physical activity impairment. The morbidity and mortality were 100% (71% for SLSS, 29% for prostration syndrome) and 29%, respectively. The pigs exhibiting SLSS had deep invasive infections in tissues and organs, and displayed more severe bacteremia and cytokine secretion in the bloodstream and organs than pigs with prostration syndrome. Moreover, the polymorphisms in the toll-like receptor 1 (TLR1) and TLR2 genes varied between the pigs affected with SLSS and prostration syndrome. Several lines of evidence indicated that SS2 infection progression into SLSS or relatively lighter prostration syndrome in pigs is closely related to the degrees of bacteremia and cytokine storm, which may be inherently determined by the diversity of innate immunity-associated genes. Furthermore, brain lesions, such as venous thrombosis, may directly contribute to the sequelae in human cases, were identified in the pigs. These results might help us to further understand the pathogenesis of SS2 in humans.

Cytokine expression in three chicken host systems infected with H9N2 influenza viruses with different pathogenicities

ABSTRACT SD/818 and SD/196 are H9N2 influenza virus strains isolated from chickens from the same farm at different times that exhibited similar genetic evolution. However, strain SD/818 exhibited higher pathogenicity in chickens than strain SD/196 and other H9N2 influenza virus epidemic strains from China. The expression of cytokines is an important host defence mechanism following viral infection and their intensity is a major determinant of viral pathogenicity. To elucidate the mechanism underlying the increased pathogenicity of strain SD/818 from the host’s perspective, viral replication and cytokine expression were dynamically studied using real-time quantitative reverse transcription PCR in chickens infected with strain SD/818 compared with chickens infected with strain SD/196 in this study. The results showed that the replication of strain SD/818 and the expressions of IL-1β, IL-6, TNF-α, IFN-α and IFN-β induced by strain SD/818 were higher than those induced by strain SD/196 in the chicken host system. Expression of these cytokines in chickens coincided with or followed virus replication. These results suggested that high-level viral replication and pro-inflammatory cytokine expression (but not decreased type I IFN expression) were associated with the higher pathogenicity of strain SD/818 in chickens.

Genetic correlation between current circulating H1N1 swine and human influenza viruses

BACKGROUND H1N1 is the main subtype influenza A virus circulating in human and swine population, and has long been a threat to economy and public health. OBJECTIVE To explore the genetic correlation between current circulating H1N1 swine and human influenza viruses. STUDY DESIGN Three new H1N1 swine influenza viruses (SIVs) were isolated and genomes sequencing were conducted followed by phylogenetic and molecular analysis of all swine and human H1N1 influenza viruses isolated in China in the past five years. RESULTS Homology and phylogenetic analysis revealed that the three isolates possessed different characteristics: the genome of A/Swine/Shandong/1112/2008 was closely related to that of classical H1N1 SIV, while A/Swine/Shandong/1123/2008 was a reassortant with NS gene from the human-like H3N2 influenza virus and other genes from the classical H1N1 SIV, and A/Swine/Fujian/0325/2008 fell into a lineage of seasonal human H1N1 influenza viruses. Genetically, 2009 H1N1 influenza A viruses (2009 H1N1) in China were contiguous to the SIV lineages rather than the seasonal H1N1 human influenza viruss lineage. Furthermore, molecular analysis among human and swine influenza viruses provided more detail information for understanding their genetic correlation. CONCLUSIONS These results suggested that in China in the past five years, the classical, avian-like and human-like H1N1 SIV existed in swine herds and the reassortment between H1N1 swine and H3N2 human influenza viruses was identified. In addition, the present data showed no evidence to support a strong correlation between the 2009 H1N1 and the swine influenza virus circulating in China.