Mutations in PB2 and HA are crucial for the increased virulence and transmissibility of H1N1 swine influenza virus in mammalian models.

Genetic analyses indicated that the pandemic H1N1/2009 influenza virus originated from a swine influenza virus (SIV). However, SIVs bearing the same constellation of genetic features as H1N1/2009 have not been isolated. Understanding the adaptation of SIVs with such genotypes in a new host may provide clues regarding the emergence of pandemic strains such as H1N1/2009. In this study, an artificial SIV with the H1N1/2009 genotype (rH1N1) was sequentially passaged in mice through two independent series, yielding multiple mouse-adapted mutants with high genetic diversity and increased virulence. These experiments were meant to mimic genetic bottlenecks during adaptation of wild viruses with rH1N1 genotypes in a new host. Molecular substitutions in the mouse-adapted variants mainly occurred in genes encoding surface proteins (hemagglutinin [HA] and neuraminidase [NA]) and polymerase proteins (polymerase basic 2 [PB2], polymerase basic 1 [PB1], polymerase acid [PA] proteins and nucleoprotein [NP]). The PB2D309N and HAL425M substitutions were detected at high frequencies in both passage lines and enhanced the replication and pathogenicity of rH1N1 in mice. Moreover, these substitutions also enabled direct transmission of rH1N1 in other mammals such as guinea pigs. PB2D309N showed enhanced polymerase activity and HAL425M showed increased stability compared with the wild-type proteins. Our findings indicate that if SIVs with H1N1/2009 genotypes emerge in pigs, they could undergo rapid adaptive changes during infection of a new host, especially in the PB2 and HA genes. These changes may facilitate the emergence of pandemic strains such as H1N1/2009.

Cross- immunity of a H9N2 live attenuated influenza vaccine against H5N2 highly pathogenic avian influenza virus in chickens.

The most commonly utilized inactivated influenza vaccines (IIVs) are usually deficient in cross immunity against divergent viruses. On the other hand, live attenuated influenza vaccines (LAIVs) are proved to be more effective in cross-protective immunity. We previously developed a H9N2 LAIV and verified its effective protection against a broad spectrum of H9N2 strains. In the present study, we evaluated its cross-immunity against H5N2 virus, a representative subtype of currently predominant H5 highly pathogenic avian influenza viruses. All chickens vaccinated with this LAIV survived from challenge of H5N2 virus in a lethal dose, and viral proliferation was effectively inhibited, as well as pathological lesions. Vaccination of this LAIV significantly activated H5N2-reactive CD4+ and CD8+ T cells in lungs. These LAIV-activated cross-reactive T cells expanded robustly following H5N2 exposure, and the increasing tendency was temporally correlated with viral clearance. Besides cellular immunity, factors of humoral immunity also play a contributing role in cross-immunity. Passively transferring H9N2 LAIV anti-serum resulted in 100% survival rate to chickens against H5N2 virus. Within components of the anti-serum, cross-binding IgGs against nucleoprotein (NP) of H5N2 virus were found of a contributing role in the cross immunity. These results indicate that this H9N2 LAIV represents a promising strategy for controlling highly pathogenic H5N2 virus in chickens. The cross immunity was partly attributed to LAIV activated H5N2-cross-reactive T cells and partly attributed to cross-binding IgGs against NP.

Generation and protective efficacy of a cold-adapted attenuated avian H9N2 influenza vaccine.

To prevent H9N2 avian influenza virus infection in chickens, a long-term vaccination program using inactivated vaccines has been implemented in China. However, the protective efficacy of inactivated vaccines against antigenic drift variants is limited, and H9N2 influenza virus continues to circulate in vaccinated chicken flocks in China. Therefore, developing a cross-reactive vaccine to control the impact of H9N2 influenza in the poultry industry remains a high priority. In the present study, we developed a live cold-adapted H9N2 influenza vaccine candidate (SD/01/10-ca) by serial passages in embryonated eggs at successively lower temperatures. A total of 13 amino acid mutations occurred during the cold-adaptation of this H9N2 virus. The candidate was safe in chickens and induced robust hemagglutination-inhibition antibody responses and influenza virus-specific CD4(+) and CD8(+) T cell immune responses in chickens immunized intranasally. Importantly, the candidate could confer protection of chickens from homologous and heterogenous H9N2 viruses. These results demonstrated that the cold-adapted attenuated H9N2 virus would be selected as a vaccine to control the infection of prevalent H9N2 influenza viruses in chickens.

Highly Pathogenic Avian Influenza H5N6 Viruses Exhibit Enhanced Affinity for Human Type Sialic Acid Receptor and In-Contact Transmission in Model Ferrets.

UNLABELLED: Since May 2014, highly pathogenic avian influenza H5N6 virus has been reported to cause six severe human infections three of which were fatal. The biological properties of this subtype, in particular its relative pathogenicity and transmissibility in mammals, are not known. We characterized the virus receptor-binding affinity, pathogenicity, and transmissibility in mice and ferrets of four H5N6 isolates derived from waterfowl in China from 2013-2014. All four H5N6 viruses have acquired a binding affinity for human-like SAα2,6Gal-linked receptor to be able to attach to human tracheal epithelial and alveolar cells. The emergent H5N6 viruses, which share high sequence similarity with the human isolate A/Guangzhou/39715/2014 (H5N6), were fully infective and highly transmissible by direct contact in ferrets but showed less-severe pathogenicity than the parental H5N1 virus. The present results highlight the threat of emergent H5N6 viruses to poultry and human health and the need to closely track their continual adaptation in humans. IMPORTANCE: Extended epizootics and panzootics of H5N1 viruses have led to the emergence of the novel 2.3.4.4 clade of H5 virus subtypes, including H5N2, H5N6, and H5N8 reassortants. Avian H5N6 viruses from this clade have caused three fatalities out of six severe human infections in China since the first case in 2014. However, the biological properties of this subtype, especially the pathogenicity and transmission in mammals, are not known. Here, we found that natural avian H5N6 viruses have acquired a high affinity for human-type virus receptor. Compared to the parental clade 2.3.4 H5N1 virus, emergent H5N6 isolates showed less severe pathogenicity in mice and ferrets but acquired efficient in-contact transmission in ferrets. These findings suggest that the threat of avian H5N6 viruses to humans should not be ignored.

Antigenic evolution of H9N2 chicken influenza viruses isolated in China during 2009-2013 and selection of a candidate vaccine strain with broad cross-reactivity.

We previously demonstrated that H9N2 subtype avian influenza viruses (AIVs) isolated from 1994 to 2008 evolved into distinct antigenic groups (C, D, and E) and then underwent antigenic drift from commercial vaccines, causing a country-wide outbreak during 2010-2013. In this study, H9N2 AIVs isolated from chickens during 2009-2013 were antigenically analyzed by performing hemagglutination inhibition and neutralization assays using a panel of polyclonal antibodies. Our findings confirmed the antigenic drift of recent H9N2 viruses from the commercial vaccine and showed that most of these antigenic variants form a novel HI antigenic group, F, with a few belonging to groups D and E. Slight antigenic variation was observed in group F viruses. Genetic analysis of amino acid sequences deduced from hemagglutinin (HA) gene sequences indicated that 9 of 15 mutations predominant in the 2009-2013 viruses can be mapped to known antigenic sites, which might be responsible for the novel antigenicity of group F. These antigenic changes make it necessary to modify the influenza vaccine to ensure efficient protection. A vaccine candidate, Ck/HeB/YT/10, was selected and provided significant protection against viruses from different antigenic groups in terms of reduction in virus shedding, suggesting broad cross-reactivity. Taken together, our results indicate that the H9N2 chicken influenza viruses in China have evolved from distinct antigenic groups into a novel group F that became dominant during the country-wide outbreak and now seems to be undergoing new antigenic divergence. Systematic surveillance and timely updating of vaccine strains are important for viral prevention and control in the future.

A duplex RT-PCR assay for detection of H9 subtype avian influenza viruses and infectious bronchitis viruses.

H9 subtype avian influenza virus (AIV) and infectious bronchitis virus (IBV) are major pathogens circulating in poultry and have resulted in great economic losses due to respiratory disease and reduced egg production. As similar symptoms are elicited by the two pathogens, it is difficult for their differential diagnosis. So far, no reverse transcription-polymerase chain reaction (RT-PCR) assay has been found to differentiate between H9 AIV and IBV in one reaction. Therefore, developing a sensitive and specific method is of importance to simultaneously detect and differentiate H9 AIV and IBV. In this study, a duplex RT-PCR (dRT-PCR) was established. Two primer sets target the hemagglutinin (HA) gene of H9 AIV and the nucleocapsid (N) gene of IBV, respectively. Specific PCR products were obtained from all tested H9 AIVs and IBVs belonging to the major clades circulating in China, but not from AIVs of other subtypes or other infectious avian viruses. The sensitivity of the dRT-PCR assay corresponding to H9 AIV, IBV and mixture of H9 AIV and IBV were at a concentration of 1×101, 1.5×101 and 1.5×101 50% egg infective doses (EID50) mL-1, respectively. The concordance rates between the dRT-PCR and virus isolation were 99.1 and 98.2%, respectively, for detection of samples from H9N2 AIV or IBV infected chickens, while the concordance rate was 99.1% for detection of samples from H9N2 AIV and IBV co-infected chickens. Thus, the dRT-PCR assay reported herein is specific and sensitive, and suitable for the differential diagnosis of clinical infections and surveillance of H9 AIVs and IBVs.

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

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.