Characterization of an Emergent Chicken H3N8 Influenza Virus in Southern China: a Potential Threat to Public Health.

Although influenza A viruses of several subtypes have occasionally infected humans, to date only those of the H1, H2, and H3 subtypes have led to pandemics and become established in humans. The detection of two human infections by avian H3N8 viruses in April and May of 2022 raised pandemic concerns. Recent studies have shown the H3N8 viruses were introduced into humans from poultry, although their genesis, prevalence, and transmissibility in mammals have not been fully elucidated. Findings generated from our systematic influenza surveillance showed that this H3N8 influenza virus was first detected in chickens in July 2021 and then disseminated and became established in chickens over wider regions of China. Phylogenetic analyses revealed that the H3 HA and N8 NA were derived from avian viruses prevalent in domestic ducks in the Guangxi-Guangdong region, while all internal genes were from enzootic poultry H9N2 viruses. The novel H3N8 viruses form independent lineages in the glycoprotein gene trees, but their internal genes are mixed with those of H9N2 viruses, indicating continuous gene exchange among these viruses. Experimental infection of ferrets with three chicken H3N8 viruses showed transmission through direct contact and inefficient transmission by airborne exposure. Examination of contemporary human sera detected only very limited antibody cross-reaction to these viruses. The continuing evolution of these viruses in poultry could pose an ongoing pandemic threat. IMPORTANCE A novel H3N8 virus with demonstrated zoonotic potential has emerged and disseminated in chickens in China. It was generated by reassortment between avian H3 and N8 virus(es) and long-term enzootic H9N2 viruses present in southern China. This H3N8 virus has maintained independent H3 and N8 gene lineages but continues to exchange internal genes with other H9N2 viruses to form novel variants. Our experimental studies showed that these H3N8 viruses were transmissible in ferrets, and serological data suggest that the human population lacks effective immunological protection against it. With its wide geographical distribution and continuing evolution in chickens, other spillovers to humans can be expected and might lead to more efficient transmission in humans.

Discovery of novel Mamastroviruses in Bactrian camels and dromedaries reveals complex recombination history.

Virus emergence may occur through interspecies transmission and recombination of viruses coinfecting a host, with potential to pair novel and adaptive gene combinations. Camels are known to harbor diverse ribonucleic acid viruses with zoonotic and epizootic potential. Among them, astroviruses are of particular interest due to their cross-species transmission potential and endemicity in diverse host species, including humans. We conducted a molecular epidemiological survey of astroviruses in dromedaries from Saudi Arabia and Bactrian camels from Inner Mongolia, China. Herein, we deployed a hybrid sequencing approach coupling deep sequencing with rapid amplification of complementary deoxyribonucleic acid ends to characterize two novel Bactrian and eight dromedary camel astroviruses, including both partial and complete genomes. Our reported sequences expand the known diversity of dromedary camel astroviruses, highlighting potential recombination events among the astroviruses of camelids and other host species. In Bactrian camels, we detected partially conserved gene regions bearing resemblance to human astrovirus types 1, 4, and 8 although we were unable to recover complete reading frames from these samples. Continued surveillance of astroviruses in camelids, particularly Bactrian species and associated livestock, is highly recommended to identify patterns of cross-species transmission and to determine any epizootic threats and zoonotic risks posed to humans. Phylogenomic approaches are needed to investigate complex patterns of recombination among the astroviruses and to infer their evolutionary history across diverse host species.

Infectivity and Transmissibility of Avian H9N2 Influenza Viruses in Pigs.

UNLABELLED: The H9N2 influenza viruses that are enzootic in terrestrial poultry in China pose a persistent pandemic threat to humans. To investigate whether the continuous circulation and adaptation of these viruses in terrestrial poultry increased their infectivity to pigs, we conducted a serological survey in pig herds with H9N2 viruses selected from the aquatic avian gene pool (Y439 lineage) and the enzootic terrestrial poultry viruses (G1 and Y280 lineages). We also compared the infectivity and transmissibility of these viruses in pigs. It was found that more than 15% of the pigs sampled from 2010 to 2012 in southern China were seropositive to either G1 or Y280 lineage viruses, but none of the sera were positive to the H9 viruses from the Y439 lineage. Viruses of the G1 and Y280 lineages were able to infect experimental pigs, with detectable nasal shedding of the viruses and seroconversion, whereas viruses of the Y439 lineage did not cause a productive infection in pigs. Thus, adaptation and prevalence in terrestrial poultry could lead to interspecies transmission of H9N2 viruses from birds to pigs. Although H9N2 viruses do not appear to be continuously transmissible among pigs, repeated introductions of H9 viruses to pigs naturally increase the risk of generating mammalian-adapted or reassorted variants that are potentially infectious to humans. This study highlights the importance of monitoring the activity of H9N2 viruses in terrestrial poultry and pigs. IMPORTANCE: H9N2 subtype of influenza viruses has repeatedly been introduced into mammalian hosts, including humans and pigs, so awareness of their activity and evolution is important for influenza pandemic preparedness. However, since H9N2 viruses usually cause mild or even asymptomatic infections in mammalian hosts, they may be overlooked in influenza surveillance. Here, we found that the H9N2 viruses established in terrestrial poultry had higher infectivity in pigs than those from aquatic birds, which suggests that adaptation of the H9N2 viruses in terrestrial poultry might have increased the infectivity of the virus to mammals. Therefore, monitoring the prevalence and evolution of H9 viruses prevalent in terrestrial birds and conducting risk assessment of their threat to mammals are critical for evaluating the pandemic potential of this virus.

[Immune Protection against H9N2 Provided by H1N1 Pre-infection in Pigs].

To explore the impact of the history of infection by the influenza A virus subtype H1N1 on secondary infection by the influenza A virus subtype H9N2, pigs non-infected and pre-infected with H1N1 were inoculated with H9N2 in parallel to compare nasal shedding and seroconversion patterns. Unlike pigs without a background of H1N1 infection, nasal shedding was not detected in pigs pre-infected with H1N1. Both groups generated antibodies against H9N2. However, levels of H1N1 antibodies in pigs pre-infected with H1N1 increased quickly and dramatically after challenge with H9N2. Cross-reaction was not observed between H1N1 antibodies and H9N2 viruses. These findings suggest that circulation of the H1N1 virus might be a barrier to the introduction and transmission of the avian H9N2 virus, thereby delaying its adaptation in pigs.

Dual E627K and D701N mutations in the PB2 protein of A(H7N9) influenza virus increased its virulence in mammalian models.

The ongoing avian H7N9 influenza outbreaks in China have caused significant human fatal cases and the virus is becoming established in poultry. Mutations with potential to increase mammalian adaptation have occurred in the polymerase basic protein 2 (PB2) and other viral genes. Here we found that dual 627K and 701N mutations could readily occur during transmission of the virus among ferrets via direct physical contact, and these mutations conferred higher polymerase activity and improved viral replication in mammalian cells, and enhanced virulence in mice. Special attention needs to be paid to patients with such mutations, as these may serve as an indicator of higher virus replication and increased pathogenicity.

2009 pandemic H1N1 influenza virus replicates in human lung tissues.

The replication activity of 2009 pandemic H1N1 influenza virus in human lung cells was evaluated in this study. Twenty-two surgically removed human lung tissue samples were infected ex vivo with pandemic H1N1 influenza virus (A/California/04/2009), seasonal human H1N1 influenza virus (A/Shantou/92/09), or a highly pathogenic H5N1 influenza virus (A/Vietnam/1194/04). Examination of nucleoprotein expression and viral RNA replication in the infected human lung tissue samples showed that whereas the replication of pandemic H1N1 influenza virus varied between tissue samples, overall this virus replicated more efficiently than seasonal H1N1 influenza virus but less efficiently than H5N1 influenza virus. Double-immunostaining for viral antigens and cellular markers indicated that pandemic H1N1 influenza virus replicates in type 2 alveolar epithelial cells.

Substitution of lysine at 627 position in PB2 protein does not change virulence of the 2009 pandemic H1N1 virus in mice.

A lysine at the 627 position (627K) of PB2 protein of influenza virus has been recognized as a determinant for host adaptation and a virulent element for some influenza viruses. While seasonal influenza viruses exclusively contained 627K, the pandemic (H1N1) 2009 possessed a glutamic acid (627E), even after circulation in humans for more than 6months. To explore the potential role of E627K substitution in PB2 in the pandemic (H1N1) 2009 virus, we compared pathogenicity and growth properties between a recombinant virus containing 627K PB2 gene and the parental A/California/4/2009 strain containing 627E. Our results showed that substitution of 627K in PB2 gene does not confer higher virulence and growth rate for the pandemic (H1N1) 2009 virus in mice and cell culture respectively, suggesting 627K is not required for human adaptation of the pandemic (H1N1) 2009 virus.