Tropism and innate host responses of influenza A/H5N6 virus: an analysis of ex vivo and in vitro cultures of the human respiratory tract.

Since their first isolation in 2013, influenza A/H5N6 viruses have spread amongst poultry across multiple provinces in China and to Laos, Vietnam and Myanmar. So far, there have been 14 human H5N6 infections with 10 fatalities.We investigated the tropism, replication competence and cytokine induction of one human and two avian H5N6 isolates in ex vivo and in vitro cultures derived from the human respiratory tract. Virus tropism and replication were studied in ex vivo cultures of human nasopharynx, bronchus and lung. Induction of cytokines and chemokines was measured in vitro in virus-infected primary human alveolar epithelial cells.Human H5N6 virus replicated more efficiently than highly pathogenic avian influenza (HPAI) H5N1 virus and as efficiently as H1N1pdm in ex vivo human bronchus and lung and was also able to replicate in ex vivo cultures of human nasopharynx. Avian H5N6 viruses replicated less efficiently than H1N1pdm in human bronchial tissues and to similar titres as HPAI H5N1 in the lung. While the human H5N6 virus had affinity for avian-like receptors, the two avian isolates had binding affinity for both avian- and human-like receptors. All three H5N6 viruses were less potent inducers of pro-inflammatory cytokines compared with H5N1 virus.Human H5N6 virus appears better adapted to infect the human airways than H5N1 virus and may pose a significant public health threat.

Transmission of H7N9 Influenza Viruses with a Polymorphism at PB2 Residue 627 in Chickens and Ferrets.

UNLABELLED: Poultry exposure is a major risk factor for human H7N9 zoonotic infections, for which the mode of transmission remains unclear. We studied the transmission of genetically related poultry and human H7N9 influenza viruses differing by four amino acids, including the host determinant PB2 residue 627. A/Silkie chicken/HK/1772/2014 (SCk1772) and A/HK/3263/14 (HK3263) replicated to comparable titers in chickens, with superior oropharyngeal over cloacal shedding; both viruses transmitted efficiently among chickens via direct contact but inefficiently via the airborne route. Interspecies transmission via the airborne route was observed for ferrets exposed to the SCk1772- or HK3263-infected chickens, while low numbers of copies of influenza viral genome were detected in the air, predominantly at particle sizes larger than 4 µm. In ferrets, the human isolate HK3263 replicated to higher titers and transmitted more efficiently via direct contact than SCk1772. We monitored "intrahost" and "interhost" adaptive changes at PB2 residue 627 during infection and transmission of the Sck1772 that carried E627 and HK3263 that carried V/K/E polymorphism at 60%, 20%, and 20%, respectively. For SCk1772, positive selection for K627 over E627 was observed in ferrets during the chicken-to-ferret or ferret-to-ferret transmission. For HK3263 that contained V/K/E polymorphism, mixed V627 and E627 genotypes were transmitted among chickens while either V627 or K627 was transmitted to ferrets with a narrow transmission bottleneck. Overall, our results suggest direct contact as the main mode for H7N9 transmission and identify the PB2-V627 genotype with uncompromised fitness and transmissibility in both avian and mammalian species. IMPORTANCE: We studied the modes of H7N9 transmission, as this information is crucial for developing effective control measures for prevention. Using chicken (SCk1772) and human (HK3263) H7N9 isolates that differed by four amino acids, including the host determinant PB2 residue 627, we observed that both viruses transmitted efficiently among chickens via direct contact but inefficiently via the airborne route. Chicken-to-ferret transmission via the airborne route was observed, along with the detection of viral genome in the air at low copy numbers. In ferrets, HK3263 transmitted more efficiently than SCk1772 via direct contact. During the transmission of SCk1772 that contained E and HK3263 that contained V/K/E polymorphism at PB2 residue 627, positive selections of E627 and K627 were observed in chickens and ferrets, respectively. In addition, PB2-V627 was transmitted and stably maintained in both avian and mammalian species. Our results support applying intervention strategies that minimize direct and indirect contact at the poultry markets during epidemics.

Comparative analysis of complete genome sequences of three avian coronaviruses reveals a novel group 3c coronavirus.

In this territory-wide molecular epidemiology study of coronaviruses (CoVs) in Hong Kong involving 1,541 dead wild birds, three novel CoVs were identified in three different bird families (bulbul CoV HKU11 [BuCoV HKU11], thrush CoV HKU12 [ThCoV HKU12], and munia CoV HKU13 [MuCoV HKU13]). Four complete genomes of the three novel CoVs were sequenced. Their genomes (26,396 to 26,552 bases) represent the smallest known CoV genomes. In phylogenetic trees constructed using chymotrypsin-like protease (3CL(pro)), RNA-dependent RNA polymerase (Pol), helicase, spike, and nucleocapsid proteins, BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13 formed a cluster distantly related to infectious bronchitis virus and turkey CoV (group 3a CoVs). For helicase, spike, and nucleocapsid, they were also clustered with a CoV recently discovered in Asian leopard cats, for which the complete genome sequence was not available. The 3CL(pro), Pol, helicase, and nucleocapsid of the three CoVs possessed higher amino acid identities to those of group 3a CoVs than to those of group 1 and group 2 CoVs. Unique genomic features distinguishing them from other group 3 CoVs include a distinct transcription regulatory sequence and coding potential for small open reading frames. Based on these results, we propose a novel CoV subgroup, group 3c, to describe this distinct subgroup of CoVs under the group 3 CoVs. Avian CoVs are genetically more diverse than previously thought and may be closely related to some newly identified mammalian CoVs. Further studies would be important to delineate whether the Asian leopard cat CoV was a result of interspecies jumping from birds, a situation analogous to that of bat and civet severe acute respiratory syndrome CoVs.

Characterization of avian influenza viruses A (H5N1) from wild birds, Hong Kong, 2004-2008.

From January 2004 through June 2008, surveillance of dead wild birds in Hong Kong, People's Republic of China, periodically detected highly pathogenic avian influenza (HPAI) viruses (H5N1) in individual birds from different species. During this period, no viruses of subtype H5N1 were detected in poultry on farms and in markets in Hong Kong despite intensive surveillance. Thus, these findings in wild birds demonstrate the potential for wild birds to disseminate HPAI viruses (H5N1) to areas otherwise free from the viruses. Genetic and antigenic characterization of 47 HPAI (H5N1) viruses isolated from dead wild birds in Hong Kong showed that these isolates belonged to 2 antigenically distinct virus groups: clades 2.3.4 and 2.3.2. Although research has shown that clade 2.3.4 viruses are established in poultry in Asia, the emergence of clade 2.3.2 viruses in nonpasserine birds from Hong Kong, Japan, and Russia raises the possibility that this virus lineage may have become established in wild birds.

Investigation of outbreaks of highly pathogenic H5N1 avian influenza in waterfowl and wild birds in Hong Kong in late 2002.

Outbreaks of highly pathogenic H5N1 avian influenza have occurred in Hong Kong in chickens and other gallinaceous poultry in 1997, 2001, twice in 2002 and 2003. High mortality rates were seen in gallinaceous birds but not in domestic or wild waterfowl or other wild birds until late 2002 when highly pathogenic H5N1 avian influenza occurred in waterfowl (geese, ducks and swans), captive Greater Flamingo (Phoenicopterus ruber) and other wild birds (Little Egret Egretta garzetta) at two waterfowl parks and from two dead wild Grey Heron (Ardea cinerea) and a Black-headed Gull (Larus ridibundus) in Hong Kong. H5N1 avian influenza virus was also isolated from a dead feral pigeon (Columba livia) and a dead tree sparrow (Passer montanus) during the second outbreak. The first waterfowl outbreak was controlled by immediate strict quarantine and depopulation 1 week before the second outbreak commenced. Control measures implemented for the second outbreak included strict isolation, culling, increased sanitation and vaccination. Outbreaks in gallinaceous birds occurred in some live poultry markets concurrently with the second waterfowl outbreak, and infection on a chicken farm was detected 1 week after the second waterfowl park outbreak was detected, on the same day the second grey heron case was detected. Subsequent virus surveillance showed the outbreaks had been contained.