Thursday, April 27, 2023

EID Dispatch: Replication of Novel Zoonotic-Like Influenza A(H3N8) Virus in Ex Vivo Human Bronchus and Lung


With China last month reporting their third human infection with avian H3N8, and recent studies suggesting that this avian virus continues to accrue mammalian adaptations (see EID Journal: Evolution of Avian Influenza Virus (H3) with Spillover into Humans, China), there is a good deal of concern over its future trajectory. 

Last week the above mentioned study identified 4 sublineages of H3 AIVs and 126 distinct genotypes, of which the H3N2 G23 genotype has recently predominated in China. The study found:
  • After reassortment with 6 internal genes of H9N2, current H3N8 AIVs seem to have the advantage of infecting humans (42). Ongoing adaptation in mammals after continuous human infections may underlie emergence of pandemic strains.
  • H3 AIV has shown the potential for cross-species transmission and was the origin of other animal influenza viruses, which caused epidemics in horses, dogs, seals, and pigs
  • The H3N8 G25 viruses had acquired human-adapted mutations after infecting humans (Appendix Figure 14), such as 228G/S in the HA gene and E627K/V in the PB2 gene, which were also present in 1968 H3N2 pandemic strains (43). This finding indicates the pandemic potential of the newly emerged H3N8 AIVs.

While concerning, the low number of human cases reported so far suggests the virus isn't quite ready for prime time.  Although there may be some cases we are missing, widespread circulation in humans seems doubtful at this point. 

We've another report today from the University of Hong Kong School of Public Health that finds the current H3N8 virus doesn't appear to replicate particularly well in human bronchus and lung cell cultures, suggesting it needs additional adaptations before it can pose a major human health threat. 

Note: This report was written before the recently announced 3rd (and fatal) infection in Guangdong Province, of which we still know little.  Hopefully we'll see a genomic analysis of that isolate in the near future. 

First some excerpts from the EID Dispatch (follow the link to read it in its entirety), after which I'll return with a postscript. 

Replication of Novel Zoonotic-Like Influenza A(H3N8) Virus in Ex Vivo Human Bronchus and Lung

Kenrie P.Y. Hui, John C.W. Ho, Ka-Chun Ng, Samuel M.S. Cheng, Ko-Yung Sit, Timmy W.K. Au, Leo L.M. Poon, John M. Nicholls, Malik Peiris, and Michael C.W. Chan


Human infection with avian influenza A(H3N8) virus is uncommon but can lead to acute respiratory distress syndrome. In explant cultures of the human bronchus and lung, novel H3N8 virus showed limited replication efficiency in bronchial and lung tissue but had a higher replication than avian H3N8 virus in lung tissue.

Avian influenza viruses (AIVs) with reassortments between AIVs from domestic poultry and wild birds sporadically cross species barriers, leading to human infections. Viruses with internal genes of H9N2, hemagglutinin, and neuraminidase acquired from wild birds constitute the zoonotic H5N1, H7N9, and H10N8 viruses (13) and can lead to severe influenza.

In 2022, two human infections with novel influenza A(H3N8) viruses were reported in Henan and Hunan Province, China (4,5). The first case was identified in a 4-year-old boy with acute respiratory distress syndrome, and the second case occurred in a 5-year-old boy with mild disease. Phylogenetic analysis revealed that the novel H3N8 viruses were triple reassortments containing the Eurasian avian H3 gene of wild-bird origin, the North American avian N8 gene derived from the wild bird AIV, and G57 genotype H9N2 internal genes from AIVs found in poultry in China (6,7).
H3N8 viruses that are genetically similar to the zoonotic H3N8 viruses reported in China (4,5) have been isolated in poultry markets in Hong Kong, China (8). Those novel avian H3N8 viruses are antigenically distant from contemporary human influenza A(H3N2) viruses, and little cross-reactive immunity to these chicken H3N8 viruses exists in the human population (8). We assessed the replication of the novel influenza A(H3N8) virus in human ex vivo bronchus and lung tissues (Appendix).

The Study

The viruses used in this study were H9N2/Y280, pH1N1, avH3N8/MP16, novel H3N8, and H5N1/483 (Appendix Table 1). The novel H3N8 virus was isolated from chickens and is genetically closely related to the virus causing zoonotic human disease in China (A/Henan/4-10CNIC/2022/H3N8) (8). Their hemagglutinin genes share a 99.1% similarity, and the neuraminidase genes share a 98.7% similarity.

The avH3N8 virus was isolated from wild bird droppings in Mai Po, Hong Kong, and is genetically unrelated to the virus causing zoonotic disease in China. The novel H3N8 virus failed to propagate in Madin-Darby canine kidney (MDCK) cells but could be propagated in eggs and titrated in chicken embryo fibroblasts (DF-1), whereas the other strains could be propagated and titrated in MDCK cells. 


Amino acid comparisons of the novel H3N8 and avH3N8 viruses demonstrated that they shared the same stalk length in the NA gene but did not have the G228S mutation that enhances binding to mammalian receptors (Appendix Table 2). The internal genes of the novel H3N8 virus were reassorted from H9N2 virus, whereas the internal genes of the avH3N8 came from H3N8, H6N1, H6N2, H3N8, H1N1, and H7N1 (Table).  

Neither virus had the E627K mutation in polymerase basic 2 that confers mammal adaptation, virulence, and transmissibility. The novel H3N8 virus had the A588V mutation in polymerase basic 2 that promotes mammal adaptation, but avian H3N8 virus did not have this mutation. This difference might contribute to higher replication of the novel H3N8 virus in human lung tissue. The S31N mutation found in the matrix protein 2 of the novel H3N8 virus provided adamantane resistance.

Although zoonotic H3N8 viruses have a dual receptor-binding affinity of α-2,3 and α-2,6 receptors (7), our findings show that this factor does not confer an advantage for replication in human bronchial tissue. Our findings demonstrated inefficient replication of the novel H3N8 virus in human bronchial tissues, which implies limited efficiency to transmit among humans. This finding is in line with a recent serologic surveillance study in which no poultry workers were positive for antibodies for the novel H3N8 virus (7), and only 2 human cases have been documented since April 2022 (4,5). The moderate replication ability of the novel H3N8 virus in human lung tissue suggests that the virus causes less severe disease than H5N1 virus.

In summary, our findings suggest that the zoonotic-like avian H3N8 virus has limited efficiency for human-to-human transmission and, at present, is unlikely to cause severe disease in humans. However, the limited cross-reactive immunity against this novel H3N8 virus in the human population (8) and the emergence of novel H3N8 viruses by continuous reassortment between AIVs in wild birds and poultry demonstrate that the zoonotic and pandemic potential of avian H3N8 viruses should be closely monitored.

While these findings may dial back some of the concern, they are based on what the virus was, not what it is today, or what it may become tomorrow.  The only real constant about viruses is that they must change in order to survive. 

A little over a month ago we looked at an extensive survey of H3N8 viruses in China (see Emerging Microbes & Inf.: Prevalence, Evolution, Replication and Transmission of H3N8 Avian Influenza Viruses), which described just how diverse and well entrenched this subtype has become in wild birds. 

H3N8 viruses have been detected in at least 64 kinds of wild birds and have evolved into several phylogenetic lineages, whereas only a few strains were detected in chickens in recent years. The H3N8 viruses analyzed in this study shared similar sequence identities and clustered into the same lineages with some strains isolated from Europe, North America and Africa, suggesting that H3N8 viruses can be transmitted globally with the migration of their natural reservoirs. 

We also found that these H3N8 viruses have undergone complicated reassortment with circulating H5N3, H7N4, H9N2, H10N4 and H10N8-like viruses isolated from Yellow River Delta wetlands [Citation21Citation33Citation34]. Of note, all the H3N8 viruses detected from chicken and humans in China in 2022 share HA and NA surface genes similar to those of duck- and wild bird-origin strains but bear an internal gene constellation from chicken H9N2 viruses [Citation29Citation31]. 

While H3N8 may still lack some of the attributes it needs to pose a bigger public health threat, its growing host range, genetic diversity, and potential for international travel via migratory birds, provide it with plenty of opportunities to hone its skills. 

All of which makes H3N8 a virus to keep our eye on.