Accelerated Evolution of H7N9 Subtype Influenza Virus under Vaccination Pressure.

No avian H7N9 outbreaks have occurred since the introduction of H7N9 inactivated vaccine in the fall of 2017. However, H7N9 is still prevalent in poultry. To surveil the prevalence, genetic characteristics, and antigenic changes of H7N9, over 7000 oropharyngeal and cloaca swab specimens were collected from live poultry markets and farms in 15 provinces of China from 2017 to 2019. A total of 85 influenza virus subtype H7N9 strains were isolated and 20 representative strains were selected for genetic analysis and antigenicity evaluation. Results indicated the decreased prevalence of low-pathogenic H7N9 strains while highly-pathogenic H7N9 strains became dominated since the introduction of vaccine. Phylogenetic analysis showed that strains from 2019 formed an independent small branch and were genetically distant to strains isolated in 2013-2018. Analysis of key amino acid sites showed that the virus strains may adapt to the host environment evolutionally through mutation. Our analysis predicted additional potential glycosylation sites for HA and NA genes in the 2019 strains. Sequence analysis of HA gene in strains isolated from 2018 to 2019 showed that there were an increased nucleotide substitution rate and an increased mutation rate in the first and second nucleotides of coding codons within the open reading frame. The hemagglutination inhibition (HI) assay showed that H7-Re1 and H7-Re2 exhibited a lower HI titer for isolates from 2019, while H7-Re3 and rLN79 showed a high HI titer. The protective effect of the vaccine decreased after 15 months of use. Overall, under vaccination pressure, the evolution of influenza virus subtype H7N9 has accelerated.

Genetic Evolution Characteristics of Genotype G57 Virus, A Dominant Genotype of H9N2 Avian Influenza Virus.

This study aimed to investigate the genetic evolution of the H9N2 avian influenza virus (AIV). Whole genome phylogenetic trees were constructed based on 306 H9N2 avian influenza strains collected in China from 2014 to 2019. The results showed that eight gene sequences were clustered separately according to their dominant clades, and a total of 10 genotypes were identified (seven of which were novel types). Among them, G57 genotype was confirmed as the most prevalent genotype with a frequency of 94%. In China, the G57 genotype of H9N2 first emerged in 2007, and then became the most common genotype in 2013. Therefore, the nucleotide substitution rates of G57 genotype in HA and NA genes collected from 2007 to 2019 were estimated, and the positive selection pressure sites in the same data set were measured. Taking 2013 as the boundary, the time period was divided into two periods: 2007-2012 and 2013-2019. From 2007 to 2012, multiple genotypes coexisted and could bear the pressures from both nature and environment; while G57 genotype was still in the adaptation stage, subjected to less selection pressure and in the process of slow evolution. However, from 2013 to 2019, G57 became the dominant genotype, and most of the external pressure reacted on it. Moreover, G57 genotype showed better adaptability than other genotypes. From 2013 to 2019, the nucleotide substitution rates of the HA gene were increased, and the positive selection pressures on HA and NA genes were stronger compared to those from 2007 to 2012. To sum up, the absolutely dominant G57 genotype exhibited a relatively constant genotype frequency and experienced adaptive evolution and natural selection simultaneously during the monitoring period. Therefore, urgent attention and diligent surveillance of H9N2 avian influenza virus are becoming increasingly important.

New molecular evolutionary characteristics of H9N2 avian influenza virus in Guangdong Province, China.

To understand the evolution of H9N2 avian influenza virus genotype and its molecular evolution rate, we systematically analyzed 72 H9N2 avian influenza virus sequences isolated from Guangdong province from 2014 to 2018. We found three genotypes (G57, G68, and G118) of the H9N2 avian influenza virus, of which G118 is a newly discovered genotype and G57 is the dominant genotype. The internal gene cassette of the G57 genotype H9N2 avian influenza virus is a stable combination that can easily transport internal genes to other novel avian influenza viruses, and the internal gene cassettes of the G68 and G118 are identical to those of G57.In addition, we estimated the nucleotide substitution rate of the HA and NA genes of the H9N2 influenza virus from 2014 to 2018.The nucleotide substitution rate of HA and NA genes showed an upward trend in 2015 and 2016. In the past two years, H9N2 avian influenza virus recombination has produced genotype G68, which disappeared in 2014 for one year. And very coincidentally, in 2015, there was a new genotype G118. We observed that the emergence of new genotypes was accompanied by a slight increase in overall nucleotide substitution rate. Therefore we hypothesize that the emergence of new genotypes could accelerate the molecular evolution rate of genes. Our research shows that the H9N2 avian influenza virus in Guangdong province has been undergoing intense evolution, demonstrating the need to strengthen influenza surveillance in the region.