Synergistic HA and NS mutations enhanced the virulence of a mouse-adapted H1N1 influenza A virus.

H1N1 reassortants between the swine Eurasian avian-like (EA) and H1N1 2009 pandemic (H1N1 pdm/09) viruses have been circulating stably in pig populations for more than ten years, and they may have contributed to increased human infections. Whether these H1N1 viruses acquire adaptive mutations to increase their pathogenicity towards a new host is unknown. To address this problem, mouse-adapted (MA) variants of swine-origin EA H1N1 influenza virus isolated from dogs (A/canine/Guangxi/LZ57/2015[LZ57-MA]) were generated by serial lung-to-lung passages in BALB/c mice. These exhibited greater virulence and replication capability than the wild-type virus (LZ57-WT). Of the six adaptive mutations, two were mapped to the ribonucleoprotein (RNP) complex (PB2-E578D and PA-T97I), two to hemagglutinin (HA-N198D and HA-A227E) and two to the non-structural protein 1 (NS1) and nuclear export protein (NS1-A53D and NEP-R42K, respectively). Reverse genetic substitution of the viral genes and mutation experiments demonstrated that the mutations in PA-T97I could enhance the polymerase activity, but a significant downregulation of activity was seen with PB2-E578D, which was consistent with a decrease in virulence. However, HA and NS, which are genes that act synergistically, were found to be determinants of virulence in mice. The reassortant viruses bearing HA mutations (N198D and A227E) were acquired during adaptation enhanced early-stage viral replication in mammalian cells. The single-point mutations in the NS genes had limited effects on virulence. Furthermore, a combination of HA (N198D and A227E) with NS(A53D) in the rLZ57-WT backbone resulted in efficient replication and a significant increase in virulence. The results suggest that these substitutions could compensate for the polymerase function and contribute to enhanced virulence, which highlights a major role for mutations in the HA and NS genes.

Co-circulation and evolution of genogroups I and II of respiratory and enteric feline calicivirus isolates in cats.

Feline calicivirus (FCV) is a highly infectious pathogen that causes upper respiratory tract disease (URTD), but the enteric FCVs raise concerns regarding their role of an enteric pathogen. In this study, between 2019 and 2020, 101 clinical samples from domestic cats with symptoms of URTD, with or without enteritis, were collected for FCV-specific detection. The FCV-positive rate reached to 42.4% (28/66) in cats with respiratory symptoms. The rates were 11.1% (3/27) and 12.5% (1/8) when faeces and serum samples were measured using reverse transcription polymerase chain reaction (RT-PCR), respectively. Ten FCV strains were successfully isolated from respiratory and enteric sources in domestic cats from Guangxi. Phylogenetic analysis based on the genome sequences of 11 isolates (including GX01-13 isolated in 2013) indicated that the newly characterized FCV strains had two recombinant events in comparison with other FCVs and were of respiratory and enteric origins. These strains displayed high genetic diversity, and they were divided into two genogroups (I and II). Of these, the GXNN02-19 isolate was grouped with previously published Chinese isolates that were identified as genogroup II, which contained three specific amino acid residues (377K, 539V and 557S) in the VP1 protein. In addition, the three enteric viruses appeared genetically heterogeneous to each other. All isolates were found to be more sensitive when exposed to low pH conditions, but they were resistant to treatment with trypsin and bile salts. Furthermore, there were no significant differences between the respiratory and enteric FCVs. Our results showed that the genetically distinct FCV strains with genogroups I and II from respiratory and enteric origins were co-circulating in this geographical area. Also, it was revealed that the potential recombinant events between the enteric and respiratory FCVs suggested an important role of enteric FCV during the evolution.