Enhanced replication of avian-origin H3N2 canine influenza virus in eggs, cell cultures and mice by a two-amino acid insertion in neuraminidase stalk

Abstract

Canine influenza virus (CIV) is a newly identified, highly contagious respiratory pathogen in dogs. Recent studies indicate that avian-origin H3N2 CIV are circulating in Chinese dogs. To investigate the effects of a two-amino acid (2-aa) insertion naturally occurring at the distal end of the neuraminidase (NA) stalk found in Chinese isolates since 2010 on virus replication and virulence, we rescued the CIV strain, A/canine/Jiangsu/06/2011(H3N2) and its NA mutant without the 2-aa insertion using reverse genetics. The NA stalk length affected virus growth in cell culture. Compared to the short stalk strain (without 2-aa insertion), the long stalk strain (with 2-aa insertion) exhibited higher peak titers and greater yields in Madin-Darby canine kidney (MDCK) cells, chicken embryo fibroblasts and canine bronchiolar epithelial cells, as well as much larger plaques in MDCK cell monolayers. Furthermore, mice inoculated with the long stalk strain showed more severe pathologic damage in lung and higher proportion of detectable viral RNA in tissues. The long stalk strain induced local IFN-γ production with faster kinetics and higher levels in mice. However, in chickens, the two viral strains showed no significant difference with nearly the same proportion of detectable viral RNA loads in tissues. These observations suggest that the 2-aa insertion in the NA stalk acquired by avian-origin H3N2 CIV helps to enhance viral replication and is likely a result of adaptive evolution in canine hosts.

Multiple Incursions and Recurrent Epidemic Fade-Out of H3N2 Canine Influenza A Virus in the United States

“…Protein-level evolution and natural selection. Putative or confirmed canineadaptive mutations that differentiate H3N2 CIV from avian reservoir viruses have been reported previously (19,26,27) and likely occurred early in the emergence of these viruses. Interestingly, our analysis reveals that since becoming established in dogs, H3N2 CIV has consistently accumulated more nonsynonymous substitutions per site than those in reservoir H3N2 avian influenza A viruses (AIVs).…”

“…Despite this abundance of amino acid diversity, an analysis of selection pressures at individual amino acid sites, while often inadequate at the level of selective sweeps, did not reveal positive selection in any U.S. CIV lineage. A similar lack of detectable adaptive evolution was observed among the CIVs circulating within Asia, with the only exceptions being positive selection of a 2-amino-acid insertion in the NA stalk (detected with the SLAC method in HyPhy), characterized previously (26), and at HA residue 453 (detected with FUBAR; posterior probability ϭ 0.98), which is of unknown phenotypic consequence. In addition, we found no evidence that any of the observed amino acid changes increased transmission rates of virus within the United States as determined by epidemiological analysis (see below).…”

Comparison of the virulence of three H3N2 canine influenza virus isolates from Korea and China in mouse and Guinea pig models

“…Sequence analysis showed a unique two amino acid insertion in the distal end of the NA stalk in JS/10 compared to KR/07 [ 10 ]. Interestingly, our previous study [ 6 ] demonstrated that the two amino acid insertion in JS/10 increased viral infectivity and led to a higher proportion of detectable viral RNA in mouse tissues. Therefore, the wider organ tropism may be partially attributed to the presence of the two amino acids.…”

“…In 2007, another canine influenza outbreak was confirmed in South Korea [ 3 ]; sequence analysis revealed that the causal agent was an avian-origin H3N2 influenza virus, which was then demonstrated to be capable of direct transmission between dogs [ 4 ]. Outbreaks of infections caused by avian-origin H3N2 canine influenza virus (CIV) have been continuously reported in South Korea [ 4 , 5 ], China [ 6 , 7 ] and Thailand [ 8 ] since 2007, and avian-origin H3N2 CIV has become the most prevalent subtype in Asia [ 1 ].…”

Morphological features and pathogenicity of mutated canine influenza viruses from China and South Korea

“…The presence of 2‐aa insertions in NA was observed in many isolates from different provinces of China, such as Zhejiang (Teng et al, ), Beijing and Liaoning (Sun et al, ). The isolate showed enhanced viral replication in mice and guinea pigs (Xie et al, ), albeit it was low in chickens, indicating inappropriateness for replication in poultry (Lin et al, ).…”