Equine and Canine Influenza H3N8 Viruses Show Minimal Biological Differences Despite Phylogenetic Divergence

Abstract

The A/H3N8 canine influenza virus (CIV) emerged from A/H3N8 equine influenza virus (EIV) around the year 2000 through the transfer of a single virus from horses to dogs. We defined and compared the biological properties of EIV and CIV by examining their genetic variation, infection, and growth in different cell cultures, receptor specificity, hemagglutinin (HA) cleavage, and infection and growth in horse and dog tracheal explant cultures. Comparison of sequences of viruses from horses and dogs revealed mutations that may be linked to host adaptation and tropism. We prepared infectious clones of representative EIV and CIV strains that were similar to the consensus sequences of viruses from each host. The rescued viruses, including HA and neuraminidase (NA) double reassortants, exhibited similar degrees of long-term growth in MDCK cells. Different host cells showed various levels of susceptibility to infection, but no differences in infectivity were seen when comparing viruses. All viruses preferred ␣2-3-over ␣2-6-linked sialic acids for infections, and glycan microarray analysis showed that EIV and CIV HA-Fc fusion proteins bound only to ␣2-3-linked sialic acids. Cleavage assays showed that EIV and CIV HA proteins required trypsin for efficient cleavage, and no differences in cleavage efficiency were seen. Inoculation of the viruses into tracheal explants revealed similar levels of infection and replication by each virus in dog trachea, although EIV was more infectious in horse trachea than CIV. IMPORTANCEInfluenza A viruses can cross species barriers and cause severe disease in their new hosts. Infections with highly pathogenic avian H5N1 virus and, more recently, avian H7N9 virus have resulted in high rates of lethality in humans. Unfortunately, our current understanding of how influenza viruses jump species barriers is limited. Our aim was to provide an overview and biological characterization of H3N8 equine and canine influenza viruses using various experimental approaches, since the canine virus emerged from horses approximately 15 years ago. We showed that although there were numerous genetic differences between the equine and canine viruses, this variation did not result in dramatic biological differences between the viruses from the two hosts, and the viruses appeared phenotypically equivalent in most assays we conducted. These findings suggest that the crossspecies transmission and adaptation of influenza viruses may be mediated by subtle changes in virus biology. Influenza A viruses are maintained in aquatic birds as intestinal infections, occasionally transfer to and become established as respiratory infections in mammals, including humans, and sometimes spread from one mammal to another (1, 2). Mammalian hosts that have been commonly seen to maintain avian-derived viruses include swine, horses, humans, mink, seals, and, recently, dogs (2-5). Host transfers between different birds, from birds to mammals, or between different mammalian hosts are relatively common but mostly result in single infe...

Cross-Species Infectivity of H3N8 Influenza Virus in an Experimental Infection in Swine

“…These results are in agreement with the findings presented in here. Feng et al showed that inoculation of both viruses into tracheal explants yielded similar levels of infection by each virus in dog tracheas, whereas equine viruses were more infectious than canine viruses in horse tracheas (27). However, in our experiments with pig trachea explants, the replication of Eq and Ca virus strains was minimal (Tables 3 and 4), indicating that factors other than sialic acid binding may play a role in infectivity and host susceptibility.…”

“…Also, they were the ones with lower binding values to pig trachea explants and therefore the ones exhibiting the lowest values of viral replication (Tables 3 and 4). Recent results from Feng et al (27) analyzing other related H3N8 canine and equine viruses indicated that canine and equine viruses show minimal biological differences despite phylogenetic divergence. In fact, both virus types bind more effectively to ␣-2,3-linked sialic acid than to ␣-2,6-linked sialic acid.…”

Temperature-Sensitive Live-Attenuated Canine Influenza Virus H3N8 Vaccine

“…Recombinant wild-type (WT) and live-attenuated (LACIV) H3N8 CIVs were generated using A/canine/ NY/dog23/2009 H3N8 plasmid-based reverse genetics techniques (72) and grown in MDCK cells at 33°C. Influenza A/Ca/IL/41915/2015 H3N2 was also grown in MDCK cells at 33°C.…”

Comparing the functions of equine and canine influenza H3N8 virus PA-X proteins: Suppression of reporter gene expression and modulation of global host gene expression

“…We utilized EIV isolates sampled close to the ancestor CIV, starting from 1990 to 2013, and CIV isolates sampled soon after the emergence in dogs, from 2004 to 2013. Sequence alignments and the generation of consensus sequences were performed as described previously (15). …”

“…We therefore sought to examine these proteins for functional differences by comparing gene suppression ability using reporter gene assays and by evaluating the details of their influence on host gene expression through RNA sequencing (RNA-seq). CIV emerged from EIV around 2000, and since then it has fixed a unique set of mutations in all its genes, including PA-X (15). By evaluating the biology of the two PA-X proteins we sought to further define PA-X function and to gain a better understanding of virus evolution and adaptation.…”