A canine influenza A(H3N2) virus emerged in the United States in February–March 2015, causing respiratory disease in dogs. The virus had previously been circulating among dogs in Asia, where it originated through the transfer of an avian-origin influenza virus around 2005 and continues to circulate. Sequence analysis suggests the US outbreak was initiated by a single introduction, in Chicago, of an H3N2 canine influenza virus circulating among dogs in South Korea in 2015. Despite local control measures, the virus has continued circulating among dogs in and around Chicago and has spread to several other areas of the country, particularly Georgia and North Carolina, although these secondary outbreaks appear to have ended within a few months. Some genetic variation has accumulated among the US viruses, with the appearance of regional-temporal lineages. The potential for interspecies transmission and zoonotic events involving this newly emerged influenza A virus is currently unknown.

Genetic sequencing, or DNA sequencing, using the Sanger technique has become widely used in the veterinary diagnostic community. This technology plays a role in verification of PCR results and is used to provide the genetic sequence data needed for phylogenetic analysis, epidemiologic studies, and forensic investigations. The Laboratory Technology Committee of the American Association of Veterinary Laboratory Diagnosticians has prepared guidelines for sample preparation, submission to sequencing facilities or instrumentation, quality assessment of nucleic acid sequence data performed, and for generating basic sequencing data and phylogenetic analysis for diagnostic applications. This guidance is aimed at assisting laboratories in providing consistent, high-quality, and reliable sequence data when using Sanger-based genetic sequencing as a component of their laboratory services.

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This consensus document presents the suggested guidelines developed by the Laboratory Technology Committee (LTC) of the American Association of Veterinary Laboratory Diagnosticians (AAVLD) for development, validation, and modification (methods comparability) of real-time PCR (rtPCR) assays. These suggested guidelines are presented with reference to the World Organisation for Animal Health (OIE) guidelines for validation of nucleic acid detection assays used in veterinary diagnostic laboratories. Additionally, our proposed practices are compared to the guidelines from the Foods Program Regulatory Subdivision of the U.S. Food and Drug Administration (FDA) and from the American Society for Veterinary Clinical Pathology (ASVCP). The LTC suggestions are closely aligned with those from the OIE and comply with version 2021-01 of the AAVLD Requirements for an Accredited Veterinary Medical Diagnostic Laboratory, although some LTC recommendations are more stringent and extend beyond the AAVLD requirements. LTC suggested guidelines are substantially different than the guidelines recently published by the U.S. FDA for validation and modification of regulated tests used for detection of pathogens in pet food and animal-derived products, such as dairy. Veterinary diagnostic laboratories that perform assays from the FDA Bacteriological Analytical Method (BAM) manual must be aware of the different standard.

Avian-origin H3N2 canine influenza virus (CIV) transferred to dogs in Asia around 2005, becoming enzootic throughout China and South Korea before reaching the United States in early 2015. To understand the posttransfer evolution and epidemiology of this virus, particularly the cause of recent and ongoing increases in incidence in the United States, we performed an integrated analysis of whole-genome sequence data from 64 newly sequenced viruses and comprehensive surveillance data. This revealed that the circulation of H3N2 CIV within the United States is typified by recurrent epidemic burst-fade-out dynamics driven by multiple introductions of virus from Asia. Although all major viral lineages displayed similar rates of genomic sequence evolution, H3N2 CIV consistently exhibited proportionally more nonsynonymous substitutions per site than those in avian reservoir viruses, which is indicative of a large-scale change in selection pressures. Despite these genotypic differences, we found no evidence of adaptive evolution or increased viral transmission, with epidemiological models indicating a basic reproductive number, , of between 1 and 1.5 across nearly all U.S. outbreaks, consistent with maintained but heterogeneous circulation. We propose that CIV's mode of viral circulation may have resulted in evolutionary cul-de-sacs, in which there is little opportunity for the selection of the more transmissible H3N2 CIV phenotypes necessary to enable circulation through a general dog population characterized by widespread contact heterogeneity. CIV must therefore rely on metapopulations of high host density (such as animal shelters and kennels) within the greater dog population and reintroduction from other populations or face complete epidemic extinction. The relatively recent appearance of influenza A virus (IAV) epidemics in dogs expands our understanding of IAV host range and ecology, providing useful and relevant models for understanding critical factors involved in viral emergence. Here we integrate viral whole-genome sequence analysis and comprehensive surveillance data to examine the evolution of the emerging avian-origin H3N2 canine influenza virus (CIV), particularly the factors driving ongoing circulation and recent increases in incidence of the virus within the United States. Our results provide a detailed understanding of how H3N2 CIV achieves sustained circulation within the United States despite widespread host contact heterogeneity and recurrent epidemic fade-out. Moreover, our findings suggest that the types and intensities of selection pressures an emerging virus experiences are highly dependent on host population structure and ecology and may inhibit an emerging virus from acquiring sustained epidemic or pandemic circulation.