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Epidemics of H3N8 and H3N2 influenza A viruses (IAVs) in dogs, along with recognition of spillover infections from IAV strains typically found in humans or other animals, have emphasized the importance of efficient laboratory testing. Given the lack of active IAV surveillance or immunization requirements for dogs, cats, or horses imported into the United States, serotype prediction and whole-genome sequencing of positive specimens detected at veterinary diagnostic laboratories are also needed. The conserved sequences at the ends of the viral genome segments facilitate universal amplification of all segments of viral genomes directly from respiratory specimens. Although several methods for genomic analysis have been reported, no optimization focusing on companion animal strains has been described, to our knowledge. We compared 2 sets of published universal amplification primers using 26 IAV-positive specimens from dogs, horses, and a cat. Libraries prepared from the resulting amplicons were sequenced using Illumina chemistry, and reference-based assemblies were generated from the data produced by both methods. Although both methods produced high-quality data, coverage profiles and base calling differed between the 2 methods. The sequence data were also used to identify the subtype of the IAV strains sequenced and then compared to standard PCR assays for neuraminidase types N2 and N8.

Two different influenza A viruses have infected and spread among dogs since 2000, and both have been widespread in dogs in North America. The H3N8 canine influenza virus arose in the United States as a variant of equine influenza virus. The H3N2 canine influenza virus arose in Asia by transfer of an avian influenza virus to dogs. Both viruses cause mild respiratory disease and are associated with outbreaks in densely housed dogs or those with frequent connections to other dogs. The 2 canine influenza viruses each caused widespread epidemics over at least several years that were associated with localized outbreaks.

Viruses are often cultured in cell lines for research and vaccine development, and those often differ from the natural hosts or tissues. Cell lines can also differ in the presence of virus receptors, such as the sialic acid (Sia) receptors used by influenza A viruses (IAV), which can vary in linkage (α2,3- or α2,6-linkage) and form (N-glycolylneuraminic acid [Neu5Gc] or N-acetylneuraminic acid [Neu5Ac]). The selective pressures resulting from passaging viruses in cell types with host-specific variations in viral receptors are still only partially understood. IAV are commonly cultured in MDCK cells which are both derived from canine kidney tubule epithelium and inherently heterogeneous. MDCK cells naturally present Neu5Ac and α2,3-linked Sia forms. Here, we examine natural MDCK variant lineages, as well as engineered variants that synthesize Neu5Gc and/or α2,6-linkages. We determined how viral genetic variation occurred within human H3N2, H1N1 pandemic and canine H3N2 IAV populations when serially passaged in MDCK cell lines that vary in cell type (MDCK-Type I or MDCK-Type II clones) and in Sia display. Deep sequencing of viral genomes showed small numbers of consensus-level mutations, mostly within the hemagglutinin (HA) gene. Both human IAV showed variants in the HA stem and the HA receptor-binding site of populations passaged in cells displaying Neu5Gc. Canine H3N2 showed variants near the receptor-binding site when passaged in cells displaying Neu5Gc or α2,6-linkages. Viruses replicated to low titres in MDCK-Type II cells, suggesting that not all cell types in heterogeneous MDCK cell populations are equally permissive to infection.

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.

In a previous study, Teleaulax amphioxeia—the preferred prey of Mesodinium in the Columbia River estuary—were undetectable within intense annual blooms, suggesting blooms are prey-limited or prey are acquired outside of bloom patches. We used a novel molecular approach specifically targeting the prey (i.e., Unique Sequence Element [USE] within the ribosomal RNA 28S D2 regions of T. amphioxeia nucleus and nucleomorph) in estuarine water samples acquired autonomously with an Environmental Sample Processor integrated within a monitoring network (ESP-SATURN). This new approach allowed for both more specific detection of the prey and better constraint of sample variability. A positive correlation was observed between abundances of M. cf. major and T. amphioxeia during bloom periods. The correlation was stronger at depth (> 8.2 m) and weak or nonexistent in the surface, suggesting that predator–prey dynamics become uncoupled when stratification is strong. We confirmed exclusive selectivity for T. amphioxeia by M. cf. major and observed the incorporation of the prey nucleus into a 4-nuclei complex, where it remained functionally active. The specific biomarker for T. amphioxeia was also recovered in M. cf. major samples from a Namibian coastal bloom, suggesting that a specific predator–prey relationship might be widespread between M. cf. major and T. amphioxeia.

New methods for deep sequence analysis provide an opportunity to follow the emergence and dynamics of virus mutations in real time. Although viruses are commonly grown in cell culture for research and for vaccine development, the cells used to grow the virus are often not derived from the same tissue or even the same host that the virus naturally replicates in. The selective pressures of culturing virusin vitroare still only partially understood. MDCK cells are the standard cell for growing influenza viruses yet are derived from the epithelium of the canine kidney and are also heterogenous. We passaged human H3N2, H1N1 pandemic, and canine H3N2 influenza A viruses (IAV) in different lineages of MDCK cells, as well as lines engineered to express variant Sia receptors, including α2,3- and α2,6-linkages orN-glycolylneuraminic acid (Neu5Gc) orN-acetylneuraminic acid (Neu5Ac) forms. MDCK-Type II cells had lower infection efficiency and virus production, and infection appeared more dependent on protease activation of the virus. When viruses were passaged in the different cells, they exhibited only small numbers of consensus-level mutations, and most were within the HA gene. Both human IAVs showed selection for single nucleotide minority variants in the HA stem across cell types, as well as low frequency variants in the HA receptor binding site of virus passaged in cells expressing Neu5Gc. Canine H3N2 also showed minority variants near the receptor-binding site in cells expressing Neu5Gc and also in those expressingα2,6-linkages.IMPORTANCEThe genetic variation and adaptability of viruses are fundamental properties that allow their evolutionary success in the face of differing host environments and immune responses. The growth of viruses in cell culture is widely used for their study and for preparing vaccines. However, the selection pressures that cell passaging imposes on viruses are often poorly understood. We used deep sequence analysis to define, in detail, how three different influenza A viruses respond to passaging in different lineages of canine MDCK cells that are commonly used for their growth, as well as in variant cells engineered to express different forms of their cell surface receptor, sialic acid. This analysis revealed that most mutations occur in the HA gene and few sequence changes in the virus population reached high proportions. This is relevant for understanding the selective pressures of virus growth in cell culture and how it shapes evolutionary patterns.

The continual emergence of novel influenza A strains from non-human hosts requires constant vigilance and the need for ongoing research to identify strains that may pose a human public health risk. Since 1999, canine H3 influenza A viruses (CIVs) have caused many thousands or millions of respiratory infections in dogs in the United States. While no human infections with CIVs have been reported to date, these viruses could pose a zoonotic risk. In these studies, the National Institutes of Allergy and Infectious Diseases (NIAID) Centers of Excellence for Influenza Research and Surveillance (CEIRS) network collaboratively demonstrated that CIVs replicated in some primary human cells and transmitted effectively in mammalian models. While people born after 1970 had little or no pre-existing humoral immunity against CIVs, the viruses were sensitive to existing antivirals and we identified a panel of H3 cross-reactive human monoclonal antibodies (hmAbs) that could have prophylactic and/or therapeutic value. Our data predict these CIVs posed a low risk to humans. Importantly, we showed that the CEIRS network could work together to provide basic research information important for characterizing emerging influenza viruses, although there were valuable lessons learned., Author summary The 2009 influenza pandemic was a stark reminder that ongoing vigilance is critical to protect the public from an influenza pandemic. The continual evolution of influenza viruses and emergence from animal reservoirs, leads to the need to quickly identify strains that pose a public health risk. In these studies, members of the National Institutes of Allergy and Infectious Diseases (NIAID) Centers for Excellence in Influenza Research and Surveillance (CEIRS) network worked together to demonstrate that the emerging canine H3 influenza viruses posed a low risk to public health and identified several therapeutic options in the event of an emergence. In addition to providing important new basic research, many lessons were learned that may be important in dealing with any emerging disease outbreak.

Influenza virus infections of carnivores-primarily in dogs and in large and small cats-have been repeatedly observed to be caused by a number of direct spillovers of avian viruses or in infections by human or swine viruses. In addition, there have also been prolonged epizootics of an H3N8 equine influenza virus in dogs starting around 1999, of an H3N2 avian influenza virus in domestic dog populations in Asia and in the United States that started around 2004, and an outbreak of an avian H7N2 influenza virus among cats in an animal shelter in the United States in 2016. The impact of influenza viruses in domesticated companion animals and their zoonotic or panzootic potential poses significant questions for veterinary and human health.

Canine parvovirus (CPV) is a highly successful pathogen that has sustained pandemic circulation in dogs for more than 40 years. Here, integrating full-genome and deep sequencing analyses, structural information, andin vitroexperimentation, we describe the macro- and micro-scale features that have accompanied CPV’s evolutionary success. Despite 40 years of viral evolution, all CPV variants are >∼99% identical in nucleotide sequence, with only a limited number (IMPORTANCERapid mutation rates and correspondingly high levels of standing intra-host diversity and accumulated inter-host diversity over epidemic scales are often cited as key features of viruses with the capacity for emergence and sustained transmission in a new host species. However, most of this information comes from studies of RNA viruses, with relatively little being known about that evolutionary processes that occur for viruses with DNA genomes. Here we provide a unique model of virus evolution, integrating both long-term global-scale and short-term intra-host evolutionary processes of a virus in a new host animal. Our analysis reveals that successful host jumping and sustained onward transmission does not necessarily depend on a high level of intra-host diversity or result in the continued accumulation of high levels of long-term evolution change. These findings indicate that all aspects of a virus’s biology and ecology are relevant when considering their adaptability.

Influenza A viruses have regularly jumped to new hosts to cause epidemics or pandemics, an evolutionary process that involves variation in the viral traits necessary to overcome host barriers and facilitate transmission. Mice are not a natural host for influenza virus, but are frequently used as models in studies of pathogenesis, often after multiple passages to achieve higher viral titers that result in clinical disease such as weight loss or death. Here we examine the processes of influenza A virus infection and evolution in mice by comparing deep sequence variation of a human H1N1 pandemic virus, a seasonal H3N2 virus, and a H3N2 canine influenza virus during experimental passage. We also compared replication and sequence variation in wild-type mice expressing N-glycolylneuraminic acid (Neu5Gc) with that seen in mice expressing only N-acetylneuraminic acid (Neu5Ac). Viruses derived from plasmids were propagated in MDCK cells and then passaged in mice up to four times. Full genome deep sequencing of the plasmids, cultured viruses, and viruses from mice at various passages revealed only small numbers of mutational changes. The H3N2 canine influenza virus showed increases in frequency of sporadic mutations in the PB2, PA, and NA segments. The H1N1 pandemic virus grew well in mice, and while it exhibited the maintenance of some minority mutations, there was no clear adaptive evolution. The H3N2 seasonal virus did not establish in the mice. Finally, there were no clear sequence differences associated with the presence or absence of Neu5Gc.SIGNIFICANCEMice are commonly used as a model to study the growth and virulence of influenza A viruses in mammals, but are not a natural host and have distinct sialic acid receptor profiles compared to humans. Using experimental infections with different subtypes of influenza A virus derived from different hosts we found that evolution of influenza A virus in mice did not necessarily proceed through the linear accumulation of host-adaptive mutations, that there was variation in the patterns of mutations detected in each repetition, and the mutation dynamics depended on the virus examined. In addition, variation in the viral receptor, sialic acid, did not effect influenza evolution in this model. Overall this shows that mice provide a useful animal model for influenza, but that host passage evolution will vary depending on the virus being tested.

Alphaherpesvirus-associated ocular infections in humans caused by human alphaherpesvirus 1 (HHV-1) remain challenging to treat due to the frequency of drug application required and the potential for the selection of drug-resistant viruses. Repurposing on-the-market drugs is a viable strategy to accelerate the pace of drug development. It has been reported that the human immunodeficiency virus (HIV) integrase inhibitor raltegravir inhibits HHV-1 replication by targeting the DNA polymerase accessory factor and limits terminase-mediated genome cleavage of human betaherpesvirus 5 (HHV-5). We have previously shown, both in vitro and in vivo, that raltegravir can also inhibit the replication of felid alphaherpesvirus 1 (FeHV-1), a common ocular pathogen of cats with a pathogenesis similar to that of HHV-1 ocular disease. In contrast to what was reported for HHV-1, we were unable to select for a raltegravir-resistant FeHV-1 strain in order to define any basis for drug action. A candidate-based approach to explore the mode of action of raltegravir against FeHV-1 showed that raltegravir did not impact FeHV-1 terminase function, as described for HHV-5. Instead, raltegravir inhibited DNA replication, similarly to HHV-1, but by targeting the initiation of viral DNA replication rather than elongation. In addition, we found that raltegravir specifically repressed late gene expression independently of DNA replication, and both activities are consistent with inhibition of ICP8. Taken together, these results suggest that raltegravir could be a valuable therapeutic agent against herpesviruses. IMPORTANCE The rise of drug-resistant herpesviruses is a longstanding concern, particularly among immunocompromised patients. Therefore, therapies targeting viral proteins other than the DNA polymerase that may be less likely to lead to drug-resistant viruses are urgently needed. Using FeHV-1, an alphaherpesvirus closely related to HHV-1 that similarly causes ocular herpes in its natural host, we found that the HIV integrase inhibitor raltegravir targets different stages of the virus life cycle beyond DNA replication and that it does so without developing drug resistance under the conditions tested. This shows that the drug could provide a viable strategy for the treatment of herpesvirus infections.

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, R0, of between 1 and 1.5 across nearly all U.S. outbreaks, consistent with maintained but heterogeneous circulation. We propose that CIV9s 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. IMPORTANCE 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.