Ian Eugene Huber Voorhees

Emergence of epidemic viruses in new hosts threatens both human and animal populations, and often involves virus evolution to overcome barriers that normally prevent efficient infection and spread in that host. After transfer the separated viruses will evolve in parallel as they spread within the original and new hosts. Here we examine the details of a virus involved in such a host-jumping event, where we define the natural evolution of feline panleukopenia virus (FPV) over 60 years, clarify the origins of the new pandemic canine parvovirus (CPV) that arose in the 1970s, and compare the separate evolution of those viruses over 47 years in cats or dogs. Several live-attenuated FPV vaccine viruses originated from early 1960s isolates or were a recombinant of an early virus, and many sequences in databases proved to be vaccine-derived. The sequences of wild viruses showed that FPV-like strains evolved at ∼25% the rate seen for CPV in dogs, and the higher rate of CPV evolution was consistent since 1979 when a genetic variant became widespread. The common ancestor of the CPV lineage was related to FPVs from Europe, and contained several unique host-adaptive capsid changes associated with canine transferrin receptor type-1 binding. Although the FPV vaccine strains are around 60 years old, little selection for antigenic variation was observed. The distinct evolutionary patterns of these closely related viruses circulating for decades in different hosts emphasizes the complex evolution associated with viral epidemic emergence and spread in endemic and new hosts. SIGNIFICANCE STATEMENT: Comparing the evolution of a virus in its reservoir host with that seen in a new host will reveal the special circumstances that allow epidemic emergence. A feline parvovirus (FPV) jumping to dogs in the mid-1970s formed canine parvovirus (CPV), which has circulated world-wide until today. The evolutionary rate of FPV in its original hosts was much lower than that of CPV in dogs, and the mutational patterns seen in the different hosts were also distinct. Early CPV isolates differed from the ancestral FPV clade in several key host range mutations. These results highlight the complex biology associated with epidemic emergence, including host-specific rates of lineage evolution and complex origins of host-adaptive mutations. (113/120)

The H3N2 canine influenza virus (CIV) emerged from an avian reservoir in Asia around 2004. As the virus has now been circulating entirely among dogs for 20 years, we here update our understanding of the evolution of virus in its new host. As a host-switched virus, H3N2 CIV will also reveal any host-adaptive changes arising during thousands of infections within its new host, and our analysis showed that the virus has evolved at a constant rate. CIV was first introduced into North America in 2015 from Korea, and we specifically examined the epidemiology of the virus among dogs in North America since then, including local outbreaks, regional die-outs, and repeated reintroduction from Asia. The H3N2 CIV now appears endemic only in China after dying out in South Korea around 2017. Virus lineages circulating in China appear to have seeded the most recent US outbreaks – with 2 or 3 introductions into North America during the past 3 years. Combining clinical reports, diagnostic testing data, and analysis of viral genomes we show that the virus spreads rapidly among dogs in kennels and shelters in different regions – likely dying out locally after all those animals become infected and immune. The overall epidemic therefore requires longer-distance dispersal of virus to initiate outbreaks in new locations. Patterns of spread in the USA may select viruses most adapted to those dense populations, which may lack the properties required for efficient long-distance transfers to other dog populations that would keep the virus in prolonged circulation. IMPORTANCE Viruses occasionally jump into new hosts to cause epidemics and may spread widely due to movement of humans or animals, or their viruses, with profound consequences for global health. The emergence and epidemiology of new epidemic viruses in companion animals provides a model for understanding disease dynamics and evolution. The H3N2 canine influenza virus arose from an avian virus, and infected dogs provide many opportunities for human exposure. H3N2 CIV transmission is dominated by fast-moving outbreaks within dense populations in animal shelters or kennels, while sustaining the epidemic likely requires movement of virus to more distant dog populations. Viral spread within North Americahas only been sustained for a few years at a time after which the virus dies out. The epidemiological and evolutionary dynamics of this virus in this structured host population shows how an acute respiratory pathogen can emerge and spread in a new host and population.

The emergence of previously unknown disease-causing viruses in mammals is in part the result of a long-term evolutionary process. Reconstructing the deep phylogenetic histories of viruses helps identify major evolutionary transitions and contextualizes the emergence of viruses in new hosts. We used a combination of total RNA sequencing and transcriptome data mining to extend the diversity and evolutionary history of the RNA virus order Articulavirales , which includes the influenza viruses. We identified instances of Articulavirales in the invertebrate phylum Cnidaria (including corals), constituting a novel and divergent family that we provisionally named the “Cnidenomoviridae.” We further extended the evolutionary history of the influenza virus lineage by identifying four divergent, fish-associated influenza-like viruses, thereby supporting the hypothesis that fish were among the first hosts of influenza viruses. In addition, we substantially expanded the phylogenetic diversity of quaranjaviruses and proposed that this genus be reclassified as a family—the “Quaranjaviridae.” Within this putative family, we identified a novel arachnid-infecting genus, provisionally named “Cheliceravirus.” Notably, we observed a close phylogenetic relationship between the Crustacea- and Chelicerata-infecting “Quaranjaviridae” that is inconsistent with virus–host codivergence. Together, these data suggest that the Articulavirales has evolved over at least 600 million years, first emerging in aquatic animals. Importantly, the evolution of the Articulavirales was likely shaped by multiple aquatic–terrestrial transitions and substantial host jumps, some of which are still observable today.

Cross-species virus transmission events can lead to dire public health emergencies in the form of epidemics and pandemics. One example in animals is the emergence of the H3N8 equine influenza virus (EIV), first isolated in 1963 in Miami, FL, USA, after emerging among horses in South America. In the early 21st century, the American lineage of EIV diverged into two ‘Florida’ clades that persist today, while an EIV transferred to dogs around 1999 and gave rise to the H3N8 canine influenza virus (CIV), first reported in 2004. Here, we compare CIV in dogs and EIV in horses to reveal their host-specific evolution, to determine the sources and connections between significant outbreaks, and to gain insight into the factors controlling their different evolutionary fates. H3N8 CIV only circulated in North America, was geographically restricted after the first few years, and went extinct in 2016. Of the two EIV Florida clades, clade 1 circulates widely and shows frequent transfers between the USA and South America, Europe and elsewhere, while clade 2 was globally distributed early after it emerged, but since about 2018 has only been detected in Central Asia. Any potential zoonotic threat of these viruses to humans can only be determined with an understanding of its natural history and evolution. Our comparative analysis of these three viral lineages reveals distinct patterns and rates of sequence variation yet with similar overall evolution between clades, suggesting epidemiological intervention strategies for possible eradication of H3N8 EIV.

Cross-species virus transmission events can lead to dire public health emergencies in the form of epidemics and pandemics. One example in animals is the emergence of the H3N8 equine influenza virus (EIV), first isolated in 1963 in Miami, Florida, USA, after emerging among horses in South America. In the early 21 ^st century the American lineage of EIV diverged into two ‘Florida’ clades that persist today, while an EIV transferred to dogs around 1999 and gave rise to the H3N8 canine influenza virus (CIV), first reported in 2004. Here, we compare CIV in dogs and EIV in horses to reveal their host-specific evolution, to determine the sources and connections between significant outbreaks, and to gain insight into the factors controlling their different evolutionary fates. H3N8 CIV only circulated in North America, was geographically restricted after the first few years, and went extinct in 2016. Of the two EIV Florida clades, clade 1 circulates widely and shows frequent transfers between the USA and South America, Europe and elsewhere, while clade 2 was globally distributed early after it emerged, but since about 2018 has only been detected in Central Asia. Any potential zoonotic threat of these viruses to humans can only be determined with an understanding of its natural history and evolution. Our comparative analysis of these three viral lineages reveals distinct patterns and rates of sequence variation yet with similar overall evolution between clades, suggesting epidemiological intervention strategies for possible eradication of H3N8 EIV. (242 words) IMPORTANCE: The emergence of viruses in new hosts is a threat to human and animal health. The H3N8 equine influenza virus (EIV) emerged in 1963 by transfer of an avian influenza virus, and the H3N8 canine influenza virus (CIV) subsequently emerged in 1999 when EIV transferred to dogs. H3N8 CIV persistently circulated in only a few locations in the USA, and has not been detected since 2016. In the same period H3N8 EIV has circulated as two separate clades, one in North America and other regions of the world, while the other currently appears to be found only in Central Asia. By comparing the hosts, epidemiology, and evolution of these influenza viruses we explain how these lineages had different evolutionary fates, and show why elucidating these evolutionary processes is key to understanding zoonotic disease and viral emergence. (137 words)

Canine parvovirus (CPV) is a small non-enveloped single-stranded DNA virus that causes serious diseases in dogs worldwide. The original strain of the virus (CPV-2) emerged in dogs during the late-1970s due to a host range switch of a virus similar to the feline panleukopenia virus (FPV) that infected another host. The virus that emerged in dogs had altered capsid receptor- and antibody-binding sites, with some changes affecting both functions. Further receptor and antibody binding changes arose when the virus became better adapted to dogs or to other hosts. Here, we use in vitro selection and deep sequencing to reveal how two antibodies with known interactions select for escape mutations in CPV. The antibodies bind two distinct epitopes, and one largely overlaps the host receptor binding site. We also engineered antibody variants with altered binding structures. Viruses were passaged with the wild type or mutated antibodies, and their genomes deep sequenced during the selective process. A small number of mutations were detected only within the capsid protein gene during the first few passages of selection, and most sites remained polymorphic or were slow to go to fixation. Mutations arose both within and outside the antibody binding footprints on the capsids, and all avoided the TfR-binding footprint. Many selected mutations matched those that have arisen in the natural evolution of the virus. The patterns observed reveal the mechanisms by which these variants have been selected in nature and provide a better understanding of the interactions between antibody and receptor selections. IMPORTANCE: Antibodies protect animals against infection by many different viruses and other pathogens, and we are gaining new information about the epitopes that induce antibody responses against viruses and the structures of the bound antibodies. However, less is known about the processes of antibody selection and antigenic escape and the constraints that apply in this system. Here, we use an in vitro model system and deep genome sequencing to reveal the mutations that arise in the virus genome during selection by each of two monoclonal antibodies or their engineered variants. High-resolution structures of each of the Fab: capsid complexes revealed their binding interactions. The engineered forms of the wild-type antibodies or mutant forms allowed us to examine how changes in antibody structure influence the mutational selection patterns seen in the virus. The results shed light on the processes of antibody binding, neutralization escape, and receptor binding, and likely have parallels for many other viruses.

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.

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. 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.

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 virus in vitro are 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 or N -glycolylneuraminic acid (Neu5Gc) or N -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 expressing α 2,6-linkages. IMPORTANCE: The 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.

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.