Brian R. Wasik

Emergence of epidemic viruses in new hosts threatens both human and other 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 y, 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 y 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 less than one-third the rate observed for CPV in dogs, and the higher rate of CPV evolution has been 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 y old, little selection for antigenic variation was observed. The distinct evolutionary patterns of these closely related viruses circulating for decades in different hosts emphasize the complex evolution associated with viral epidemic emergence and spread in endemic and new hosts.

Sustained mammal-to-mammal transmission of high pathogenicity H5N1avian influenza viruses is reshaping the host range of these pathogens. One of the longest-running mammalian transmission chains involves the B3.13 genotype circulating in U.S. dairy cattle which was detected early in 2024. Genomic analysis revealed selection and rapid fixation of haemagglutinin mutations D104G and V147M. We demonstate, via glycomic profiling, that bovine tissues, including the mammary gland, are enriched in N- and O-linked glycans capped with N-glycolylneuraminic acid (NeuGc), a sialic acid absent in humans and birds, which instead express only N-acetylneuraminic acid (NeuAc). Early cattle H5 viruses poorly recognized NeuGc, but D104G and V147M enabled efficient engagement of both NeuAc- and NeuGc-containing receptors. These mutations enhanced replication in bovine mammary tissue without major attenuation of replication in human lung and primary nasal epithelial cells. NeuGc-driven receptor adaptation therefore promotes viral fitness in cattle while potentially limiting immediate zoonotic risk. Deep mutational scanning further identifies alternative haemagglutinin substitutions that confer NeuGc usage and represent surveillance markers for emerging cattle H5 lineages.

The intestinal mucus network, primarily composed of O -glycosylated MUC2 mucin polymers, is essential for protecting the gastrointestinal tract from microbial threats. Sialic acid (Sia), a terminal monosaccharide on complex O -glycans, plays a key role in maintaining mucus integrity and is frequently modified by Casd1-dependent O -acetylation ( O Ac). Despite its prevalence, the biological significance of sialic acid O Ac ( O Ac-Sia) modifications in human and murine mucus remains unclear. We hypothesized that O Ac-Sia variants on mucus interact with the microbiota and are required for optimal mucus barrier function and host-microbe homeostasis in the colon. To test this, we profiled O Ac-Sia on human and mouse MUC2 in situ using viral-derived probes with bacterial FISH and confocal microscopy; generated intestinal epithelial cell (IEC)-specific Casd1 null mice (IEC Casd1 ^-/- ); performed sialylomic and O -glycomic HPLC-MS analyses; assessed microbial communities by 16S rRNA sequencing with quantitative microbial profiling (QMP); and evaluated disease susceptibility using DSS colitis and Citrobacter rodentium infection models. Results revealed that both human and murine mucins are extensively O -acetylated and interact with the microbiota, suggesting biological relevance. IEC Casd1 ^-/- mice were viable and displayed a complete loss of mucin O Ac-Sia, indicating Casd1 is the sole contributor to O Ac-status. Unexpectedly, mucus function was intact in IEC Casd1 ^-/- mice, with no difference in structure or quality vs. WT co-housed littermates.16S rRNA analysis showed a modest but significant sex-specific reduction of microbial loads in male IEC Casd1 ^-/- mice, and a clear trend toward reduced Turicibacter spp. vs. WT mice in both male and females, without impacting overall short-chain fatty acid (SCFA) production. DSS treatment led to more severe and extensive tissue damage in IEC Casd1 ^-/- mice. C. rodentium infection led to increased damage in the cecum and distal colon of IEC Casd1 ^-/- mice without affecting pathogen load, suggesting that O Ac-Sia status has a role in tolerance defense. These findings establish intestinal epithelial Sia O -acetylation as a component dispensable for mucus and host-microbe homeostasis at baseline, but important in limiting damage to mucosal inflammatory insults.

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)

Human and avian influenza A viruses bind to sialic acid (Sia) receptors on cells as their primary receptors, and this results in endocytic uptake of the virus. While the role of Sia on glycoproteins and/or glycolipids for virus entry is crucial, the roles of the carrier proteins are still not well understood. Furthermore, it is still unclear how receptor binding leads to infection, including whether the receptor plays a structural or other roles beyond being a simple tether. To enable the investigation of the receptor binding and cell entry processes in a more controlled manner, we have designed a protein receptor for pandemic H1 influenza A viruses. The engineered receptor possesses the binding domains of an anti-HA antibody prepared as a single-chain variable fragment (scFv) fused with the stalk, transmembrane, and cytoplasmic sequences of the feline transferrin receptor type-1 (fTfR). When expressed in cells that lack efficient display of Sia due to a knockout of the Slc35A1 gene, which encodes for the solute carrier family 35 transporter (SLC35A1), the anti-H1 receptor was displayed on the cell surface, bound virus, or hemagglutinin proteins, and the virus was efficiently endocytosed into the cells. Infection occurred at similar levels to those seen after reintroducing Sia expression, and lower affinity receptor mutants displayed enhanced infections. Treatment with clathrin-mediated endocytosis (CME) inhibitors significantly reduced viral entry, indicating that virus rescue by the antibody-based receptor follows a similar internalization route as Sia-expressing cells. IMPORTANCE: Influenza A viruses primarily circulate among avian reservoir hosts but can also jump species, causing outbreaks in mammals, including humans. A key interaction of the viruses is with host cell sialic acids, which vary in chemical form, in their linkages within the oligosaccharide, and in their display on various surface glycoproteins or glycolipids with differing properties. Here, we report a new method for examining the processes of receptor binding and uptake into cells during influenza A virus infection, by use of an engineered HA-binding membrane glycoprotein, where antibody variable domains are used to bind the virus, and the transferrin receptor uptake structures mediate efficient entry. This will allow us to test and manipulate the processes of cell binding, entry, and infection.

The H3N2 canine influenza virus (CIV) emerged from an avian reservoir in Asia to circulate entirely among dogs for the last 20 years. The virus was first seen circulating outside Asian dog populations in 2015, in North America. Utilizing viral genomic data in addition to clinical reports and diagnostic testing data, we provide an updated analysis of the evolution and epidemiology of the virus in its canine host. CIV in dogs in North America is marked by a complex life history – including local outbreaks, regional lineage die-outs, and repeated reintroductions of the virus (with diverse genotypes) from different regions of Asia. Phylogenetic and Bayesian analysis reveal multiple CIV clades, and viruses from China have seeded recent North American outbreaks, with 2 or 3 introductions in the past 3 years. Genomic epidemiology confirms that within North America the virus spreads very rapidly among dogs in kennels and shelters in different regions – but then dies out locally. The overall epidemic therefore requires longer-distance dispersal of virus to maintain outbreaks over the long term. With a constant evolutionary rate over 20 years, CIV still appears best adapted to transmission in dense populations and has not gained properties for prolonged circulation among dogs.

Human and avian influenza A viruses bind to sialic acid (Sia) receptors on cells as their primary receptors, and this results in endocytic uptake of the virus. While the role of Sia on glycoproteins and/or glycolipids for virus entry is crucial, the roles of the carrier proteins are still not well understood. Furthermore, it is still unclear how receptor binding leads to infection, including whether the receptor plays a structural or other roles beyond being a simple tether. To enable the investigation of the receptor binding and cell entry processes in a more controlled manner, we have designed a protein receptor for pandemic H1 influenza A viruses. The engineered receptor possesses the binding domains of an anti-HA antibody prepared as a single chain variable fragment (scFv) fused with the stalk, transmembrane and cytoplasmic sequences of the feline transferrin receptor type-1 (fTfR). When expressed in cells that lack efficient display of Sia due to a knockout of the Slc35A1 gene which encodes for the Solute Carrier Family 35 transporter (SLC35A1), the anti-H1 receptor was displayed on the cell surface, bound virus or hemagglutinin proteins, and the virus was efficiently endocytosed into the cells. Infection occurred at similar levels to those seen after Sia reconstitution, and treatment with clathrin-mediated endocytosis (CME) inhibitors significantly reduced viral entry. IMPORTANCE.: Influenza A viruses mostly circulate among avian reservoirs, and also can jump hosts to cause epidemics in mammals, including among humans. A key interaction of the viruses is with host cell Sia, which vary in chemical form, in their linkages within the oligosaccharide, and in the attachment to surface glycoproteins or glycolipids with different properties. Here we report a new method for examining the processes of receptor binding and uptake into cells during influenza A virus infection, by use of an engineered HA-binding membrane glycoprotein, where an antibody is used as the binding domain and the transferrin receptor uptake structures mediate efficient entry, which should allow us to test and manipulate the processes of cell binding, entry, and infection.

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.

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.