Unrecognized diversity of mammalian orthoreoviruses in North American bats.

Bats have recently been identified as potential reservoir hosts for mammalian orthoreoviruses (MRVs) throughout Europe and China. Here we present the first evolutionary and biological characterization of bat-borne MRVs in North America, including phylogenomic analysis, in vitro relative infectivity in bat and other mammalian cell cultures, host cell receptor specificity, and epifluorescence microscopy of viral factory formation. Through genetic and phylogenetic comparisons, we show that two divergent MRV serotype 2 (T2) strains - isolated from a silver-haired bat (Lasionycteris noctivagans) and a big brown bat (Eptesicus fuscus) from Pennsylvania, USA - provide an evolutionary link to an MRV strain (T2W) recovered from an 8-week-old infant who died in Winnipeg, Manitoba, Canada in 1997. Although these findings suggest North American bats may represent a previously unrecognized source for the cross-species transmission of MRVs to other animals, including humans, the ecology and epidemiology of MRVs in wildlife remain enigmatic.

Distribution of O-Acetylated Sialic Acids among Target Host Tissues for Influenza Virus.

Sialic acids (Sias) are important glycans displayed on the cells and tissues of many different animals and are frequent targets for binding and modification by pathogens, including influenza viruses. Influenza virus hemagglutinins bind Sias during the infection of their normal hosts, while the encoded neuraminidases and/or esterases remove or modify the Sia to allow virion release or to prevent rebinding. Sias naturally occur in a variety of modified forms, and modified Sias can alter influenza virus host tropisms through their altered interactions with the viral glycoproteins. However, the distribution of modified Sia forms and their effects on pathogen-host interactions are still poorly understood. Here we used probes developed from viral Sia-binding proteins to detect O-acetylated (4-O-acetyl, 9-O-acetyl, and 7,9-O-acetyl) Sias displayed on the tissues of some natural or experimental hosts for influenza viruses. These modified Sias showed highly variable displays between the hosts and tissues examined. The 9-O-acetyl (and 7,9-) modified Sia forms were found on cells and tissues of many hosts, including mice, humans, ferrets, guinea pigs, pigs, horses, dogs, as well as in those of ducks and embryonated chicken egg tissues and membranes, although in variable amounts. The 4-O-acetyl Sias were found in the respiratory tissues of fewer animals, being primarily displayed in the horse and guinea pig, but were not detected in humans or pigs. The results suggest that these Sia variants may influence virus tropisms by altering and selecting their cell interactions. IMPORTANCE Sialic acids (Sias) are key glycans that control or modulate many normal cell and tissue functions while also interacting with a variety of pathogens, including many different viruses. Sias are naturally displayed in a variety of different forms, with modifications at several positions that can alter their functional interactions with pathogens. In addition, Sias are often modified or removed by enzymes such as host or pathogen esterases or sialidases (neuraminidases), and Sia modifications can alter those enzymatic activities to impact pathogen infections. Sia chemical diversity in different hosts and tissues likely alters the pathogen-host interactions and influences the outcome of infection. Here we explored the display of 4-O-acetyl, 9-O-acetyl, and 7,9-O-acetyl modified Sia forms in some target tissues for influenza virus infection in mice, humans, birds, guinea pigs, ferrets, swine, horses, and dogs, which encompass many natural and laboratory hosts of those viruses.

Parvovirus Capsid Structures Required for Infection: Mutations Controlling Receptor Recognition and Protease Cleavages.

Parvovirus capsids are small but complex molecular machines responsible for undertaking many of the steps of cell infection, genome packing, and cell-to-cell as well as host-to-host transfer. The details of parvovirus infection of cells are still not fully understood, but the processes must involve small changes in the capsid structure that allow the endocytosed virus to escape from the endosome, pass through the cell cytoplasm, and deliver the single-stranded DNA (ssDNA) genome to the nucleus, where viral replication occurs. Here, we examine capsid substitutions that eliminate canine parvovirus (CPV) infectivity and identify how those mutations changed the capsid structure or altered interactions with the infectious pathway. Amino acid substitutions on the exterior surface of the capsid (Gly299Lys/Ala300Lys) altered the binding of the capsid to transferrin receptor type 1 (TfR), particularly during virus dissociation from the receptor, but still allowed efficient entry into both feline and canine cells without successful infection. These substitutions likely control specific capsid structural changes resulting from TfR binding required for infection. A second set of changes on the interior surface of the capsid reduced viral infectivity by >100-fold and included two cysteine residues and neighboring residues. One of these substitutions, Cys270Ser, modulates a VP2 cleavage event found in ∼10% of the capsid proteins that also was shown to alter capsid stability. A neighboring substitution, Pro272Lys, significantly reduced capsid assembly, while a Cys273Ser change appeared to alter capsid transport from the nucleus. These mutants reveal additional structural details that explain cell infection processes of parvovirus capsids. IMPORTANCE: Parvoviruses are commonly found in both vertebrate and invertebrate animals and cause widespread disease. They are also being developed as oncolytic therapeutics and as gene therapy vectors. Most functions involved in infection or transduction are mediated by the viral capsid, but the structure-function correlates of the capsids and their constituent proteins are still incompletely understood, especially in relation to identifying capsid processes responsible for infection and release from the cell. Here, we characterize the functional effects of capsid protein mutations that result in the loss of virus infectivity, giving a better understanding of the portions of the capsid that mediate essential steps in successful infection pathways and how they contribute to viral infectivity.

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.

The influenza PA-X protein is translated from the PA open reading frame from frameshifting and suppresses cellular gene expression due to its ribonuclease activity. We further defined the functional roles of PA-X by comparing PA-X proteins from two related viruses - equine influenza (EIV) and canine influenza (CIV) H3N8 - that differ in a C-terminal truncation and internal mutations. In vitro reporter gene assays revealed that both proteins were able to suppress gene expression. Interestingly, EIV PA-X demonstrated ~50% greater activity compared to CIV PA-X, and we identified the mutations that caused this difference. We used RNA-seq to evaluate the effects of PA-X on host gene expression after transfection into cultured cells. There were no significant differences in this property between EIV and CIV PA-X proteins, but expression of either resulted in the up-regulation of genes when compared to controls, most notably immunity-related proteins, trafficking proteins, and transcription factors.

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

UNLABELLED: 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 α2-3- over α2-6-linked sialic acids for infections, and glycan microarray analysis showed that EIV and CIV HA-Fc fusion proteins bound only to α2-3-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. IMPORTANCE: Influenza 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 cross-species transmission and adaptation of influenza viruses may be mediated by subtle changes in virus biology.