Circumstantial evidence has linked a new group of nidoviruses with respiratory disease in pythons, lizards, and cattle. We conducted experimental infections in ball pythons (Python regius) to test the hypothesis that ball python nidovirus (BPNV) infection results in respiratory disease. Three ball pythons were inoculated orally and intratracheally with cell culture isolated BPNV and two were sham inoculated. Antemortem choanal, oroesophageal, and cloacal swabs and postmortem tissues of infected snakes were positive for viral RNA, protein, and infectious virus by qRT-PCR, immunohistochemistry, western blot and virus isolation. Clinical signs included oral mucosal reddening, abundant mucus secretions, open-mouthed breathing, and anorexia. Histologic lesions included chronic-active mucinous rhinitis, stomatitis, tracheitis, esophagitis and proliferative interstitial pneumonia. Control snakes remained negative and free of clinical signs throughout the experiment. Our findings establish a causal relationship between nidovirus infection and respiratory disease in ball pythons and shed light on disease progression and transmission.

The aim of this study of serpentovirus infection in captive snakes was to assess the susceptibility of different types of snakes to infection and disease, to survey viral genetic diversity, and to evaluate management practices that may limit infection and disease. Antemortem oral swabs were collected from 639 snakes from 12 US collections, including 62 species, 28 genera, and 6 families: Pythonidae (N = 414 snakes; pythons were overrepresented in the sample population), Boidae (79), Colubridae (116), Lamprophiidae (4), Elapidae (12), and Viperidae (14). Infection was more common in pythons (38%; 95% CI: 33.1–42.4%), and in boas (10%; 95% CI: 5.2–18.7%) than in colubrids (0.9%, 95% CI: <0.01–4.7%); infection was not detected in other snake families (lamprophiids 0/4, 95% CI: 0–49%; elapids 0/12, 95% CI: 0–24.2%; and vipers 0/14, 95% CI: 0–21.5%), but more of these snakes need to be tested to confirm these findings. Clinical signs of respiratory disease were common in infected pythons (85 of 144). Respiratory signs were only observed in 1 of 8 infected boas and were absent in the single infected colubrid. Divergent serpentoviruses were detected in pythons, boas, and colubrids, suggesting that different serpentoviruses might vary in their ability to infect snakes of different families. Older snakes were more likely to be infected than younger snakes (p-value < 0.001) but males and females were equally likely to be infected (female prevalence: 23.4%, 95% CI 18.7–28.9%; male prevalence: 23.5%, 95% CI 18–30.1%; p-value = 0.144). Neither age (p-value = 0.32) nor sex (p-value = 0.06) was statistically associated with disease severity. Longitudinal sampling of pythons in a single collection over 28 months revealed serpentovirus infection is persistent, and viral clearance was not observed. In this collection, infection was associated with significantly increased rates of mortality (p-value = 0.001) with death of 75% of infected pythons and no uninfected pythons over this period. Offspring of infected parents were followed: vertical transmission either does not occur or occurs with a much lower efficiency than horizontal transmission. Overall, these findings confirm that serpentoviruses pose a significant threat to the health of captive python populations and can cause infection in boa and colubrid species.

Nonenveloped viral capsids are metastable structures that undergo conformational changes during virus entry that lead to interactions of the capsid or capsid fragments with the cell membrane. For members of the Caliciviridae, neither the nature of these structural changes in the capsid nor the factor(s) responsible for inducing these changes is known. Feline junctional adhesion molecule A (fJAM-A) mediates the attachment and infectious viral entry of feline calicivirus (FCV). Here, we show that the infectivity of some FCV isolates is neutralized following incubation with the soluble receptor at 37°C. We used this property to select mutants resistant to preincubation with the soluble receptor. We isolated and sequenced 24 soluble receptor-resistant (srr) mutants and characterized the growth properties and receptor-binding activities of eight mutants. The location of the mutations within the capsid structure of FCV was mapped using a new 3.6-Å structure of native FCV. The srr mutations mapped to the surface of the P2 domain were buried at the protruding domain dimer interface or were present in inaccessible regions of the capsid protein. Coupled with data showing that both the parental FCV and the srr mutants underwent increases in hydrophobicity upon incubation with the soluble receptor at 37°C, these findings indicate that FCV likely undergoes conformational change upon interaction with its receptor. Changes in FCV capsid conformation following its interaction with fJAM-A may be important for subsequent interactions of the capsid with cellular membranes, membrane penetration, and genome delivery.The interactions between viruses and receptors on the surface of host cells strongly influence viral pathogenesis and regulate morbidity and mortality in the host. Virus-receptor interactions determine the types of cells that can be infected, the pathway of entry into the cell, and the efficiency of productive infection. Interactions between nonenveloped virus capsids and their receptor(s) trigger one or more steps required for infectious entry. These steps can include interaction with other receptors, exposure to low pH or endosomal proteases, or other factors. Ultimately, one or more of these interactions induce changes in capsid conformation that result in the exposure of hydrophobic regions or release of a lipidseeking factor that can interact with and disrupt the limiting cellular membrane to allow the capsid and/or the genome to be delivered to the interior of the cell (reviewed in reference 60).The Caliciviridae are small nonenveloped viruses containing a positive-sense RNA genome (ϳ7 to 8 kb). Several important disease-causing members of the Caliciviridae, including human noroviruses and rabbit hemorrhagic disease virus, cannot be propagated in tissue culture systems (19,56). This has slowed progress on studies of the mechanisms of cellular entry of these viruses. In contrast, feline caliciviruses (FCVs) propagate readily in tissue culture, and two cell surface receptor molecules, feline junctional adhesion molec...

Canine distemper virus (CDV) has recently emerged as an extinction threat for the endangered Amur tiger (Panthera tigris altaica). CDV is vaccine-preventable, and control strategies could require vaccination of domestic dogs and/or wildlife populations. However, vaccination of endangered wildlife remains controversial, which has led to a focus on interventions in domestic dogs, often assumed to be the source of infection. Effective decision making requires an understanding of the true reservoir dynamics, which poses substantial challenges in remote areas with diverse host communities. We carried out serological, demographic, and phylogenetic studies of dog and wildlife populations in the Russian Far East to show that a number of wildlife species are more important than dogs, both in maintaining CDV and as sources of infection for tigers. Critically, therefore, because CDV circulates among multiple wildlife sources, dog vaccination alone would not be effective at protecting tigers. We show, however, that low-coverage vaccination of tigers themselves is feasible and would produce substantive reductions in extinction risks. Vaccination of endangered wildlife provides a valuable component of conservation strategies for endangered species.

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.

During the past decade, several outbreaks of severe systemic disease associated with Feline calicivirus (FCV) have occurred in the USA and the UK. This new disease has caused high mortality in the affected animals and has been termed virulent systemic (VS)-FCV disease. Currently, there are no genetic or in vitro diagnostic methods to distinguish viruses isolated from cases of VS-FCV disease from other isolates. Here, five in vitro properties, as well as the capsid and proteinase-polymerase (pro-pol) sequences, of a set of FCV isolates that included seven isolates from five distinct VS-FCV outbreaks ('VS isolates') were investigated. Although all of the FCV isolates investigated had similar kinetics of growth under single-cycle conditions, VS isolates infected tissue-culture cells more efficiently under multiple-cycle growth conditions. Moreover, it was found that cells infected with VS isolates showed cytopathic effects earlier than cells infected with non-VS isolates, although no difference in relative ATP levels were noted at times when morphological changes were first seen. Both VS-and other (non-VS) isolates of FCV demonstrated similar temperature stabilities. Phylogenetic analyses and alignments of the capsid and pro-pol regions of the genome did not reveal any conserved changes that correlated with virulence, and the VS isolates did not segregate into a unique clade. These results suggest that VS isolates have arisen independently several times since first being described and can spread more efficiently in tissue culture than other isolates when infected at low multiplicity.

BackgroundRespiratory infections are important causes of morbidity and mortality in reptiles; however, the causative agents are only infrequently identified.FindingsPneumonia, tracheitis and esophagitis were reported in a collection of ball pythons (Python regius). Eight of 12 snakes had evidence of bacterial pneumonia. High-throughput sequencing of total extracted nucleic acids from lung, esophagus and spleen revealed a novel nidovirus. PCR indicated the presence of viral RNA in lung, trachea, esophagus, liver, and spleen. In situ hybridization confirmed the presence of intracellular, intracytoplasmic viral nucleic acids in the lungs of infected snakes. Phylogenetic analysis based on a 1,136 amino acid segment of the polyprotein suggests that this virus may represent a new species in the subfamily Torovirinae.ConclusionsThis report of a novel nidovirus in ball pythons may provide insight into the pathogenesis of respiratory disease in this species and enhances our knowledge of the diversity of nidoviruses.

The feline junctional adhesion molecule A (fJAM-A) is a functional receptor for feline calicivirus (FCV).fJAM-A is a member of the immunoglobulin superfamily (IgSF) and consists of two Ig-like extracellular domains (D1 and D2), a membrane-spanning domain, and a short cytoplasmic tail. To identify regions of fJAM-A that interact with FCV, we purified recombinant fJAM-A ectodomain and D1 and D2 domains. We found that preincubation of FCV with the ectodomain or D1 was sufficient to inhibit FCV infection in plaque reduction assays. In enzyme-linked immunosorbent assays, FCV binding to fJAM-A ectodomain was concentration dependent and saturable; however, FCV bound D1 alone weakly and was unable to bind D2. To characterize FCV binding to surface-expressed fJAM-A, we transfected truncated and chimeric forms of fJAM-A into a nonpermissive cell line and assayed binding by flow cytometry. Only D1 was necessary for FCV binding to cells; all other domains could be replaced. Using a structure-guided mutational approach, we identified three mutants of fJAM-A within D1 (D42N, K43N, and S97A) that exhibited significantly decreased capacities to bind FCV. In contrast to our finding that D1 mediated FCV binding, we found that all domains of fJAM-A were necessary to confer susceptibility to FCV infection. Furthermore, surface expression of fJAM-A was not sufficient to permit FCV infection by all of the isolates we investigated. This indicates that (i) other cellular factors are required to permit productive FCV infection and (ii) individual FCV isolates differ in the factors they require.