Robert J. Ossiboff

Conformational Changes in the Capsid of a Calicivirus upon Interaction with Its Functional Receptor

ABSTRACT 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.

Identification of regions and residues in feline junctional adhesion molecule required for feline calicivirus binding and infection.

ABSTRACT 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.

Lability in Host Defenses: Terrestrial Frogs Die from Chytridiomycosis under Enzootic Conditions

Chytridiomycosis-induced mortalities often occur upon the emergence of Batrachochytrium dendrobatidis (Bd) in naïve amphibian populations. We report chytridiomycosis-associated mortalities in the wild of the coqui (Eleutherodactylus coqui), a declining direct-developing frog with persistent Bd infections. These findings provide additional evidence of decreased host defenses during cool-dry seasons in Puerto Rico.

Identification of a novel nidovirus in an outbreak of fatal respiratory disease in ball pythons (Python regius)

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.

A Mycoplasma Species of Emydidae Turtles in the Northeastern USA

Abstract Mycoplasma infections can cause significant morbidity and mortality in captive and wild chelonians. As part of a health assessment of endangered bog turtles (Glyptemys muhlenbergii) in the northeastern US, choanal and cloacal swabs from these and other sympatric species, including spotted turtles (Clemmys guttata), eastern box turtles (Terrapene carolina carolina), wood turtles (Glyptemys insculpta), and common snapping turtles (Chelydra serpentina) from 10 sampling sites in the states (US) of Delaware, New Jersey, and Pennsylvania, were tested by PCR for Mycoplasma. Of 108 turtles tested, 63 (58.3%) were PCR positive for Mycoplasma including 58 of 83 bog turtles (70%), three of three (100%) eastern box turtles, and two of 11 (18%) spotted turtles; all snapping turtles (n = 7) and wood turtles (n = 4) were negative. Sequence analysis of portions of the 16S–23S intergenic spacer region and the 16S ribosomal RNA gene revealed a single, unclassified species of Mycoplasma that has been previously reported in eastern box turtles, ornate box turtles (Terrapene ornata ornata), western pond turtles (Emys marmorata), and red-eared sliders (Trachemys scripta elegans). We document a high incidence of Mycoplasma, in the absence of clinical disease, in wild emydid turtles. These findings, along with wide distribution of the identified Mycoplasma sp. across a broad geographic region, suggest this bacterium is likely a commensal inhabitant of bog turtles, and possibly other species of emydid turtles, in the northeastern US.

Three Novel Herpesviruses of Endangered Clemmys and Glyptemys Turtles

The rich diversity of the world’s reptiles is at risk due to significant population declines of broad taxonomic and geographic scope. Significant factors attributed to these declines include habitat loss, pollution, unsustainable collection and infectious disease. To investigate the presence and significance of a potential pathogen on populations of critically endangered bog turtles (Glyptemys muhlenbergii) as well sympatric endangered wood (G. insculpta) and endangered spotted (Clemmys guttata) turtles in the northeastern United States, choanal and cloacal swabs collected from 230 turtles from 19 sites in 5 states were screened for herpesvirus by polymerase chain reaction. We found a high incidence of herpesvirus infection in bog turtles (51.5%; 105/204) and smaller numbers of positive wood (5) and spotted (1) turtles. Sequence and phylogenetic analysis revealed three previously uncharacterized alphaherpesviruses. Glyptemys herpesvirus 1 was the predominant herpesvirus detected and was found exclusively in bog turtles in all states sampled. Glyptemys herpesvirus 2 was found only in wood turtles. Emydid herpesvirus 2 was found in a small number of bog turtles and a single spotted turtle from one state. Based on these findings, Glyptemys herpesvirus 1 appears to be a common infection in the study population, whereas Glyptemys herpesvirus 2 and Emydid herpesvirus 2 were not as frequently detected. Emydid herpesvirus 2 was the only virus detected in more than one species. Herpesviruses are most often associated with subclinical or mild infections in their natural hosts, and no sampled turtles showed overt signs of disease at sampling. However, infection of host-adapted viruses in closely related species can result in significant disease. The pathogenic potential of these viruses, particularly Emydid herpesvirus 2, in sympatric chelonians warrants additional study in order to better understand the relationship of these viruses with their endangered hosts.

Emydid herpesvirus 1 infection in northern map turtles (Graptemys geographica) and painted turtles (Chrysemys picta)

A captive, juvenile, female northern map turtle (Graptemys geographica) was found dead following a brief period of weakness and nasal discharge. Postmortem examination identified pneumonia with necrosis and numerous epithelial, intranuclear viral inclusion bodies, consistent with herpesviral pneumonia. Similar intranuclear inclusions were also associated with foci of hepatocellular and splenic necrosis. Polymerase chain reaction (PCR) screening of fresh, frozen liver for the herpesviral DNA-dependent DNA polymerase gene yielded an amplicon with 99.2% similarity to recently described emydid herpesvirus 1 (EmyHV-1). Molecular screening of turtles housed in enclosures that shared a common circulation system with the affected map turtle identified 4 asymptomatic, EmyHV-1 PCR-positive painted turtles (Chrysemys picta) and 1 asymptomatic northern map turtle. Herpesvirus transmission between painted and map turtles has been previously suggested, and our report provides the molecular characterization of a herpesvirus in asymptomatic painted turtles that can cause fatal herpesvirus-associated disease in northern map turtles.

Characterization of a continuous feline mammary epithelial cell line susceptible to feline epitheliotropic viruses.

Abstract Mucosal epithelial cells are the primary targets for many common viral pathogens of cats. Viral infection of epithelia can damage or disrupt the epithelial barrier that protects underlying tissues. In vitro cell culture systems are an effective means to study how viruses infect and disrupt epithelial barriers, however no true continuous or immortalized feline epithelial cell culture lines are available. A continuous cell culture of feline mammary epithelial cells (FMEC UCD-04-2) that forms tight junctions with high transepithelial electrical resistance (>2000Ωcm−1) 3–4 days after reaching confluence was characterized. In addition, it was shown that FMECs are susceptible to infection with feline calicivirus (FCV), feline herpesvirus (FHV-1), feline coronavirus (FeCoV), and feline panleukopenia virus (FPV). These cells will be useful for studies of feline viral disease and for in vitro studies of feline epithelia.

Respiratory disease in ball pythons (Python regius) experimentally infected with ball python nidovirus

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.

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

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. ABSTRACT 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.