Studies of Virus Receptor Interactions and Virulence Determinants of Feline Calicivirus

Chapter 2 of the dissertation was previously published: Ossiboff RJ, Sheh A, Shotton J, Pesavento PA, Parker JS (2007) Feline caliciviruses (FCVs) isolated from cats with virulent systemic disease possess in vitro phenotypes distinct from those of other FCV isolates. J Gen Virol 88: 506-527. Chapter 3 of the dissertation was also previously published: Ossiboff RJ, Parker JS (2007) Identification of regions and residues in feline junctional adhesion molecule required for feline calicivirus binding and infection. J Virol 81: 13608-13621.The virus-receptor interaction is critical for establishing infection in host cells. The interaction (i) allows the virion to attach specifically to the host cell membrane, (ii) is often required to permit the process of infection further downstream, and (iii) strongly influences viral tropism and pathogenesis. Feline calicivirus (FCV) is a common feline pathogen. Infection is generally asymptomatic or causes mild upper respiratory signs and oral ulceration; morbidity is high while mortality is low. During the last decade however, there have been outbreaks of extremely virulent viruses that cause systemic disease and result in mortality rates as high as 50%. One objective of these studies was to characterize in vitro properties of FCV isolates of high and low virulence and to identify in vitro correlates to virulence. FCV, a small nonenveloped virus containing a positive sense RNA genome, is known to bind two cellular receptors, feline junctional adhesion molecule A (fJAM-A) and ?2,6 sialic acid. A second objective was to elucidate the details of the FCV-fJAM-A interaction by determining the regions of fJAM-A required for FCV binding and selecting FCV mutants resistant to neutralization by soluble fJAM-A. Analysis of FCV in vitro properties demonstrated that highly virulent isolates were able to spread more efficiently in tissue culture than other isolates investigated. Highly virulent isolates were also more susceptible to neutralization by soluble fJAM A than other isolates. Lastly, sequence analysis of the FCV structural protein did not identify conserved sequence unique to highly virulent viruses. Mutagenesis studies identified that while the binding of FCV to fJAM-A requires the extracellular membrane-distal loop, all domains of the protein were required for successful infection. Selection of FCV mutants resistant to receptor neutralization and measurements of protein hydrophobicity provided genetic and biological evidence indicating that FCV undergoes a significant conformational change upon interaction with fJAM-A. This work shows that FCV interaction with fJAM-A results in changes to the capsid likely to be important for subsequent steps in infection, and suggests a determinant of virulence in FCV is accelerated entry post-binding

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

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 (>2000Omegacm(-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

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

Divergent Serpentoviruses in Free-Ranging Invasive Pythons and Native Colubrids in Southern Florida, United States

Burmese python (Python bivittatus) is an invasive snake that has significantly affected ecosystems in southern Florida, United States. Aside from direct predation and competition, invasive species can also introduce nonnative pathogens that can adversely affect native species. The subfamily Serpentovirinae (order Nidovirales) is composed of positive-sense RNA viruses primarily found in reptiles. Some serpentoviruses, such as shingleback nidovirus, are associated with mortalities in wild populations, while others, including ball python nidovirus and green tree python nidovirus can be a major cause of disease and mortality in captive animals. To determine if serpentoviruses were present in invasive Burmese pythons in southern Florida, oral swabs were collected from both free-ranging and long-term captive snakes. Swabs were screened for the presence of serpentovirus by reverse transcription PCR and sequenced. A total serpentovirus prevalence of 27.8% was detected in 318 python samples. Of the initial swabs from 172 free-ranging pythons, 42 (24.4%) were positive for multiple divergent viral sequences comprising four clades across the sampling range. Both sex and snout-vent length were statistically significant factors in virus prevalence, with larger male snakes having the highest prevalence. Sampling location was statistically significant in circulating virus sequence. Mild clinical signs and lesions consistent with serpentovirus infection were observed in a subset of sampled pythons. Testing of native snakes (n = 219, 18 species) in part of the python range found no evidence of python virus spillover; however, five individual native snakes (2.3%) representing three species were PCR positive for unique, divergent serpentoviruses. Calculated pairwise uncorrected distance analysis indicated the newly discovered virus sequences likely represent three novel genera in the subfamily Serpentovirinae. This study is the first to characterize serpentovirus in wild free-ranging pythons or in any free-ranging North America reptile. Though the risk these viruses pose to the invasive and native species is unknown, the potential for spillover to native herpetofauna warrants further investigation

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

Background: Respiratory infections are important causes of morbidity and mortality in reptiles; however, the causative agents are only infrequently identified. Findings: Pneumonia, 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. Conclusions: This 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

Sources of Hepatitis E Virus Genotype 3 in the Netherlands

Four subtypes have been detected, and pathogenicity, zoonotic potential, or stability may vary between subtypes

Distemper, extinction, and vaccination of the Amur tiger

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

Understanding virulence determinants of feline calicivirus

Feline calicivirus (FCV) is a common feline pathogen associated with a variety of clinical syndromes. Typically, FCV infection is either asymptomatic or causes mild upper respiratory signs, oral ulceration, and fever; fatal disease is unusual. In 1998 however, an outbreak of atypical FCV caused severe systemic disease. Since that report, there have been multiple other documented outbreaks of extremely virulent FCV that have resulted in mortality rates as high as 50%. The extremely virulent nature of these viruses as well as the reported speed with which they can spread within veterinary hospitals and shelters makes their early identification imperative for limiting morbidity and mortality. Sequence analysis of the FCV structural protein did not identify conserved sequence unique to highly virulent viruses, suggesting highly virulent isolates have arisen independently. Analysis of FCV in vitro properties including growth kinetics and neutralization assays suggests a determinant of virulence in FCV is accelerated entry post-binding

Identification of Regions and Residues in Feline Junctional Adhesion Molecule Required for Feline Calicivirus Binding and Infection▿

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