Inhibition of Vesivirus infections in mammalian tissue culture with antisense morpholino oligomers.

Caliciviruses infect and cause disease in animals and humans. They are nonenveloped, positive-stranded RNA viruses with a genome of approximately 7.5 kb that encodes viral proteins in three open reading frames (ORF). Antisense oligomers targeting one of the three ORF of caliciviruses of the genus Vesivirus significantly inhibit viral replication in tissue culture. Porcine kidney and African green monkey kidney cells were infected with Vesivirus isolates SMSV-13 and PCV Pan-1. Phosphorodiamidate morpholino oligomers (PMO) with sequence complementary to the AUG translation start site regions of ORF1, ORF2, and ORF3 were evaluated for their effect on viral titer. Scrape-loading delivered PMO to 50%-70% of the cells of the two cell lines, as measured by fluorescence microscopy and flow cytometry. A PMO targeting ORF3 caused a significant increase in viral titer. A PMO targeting ORF2, a scrambled PMO control sequence, and an unrelated PMO antisense sequence did not alter viral titer. Various PMO sequences antisense to an upstream region of ORF1 were effective in reducing viral titer up to 80% in a dose-dependent and sequence-specific manner. The extent of viral titer reduction was proportional to the delivery of PMO to cells. These observations demonstrate that antisense PMO can disrupt caliciviral gene function in a nucleic acid sequence-specific manner and are potentially effective antiviral agents.

Isolation of reptilian calicivirus Crotalus type 1 from feral pinnipeds.

Ten virus isolates were obtained from three species of marine mammals sampled on San Miguel Island (California, USA) and 1,200 km north on Rogue Reef (Oregon, USA) during tagging operations in 1986-87. Seven of these 10 were derived from 30 sampled Steller sea lion (Eumetopias jubatus pups, while two of 10 were isolated from one of 19 sampled California sea lion (Zalophus californianus californianus pups, and the remaining isolate was derived from 30 sampled northern fur seal (Callorhinus ursinus) pups. All 10 isolates were identified as belonging to a single serotype, reptilian calicivirus Crotalus type 1 (RCV Cro-1), previously isolated from both healthy and diseased snakes and frogs in a California zoologic collection. The marine samples also showed that nine of 30 Steller sea lion pups, one of 19 California sea lion pups and zero of 30 fur seal pups were producing type specific neutralizing antibodies to RCV Cro-1. This represents the first reported instance of the isolation from marine sources of calicivirus originally isolated from a terrestrial species.

Calicivirus emergence from ocean reservoirs: zoonotic and interspecies movements.

Caliciviral infections in humans, among the most common causes of viral-induced vomiting and diarrhea, are caused by the Norwalk group of small round structured viruses, the Sapporo caliciviruses, and the hepatitis E agent. Human caliciviruses have been resistant to in vitro cultivation, and direct study of their origins and reservoirs outside infected humans or water and foods (such as shellfish contaminated with human sewage) has been difficult. Modes of transmission, other than direct fecal-oral routes, are not well understood. In contrast, animal viruses found in ocean reservoirs, which make up a second calicivirus group, can be cultivated in vitro. These viruses can emerge and infect terrestrial hosts, including humans. This article reviews the history of animal caliciviruses, their eventual recognition as zoonotic agents, and their potential usefulness as a predictive model for noncultivatable human and other animal caliciviruses (e.g., those seen in association with rabbit hemorrhagic disease).

In vitro isolation and characterization of a calicivirus causing a vesicular disease of the hands and feet.

We report that a calicivirus of oceanic origin, San Miguel sea lion virus serotype 5 (SMSV-5), is a human pathogen. This biotype was isolated originally from blisters on the flippers of northern fur seals (Callorhinus ursinus) and replicates readily in primate and human cell lines. It infects a phylogenetically diverse array of hosts (poikilotherms to primates) and induces type-specific neutralizing antibodies in exposed humans. Group antibody against a pooled antigen of SMSV-5 and two other serotypes was also observed in 18% of 300 blood donors from a population in the northwestern United States. The human calicivirus isolate designated SMSV-5 Homosapien-1 (SMSV-5 Hom-1) was recovered from a laboratory worker with systemic illness, including vesicular lesions on all four extremities. We believe this newly described human disease represents a paradigmatic shift in calicivirus disease recognition.

Detection of a non-cultivatable calicivirus from the white tern (Gygis alba rothschildi).

In April 1992, on Tern Island, French Frigate Shoals, Hawaii (USA), researchers observed a hand-reared white tern hatchling (Gygis alba rothschildi) develop vesicular lesions on the webbing between its toes, 6 days after falling out of its nest. Vesicular fluid collected from the foot lesions contained virus-like particles having typical calicivirus morphology. Calicivirus RNA was detected in the vesicular fluid by dot hybridization with a group-specific calicivirus copy DNA probe. Attempts to cultivate the virus in African green monkey kidney cells and porcine kidney cells were unsuccessful. This is the first report of a calicivirus infection associated with vesicular disease in a wild avian species.

New marine calicivirus serotype infective for swine.

A new serotype of calicivirus was isolated from California sea lions (Zalophus californianus) with severe vesicular disease. Neutralizing antibodies were found in 27 of 82 (32.9%) serum samples from California sea lions and in 15 of 146 (10.3%) serum samples from Steller sea lions (Eumetopias jubatus) tested. The seropositive animals were widely dispersed along the margins of the eastern Pacific basin, from the Bering Sea to the Santa Barbara Channel. Seropositive samples were found from as early as 1976 through the present time. This new calicivirus serotype, San Miguel sea lion virus type 13, was inoculated into weaned pigs, resulting in induction of severe vesicular disease, which spread to all pigs, including uninoculated pen contacts. Virus was continually shed by most of the pigs throughout the 2-week duration of the experiment.

Mortality of harbor seal pups at different sites in the inland waters of Washington.

We examined the mortality rates and causes of death of harbor seal (Phoca vitulina) pups in three regions of the inland waters of Washington (USA) in 1984. One hundred eight pups were collected during 239 searches of the shoreline areas near harbor seal haulout sites or through public reports. Minimum neonatal (up to 1 mo after birth) mortality rates at these regions ranged from 12% to 26% of the pups born. Neonatal mortality was highest in the Strait of Juan de Fuca; 33 of the estimated 105 (31%) pups born at the primary site died. Causes of death varied by location. In southern Puget Sound predation by coyotes (Canis latrans) was the primary cause of death, accounting for eight of 43 (19%) of the dead pups examined; starvation was the next most common cause of death. Mortality at study sites in the Strait of Juan de Fuca was related to premature parturition; 19 of 49 (39%) of the pups found dead were born prematurely. Nine species of bacteria were identified in samples taken from 42 pups; Proteus sp. and Escherichia coli were the most common.

First isolation of a calicivirus from the Steller sea lion (Eumetopias jubatus).

A calicivirus was isolated from the rectum of a Steller sea lion (Eumetopias jubatus) pup on Rogue Reef, off the southern Oregon coast. Based on the results of neutralization tests with specific typing antisera, the isolate was identified as San Miguel sea lion virus serotype 6 (SMSV-6). Blood obtained from nine of 37 pups (24%) during virus sample collection procedures had specific neutralizing antibodies to SMSV-6. The isolation of SMSV-6 from a Steller sea lion represents, to our knowledge, the first isolation of any virus from this widely distributed marine mammal species, and serves to reconfirm the host-nonspecificity of yet another calicivirus of marine origin.

Prevalence and distribution of serum neutralizing antibodies to Tillamook (bovine) calicivirus in selected populations of marine mammals.

Neutralizing antibodies to Tillamook calicivirus (TCV) were found in sera collected from California sea lions (Zalophus c. californianus Lesson) in 1983 and 1984 and in sera collected from Steller sea lions (Eumetopias jubatus Schreber) in 1976 and 1985. The combined prevalence of antibodies for these two species was 10/228 = 4.38%. Titers ranged from 1:20 (five animals), to 1:40 (four animals), to 1:80 (one animal) by standard microtiter neutralization assay. The seropositive pinnipeds were dispersed widely along the margins of the eastern Pacific rim, from the Bering Sea to the Santa Barbara Channel. Antibodies to TCV were not found in sera collected from northern fur seals (Callorhinus ursinus L.), Pacific walruses (Odobenus rosmarus divergens Illiger), seals of the family Phocidae, or several cetacean species. Tillamook calicivirus was isolated originally in 1981 from dairy calves in Oregon; the finding of neutralizing antibodies in two widely distributed species of sea lions suggests the possibility of a marine origin for this agent.

Serology and virology of the bowhead whale (Balaena mysticetus L.).

Sera from four bowhead whales (Balaena mysticetus L.) were examined for the presence of specific antibodies, and tissue and swab samples from six and four animals respectively were processed for isolation of viruses and for initiation of bowhead whale cell cultures. All sera were negative for antibodies to nine serovars of Leptospira interrogans and to 21 orthomyxovirus subtypes and a paramyxovirus (Newcastle disease virus). All sera were positive, however, for neutralizing antibodies to one or more calicivirus serotypes. Two untyped adenoviruses were isolated from colon samples of two different whales, but neutralizing antibodies to the agents could not be demonstrated in any sera. Three primary bowhead whale cell cultures were derived from kidney (two cultures) and testis (one culture), from three individual whales.

First isolation of calicivirus from reptiles and amphibians.

Calicivirus isolations were made from 4 poikilothermic species in a zoologic collection. Viruses were recovered from 8 asymptomatic Aruba Island rattlesnakes (Crotalus unicolor; rectal swab samples) and from 8 symptomatic animals (4 Aruba Island rattlesnakes, 2 Bell's horned frogs [Ceratophrys orata], 1 rock rattlesnake [C lepidus], and 1 eyelash viper [Bothrops schlegeli] tissue samples obtained at necropsy). On the basis of cross-neutralization test results, the 16 isolates were antigenically indistinguishable and were considered to represent a unique calicivirus serotype, tentatively designated reptilian calicivirus Crotalus type 1. These isolations could not be associated causally with any specific disease entity either in naturally infected poikilotherms or in experimentally infected snakes and pigs.

Antibodies to marine caliciviruses in the Pacific walrus (Odobenus rosmarus divergens Illiger).

Sera from 155 Pacific walruses (Odobenus rosmarus divergens Illiger), sampled in the Chukchi Sea during the summer of 1983, were tested for serum neutralizing (SN) antibodies to six marine calicivirus serotypes. Serotypes tested included San Miguel sea lion virus (SMSV) types 1, 5, 8, and 10, previously isolated from northern fur seals (Callorhinus ursinus Linné) in the Bering Sea; walrus calicivirus (WCV), previously isolated from walrus feces collected off sea ice in the Chukchi Sea; and Tillamook calicivirus (TCV), a bovine isolate from Oregon of suspected marine origin. No antibodies were found to SMSV-1, SMSV-10, or TCV. Antibodies to SMSV-5 were found in two animals (titers 1:20 and 1:160); antibodies to SMSV-8 were found in four animals (all 1:20); and antibodies to WCV were found in one animal (titer 1:40). Antibodies to WCV have been found in the Pacific walrus previously; however, this represents the first report of antibodies to any of the SMSV serotypes in this marine mammal.

A simple, rapid method for preparation of virus isolates from cell culture for electron microscopy.

A simple procedure for the rapid preparation of virus isolates from cell culture for negative-contrast electron microscopy was devised. Using only conventional centrifugation steps (i.e. without ultracentrifugation), the procedure produced consistent, fine-quality preparations of a variety of virus types differing in size/shape and buoyant density.

Calicivirus isolation from three species of primates: an incidental finding.

Calicivirus isolations were made from 3 species of subhuman primates. Viruses were recovered from gingival lesions associated with periodontal disease in a spider monkey, from the oropharynx of a healthy silver leaf langur, and from the spleen of a lowland gorilla that had died of systemic coccidioidomycosis. Based on the results of cross-neutralization tests, all 3 isolates were serologically indistinguishable from a primate calicivirus Pan paniscus type 1. These isolations appeared to be incidental in nature and could not be associated causally with any specific disease entity.

Isolation and identification of caliciviruses from dogs with enteric infections.

Caliciviruses were isolated from 7 dogs and 1 captured coyote with enteritis. There was a high fatality rate in dogs 4 to 16 weeks of age. The occurrence in these dogs of concurrent infection with known enteric pathogens such as Salmonella sp, canine parvovirus, canine coronavirus, and canine rotavirus did not allow making any conclusions regarding the pathogenicity of this newly recognized calicivirus. The caliciviruses were characterized by electron microscopy and were further identified as being closely related to feline calicivirus by immunoelectron microscopy with specific antibody.

Characterization of a new calicivirus isolated from feces of a dog.

Canine calicivirus (CaCV), isolated from feces of a dog with diarrhea, was readily propagated in cultures of canine cells and in a dolphin cell line. Serologic evidence indicated many dogs in at least one geographic area had been infected with CaCV, but its role as an etiologic agent of disease was not established. In cell culture most CaCV virions were strongly cell-associated making purification difficult. CaCV was established as a member of the Caliciviridae by morphology and physicochemical properties of virions (density, sedimentation rate, single major polypeptide, RNA genome size), although some of the properties differed slightly from those of previously described caliciviruses; evidence was also obtained for caliciviral RNA species in infected cells. Based on tests with antisera to numerous caliciviruses and presumed caliciviruses, CaCV appeared to be not closely related to any previously described virus except the stunting syndrome agent of chickens.

Regression of cetacean tattoo lesions concurrent with conversion of precipitin antibody against a poxvirus.

Tattoo lesions linked to the cetacean poxvirus of bottlenose dolphins regressed without treatment. Two types of regression were observed: (1) The tattoo lesions became raised and blanched, then disappeared along with sloughing skin. (2) When an incision was made through a tattoo lesion, the tattoo disappeared in a zone around the incision. Poxviruses removed from the raised, blanched skin lesions and from typical tattoo lesions were reacted with dolphin serums and examined by immunoelectron microscopy. Antibody was not detected against either of these poxvirus preparations when the dolphins had only typical tattoo lesions. However, after the raised, blanched lesions appeared, serums obtained during the acute or convalescent stages were positive for the poxvirus separated from the lesions. Regression of the typical tattoo lesions was concurrent with antibody conversion.

Calicivirus-induced vesicular disease in cetaceans and probable interspecies transmission.

A calicivirus isolated from cetaceans is a new serotype designated cetacean calicivirus Tursiops-1 (CCV-Tur-1). It appears to have spread from an initially infected Atlantic bottlenose dolphin to a California sea lion, and was then carried by the sea lion to a second facility several miles away, where a second dolphin became infected and developed vesicular skin lesions that eroded, leaving shallow ulcers. Cetaceans and pinnipeds belong to separate orders, so this finding of interspecies transmission demonstrates the potentially broad host spectrum for yet another calicivirus.

Calicivirus isolation and persistence in a pygmy chimpanzee (Pan paniscus).

What may be the first calicivirus isolate from any primate species, including man, was recovered from a herpesvirus-like lip lesion on a pygmy chimpanzee and then, 6 months later, from the throat of the same animal. The infected individual and its cage mates had circulating antibodies that were type-specific for this calicivirus. The agent was antigenically different from 30 other calicivirus serotypes and is tentatively designated primate calicivirus Pan paniscus type 1 (PCV-Pan 1).