Abstract

Caliciviruses of ocean origin have been implicated as the Foreign Animal Disease Agent which devastated the swine industry of the United States 35 years ago. The origins of those viruses are believed to have been in raw fish and fish scraps fed to swine. Caliciviruses isolated from an ocean fish (Girella nigricans) do cause disease in both swine and pinnipeds. The disease conditions in pinnipeds can mimic those of swine and include vesicles around the feet and mouth, abortion, encephalitis, and myocarditis. Recently, one marine calicivirus called San Miguel Sea lion virus Type 5 (SMSV-5) which was originally isolated from blisters on the flippers of Northern fur seals was reisolated from blisters on the hands and feet of a human. Today, over 32 serotypes of calicivirus which are known to have an ocean presence have been isolated. Several additional types have been observed but not isolated and these include the Norwalk agents of man which cause gastroenteritis, the human enteric caliciviruses which are rarely or never replicated in vitro and the human Non A Non B hepatitis (NANBH) virus which has been classed morphologically as a calicivirus but has not been isolated. Although all members of the above-mentioned human calicivirus-like pathogens are transmitted by the fecal oral route, the original outbreaks of Norwalk, and perhaps human calicivirus, very frequently occur when raw or poorly-cooked shellfish are eaten. Recently, a calicivirus of walrus origin has been shown to cause hepatitis in swine. This is a newly-described disease manifestation for the caliciviruses of ocean origin and one that clearly suggests possible linkages to NANBH of man.

Introduction

Between the years 1932-1952, 11 types of caliciviruses were shown to cause a new disease of swine named vesicular exanthema (VE) which spread throughout California but for the entire 20-year period remained confined to that state. New outbreaks of VE were associated with feeding raw garbage; however, the calicivirus-laden garbage components other than scraps of infected pork were never identified. Between 1952 and 1956, two additional serotypes of the virus were isolated and the disease spread throughout all the major pork-producing areas of the United States. Disease control measures using quarantine, garbage cooking, and slaughter began in 1954 and eradication was complete by the winter of 1956. Prior to 1972, the point sources of origin for this new virus of swine remained unknown and swine were thought to be the only naturally occurring host. Furthermore, during the 17-year period from the last recorded outbreaks of VE in 1956 until 1972 not a single case of caliciviral disease was reported in any species except cats which commonly were infected with the ubiquitous feline calicivirus.

Beginning in 1972, a rapidly unfolding chain of events demonstrated that caliciviruses of ocean origin could and did infect several species of pinnipeds and that these viruses caused vesicular exanthema in swine. Ocean caliciviruses also infected fish and the fish isolates caused vesicular exanthema in exposed swine. From 1972 to 1989 between 19 and 22 distinct serotypes of these viruses had been isolated (San Miguel sea lion virus Type 3 remains an unproven new type and the ocean origins of mink calicivirus and primate calicivirus remain unproven). During that same period, caliciviruses were isolated from a total of 19 different species not previously known to harbor caliciviruses, including domestic cattle. Furthermore, a vesicular exanthema of swine virus type originally isolated during the California outbreaks of VE in swine was reisolated from a marine mammal and another calicivirus originally isolated from an aborted sea lion fetus was re-isolated from a herd of domestic swine.

Prior to 1972 caliciviruses were thought to be host specific, but today know them to have unusually broad host ranges where a single serotype can infect fish, pinnipeds, swine, and man. It is this host nonspecificity and proven zoonotic capability which is being addressed in this report.

Methods and Results

Most of the caliciviruses of ocean origin were isolated originally on one of two cell lines. These were pig kidney cells (PK15) and African green monkey kidney cells (Vero). Both are continuous cell lines susceptible to infection by a wide range of viruses but the caliciviruses usually preferentially replicate in the Vero cells. Marine mammal cells generally have been unrewarding for calicivirus propagation and isolation. In vitro studies with a variety of cell lines have shown that human and primate cells are often preferred, dog and cat cells usually become infected, cell lines derived from herbivores occasionally become infected, and rodent cell lines almost never support viral replication. This preference for human and primate derived cell lines was an early clue to the broad host range and the possible zoonotic quality of these agents. For a listing of virus isolates see Table I.

Table 1: Caliciviruses of Probable Marine Origin

Serologic studies using virus neutralization tests have revealed a diversity of antigenic strains of ocean caliciviruses. Each new serotype has been identified on the basis of 20 antibody units of known heterologous typing serum tested against 100 tissue culture infective doses of the virus. Because of the antigenic distance between types, each of the many calicivirus serotypes active in the sea can affect the host as a new disease agent where cross-reactivity or previous exposure to one serotype affords little or no protection against exposure to a new serotype. See Table II for summary of serologic data.

Table II: Species Showing Type Specific Serum Antibodies to Marine Caliciviruses*

Our retrospective serologic studies have shown that multiple virus types have a presence throughout the Pacific Basin, Bering, Chukchi, Beaufort, and Eastern Siberian Seas. Furthermore, feral mammals from insular populations routinely carry neutralizing antibodies to a variety of marine calicivirus types. A calicivirus first isolated from cattle along the Oregon Coast subsequently was shown to be neutralized by marine mammal sera collected along the Pacific shores of the continental United States and Alaska, thus confirming the ocean presence of this cattle virus. A reptilian calicivirus originally isolated from a San Diego Zoological collection subsequently has been re-isolated from two species of pinnipeds off the Oregon and California coasts and serologic surveys show this virus to have widespread distribution in the ocean. Perhaps more perplexing is the finding of antibodies against VE virus types J56 and K56 in two of four bowhead whales sampled in 1980 in Barrow, Alaska. The J and K viruses had been isolated only once previously and that fed raw garbage in Secaucus, New Jersey in 1956.

From time to time researchers working with marine caliciviruses have been tested for and shown to be positive for neutralizing antibodies against several virus types.

Experimental infectivity studies have been undertaken to understand more completely the pathogenesis of these agents in a variety of host species. opal eye fish (Girella nigricans) taken from tidal pools were infected per os, intraperitoneally, and by feeding lungworm larva (Parafilaroides decorum) which were bathed first in tissue culture media containing calicivirus and then rinsed three times. Virus was isolated from all three groups of fish 30 days later and these same fish preparations were given to Northern fur seal pups which subsequently developed typical caliciviral vesicular lesions on their flippers. Fluid from these lesions yielded virus isolates of the original type given the fish.

Caliciviruses first isolated from ocean-caught fish were used to expose domestic swine and not only did the inoculated swine develop VE but the disease spread horizontally to uninoculated pen-contact pigs. Most, but not all, of the marine caliciviruses isolates do cause vesicular disease of varying severity in exposed pigs and, in some cases, the disease is far more severe than that caused by some of the 13 serotypes of vesicular exanthema virus originally isolated from swine. In certain other cases, even where vesicular disease did not occur, moderate-to-marked hepatocellular degeneration was seen in both the inoculated and pen-contact swine.

Although the bovine calicivirus isolate from cattle did not produce marked clinical disease in experimentally-infected calves, the calves continued to shed virus for up to seven weeks even in the presence of high neutralizing antibody titers and swine exposed to the bovine calicivirus developed typical vesicular exanthema. A second virus type developed vesicular lesions at inoculation sites with secondary spread to the feet and then horizontal spread to a pen-contact calf which also developed severe vesicles around the coronary bands of the feet.

There are several caliciviruses or calicivirus-like viruses which infect people and are thought to be host specific for man. These viruses rarely, if ever, are isolated and are seen generally by direct electromicroscopic examination of diarrheic stools. The first of these is the Norwalk agent first seen in Norwalk, Virginia, in 1968. It is associated primarily with outbreaks of gastroenteritis of adults and in nursing homes, cruise ships, and institutions. Frequently oysters and mussels are implicated as the point source for such outbreaks. A so-called human calicivirus is associated with diarrhea of children and appears widespread. A third virus of interest is the enterically transmitted Non-A Non-B hepatitis (NANBH) virus which has not been cultivated but has the physio-chemical characteristics of a typical calicivirus. None of these human agents described here have a proven connection with the caliciviruses of ocean origin which we are reporting; however, oysters and mussels may yet be shown to be primary reservoirs.

We do wish to report, however, that one of the marine caliciviruses is a proven pathogen for man. The virus, first isolated in 1973 from blisters on the flippers of Northern fur seals in the Pribilof Islands, was re-isolated recently from blisters occurring on the palms of the hands and the soles of the feet of an ill researcher. The clinical manifestation of this virus in man mimicked that seen in experimentally infected swine and naturally infected seals, thus, the epitheliotropic vesiculogenic nature of this calicivirus of ocean origin has been manifested in seals, swine, and people.

The findings that a marine calicivirus first isolated from a walrus can cause hepatitis in swine and that the NANBH virus is a calicivirus of unknown point source in nature has not been examined thoroughly in order to determine the relatedness, if any, between these two agents.

Discussion

Although both the vesiculogenic and hepatotropic nature of the marine caliciviruses have been mentioned, the caliciviruses of ocean origin and of cats have been implicated in a wide range of disease conditions including abortion, agalactia, pneumonia, myositis, myocarditis and encephalitis. The known disease potential of caliciviruses when examined across widely divergent phylogenetic lines appears to be consistent and for this reason, a variety of disease manifestations resulting from calicivirus infections in various species would be expected to occur also in man. Further evidence of this possibility is as follows: caliciviruses are known to be abortogenic in cats, swine, California sea lions, Steller Sea lions, and perhaps Northern fur seals. They are pneumogenic in cats, Northern fur seals and perhaps swine and vesiculogenic in three species of seals, swine, cats, cattle, and man. By all rules of extrapolation, we should expect caliciviruses to emerge as serious pathogens of man capable of causing a variety of severe disease conditions, including pneumonia, abortion, myocarditis, and encephalitis.

Another calicivirus characteristic, which makes them more dangerous than originally believed, is their abundant antigenic diversity, their expression of unpredictable virulence type and their unusually broad host range. The outcome of our epidemiologic studies and the historical accounts of calicivirus disease outbreaks show that once these agents become introduced into a new host species, they establish themselves and spread horizontally to others of the same species as well as new host species.

Evidence shows that these agents frequently bridge the land-sea interface; yet, we believe them to be uniquely marine in their origins. Some bits of information suggest that they replicate well at reduced temperatures (150 C) as occurs in ocean fish and that at higher temperatures (39°C), such as occurs in the mammalian body, a cytolytic cycle is triggered thus disrupting cells causing vesicular lesions and perhaps other disease manifestations. The ability of these agents to establish persistent infections and thereby be transported long distances by their ocean hosts is suggested by the wide distribution of most serotypes. The mammals of the sea are thought to be somewhat incidental or secondary hosts while the great variety of antigenic virus types being maintained in nature is thought to occur because of infections in numerically large populations such as fish. In this scenario, virus spread would occur up through the foodweb to the top-order predators such as game fish and marine mammals then proceed horizontally wherever infected fish, marine mammals, or infected terrestrial species may migrate or be transported.

Today there is evidence of repeated introductions of these agents into new terrestrial host species. Implicated in this process is any mechanism whereby primarily fish or fish excreta or, perhaps of less significance, marine mammals are taken from the sea and moved inland. We should be reminded that there are no restrictions on the worldwide transport of wild captured California sea lions and these, without exception, have been exposed to caliciviruses in their native habitat. Furthermore, prevailing ocean winds are well-known scavengers that can carry microbial-laden aerosols inland from the sea. Perhaps of more significance is the distribution of fisheries' products to be consumed raw, or to be processed without specific methods to inactivate caliciviruses and then to be used as protein supplements or foods.

These considerations are important in our future management decisions of aquaria, seaquaria, fisheries products, marine mammals, and aquaculture industries, particularly as these decisions relate to possible ocean fish scrap or meat scrap protein supplements and the movements of brood stock, products, and displays. One should bear in mind that these agents are zoonotic and animal handlers as well as the consuming public may be exposed.

The implication for human health falls roughly into four categories. First is the broadening host range of the marine caliciviruses and the possibility of human exposure through contact with increasing numbers of species. Second is the habit, or fad of our eating raw fish as a delicacy. Third is the already implied threat of eating raw or only partially cooked filter feeders (bivalves) that could concentrate caliciviruses from naturally infected waters, fecally contaminated waters or perhaps as primary hosts fully capable of replicating their own repertoire of caliciviruses. Finally, we now must consider that caliciviruses may find their way into our food supply by way of infected cattle, swine, or fish. Tests on whole, ground frozen carcasses of calicivirus-infected Northern fur seals contained 103 infected virus per gram and based on this data one could expect infected meats from any source to retain active virus at a similar level.

The implication of this series of studies is very clear. This is an emerging disease issue for not only the many animal species under our care and management but for mankind as a producer and consumer of animal products.

References

1.  Barlough JB, Berry ES, Skilling DB, and Smith AW (1986) The Marine Calicivirus Story - part I and Part II. The Comp of Cont Ed for the Practicing Veteriarians. Vol 8, (9) F5-F14 and (10) F75-81.

2.  Smith AW 1981, Marine Reservoirs of Caliciviruses in Steele JH (ed) CRC Handbook Series in Zoonoses, Vol II, Sect 8 Boca Raton FL, CRC Press Inc. pp 182-190.

3.  Smith AW and Akers TG (1976) Vesicular Exanthema of Swine JAVMA 169:700-703.

4.  Bankowski RA: Vesicular Exanthema (1965) Adv Vet Sci 10:23-64.