Caliciviruses are known to infect, and cause disease in, a broad spectrum of phylogenetically diverse animal species in terrestrial as well as marine environments. San Miguel Sea Lion Virus - Type 5 (SMSV-5), first isolated from a northern fur seal (Callorhinus ursinus), has been shown to infect marine mammals throughout the pacific basin, causing blisters on the flippers of affected animals. It was subsequently isolated from food animals and man, appearing to move readily within and among species. An Escherichia cold library of cDNA synthesized from the single-stranded RNA genome of SMSV-5 has been produced using the plasmid vector pcDNA II. These synthesized probes are to be used to detect calicivirus carrier animals and viral contaminated food products of ocean origin. One such clone (5RT-12) of approximately 1600 base pairs in length was isolated. An Xba I/Hind III restriction endonuclease cleaved fragment of 5RT-12 was utilized in cDNA-RNA hybridization reactions against RNA isolated from 27 different caliciviruses and two non-caliciviruses. Twenty of the 27 caliciviruses hybridized with the radiolabeled 5RT-12 probe, while the non-calicivirus samples did not react. A semi-quantitative hybridization study was also performed to determine if the 5RT-12 probe was able to identify SMSV-5 in experimentally-5 inoculated clam and salmon tissue. In salmon tissue, 2000 molecules (4 x 10^-15 g) of infective calicivirus RNA could be detected using a dot hybridization method. It appeared that ribonucleases associated with the whole clam homogenate prevented detection of viral RNA in undiluted clam samples.

Introduction

The family Caliciviridae comprises a group of morphologically distinct viruses with an unusually diverse host spectrum, both phylogenetically and spatially. These agents have been shown to cause disease in terrestrial and marine animals. Insects, amphibians, reptiles, fish, mammals, and humans have been shown to be susceptible to calicivirus infection (1,2,5,9). Caliciviruses are being implicated in a growing number of syndromes, including: hepatic, enteric, hemorrhagic, and vesicular diseases (1,2,6,7). San Miguel Sea Lion Virus, Vesicular Exanthema of Swine Virus, Feline Calicivirus, Rabbit Hemorrhagic Disease Virus, Hepatitis E Virus, and the Norwalk Agent are members of this rapidly expanding family.

San Miguel Sea Lion Virus was the first virus isolated from a pinniped species. Of even greater significance, however, was SMSV's pathogenic, morphologic, and physicochemical indistinguishability from Vesicular Exanthema of Swine Virus (VESV).

VESV was eradicated, at great cost (39 million dollars), from the United States in 1956. The vesicular lesions on the feet and around the mouth of SMSV experimentally infected swine were identical to VESV lesions (1,2). Since the first isolation of SMSV in 1972, 18 additional serotypes of marine caliciviruses have been isolated from a variety of marine mammals and one species of fish (12).

SMSV-5 was first isolated from a northern fur seal (Callorhinus ursinus) on St. Paul Island, Alaska, in 1973 (10). This virus has an unusually broad host range. SMSV-5 has been postulated to be carried by a nematode larva, (Parafilaroides decorous), to a poikilothermic host, (Girella nigricans), and subsequently transmitted to, and cause disease in, a homothermic host (Callorhinus ursinus), experimentally infected by feeding virus contaminated fish (13). In 1985, SMSV-5 was proven to be zoonotic when a research worker developed clinical signs identical to calicivirus vesicular disease in other animals. The worker developed blisters on the thick skinned areas of the hands and feet, and SMSV-5 was isolated from the vesicular fluid (9).

The potential transmission of calicivirus disease through contaminated marine food products puts at risk virtually all animals: domestic, captive exotic, and human. Reliable diagnostic reagents for detecting virus from ocean sources are essential. Detection of caliciviruses is accomplished by directly observing the uniquely shaped viral particles in negative contrast electron micrographs of clinical or field samples, or, if the investigators are fortunate, the virus will grow in a laboratory cell line and cause detectable cytopathic effect. If the virus is of insufficient concentration to be detected by electron microscopy or is unable to grow or cause cytolysis in vitro, there is no method at present to reliably confirm the presence of calicivirus contamination in samples. By developing a nucleic acid probe, the presence of calicivirus can be specifically detected from samples suspected of harboring very small numbers of the agent. Nucleic acid probes use the genome sequence of the virus as a detection tool rather than the products of the genetic code. Since the complexity of the calicivirus genome is limited, a piece of the viral RNA, transcribed to its complementary DNA may be found that could be specific for all or most of the 33 distinct calicivirus types known.

Because of its exceptionally broad host range, SMSV-5 was chosen to be the template for the production of a copy DNA (cDNA) library, which is being used to develop a hybridization probe. Furthermore, the cDNA library will be used to advance the understanding of the molecular biology and replication strategy of this unusual family of viruses.

Methods

Roller bottles of SMSV-5 were grown in vero monkey kidney cells according to the method of Smith, et.al. (11). The virus was purified by lipid solvent extraction and cesium chloride gradient centrifugation according to a modification of the method of Schaffer and Soergel (8). Purified virus particles were checked by electron microscopy for contaminating cell debris and the viral RNA was subsequently extracted using guanidinium isothiocyanate (3).

Complementary double-stranded DNA was synthesized from the SMSV-5 RNA template utilizing the enzyme reverse transcriptase and a modification of the Gubler Hoffman procedure (4). The first strand DNA synthesis reaction was primed using a combination of random hexa nucleotide and oligo-dT fragments. Non-palindromic linkers were ligated onto the ends of the cDNA molecules. The double stranded cDNA fragments were sized in a 1% agarose electrophoresis gel and molecules greater than 1500 bases in length were purified. Ligation into the plasmid vector pcDNA II followed by transformation into a DH1 strain of E. cold completed the SMSV-5 cDNA library.

RNA was extracted from separate flasks of vero monkey kidney cells that were infected with 27 different caliciviruses, two non-Caliciviridae viruses, and uninfected vero cells. Approximately 200 ng of each RNA sample was applied to a nylon membrane, dried, and irradiated with ultra-violet light. A purified plasmid clone from the SMSV-5 cDNA library, 5RT-12, was digested with the restriction endonucleases Xba I and Hind III, nick-translated with 32, and hybridized against the 29 viral RNA samples. The results were visualized by autoradiography.

Salmon muscle and whole clams were homogenized separately in tissue culture media at room temperature. The final concentrations for the salmon and clam tissue were 333 mg/ml and 500 mg/ml respectively. Serial log dilutions of SMSV-5, with an original titer of 4 x 10^7.5, were prepared to 10^-8. Equal numbers of infectious virus, starting with 1.05 x 10⁷particles, from each dilution, were added to 0.25 g of homogenized tissue and the RNA of the tissue-virus mixture was extracted from 100 ul of each sample. A known fraction of the extracted RNA was applied to nylon membrane and probed with 5RT-12. Serial log dilutions, starting with 0.25 g, of salmon and clam tissue were also prepared, and each dilution was inoculated with 1.05 x 10⁷ infectious particles of SMSV-5. The RNA of these samples was extracted and probed in an identical manner. Uninoculated tissue controls were also prepared.

Results and Discussion

The SMSV-5 cDNA library synthesized using random and oligo-dT primers in combination yielded approximately one million ampicillin resistant colonies per milliliter. One of these clones, 5RT-12, approximately 1600 base pairs in length, was utilized as a probe in the subsequent hybridization studies.

Results of hybridizing 5RT-12 against a variety of viral infected vero cell RNA is shown in Figure 1. Strong hybridization is evident with SMSV-5 infected cells while uninfected cellular RNA does not react. Furthermore, 5RT-12 reacts with a majority of the calicivirus infected cells while excluding the two non-Caliciviridae samples, a marine rotavirus, isolated from C. ursinus, and putative marine retrovirus, isolated from a walrus, Odobenus rosmarus. Of particular interest is the positive reaction seen with nucleic acid extracted from the virus that was isolated from vesicles of the research worker infected with SMSV-5 in 1985 (9).

All SMSV serotypes and Cetacean Calicivirus hybridize with the cDNA probe except for SMSV-8. Terrestrial animal isolates, Primate, Reptile, and Tillamook Caliciviruses, in addition to the human vesicular agent also hybridize. Two additional marine caliciviruses (Grey Whale and Walrus Caliciviruses), as well as four terrestrial animal isolates (Cheetah, Feline, Canine, and Mink Caliciviruses) fail to react.

The 5RT-12 probe specifically detects experimentally inoculated SMSV-5 in marine food products (Figure 2) even at a 1/10 dilution of virus in 0.25 g of salmon muscle. The amount of viral RNA extracted and applied to the nylon membrane, at this dilution, is estimated to represent 2000 infectious virus particles. Since Caliciviruses possess one Strand of RNA per virus particle, 2000 particles represents approximately 4 x 10^-15 g of nucleic acid. Non-infectious virus particles are not included in this calculation and may contribute significant amounts of viral RNA in the hybridization reaction.

The probe did not detect virus RNA in 0.25 g of whole clam tissue (Figure 2). When the clam tissue was diluted 100 fold while keeping the virus inoculum constant, a positive reaction occurred. This indicates that higher concentrations of clam tissue are interfering with the viral RNA extraction process. Since the clam tissue comprised the entire animal, except the shell, ribonucleases associated with the animal's digestive system may have degraded the viral RNA prior to hybridization with the cDNA probe.

Figure 1. 5RT-12 Dot Hybridization of Calicivirus RNA; SMSV, San Miguel Sea Lion Virus; CCV, Cetacean Calicivirus; GWCV, Grey Whale Calicivirus; WCV, Walrus Calicivirus; HVCV, Human Vascular Calicivirus; RCV, Reptile Calicivirus; TCV, Tillamook Calicivirus; PCV, Primate Calicivirus; ChCV, Cheetah Calicivirus; FCV, Feline Calicivirus; CaCV, Canine Calicivirus; MCV, Mink Calicivirus. The non-caliciviruses are Rota and Retro. Uninfected vero monkey kidney cells (vero) were used as a negative control. 0= No hybridization observed; 1= Positive hybridization.

Figure 2A.

A: Log Dilution of Tissue.

Figure 2. A: SMSV-5 Detection by 5RT-12 in Clam and Salmon tissue. The virus concentration was held constant while serial log dilutions were carried out on the tissues. Uninoculated dilutions were used as negative controls. 0= No hybridization observed; 1= Positive hybridization B: SMSV-5 Detection by 5RT-12 in Clam and Salmon tissue. The tissue concentration was held constant while serial log dilutions were carried out on the virus. Uninoculated tissues were used as negative controls. 0= No hybridization observed; 1= Positive hybridization.

B: Log Dilution of Virus

The SMSV-5 cDNA molecular hybridization probe, 5RT-12, specifically detects a large percentage of Caliciviridae members in experimentally infected tissue culture cells. Furthermore, 5RT-12 could be useful in identifying SMSV-5 contamination of ocean origin food products. Caliciviruses are being implicated in an ever widening spectrum of disease syndromes associated with phylogenetically diverse organisms of land and sea. The synthesis and identification of this recombinate hybridization probe is the first step in the development of a rapid, sensitive tool for the identification of calicivirus presence in food products, livestock, wild animal populations, and asymptomatic human carriers.

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