Abstract

San Miguel Sea lion Virus - Type 5 (SMSV-5) was first isolated from Northern Fur Seals (Callorhinus ursinus) in the Pribilof Islands, and subsequently has been shown to possess a diverse spatial and phylogenetic host range, infecting food animals, marine mammals, fish, and humans. The virus is spread throughout the Pacific basin and causes blisters on the flippers of marine mammals and on the feet and mouths of inoculated swine. In man it causes vesicles on the thick skinned areas of the hands and feet. SMSV-5 appears to spread readily within and between species. Libraries of cDNA synthesized from the single-stranded RNA genome of SMSV-5 have been produced in order to obtain a nucleic acid diagnostic probe. This probe will be utilized in cDNA-RNA hybridization reactions to rapidly and sensitively detect SMSV-5 carrier animals and viral contamination in human and animal food products of ocean origin. Three E. coli libraries have been generated using the plasmid vector pTZ18R-Bstx 1. Two libraries, greater than 1000 and 5000 base pairs in length, of cDNA synthesized by internal random priming of the viral genome produced approximately JX106 and 6x105 recombinants, respectively. One library, greater than 5000 base pairs in length, of cDNA synthesized by oligo-dT priming of the viral genome produced approximately 1.5x105 recombinants. An insert of approximately 3600 base pairs has been isolated from the random primed library. This insert has been used in hybridization studies against SMSV-5 RNA.

Introduction

San Miguel Sea Lion Virus (SMSV) is a member of the Caliciviridae family, which also includes Vesicular Exanthema of Swine Virus (VESV), Feline Calicivirus, and the Norwalk Agent. Caliciviruses were named for their characteristic soccer ball-like appearance caused by cups, or calices, seen on the surface when the virus is viewed using negative-contrast electron microscopy. All caliciviruses possess a single-stranded RNA genome and one major capsid polypeptide [9]. The host range of caliciviruses is unusually diverse, including: insects, amphibians, reptiles, fish, and mammals of both ocean and terrestrial origin [3].

SMSV was the first virus isolated from a pinniped species and the second isolation of a virus from a marine mammal. It has been shown to be morphologically and physicochemically indistinguishable from VESV and in experimentally infected swine it causes vesicular lesions on the feet and mouths, clinically identical to VES (2]. Seventeen serotypes of SMSV or related marine caliciviruses have been isolated from marine mammals since they were first detected in 1972 [17].

SMSV-5 was first isolated from a Northern Fur Seal (Callorhinus ursinus) on St. Paul Island, Alaska, in 1973 [14]. It has been shown that the virus may be carried by a nematode larvae, (Parafilaroides decorus), 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 [18]. Furthermore, SMSV-5 has been shown to replicate in an experimentally infected teleost, the opaleye, (Girella nigricans) [19]. For many years it has been thought that SMSV should be zoonotic. This was based on the evidence of the virus growing in cell lines of primate and human origin, the ability of SMSV-5 to cause disease in experimentally inoculated African Green Monkeys, (Cercopithecus aethiops), and the production of specific serum neutralizing antibodies against the virus in researchers exposed to caliciviruses [3,15]. In 1985 the virus was proven zoonotic when a research worker developed clinical signs identical to calicivirus vesicular disease in other animals. The worker presented blisters on the thick skinned areas of the hands and feet, and SMSV-5 was isolated from the vesicular fluid [13]. Because of its exceptionally broad host range, SMSV-5 genome RNA was chosen to be the template for the production of copy DNA (cDNA) libraries, which will be used to better understand the molecular biology of this unusual family of viruses. Moreover, the libraries can be used to develop a diagnostic hybridization probe which can be used to detect calicivirus contamination in food products as well as virus infection in clinical patients, both animal and human.

Very little is known about the molecular biology of caliciviruses. The viral capsid consists of 120 to 180 copies of a single polypeptide of about 60,000 to 70,000 daltons molecular weight [1,4,12]. Two other viral encoded proteins are present in infected cells, a small (10,000 - 15,000 daltons) polypeptide covalently linked to the RNA genome of the virus in a fashion similar to the picornavirus family, and an unstudied protein which is assumed to be the viral replicase [4,10]. The genome of caliciviruses is a single strand of RNA which can act as a messenger within a cell. The viral RNA is polyadenylated and possesses the covalently linked protein [4,10]. Upon infection in a susceptible cell, the virus produces three types of RNA molecules: a 37S molecule (the genomic size), a 22S molecule, and an 18S molecule [4,6,7]. The 22S subgenomic RNA molecule has been shown to act as a messenger for the production of the capsid polypeptide [4]. This replication strategy is markedly different from the post-translational cleavage method of the closely related picornaviruses even though both virus families have polyadenylated and protein linked genomes.

Detection of caliciviruses is accomplished by either 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 of either clinical or food origin. By developing a nucleic acid probe, the presence of calicivirus can be specifically detected from samples suspected of harboring very small numbers of the disease agent. Nucleic acid probes use the genome sequence code of the virus as a detection tool rather than the products of the genetic code. Since the complexity of the calicivirus genome is limited to three genes, a piece of the viral RNA may be found that could be specific for all or most members of this unusual virus family. SMSV-5 was chosen as the template virus because of its diverse host range. It is capable of infecting virtually all known hosts infected by all the other members of the calicivirus family [3].

Methods

Roller bottles of SMSV-5 were grown in Vero monkey kidney cells according to the method of Smith, et. al. (16]. When the viral induced lysis of all the cells was complete, the virus was purified by lipid solvent extraction and cesium chloride gradient centrifugation according to a modification of the method of Schaffer and Soergel [11]. Purified virus particles were checked by electron microscopy for contaminating cell debris and the RNA was subsequently extracted using guanidinium isothiocyanate [5].

Copy DNA (cDNA) was replicated from the SMSV-5 RNA template using a modification of the Gubler-Hoffman procedure [8]. First, a single strand of complementary DNA was produced using the enzyme reverse transcriptase. The reverse transcription reaction was primed by either an internal random hexanucleotide or an oligo-dT fragment. Both methods of first strand synthesis priming were used, because calicivirus RNA is polyadenylated. After the first DNA strand has been synthesized, it exists as an RNA-cDNA hybrid. Ribonuclease H was used to cleave the RNA strand in the RNA-cDNA hybrid and E. coli DNA polymerase I used the nicked fragments of RNA as primer to synthesize the second strand of cDNA. Any nicks in the double-stranded cDNA molecule were repaired with T4 DNA ligase and the cDNA molecule was made blunt-ended with T4 DNA polymerase as a prerequisite for the addition of BstX 1 nonpalindromic linkers. After the addition of the linkers, the cDNA was sized in a 1% agarose gel in which fragments of the desired size were cut out of the gel and electroeluted. The cDNA fragments were ligated into the pTZ18R-BstX 1 plasmid vector and transformed into E. coli DHF1aF' cells. The transformed bacteria were plated out onto ampicillin agar plates and the transformation efficiency was determined. Insert containing colonies were screened to detect large viral genome cDNA inserts, and the size of the cDNA was determined by excising the insert from the plasmid with the restriction endonuclease Xho I and coelectrophoresing in agarose with a known molecular weight standard ladder. The purified insert was nick translated with 32P and used in hybridization reactions against itself and viral RNA.

Results and Discussion

Three libraries of SMSV-5 cDNA were constructed. Two of the libraries were primed using the internal random hexanucleotide primer for first-strand cDNA synthesis. One of the random libraries was sized to include any cDNA fragments above 1000 bases in length and the other random library was sized to include any cDNA fragments above 5000 bases. The yields for these libraries were JX106 and 6x105 ampicillin resistant colonies per milliliter, respectively. The other library was primed using an oligo-dT primer, and was sized to include any cDNA fragments above 5000 bases in length. The yield for the oligo-dT primed library was 1.5x105 ampicillin colonies per milliliter.

Figure 1. Xho I Restriction Digest of the SMSV-5 cDNA Insert 05L1C39. The ethidium bromide stained, 1% agarose gel was electrophoreses in TBE buffer at 100V for approximately one hour. Lane 1, Xho 1 digest of plasmid pTZ18R-BstX 1 containing the SMSV-5 cDNA insert 05L1C39 of approximately 3.6 kilobases (Kb) in length; Lane 2, plasmid pTZ18R-BstX 1; Lane 3, Hind III digest of lambda DNA. Marker sizes are in Kb.

The actual number of large insert-containing transformants will be a small percentage of the actual transformation yield since contamination by linkers blunt-end ligated together during the linker addition reaction can migrate in the complex of larger inserts during agarose electrophoresis and be preferentially ligated into the plasmid vector prior to transformation since smaller DNA pieces will more easily ligate into a plasmid than longer DNA fragments.

An insert (05LlC39) was isolated from the 1000 base random library and found to be approximately 3600 bases in length (Figure 1). Based on a molecular weight for the calicivirus genome of 2.6x106 daltons, the insert which has been isolated is approximately half the size of the SMSV-5 RNA [11]. Preliminary hybridization reactions have shown that 05LIC39 will hybridize to itself and to SMSV-5 RNA, but not to vero cell nucleic acid.

Further hybridization studies will be needed to determine the ability of this insert to detect other members of the calicivirus family and to determine its sensitivity in detecting viral contamination in food products and viral infection in clinical specimens. Of importance is whether this insert will recognize SMSV-5 isolated from the vesicles of the infected research worker.

Caliciviruses are being isolated from a broad spectrum of phylogenetically diverse organisms. These agents are capable of causing serious disease in animal and human populations, creating economic losses and life-threatening situations. Because many of these viruses cannot be cultivated in the laboratory, and their detection is often difficult, new diagnostic tools must be developed to rapidly identify their presence in food products, livestock, wild animal populations, and asymptomatic human carriers. The molecular cloning of SMSV-5, a zoonotic agent with a extremely wide host range, is a first step in the development of a rapid calicivirus identification method. Furthermore, the cDNA libraries will also enable researchers to study and better understand the poorly defined life cycle of this unique group of viruses.

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