RNA synthesized in calicivirus-infected cells is atypical of picornaviruses.

RNA labeled with [3H]uridine from Vero cells infected with San Miguel sea lion virus in the presence of actinomycin D was analyzed by glycerol density gradient sedimentation and polyacrylamide gel electrophoresis. The predominant single-stranded RNA (36S, 2.6 x 10(6) molecular weight) was genome size. There was also a prominent 22S, 1.1 x 10(6)-molecular weight, single-stranded component and one or more double-stranded or partially double-stranded classes. Replicative forms, sedimenting at 18S, contained single-stranded RNA corresponding to the larger-molecular-weight class. All classes of intracellular RNA and virion RNA were polyadenylated. These findings and results with pig kidney cells infected with vesicular exanthema of swine virus and feline cells infected with feline calicivirus indicate that caliciviruses exhibit a strategy of replication different from typical picornaviruses and supports removal of the caliciviruses from the family Picornaviridae.     

Proteins of Yaba Monkey Tumor Virus I. Structural Proteins

Yaba virus proteins were characterized by polyacrylamide gel electrophoresis. Electrophoresis of Yaba virion (proteins) dissociated by sodium dodecyl sulfate and 2-mercaptoethanol in continuous and discontinuous buffer systems yielded 37 polypeptide species by staining and by counting bands of radioactively labeled polypeptides. The molecular weights of the viral polypeptide species were found to range from 10,000 to 220,000 by comparing the relative distance of migration of viral proteins with proteins of known molecular weights. Two polypeptides were removed from purified virions by nonionic detergent nonidet P -40 treatment, and the amount of one polypeptide was reduced. Purified cores yielded 21 polypeptide species, none of which was labeled with radioactive glucosamine.     

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.     

Crystallization and preliminary crystallographic analysis of San Miguel sea lion virus: an animal calicivirus

The Caliciviridae is a family of nonenveloped, icosahedral, positive-sense single-stranded RNA viruses. This family of viruses consists of both animal and human pathogens. Adapting human caliciviruses to cell culture has not been successful, whereas some animal caliciviruses, including San Miguel sea lion virus, have been successfully propagated in vitro. Here we report the crystallization of San Miguel sea lion virus serotype 4 (SMSV4) and the preliminary X-ray crystallographic analysis of the crystals. SMSV4 have been crystallized using the hanging-drop method. These crystals diffracted to approximately 3A resolution using a synchrotron radiation source. A single crystal under cryo-conditions yielded a complete set of diffraction data. Data processing of the diffraction patterns showed that SMSV crystals belong to I23 space group with cell dimensions a=b=c=457 A. The crystallographic asymmetric unit includes five icosahedral asymmetric units, each consisting of three capsid protein subunits. In the space group I23, given the icosahedral symmetry and the size of the virus particle, the location of the particle is constrained to be at the point where the crystallographic 2- and 3-fold axes intersect. The orientation of the virus particle in the unit cell was ascertained by self-rotation function calculations.     

Antibodies to marine caliciviruses in the Steller sea lion (Eumetopias jubatus Schreber)

Sera from 145 Steller sea lions (76 adults, three subadults, 37 pups, and 29 fetuses) were tested for neutralizing antibodies to nine marine calicivirus serotypes. Antibodies were found to San Miguel sea lion virus (SMSV) types 1, 5, 6, 7, 8, 10 and 13, and to Tillamook (bovine) calicivirus, but no antibodies were found to the walrus calicivirus. Titers (microtiter neutralization assay) ranged from 1:20 to 1:320, with many positive reactions at the higher dilutions (greater than or equal to 1:80). Antibodies to SMSV's 5 and 10 were most common among animals sampled in Alaskan waters, while antibodies to SMSV-6 were most common among pups from the southern Oregon coast. These data provide evidence that Steller sea lions, like their California sea lion (Zalophus c. californianus Lesson) counterparts, have experienced widespread exposure to multiple serotypes of marine caliciviruses.     

Bovine coronavirus structural proteins.

The tissue culture-adapted strain (Mebus) of bovine coronavirus was grown in the presence of isotopically labeled amino acids, glucosamine, or orthophosphate for the purpose of analyzing the virion structural proteins. Five species of polypeptides were identified when purified virions were solubilized in urea and sodium dodecyl sulfate and resolved by polyacrylamide gel electrophoresis. Four species were glycosylated and had apparent molecular weights of 140,000, 120,000, 100,000, and 26,000. The glycoproteins were susceptible to proteolytic cleavage and enzymatic iodination when intact virions were studied and are thus at least partially external to the virion envelope. The 140,000-molecular-weight glycoprotein is apparently a dimer of 65,000-molecular-weight glycopolypeptides held together by disulfide linkages. Species 5 was phosphorylated and had an apparent molecular weight of 52,000. In the intact virion, it was unaffected by protease and was not enzymatically iodinated. It is therefore apparently an internal protein.     

Pathology and preliminary characterization of a parapoxvirus isolated from a California sea lion (Zalophus californianus)

Cutaneous pox-like lesions are a common complication in the rehabilitation of pinnipeds. However, the exact identity, taxonomy, and host range of pinniped parapoxviruses remain unknown. During a poxvirus outbreak in May 2003 in California sea lions (Zalophus californianus) at a marine mammal rehabilitation facility, multiple raised, firm, 1-3-cm skin nodules from the head, neck, and thorax of one sea lion weanling pup that spontaneously died were collected. Histologically, the nodules were characterized by inflammation and necrosis of the dermis and epidermis, acanthosis, and ballooning degeneration of the stratum spinosum. Large, coalescing eosinophilic cytoplasmic inclusions were observed in the ballooned cells. A parapoxvirus (sea lion poxvirus 1, SLPV-1) was isolated on early passage California sea lion kidney cells inoculated with a tissue homogenate of a skin nodule. The morphology of the virions on electron microscopy was consistent with that of parapoxviruses. Partial sequencing of the genomic region encoding the putative major virion envelope antigen p42K confirmed the assignment of the sea lion poxvirus to the genus Parapoxvirus. Although SLPV-1 is most closely related to the poxvirus of harbor seals of the European North Sea, it is significantly different from orf virus, bovine papular stomatitis virus, pseudocowpox virus and the parapoxvirus of New Zealand red deer.     

Translation of bovine leukemia virus virion RNAs in heterologous protein-synthesizing systems.

Bovine leukemia virus 60 to 70S RNA was heat denatured, the polyadenylic acid-containing species were separated by velocity sedimentation, and several size classes were translated in a micrococcal nuclease-treated cell-free system from rabbit reticulocytes. The major RNA species sedimented at 38S and migrated as a single component of molecular weight 2.95 x 10(6) when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The predominant polypeptides of the in vitro translation of bovine leukemia virus 38S RNA were products with molecular weights of 70,000 and 45,000; minor components with molecular weights of 145,000 and 18,000 were also observed. Two lines of evidence indicate that the 70,000- and 45,000-molecular weight polypeptides represent translation products of the gag gene of the bovine leukemia virus genome (Pr70gag and Pr45gag). First, they are specifically precipitated by a monospecific antiserum to the major internal protein, p24, and second, they are synthesized and correctly processed into virion proteins p24, p15, and p10 in Xenopus laevis oocytes microinjected with bovine leukemia virus 38S RNA. The 145,000-molecular weight polypeptide was immunoprecipitated by the anti-p24 serum and not by an antiserum to the major envelope glycoprotein, gp60. It contained all the tryptic peptides of Pr70gag and additional peptides unique to it, and thus represents in elongation product of Pr70gag in an adjacent gene, presumably the pol gene. The 18,000-molecular weight product was antigenically unrelated to p24 and gp60 and shared no peptides in common with Pr70gag, Pr45gag, or the 145,000-molecular weight polypeptide. It was maximally synthesized on a polyadenylic acid-containing virion 16 to 18S RNA, and we present evidence that this RNA is a 3' end-derived subgenomic fragment of the bovine leukemia virus genome rather than a contaminating cellular RNA.     

Prevalence and characterization of Salmonella spp. among marine animals in the Channel Islands, California

Salmonella enterica is a zoonotic pathogen that has been isolated from free-ranging marine mammals throughout the world, with animals in the Channel Islands of California (USA) showing the highest prevalence. The goal of this study was to determine prevalence, antimicrobial sensitivity and genetic similarity using pulsed-field gel electrophoresis (PFGE) of Salmonella in several non-domestic animal species on San Miguel and San Nicolas Islands. Fecal samples were collected from 90 California sea lion Zalophus californianus pups, 30 northern elephant seal Mirounga angustirostris pups and 87 western gulls Larus occidentalis in the Channel Islands and 59 adult male sea lions in Puget Sound, WA (USA). Salmonella were isolated, identified and serotyped, followed by antimicrobial susceptibility testing and PFGE. Of the California sea lion pups that were sampled on the islands, 21% (n = 19) were positive for Salmonella, whereas no adults males in Puget Sound were positive. Of the northern elephant seal pups sampled, 87% (n = 26) were harboring Salmonella. Only 9% (n = 8) of western gulls were shedding Salmonella, with one of these gulls harboring the only antimicrobial resistant isolate. The serotypes found in these animals were Enteritidis, Montevideo, Newport, Reading, and Saint Paul. The only serotype that showed variation on PFGE was Newport. The pinnipeds of the Channel Islands harbor Salmonella at a higher prevalence than pinnipeds from other geographic areas observed in previous studies. Researchers and veterinarians should exercise increased caution when working with these animals due to the zoonotic potential of Salmonella.     

Purification and physicochemical and immunological analysis of chicken alpha-fetoprotein

Chicken alpha-fetoprotein was isolated from 12 to 13-day-old embryonic chicken serum by column chromatography on CM-Sephadex C-50. Hydroxyapatite and DEAE-Sephadex A-25. The purified protein was homogeneous based on polyacrylamide gel electrophoresis, immunoelectrophoresis and isoelectric focusing. The purified protein had the following physicochemical and immunological properties. (1) It was a glycoprotein with a single polypeptide chain. (2) The molecular weight of the protein was estimated at 71,000 by SDS-polyacrylamide gel electrophoresis. (3) The isoelectric point of the protein was 4.90. (4) The amino acid composition of the protein was similar to those of mammalian alpha-fetoproteins. (5) The protein showed no steroid-binding capacity. (6) It was immunologically distinct from mammalian alpha-fetoprotein. (7) No immunological cross-reaction was observed between the protein and chicken albumin.     

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.     

Epizootic vesicular disease in captive California sea lions

An epizootic of vesicular disease occurred in a group of semi-domesticated California sea lions (Zalophus californianus) during the months of April and May 1997. Ten castrated mature male sea lions, ages 12 to 19 yr, were housed in three adjacent open-ocean net enclosures in San Diego Bay (California, USA). Four animals (40%) developed oral and extremity vesicles, anorexia, and were reluctant to perform learned behaviors. One animal developed vesicles but maintained a normal appetite and behavior. The remaining animals showed no clinical signs of infection. Virus (designated FADDL 7005) was isolated from four of the five animals that developed vesicles. Serum antibody titers to FADDL 7005, a previously untyped calicivirus, were demonstrated in animals that showed any combination of clinical signs and in two animals that did not show any clinical signs. No virus was isolated from five fecal samples collected from four of the group animals. Clinical signs lasted 4 to 20 days in affected animals. All affected animals recovered from infection. An experimental swine was inoculated with FADDL 7005 and developed vesicular disease, which was transmitted to another experimental swine upon contact. It is proposed that FADDL 7005 is a new San Miguel sea lion virus.     

Structural components of influenza C virions.

The genome RNA species of influenza type C virions were analyzed by polyacrylamide gel electrophoresis. The pattern obtained was found to resemble those of other influenza viruses. Six RNA species were resolved, with estimated sizes ranging from 0.37 X 10(6) to 1.25 X 10(6) daltons. The internal ribonucleoproteins of influenza C virions were found to sediment heterogeneously in glycerol velocity gradients as demonstrated previously with influenza A/WSN virus. The ribonucleoproteins possessed diameters of 12 to 15 nm, with lengths ranging from 30 to 100 nm. Of the three major virion polypeptides (molecular weights, 88,000, 66,000, and 26,000), only the largest is glycosylated. Similar polypeptide species were present in influenza C virions of five different strains. All three major proteins of influenza C virions possess electrophoretic mobilities distinguishable from those of the major polypeptides of influenza A/WSN. The 66,000-dalton protein is associated with the ribonucleoprotein components. Two additional glycosylated polypeptides, with estimated molecular weights of 65,000 and 30,000, were detected in virions grown in embryonated eggs, but not in virus particles obtained from chicken embryo fibroblasts.     

In Vitro Translation of Uukuniemi Virus-Specific RNAs: Identification of a Nonstructural Protein and a Precursor to the Membrane Glycoproteins

We isolated the virus-specific RNA species from Uukuniemi virus-infected chicken embryo cells and fractionated them by sucrose gradient centrifugation. In addition to three RNA species cosedimenting with the three viral RNA segments L (29S), M (23S), and S (17S), a fourth major RNA species, sedimenting at about 12S (S2), was found early in the infection. Annealing experiments indicated that the cytoplasmic L and M RNA species consisted of both plus and minus strands, with the plus strands in slight excess. Most of the S1 RNA was of negative polarity, whereas S2 was of positive polarity. The S2 RNA specifically annealed to the virion S RNA segment, indicating that it is transcribed from this segment. In vitro translation of the individual RNA species in micrococcal nuclease-treated cell-free reticulocyte extracts showed that an mRNA cosedimenting with the virion M RNA directed the synthesis of a virus-specific 110,000-dalton polypeptide (p110). This polypeptide could be immunoprecipitated with antiserum prepared against purified virions. When translation was carried out in the presence of dog pancreas microsomes, p110 was absent. Instead, an immunoprecipitable polypeptide band, with a molecular weight of about 70,000 and migrating between the virion surface glycoproteins G1 and G2, was observed. It is thus likely that the glycoproteins are synthesized as a precursor (p110), which during translation is cleaved roughly in the middle to yield G1 and G2. The 12S RNA species directed the synthesis of the nucleocapsid protein and a novel polypeptide with an apparent molecular weight of about 30,000. The latter was not precipitated with antivirion serum and was absent from lysates programmed with the corresponding RNA fraction from a mock-infected extract. Since, in addition, it was not found in purified virions and was present in the cytoplasm of infected cells but not in uninfected cells, it probably represents a nonstructural polypeptide.     

Proton magnetic resonance studies of the binding of an anionic surfactant with a benzene ring to a protein polypeptide with special reference to SDS-polyacrylamide gel electrophoresis.

The binding of an anionic surfactant to a protein polypeptide has been studied by the proton magnetic resonance (PMR) technique to form a part of our studies on the principles of SDS-polyacrylamide gel electrophoresis. Sodium 4-(p-butylphenyl) butane-1-sulfonate (CH3-(CH2)3-0-(CH2)4-SO3-Na+) was employed as an anionic surfactant, and reduced and carbosyamidomethylated (RCAM) bovine serum albumin as a typical protein polypeptide. The binding isotherm of the surfactant to RCAM bovine serum albumin was similar to that of sodium dodecyl sulfate (SDS). The surfactant could replace SDS in SDS-polyacrylamide gel electrophoresis without affecting the wellknown mode of spearation of protein bands. These results gave a sound basis for the assumption that the investigation of the complex between a surfactant with a benzene ring and RCAM bovine serum albumin would provide useful knowledge concerning the principles of SDS-polyacrylamide gel electrophoresis. Aggregation of the aromatic surfactant necessarily brings benzene rings together. A benzene ring is a strong source of the ring current effect on chemical shifts in nuclear magnetic resonance (NMR). Chemical shifts of the surfactant in NMR are, therefore, sensitive to whether the surfactant molecules are single-molecularly dissolved or aggregated. Full advantage was taken of the above fact in the present PMR study of the binding of the surfactant to RCAM bovine serum albumin. The chemical shifts of the phenyl and methyl protons both for the single-molecular and micellar aggregated states were estimated from measurements of the shifts as a function of the surfactant concentration. They shifted to a higher magnetic field on micelle formation, due to the increase of the ring current effect. Corresponding measurements for the complex between the surfactant and RCAM bovine serum albumin gave estimates of the chemical shifts of the phenyl and methyl groups of the surfactant bound to the protein polypeptide. They were found to shift to a magnetic field somewhat higher than that for the micellar state throughout the concentration range of the surfactant examined. These results strongly suggest that the surfactant molecules bind to the protein polypeptide in the form of micelle-like clusters, and that PMR of the groups are further influenced by the diagmagnetic effect of the protein polypeptide present as a core. No appreciable change in the mode of binding, corresponding to the steep increase in the amount of binding in the binding isotherm, was observed from the PMR studies. Taking the observed similarity between SDS and the aromatic surfactant in the binding and the gel electrophoresis into consideration, the present results strongly suggest that SDS also binds to protein polypeptides in the form of micelle-like clusters under the conditions of SDS-polyacrylamide gel electrophoreses, and support our "necklace model".     

Cryo-electron microscopy reconstructions of two types of wild rabbit hemorrhagic disease viruses characterized the structural features of Lagovirus

Rabbit hemorrhagic disease was described in China in 1984 and can cause hemorrhagic necrosis of the liver within two or three days after infection. The etiological agent, rabbit hemorrhagic disease virus (RHDV), belongs to the Lagovirus genus in the Caliciviridae family. Compared to other calicivirus, such as rNV and SMSV, the structure of Lagovirus members is not well characterized. In this report, structures of two types of wild RHDV particles, the intact virion and the core-like particle (CLP), were reconstructed by cryo-electron microscopy at 11Å and 17Å, respectively. This is the first time the 3D structure of wild caliciviruses CLP has been provided, and the 3D structure of intact RHDV virion is the highest resolution structure in Lagovirus. Comparison of the intact virion and CLP structures clearly indicated that CLP was produced from the intact virion with the protrusion dissociated. In contrast with the crystal structures of recombinant Norovirus and San Miguel sea lion virus, the capsomers of RHDV virion exhibited unique structural features and assembly modes. Both P1 and P2 subdomains have interactions inside the AB capsomer, while only P2 subdomains have interaction inside CC capsomer. The pseudo atomic models of RHDV capsomers were constructed by homology modeling and density map fitting, and the rotation of RHDV VP60 P domain with respect to its S domain, compared with SMSV, was observed. Collectively, our cryo-electron microscopic studies of RHDV provide close insight into the structure of Lagovirus, which is important for functional analysis and better vaccine development in the future.     

Structural Proteins of Simian Virus 40

Sodium dodecyl sulfate acrylamide gel electrophoresis of the solubilized proteins from purified simian virus 40 (SV40) virions revealed two major and two minor structural polypeptide components. The major components which comprise over 75% of the total virion were shown to be the capsid proteins by immunological and isoelectric focusing fractionation analysis. These two polypeptides have estimated molecular weights of 45,000 daltons as determined by gel electrophoresis. One of the two minor components was identified as the nucleocapsid protein and has an approximate molecular weight of 16,000. The other unidentified minor component has an average molecular weight of 29,000.     

Molecular biology of rotaviruses. I. Characterization of basic growth parameters and pattern of macromolecular synthesis

The United Kingdom tissue-adapted bovine rotavirus growing in African green monkey kidney (BSC-1) cells was selected as a model system with which to study the detailed molecular virology of rotavirus replication. Study of the kinetics of infectious virus production revealed a fairly rapid replication cycle, with maximum yield of virus after 10 to 12 h at 37 degrees C. Progeny genome synthesis was first detected during the virus latent period at 2 to 3 h postinfection. Study of the kinetics of viral polypeptide synthesis showed that virus rapidly inhibited cellular polypeptide synthesis such that by 4 h postinfection, only virus-induced polypeptides, 15 of which were detected, were being synthesized. No qualitative changes in the pattern of viral polypeptide synthesis were observed during infection, although, based on kinetic synthesis, three quantitative classes of polypeptides were defined. Pulse-chase analysis revealed three post-translational changes in viral proteins, two of which were shown to be due to glycosylation. Tunicamycin inhibition studies were used to identify the putative non-glycosylated precursors of the two glycoproteins. Comparison of the infected-cell polypeptides with those present in purified virions revealed that mot of the virus-induced proteins were incorporated into virions, with only VP9 being a truly nonstructural protein. Some localization of the various polypeptides within the purified virion was achieved by producing viral cores.     

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of bovine serum albumin oligomers produced by lipid peroxidation.

The oligomers of bovine serum albumin were produced by controlled reaction with peroxidizing linoleic acid to examine their possible utility as calibration proteins insodium dodecyl sulfate-polyacrylamide gel electrophoresis. The polymerization was effected in reaction mixtures containing linoleic acid undergoing peroxidation in the presence of ascorbic acid, and conditions that yield soluble oligomers with a wide molecular weight distribution were established. The interaction of these soluble oligomers with sodium dodecyl sulfate exhibited a binding isotherm indistinguishable from that obtained with bovine serum albumin. Furthermore, sodium dodecy sulfate-polyacrylamide gel electrophoresis of the albumin oligomers conformed to the empirical relation of molecular weight to mobility that pertains to the use of these oligomers as standard molecular weight markers.