Enzyme-linked immunosorbent assay of antibodies to rabbit haemorrhagic disease virus and determination of its major structural proteins.

An ELISA was developed for the determination of antibodies to rabbit haemorrhagic disease virus (RHDV) in whole blood and blood serum of rabbits. Naturally acquired antibodies were detected in 19.4% of blood samples collected from 1461 rabbits in 43 farms apparently free of the disease, 19.7% samples were doubtful and 60.9% of the rabbits were free of antibodies to RHDV. Their presence has a considerable effect on the resistance of rabbits to infection with RHDV. Antibodies were also found in rabbit blood serum samples collected up to 12 years before the first outbreaks of RHD were reported. Up to 14 viral protein antigens were determined by PAGE and Western blot analysis, of which three with Mr values of 61K, 38K and 52K were major proteins, the 61K being dominant. Our hyperimmune sera, a Chinese reference serum and sera with positive antibody titres, including those collected several years before the first outbreaks of RHD, reacted identically with these antigens in the Western blot analysis. The data obtained suggest that naturally acquired antibodies are a product of a specific response to prior infection with an avirulent strain of the virus.

Biochemical characterization of porcine enteric calicivirus: analysis of structural and nonstructural viral proteins.

In this report, the molecular weight and antigenicity of the proteins of a porcine enteric calicivirus (PEC) were characterized. The PEC virions were purified from intestinal contents of infected pigs and from infected cell culture lysates. The average buoyant density of the purified virus was 1.37 gm/cm3 in cesium chloride. One major structural protein with a molecular weight of approximately 58 k was found in the gut and cell culture-passaged PEC using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Using immunoblotting techniques only one immunoreactive protein (58 k) ws identified. The PEC and a prototype calicivirus, feline calicivirus (FCV) were propagated in pig kidney and feline kidney (Crandell) cell lines, respectively and intrinsically labeled using [35S]methionine at various times post-inoculation (PI). SDS-PAGE of the radiolabeled proteins indicated the presence of the major structural protein (58 k) and one probable nonstructural protein (28 k) synthesized in the PEC-infected cell lysates by 12 h PI. Other minor protein bands were also evident by 24 h PI (32 k and 82 k). Only the 58 k major protein was detected by radioimmunoprecipitation (RIP) analysis using hyperimmune anti-PEC serum. SDS-PAGE and RIP analysis of FCV-infected cell lysates using hyperimmune anti-FCV serum identified a single major protein of approximately 64 k. No antigenic relationship between PEC and FCV proteins was detected by RIP analysis. The single major structural protein of PEC, the morphological appearance and size of the virus, and its average density of 1.37 gm/cm3 in cesium chloride are consistent with properties of other members of the family Caliciviridae.

Immunohistochemical detection of feline calicivirus in formalin-fixed, paraffin-embedded specimens.

An immunohistochemical technique was developed for detection of feline calicivirus (FCV) in formalin-fixed, paraffin-embedded cultured cells and tissues. Initial trials with cultured cells indicated that the indirect immunoperoxidase method using rabbit antiserum to FCV strain 255, and horseradish peroxidase-labelled antibodies to rabbit immunoglobulin G lacked sensitivity and showed excessive diffuse background staining despite trypsin digestion of sections before staining. An amplified indirect immunoperoxidase technique using commercially available biotinylated antirabbit antibodies and avidin-biotin-peroxidase or streptavidin-peroxidase (SP) complexes proved highly successful. When optimal conditions, including those for trypsinization, inactivation of endogenous peroxidase and blocking were determined, the SP technique was preferred. Applied to tissue of cats in the acute phase of FCV infection, the technique provided clear identification of cells containing FCV antigens in sections in which histological detail was well preserved.

The candidate caliciviruses.

The Caliciviridae are a family of small (35-40 nm) RNA viruses with a characteristic cupped morphology. They are unique in possessing only a single major structural polypeptide, of Mr 60,000-71,000. The use of electron microscopy to investigate diarrhoeal diseases has revealed viral particles with the size and structure of the caliciviruses in the faeces of humans, domestic and farm animals, birds, reptiles and insects. In vivo experiments indicate that they are species specific and have confirmed that they replicate in the gut, which often results in the host developing diarrhoea and failing to thrive. Biochemical characterization of these agents has been hampered by a failure to produce sufficient yields of virus in vitro. However, fluorescence and radiolabelling experiments indicate that the human, canine and chicken viruses replicate in the cytoplasm and possess an RNA genome. A major structural polypeptide (Mr 60,000-71,000) has been identified in the human, canine and insect viruses. Diagnosis of the candidate caliciviruses is dependent on electron microscopy and fluorescence labelling, with the exception of the human agents, for which radioimmunoassays have been developed. There is little epidemiological information on these agents but there is increasing evidence that the human caliciviruses are a common cause of outbreaks of diarrhoea and vomiting in infants, adults and the elderly.

Characterization of the Snow Mountain agent of viral gastroenteritis.

Snow Mountain agent (SMA) is a 27- to 32-nm virus which is the etiologic agent of outbreaks of acute gastroenteritis in Colorado and Vermont. SMA is morphologically similar to but antigenically distinct from the Norwalk and Hawaii agents of viral gastroenteritis but, like those agents, has not been cultivated in vitro. We purified and characterized SMA directly from human stool specimens containing the virus. The density of the SMA virion was 1.29 g/cm3 and 1.21 to 1.22 g/cm3 on potassium tartrate-glycerol gradients and 1.33 to 1.34 g/cm3 on cesium chloride gradients. SMA had an S value of 170 to 183S on a sucrose velocity gradient. The purified virion was iodinated, immunoprecipitated with acute and convalescent sera from volunteers challenged with SMA, and analyzed on polyacrylamide gels. The virion contains one major structural protein of 62,000 molecular weight, which is similar in size to the 59,000-molecular-weight protein found in the Norwalk virion. The biophysical properties and single structural protein of SMA most closely resemble those of the calicivirus group.

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.

The polypeptide of a human calicivirus.

Viral particles morphologically resembling animals caliciviruses in the faeces of a patient with acute gastroenteritis were purified, radiolabeled with [125I], and analyzed by SDS-PAGE. A single major structural protein with a mol. mass 62,000 daltons was identified by immunoprecipitation technique. The finding is consistent with human calicivirus-like particles associated with gastroenteritis being a member of the family Caliciviridae.

A protein, VPg, covalently linked to 36S calicivirus RNA.

Proteins associated with 36S virus RNA from Vero cells infected with San Miguel sea lion virus, type 2 (SMSV-2), were labelled with 125I. One protein, VPg, remained linked to RNA when subjected to deproteinization techniques. VPg labelled wtih 32P was observed on 36S RNA from purified virions; the quantity of label was compatible with two phosphates per genome. The estimated mol. wt. of SMSV-2 VPg was 15 000.

Feline calicivirus induced polypeptides.

Analysis of feline calicivirus-infected cell extract for large and low molecular weight proteins revealed the presence, in submolar amounts, of a polypeptide of molecular weight 80,000 daltons which had no precursor-product relationship to the capsid proteins (mol. wt 68,000 and 14,000) synthesized in infected cells. Two other highly labelled non-structural polypeptides of molecular weights 80,000 and 40,000 daltons yet to be identified were also described.

Effect of storage on the integrity of purified feline calicivirus particles.

The effect of storage on the integrity and infectivity of purified feline calicivirus (FCV) particles at varying temperatures (-70 degrees C, -20 degrees C and 4 degrees C) was examined. The results showed that over a period of 8 weeks the purified virus was best preserved at 4 degrees C.

Presence of a covalently linked protein on calicivirus RNA.

The infective RNA of the calicivirus, vesicular exanthema virus, has been shown to contain a protein which is apparently linked to the RNA by a covalent bond. The protein remained bound to the RNA after boiling with SDS-mercaptoethanol-urea or treating with formamide-dimethylsulphoxide but was removed by incubating with proteinase K. The mol. wt. of the protein was estimated to be about 1o X 1O(3) by electrophoresis in highly cross-linked polyacrylamide gels. The infectivity of the RNA was destroyed by removal of the protein with proteinase K.

Relationship of San Miguel sea lion virus to other members of the calicivirus group.

San Miguel sea lion virus (SMSV) is indistinguishable from vesicular exanthema virus (VEV) and feline calicivirus (FCV) in its morphology and in possessing a single capsid polypeptide with a molecular weight of approximately 65 X 10(3). Neutralization tests readily differentiate the three viruses, but immunodiffusion tests show that SMSV is closely related serologically to VEV but not to FCV. Although the RNAs of the three caliciviruses have similar base compositions, homology tests show that SMSV is closely related to VEV but is not related to FCV. Tryptic peptide maps of the single major polypeptide comprising the capsid of each virus also show that SMSV and VEV are more closely related to each other than to FCV.

Feline calicivirus: purification of virus and extraction and characterisation of its ribonucleic acid.

Purification of feline calicivirus was achieved by cycles of differential centrifugation and two cycles of sucrose gradient centrifugation. Feline calicivirus grown in the presence of Actinomycin D and 3H-uridine-5, sediments in 15% to 45% sucrose gradients and forms a peak of radioactivity which corresponds with the peak of infectivity. Ribonucleic acid (RNA) extracted from the peak radioactive fractions taken from the sucrose gradient sedimented as a single peak ahead of the 28S peak of cellular RNA. It was sensitive to ribonuclease and was presumed to be single stranded feline calicivirus RNA with sedimentation of 32S-35S. A single peak of radioactivity at 35S was extracted from purified virus by heating at 60 degrees for two minutes in 1% sodium dodecyl sulphate (SDS), or by heating at 37 degrees for 5 minutes at 1% SDS. Virus extracted at 37 degrees for 10 minutes in 1% SDS showed also a small peak at 16S and by 15 minutes at 37 degrees only a broad peak at 16S occurred. All peaks were susceptible to ribonuclease. A component sedimenting at 18S which was resistent to degradation by ribonuclease under the conditions outlined by Baltimore (4) and presumed to be double-stranded RNA was present in kitten kidney cells infected with feline calicivirus.