The cloning, sequencing and expression of a major antigenic region from the feline calicivirus capsid protein.
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RNA purified from the feline calicivirus (FCV) F9 vaccine strain was used to prepare a cDNA library in the expression vector lambda gt11. The library was screened for expression of FCV antigen using a rabbit antiserum prepared against purified FCV. A 330 bp cDNA clone was identified and used as a probe to obtain a larger overlapping clone of 1369 bp. Comparative sequence analysis with the CFI and F4 strains showed that the clones were derived from the 3' open reading frame encoding the capsid protein. The region encoded by the 330 bp clone was shown to be variable in the three strains compared, and therefore the probable location of major antigenic variation. This clone was expressed in a bacterial system and antiserum to the recombinant protein was used in immunoblots to confirm that this clone was derived from the gene encoding the capsid protein. From these immunoblots, several other capsid-related polypeptides were identified. Comparison with immunoblots using post-vaccination cat sera showed the antibody response in the cat was directed mainly against the capsid protein. Antiserum to the recombinant protein was shown to be effective in neutralizing the infectivity of FCV, indicating that at least one major neutralizing epitope had been cloned. (+info)
In vitro proteolytic processing of the MD145 norovirus ORF1 nonstructural polyprotein yields stable precursors and products similar to those detected in calicivirus-infected cells.
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The MD145-12 strain (GII/4) is a member of the genus Norovirus in the Caliciviridae and was detected in a patient with acute gastroenteritis in a Maryland nursing home. The open reading frame 1 (ORF1) (encoding the nonstructural polyprotein) was cloned as a consensus sequence into various expression vectors, and a proteolytic cleavage map was determined. The virus-encoded cysteine proteinase mediated at least five cleavages (Q(330)/G(331), Q(696)/G(697), E(875)/G(876), E(1008)/A(1009), and E(1189)/G(1190)) in the ORF1 polyprotein in the following order: N-terminal protein; nucleoside triphosphatase; 20-kDa protein (p20); virus protein, genome linked (VPg); proteinase (Pro); polymerase (Pol). A time course analysis of proteolytic processing of the MD145-12 ORF1 polyprotein in an in vitro coupled transcription and translation assay allowed the identification of stable precursors and final mapped cleavage products. Stable precursors included p20VPg (analogous to the 3AB of the picornaviruses) and ProPol (analogous to the 3CD of the picornaviruses). Less stable processing intermediates were identified as p20VPgProPol, p20VPgPro, and VPgPro. The MD145-12 Pro and ProPol proteins were expressed in bacteria as active forms of the proteinase and used to further characterize their substrate specificities in trans cleavage assays. The MD145-12 Pro was able to cleave its five mapped cleavage sites in trans and, in addition, could mediate trans cleavage of the Norwalk virus (GI/I) ORF1 polyprotein into a similar proteolytic processing profile. Taken together, our data establish a model for proteolytic processing in the noroviruses that is consistent with nonstructural precursors and products identified in studies of caliciviruses that replicate in cell culture systems. (+info)
Inter- and intragenus structural variations in caliciviruses and their functional implications.
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The family Caliciviridae is divided into four genera and consists of single-stranded RNA viruses with hosts ranging from humans to a wide variety of animals. Human caliciviruses are the major cause of outbreaks of acute nonbacterial gastroenteritis, whereas animal caliciviruses cause various host-dependent illnesses with a documented potential for zoonoses. To investigate inter- and intragenus structural variations and to provide a better understanding of the structural basis of host specificity and strain diversity, we performed structural studies of the recombinant capsid of Grimsby virus, the recombinant capsid of Parkville virus, and San Miguel sea lion virus serotype 4 (SMSV4), which are representative of the genera Norovirus (genogroup 2), Sapovirus, and Vesivirus, respectively. A comparative analysis of these structures was performed with that of the recombinant capsid of Norwalk virus, a prototype member of Norovirus genogroup 1. Although these capsids share a common architectural framework of 90 dimers of the capsid protein arranged on a T=3 icosahedral lattice with a modular domain organization of the subunit consisting of a shell (S) domain and a protrusion (P) domain, they exhibit distinct differences. The distally located P2 subdomain of P shows the most prominent differences both in shape and in size, in accordance with the observed sequence variability. Another major difference is in the relative orientation between the S and P domains, particularly between those of noroviruses and other caliciviruses. Despite being a human pathogen, the Parkville virus capsid shows more structural similarity to SMSV4, an animal calicivirus, suggesting a closer relationship between sapoviruses and animal caliciviruses. These comparative structural studies of caliciviruses provide a functional rationale for the unique modular domain organization of the capsid protein with an embedded flexibility reminiscent of an antibody structure. The highly conserved S domain functions to provide an icosahedral scaffold; the hypervariable P2 subdomain may function as a replaceable module to confer host specificity and strain diversity; and the P1 subdomain, located between S and P2, provides additional fine-tuning to position the P2 subdomain. (+info)
Calicivirus 3C-like proteinase inhibits cellular translation by cleavage of poly(A)-binding protein.
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Caliciviruses are single-stranded RNA viruses that cause a wide range of diseases in both humans and animals, but little is known about the regulation of cellular translation during infection. We used two distinct calicivirus strains, MD145-12 (genus Norovirus) and feline calicivirus (FCV) (genus Vesivirus), to investigate potential strategies used by the caliciviruses to inhibit cellular translation. Recombinant 3C-like proteinases (r3CL(pro)) from norovirus and FCV were found to cleave poly(A)-binding protein (PABP) in the absence of other viral proteins. The norovirus r3CL(pro) PABP cleavage products were indistinguishable from those generated by poliovirus (PV) 3C(pro) cleavage, while the FCV r3CL(pro) products differed due to cleavage at an alternate cleavage site 24 amino acids downstream of one of the PV 3C(pro) cleavage sites. All cleavages by calicivirus or PV proteases separated the C-terminal domain of PABP that binds translation factors eIF4B and eRF3 from the N-terminal RNA-binding domain of PABP. The effect of PABP cleavage by the norovirus r3CL(pro) was analyzed in HeLa cell translation extracts, and the presence of r3CL(pro) inhibited translation of both endogenous and exogenous mRNAs. Translation inhibition was poly(A) dependent, and replenishment of the extracts with PABP restored translation. Analysis of FCV-infected feline kidney cells showed that the levels of de novo cellular protein synthesis decreased over time as virus-specific proteins accumulated, and cleavage of PABP occurred in virus-infected cells. Our data indicate that the calicivirus 3CL(pro), like PV 3C(pro), mediates the cleavage of PABP as part of its strategy to inhibit cellular translation. PABP cleavage may be a common mechanism among certain virus families to manipulate cellular translation. (+info)
Inactivation of caliciviruses.
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The viruses most commonly associated with food- and waterborne outbreaks of gastroenteritis are the noroviruses. The lack of a culture method for noroviruses warrants the use of cultivable model viruses to gain more insight on their transmission routes and inactivation methods. We studied the inactivation of the reported enteric canine calicivirus no. 48 (CaCV) and the respiratory feline calicivirus F9 (FeCV) and correlated inactivation to reduction in PCR units of FeCV, CaCV, and a norovirus. Inactivation of suspended viruses was temperature and time dependent in the range from 0 to 100 degrees C. UV-B radiation from 0 to 150 mJ/cm(2) caused dose-dependent inactivation, with a 3 D (D = 1 log(10)) reduction in infectivity at 34 mJ/cm(2) for both viruses. Inactivation by 70% ethanol was inefficient, with only 3 D reduction after 30 min. Sodium hypochlorite solutions were only effective at >300 ppm. FeCV showed a higher stability at pH <3 and pH >7 than CaCV. For all treatments, detection of viral RNA underestimated the reduction in viral infectivity. Norovirus was never more sensitive than the animal caliciviruses and profoundly more resistant to low and high pH. Overall, both animal viruses showed similar inactivation profiles when exposed to heat or UV-B radiation or when incubated in ethanol or hypochlorite. The low stability of CaCV at low pH suggests that this is not a typical enteric (calici-) virus. The incomplete inactivation by ethanol and the high hypochlorite concentration needed for sufficient virus inactivation point to a concern for decontamination of fomites and surfaces contaminated with noroviruses and virus-safe water. (+info)
A chimeric bovine enteric calicivirus: evidence for genomic recombination in genogroup III of the Norovirus genus of the Caliciviridae.
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The Norovirus genus of the Caliciviridae encompasses viruses that cause outbreaks of gastroenteritis in human and viruses that have been associated with diarrhea in cattle. The two bovine noroviruses, Bo/Newbury2/76/UK and Bo/Jena/80/DE, represent two distinct genetic clusters in the newly described genogroup III. In the present study, Jena-like polymerase sequences were identified for the first time in the UK, but one of these, Bo/Thirsk10/00/UK, was a chimeric virus. Bo/Thirsk10/00/UK had a Jena-like polymerase gene but Newbury2-like capsid and ORF3 genes by comparison of their genome organization, nucleotide, and amino acid identities and phylogenetic analyses. The present study is one of few studies to clearly demonstrate the existence of chimeric genomes in the Norovirus genus and the first, to our knowledge, to identify a chimeric genome in genogroup III. It provides additional support that genomic recombination is part of the natural evolution of noroviruses and is relevant to the diagnosis and immunological control of norovirus diarrhea outbreaks. (+info)
Human caliciviruses as a cause of severe gastroenteritis in Peruvian children.
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To define the role of human caliciviruses (HuCVs) in severe childhood gastroenteritis, fecal and paired serum samples from 233 Peruvian children hospitalized with gastroenteritis (case patients) and fecal samples from 248 control subjects were evaluated. Overall, 128 case patients (55%) demonstrated HuCV infection by either fecal (n=81 [35%]) or serological (n=96 [41%]) testing. HuCVs were more prevalent in fecal samples from case patients than those from control subjects (35% vs. 13%; P<.001). HuCV infection was more prevalent among case patients without another pathogen than in those who had a coinfecting pathogen (77% [40/52] vs. 49% [88/181]; P<.001). HuCVs appear to be an important cause of gastroenteritis in Peruvian children. (+info)
An insect picornavirus may have genome organization similar to that of caliciviruses.
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Computer-assisted analysis of the amino acid sequence of the product encoded by the sequenced 3' portion of the cricket paralysis virus (CrPV), an insect picornavirus, genome showed that this protein is homologous not to the RNA-directed RNA polymerases, as originally suggested, but to the capsid proteins of mammalian picornaviruses. Alignment of the CrPV protein sequence with those of picornavirus and calicivirus capsid proteins demonstrated that the sequenced portion of the insect picornavirus genome encodes the C-terminal part of VP3 and the entire VP1. Thus CrPV seems to have a genome organization distinct from that of other picornaviruses but closely resembling that of caliciviruses, with the capsid proteins encoded in the 3' part of the genome. On the other hand, the tentative phylogenetic trees generated from the VP3 alignment revealed grouping of CrPV with hepatitis A virus, a true picornavirus, not with caliciviruses. Thus CrPV may be a picornavirus with a calicivirus-like genome organization. Different options for CrPV genome expression are discussed. (+info)