Characterization of an enteropathogenic bovine calicivirus representing a potentially new calicivirus genus

Bovine enteric caliciviruses (BEC) are associated with diarrhea in young calves. The BEC strains detected in Europe form a third genogroup within the genus "Norwalk-like viruses" (NLV) of the family Caliciviridae. In this report, we present sequence, clinical, and histological data characterizing a novel enteropathogenic BEC strain, NB, detected in fecal specimens from calves in the United States. The complete RNA genome of the NB virus is 7,453 bases long and is organized into two open reading frames (ORFs). ORF-1 is 2,210 amino acids long and encodes a large nonstructural polyprotein contiguous with the major capsid protein (VP1), similar to the lagoviruses and "Sapporo-like viruses" (SLV). The conserved calicivirus motifs were identified in the nonstructural proteins. ORF-2 is located at the 3' end of the genome and encodes a small basic protein (VP2) of 225 amino acids. The 5' and 3' untranslated regions are 74 and 67 bases long, respectively. Among caliciviruses, NB virus shows amino acid identities of 14.1 to 22.6% over the entire ORF-1 nonstructural-protein sequence with NLV, SLV, vesivirus, and lagovirus strains, while the overall sequence identity of the complete NB VP-1 with other caliciviruses is low, varying between 14.6 and 26.7%. Phylogenetic analysis of the complete VP1 protein, including strains from all four calicivirus genera, showed the closest grouping of NB virus to be with viruses in the genus Lagovirus, which cause liver infections and systemic hemorrhage in rabbits. In gnotobiotic calves, however, NB virus elicited only diarrhea and intestinal lesions that were most severe in the upper small intestine (duodenum and jejunum), similar to the NLV BEC strains. The tissues of major organs, including the lung, liver, kidney, and spleen, had no visible microscopic lesions.     

Molecular Characterization of a Bovine Enteric Calicivirus: Relationship to the Norwalk-Like Viruses

Jena virus (JV) is a noncultivatable bovine enteric calicivirus associated with diarrhea in calves and was first described in Jena, Germany. The virus was serially passaged 11 times in colostrum-deprived newborn calves and caused diarrheal disease symptoms at each passage. The complete JV genome sequence was determined by using cDNA made from partially purified virus obtained from a single stool sample. JV has a positive-sense single-stranded RNA genome which is 7,338 nucleotides in length, excluding the poly(A) tail. JV genome organization is similar to that of the human Norwalk-like viruses (NLVs), with three separate open reading frames (ORFs) and a 24-nucleotide sequence motif located at the 5′ terminus of the genome and at the start of ORF 2. The polyprotein (ORF 1) consists of 1,680 amino acids and has the characteristic 2C helicase, 3C protease, and 3D RNA polymerase motifs also found in the NLVs. However, comparison of the N-terminal 100 amino acids of the JV polyprotein with those of the group 1 and group 2 NLVs showed a considerable divergence in sequence. The capsid protein (ORF 2) at 519 amino acids is smaller than that of all other caliciviruses. JV ORF 2 was translated in vitro to produce a 55-kDa protein that reacted with postinfection serum but not preinfection serum. Phylogenetic studies based on partial RNA polymerase sequences indicate that within the Caliciviridae JV is most closely related to the group 1 NLVs.     

An insect picornavirus may have genome organization similar to that of caliciviruses.

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.     

Genetic map of the calicivirus rabbit hemorrhagic disease virus as deduced from in vitro translation studies.

The 7.5-kb plus-stranded genomic RNA of rabbit hemorrhagic disease virus contains two open reading frames of 7 kb (ORF1) and 351 nucleotides (ORF2) that cover nearly 99% of the genome. The aim of the present study was to identify the proteins encoded in these open reading frames. To this end, a panel of region-specific antisera was generated by immunization of rabbits with bacterially expressed fusion proteins that encompass in total 95% of the ORF1 polyprotein and almost the complete ORF2 polypeptide. The antisera were used to analyze the in vitro translation products of purified virion RNA of rabbit hemorrhagic disease virus. Our studies show that the N-terminal half of the ORF1 polyprotein is proteolytically cleaved to yield three nonstructural proteins of 16, 23, and 37 kDa (p16, p23, and p37, respectively). In addition, a cleavage product of 41 kDa which is composed of VPg and a putative nonstructural protein of approximately 30 kDa was identified. Together with the results of previous studies which identified a trypsin-like cysteine protease (TCP) of 15 kDa, a putative RNA polymerase (pol) of 58 kDa, and the major capsid protein VP60, our data establish the following gene order in ORF1: NH2-p16-p23-p37 (helicase)-p30-VPg-TCP-pol-VP60-COOH. Immunoblot analyses showed that a minor structural protein of 10 kDa is encoded in ORF2. The data provide the first complete genetic map of a calicivirus. The map reveals a remarkable similarity between caliciviruses and picornaviruses with regard to the number and order of the genes that encode the nonstructural proteins.     

European brown hare syndrome virus: relationship to rabbit hemorrhagic disease virus and other caliciviruses.

Monoclonal antibodies directed against the capsid protein of rabbit hemorrhagic disease virus (RHDV) were used to identify field cases of European brown hare syndrome (EBHS) and to distinguish between RHDV and the virus responsible for EBHS. Western blot (immunoblot) analysis of liver extract of an EBHS virus (EBHSV)-infected hare revealed a single major capsid protein species of approximately 60 kDa that shared epitopes with the capsid protein of RHDV. RNA isolated from the liver of an EBHSV-infected hare contained two viral RNA species of 7.5 and 2.2 kb that comigrated with the genomic and subgenomic RNAs of RHDV and were recognized by labeled RHDV cDNA in Northern (RNA) hybridizations. The nucleotide sequence of the 3' 2.8 kb of the EBHSV genome was determined from four overlapping cDNA clones. Sequence analysis revealed an open reading frame that contains part of the putative RNA polymerase gene and the complete capsid protein gene. This particular genome organization is shared by RHDV but not by other known caliciviruses. The deduced amino acid sequence of the capsid protein of EBHSV was compared with the capsid protein sequences of RDDV and other caliciviruses. The amino acid sequence comparisons revealed that EBHSV is closely related to RHDV and distantly related to other caliciviruses. On the basis of their genome organization, it is suggested that caliciviruses be divided into three groups.     

Inactivation of feline calicivirus and adenovirus type 40 by UV radiation

Little information regarding the effectiveness of UV radiation on the inactivation of caliciviruses and enteric adenoviruses is available. Analysis of human calicivirus resistance to disinfectants is hampered by the lack of animal or cell culture methods that can determine the viruses' infectivity. The inactivation kinetics of enteric adenovirus type 40 (AD40), coliphage MS-2, and feline calicivirus (FCV), closely related to the human caliciviruses based on nucleic acid organization and capsid architecture, were determined after exposure to low-pressure UV radiation in buffered demand-free (BDF) water at room temperature. In addition, UV disinfection experiments were also carried out in treated groundwater with FCV and AD40. AD40 was more resistant than either FCV or coliphage MS-2 in both BDF water and groundwater. The doses of UV required to achieve 99% inactivation of AD40, coliphage MS-2, and FCV in BDF water were 109, 55, and 16 mJ/cm(2), respectively. The doses of UV required to achieve 99% inactivation of AD40, coliphage MS-2, and FCV in groundwater were slightly lower than those in BDF water. FCV was inactivated by 99% by 13 mJ/cm(2) in treated groundwater. A dose of 103 mJ/cm(2) was required for 99% inactivation of AD40 in treated groundwater. The results of this study indicate that if FCV is an adequate surrogate for human caliciviruses, then their inactivation by UV radiation is similar to those of other single-stranded RNA enteric viruses, such as poliovirus. In addition, AD40 appears to be more resistant to UV disinfection than previously reported.     

Inactivation of Feline Calicivirus and Adenovirus Type 40 by UV Radiation

Little information regarding the effectiveness of UV radiation on the inactivation of caliciviruses and enteric adenoviruses is available. Analysis of human calicivirus resistance to disinfectants is hampered by the lack of animal or cell culture methods that can determine the viruses' infectivity. The inactivation kinetics of enteric adenovirus type 40 (AD40), coliphage MS-2, and feline calicivirus (FCV), closely related to the human caliciviruses based on nucleic acid organization and capsid architecture, were determined after exposure to low-pressure UV radiation in buffered demand-free (BDF) water at room temperature. In addition, UV disinfection experiments were also carried out in treated groundwater with FCV and AD40. AD40 was more resistant than either FCV or coliphage MS-2 in both BDF water and groundwater. The doses of UV required to achieve 99% inactivation of AD40, coliphage MS-2, and FCV in BDF water were 109, 55, and 16 mJ/cm², respectively. The doses of UV required to achieve 99% inactivation of AD40, coliphage MS-2, and FCV in groundwater were slightly lower than those in BDF water. FCV was inactivated by 99% by 13 mJ/cm² in treated groundwater. A dose of 103 mJ/cm² was required for 99% inactivation of AD40 in treated groundwater. The results of this study indicate that if FCV is an adequate surrogate for human caliciviruses, then their inactivation by UV radiation is similar to those of other single-stranded RNA enteric viruses, such as poliovirus. In addition, AD40 appears to be more resistant to UV disinfection than previously reported.     

The 3' end of Norwalk virus mRNA contains determinants that regulate the expression and stability of the viral capsid protein VP1: a novel function for the VP2 protein

Norwalk virus (NV) is the prototype strain of a group of noncultivable human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. The capsid protein VP1 is synthesized from a subgenomic RNA that contains two open reading frames (ORFs), ORF2 and ORF3, and the 3' untranslated region (UTR). ORF2 and ORF3 code for the capsid protein (VP1) and a small structural basic protein (VP2), respectively. We discovered that the yields of virus-like particles (VLPs) composed of VP1 are significantly reduced when this protein is expressed from ORF2 alone. To determine how the 3' terminus of the NV subgenomic RNA regulates VP1 expression, we compared VP1 expression levels by using recombinant baculovirus constructs containing different 3' elements. High VP1 levels were detected by using a recombinant baculovirus that contained ORF2, ORF3, and the 3'UTR (ORF2+3+3'UTR). In contrast, expression of VP1 from constructs that lacked the 3'UTR (ORF2+3), ORF3 (ORF2+3'UTR), or both (ORF2 alone) was highly reduced. Elimination of VP2 synthesis from the subgenomic RNA by mutation resulted in VP1 levels similar to those obtained with the ORF2 construct alone, suggesting a cis role for VP2 in upregulation of VP1 expression levels. Comparisons of the kinetics of RNA and capsid protein expression levels by using constructs with or without ORF3 or the 3'UTR revealed that the 3'UTR increased the levels of VP1 RNA, whereas the presence of VP2 resulted in increased levels of VP1. Furthermore, VP2 increased VP1 stability and protected VP1 from disassembly and protease degradation. The increase in VP1 expression levels caused by the presence of VP2 in cis was also observed in mammalian cells.     

Mutagenesis of tyrosine 24 in the VPg protein is lethal for feline calicivirus

The genome of feline calicivirus (FCV) is an approximately 7.7-kb single-stranded positive-sense RNA molecule that is polyadenylated at its 3' end and covalently linked to a VPg protein (calculated mass, 12.6 kDa) at its 5' end. We performed a mutational analysis of the VPg protein in order to identify amino acids potentially involved in linkage to the genome and replication. The tyrosine residues at positions 12, 24, 76, and 104 were changed to alanines by mutagenesis of an infectious FCV cDNA clone. Viruses were recovered when Tyr-12, Tyr-76, or Tyr-104 of the VPg protein was changed to alanine, but virus was not recovered when Tyr-24 was changed to alanine. Growth properties of the recovered viruses were similar to those of the parental virus. We examined whether the amino acids serine, threonine, and phenylalanine could substitute for the tyrosine at position 24, but these mutations were lethal as well. A tyrosine at this relative position is conserved among all calicivirus VPg proteins examined thus far, suggesting that the VPg protein of caliciviruses, like those of picornaviruses and potyviruses, utilizes tyrosine in the formation of a covalent bond with RNA.     

Cryo-EM Structure of a Novel Calicivirus,Tulane Virus

Tulane virus (TV) is a newly isolated cultivatable calicivirus that infects juvenile rhesus macaques. Here we report a 6.3 Å resolution cryo-electron microscopy structure of the TV virion. The TV virion is about 400 Å in diameter and consists of a T = 3 icosahedral protein capsid enclosing the RNA genome. 180 copies of the major capsid protein VP1 (∼57 KDa) are organized into two types of dimers A/B and C/C and form a thin, smooth shell studded with 90 dimeric protrusions. The overall capsid organization and the capsid protein fold of TV closely resemble that of other caliciviruses, especially of human Norwalk virus, the prototype human norovirus. These close structural similarities support TV as an attractive surrogate for the non-cultivatable human noroviruses. The most distinctive feature of TV is that its C/C dimers are in a highly flexible conformation with significantly reduced interactions between the shell (S) domain and the protruding (P) domain of VP1. A comparative structural analysis indicated that the P domains of TV C/C dimers were much more flexible than those of other caliciviruses. These observations, combined with previous studies on other caliciviruses, led us to hypothesize that the enhanced flexibility of C/C dimer P domains are likely required for efficient calicivirus-host cell interactions and the consequent uncoating and genome release. Residues in the S-P1 hinge between the S and P domain may play a critical role in the flexibility of P domains of C/C dimers.     

兔出血症病毒（RHDV）WHNRH株的分离鉴定及基因组全序列的测定与分析

从发病长毛兔中分离鉴定了兔病毒性出血症病毒WHNRH株。参考GenBank中已登录的RHDV毒株序列对RHDV WHNRH分离株进行了全基因组序列测定与分析。设计5对扩增区段相互重叠的RHDV特异性引物,扩增除5′和3′末端以外的序列,采用设计锚引物的5′RACE方法以及针对RHDV 3′末端的polyA结构设计引物获得了RHDV WHNRH株的5′和3′末端序列。胶回收各PCR产物,连接pMD 18-T克隆载体,测得RHDV WHNRH分离株的基因组全长为7437nt(不包括polyA),与GenBank公布的全部共6株RHDV全基因序列进行同源性比较分析,同源性在89.0%~97.1%之间,ORF1同源性为89.0%~97.1%,编码氨基酸序列的同源性为95.2%~98.7%;ORF2的核酸苷序列同源性为92.1%~97.7%,编码氨基酸序列的同源性为94.1%~96.6%。     

Conditional rather than absolute requirements of the capsid coding sequence for initiation of methionine-independent translation in Plautia stali intestine virus

The positive-stranded RNA genome of Plautia stali intestine virus (PSIV) has an internal ribosome entry site (IRES) in an intergenic region (IGR). The IGR-IRES of PSIV initiates translation of the capsid protein by using CAA, the codon for glutamine. It was previously reported (J. Sasaki and N. Nakashima, J. Virol. 73:1219-1226, 1999) that IGR-IRES extended by several nucleotides into the capsid open reading frame (ORF). Despite the fact that the secondary structure model of the IGR-IRES is highly conserved, we were unable to find structural similarities in the 5' region of the capsid ORFs in related viruses. Therefore, we reevaluated the role of the capsid ORF in IGR-IRES-mediated translation in PSIV. Mutation of the CAA codon with various triplets did not inhibit IGR-IRES-mediated translation. N-terminal amino acid analyses of mutated products showed that the IGR-IRES could initiate translation by using various elongator tRNAs. By replacement of the capsid ORF with exogenous coding sequences having AUG deleted, translation products were produced in most cases, but capsid-exogenous fusion proteins were produced more efficiently than were the translation products. These data indicate that the 5' part of the capsid ORF is not an absolute requirement for the IGR-IRES-mediated translation. RNA structure probing analyses showed that the 5' part of the capsid ORF was a single strand, while that of exogenous reading frames was structured. Exogenous sequences also caused structural distortion in the 3' part of the IGR-IRES. We hypothesize that the single-stranded capsid ORF helps to form the tertiary structure of the IGR-IRES and facilitates precise positioning of ribosomes.     

Evolution of human calicivirus RNA in vivo: accumulation of mutations in the protruding P2 domain of the capsid leads to structural changes and possibly a new phenotype

In the present study we report on evolution of calicivirus RNA from a patient with chronic diarrhea (i.e., lasting >2 years) and viral shedding. Partial sequencing of open reading frame 1 (ORF1) from 12 consecutive isolates revealed shedding of a genogroup II virus with relatively few nucleotide changes during a 1-year period. The entire capsid gene (ORF2) was also sequenced from the same isolates and found to contain 1,647 nucleotides encoding a protein of 548 amino acids with similarities to the Arg320 and Mx strains. Comparative sequence analysis of ORF2 revealed 32 amino acid changes during the year. It was notable that the vast majority of the cumulative amino acid changes (8 of 11) appeared within residues 279 to 405 located within the hypervariable domain (P2) of the capsid protein and hence were subject to immune pressure. An interesting and novel observation was that the accumulated amino acid changes in the P2 domain resulted in predicted structural changes, including disappearance of a helix structure, and thus a possible emergence of a new phenotype. FUT2 gene polymorphism characterization revealed that the patient is heterozygous at nucleotide 428 and thus Secretor(+), a finding in accordance with the hypothesis of FUT2 gene polymorphism and calicivirus susceptibility. To our knowledge, this is the first report of RNA evolution of calicivirus in a single individual, and our data suggest an immunity-driven mechanism for viral evolution. We also report on chronic virus excretion, immunoglobulin treatment, and modification of clinical symptoms; our observations from these studies, together with the FUT2 gene characterization, may lead to a better understanding of calicivirus pathogenesis.     

The ORF2 protein of hepatitis E virus binds the 5' region of viral RNA

Hepatitis E virus (HEV) is a major human pathogen in much of the developing world. It is a plus-strand RNA virus with a 7.2-kb polyadenylated genome consisting of three open reading frames, ORF1, ORF2, and ORF3. Of these, ORF2 encodes the major capsid protein of the virus and ORF3 encodes a small protein of unknown function. Using the yeast three-hybrid system and traditional biochemical techniques, we have studied the RNA binding activities of ORF2 and ORF3, two proteins encoded in the 3' structural part of the genome. Since the genomic RNA from HEV has been postulated to contain secondary structures at the 5' and 3' ends, we used these two terminal regions, besides other regions within the genome, in this study. Experiments were designed to test for interactions between the genomic RNA fusion constructs with ORF2 and ORF3 hybrid proteins in a yeast cellular environment. We show here that the ORF2 protein contains RNA binding activity. The ORF2 protein specifically bound the 5' end of the HEV genome. Deletion analysis of this protein showed that its RNA binding activity was lost when deletions were made beyond the N-terminal 111 amino acids. Finer mapping of the interacting RNA revealed that a 76-nucleotide (nt) region at the 5' end of the HEV genome was responsible for binding the ORF2 protein. This 76-nt region included the 51-nt HEV sequence, conserved across alphaviruses. Our results support the requirement of this conserved sequence for interaction with ORF2 and also indicate an increase in the strength of the RNA-protein interaction when an additional 44 bases downstream of this 76-nt region were included. Secondary-structure predictions and the location of the ORF2 binding region within the HEV genome indicate that this interaction may play a role in viral encapsidation.     

Inactivation of caliciviruses

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.     

A bipartite sequence motif induces translation reinitiation in feline calicivirus RNA

The mechanism leading to reinitiation of translation after termination of protein synthesis in eukaryotes has not yet been resolved in detail. One open question concerns the way the post-termination ribosome is tethered to the mRNA to allow binding of the necessary initiation factors. In caliciviruses, a family of positive strand RNA viruses, the capsid protein VP2 is translated via a termination/reinitiation process. VP2 of the feline calicivirus is encoded in the 3'-terminal open reading frame 3 (ORF3) that overlaps with the preceding ORF2 by four nucleotides. In transient expression studies, the efficiency of VP2 expression was 20 times lower than that of the ORF2 proteins. The close vicinity of the ORF2 termination signal and the ORF3 AUG codon was crucial, whereas the AUG could be replaced by alternative codons. Deletion mapping revealed that the 3'-terminal 69 nucleotides of ORF2 are crucial for VP2 expression. This sequence contains two essential sequence motifs. The first motif is conserved among caliciviruses and complementary to part of the 18 S rRNA. In conclusion, VP2 is expressed in a translation termination/reinitiation process that is special because it requires a sequence element that could prevent dissociation of post-termination ribosomes via hybridization with 18 S rRNA.