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The family Caliciviridae includes viruses with single-stranded, positive-sense RNA genomes of 7.4-8.3 kb. The most clinically important representatives are human noroviruses, which are a leading cause of acute gastroenteritis in humans. Virions are non-enveloped with icosahedral symmetry. Members of seven genera infect mammals ( Lagovirus , Norovirus , Nebovirus , Recovirus , Sapovirus , Valovirus and Vesivirus ), members of two genera infect birds ( Bavovirus and Nacovirus ), and members of two genera infect fish ( Minovirus and Salovirus ). This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Caliciviridae , which is available at ictv.global/report/caliciviridae.

Single particle cryo-electron microscopy (cryo-EM) is an emerging powerful tool for structural studies of macromolecular assemblies (i.e., protein complexes and viruses). Although single particle cryo-EM requires less concentrated and smaller amounts of samples than X-ray crystallography, it remains challenging to study specimens that are low-abundance, low-yield, or short-lived. The recent development of affinity grid techniques can potentially further extend single particle cryo-EM to these challenging samples by combining sample purification and cryo-EM grid preparation into a single step. Here we report a new design of affinity cryo-EM approach, cryo-SPIEM, that applies a traditional pathogen diagnosis tool Solid Phase Immune Electron Microscopy (SPIEM) to the single particle cryo-EM method. This approach provides an alternative, largely simplified and easier to use affinity grid that directly works with most native macromolecular complexes with established antibodies, and enables cryo-EM studies of native samples directly from cell cultures. In the present work, we extensively tested the feasibility of cryo-SPIEM with multiple samples including those of high or low molecular weight, macromolecules with low or high symmetry, His-tagged or native particles, and high- or low-yield macromolecules. Results for all these samples (non-purified His-tagged bacteriophage T7, His-tagged Escherichiacoli ribosomes, native Sindbis virus, and purified but low-concentration native Tulane virus) demonstrated the capability of cryo-SPIEM approach in specifically trapping and concentrating target particles on TEM grids with minimal view constraints for cryo-EM imaging and determination of 3D structures., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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.

The caliciviruses are by far the major cause of non-bacterial gastroenteritis, highly infectious, and have a rapid and severe onset of symptoms. Studies on this family of viruses have been hampered by the lack of animal model and tissue culture system. However, recent advances in protein expression systems and the development of a mouse norovirus animal model has led to rapid advances in our understanding of these viruses with regard to structure and the host immune response. Our current understanding of this important family of viruses is reviewed here.

Caliciviruses are a well-established cause of respiratory, vesicular and hemorrhagic diseases in animals. In addition, these viruses are an important cause of enteric diseases in humans. Recently, molecular analysis of several porcine enteric caliciviruses indicated that they are closely related to human enteric caliciviruses. The objective of this work was to determine the frequency, age distribution, and association with diarrhea of enteric calicivirus infections in piglets and to partially characterize the detected isolates. A total of 203 stool samples from animals 0 to 9 weeks of age, collected between 1993 and 2003 in seven porcine farms located in the central region of Venezuela were tested for enteric caliciviruses by reverse transcription-polymerase chain reaction (RT-PCR) amplification using primers designed to detect both norovirus and sapovirus. Selected amplicons were sequenced to establish phylogenetic relationships with reference strains. Calicivirus were detected in 18% (36/204) of the samples. Viruses were detected more frequently in animals between 3 and 4 weeks of age, and were detected in samples from animals with diarrhea and without diarrhea with equal frequencies (14 versus 19%, p>0.5). Phylogenetic studies based on partial RNA polymerase gene sequences indicated that the Venezuelan isolates were most closely related (75-95% identity) to the sapovirus Cowden reference strain. These results provide evidence that caliciviruses of the genus sapovirus circulate frequently in piglets but further studies are needed to clarify their importance as cause of diarrhea.

The pathogenic bovine enteric virus, Newbury agent-1 (Bo//Newbury1/1976/UK), first identified in 1976, was characterized as a possible calicivirus by morphology, buoyant density in CsCl and the presence of a single capsid protein but genomic sequence could not be obtained. In the present study, the complete genome sequence of Newbury1 was determined and classified Newbury1 in a new genus of the Caliciviridae. The Newbury1 genome, of 7454 nucleotides, had two predicted open reading frames (ORFs). ORF1 encoded the non-structural and contiguous capsid proteins. ORF2 encoded a basic protein characteristic of the family Caliciviridae. Compared to the 4 recognized Caliciviridae genera, Norovirus, Sapovirus, Lagovirus and Vesivirus, Newbury1 had less than 39% amino acid (47% nucleotide) identity in the complete 2C-helicase, 3C-protease, 3D-polymerase and capsid regions but had 89% to 98% amino acid (78% to 92% nucleotide) identity to the recently characterized NB virus in these regions. By phylogenetic analyses, Newbury1 and NB viruses formed a distinct clade independent of the 4 recognized genera. However, amino acid identities showed that Newbury1 and the NB virus were distinct polymerase types (90% amino acid identity), but their complete capsid proteins were almost identical (98% amino acid identity). Analyses of contemporary viruses showed that the two polymerase genotypes, Newbury1 and NB, were circulating in UK cattle and antibody to Newbury1-like viruses was common in cattle sera. The present study defined the existence of a new genus in the Caliciviridae that we propose be named Becovirus or Nabovirus to distinguish the new clade from bovine noroviruses.

Caliciviruses, grouped into four genera, are important human and veterinary pathogens with a potential for zoonosis. In these viruses, capsid-related functions such as assembly, antigenicity, and receptor interactions are predominantly encoded in a single protein that forms an icosahedral capsid. Understanding of the immunologic functions and pathogenesis of human caliciviruses in the Norovirus and Sapovirus genera is hampered by the lack of a cell culture system or animal models. Much of our understanding of these viruses, including the structure, has depended on recombinant capsids. Here we report the atomic structure of a native calicivirus from the Vesivirus genus that exhibits a broad host range possibly including humans and map immunological function onto a calicivirus structure. The vesivirus structure, despite a similar architectural design as seen in the recombinant norovirus capsid, exhibits novel features and indicates how the unique modular organization of the capsid protein with interdomain flexibility, similar to an antibody structure with a hinge and an elbow, integrates capsid-related functions and facilitates strain diversity in caliciviruses. The internally located N-terminal arm participates in a novel network of interactions through domain swapping to assist the assembly of the shell domain into an icosahedral scaffold, from which the protruding domain emanates. Neutralization epitopes localize to three hypervariable loops in the distal portion of the protruding domain surrounding a region that exhibits host-specific conservation. These observations suggest a mechanism for antigenic diversity and host specificity in caliciviruses and provide a structural framework for vaccine development.

Background and Aim of the Work: Since the introduction of the electron microscope and its subsequent development, virology has made a great step forward by the improvement of the basic knowledge on viral structure, as well as by broad application of electron microscopy (EM) to viral diagnosis. In this report, we describe a five-year experience in the use of EM for the diagnosis of enteric viral infections., Methods: Three thousand four hundred and ninety stool specimens were analyzed at the Virology Unit (Section of Microbiology, Department of Pathology and Laboratory Medicine, University of Parma, Italy) during a five-year period, from January 1999 to January 2004. The faecal extracts were subjected to EM after negative staining and were simultaneously cultured to evidence the presence of cytopathogenic agents., Results: EM directly applied to the above specimens allowed the detection of several enteric viral agents, particularly evidencing those normally hard to cultivate (thus easily lost with culture methods). It also enabled diagnosis of dual gut infections, such as those from rotavirus and calicivirus. On the other hand, EM-based identification of viral agents after cell culture and ultracentrifugation of cytopathogenic agent-containing cellular extracts, allowed the identification of cultivable agents, such as picornaviruses, which can escape the direct EM detection if low concentrated., Conclusions: A rationalized use of EM on selected samples, such as stool, appears suitable in epidemiological or clinical conditions when a very rapid diagnosis is required to save time, including cases of suspected emerging viral infections.

Background: Many paediatric illnesses are caused by viral agents, for example, acute gastroenteritis. Electron microscopy can provide images of viral particles and can be used to identify the agents., Objectives: The use of electron microscopy as a diagnostic tool is limited by the need for high level of expertise in interpreting these images and the time required. A semi-automated method is proposed in this paper., Study Design: The method is based on bispectal features that capture contour and texture information while providing robustness to shift, rotation, changes in size and noise. The magnification or true size of the viral particles need not be known precisely, but if available can be used additionally for improved classification. Viral particles from one or more images are segmented and analyzed to verify whether they belong to a particular class (such as Adenovirus, Rotavirus, etc.) or not. Two experiments were conducted-depending on the populations from which virus particle images were collected for training and testing, respectively. In the first, disjoint subsets from a pooled population of virus particles obtained from several images were used. In the second, separate populations from separate images were used. The performance of the method on viruses of similar size was separately evaluated using Astrovirus, HAV and Poliovirus. A Gaussian Mixture Model was used for the probability density of the features. A threshold on the log-likelihood is varied to study false alarm and false rejection trade-off. Features from many particles and/or likelihoods from independent tests are averaged to yield better performance., Results: An equal error rate (EER) of 2% is obtained for verification of Rotavirus (tested against three other viruses) when features from 15 viral particle images are averaged. It drops further to less than 0.2% when scores from two tests are averaged to make a decision. For verification of Astrovirus (tested against two others of the same size) the EER was less than 2% when 20 particles and two tests were used., Conclusion: Bispectral features and Gaussian mixture modelling of their probability density are shown to be effective in identifying viruses from electron microscope images. With the use of digital imaging in electron microscopes, this method can be fully automated.

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.

Young rabbits are naturally resistant to rabbit haemorrhagic disease (RHD) caused by the same calicivirus that kills, within 3 days, nearly all adult animals. We have investigated changes in blood leukocytes, and in the morphology and biochemistry of the liver (the organ where caliciviruses replicate) of young rabbits undergoing benign infection by the RHD virus. Four-week-old rabbits were infected with a calicivirus inoculum having a titre of 2(12) haemagglutination units either sacrificed 18, 24, 48 and 72 h later, or kept for follow-up studies up to 21 days after inoculation. The infection caused an acute and transient decrease in blood heterophils, and sustained enhancement in hepatic transaminases. Inflammatory infiltrates of the liver were seen in all animals after 24 h of infection; they had a predominant midlobular location. Hepatocytes could present different degrees of cell damage, including cell death; these lesions were limited to the liver cells located around the inflammatory infiltrates. Liver transaminases peaked 24-48 h after calicivirus infection; this was the same timing when liver infiltration and hepatocyte damage were more evident. No alterations of other parameters of liver biochemistry were observed. We conclude that calicivirus infection of young rabbits causes a subclinical disorder characterised by an acute and transient decrease in circulating heterophils, and focal liver damage that is expressed by intralobular infiltration by heterophils, initially, and, later on, by mononuclear cells. Our finding of persistence of increased values of liver transaminases suggests chronicity of the infection in young rabbits. We propose that, although resistant to RHD, young rabbits infected by calicivirus may be long-term carriers of the infectious agent and, thus, become a major source of transmission of the virus.

Noroviruses (NV) are transmitted by fecally contaminated food, vomit, and person-to-person contact. They are one of the main causes of non-bacterial acute gastroenteritis in nursing, old people and children's homes. NV outbreaks are characterized by a short incubation period (12-48 h), nausea, vomiting and diarrhea, and high secondary attack rates. The illness is generally mild and self-limiting. The aim of diagnostic procedures in viral gastroenteritis is to avoid nosocomial infections on the one hand and unnecessary antibiotic treatment on the other. Diagnostic procedures for NV are based on the detection of virus in stool samples by (immune) transmission electron microscopy (TEM), antigen ELISA, or polymerase chain reaction (PCR). In our study, a total of 244 stool samples obtained from 227 patients between March and May 2002 were tested by TEM, antigen ELISA and in-house PCR. Our data showed that PCR has the highest sensitivity (94.1%), followed by TEM (58.3%), and ELISA (31.3%), while specificity was highest for TEM (98.0%), followed by ELISA (94.9%), and PCR (92.4%). All three methods tested (TEM, ELISA and PCR) are useful for epidemiological investigations in gastroenteritis outbreaks; however, to maximize diagnostic validity for individual cases, at least two of the methods should be combined., (Copyright 2003 S. Karger AG, Basel)

Enteric caliciviruses are emerging pathogens responsible for diarrhea or gastroenteritis in their respective hosts. In this report, mink enteric caliciviruses (MEC) were detected in feces from diarrheic mink by both immune electron microscopy (IEM) and RT-PCR using a broadly reactive primer pair (p289/290) targeting the highly conserved RNA polymerase regions of the enteric caliciviruses, Norwalk-like viruses (NLVs) and Sapporo-like viruses (SLVs). The MEC possess classical caliciviral morphology with typical cup-shaped depressions on the viral surface. Sequence analyses based on nucleotide and predicted amino acid (aa) sequences of the RT-PCR products indicated that MEC is most closely related genetically to SLVs of humans and animals. The MEC shared the highest aa identities (64-71%) in the RNA polymerase region with both human SLVs and the porcine enteric calicivirus (PEC) Cowden strain SLV, indicating that MEC may belong to an individual genogroup or subgroup in the SLV genus. The MEC shared only limited aa identities in the RNA polymerase region with vesiviruses (40-51%) and NLVs (29-33%). The RNA polymerase regions of the cultivable, non-enteric mink caliciviruses (MCV) were also amplified by RT-PCR using the primer pair Pol1/Pol3 based on sequences of vesiviruses, and the primer pair p289/290. Sequence analysis indicated that these MCV shared higher aa identities in the RNA polymerase region with vesiviruses (58-81%) than with SLVs (43-51%) including the MEC, lagoviruses (35-37%) and NLVs (27-35%), suggesting that they are most closely related genetically to vesiviruses. The MEC associated with diarrhea in mink are morphologically similar to but are genetically distinct from the cultivable MCV and likely represent a new member of the SLV genus.

Gastroenteritis is one of the most common illnesses of humans, and many different viruses have been causally associated with this disease. Of those enteric viruses that have been established as etiologic agents of gastroenteritis, only the human caliciviruses cannot be cultivated in vitro. The cloning of Norwalk virus and subsequently of other human caliciviruses has led to the development of several new diagnostic assays. Antigen detection enzyme immunoassays (EIAs) using polyclonal hyperimmune animal sera and antibody detection EIAs using recombinant virus-like particles have supplanted the use of human-derived reagents, but the use of these assays has been restricted to research laboratories. Reverse transcription-PCR assays for the detection of human caliciviruses are more widely available, and these assays have been used to identify virus in clinical specimens as well as in food, water, and other environmental samples. The application of these newer assays has significantly increased the recognition of the importance of human caliciviruses as causes of sporadic and outbreak-associated gastroenteritis.

Specimens (n=287) from 59 gastroenteritis outbreaks collected from February 1997 to March 1999 were analyzed by reverse transcriptase-polymerase chain reaction. The majority of outbreaks (88%) were associated with Norwalk-like viruses. Molecular analyses of strains from 46 outbreaks showed the cocirculation during the 1998-1999 winter of 2 genogroup II clusters, accounting for 57% and 28% of outbreaks, respectively. An important genetic diversity was observed during this 2-year period. Thirteen different genogroup II strains and 3 different genogroup I strains were found. Genogroup I strains, although from the same cluster, were highly divergent (9%-16%). Epidemiologic and molecular data indicate that several introductions did not result in any major shift of prominent strains, whereas 1 apparently established itself. Some point mutations allowed corroboration of epidemiologic links and strongly suggest that, in several instances, sharing staff and/or transfer of patients between health care institutions can create a significant risk for Norwalk-like virus dissemination.

A significant global problem is the microbiological contamination of foods and water. The microorganisms associated with about half of the foodborne disease outbreaks still go unrecognized, primarily as a result of inadequate diagnostic methods and sampling. A significant amount of food- and waterborne diseases are associated with viruses, information that has been obtained only in recent years. Improved diagnostic methods have established that caliciviruses are the most important non-bacterial pathogens associated with food- and waterborne outbreaks, and are the major cause of seafood-associated gastroenteritis.

A new type of human calicivirus (HuCV) showing the classic cup-shaped surface morphology was identified in the stool sample from a child with symptoms of acute gastroenteritis in Seoul, Korea (SK virus). Genomic RNA was extracted directly from the stool sample, and the nucleotide sequence of 3.2 kb of the 3' end of SK virus was determined from cDNA. This region spanned sequences from the RNA-dependent RNA polymerase (RDRP) region in the open reading frame 1 (ORF1) to the 3' poly A tail. The non-structural and capsid protein coding sequences were fused in a single ORF as observed in Manchester type (Genogroup III). However, ORF2 of Manchester virus was missing in SK virus. In RDRP region, SK virus showed amino acid and nucleotide identities of 74-75% and 68-69% respectively, with those of Manchester virus, while showed 34-46% and 55-60% identities respectively with those of other human caliciviruses. However, capsid protein of SK virus showed a partial (29-46%) amino acid identity with those of other caliciviruses including Manchester type. The closest resemblance in amino acid (97-99%) and nucleotide sequence (85-86%) identities were found in RDRP region with Vanderbijlpark and Pretoria isolates recently found in South Africa. These results suggest that SK virus together with Vanderbijlpark and Pretoria isolates belong to a new type different from Manchester virus.

The hypothesis that the enteric bovine calici-like virus Newbury agent (NA-2) belongs to the family Caliciviridae was examined by genome sequence analysis. Use of solid-phase immune electron microscopy allowed samples with good levels of virus to be identified and amplification of the genome was achieved by reverse transcription-polymerase chain reaction. Examination of a 216-amino-acid sequence in the RNA-dependent RNA polymerase gene and a 116-amino-acid sequence in the capsid gene showed that NA-2 had the closest deduced amino acid identity (77 to 80% for the polymerase region and 67 to 73% for the capsid region) to the morphologically indistinguishable human SRSVs (small round structured viruses) of genogroup 1, which are classified as members of the Caliciviridae. It had a weak relationship (<34.5% deduced amino acid identity) in both the polymerase and the capsid regions to animal caliciviruses, all of which have classical morphology. This is the first genomic data from a nonhuman virus with SRSV morphology. It confirms the hypothesis that the bovine enteric calici-like virus NA-2 is a member of the family Caliciviridae and endorses the observation to date that viruses with SRSV morphology are genomically distinct., (Copyright 1999 Academic Press.)

Ten virus isolates were obtained from three species of marine mammals sampled on San Miguel Island (California, USA) and 1,200 km north on Rogue Reef (Oregon, USA) during tagging operations in 1986-87. Seven of these 10 were derived from 30 sampled Steller sea lion (Eumetopias jubatus pups, while two of 10 were isolated from one of 19 sampled California sea lion (Zalophus californianus californianus pups, and the remaining isolate was derived from 30 sampled northern fur seal (Callorhinus ursinus) pups. All 10 isolates were identified as belonging to a single serotype, reptilian calicivirus Crotalus type 1 (RCV Cro-1), previously isolated from both healthy and diseased snakes and frogs in a California zoologic collection. The marine samples also showed that nine of 30 Steller sea lion pups, one of 19 California sea lion pups and zero of 30 fur seal pups were producing type specific neutralizing antibodies to RCV Cro-1. This represents the first reported instance of the isolation from marine sources of calicivirus originally isolated from a terrestrial species.

Modifications are described for the center and angular parameter estimation algorithms of common line-based particle image orientation determination which is an essential step in the three-dimensional reconstruction of icosahedral virus particles. The modifications incorporate a variety of image processing, pattern recognition, and statistical tools resulting in objective and automated orientation estimation algorithms. The modified algorithms were tested using electron cryo-microscopic particle images of three different virus specimens, with sizes 400-1250 A in diameter, covering a broad range of defocus values. Evaluation of these modified algorithms shows significant improvement over the previous algorithms. The center and angular parameters were estimated with higher accuracy allowing the identification of a larger number of particle orientations. Usage of the modified estimation algorithms resulted in the identification of particle orientations which could not to be identified using the algorithms before modification. Furthermore, these improvements have resulted in the determination of a better quality and a higher resolution three-dimensional reconstruction. The improved algorithms have been developed into a software package which can be obtained via the world wide web at http://ncmi.bioch.bcm.tmc.edu/pthuman.

In December 1992, 17 African lions and 7 Siberian tigers in a Safari park in Japan became sick with characteristic clinical symptoms of acute vesicular formations on tongue and snout. The disease was highly contagious since all of these animals showed similar symptoms within two days after the onset of the first case. Swabs were taken from affected animals in rubbing tongues, snouts and some from rectums. Cytopathic viruses were isolated on CRFK cell culture by virological tests. The physicochemical property of a representative virus strain, named Arthur/L, isolated from a male lion was identified as a member of Caliciviridae. However, seroneutralization test indicated that this virus strain was antigenically distinct from Japanese isolates of feline caliciviruses used for comparison. Viral capsid proteins of the present isolate, Arthur/L, and of a feline calicivirus, strain FC7, were compared in an electrophoresis in SDS-PAGE gel. The major viral capsid polypeptide of them were proved to be significantly different in molecular weight. The polypeptide of FC7 was estimated to be ca. 63 KDa whereas that of Arthur/L consisted of 2 components of ca. 65 and 62 KDa. The viral proteins of these two strains were also proved to be distinct by an immunoblotting test.

Caliciviruses are important veterinary and human pathogens. The viruses gain their name from characteristic cup-shaped structures seen on the virion surface by negative stain electron microscopy. In humans caliciviruses are a major cause of diarrhoeal disease. There are two fundamentally different genome structures amongst human caliciviruses. The Norwalk-like or small round structured viruses (SRSVs) are viruses that have an amorphous structure when viewed by EM, they have a genome composed of 3 major open reading frames (ORFs). These viruses cause epidemic gastroenteritis amongst all age groups. In contrast, the 'classic' human caliciviruses (HuCVs) display the typical calicivirus surface structure and have their capsid ORF fused to and contiguous with the non structural proteins forming one giant polyprotein. HuCVs are predominantly associated with paediatric infections and are only a minor cause of disease in humans. Spread of disease for both SRSVs and HuCVs is usually by faecal oral transmission. SRSVs are a major cause of foodborne gastroenteritis especially linked to the consumption of sewage-contaminated shellfish. However, there is no evidence that these viruses replicate in shellfish or that they originate from an animal source.

Human calicivirus Sapporo (SV) has typical calicivirus morphology and causes acute gastroenteritis in children. The nucleotide sequence of 3.2 kb of the 3' end of SV was determined from a cloned cDNA. The 3' end of the SV genome is predicted to encode the RNA-dependent RNA polymerase region, the capsid protein and two small open reading frames. The nonstructural and capsid protein coding sequences in the SV genome are fused in a single open reading frame. The organization of these proteins in the SV sequence is similar to that of rabbit hemorrhagic disease virus and the recently described Manchester virus, and distinct from the genome organization of the prototype human calicivirus, Norwalk virus, that lacks typical calicivirus morphology and has been described as a small round structured virus (SRSV). Sequence analysis of the predicted capsid region showed that the SV capsid is longer by approximately 30 amino acids than the capsid of any of the SRSVs, and multiple sequence alignments showed that these additional amino acids are located in the variable region of the capsid protein. Expression of the capsid protein of SV in insect cells resulted in the self-assembly of virus-like particles that have a morphology similar to that of the native virus. This result shows that calicivirus morphology is determined by the primary sequence of the capsid protein.

In April 1992, on Tern Island, French Frigate Shoals, Hawaii (USA), researchers observed a hand-reared white tern hatchling (Gygis alba rothschildi) develop vesicular lesions on the webbing between its toes, 6 days after falling out of its nest. Vesicular fluid collected from the foot lesions contained virus-like particles having typical calicivirus morphology. Calicivirus RNA was detected in the vesicular fluid by dot hybridization with a group-specific calicivirus copy DNA probe. Attempts to cultivate the virus in African green monkey kidney cells and porcine kidney cells were unsuccessful. This is the first report of a calicivirus infection associated with vesicular disease in a wild avian species.

An epidemiologic investigation of a gastroenteritis outbreak in December 1994 indicated that salad consumption during lunch was linked with illness on 2 days (5 December: odds ratio [OR]=3.1, 95% confidence interval [CI]=2.0-5.0; 6 December: OR=3.1, 95% CI=1.9-4.9). Single stool or vomitus specimens from ill students and staff (case-patients) were examined for bacterial and viral pathogens. Small round-structured viruses (SRSVs) were detected by electron microscopy in stool specimens from 9 of 19 case-patients and in vomitus specimens from 3 of 5 case-patients. By reverse transcription-polymerase chain reaction (RT-PCR), the SRSVs were shown to be G-2/P2-B type strain. The nucleotide sequences of RT-PCR products from vomitus and stool specimens of ill students were identical to stool specimens from the ill salad chef. These findings suggest that a single SRSV strain was the etiologic agent in the outbreak that was possibly transmitted to students through consumption of contaminated salad. Epidemiologic investigation in conjunction with molecular diagnostics may enable early identification of sources of infection and improve outbreak control.

The use of a recently developed EIA using antisera raised against purified baculovirus expressed recombinant Mexico virus (rMx) capsid protein is described for screening for human calicivirus in stools. The results show that MX-like viruses have been circulating in the UK periodically since 1983 and were an occasional cause of sporadic cases of diarrhoea in infants and outbreaks of infection among elderly patients in hospitals and old people's homes. Further evidence is presented that some strains of caliciviruses with characteristic surface morphology (HuCVs) and some with an indistinct appearance, small round structured viruses (SRSVs) are antigenically related to MxV. Tests on SRSVs from four unrelated outbreaks typed as UK3 failed to react in the Mx EIA or recombinant Norwalk virus (rNV) EIA. A 2-month survey of 206 children treated in two London hospitals for diarrhoea showed that only one was positive for MxV, a child known to be infected with HIV-1. None of the samples reacted in the rNV EIA.

Infections caused by caliciviruses, i.e., vesicular exanthema virus of swine were recognised as a major cause of economic loss in the 1930s. However, it was not until the application of electronmicroscopy in the 1970s that caliciviruses and astroviruses were recognised and proven to be a cause of diarrhoea and vomiting. The following review briefly describes the steps which have led to the development of diagnostic tests and enabled the characterization of several members of the Caliciviridae and Astroviridae. In the past five years this has culminated in the sequencing of their genomes and the expression of viral proteins. This in turn has led to the development of improved diagnostic tests e.g., RT-PCR and enzyme immunoassays, and may pave the way towards producing effective vaccines in the future.

Caliciviruses were isolated from feces of skunks imported from the north central United States to Canada. Virus isolation was accomplished using adenovirus-transformed human kidney (293) cells, swine testes and Vero cells. Plaque size variants were presented, but there was no apparent difference in virus morphology by negative stain or immune electron microscopy. Pigs infected with skunk calicivirus had a slightly elevated body temperature at 3 days postinfection. Although the infected animals seroconverted, no overt clinical signs were observed. Purified infectious genomic skunk calicivirus RNA behaved exactly as San Miguel sea lion virus (SMSV) 1 and 4 genomic RNA in cell culture transfection studies. Of the cell types examined, only primary porcine kidney, 293 and Vero cells supported viral replication. No viral replication was detected in cells of bovine, equine, ovine, caprine or feline origin. The skunk caliciviruses contained a single capsid protein with a relative mobility similar to SMSV virus 1 and 4 capsid proteins. The capsid protein was positive by Western blot analysis with SMSV and vesicular exanthema of swine virus (VESV) antisera. Purified RNA from skunk calicivirus infected cells was subjected to reverse transcription followed by polymerase chain reaction. Nucleotide sequences were identified that had greater than 85% similarity to the 2C and RNA polymerase gene regions of SMSV 1 and 4 and VESV A48. Predicted amino acid sequences of these regions were greater than 95% similar and the partial coding sequence of the polymerase gene contained the YGDD sequence common to positive-strand RNA virus polymerases.

Two outbreaks of gastroenteritis in the UK which occurred nine days apart at Lymington and Southampton hospitals were investigated. The clinical and epidemiological features of both outbreaks were characteristic of small round-structured virus (SRSV) infection with rapid onset of diarrhoea and/or nausea and vomiting and propagation of the outbreaks by secondary spread. SRSV particles were observed by immune electron microscopy (EM) in 60% of faecal samples from both outbreaks and no other pathogens were detected. The index case for the second outbreak was a patient who was admitted with diarrhoea and vomiting after being discharged from Lymington hospital during the first outbreak. The possibility that the two outbreaks were caused by the same strain of SRSV was investigated by the polymerase chain reaction (PCR). New inosine-containing PCR primers were designed to amplify the RNA polymerase region of SRSV cDNA from genetic groups I and II. The PCR using the group II primers achieved a higher detection rate for SRSVs in faecal samples (68% of samples positive from both outbreaks) than immune EM. SRSVs were not detected using the group I primers or using conventional degenerate PCR primers. The nucleotide sequences of PCR amplicons from both outbreaks were identical providing molecular epidemiological evidence for the involvement of a single SRSV strain. Comparison of the RNA polymerase region of this virus with the equivalent regions of genetic group I (69.4-75.0% amino acid identify) and genetic group II (88.9-100% amino acid and 77.1-88.1% nucleotide identity) SRSVs revealed that the causative SRSV was a distinct member of genetic group II.

Classic human enteric caliciviruses (HuCVs) have a distinctive morphology and are primarily associated with pediatric acute gastroenteritis. Although morphologically distinct from the small round structured viruses (SRSVs), the classic HuCVs are thought to be closely related and were anticipated to have a similar genome organisation. We report the first genome sequence and molecular characterisation of a classic human enteric calicivirus associated with a case of acute vomiting and diarrhoea in an infant. The RNA genome (7266 nt) is smaller than the genome of SRSVs from the two genetic groups and has a unique arrangement of open reading frames. Further analysis of the 3' terminal 3 kb from a second unrelated isolate confirmed this genomic organisation. Analysis of capsid and RNA polymerase sequences together with the unique genomic organisation of classic HuCV suggest these viruses are more closely related to the animal caliciviruses than the enteric SRSV group of viruses.

The Caliciviridae comprise a new family of single-stranded RNA viruses. While human caliciviruses cause gastroenteritis, the animal caliciviruses cause a wide range of diseases. We have determined the three-dimensional structure of a primate calicivirus using electron cryomicroscopy and computer image-processing techniques. Calicivirus is one of the rare animal viruses whose capsid is made of a single structural protein. The three-dimensional structure of the virus is distinct from that of any other animal virus. However, there are several architectural similarities with plant viruses such as tomato bushy stunt virus and turnip crinkle virus. The calicivirions are 405 A in diameter and exhibit T = 3 icosahedral symmetry. The main features of the three-dimensional structure are the 32 large surface hollows, 50 A deep and 90 A wide, at the icosahedral 5-fold and 3-fold axes, and the 90 distinctive arch-like capsomeres surrounding these hollows at the local and strict 2-fold axes. Each capsomere is a dimer of the capsid protein. Despite noticeable differences, the three quasi-equivalent subunits show common structural features: the upper bilobed domain, the central stem domain, and the lower shell domain. The 2-fold related capsid proteins interact through the bilobed domains to form the top of the arch. The structural differences between the connectors of the stem and the shell domain among the three subunits suggest the presence of a hinge region that may facilitate the capsid protein to adapt to the three quasi-equivalent environments of the T = 3 icosahedral structure. The shell domains of the pentavalent and hexavalent capsid proteins associate to form a continuous shell between the radii of 115 and 150 A. A beta-barrel structure has been suggested for the shell domain. The mass density in the inner shell between the radius of 85 and 110 A may contain a portion of the capsid protein interacting with the RNA. The features between the 45 and 85 A radius are suggestive of ordered RNA.

Astroviruses and caliciviruses are two families defined initially by their characteristic morphology. Many of these viruses have been difficult to grow in culture. Molecular biology has now provided a valuable insight into the nature of these viruses, and in many respects knowledge of genome structure now outstrips that of more classical virological features. However these advances have allowed a more detailed approach to virus classification and have led to the establishment of the Astroviridae as a distinct virus family.

Signs and pathologic changes of European brown hare syndrome (EBHS) were reproduced in four hares (Lepus europaeus) after experimental inoculation of a liver suspension from hares from Belgium, which naturally died of EBHS. Virus particles were demonstrated by electron microscopy in the liver of an experimentally infected hare. They were spherical with a diameter of 28 to 30 nm. After density gradient centrifugation, virus particles were detected in the density zone of 1.34 g/ml. Based on immunoelectron microscopy with a convalescent serum, we identified the virus as the cause of EBHS. Clinical signs were not produced in three seronegative domestic rabbits after subcutaneous inoculation of the EBHS virus. Although low levels of antibodies against EBHS virus were found in the serum of these three rabbits 4 weeks after the inoculation of EBHS virus, the rabbits were not protected when challenged with viral hemorrhagic disease (VHD) virus. The different pathogenicity of the Belgian EBHS and VHD virus isolates in rabbits and the lack of protection in rabbits with circulating EBHS virus antibodies against a challenge with VHD virus indicates that both Belgian virus isolates form two different virus populations.

Electron- and immunoelectron-microscopic methods were used for detection and investigation of rabbit haemorrhagic disease virus (RHDV). The observed virus particles were uncoated, with an icosahedral symmetry, with a 35-40 nm dia and capsids built up by 32 capsomers with a central hollow part and a 3.5-5 nm dia. Particles with empty capsids could also be seen among them. Single virions visible in 5-fold symmetry, exhibited 10 projections on the surface. Virions with a 33-34 nm dia and cup-shaped surface depressions were a rare finding. Some of these particles were indistinguishable from the caliciviruses, but in most of them the cup-shaped depressions were single or no more then 2-3 in number, localized predominantly unilaterally or asymmetrically. Particles 25-29 nm large with full or empty capsids without capsomers could also be seen. Most probably they were some immature forms in the process of development of the RHDV.

Five monoclonal antibodies (MoAbs) to rabbit haemorrhagic disease virus (RHDV), prepared and tested in ELISA, immunoperoxidase (IP) and immunofluorescence (IF) test previously, reacted specifically in immunoelectron microscopy (IEM), too. No differences in binding of individual MoAbs with full or empty RHDV particles were found by IEM.

The lesions of acute feline calicivirus infection are of a transient vesiculo-ulcerative nature and involve, to varying degrees, the palate, tongue, gingiva, lips, nasal philtrum, and oral fauces. Chronic ulceroproliferative faucitis is a specific but uncommon sequelae to persistent feline calicivirus oral carriage.

In the spring 1986, an acute infectious disease occurred in Wuhan Second Producing Medical Manufactory, and the rabbit almost died. We tested the mortal symptom and confirmed rabbit Hemorrhagic Disease (RHD) as same as Huang Yinyao report. Hubei Traditional Chinese Medicine Institute appear this RHD also. After we purified virus of above two source by low speed, high speed and sucrose density gradient centrifugation, they can react with antiserum of RHDV from Nanjing Agricultural University in agar gel immunodiffusion tests. These results proved that they belong to the same serotype. Data indicate RHDV have difference morphological superstructure, viral polypeptides and especially RHDV can't react with antiserum of standard Parvovirus of rabbit and so on, so we suggest RHDV is a new virus.

Formalin fixed liver, spleen, kidney, heart, lung, duodenum and appendix tissues from nine rabbits, experimentally infected with rabbit haemorrhagic disease virus (RHDV), were investigated for evidence of RHDV antigen by the direct avidin-biotin peroxidase complex immunohistochemical method. In all the rabbits examined, RHDV antigen was detected in degenerative and necrotic hepatocytes of the liver tissues. The area involved coincided with histopathological lesions on serial liver sections. The RHDV antigen was expressed in the cytoplasm of the hepatocytes, suggesting that RHDV replicated in these cells. RHDV antigen was also detected in the spleen. The results of immunohistochemistry were supported by the demonstration of RHDV protein by Western blot analysis and of RHDV particles by protein A-gold immunoelectron microscopy in the liver homogenate from all the rabbits that were examined.

A total of 56 liver specimens from rabbits with symptoms of rabbit haemorrhagic disease were tested for virus by electron microscopy (EM) and haemagglutination (HA). Both methods simultaneously gave positive or negative results in 28 or 22 cases, respectively. Divergent results were obtained in only 6 samples. Five of them were positive by EM but negative by HA and in one specimen with a HA-titer of 1:32 virus could not be detected.

The causative agent of rabbit viral haemorrhagic disease (VHD) was purified by CsCl density gradient centrifugation from liver homogenates of rabbits infected with a strain of VHD virus isolated in France. Electron microscopy observations revealed that the virions were icosahedral with a diameter of 33-35 nm, and with an average buoyant density of 1.33. Structural proteins were investigated by SDS-polyacrylamide gel electrophoresis and western blotting. The results indicated the presence of a major structural protein of 64 kDa and other minor proteins of molecular weight of 43, 36 and 32 kDa, the last presumably resulting from the proteolytic degradation of VP 64. These results correspond with the description made by the different authors who identify the VHD virus as a member of the Caliciviridae family.

The virus recovered from cases of European brown hare syndrome in the U.K. contains one major capsid protein of approximately 60 k molecular weight and morphologically resembles known caliciviruses. It has been compared with a European isolate of rabbit haemorrhagic disease calicivirus and, although it shows some antigenic similarity, it is not identical. In transmission and protection studies the virus from U.K. hares failed to produce disease in rabbits and did not effectively protect against subsequent challenge with the rabbit calicivirus.

Causative agent of rabbit haemorrhagic disease (RHD) was purified by CsCl density gradient centrifugation from the liver homogenate of rabbits infected with RHD virus which originated from Korea. The viral particles were 35-40 nm in diameter, and had hollow depressions on their surface. Protein A-gold immunoelectron microscopy clearly showed that the convalescent antisera of diseased rabbits reacted specifically with the virus particles. SDS-PAGE and Western blot analyses demonstrated that the structural protein of the virus was composed of a single major polypeptide of 63 kD. These findings indicate that the causative agent of RHD, tentatively named as picornavirus in Korea, belongs to calicivirus.

The major subgenomic RNA of the calicivirus rabbit hemorrhagic disease virus which codes for the viral capsid protein has been cloned as cDNA. The nucleotide sequence of this mRNA was shown to be identical to the 3' terminal region of the genomic RNA. The 5' end of the mRNA corresponds to position 5296 of the genomic sequence; except for two differences the first 16 nucleotides of genomic and subgenomic RNAs are identical. After isolation from liver tissue viral genomic and subgenomic RNAs were found to be resistant to RNase degradation. This protection was due to RNA packaging into particles. Sucrose density gradient centrifugation of liver homogenates allowed separation of such particles containing either genomic RNA or subgenomic RNA. Genomic and subgenomic RNAs are protein-linked and for the genomic molecule this interaction is localized within the first 179 nucleotides. After radioactive labeling of purified RNA and subsequent RNase treatment a protein of 15 kDa was identified.

Since October 1986 an unusually high mortality has been observed both in wild European brown hares (Lepus europaeus) and in hare farms in Italy. Pathological alterations, including severe hepatosis, nephrosis, congestion and hemorrhages of tracheal mucosa and lungs, and splenic vascular congestion, were observed in 179 of 381 hares necropsied from 1986 to 1988. Jaundice also was seen in 30% of these hares. Histologically, the liver damage was characterized by coagulative necrosis, mainly located around the portal areas, or by degenerative changes. Hyperemia, focal hemorrhages and periportal mononuclear cell infiltration were also present. The epithelium of renal tubules showed the presence of various degrees of vacuolar degeneration and necrosis, and eosinophilic granular hyaline casts or homogenous proteinaceous material were found within the lumen of tubules. Only the adult hares were affected. In wild hare populations night counts revealed a reduction of the number of observed wild hares during the winter period which ranged from 27 to 40%, whereas in hare farms the mortality ranged from 30 to 90%. Bacteriological, parasitological, and toxicological investigations were unable to confirm the primary cause of these deaths. Negative stain electron microscopy and immunoelectronmicroscopy conducted since October 1988 on liver and spleen homogenates from hares with acute hepatosis revealed the presence of viral particles with morphological aspects resembling those of calicivirus, antigenically related to the etiological agent of viral haemorrhagic disease of rabbits.