ICTV Virus Taxonomy Profile: Caliciviridae.

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-step antibody-based affinity cryo-electron microscopy for imaging and structural analysis of macromolecular assemblies.

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.

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 Šresolution cryo-electron microscopy structure of the TV virion. The TV virion is about 400 Šin 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.

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.

Molecular detection of porcine enteric caliciviruses in Venezuelan farms.

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.

X-ray structure of a native calicivirus: structural insights into antigenic diversity and host specificity.

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.

Genomic characterization of the unclassified bovine enteric virus Newbury agent-1 (Newbury1) endorses a new genus in the family Caliciviridae.

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.

Identification of gastroenteric viruses by electron microscopy using higher order spectral features.

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.

Electron microscopy as a reliable tool for rapid and conventional detection of enteric viral agents: a five-year experience report.

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.

Inter- and intragenus structural variations in caliciviruses and their functional implications.

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.

Transient decrease in blood heterophils and sustained liver damage caused by calicivirus infection of young rabbits that are naturally resistant to rabbit haemorrhagic disease.

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.

Laboratory diagnosis of norovirus: which method is the best?

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.

Detection and molecular characterization of cultivable caliciviruses from clinically normal mink and enteric caliciviruses associated with diarrhea in mink.

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.

Diagnosis of noncultivatable gastroenteritis viruses, the human caliciviruses.

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.

Diagnosis of foodborne viral infections in patients.

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.

Genetic diversity and molecular epidemiology of Norwalk-like viruses.

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.

The new genotypic human calicivirus isolated in Seoul.

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 new genotypic human calicivirus isolated in Seoul

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 bovine Newbury agent-2 is genetically more closely related to human SRSVs than to animal caliciviruses.

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.