Immune Response Modulation by Caliciviruses.

Noroviruses and Sapoviruses, classified in the Caliciviridae family, are small positive-stranded RNA viruses, considered nowadays the leading cause of acute gastroenteritis globally in both children and adults. Although most noroviruses have been associated with gastrointestinal disease in humans, almost 50 years after its discovery, there is still a lack of comprehensive evidence regarding its biology and pathogenesis mainly because they can be neither conveniently grown in cultured cells nor propagated in animal models. However, other members of this family such as Feline calicivirus (FCV), Murine norovirus (MNV), Rabbit hemorrhagic disease virus (RHDV), and Porcine sapovirus (PS), from which there are accessible propagation systems, have been useful to study the calicivirus replication strategies. Using cell cultures and animal models, many of the functions of the viral proteins in the viral replication cycles have been well-characterized. Moreover, evidence of the role of viral proteins from different members of the family in the establishment of infection has been generated and the mechanism of their immunopathogenesis begins to be understood. In this review, we discuss different aspects of how caliciviruses are implicated in membrane rearrangements, apoptosis, and evasion of the immune responses, highlighting some of the pathogenic mechanisms triggered by different members of the Caliciviridae family.

Bat Caliciviruses and Human Noroviruses Are Antigenically Similar and Have Overlapping Histo-Blood Group Antigen Binding Profiles.

Emerging zoonotic viral diseases remain a challenge to global public health. Recent surveillance studies have implicated bats as potential reservoirs for a number of viral pathogens, including coronaviruses and Ebola viruses. Caliciviridae represent a major viral family contributing to emerging diseases in both human and animal populations and have been recently identified in bats. In this study, we blended metagenomics, phylogenetics, homology modeling, and in vitro assays to characterize two novel bat calicivirus (BtCalV) capsid sequences, corresponding to strain BtCalV/A10/USA/2009, identified in Perimyotis subflavus near Little Orleans, MD, and bat norovirus. We observed that bat norovirus formed virus-like particles and had epitopes and receptor-binding patterns similar to those of human noroviruses. To determine whether these observations stretch across multiple bat caliciviruses, we characterized a novel bat calicivirus, BtCalV/A10/USA/2009. Phylogenetic analysis revealed that BtCalV/A10/USA/2009 likely represents a novel Caliciviridae genus and is most closely related to "recoviruses." Homology modeling revealed that the capsid sequences of BtCalV/A10/USA/2009 and bat norovirus resembled human norovirus capsid sequences and retained host ligand binding within the receptor-binding domains similar to that seen with human noroviruses. Both caliciviruses bound histo-blood group antigens in patterns that overlapped those seen with human and animal noroviruses. Taken together, our results indicate the potential for bat caliciviruses to bind histo-blood group antigens and overcome a significant barrier to cross-species transmission. Additionally, we have shown that bat norovirus maintains antigenic epitopes similar to those seen with human noroviruses, providing further evidence of evolutionary descent. Our results reiterate the importance of surveillance of wild-animal populations, especially of bats, for novel viral pathogens.IMPORTANCE Caliciviruses are rapidly evolving viruses that cause pandemic outbreaks associated with significant morbidity and mortality globally. The animal reservoirs for human caliciviruses are unknown; bats represent critical reservoir species for several emerging and zoonotic diseases. Recent reports have identified several bat caliciviruses but have not characterized biological functions associated with disease risk, including their potential emergence in other mammalian populations. In this report, we identified a novel bat calicivirus that is most closely related to nonhuman primate caliciviruses. Using this new bat calicivirus and a second norovirus-like bat calicivirus capsid gene sequence, we generated virus-like particles that have host carbohydrate ligand binding patterns similar to those of human and animal noroviruses and that share antigens with human noroviruses. The similarities to human noroviruses with respect to binding patterns and antigenic epitopes illustrate the potential for bat caliciviruses to emerge in other species and the importance of pathogen surveillance in wild-animal populations.

Critical role of RIG-I and MDA5 in early and late stages of Tulane virus infection.

Human noroviruses are a major cause of acute gastroenteritis worldwide, but the lack of a robust cell culture system or small animal model have hampered a better understanding of innate immunity against these viruses. Tulane virus (TV) is the prototype virus of a tentative new genus, Recovirus, in the family Caliciviridae. Its epidemiology and biological properties most closely resemble human norovirus. The host innate immune response to RNA virus infection primarily involves pathogen-sensing toll-like receptors (TLRs) TLR3 and TLR7 and retinoic acid-inducible gene I-like receptor RIG-I and melanoma differentiation associated gene 5 (MDA5). In this study, by using siRNA knockdown, we report that TV infection in LLC-MK2 cells results in an early [3 h post infection (h p.i.), P<0.05] RIG-I-dependent and type I interferon-mediated antiviral response, whereas an MDA5-mediated antiviral effect was observed at later (12 h p.i.; P<0.05) stages of TV replication. Induction of RIG-I and MDA5 was critical for inhibition of TV replication. Furthermore, pre-activation of the RIG-I/MDA5 pathway prevented TV replication (>900-fold decrease; P<0.05), suggesting that RIG-I and MDA5 ligands could be used to develop novel preventive and therapeutic measures against norovirus.

Assessment of the IFN-ß response to four feline caliciviruses: Infection in CRFK cells.

Feline calicivirus (FCV) is a highly contagious pathogen with a widespread distribution. Although the cat genome has been sequenced, little is known about innate immunity in cats, which limits the understanding of FCV pathogenesis. To investigate the IFN-ß response during FCV infection in CRFK cells, we first cloned and identified the feline IFN-ß promoter sequence and the positive regulatory domain (PRD) motifs, which shared a high similarity with human and porcine IFN-ß promoters. Next, we found that infections with FCV strains F9, Bolin and HRB-SS at the 100 or 1000 TCID50 doses could not activate the IFN-ß promoter at 12 and 24h post-infection. Only strain 2280 infection at a 1000 TCID50 dose could induce the IFN-ß promoter mainly through IRF3 and partially through NF-κB, at 24h post-infection. However, the IFN response occurred much later and was smaller in magnitude compared with that following Sendai virus (SeV) infection. Further, we found that induction of the IFN-ß promoter by FCV 2280 infection depended on dsRNA and not on viral proteins. Finally, we examined whether the IFN-ß response had an antiviral effect against FCV replication. The over-expression of IFN-ß before exposure to the virus reduced viral yields by a range of 2.2-3.2 log10TCID50, but its over-expression at 12h post-infection did not inhibit FCV replication. Our results indicate that some FCV strains cannot induce IFN-ß expression in vitro, which may be a potential factor for FCV survival in cats. Whether this is important in evading the host interferon response in vivo must be investigated.

Meeting report VLPNPV: Session 3: Immune responses.

Virus-like particles (VLPs) and nano-particles (NP) are increasingly considered for both prophylactic and therapeutic vaccines for a wide variety of human and animal diseases. Indeed, 2 VLPs have already been licensed for use in humans, the human papilloma virus vaccine and the hepatitis B virus vaccine. (1) Reflecting this increased interest, a second international conference with a specific focus on VLPs and NP was held at the Salk Institute for Biological Studies in La Jolla, California, in June 2014. Approximately 100 attendees, hailing from many nations, came from academic institutions, research institutes, and biotech companies. A wide variety of topics were discussed, ranging from development and characterization of specific VLP and NP vaccine candidates to methods of production of these particles. Session three was focused on the general question of immune responses to VLPs.

Prevalence of recovirus-neutralizing antibodies in human serum samples.

To investigate recovirus infections and their association with zoonosis, the prevalence of the virus-neutralizing antibody against three recovirus serotypes was tested in the general population and in zookeepers. Neutralizing antibodies were detected in a significantly higher number of zookeepers than in the general population but with significantly lower titers than in macaques.

Relationship between genotypes and serotypes of genogroup 1 recoviruses: a model for human norovirus antigenic diversity.

Human norovirus (NoV) research greatly relies on cell culture-propagable surrogate caliciviruses, including murine NoVs and the prototype 'recovirus' (ReCV), Tulane virus. However, the extreme biological diversity of human NoVs cannot be modelled by a uniform group of viruses or single isolate. Based on a diverse group of recently described ReCVs, a more advanced model reflecting human NoV biological diversity is currently under development. Here, we have reported the genotypic and serotypic relationships among 10 G1 ReCV isolates, including Tulane virus and nine other recent cell culture-adapted strains. Based on the amino acid sequences of virus capsid protein, VP1, and classification constraints established for NoVs, G1 ReCVs were separated into three genotypes, with variable organization of the three open reading frames. Interestingly, cross-neutralization plaque assays revealed the existence of four distinct serotypes, two of which were detected among the G1.2 strains. The amino acid (aa) difference between the two G1.2 ReCV serotypes (12%) was less than the minimum 13% difference established between NoV genotypes. Interestingly, one of the G1.3 ReCVs was equally neutralized by antisera raised against the G1.3 (6% aa difference) and G1.1 (25% aa difference) representative strains. These results imply the existence of a large number of human NoV serotypes, but also shared cross-neutralization epitopes between some strains of different genotypes. In conclusion, the newly developed ReCV surrogate model can be applied to address biologically relevant questions pertaining to enteric CV diversity.

Impact of rotavirus vaccine on rotavirus genotypes and caliciviruses circulating in French cattle.

Group A rotaviruses are a leading cause of neonatal calf diarrhoea worldwide and prevention of this disease includes vaccination against these viruses. In order to highlight the potential selection of rotavirus genotypes due to immune pressure driven by vaccination, the aim of this study was to compare group A rotavirus genotypes circulating in French diarrhoeic calves in rotavirus vaccinated herds (G6P[5] vaccine) with those in non-vaccinated herds during one calving season in 2010. This study showed a high prevalence of rotavirus in both groups with no significant difference between the two. No significant differences regarding G, P and G/P rotavirus genotype distribution between the two groups were observed, with G6, P[5] and G6P[5] genotypes being by far the most prevalent. Moreover, sequence analyses of the VP7 and VP4 partial coding genes of the G6P[5] strains from this study did not allow us to distinguish them according to their origin. This study also showed that other pathogens responsible for calf diarrhoea, such as genogroup III noroviruses and neboviruses, were not more frequently associated with calf diarrhoea in vaccinated herds. Altogether, these results suggest that the studied vaccine did not promote the emergence of rotavirus genotypes or variants different from those of the vaccine or other viruses responsible for calf diarrhoea, such as caliciviruses.

A sensitive and specific blocking ELISA for the detection of rabbit calicivirus RCV-A1 antibodies.

BACKGROUND: Antibodies to non-pathogenic rabbit caliciviruses (RCVs) cross-react in serological tests for rabbit hemorrhagic disease virus (RHDV) and vice versa, making epidemiological studies very difficult where both viruses occur. It is important to understand the distribution and interaction of the two viruses because the highly pathogenic RHDV has been used as a biocontrol agent for wild rabbits in Australia and New Zealand for the past 17 years. The presence of the benign RCV Australia 1 (RCV-A1) is considered a key factor for the failure of RHDV mediated rabbit control in cooler, wetter areas of Australia. RESULTS: A highly sensitive and specific blocking ELISA was developed for the detection of RCV-A1 antibodies. When sera from rabbits with a known infection history for either RCV-A1 or RHDV were tested, this assay showed 100% sensitivity and no cross-reactivity with RHDV sera (100% specificity). CONCLUSIONS: This new ELISA not only allows the detection of RCV-A1 at a population level, but also permits the serological status of individual rabbits to be determined more reliably than previously described methods. This robust and simple to perform assay is therefore the tool of choice for studying RCV-A1 epidemiology in Australian wild rabbit populations.

Experimental inoculation of juvenile rhesus macaques with primate enteric caliciviruses.

BACKGROUND: Tissue culture-adapted Tulane virus (TV), a GI.1 rhesus enteric calicivirus (ReCV), and a mixture of GII.2 and GII.4 human norovirus (NoV)-containing stool sample were used to intrastomacheally inoculate juvenile rhesus macaques (Macaca mulatta) in order to evaluate infection caused by these viruses. METHODOLOGY & FINDINGS: Two of the three TV-inoculated macaques developed diarrhea, fever, virus-shedding in stools, inflammation of duodenum and 16-fold increase of TV-neutralizing (VN) serum antibodies but no vomiting or viremia. No VN-antibody responses could be detected against a GI.2 ReCV strain FT285, suggesting that TV and FT285 represent different ReCV serotypes. Both NoV-inoculated macaques remained asymptomatic but with demonstrable virus shedding in one animal. Examination of duodenum biopsies of the TV-inoculated macaques showed lymphocytic infiltration of the lamina propria and villous blunting. TV antigen-positive (TV+) cells were detected in the lamina propria. In most of the TV+ cells TV co-localized perinuclearly with calnexin--an endoplasmic reticulum protein. A few CD20+TV+ double-positive B cells were also identified in duodenum. To corroborate the authenticity of CD20+TV+ B cells, in vitro cultures of peripheral blood mononuclear cells (PBMCs) from healthy macaques were inoculated with TV. Multicolor flow cytometry confirmed the presence of TV antigen-containing B cells of predominantly CD20+HLA-DR+ phenotype. A 2-log increase of viral RNA by 6 days post inoculation (p<0.05) suggested active TV replication in cultured lymphocytes. CONCLUSIONS/SIGNIFICANCE: Taken together, our results show that ReCVs represent an alternative cell culture and animal model to study enteric calicivirus replication, pathogenesis and immunity.

Seroprevalence of St-Valerien-like caliciviruses in Italian swine.

St-Valérien-like viruses are newly recognized porcine caliciviruses recently detected in North America and Europe. In this study, baculovirus-expressed virus-like particles of the St-Valérien strain 25A/ITA were generated and used for the development of an antibody-detection ELISA kit to assess the seroprevalence of these novel caliciviruses in swine. Antibodies specific for St-Valérien-like virus were detected in 63 (10.3 %) of 614 serum samples tested with titres ranging from 1 : 50 (28.6 %) to 1 : 800 (40.7 %). These results indicate that St-Valérien-like infections are common among domestic pigs, italy.

Genetic diversity and histo-blood group antigen interactions of rhesus enteric caliciviruses.

Recently, we reported the discovery and characterization of Tulane virus (TV), a novel rhesus calicivirus (CV) (T. Farkas, K. Sestak, C. Wei, and X. Jiang, J. Virol. 82:5408-5416, 2008). TV grows well in tissue culture, and it represents a new genus within Caliciviridae, with the proposed name of Recovirus. We also reported a high prevalence of CV antibodies in macaques of the Tulane National Primate Research Center (TNPRC) colony, including anti-norovirus (NoV), anti-sapovirus (SaV), and anti-TV (T. Farkas, J. Dufour, X. Jiang, and K. Sestak, J. Gen. Virol. 91:734-738, 2010). To broaden our knowledge about CV infections in captive nonhuman primates (NHP), 500 rhesus macaque stool samples collected from breeding colony TNPRC macaques were tested for CVs. Fifty-seven (11%) samples contained recovirus isolates. In addition, one NoV was detected. Phylogenetic analysis classified the recovirus isolates into two genogroups and at least four genetic types. The rhesus NoV isolate was closely related to GII human NoVs. TV-neutralizing antibodies were detected in 88% of serum samples obtained from primate caretakers. Binding and plaque reduction assays revealed the involvement of type A and B histo-blood group antigens (HBGA) in TV infection. Taken together, these findings indicate the zoonotic potential of primate CVs. The discovery of a genetically diverse and prevalent group of primate CVs and remarkable similarities between rhesus enteric CVs and human NoVs opens new possibilities for research involving in vitro and in vivo models of human NoV gastroenteritis.

Detection of norovirus-, sapovirus- and rhesus enteric calicivirus-specific antibodies in captive juvenile macaques.

The objective of this study was to determine the prevalence of anti-norovirus (NoV), -sapovirus (SaV) and -Tulane virus (TV) antibodies in rhesus macaques of the Tulane National Primate Research Center and to evaluate the antigenic relationship between these viruses. A high prevalence of NoV-binding (51-61 %) and SaV-binding (50-56 %) antibodies and TV-neutralizing (69 %) antibodies were detected. Serum samples obtained during a human NoV outbreak and a multivalent anti-NoV hyperimmune serum were not able to neutralize TV infectivity. Conversely, low levels of cross-reactivity between the prototype TV and NoVs, but not between the TV and SaVs were detected by ELISA. These data indicate the preservation of some cross-reactive B-cell epitopes between the rhesus and human caliciviruses (CVs). The high prevalence of human and rhesus CV-specific serum antibodies suggests the frequent exposure of colony macaques to enteric CVs including the possibility of CV transmission between human and non-human primate hosts.

[Human caliciviruses and an immune response].

The review deals with calicivirus infection that has long been known in animals, but also just recently disclosed in humans. Human caliciviruses induce outbreaks of acute gastroenteritis in all countries of the world, which makes this immunobiological research very actual. The history of this discovery, evolution of caliciviruses, the host resistance and immune response mechanisms are described in this review.

New variants of European brown hare syndrome virus strains in free-ranging European brown hares (Lepus europaeus) from Slovakia.

Investigations regarding European brown hare syndrome virus (EBHSV) in European brown hares (Lepus europaeus) in Slovakia were undertaken in order to detect the possible presence of EBHSV and to evaluate its phylogenetic position. Liver and/or serum samples were obtained from 135 European brown hares shot by hunters in eight regional hunting areas. From 36 animals corresponding liver and serum samples were available; from the remaining 49 and 50 animals only liver or serum samples were examined, respectively. Samples were tested for antibodies against EBHSV and for viral RNA by reverse transcriptase-polymerase chain reaction (RT-PCR) and RT-PCR products were subsequently sequenced. Additionally, matrilinear hare haplotypes were analyzed in order to detect potential familial susceptibility to EBHSV. Sixty-three of 86 sera contained antibodies against EBHSV, whereas 15 of 85 liver samples were PCR positive. Of the latter, 14 were sequenced, revealing three new strains of EBHSV. Fifteen different matrilinear haplotypes were identified, but no correlation was found between haplotype and susceptibility to EBHSV infection. Our findings confirmed the existence of EBHSV in Slovakia and reinforce the need for determining EBHSV status when importing hares for restocking.

Porcine enteric caliciviruses: genetic and antigenic relatedness to human caliciviruses, diagnosis and epidemiology.

Porcine enteric caliciviruses include sapoviruses and noroviruses. Porcine sapoviruses infect pigs of all ages and cause diarrhea in young pigs, whereas porcine noroviruses were detected exclusively from adult pigs without clinical signs. Importantly, certain porcine norovirus strains were genetically and antigenically related to human noroviruses. This raises public health concerns that pigs may be reservoirs for emergence of epidemic human norovirus strains. This article reviews the discovery of porcine noroviruses and sapoviruses, their classification, diagnosis, epidemiology and genetic and antigenic relatedness to human caliciviruses.

High efficiency cross-reactive monoclonal antibody production by oral immunization with recombinant norwalk virus-like particles.

Monoclonal antibodies (MAbs) are important for in-depth antigenic characterization and diagnosis of infections with human caliciviruses that cause almost all outbreaks of nonbacterial gastroenteritis. We compared different routes of immunization with nonreplicating virus-like particles (VLPs) from recombinant Norwalk virus (rNV) and recombinant Mexico virus (rMX) administered to BALB/c mice to determine the efficiency of hybridoma production. Oral immunization with VLPs without adjuvant resulted in high yields of MAb-secreting hybridomas (90%) to these VLPs of IgG (61%), IgM (29%) and IgA (10%) isotypes. Fusions with mesenteric lymph node lymphocytes yielded MAbs of various subclasses including IgG2a, IgG3, IgM and IgA. These results suggest that an immunization route that mimics the natural route of viral infection pathway may facilitate MAb technology by increasing the yields of antibody secreting hybridoma cells.

Comparison of two polymer-based immunohistochemical detection systems: ENVISION+ and ImmPRESS.

The non-specific background reaction produced in avidin-biotin-based immunohistochemistry, particularly after harsh antigen retrieval procedures, has promoted the use of non-avidin-biotin systems, yet there are few reports comparing the performance of non-avidin-biotin, polymer-based methods. In this study we compare two of these methods, ENVISION+trade mark and ImmPRESS, in animal tissues. We examined the immunoreactivity of 18 antigens in formalin-fixed, paraffin-embedded tissues. Antigens were located in the cytoplasmic membrane (CD11d, CD18 and CD79a), cytoplasm (calretinin, COX-1, COX-2, Glut-1, HepPar 1, KIT, Melan A, tryptase and uroplakin III) or nucleus (MUM-1, PGP 9.5 and thyroid transcription factor 1). We also evaluated three infectious agents (Aspergillus, calicivirus and West Nile virus). The staining with ENVISION+ or ImmPRESS was performed simultaneously for each antigen. The intensity of the reaction and background staining were scored. ImmPRESS yielded similar or higher reaction intensity than ENVISION+trade mark in 16/18 antigens. ImmPRESS produced abundant background with the other two antigens (calretinin and COX-2), which hindered interpretation of the specific reaction. The cost of ImmPRESS was 25% lower than for ENVISION+trade mark. Based on these results, ImmPRESS is a good polymer-based detection system for routine immunohistochemistry.

Detection and characterization of human caliciviruses in hospitalized children with acute gastroenteritis in Blantyre, Malawi.

The human caliciviruses (HuCVs), including Norovirus and Sapovirus, are recognized causes of acute gastroenteritis in children and adults. A 1-year study was undertaken in Blantyre, Malawi, to examine the prevalence, and genetic diversity, of human caliciviruses (HuCVs) amongst children under 5 years of age hospitalized with acute gastroenteritis. Using the reverse transcription-polymerase chain reaction (RT-PCR), combined with nucleotide sequencing of the RT-PCR products, HuCVs were detected in 34/398 (8.5%) of children. Twelve (35.3%) of the children were co-infected with additional enteric viruses (predominantly rotavirus). The HuCVs comprised 26 Noroviruses (6.5%) and 8 Sapoviruses (2.0%). Each of the Noroviruses belonged to genogroup II, and could be further classified into six genotypes, including GII/3 (18 strains), GII/4 (2 strains), GII/11 (1 strain), GII/13 (1 strain), GII/16 (2 strains), and a putative new genotype GII/20 (2 strains). Each of the Sapoviruses belonged to genogroup GIII. HuCVs are the second most commonly identified viral enteropathogens (after rotavirus) among hospitalized children with gastroenteritis in Malawi.

Serologic survey for selected virus infections in polar bears at Svalbard.

Polar bears (Ursus maritimus) were chemically immobilized and sampled at Svalbard, Norway, and on the pack ice in the Barents Sea from late March to mid-May between 1990 and 1998. Plasma samples were tested for the presence of antibodies to canine distemper virus (CDV), calicivirus, phocid herpesvirus type 1 (PhHV-1), and rabies virus. A seroprevalence of 8% to CDV and 2% to calicivirus were found, whereas no antibodies were detected against PhHV-1 or rabies virus. This serologic survey indicates that polar bears in this region are exposed to morbillivirus and calicivirus, although the nature of these viruses and infections are unknown. Morbillivirus and calicivirus are potential pathogens in seals, but it is unknown whether they may cause health problems in polar bears.