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Caliciviruses have positive-sense RNA genomes, typically with short 5′-untranslated regions (5′UTRs) that precede the long open reading frame 1 (ORF1). Exceptionally, some avian caliciviruses have long 5′UTRs containing a picornavirus-like internal ribosomal entry site (IRES), which was likely acquired by horizontal gene transfer. Here, we identified numerous additional avian calicivirus genomes with IRESs, predominantly type 2, and determined that many of these genomes contain a ~200–300 codon-long ORF (designated ORF1*) that overlaps the 5′-terminal region of ORF1. The activity of representative type 2 IRESs from grey teal calicivirus (GTCV) and Caliciviridae sp. isolate yc-13 (RaCV1) was confirmed by in vitro translation. Toeprinting showed that in cell-free extracts and in vitro reconstituted reactions, ribosomal initiation complexes assembled on the ORF1* initiation codon and at one or two AUG codons in ORF1 at the 3′-border and/or downstream of the IRES. Initiation at all three sites required eIF4A and eIF4G, which bound to a conserved region of the IRES; initiation on the ORF1* and principal ORF1 initiation codons involved eIF1/eIF1A-dependent scanning from the IRES's 3′-border. Initiation on these IRESs was enhanced by the IRES trans-acting factors (ITAFs) Ebp1/ITAF45, which bound to the apical subdomain Id of the IRES, and PTB (GTCV) or PCBP2 (RaCV1). [ABSTRACT FROM AUTHOR]

An intrinsically disordered protein (IDP) or region (IDR) lacks or has little protein structure but still maintains function. This lack of structure creates flexibility and fluidity, allowing multiple protein conformations and potentially transient interactions with more than one partner. Caliciviruses are positive-sense ssRNA viruses, containing a relatively small genome of 7.6–8.6 kb and have a broad host range. Many viral proteins are known to contain IDRs, which benefit smaller viral genomes by expanding the functional proteome through the multifunctional nature of the IDR. The percentage of intrinsically disordered residues within the total proteome for each calicivirus type species can range between 8 and 23%, and IDRs have been experimentally identified in NS1-2, VPg and RdRP proteins. The IDRs within a protein are not well conserved across the genera, and whether this correlates to different activities or increased tolerance to mutations, driving virus adaptation to new selection pressures, is unknown. The function of norovirus NS1-2 has not yet been fully elucidated but includes involvement in host cell tropism, the promotion of viral spread and the suppression of host interferon-λ responses. These functions and the presence of host cell-like linear motifs that interact with host cell caspases and VAPA/B are all found or affected by the disordered region of norovirus NS1-2. The IDRs of calicivirus VPg are involved in viral transcription and translation, RNA binding, nucleotidylylation and cell cycle arrest, and the N-terminal IDR within the human norovirus RdRP could potentially drive liquid–liquid phase separation. This review identifies and summarises the IDRs of proteins within the Caliciviridae family and their importance during viral replication and subsequent host interactions. [ABSTRACT FROM AUTHOR]

Background and Objectives: The ocular surface is perpetually exposed to the external environment, rendering it susceptible to microbial contamination. The ocular surface microbiota consists of non-pathogenic microorganisms that inhabit the conjunctiva and cornea. This study's objective was to extensively review the prevalence of bacterial and fungal organisms in the conjunctiva of healthy and diseased cats. (Herpes-and Calici-infected groups). Materials and Methods: The current study was performed on 240 cats that had visited veterinary health centers (Tehran, Iran) for examination. Sterile swabs from each cat's eyes were investigated for microbiological assessment. After sample collection, viral pathogens (Herpes and Calici viruses) were isolated and identified using the PCR method. The ages of the investigated group were 3.76, 3.93, and 4.15 months. Results: The highest frequency of bacteria in the normal, Herpes-infected/Calici-infected, and Herpes/Calici-infected groups were associated with Staphylococcus intermedius and Streptococcus agalactiae, Staphylococcus epidermidis, and Staphylococcus intermedius, respectively. In addition, it was found that the high prevalence of fungal microorganisms in the isolated samples was related to yeasts, Aspergillus (Aspergillus fumigatus, Aspergillus niger), and Penicillium species. Conclusion: Bacterial prevalence was significantly higher in all groups than the prevalence of fungi in the eyes of cats. The statistical comparison between the study groups regarding microbial and fungal frequency showed that significant differences were found between them, such that the frequency was higher in all disease groups, against the control group. In addition, a significant relation was observed between the Herpes-infected and Calici-infected groups regarding microbial and fungal prevalence. [ABSTRACT FROM AUTHOR]

In virology, the term reverse genetics refers to a set of methodologies in which changes are introduced into the viral genome and their effects on the generation of infectious viral progeny and their phenotypic features are assessed. Reverse genetics emerged thanks to advances in recombinant DNA technology, which made the isolation, cloning, and modification of genes through mutagenesis possible. Most virus reverse genetics studies depend on our capacity to rescue an infectious wild-type virus progeny from cell cultures transfected with an "infectious clone". This infectious clone generally consists of a circular DNA plasmid containing a functional copy of the full-length viral genome, under the control of an appropriate polymerase promoter. For most DNA viruses, reverse genetics systems are very straightforward since DNA virus genomes are relatively easy to handle and modify and are also (with few notable exceptions) infectious per se. This is not true for RNA viruses, whose genomes need to be reverse-transcribed into cDNA before any modification can be performed. Establishing reverse genetics systems for members of the Caliciviridae has proven exceptionally challenging due to the low number of members of this family that propagate in cell culture. Despite the early successful rescue of calicivirus from a genome-length cDNA more than two decades ago, reverse genetics methods are not routine procedures that can be easily extrapolated to other members of the family. Reports of calicivirus reverse genetics systems have been few and far between. In this review, we discuss the main pitfalls, failures, and delays behind the generation of several successful calicivirus infectious clones. [ABSTRACT FROM AUTHOR]

Feline Calicivirus (FCV) is a major cause of oral lesions in cats with respiratory tract disease (RTD). FCV is a single-stranded, positive-polarity RNA virus that encodes three open reading frames (ORFs). Active virus excretion occurs through the saliva of cats infected with FCV, which belongs to the Vesivirus genus of the Caliciviridae family. Oral mucosal lesions caused by infectious agents in RTD lead to significant impairment in the quality of life of cats. RTD, which also affects the oral mucosa, is a common problem in cats. Ten cats of different ages, breeds, and genders with ocular lesions were used in this study. At the time of sample collection, the veterinarian performed general and oral examinations on each animal. On oral examination,varying degrees of gingivitis, stomatitis, and ulceration symptoms were noted. Samples were extracted using a commercial viral nucleic acid isolation kit. Three out of ten samples (30%) were found to be positive for FCV using RT-PCR. T In conclusion, the high sensitivity, specificity, and potential for field sample testing make RT-PCR a very important and inevitable method for research and clinical diagnosis related to FCV infection in cats with oral lesions. [ABSTRACT FROM AUTHOR]

Simple Summary: Feline upper respiratory infections (URI) are often endemic in animal shelters. Infections lead to increased morbidity, longer duration of stay at the shelter and, subsequently, reduced welfare and quality of life of affected cats. They impede the shelter's ability to use already stretched resources and space optimally and can also result in increased rates of euthanasia of seriously affected cats. Despite the clear association of infection rates and shelter animal welfare, data on the prevalence of disease and the relative contribution of potential risk factors remain unclear, and no systematically synthesised research exists. In this scoping review, we provide a comprehensive description of the literature on feline URI in multiple contexts and explore whether the literature lends itself to be systematically synthesised. We describe patterns in spatial locations of studies, the range of pathogens and diagnostic tests, cohort characteristics and the findings of risk factor analyses. Assessing the impact of risk factors has the potential to alleviate the severity of disease, especially in shelters; however, the results were not easily pooled as the studies used inconsistent approaches. We present recommendations for ongoing epidemiological research on feline URI to provide a more structured framework and define research questions for future systematic reviews. Feline upper respiratory tract infections (URI) are of concern, especially in animal shelters. This scoping review identifies epidemiological literature on URI as caused by feline herpesvirus (FHV), feline calicivirus (FCV), Chlamydia felis, Mycoplasma felis and Bordetella bronchiseptica. Four databases were searched, studies were screened, and data were extracted on a standardised template. We described patterns in spatial locations of the studies, the range of pathogens and diagnostic tests, cohort characteristics and the findings of risk factor analyses. A total of 90 articles were selected for final data extraction. There was diversity in sampling methods, precluding quantitative meta-analysis of prevalence reports. FHV was most frequently studied (n = 57/90). The most popular sampling site was conjunctival swabbing (n = 43). Most studies (n = 57) used polymerase chain reaction (PCR) to confirm diagnosis. Approximately one-third (n = 32/90) of the studies included sheltered felines. This review explores the current state of knowledge on the epidemiology and risk factors of feline URI. Assessing the impact of risk factors has the potential to alleviate the severity of disease, especially in shelters; however, the results were not easily pooled as the studies used inconsistent approaches. We present recommendations for ongoing epidemiological research on feline URI to provide a more structured framework and define research questions for future systematic reviews. [ABSTRACT FROM AUTHOR]

Feline calicivirus (FCV) is one of the few members of the Caliciviridae family that grows well in cell lines and, therefore, serves as a surrogate to study the biology of other viruses in the family. Conley et al. (14) demonstrated that upon the receptor engagement to the capsid, FCV VP2 forms a portal-like assembly, which might provide a channel for RNA release. However, the process of calicivirus RNA release is not yet fully understood. Our findings suggest that the separation of the FCV capsid from its genome RNA (gRNA) occurs rapidly in the early endosomes of infected cells. Using a liposome model decorated with the FCV cell receptor fJAM-A, we demonstrate that FCV releases its gRNA into the liposomes by penetrating membranes under low pH conditions. Furthermore, we found that VP2, which is rich in hydrophobic residues at its N-terminus, functions as the pore-forming protein. When we substituted the VP2 N-terminal hydrophobic residues, the gRNA release efficacy of the FCV mutants decreased. In conclusion, our results suggest that in the acidic environment of early endosomes, FCV VP2 functions as the pore-forming protein to mediate gRNA release into the cytoplasm of infected cells. This provides insight into the mechanism of calicivirus genome release. [ABSTRACT FROM AUTHOR]

Cat breeding has become very popular nowadays. The presence of these animals in catteries, especially in conditions of imperfect compliance with sanitary and hygienic requirements, often causes outbreaks of viral diseases, the causative agents of which are transmitted mainly by the aerogenic transmission mechanism. Calicivirosis is the most common kennel disease; it can occur as a mono-infection and can also be associated with other viral respiratory diseases. The study aimed to evaluate the epizootic features and clinical course of feline calicivirus in catteries. It was found that the disease's stationary focus was in the experimental cattery. Depending on the year's season, the morbidity increase begins in April, peaks (40 %) in July, and gradually decreases until September. The most vulnerable age group is kittens aged 2–6 months due to imperfect immune mechanisms at this age. The disease occurs mainly manifestly in this age group, with characteristic pronounced clinical signs. Sick cats have sub febrile or pyretic fever, lack of appetite, and salivation with viscous saliva. Ulcers develop on the mucous membranes of the oral cavity. Also, erosions and ulcers are noted in the nasal mirror and on the mucous membrane of the larynx. There was conjunctivitis with serous to catarrhal-purulent exudate. Laboratory tests revealed leukopenia, acceleration of erythrocyte sedimentation rate, left shift of the nucleus, lymphocytosis, and monocytosis, which indirectly determine the viral disease and stimulation of immune functions, and the development of a systemic inflammatory response. Biochemical tests revealed the development of intoxication syndrome, hepatitis liver failure, and renal failure. The data obtained prove the systemic nature of the impact of calicivirus infection, the development of complications in the form of a systemic inflammatory response, which affects various organs and systems, particularly the liver and excretory system. The development of multiorgan pathology in feline calicivirus infection requires an integrated approach to treatment, including pathogenetic and symptomatic measures in the treatment protocol. [ABSTRACT FROM AUTHOR]

Background and Objectives: The ocular surface is perpetually exposed to the external environment, rendering it susceptible to microbial contamination. The ocular surface microbiota consists of non-pathogenic microorganisms that inhabit the conjunctiva and cornea. This study's objective was to extensively review the prevalence of bacterial and fungal organisms in the conjunctiva of healthy and diseased cats. (Herpes- and Calici-infected groups). Materials and Methods: The current study was performed on 240 cats that had visited veterinary health centers (Tehran, Iran) for examination. Sterile swabs from each cat's eyes were investigated for microbiological assessment. After sample collection, viral pathogens (Herpes and Calici viruses) were isolated and identified using the PCR method. The ages of the investigated group were 3.76, 3.93, and 4.15 months. Results: The highest frequency of bacteria in the normal, Herpes-infected/Calici-infected, and Herpes/Calici-infected groups were associated with Staphylococcus intermedius and Streptococcus agalactiae, Staphylococcus epidermidis, and Staphylococcus intermedius, respectively. In addition, it was found that the high prevalence of fungal microorganisms in the isolated samples was related to yeasts, Aspergillus (Aspergillus fumigatus, Aspergillus niger), and Penicillium species. Conclusion: Bacterial prevalence was significantly higher in all groups than the prevalence of fungi in the eyes of cats. The statistical comparison between the study groups regarding microbial and fungal frequency showed that significant differences were found between them, such that the frequency was higher in all disease groups, against the control group. In addition, a significant relation was observed between the Herpes-infected and Calici-infected groups regarding microbial and fungal prevalence.

Caliciviruses have positive-sense RNA genomes, typically with short 5′-untranslated regions (5′UTRs) that precede the long open reading frame 1 (ORF1). Exceptionally, some avian caliciviruses have long 5′UTRs containing a picornavirus-like internal ribosomal entry site (IRES), which was likely acquired by horizontal gene transfer. Here, we identified numerous additional avian calicivirus genomes with IRESs, predominantly type 2, and determined that many of these genomes contain a ~200–300 codon-long ORF (designated ORF1*) that overlaps the 5′-terminal region of ORF1. The activity of representative type 2 IRESs from grey teal calicivirus (GTCV) and Caliciviridae sp. isolate yc-13 (RaCV1) was confirmed by in vitro translation. Toeprinting showed that in cell-free extracts and in vitro reconstituted reactions, ribosomal initiation complexes assembled on the ORF1* initiation codon and at one or two AUG codons in ORF1 at the 3′-border and/or downstream of the IRES. Initiation at all three sites required eIF4A and eIF4G, which bound to a conserved region of the IRES; initiation on the ORF1* and principal ORF1 initiation codons involved eIF1/eIF1A-dependent scanning from the IRES’s 3′-border. Initiation on these IRESs was enhanced by the IRES trans-acting factors (ITAFs) Ebp1/ITAF45, which bound to the apical subdomain Id of the IRES, and PTB (GTCV) or PCBP2 (RaCV1).

Feline calicivirus (FCV) is a major cause of upper respiratory disease in cats and is often used as a model for human norovirus, making it of great veterinary and human medical importance. However, questions remain regarding the route of entry of FCV in vivo. Increasing work has shown that extracellular vesicles (EVs) can be active in viral infectivity, yet there is no work examining the role of EVs in FCV infection. Here, we begin to address this knowledge gap by characterizing EVs produced by a feline mammary epithelial cell line (FMEC). We have confirmed that EVs are produced by infected and mock-infected FMECs and that both virions and EVs are coisolated with standard methods of virus purification. We also show that they can be enriched differentially by continuous iodixanol density gradient. EVs were enriched at a density of 1.10 g/mL confirmed by tetraspanin expression, size profile, and transmission electron microscopy (TEM). Maximum enrichment of FCV at a density of 1.18 g/mL was confirmed by titration, quantitative reverse transcriptase PCR (q-RT PCR), and TEM. However, infectious virus was recovered from nearly all samples. When used to infect in vitro epithelium, both EV-rich and virus-rich fractions had the same levels of infectiousness as determined by percentage of wells infected or titer achieved postinfection. These findings highlight the importance of coisolates during viral purification, showing that EVs may represent a parallel route of entry that has previously been overlooked. Additional experiments are necessary to explore the role of EVs in FCV infection. IMPORTANCE Feline calicivirus (FCV) is a common cause of upper respiratory infection in cats. Both healthy and infected cells produce small particles called extracellular vesicles (EVs), which are nanoparticles that act as messengers between cells and can be hijacked during viral infection. Historically, the role of EVs in viral infection has been overlooked, and subsequently no group has studied the role of EVs in FCV infection. We hypothesized that EVs may play a role in FCV infection. Here, we show that EVs are copurified with FCV when collecting virus. To study their individual effects, we successfully enrich for viral particles and EVs separately by taking advantage of their different densities. Our initial studies show that EV-enriched versus virus-enriched fractions are equally able to infect cells in culture. These findings highlight the need to both consider the purity of virus after purification and to further study EVs' role in natural FCV infection.

The incidence of reporting caliciviruses in wild birds is less common than in other animals, and the majority of cases remain unclassified. A strain of calicivirus was discovered in this study in the feces of cormorants collected at Xiamen Horticulture Expo Garden in 2021 and was named Cormcali01. The genome of Cormcali01 was 8,561 bp in length which contained characteristic motifs present in other caliciviruses. Furthermore, it demonstrated a significant deviation from all existing calicivirus nucleotide sequences, exhibiting the highest amino acid identity (47.34%) to the unclassified Ruddy turnstone calicivirus A. A pairwise comparison of the VP1 protein showed that Cormcali01 had the highest amino acid identity of 43.90% with the unassigned Ruddy turnstone calicivirus A. Phylogenetic analysis demonstrated that VP1 of Cormcali01 clustered with unassigned caliciviruses. Therefore, based on phylogenetic analysis and pairwise comparison, Cormcali01 should be affiliated with the unassigned calicivirus, which were suggested to comprise a new calicivirus genus, the Sanovirus genus. After investigating the prevalence of Cormcali01, we discovered that 22.22% of fecal samples (10/45) were tested positive. These findings expand our understanding of the genetic variation of caliciviruses and provide valuable epidemiological information regarding a potential outbreak of calicivirus disease in birds. [ABSTRACT FROM AUTHOR]

Australia has multiple lagoviruses with differing pathogenicity. The circulation of these viruses was traditionally determined through opportunistic sampling events. In the lead up to the nationwide release of RHDVa-K5 (GI.1aP-GI.1a) in 2017, an existing citizen science program, RabbitScan, was augmented to allow members of the public to submit samples collected from dead leporids for lagovirus testing. This study describes the information obtained from the increased number of leporid samples received between 2015 and 2022 and focuses on the recent epidemiological interactions and evolutionary trajectory of circulating lagoviruses in Australia between October 2020 and December 2022. A total of 2771 samples were tested from January 2015 to December 2022, of which 1643 were lagovirus-positive. Notable changes in the distribution of lagovirus variants were observed, predominantly in Western Australia, where RHDV2-4c (GI.4cP-GI.2) was detected again in 2021 after initially being reported to be present in 2018. Interestingly, we found evidence that the deliberately released RHDVa-K5 was able to establish and circulate in wild rabbit populations in WA. Overall, the incorporation of citizen science approaches proved to be a cost-efficient method to increase the sampling area and enable an in-depth analysis of lagovirus distribution, genetic diversity, and interactions. The maintenance of such programs is essential to enable continued investigations of the critical parameters affecting the biocontrol of feral rabbit populations in Australia, as well as to enable the detection of any potential future incursions. [ABSTRACT FROM AUTHOR]

An intrinsically disordered protein (IDP) or region (IDR) lacks or has little protein structure but still maintains function. This lack of structure creates flexibility and fluidity, allowing multiple protein conformations and potentially transient interactions with more than one partner. Caliciviruses are positive-sense ssRNA viruses, containing a relatively small genome of 7.6–8.6 kb and have a broad host range. Many viral proteins are known to contain IDRs, which benefit smaller viral genomes by expanding the functional proteome through the multifunctional nature of the IDR. The percentage of intrinsically disordered residues within the total proteome for each calicivirus type species can range between 8 and 23%, and IDRs have been experimentally identified in NS1-2, VPg and RdRP proteins. The IDRs within a protein are not well conserved across the genera, and whether this correlates to different activities or increased tolerance to mutations, driving virus adaptation to new selection pressures, is unknown. The function of norovirus NS1-2 has not yet been fully elucidated but includes involvement in host cell tropism, the promotion of viral spread and the suppression of host interferon-λ responses. These functions and the presence of host cell-like linear motifs that interact with host cell caspases and VAPA/B are all found or affected by the disordered region of norovirus NS1-2. The IDRs of calicivirus VPg are involved in viral transcription and translation, RNA binding, nucleotidylylation and cell cycle arrest, and the N-terminal IDR within the human norovirus RdRP could potentially drive liquid–liquid phase separation. This review identifies and summarises the IDRs of proteins within the Caliciviridae family and their importance during viral replication and subsequent host interactions.

ObjectiveTo identify associations between microbes and host genes in cats with feline chronic gingivostomatitis (FCGS), a debilitating inflammatory oral mucosal disease with no known cause, compared with healthy cats and cats with periodontitis (control cats).Animals19 control cats and 23 cats with FCGS.ProceduresAt least 1 caudal oral mucosal swab specimen was obtained from each cat. Each specimen underwent unbiased metatranscriptomic next-generation RNA sequencing (mNGS). Filtered mNGS reads were aligned to all known genetic sequences from all organisms and to the cat transcriptome. The relative abundances of microbial and host gene read alignments were compared between FCGS-affected cats and control cats and between FCGS-affected cats that did and did not clinically respond to primary treatment. Assembled feline calicivirus (FCV) genomes were compared with reverse transcription PCR (RT-PCR) primers commonly used to identify FCV.ResultsThe only microbe strongly associated with FCGS was FCV, which was detected in 21 of 23 FCGS-affected cats but no control cats. Problematic base pair mismatches were identified between the assembled FCV genomes and RT-PCR primers. Puma feline foamy virus was detected in 9 of 13 FCGS-affected cats that were refractory to treatment and 5 healthy cats but was not detected in FCGS-affected cats that responded to tooth extractions. The most differentially expressed genes in FCGS-affected cats were those associated with antiviral activity.Conclusions and clinical relevanceResults suggested that FCGS pathogenesis has a viral component. Many FCV strains may yield false-negative results on RT-PCR-based assays. Coinfection of FCGS-affected cats with FCV and puma feline foamy virus may adversely affect response to treatment.

Feline calicivirus (FCV) causes upper respiratory tract disease (URTD) and sporadic outbreaks of virulent systemic disease (FCV-VSD). The basis for the increased pathogenicity of FCV-VSD viruses is incompletely understood, and antivirals for FCV-VSD have yet to be developed. We investigated the clinicoepidemiology and viral features of three FCV-VSD outbreaks in Australia and evaluated the in vitro efficacy of nitazoxanide (NTZ), 2'-C-methylcytidine (2CMC) and NITD-008 against FCV-VSD viruses. Overall mortality among 23 cases of FCV-VSD was 39%. Metagenomic sequencing identified five genetically distinct FCV lineages within the three outbreaks, all seemingly evolving in situ in Australia. Notably, no mutations that clearly distinguished FCV-URTD from FCV-VSD phenotypes were identified. One FCV-URTD strain likely originated from a recombination event. Analysis of seven amino-acid residues from the hypervariable E region of the capsid in the cultured viruses did not support the contention that properties of these residues can reliably differentiate between the two pathotypes. On plaque reduction assays, dose-response inhibition of FCV-VSD was obtained with all antivirals at low micromolar concentrations; NTZ EC50, 0.4-0.6 µM, TI = 21; 2CMC EC50, 2.7-5.3 µM, TI > 18; NITD-008, 0.5 to 0.9 µM, TI > 111. Investigation of these antivirals for the treatment of FCV-VSD is warranted.

In virology, the term reverse genetics refers to a set of methodologies in which changes are introduced into the viral genome and their effects on the generation of infectious viral progeny and their phenotypic features are assessed. Reverse genetics emerged thanks to advances in recombinant DNA technology, which made the isolation, cloning, and modification of genes through mutagenesis possible. Most virus reverse genetics studies depend on our capacity to rescue an infectious wild-type virus progeny from cell cultures transfected with an “infectious clone”. This infectious clone generally consists of a circular DNA plasmid containing a functional copy of the full-length viral genome, under the control of an appropriate polymerase promoter. For most DNA viruses, reverse genetics systems are very straightforward since DNA virus genomes are relatively easy to handle and modify and are also (with few notable exceptions) infectious per se. This is not true for RNA viruses, whose genomes need to be reverse-transcribed into cDNA before any modification can be performed. Establishing reverse genetics systems for members of the Caliciviridae has proven exceptionally challenging due to the low number of members of this family that propagate in cell culture. Despite the early successful rescue of calicivirus from a genome-length cDNA more than two decades ago, reverse genetics methods are not routine procedures that can be easily extrapolated to other members of the family. Reports of calicivirus reverse genetics systems have been few and far between. In this review, we discuss the main pitfalls, failures, and delays behind the generation of several successful calicivirus infectious clones.

Copyright of Summa, Animali da Reddito is the property of Point Veterinaire Italie s.r.l. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Summa, Animali da Reddito is the property of Point Veterinaire Italie s.r.l. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

ObjectivesThe aim of this study was to assess the effects of famciclovir administration in cats with spontaneously acquired acute upper respiratory tract disease.MethodsTwenty-four kittens with clinical signs of acute upper respiratory tract disease were randomly allocated to receive doxycycline (5 mg/kg PO q12h) alone (group D; n = 12) or with famciclovir (90 mg/kg PO q12h; group DF; n = 12) for up to 3 weeks. Clinical disease severity was scored at study entry and daily thereafter. Oculo-oropharyngeal swabs collected at study entry and exit were assessed using quantitative PCR for nucleic acids of feline herpesvirus type 1 (FHV-1), feline calicivirus (FCV), Chlamydia felis, Bordetella bronchiseptica and Mycoplasma felis.ResultsThe median (range) age of cats was 1.5 (1-6) months in group D vs 1.6 (1-5) months in group DF (P = 0.54). Pathogens detected in oculo-oropharyngeal swabs at study entry included FCV (n = 13/24; 54%), M felis (n = 8/24; 33%), FHV-1 (n = 7/24; 29%), C felis (n = 7/24; 29%) and B bronchiseptica (n = 3/24; 12%). Median (range) duration of clinical signs was 11.5 (3-21) days in group DF and 11 (3-21) days in group D (P = 0.75). Median (range) total disease score at the end of the study did not differ between groups (group D 1 [1-1] vs group DF 1 [1-3]; P = 0.08).Conclusions and relevanceThis study revealed no significant difference in response to therapy between cats treated with doxycycline alone or with famciclovir; cats improved rapidly in both groups. However, identification of FHV-1 DNA was relatively uncommon in this study and clinical trials focused on FHV-1-infected cats are warranted to better evaluate famciclovir efficacy.

Chimeric virus-like particles (cVLPs) show great potential in improving public health as they are safe and effective vaccine candidates. The capsid protein of caliciviruses has been described previously as a self-assembling, highly immunogenic delivery platform. The ability to significantly induce cellular and humoral immunity can be used to boost the immune response to low immunogenic foreign antigens displayed on the surface of VLPs. Capsid proteins of caliciviruses despite sequence differences share similar architecture with structural loops that can be genetically modified to present foreign epitopes on the surface of cVLPs. Here, based on the VP1 protein of norovirus (NoV), we investigated the impact of the localization of the epitope in different structural loops of the P domain on the immunogenicity of the presented epitope. In this study, three distinct loops of NoV VP1 protein were genetically modified to present a multivalent influenza virus epitope consisting of a tandem repeat of M2/NP epitopes. cVLPs presenting influenza virus-conserved epitopes in different localizations were produced in the insect cells and used to immunize BALB/c mice. Specific reaction to influenza epitopes was compared in sera from vaccinated mice to determine whether the localization of the foreign epitope has an impact on the immunogenicity. [ABSTRACT FROM AUTHOR]

Norovirus is a major human pathogen that can cause severe gastroenteritis in vulnerable populations. The extensive viral diversity presented by human noroviruses constitutes a major roadblock for the development of effective vaccines. In addition to the large number of genotypes, antigenically distinct variants of GII.4 noroviruses have chronologically emerged over the last 3 decades. The last variant to emerge, Sydney_2012, has been circulating at high incidence worldwide for over a decade. We analyzed 1449 capsid sequences from GII.4 Sydney_2012 viruses to determine genetic changes indicative of antigenic diversification. Phylogenetic analyses show that Sydney_2012 viruses scattered within the tree topology with no single cluster dominating during a given year or geographical location. Fourteen residues presented high variability, 7 of which mapped to 4 antigenic sites. Notably,; 52% of viruses presented mutations at 2 or more antigenic sites. Mutational patterns showed that residues 297 and 372, which map to antigenic site A, changed over time. Virus-like particles (VLPs) developed from wild-type Sydney_2012 viruses and engineered to display all mutations detected at antigenic sites were tested against polyclonal sera and monoclonal antibodies raised against Sydney_2012 and Farmington_Hills_2002 VLPs. Minimal changes in reactivity were detected with polyclonal sera and only 4 MAbs lost binding, with all mapping to antigenic site A. Notably, reversion of residues from Sydney_2012 reconstituted epitopes from ancestral GII.4 variants. Overall, this study demonstrates that, despite circulating for over a decade, Sydney_2012 viruses present minimal antigenic diversification and provides novel insights on the diversification of GII.4 noroviruses that could inform vaccine design. IMPORTANCE GII.4 noroviruses are the major cause of acute gastroenteritis in all age groups. This predominance has been attributed to the continued emergence of phylogenetically discrete variants that escape immune responses to previous infections. The last GII.4 variant to emerge, Sydney_2012, has been circulating at high incidence for over a decade, raising the question of whether this variant is undergoing antigenic diversification without presenting a major distinction at the phylogenetic level. Sequence analyses that include .1400 capsid sequences from GII.4 Sydney_2012 showed changes in 4 out of the 6 major antigenic sites. Notably, while changes were detected in one of the most immunodominant sites over time, these resulted in minimal changes in the antigenic profile of these viruses. This study provides new insights on the mechanism governing the antigenic diversification of GII.4 norovirus that could help in the development of crossprotective vaccines to human noroviruses. [ABSTRACT FROM AUTHOR]

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Since their introduction in 1859, European rabbits (Oryctolagus cuniculus) have had a devastating impact on agricultural production and biodiversity in Australia, with competition and land degradation by rabbits being one of the key threats to agricultural and biodiversity values in Australia. Biocontrol agents, with the most important being the rabbit haemorrhagic disease virus 1 (RHDV1), constitute the most important landscape-scale control strategies for rabbits in Australia. Monitoring field strain dynamics is complex and labour-intensive. Here, using phylodynamic models to analyse the available RHDV molecular data, we aimed to: investigate the epidemiology of various strains, use molecular data to date the emergence of new variants and evaluate whether different strains are outcompeting one another. We determined that the two main pathogenic lagoviruses variants in Australia (RHDV1 and RHDV2) have had similar dynamics since their release, although over different timeframes (substantially shorter for RHDV2). We also found a strong geographic difference in their activities and evidence of overall competition between the two viruses. [ABSTRACT FROM AUTHOR]

In order to survey noroviruses in our environment, it is essential that both wet-lab and computational methods are fit for purpose. Using a simulated sequencing data set, denoising-based (DADA2, Deblur and USEARCH-UNOISE3) and clustering-based pipelines (VSEARCH and FROGS) were compared with respect to their ability to represent composition and sequence information. Open source classifiers (Ribosomal Database Project [RDP], BLASTn, IDTAXA, QIIME2 naive Bayes, and SINTAX) were trained using three different databases: a custom database, the NoroNet database, and the Human calicivirus database. Each classifier and database combination was compared from the perspective of their classification accuracy. VSEARCH provides a robust option for analyzing viral amplicons based on composition analysis; however, all pipelines could return OTUs with high similarity to the expected sequences. Importantly, pipeline choice could lead to more false positives (DADA2) or underclassification (FROGS), a key aspect when considering pipeline application for source attribution. Classification was more strongly impacted by the classifier than the database, although disagreement increased with norovirus GII.4 capsid variant designation. We recommend the use of the RDP classifier in conjunction with VSEARCH; however, maintenance of the underlying database is essential for optimal use. [ABSTRACT FROM AUTHOR]

Rabbit haemorrhagic disease (RHD) is a signifcant and debilitating viral disease affecting lagomorphs. In September 2020, Singapore reported its frst cases of RHD virus (RHDV) infection in domesticated rabbits. The initial fndings reported that the outbreak strain belonged to genotype GI.2 (RHDV2/RHDVb), and epidemiological investigations could not identify the defnitive source of the virus origin. Further recombination detection and phylogenetic analyses of the Singapore outbreak strain revealed that the RHDV was a GI.2 structural (S)/GI.4 non-structural (NS) recombinant variant. Sequence analyses on the National Centre for Biotechnology Information (NCBI) database showed high homology to recently emerged Australian variants, which were prevalent in local Australian lagomorph populations since 2017. Time-structured and phylogeographic analyses for the S and NS genes revealed a close genetic relationship between the Singapore RHDV strain and the Australian RHDV variants. More thorough epidemiological inquiries are necessary to ascertain how an Australian RHDV was introduced into the Singapore rabbit population, and opportune development of RHDV diagnostics and vaccines will be important to safeguard lagomorphs from future RHDV infection and disease management. [ABSTRACT FROM AUTHOR]

Barramundi aquaculture is at risk of severe disease outbreaks and massive production losses. Here we used bioinformatics to screen 84 farmed barramundi transcriptomes to identify novel viruses that could threaten barramundi aquaculture and to establish a barramundi aquaculture virome. We discovered five novel viruses: latid herpesvirus 1 (LatHV-1) from the Alloherpesviridae family, barramundi parvovirus 1 (BParV1) from the Parvoviridae family, barramundi calicivirus 1 (BCaV1) from the Caliciviridae family, and barramundi associated picorna-like virus 1 and 2 (BPicV1 and BPicV2) from the Picornaviridae family. LatHV-1, BCaV1, and BParV1 are closely related to pathogenic viruses found in other fish species that can cause mass mortality in farms. To aid in future viral surveillance, we also designed and successfully tested an RT-PCR assay for the detection of BCaV1. Overall, we discovered a range of pathogenic viruses in barramundi aquaculture, paving the way for developing effective detection methods to assist early outbreak management. • Novel viruses were discovered in farmed barramundi transcriptomes. • RT-PCR detection assay was developed and trialled for barramundi calicivirus 1. • Novel picorna-like viruses were discovered in barramundi farm ecosystems. [ABSTRACT FROM AUTHOR]

Since 2020, another type of epidemic disease, 'red lower jaw disease' (RLJD), has broken out in yellow catfish Pelteobagrus fulvidraco every spring in China, causing large-scale deaths and significant economic losses. This new disease has the characteristics of strong infectivity, high mortality and a single host. To prevent and control RLJD, epidemiological investigation, transmission electron microscopy (TEM), virome sequencing, Koch's rule verification and epidemic factor investigation were studied in this article. The clinical symptoms of diseased yellow catfish were pale gill filaments, hyperaemia and swelling of the spleen and kidney. TEM results revealed that a large number of 35–40 nm spherical virus particles without envelope structure were found in the cytoplasm of spleen cells. The liver, spleen and kidney of the diseased fish had serious lesions. After virome sequencing, the calicivirus reads in the unique virus reads accounted for 99.5% of the unique virus reads in the diseased fish, which provided a relevant reference value for identifying the pathogen of RLJD. The full-length sequence of yellow catfish calicivirus (YCCV) obtained at 7405 bp, including the RNA-helicase domain, RdRp-1 domain and d1iqqa domain. At the same time, both PCR and TaqMan fluorescence quantitative PCR detection method of YCCV were established. Through the artificial infection test, YCCV was verified as the pathogen for RLJD. It was also found that YCCV mainly harmed the fish within 1 year, whereas the fish above 2 years had good resistance. The water temperature of the RLJD outbreak was mainly concentrated at 18 °C–24 °C. In the current study, after YCCV was verified as the pathogen of RLJD, epidemic factors including fish age and water temperature were studied, to the best of our knowledge, for the first time to provide a relevant theoretical basis for the pathogenic mechanism and prevention and control measures of yellow catfish RLJD. • The pathogen of Pelteobagrus fulvidraco red lower jaw disease is yellow catfish calicivirus. • The calicivirus can cause typical clinical symptoms and serious tissue lesions. • P. fulvidraco is the unique host of yellow catfish calicivirus. • The infection of calicivirus is influenced by yellow catfish age and water temperature. [ABSTRACT FROM AUTHOR]

Australia has multiple lagoviruses with differing pathogenicity. The circulation of these viruses was traditionally determined through opportunistic sampling events. In the lead up to the nationwide release of RHDVa-K5 (GI.1aP-GI.1a) in 2017, an existing citizen science program, RabbitScan, was augmented to allow members of the public to submit samples collected from dead leporids for lagovirus testing. This study describes the information obtained from the increased number of leporid samples received between 2015 and 2022 and focuses on the recent epidemiological interactions and evolutionary trajectory of circulating lagoviruses in Australia between October 2020 and December 2022. A total of 2771 samples were tested from January 2015 to December 2022, of which 1643 were lagovirus-positive. Notable changes in the distribution of lagovirus variants were observed, predominantly in Western Australia, where RHDV2-4c (GI.4cP-GI.2) was detected again in 2021 after initially being reported to be present in 2018. Interestingly, we found evidence that the deliberately released RHDVa-K5 was able to establish and circulate in wild rabbit populations in WA. Overall, the incorporation of citizen science approaches proved to be a cost-efficient method to increase the sampling area and enable an in-depth analysis of lagovirus distribution, genetic diversity, and interactions. The maintenance of such programs is essential to enable continued investigations of the critical parameters affecting the biocontrol of feral rabbit populations in Australia, as well as to enable the detection of any potential future incursions.

Chimeric virus-like particles (cVLPs) show great potential in improving public health as they are safe and effective vaccine candidates. The capsid protein of caliciviruses has been described previously as a self-assembling, highly immunogenic delivery platform. The ability to significantly induce cellular and humoral immunity can be used to boost the immune response to low immunogenic foreign antigens displayed on the surface of VLPs. Capsid proteins of caliciviruses despite sequence differences share similar architecture with structural loops that can be genetically modified to present foreign epitopes on the surface of cVLPs. Here, based on the VP1 protein of norovirus (NoV), we investigated the impact of the localization of the epitope in different structural loops of the P domain on the immunogenicity of the presented epitope. In this study, three distinct loops of NoV VP1 protein were genetically modified to present a multivalent influenza virus epitope consisting of a tandem repeat of M2/NP epitopes. cVLPs presenting influenza virus-conserved epitopes in different localizations were produced in the insect cells and used to immunize BALB/c mice. Specific reaction to influenza epitopes was compared in sera from vaccinated mice to determine whether the localization of the foreign epitope has an impact on the immunogenicity.

Background: Human norovirus (HuNoV) is the leading viral cause of diarrhea, with GII.4 as the predominant genotype of HuNoV outbreaks globally. However, new genogroup variants emerge periodically, complicating the development of anti-HuNoV vaccines; other prophylactic or therapeutic medications specifically for HuNoV disease are lacking. Passive immunization using oral anti-HuNoV antibodies may be a rational alternative. Here, we explore the feasibility of using avian immunoglobulins (IgY) for preventing HuNoV infection in vitro in a human intestinal enteroid (HIE) model. Methods: Hens were immunized with virus-like particles (VLP) of a GII.4 HuNoV strain (GII.4/CHDC2094/1974/US) by intramuscular injection. The resulting IgY was evaluated for inhibition of binding to histo-blood group antigens (HBGA) and viral neutralization against representative GII.4 and GII.6 clinical isolates, using an HIE model. Results: IgY titers were detected by three weeks following initial immunization, persisting at levels of 1:221 (1:2,097,152) from 9 weeks to 23 weeks. Anti-HuNoV IgY significantly (p < 0.05) blocked VLP adhesion to HBGA up to 1:12,048 dilution (0.005 mg/mL), and significantly (p < 0.05) inhibited replication of HuNoV GII.4[P16] Sydney 2012 in HIEs up to 1:128 dilution (0.08 mg/mL). Neutralization was not detected against genotype GII.6. Conclusions: We demonstrate the feasibility of IgY for preventing infection of HIE by HuNoV GII.4. Clinical preparations should cover multiple circulating HuNoV genotypes for comprehensive effects. Plans for animal studies are underway. [ABSTRACT FROM AUTHOR]

Noroviruses are the leading cause of severe childhood diarrhea and foodborne disease worldwide. While they are a major cause of disease in all age groups, infections in the very young can be quite severe with annual estimates of 50,000-200,000 fatalities in children under 5 years old. In spite of the remarkable disease burden associated with norovirus infections in people, very little is known about the pathogenic mechanisms underlying norovirus diarrhea, principally because of the lack of tractable small animal models. We recently demonstrated that wild-type neonatal mice are susceptible to murine norovirus (MNV)-induced acute self-resolving diarrhea in a time course mirroring human norovirus disease. Using this robust pathogenesis model system, we demonstrate that virulence is regulated by the responsiveness of the viral capsid to environmental cues that trigger contraction of the VP1 protruding (P) domain onto the particle shell, thus enhancing receptor binding and infectivity. The capacity of a given MNV strain to undergo this contraction positively correlates with infection of cells expressing low abundance of the virus receptor CD300lf, supporting a model whereby virion contraction triggers infection of CD300lflo cell types that are responsible for diarrhea induction. These findings directly link environmentally-influenced biophysical features with norovirus disease severity. [ABSTRACT FROM AUTHOR]

Here, we report the coding-complete genomic sequences of two chicken caliciviruses from US poultry flocks in 2003 and 2004. They show the same genomic organization as that of other members of the Bavovirus genus and have the highest nucleotide identity (~88%) with strains from clinically normal chickens from Germany in 2004 and Netherlands in 2019., Competing Interests: The authors declare no conflict of interest.

Abstract Background Rabbit haemorrhagic disease virus (RHDV) is a Lagovirus, a subgroup of the family Caliciviridae. RHDV2 is a variant first described in France in 2010, and has since spread globally. It has been reported in several Lagomorph species (rabbits, hares, and their relatives) as well as other mammals including voles and shrews. The disease has raised international concerns for its potential impact on population abundance trajectories, particularly as 25% of Lagomorphs are currently Red-Listed by the International Union for the Conservation of Nature (IUCN). The Irish hare (Lepus timidus hibernicus) is a subspecies of the mountain hare, L. timidus, and is endemic to Ireland, making it an Evolutionarily Significant Unit of intrinsic value. Case presentation The first case of RHDV2 was detected in a wild Irish hare in July 2019. The individual exhibited atypical neurological behaviour (running in circles) prior to death. On necropsy, pink tinged foam was seen in the trachea and congestion was noted in the lungs, but there was no evidence of haemorrhages in any other organ. Both the liver and spleen were tested by reverse transcription real time qPCR confirming high levels of RHDV2 RNA. Histopathology confirmed multifocal necrotising hepatitis. Conclusion The Irish hare is susceptible to RHDV2 infection. Further investigation is warranted to explore the clinical, epidemiological, and population biology implications.

Noroviruses are the leading cause of severe childhood diarrhea and foodborne disease worldwide. While they are a major cause of disease in all age groups, infections in the very young can be quite severe with annual estimates of 50,000-200,000 fatalities in children under 5 years old. In spite of the remarkable disease burden associated with norovirus infections in people, very little is known about the pathogenic mechanisms underlying norovirus diarrhea, principally because of the lack of tractable small animal models. We recently demonstrated that wild-type neonatal mice are susceptible to murine norovirus (MNV)-induced acute self-resolving diarrhea in a time course mirroring human norovirus disease. Using this robust pathogenesis model system, we demonstrate that virulence is regulated by the responsiveness of the viral capsid to environmental cues that trigger contraction of the VP1 protruding (P) domain onto the particle shell, thus enhancing receptor binding and infectivity. The capacity of a given MNV strain to undergo this contraction positively correlates with infection of cells expressing low abundance of the virus receptor CD300lf, supporting a model whereby virion contraction triggers infection of CD300lflo cell types that are responsible for diarrhea induction. These findings directly link environmentally-influenced biophysical features with norovirus disease severity.

Background: Rabbit haemorrhagic disease virus (RHDV) is a Lagovirus, a subgroup of the family Caliciviridae. RHDV2 is a variant first described in France in 2010, and has since spread globally. It has been reported in several Lagomorph species (rabbits, hares, and their relatives) as well as other mammals including voles and shrews. The disease has raised international concerns for its potential impact on population abundance trajectories, particularly as 25% of Lagomorphs are currently Red-Listed by the International Union for the Conservation of Nature (IUCN). The Irish hare (Lepus timidus hibernicus) is a subspecies of the mountain hare, L. timidus, and is endemic to Ireland, making it an Evolutionarily Significant Unit of intrinsic value. Case presentation: The first case of RHDV2 was detected in a wild Irish hare in July 2019. The individual exhibited atypical neurological behaviour (running in circles) prior to death. On necropsy, pink tinged foam was seen in the trachea and congestion was noted in the lungs, but there was no evidence of haemorrhages in any other organ. Both the liver and spleen were tested by reverse transcription real time qPCR confirming high levels of RHDV2 RNA. Histopathology confirmed multifocal necrotising hepatitis. Conclusion: The Irish hare is susceptible to RHDV2 infection. Further investigation is warranted to explore the clinical, epidemiological, and population biology implications. [ABSTRACT FROM AUTHOR]

Keywords: calicivirus; emerging diseases; nidovirus; RNA sequencing; transcriptomics; viral discovery EN calicivirus emerging diseases nidovirus RNA sequencing transcriptomics viral discovery 931 938 8 05/13/22 20220601 NES 220601 INTRODUCTION Agnatha is an ancient superclass of jawless fish that gave rise to all other vertebrates after diverging from chordates ~535 million years ago (mya) (Janvier, 1981). Lamprey and hagfish viruses range from simpler viruses such as caliciviruses, to complex viruses such as bafiniviruses. As a subset of fish viruses, agnathan viruses are even more underexplored than teleost viruses. [Extracted from the article]

ABSTRACT Rabbit hemorrhagic disease (RHD) and European brown hare syndrome (EBHS) are highly contagious diseases caused by lagoviruses in the Caliciviridae family. These infectious diseases are associated with high mortality and a serious threat to domesticated and wild rabbits and hares, including endangered species such as riparian brush rabbits (Sylvilagus bachmani riparius). In the United States (U.S.), only isolated cases of RHD had been reported until Spring 2020. However, RHD caused by GI.2/rabbit hemorrhagic disease virus (RHDV)2/b was unexpectedly reported in April 2020 in New Mexico and has subsequently spread to several U.S. states, infecting wild rabbits and hares and making it highly likely that RHD will become endemic in the U.S. Vaccines are available for RHD; however, there is no specific treatment for this disease. Lagoviruses encode a 3C-like protease (3CLpro), which is essential for virus replication and a promising target for antiviral drug development. We have previously generated focused small-molecule libraries of 3CLpro inhibitors and demonstrated the in vitro potency and in vivo efficacy of some protease inhibitors against viruses encoding 3CLpro, including caliciviruses and coronaviruses. Here, we report the development of the enzyme and cell-based assays for the 3CLpro of GI.1c/RHDV, recombinant GI.3P-GI.2 (RHDV2/b), and GII.1/European brown hare syndrome virus (EBHSV) as well as the identification of potent lagovirus 3CLpro inhibitors, including GC376, a protease inhibitor being developed for feline infectious peritonitis. In addition, structure-activity relationship study and homology modeling of the 3CLpro and inhibitors revealed that lagovirus 3CLpro share similar structural requirements for inhibition with other calicivirus 3CLpro. IMPORTANCE Rabbit hemorrhagic disease (RHD) and European brown hare syndrome (EBHS) are viral diseases that affect lagomorphs with significant economic and ecological impacts. RHD vaccines are available, but specific antiviral treatment for these viral infections would be a valuable addition to the current control measures. Lagoviruses encode 3C-like protease (3CLpro), which is essential for virus replication and an attractive target for antiviral drug discovery. We have screened and identified potent small-molecule inhibitors that block lagovirus 3CLpro in the enzyme- and cell-based assays. Our results suggest that these compounds have the potential for further development as antiviral drugs for lagoviruses.

Abstract Bile acids (BAs) are evolutionally conserved molecules synthesized in the liver from cholesterol to facilitating the absorption of fat-soluble nutrients. In the intestines, where enteric viruses replicate, BAs also act as signaling molecules that modulate various biological functions via activation of specific receptors and cell signaling pathways. To date, BAs present either pro-viral or anti-viral effects for the replication of enteric viruses in vivo and in vitro. In this review, we summarized current information on biosynthesis, transportation and metabolism of BAs and the role of BAs in replication of enteric caliciviruses, rotaviruses, and coronaviruses. We also discussed the application of BAs for cell culture adaptation of fastidious enteric caliciviruses and control of virus infection, which may provide novel insights into the development of antivirals and/or disinfectants for enteric viruses.

Caliciviruses (Caliciviridae) and astroviruses (Astroviridae) are among the leading cause of non-bacterial foodborne disease and gastroenteritis in human. These non-enveloped RNA viruses infect a wide range of vertebrate species including rodents. Rodents are among the most important hosts of infectious diseases globally and are responsible for over 80 zoonotic pathogens that affect humans. Therefore, screening pathogens in rodents will be is necessary to prevent cross-species transmission to prevent zoonotic outbreaks. In the present study, we screened caliciviruses and astroviruses in order to describe their diversity and whether they harbor strains that can infect humans. RNA was then extracted from intestine samples of 245 rodents and retrotranscribed in cDNA to screen caliciviruses and astroviruses by PCRs. All the samples tested negative for caliciviruses and while astroviruses were detected in 18 (7.3%) samples of Rattus rattus species. Phylogenetic analyses based on the RdRp gene showed that all the sequences belonged to Mamastrovirus genus in which they were genetically related to R. rattus related AstVs previously detected in Gabon or in Rattus spp. AstV from Kenya and Asia. These findings suggested that transportation such as land and railway, as well national and international trade, are likely to facilitate spread of AstVs by the dissemination of rodents. • All Rodent were tested negative for caliciviruses. • Rattus rattus is the only species infected by astroviruses. • No rodents were infected with human astroviruses. • Transportation such as land and railway, as well national and international trade, are likely to facilitate spread of enteric viruses by the dissemination of rodents. [ABSTRACT FROM AUTHOR]

Human noroviruses are the most common viral agents of acute gastroenteritis. Recently, human intestinal enteroids were shown to be permissive for norovirus infection. We tested their suitability as a system to study norovirus neutralization. Hyperimmune sera raised against virus-like particles (VLPs) representing different genotypes showed highly specific neutralization activity against GII.4 and GII.6 noroviruses. Carbohydrate blocking assays and neutralization exhibited similar patterns in antibody responses. Notably, sera produced against chimeric VLPs that presented swapped structural shell and protruding (P) domains, from different genotypes showed that neutralization is primarily mediated by antibodies mapping to the P domain of the norovirus capsid protein. This study provides empirical information on the antigenic differences among genotypes as measured by neutralization, which could guide vaccine design. [ABSTRACT FROM AUTHOR]

Norovirus is a major cause of acute gastroenteritis. Human noroviruses present >30 different genotypes, with a single genotype (GII.4) predominating worldwide. Concurrent outbreaks of norovirus are often associated with the emergence of new viruses. While different hypotheses have been presented, the source of new mutations in noroviruses is still unknown. In this study, we applied high-resolution sequencing to determine the intra-host viral diversity presented by noroviruses during the acute and shedding phase of infection in children. Profiling viral intra-host diversification at nearly full genome level indicated that GII.4 viruses presented dynamic intra-host variation, while non-GII.4 viruses presented minimal variation throughout the infection. Notably, the intra-host genetic variation during the shedding phase recapitulates the genetic diversity observed at the global level, particularly those mapping at the VP1 antigenic sites. Thus the intra-host evolution in healthy children explains the source of norovirus mutations that results in diversification at the global scale.

ABSTRACT Human norovirus (HNoV) is a global health and socioeconomic burden, estimated to infect every individual at least five times during their lifetime. The underlying mechanism for the potential lack of long-term immune protection from HNoV infections is not understood and prompted us to investigate HNoV susceptibility of primary human B cells and its functional impact. Primary B cells isolated from whole blood were infected with HNoV-positive stool samples and harvested at 3 days postinfection (dpi) to assess the viral RNA yield by reverse transcriptase quantitative PCR (RT-qPCR). A 3- to 18-fold increase in the HNoV RNA yield was observed in 50 to 60% of donors. Infection was further confirmed in B cells derived from splenic and lymph node biopsy specimens. Next, we characterized infection of whole-blood-derived B cells by flow cytometry in specific functional B cell subsets (naive CD27− IgD+, memory-switched CD27+ IgD−, memory-unswitched CD27+ IgD+, and double-negative CD27− IgD− cells). While the susceptibilities of the subsets were similar, changes in the B cell subset distribution upon infection were observed, which were also noted after treatment with HNoV virus-like particles and the predicted recombinant NS1 protein. Importantly, primary B cell stimulation with the predicted recombinant NS1 protein triggered B cell activation and induced metabolic changes. These data demonstrate that primary B cells are susceptible to HNoV infection and suggest that the NS1 protein can alter B cell activation and metabolism in vitro, which could have implications for viral pathogenesis and immune responses in vivo. IMPORTANCE Human norovirus (HNoV) is the most prevalent causative agent of gastroenteritis worldwide. Infection results in a self-limiting disease that can become chronic and severe in the immunocompromised, the elderly, and infants. There are currently no approved therapeutic and preventative strategies to limit the health and socioeconomic burdens associated with HNoV infections. Moreover, HNoV does not elicit lifelong immunity as repeat infections are common, presenting a challenge for vaccine development. Given the importance of B cells for humoral immunity, we investigated the susceptibility and impact of HNoV infection on human B cells. We found that HNoV replicates in human primary B cells derived from blood, spleen, and lymph node specimens, while the nonstructural protein NS1 can activate B cells. Because of the secreted nature of NS1, we put forward the hypothesis that HNoV infection can modulate bystander B cell function with potential impacts on systemic immune responses.

Akt (protein kinase B) is a key signaling protein in eukaryotic cells that controls many cellular processes, such as glucose metabolism and cell proliferation, for survival. As obligate intracellular pathogens, viruses modulate host cellular processes, including Akt signaling, for optimal replication. The mechanisms by which viruses modulate Akt and the resulting effects on the infectious cycle differ widely depending on the virus. In this study, we explored the effect of Akt serine 473 phosphorylation (p-Akt) during murine norovirus (MNV) infection. p-Akt increased during infection of murine macrophages with acute MNV-1 and persistent CR3 and CR6 strains. Inhibition of Akt with MK2206, an inhibitor of all three isoforms of Akt (Akt1/2/3), reduced infectious virus progeny of all three virus strains. This reduction was due to decreased viral genome replication (CR3), defective virus assembly (MNV-1), or altered cellular egress (CR3 and CR6) in a virus strain-dependent manner. Collectively, our data demonstrate that Akt activation increases in macrophages during the later stages of the MNV infectious cycle, which may enhance viral infection in unique ways for different virus strains. The data, for the first time, indicate a role for Akt signaling in viral assembly and highlight additional phenotypic differences between closely related MNV strains. [ABSTRACT FROM AUTHOR]

The diversity of lagoviruses (Caliciviridae) in Australia has increased considerably in recent years. By the end of 2017, five variants from three viral genotypes were present in populations of Australian rabbits, while prior to 2014 only two variants were known. To understand the evolutionary interactions among these lagovirus variants, we monitored their geographical distribution and relative incidence over time in a continental-scale competition study. Within 3 years of the incursion of rabbit haemorrhagic disease virus 2 (RHDV2, denoted genotype GI.1bP-GI.2 [polymerase genotype]P-[capsid genotype]) into Australia, two novel recombinant lagovirus variants emerged: RHDV2-4e (genotype GI.4eP-GI.2) in New South Wales and RHDV2-4c (genotype GI.4cP-GI.2) in Victoria. Although both novel recombinants contain non-structural genes related to those from benign, rabbit-specific, enterotropic viruses, these variants were recovered from the livers of both rabbits and hares that had died acutely. This suggests that the determinants of host and tissue tropism for lagoviruses are associated with the structural genes, and that tropism is intricately connected with pathogenicity. Phylogenetic analyses demonstrated that the RHDV2-4c recombinant emerged independently on multiple occasions, with five distinct lineages observed. Both the new RHDV2-4e and -4c recombinant variants replaced the previous dominant parental RHDV2 (genotype GI.1bP-GI.2) in their respective geographical areas, despite sharing an identical or near-identical (i.e. single amino acid change) VP60 major capsid protein with the parental virus. This suggests that the observed replacement by these recombinants was not driven by antigenic variation in VP60, implicating the non-structural genes as key drivers of epidemiological fitness. Molecular clock estimates place the RHDV2-4e recombination event in early to mid-2015, while the five RHDV2-4c recombination events occurred from late 2015 through to early 2017. The emergence of at least six viable recombinant variants within a 2-year period highlights the high frequency of these events, detectable only through intensive surveillance, and demonstrates the importance of recombination in lagovirus evolution. [ABSTRACT FROM AUTHOR]

Copyright of Veterinary Journal of Mehmet Akif Ersoy University is the property of Veterinary Journal of Mehmet Akif Ersoy University and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)