Recent Progress in Torovirus Molecular Biology.

Torovirus (ToV) has recently been classified into the new family Tobaniviridae, although it belonged to the Coronavirus (CoV) family historically. ToVs are associated with enteric diseases in animals and humans. In contrast to CoVs, which are recognised as pathogens of veterinary and medical importance, little attention has been paid to ToVs because their infections are usually asymptomatic or not severe; for a long time, only one equine ToV could be propagated in cultured cells. However, bovine ToVs, which predominantly cause diarrhoea in calves, have been detected worldwide, leading to economic losses. Porcine ToVs have also spread globally; although they have not caused serious economic losses, coinfections with other pathogens can exacerbate their symptoms. In addition, frequent inter- or intra-recombination among ToVs can increase pathogenesis or unpredicted host adaptation. These findings have highlighted the importance of ToVs as pathogens and the need for basic ToV research. Here, we review recent progress in the study of ToV molecular biology including reverse genetics, focusing on the similarities and differences between ToVs and CoVs.

Characterization of Localization and Export Signals of Bovine Torovirus Nucleocapsid Protein Responsible for Extensive Nuclear and Nucleolar Accumulation and Their Importance for Virus Growth.

Torovirus (ToV) has recently been classified into the new family Tobaniviridae, although historically, it belonged to the Coronavirus (CoV) family. The nucleocapsid (N) proteins of CoVs are predominantly localized in the cytoplasm, where the viruses replicate, but in some cases the proteins are partially located in the nucleolus. Many studies have investigated the subcellular localization and nucleocytoplasmic trafficking signals of the CoV N proteins, but little is known about ToV N proteins. Here, we studied the subcellular localization of the bovine ToV (BToV) N protein (BToN) and characterized its nucleocytoplasmic trafficking signals. Unlike other CoVs, BToN in infected cells was transported mainly to the nucleolus during early infection but was distributed predominantly in the nucleoplasm rather than in the nucleolus during late infection. Interestingly, a small quantity of BToN was detected in the cytoplasm during infection. Examination of a comprehensive set of substitution or deletion mutants of BToN fused with enhanced green fluorescent protein (EGFP) revealed that clusters of arginine (R) residues comprise nuclear/nucleolar localization signals (NLS/NoLS), and the C-terminal region served as a chromosomal maintenance 1 (CRM1)-independent nuclear export signal (NES). Moreover, recombinant viruses with mutations in the NLS/NoLS, but retaining nuclear accumulation, were successfully rescued and showed slightly reduced growth ability, while the virus that lost the NLS/NoLS-mediated nuclear accumulation of BToN was not rescued. These results indicate that BToN uniquely accumulates mainly in nuclear compartments during infection, regulated by an R-rich NLS/NoLS and a CRM1-independent NES, and that the BToN accumulation in the nuclear compartment driven by NLS/NoLS is important for virus growth.IMPORTANCE ToVs are diarrhea-causing pathogens detected in many species, including humans. BToV has spread worldwide, leading to economic loss, and there is currently no treatment or vaccine available. Positive-stranded RNA viruses, including ToVs, replicate in the cytoplasm, and their structural proteins generally accumulate in the cytoplasm. Interestingly, BToN accumulated predominantly in the nucleus/nucleolus during all infectious processes, with only a small fraction accumulating in the cytoplasm despite being a major structural protein. Furthermore, we identified unique nucleocytoplasmic trafficking signals and demonstrated the importance of NLS/NoLS for virus growth. This study is the first to undertake an in-depth investigation of the subcellular localization and intracellular trafficking signals of BToN. Our findings additionally suggest that the NLS/NoLS-mediated nuclear accumulation of BToN is important for virus replication. An understanding of the unique features of BToV may provide novel insights into the assembly mechanisms of not only ToVs but also other positive-stranded RNA viruses.

Activation of the autophagy pathway by Torovirus infection is irrelevant for virus replication.

Autophagy is a conserved eukaryotic process that mediates lysosomal degradation of cytoplasmic macromolecules and damaged organelles, also exerting an important role in the elimination of intracellular pathogens. Despite the antiviral role of autophagy, many studies suggest that some positive-stranded RNA viruses exploit this pathway to facilitate their own replication. In this study, we demonstrate that the equine torovirus Berne virus (BEV), the prototype member of the Torovirus genus (Coronaviridae Family, Nidovirales Order), induces autophagy at late times post-infection. Conversion of microtubule associated protein 1B light chain 3 (LC3) from cytosolic (LC3 I) to the membrane associated form (LC3 II), a canonical marker of autophagosome formation, is enhanced in BEV infected cells. However, neither autophagy induction, via starvation, nor pharmacological blockade significantly affect BEV replication. Similarly, BEV infection is not altered in autophagy deficient cells lacking either Beclin 1 or LC3B protein expression. Unexpectedly, the cargo receptor p62, a selective autophagy receptor, aggregates within the region where the BEV main protease (Mpro) localizes. This finding, coupled with observation that BEV replication also induces ER stress at the time when selective autophagy is taking place, suggests that the autophagy pathway is activated in response to the hefty accumulation of virus-encoded polypeptides during the late phase of BEV infection.

[A review of porcine torovirus research: etiology and epidemiology].

Porcine Torovirus (PToV) is widely distributed in the world with high prevalence rate in swinery. Due to the high detection rate in diarrhea pigs, PToV is thought to be a potential pathogen of swine diarrhea. In recent years, epidemic outbreaks of diarrhea with high morbidity and mortality in China have caused great economic losses. Intertypic recombination events and antigenic cross-reactivity among toroviruses implies potential zoonotic transmission of PToV. The review represented the development history of PToV and made a brief summary of the features in genome and protein epidemiology and laboratory diagnosis of the PToV, and so on.

Equine torovirus (BEV) induces caspase-mediated apoptosis in infected cells.

Toroviruses are gastroenteritis causing agents that infect different animal species and humans. To date, very little is known about how toroviruses cause disease. Here, we describe for the first time that the prototype member of this genus, the equine torovirus Berne virus (BEV), induces apoptosis in infected cells at late times postinfection. Observation of BEV infected cells by electron microscopy revealed that by 24 hours postinfection some cells exhibited morphological characteristics of apoptotic cells. Based on this finding, we analyzed several apoptotic markers, and observed protein synthesis inhibition, rRNA and DNA degradation, nuclear fragmentation, caspase-mediated cleavage of PARP and eIF4GI, and PKR and eIF2α phosphorylation, all these processes taking place after peak virus production. We also determined that both cell death receptor and mitochondrial pathways are involved in the apoptosis process induced by BEV. BEV-induced apoptosis at late times postinfection, once viral progeny are produced, could facilitate viral dissemination in vivo and contribute to viral pathogenesis.

Longitudinal serological and virological study on porcine torovirus (PToV) in piglets from Spanish farms.

A study was performed to evaluate porcine torovirus (PToV) seroprevalence and infection in three multi-site farms from the North-eastern region of Spain. Serum samples from 120 piglets and faecal samples from 36 piglets were longitudinally collected at 1, 3, 7, 11 and 15 weeks of age. Serum samples from their dams (n=30) were also taken 1-week post-farrowing. PToV antibodies in serum were monitored by ELISA, while viral infection was assessed by real-time RT-PCR in faeces. A high seroprevalence (about 100%) was observed in animals older than 11 weeks and in adult sows. Moreover, all 1-week-old animals were seropositive, indicating maternal antibody transference through colostrum. The antibody titers declined to close to or below the ELISA cut-off value by the age of weaning (3 weeks of age). Development of a significant antibody response to PToV occurred before 7 weeks of age in about 50% of piglets, and the remaining animals developed the response by weeks 11 or 15. These results indicate that PToV infection occurred soon after weaning. Although the prevalence of infection in suckling piglets varied among the studied farms, PToV prevalences in 7 and 11-week-old pigs were between 50-67% and 58-75%, respectively, in all farms. Sequencing results indicated that more than one PToV strains were circulating in the studied farms. Present data suggest that PToV was endemic on the studied farms, and provide new insights on the epidemiology of PToV.

Structure, function and evolution of the hemagglutinin-esterase proteins of corona- and toroviruses.

Virus attachment to host cells is mediated by dedicated virion proteins, which specifically recognize one or, at most, a limited number of cell surface molecules. Receptor binding often involves protein-protein interactions, but carbohydrates may serve as receptor determinants as well. In fact, many different viruses use members of the sialic acid family either as their main receptor or as an initial attachment factor. Sialic acids (Sias) are 9-carbon negatively-charged monosaccharides commonly occurring as terminal residues of glycoconjugates. They come in a large variety and are differentially expressed in cells and tissues. By targeting specific Sia subtypes, viruses achieve host cell selectivity, but only to a certain extent. The Sia of choice might still be abundantly present on non-cell associated molecules, on non-target cells (including cells already infected) and even on virus particles themselves. This poses a hazard, as high-affinity virion binding to any of such "false'' receptors would result in loss of infectivity. Some enveloped RNA viruses deal with this problem by encoding virion-associated receptor-destroying enzymes (RDEs). These enzymes make the attachment to Sia reversible, thus providing the virus with an escape ticket. RDEs occur in two types: neuraminidases and sialate-O-acetylesterases. The latter, originally discovered in influenza C virus, are also found in certain nidoviruses, namely in group 2 coronaviruses and in toroviruses, as well as in infectious salmon anemia virus, an orthomyxovirus of teleosts. Here, the structure, function and evolution of viral sialate-O-acetylesterases is reviewed with main focus on the hemagglutinin-esterases of nidoviruses.

Association of enteric shedding of bovine torovirus (Breda virus) and other enteropathogens with diarrhea in veal calves.

OBJECTIVE: To determine the prevalence, fecal shedding pattern, and association of bovine torovirus (BoTV) with diarrhea in veal calves at time of arrival and periodically throughout the first 35 days after their arrival on a veal farm. ANIMALS: 62 veal calves. PROCEDURE: Fecal samples collected on days 0, 4, 14, and 35 after arrival were tested for BoTV by use of ELISA and reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Paired serum samples obtained from blood collected on days 0 and 35 were analyzed for BoTV antibodies with a hemagglutination inhibition assay. Fecal samples were also screened for other enteric pathogens, including rotavirus, coronavirus, and Cryptosporidium spp. RESULTS: Fecal shedding of BoTV was detected in 15 of 62 (24%) calves by use of ELISA and RT-PCR assay, with peak shedding on day 4. A significant independent association between BoTV shedding and diarrhea was observed. In addition, calves shedding > or = 2 enteric pathogens were more likely to have diarrhea than calves shedding < or = 1 pathogen. Calves that were seronegative or had low antibody titers against BoTV (< or = 1:10 hemagglutination inhibition units) at arrival seroconverted to BoTV (> 4-fold increase in titer); these calves were more likely to shed virus than calves that were seropositive against BoTV at arrival. CONCLUSIONS AND CLINICAL RELEVANCE: Shedding of BoTV was strongly associated with diarrhea in neonatal veal calves during the first week after arrival at the farm. These data provide evidence that BoTV is an important pathogen of neonatal veal calves.

Enteric and nasal shedding of bovine torovirus (Breda virus) in feedlot cattle.

OBJECTIVE: To assess fecal and nasal shedding patterns of bovine torovirus (BoTV) in cattle at time of arrival and periodically throughout the first 21 days after arrival at a feedlot. ANIMALS: 57 steers. PROCEDURE: Fecal and nasal-swab samples collected on days 0, 4, 14, and 21 after arrival were tested for BoTV, using ELISA. A subset of samples from calves testing positive and negative for BoTV was analyzed, using reverse transcriptase-polymerase chain reaction (RT-PCR). Paired serum samples were collected on days 0 and 21 and tested for BoTV antibodies, using a hemagglutination inhibition assay. RESULTS: Overall rate of fecal shedding of BoTV was 21 of 57 (37%) by ELISA and 40 of 42 (95%) by RT-PCR with peak shedding on day 4. Diarrhea was more common in calves shedding BoTV than those not shedding the virus (odds ratio, 1.72). Overall rate of nasal shedding of BoTV was 15 of 57 (26%) by ELISA and 42 of 42 (100%) by RT-PCR, with peak shedding on day 0. Specificity of the RT-PCR product was confirmed by sequence analysis. Approximately 93% of the calves seroconverted to BoTV (> 4-fold increase in titer). Differences were not detected between calves shedding BoTV and nonshedders in relation to disease and treatments, perhaps because of the low number of cattle in the study. CONCLUSIONS AND CLINICAL RELEVANCE: This study confirmed BoTV infections in feedlot cattle, including BoTV antigen and viral RNA in nasal secretions, and the shedding pattern during the first 21 days after arrival in a feedlot.

Gastroenteritis viruses: an overview.

Acute gastroenteritis is among the most common illnesses of humankind, and its associated morbidity and mortality are greatest among those at the extremes of age, children and the elderly. In developing countries, gastroenteritis is a common cause of death in children < 5 years that can be linked to a wide variety of pathogens. In developed countries, while deaths from diarrhoea are less common, much illness leads to hospitalization or doctor visits. Much of the gastroenteritis in children is caused by viruses belonging to four distinct families--rotaviruses, caliciviruses, astroviruses and adenoviruses. Other viruses, such as the toroviruses, picobirnaviruses, picornavirus (the Aichi virus), and enterovirus 22, may play a role as well. Viral gastroenteritis occurs with two epidemiologic patterns, diarrhoea that is endemic in children and outbreaks that affect people of all ages. Viral diarrhoea in children is caused by group A rotaviruses, enteric adenoviruses, astroviruses and the caliciviruses; the illness affects all children worldwide in the first few years of life regardless of their level of hygiene, quality of water, food or sanitation, or type of behaviour. For all but perhaps the caliciviruses, these infections provide immunity from severe disease upon reinfection. Epidemic viral diarrhoea is caused primarily by the Norwalk-like virus genus of the caliciviruses. These viruses affect people of all ages, are often transmitted by faecally contaminated food or water, and are therefore subject to control by public health measures. The tremendous antigenic diversity of caliciviruses and short-lived immunity to infection permit repeated episodes throughout life. In the past decade, the molecular characterization of many of these gastroenteritis viruses has led to advances both in our understanding of the pathogens themselves and in development of a new generation of diagnostics. Application of these more sensitive methods to detect and characterize individual agents is just beginning, but has already opened up new avenues to reassess their disease burden, examine their molecular epidemiology, and consider new directions for their prevention and control through vaccination, improvements in food and water quality and sanitary practices.