Detection and Genetic Characterization of Bovine Torovirus in Uruguay.

Bovine torovirus (BToV) is an enteric pathogen that may cause diarrhea in calves and adult cattle, which could result in economic losses due to weight loss and decreased milk production. This study aimed to report the presence, the genetic characterization and the evolution of BToV in calves in Uruguay. BToV was detected in 7.9% (22/278) of fecal samples, being identified in dairy (9.2%, 22/239) but not beef (0.0%, 0/39) calves. BToV was detected in both diarrheic (14%, 6/43) and non-diarrheic (13.2%, 5/38) dairy calves. In addition, BToV was detected in the intestinal contents of 14.9% (7/47) of naturally deceased dairy calves. A complete genome (28,446 nucleotides) was obtained, which was the second outside Asia and the first in Latin America. In addition, partial S gene sequences were obtained to perform evolutionary analyses. Nucleotide and amino acid substitutions within and between outbreaks/farms were observed, alerting the continuous evolution of the virus. Through Bayesian analysis using BEAST, a recent origin (mid-60s) of BToV, possibly in Asia, was estimated, with two introductions into Uruguay from Asia and Europe in 2004 and 2013, respectively. The estimated evolutionary rate was 1.80 × 10-3 substitutions/site/year. Our findings emphasize the importance of continued surveillance and genetic characterization for the effective management and understanding of BToV's global epidemiology and evolution.

Progress of research on coronaviruses and toroviruses in large domestic animals using reverse genetics systems.

The generation of genetically engineered recombinant viruses from modified DNA/RNA is commonly referred to as reverse genetics, which allows the introduction of desired mutations into the viral genome. Reverse genetics systems (RGSs) are powerful tools for studying fundamental viral processes, mechanisms of infection, pathogenesis and vaccine development. However, establishing RGS for coronaviruses (CoVs) and toroviruses (ToVs), which have the largest genomes among vertebrate RNA viruses, is laborious and hampered by technical constraints. Hence, little research has focused on animal CoVs and ToVs using RGSs, especially in large domestic animals such as pigs and cattle. In the last decade, however, studies of porcine CoVs and bovine ToVs using RGSs have been reported. In addition, the coronavirus disease-2019 pandemic has prompted the development of new and simple CoV RGSs, which will accelerate RGS-based research on animal CoVs and ToVs. In this review, we summarise the general characteristics of CoVs and ToVs, the RGSs available for CoVs and ToVs and the progress made in the last decade in RGS-based research on porcine CoVs and bovine ToVs.

A novel spike subunit 1-based enzyme-linked immunosorbent assay reveals widespread porcine torovirus infection in eastern China.

Toroviruses (ToVs), closely related but genetically distinct from coronaviruses, are known to infect horses, cows, pigs, goats and humans, mainly causing enteritic disorders. However, due to the lack of an adaptive culture system, porcine ToV (PToV) has received less attention. In this study, we developed a novel serological detection method based on the PToV envelope spike subunit 1 (S1) protein for the first time, and compared it to an existing indirect enzyme-linked immunosorbent assay (ELISA) based on the nucleocapsid protein. By using the S1-based ELISA, we carried out the first seroepidemiological survey of PToV in China, assaying both specific IgG and IgA responses in 1,037 serum samples collected from diarrheic pigs in eastern China. There was a relatively high incidence of seropositivity in pigs of different ages, especially one-week-old piglets and sows (78% and 43%), the former probably reflecting maternal antibodies. Furthermore, 3/20 (15%) of faecal samples collected from one PToV-seropositive swine herd in Zhejiang province tested positive by RT-PCR. The complete PToV genome was sequenced from one of these samples, and its phylogenetic relationship with other full-length PToV sequences available in GenBank was determined. Our data provide the first serological evidence for PToV infection in pigs from China, which will help elucidate the potential pathogenicity of PToV in pigs.

Reverse Genetics with a Full-Length Infectious cDNA Clone of Bovine Torovirus.

Historically part of the coronavirus (CoV) family, torovirus (ToV) was recently classified in the new family Tobaniviridae. While reverse genetics systems have been established for various CoVs, none exist for ToVs. Here, we developed a reverse genetics system using an infectious full-length cDNA clone of bovine ToV (BToV) in a bacterial artificial chromosome (BAC). Recombinant BToV harboring genetic markers had the same phenotype as wild-type (wt) BToV. To generate two types of recombinant virus, the hemagglutinin-esterase (HE) gene was edited, as cell-adapted wtBToV generally loses full-length HE (HEf), resulting in soluble HE (HEs). First, recombinant viruses with HEf and hemagglutinin (HA)-tagged HEf or HEs genes were rescued. These exhibited no significant differences in their effect on virus growth in HRT18 cells, suggesting that HE is not essential for viral replication in these cells. Thereafter, we generated a recombinant virus (rEGFP) wherein HE was replaced by the enhanced green fluorescent protein (EGFP) gene. rEGFP expressed EGFP in infected cells but showed significantly lower levels of viral growth than wtBToV. Moreover, rEGFP readily deleted the EGFP gene after one passage. Interestingly, rEGFP variants with two mutations (C1442F and I3562T) in nonstructural proteins (NSPs) that emerged during passage exhibited improved EGFP expression, EGFP gene retention, and viral replication. An rEGFP into which both mutations were introduced displayed a phenotype similar to that of these variants, suggesting that the mutations contributed to EGFP gene acceptance. The current findings provide new insights into BToV, and reverse genetics will help advance the current understanding of this neglected pathogen. IMPORTANCE ToVs are diarrhea-causing pathogens detected in various species, including humans. Through the development of a BAC-based BToV, we introduced the first reverse genetics system for Tobaniviridae. Utilizing this system, recombinant BToVs with a full-length HE gene were generated. Remarkably, although clinical BToVs generally lose the HE gene after a few passages, some recombinant viruses generated in the current study retained the HE gene for up to 20 passages while accumulating mutations in NSPs, which suggested that these mutations may be involved in HE gene retention. The EGFP gene of recombinant viruses was unstable, but rEGFP into which two NSP mutations were introduced exhibited improved EGFP expression, gene retention, and viral replication. These data suggested the existence of an NSP-based acceptance or retention mechanism for exogenous RNA or HE genes. Recombinant BToVs and reverse genetics are powerful tools for understanding fundamental viral processes, pathogenesis, and BToV vaccine development.

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.

Development and use of a reverse transcription insulated isothermal PCR assay for detection and characterization of bovine torovirus in yaks.

Bovine torovirus (BToV) is an important diarrhea-causing pathogen affecting bovines. To facilitate BToV detection, a reverse transcription insulated isothermal PCR (RT-iiPCR) assay was developed that targets the BToV M gene with high specificity and reproducibility. The assay has a limit of detection of 23 copies/µL. Out of 69 diarrheic fecal samples from yaks collected on six farms in Tibet and Sichuan provinces in China, 11.59% (8/69) tested positive for BToV using this assay. The full-length spike (S) and hemagglutinin-esterase (HE) genes of three positive samples were subsequently sequenced. Notably, an identical recombination event was identified in the S1 subunit of the S protein of three isolates. All of the HE genes were found to belong to genotype III and shared the same unique aa variation (P44S) in the esterase domain. This study is the first confirmation of BToV in yaks and the first report of an S gene recombination event in BToV. Our findings will enhance the current understanding of the molecular characteristics and genetic evolution of BToV.

First report and genetic characterization of bovine torovirus in diarrhoeic calves in China.

BACKGROUND: Coronaviruses are notorious pathogens that cause diarrheic and respiratory diseases in humans and animals. Although the epidemiology and pathogenicity of coronaviruses have gained substantial attention, little is known about bovine coronavirus in cattle, which possesses a close relationship with human coronavirus. Bovine torovirus (BToV) is a newly identified relevant pathogen associated with cattle diarrhoea and respiratory diseases, and its epidemiology in the Chinese cattle industry remains unknown. RESULTS: In this study, a total of 461 diarrhoeic faecal samples were collected from 38 different farms in three intensive cattle farming regions and analysed. Our results demonstrated that BToV is present in China, with a low prevalence rate of 1.74% (8/461). The full-length spike genes were further cloned from eight clinical samples (five farms in Henan Province). Phylogenetic analysis showed that two different subclades of BToV strains are circulating in China. Meanwhile, the three BToV strains identified from dairy calves, 18,307, 2YY and 5YY, all contained the amino acid variants R614Q, I801T, N841S and Q885E. CONCLUSIONS: This is the first report to confirm the presence of BToV in beef and dairy calves in China with diarrhea, which extend our understanding of the epidemiology of BToVs worldwide.

Characterization of Self-Processing Activities and Substrate Specificities of Porcine Torovirus 3C-Like Protease.

The 3C-like protease (3CLpro) of nidovirus plays an important role in viral replication and manipulation of host antiviral innate immunity, which makes it an ideal antiviral target. Here, we characterized that porcine torovirus (PToV; family Tobaniviridae, order Nidovirales) 3CLpro autocatalytically releases itself from the viral precursor protein by self-cleavage. Site-directed mutagenesis suggested that PToV 3CLpro, as a serine protease, employed His53 and Ser160 as the active-site residues. Interestingly, unlike most nidovirus 3CLpro, the P1 residue plays a less essential role in N-terminal self-cleavage of PToV 3CLpro Substituting either P1 or P4 residue of substrate alone has little discernible effect on N-terminal cleavage. Notably, replacement of the two residues together completely blocks N-terminal cleavage, suggesting that N-terminal self-cleavage of PToV 3CLpro is synergistically affected by both P1 and P4 residues. Using a cyclized luciferase-based biosensor, we systematically scanned the polyproteins for cleavage sites and identified (FXXQ↓A/S) as the main consensus sequences. Subsequent homology modeling and biochemical experiments suggested that the protease formed putative pockets S1 and S4 between the substrate. Indeed, mutants of both predicted S1 (D159A, H174A) and S4 (P62G/L185G) pockets completely lost the ability of cleavage activity of PToV 3CLpro In conclusion, the characterization of self-processing activities and substrate specificities of PToV 3CLpro will offer helpful information for the mechanism of nidovirus 3C-like proteinase's substrate specificities and the rational development of the antinidovirus drugs.IMPORTANCE Currently, the active-site residues and substrate specificities of 3C-like protease (3CLpro) differ among nidoviruses, and the detailed catalytic mechanism remains largely unknown. Here, porcine torovirus (PToV) 3CLpro cleaves 12 sites in the polyproteins, including its N- and C-terminal self-processing sites. Unlike coronaviruses and arteriviruses, PToV 3CLpro employed His53 and Ser160 as the active-site residues that recognize a glutamine (Gln) at the P1 position. Surprisingly, mutations of P1-Gln impaired the C-terminal self-processing but did not affect N-terminal self-processing. The "noncanonical" substrate specificity for its N-terminal self-processing was attributed to the phenylalanine (Phe) residue at the P4 position in the N-terminal site. Furthermore, a double glycine (neutral) substitution at the putative P4-Phe-binding residues (P62G/L185G) abolished the cleavage activity of PToV 3CLpro suggested the potential hydrophobic force between the PToV 3CLpro and P4-Phe side chains.

First detection and genomic characteristics of bovine torovirus in dairy calves in China.

Bovine torovirus (BToV) is a diarrhea-causing pathogen. In this study, 92 diarrheic fecal samples from five farms in four provinces in China were collected and tested for BToV using a RT-PCR assay, and 21.73% samples were found to be BToV positive. Moreover, two complete BToV genome sequences (MN073058 and MN073059) were obtained from the clinical samples, which were 28,297 and 28,301 nucleotides in length, respectively. Sequence analysis showed that the two isolates shared 10 identical amino acid mutations in the S protein compared to the complete S sequences of BToV available in the GenBank database. In addition, seven consecutive amino acid mutations were found from aa 1,486 to 1,492 in the S protein of isolate MN073058. Moreover, the two isolates shared one identical amino acid mutation in the receptor binding sites of the HE protein. To the best of our knowledge, this is the first report on the epidemic and genomic characterization of BToV in China, which is helpful for further understanding the genetic evolution of BToV.

Complete genome sequencing and genetic analysis of a Japanese porcine torovirus strain detected in swine feces.

We sequenced the complete genome of a porcine torovirus (PToV) strain from Japan for the first time. Whole-genome analysis revealed that this strain (Iba/2018) has a mosaic sequence composed of at least three genome backgrounds, related to US, Chinese and German PToV strains. Clear recombination breakpoints were detected in the M and HE coding regions. A similarity plot and structural analysis demonstrated that the HE coding region exhibits the highest diversity, and the most sequence variation was found in the lectin domain. PToVs were divided into two lineages in the HE region, whereas clear lineages were not found in other regions.

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.

Full-length and defective enterovirus G genomes with distinct torovirus protease insertions are highly prevalent on a Chinese pig farm.

Recombination occurs frequently between enteroviruses (EVs) which are classified within the same species of the Picornaviridae family. Here, using viral metagenomics, the genomes of two recombinant EV-Gs (strains EVG 01/NC_CHI/2014 and EVG 02/NC_CHI/2014) found in the feces of pigs from a swine farm in China are described. The two strains are characterized by distinct insertion of a papain-like protease gene from toroviruses classified within the Coronaviridae family. According to recent reports the site of the torovirus protease insertion was located at the 2C/3A junction region in EVG 02/NC_CHI/2014. For the other variant EVG 01/NC_CHI/2014, the inserted protease sequence replaced the entire viral capsid protein region up to the VP1/2A junction. These two EV-G strains were highly prevalent in the same pig farm with all animals shedding the full-length genome (EVG 02/NC_CHI/2014) while 65% also shed the capsid deletion mutant (EVG 01/NC_CHI/2014). A helper-defective virus relationship between the two co-circulating EV-G recombinants is hypothesized.

Viral enteritis in calves.

A complex community of bacteria, viruses, fungi, protists, and other microorganisms inhabit the gastrointestinal tract of calves and play important roles in gut health and disease. The viral component of the microbiome (the virome) is receiving increasing attention for its role in neonatal calf diarrhea (NCD). Rotavirus and coronavirus have for a long time been associated with NCD and commercial vaccines have been produced against these agents. Recently, several other viruses which may play a role in diarrhea have been discovered in calf fecal samples, mostly by sequence-based methods. These viruses include torovirus, norovirus, nebovirus, astrovirus, kobuvirus, and enterovirus. Most studies have involved epidemiologic investigations seeking to show association with diarrhea for each virus alone or in combination with potential pathogens. However, determining the contribution of these viruses to calf diarrhea has been challenging and much uncertainty remains concerning their roles as primary pathogens, co-infection agents, or commensals.

Entérite virale chez les veaux. Une communauté complexe de bactéries, de virus, de champignons, de protistes et d'autres micro-organismes habitent dans le tube gastro-intestinal des veaux et joue des rôles importants dans la santé et les pathologies du tractus digestif. La composante virale du microbiome (le virome) reçoit de plus en plus d'attention pour son rôle dans la diarrhée néonatale du veau (DNV). Le rotavirus et le coronavirus sont depuis longtemps associés à la DNV et des vaccins ont été produits contre ces agents. Récemment, plusieurs autres virus, qui peuvent jouer un rôle dans la diarrhée, ont été découverts dans des échantillons de fèces des veaux, surtout par des méthodes de séquençage. Ces virus incluent le torovirus, le norovirus, le nébovirus, l'astrovirus, le kobuvirus et l'entérovirus. La plupart des études ont comporté des enquêtes épidémiologiques pour découvrir l'association de chaque virus avec la diarrhée, seul ou en combinaison avec des agents pathogènes potentiels. Cependant, la détermination de la contribution de ces virus à la diarrhée du veau a été difficile et il reste encore beaucoup d'incertitude concernant leurs rôles en tant qu'agents pathogènes primaires, agents de co-infection ou commensaux.(Traduit par Isabelle Vallières).

Rapid and sensitive detection of porcine torovirus by a reverse transcription loop-mediated isothermal amplification assay (RT-LAMP).

Porcine torovirus (PToV) is associated with swine gastroenteritis, but its pathogenesis is uncertain because there is limited information regarding PToV due to its difficulty to adapt in vitro. This study has developed a rapid one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of PToV. A set of four primers specific to six regions within the PToV's highly conserved fragment of the M gene was designed for use with the RT-LAMP assay. The RT-LAMP assay was sensitive with a detection limit of 1 × 10(1)copies/µL, which was 100-fold higher than reverse-transcription PCR. No cross-reaction was observed with other similar viruses. A total of 175 clinical specimens were collected from the Sichuan province, and PToV was detected by the established RT-LAMP assay with a positive rate of 39.2% (69/175). This study developed the first rapid, sensitive, simple, cost-effective and accurate method for the detection of PToV. The results show that the RT-LAMP assay is highly feasible in clinical settings.

Development of a novel detection system for microbes from bovine diarrhea by real-time PCR.

Diarrhea in cattle is one of the most economically costly disorders, decreasing milk production and weight gain. In the present study, we established a novel simultaneous detection system using TaqMan real-time PCR designed as a system for detection of microbes from bovine diarrhea using real-time PCR (referred to as Dembo-PCR). Dembo-PCR simultaneously detects a total of 19 diarrhea-causing pathogens, including viruses, bacteria and protozoa. Specific primer-probe sets were newly designed for 7 pathogens and were synthesized on the basis of previous reports for 12 pathogens. Assays were optimized to react under the same reaction conditions. The PCR efficiency and correlation coefficient (R(2)) of standard curves for each assay were more than 80% and 0.9766, respectively. Furthermore, the sensitivity of Dembo-PCR in fecal sample analysis was measured with feces spiked with target pathogens or synthesized DNA that included specific nucleotide target regions. The resulting limits of detection (LOD) for virus-spiked samples, bacteria and DNA fragments were 0.16-1.6 TCID50 (PFU/reaction), 1.3-13 CFU/reaction and 10-100 copies/reaction, respectively. All reactions showed high sensitivity in pathogen detection. A total of 8 fecal samples, collected from 6 diarrheic cattle, 1 diarrheic calf and 1 healthy cow, were tested using Dembo-PCR to validate the assay's clinical performance. The results revealed that bovine coronavirus had infected all diarrheic adult cattle and that bovine torovirus had infected the diarrheic calf. These results suggest that Dembo-PCR may be a powerful tool for diagnosing infectious agents in cattle diarrhea.

Detection and molecular characterisation of bovine corona and toroviruses from Croatian cattle.

BACKGROUND: Bovine coronavirus (BCoV) together with bovine torovirus (BToV), both members of the Coronaviridae family, order Nidovirales are the most common viral enteric pathogens. Although studied separately, their joint occurrence and the molecular diversity in cattle in Croatia have not been investigated. METHODS: A survey is carried out on 101 fecal samples from diarrheic young and adult cattle during the 3-year period from i) one large dairy herd, ii) four small herds and iii) three nasal and paired fecal samples from calves with symptoms of respiratory disease. Samples were submitted to RT-PCR and sequencing for BCoV Nucleocapsid gene, BCoV Spike gene and BToV Spike gene. RESULTS: BCoV was detected in 78.8 % of fecal samples from symptomatic cattle and three nasal and paired fecal samples from calves with respiratory symptoms. BToV was detected in 43.2 % of fecal samples from symptomatic cattle and a fecal sample from calves with respiratory symptoms. Molecular characterisation of those viruses revealed some nucleotide and aminoacid differences in relation to reference strains. CONCLUSIONS: BToV should be regarded as a relevant pathogen for cattle that plays a synergistic role in mixed enteric infections.

Molecular epidemiology of Porcine torovirus (PToV) in Sichuan Province, China: 2011-2013.

BACKGROUND: Porcine torovirus (PToV) is a member of the genus Torovirus which is responsible for gastrointestinal disease in both human beings and animals with particular prevalence in youth. Torovirus infections are generally asymptomatic, however, their presence may worsen disease consequences in concurrent infections with other enteric pathogens. METHODS: A total of 872 diarrheic fecal samples from pigs of different ages were collected from 12 districts of Sichuan Province in the southwest of China. RT-PCR was done with PToV S gene specific primers to detect the presence of PToV positive samples. M gene specific primers were used with the PToV positive samples and the genes were sequenced. A phylogenetic tree was constructed based on the M gene nucleotide sequences from the 19 selected novel Sichuan strains and 21 PToV and BToV M gene sequences from GenBank. RESULTS: A total of 331 (37.96%, 331/872) samples were found to be positive for PToV and the highest prevalence was observed in piglets aged from 1 to 3 weeks old. Through phylogenetic inference the 40 PToV M gene containing sequences were placed into two genotypes (I & II). The 19 novel Sichuan strains of genotype I showed strong correlations to two Korean gene sequences (GU-07-56-11 and GU-07-56-22). Amino-acid sequence analysis of the 40 PToV M gene strains revealed that the M gene protein was highly conserved. CONCLUSIONS: This study uncovered the presence of PToV in Sichuan Province, and demonstrated the need for continuous surveillance PToV of epidemiology.

Detection of bovine torovirus in fecal specimens from calves with diarrhea in Turkey.

Bovine torovirus (BToV), a member of the family Coronaviridae, is an established gastrointestinal infectious agent in cattle. In this study, we performed a survey to detect BToV in Turkey between 2009 and 2011 using 235 fecal samples from neonatal calves with diarrhea that were analyzed by the nested reverse transcription (RT) PCR method using primers located in the consensus sequences of the BToV membrane (M) gene. The BToV M gene was detected in 4.7 % (11/235) of the samples using the nested RT-PCR method. The nucleotide sequences of partial M fragments from the BToV isolates, including the newly identified Turkish isolates, showed more than 96 % identity. The result indicates that BToV is one of the pathogens that contribute to neonatal calf diarrhea cases in Turkey.

Lineage specific antigenic differences in porcine torovirus hemagglutinin-esterase (PToV-HE) protein.

Hemagglutinin-esterases (HE) are viral envelope proteins present in some members from the toro-, corona- and orthomyxovirus families, all related with enteric and/or respiratory tract infections. HE proteins mediate reversible binding to sialic acid receptor determinants, very abundant glycan residues in the enteric and respiratory tracts. The role of the HE protein during the torovirus infection cycle remains unknown, although it is believed to be important in the natural infection process. The phylogenetic analysis of HE coding sequences from porcine torovirus (PToV) field strains revealed the existence of two distinct HE lineages. In a previous study, PToV virus strains with HE proteins from the two lineages were found coexisting in a pig herd, and they were even obtained from the same animal at two consecutive sampling time points. In this work, we report antigenic differences between the two HE lineages, and discuss the possible implications that the coexistence of viruses belonging to both lineages might have on the spread and sustainment of PToV infection in the farms.

Evolution and homologous recombination of the hemagglutinin-esterase gene sequences from porcine torovirus.

The objective of the present study was to gain new insights into the evolution, homologous recombination, and selection pressures imposed on the porcine torovirus (PToV), by examining the changes in the hemagglutinin-esterase (HE) gene. The most recent common ancestor of PToV was estimated to have emerged 62 years ago based upon HE gene sequence data obtained from PToV isolates originating from Spain, South Korea, Netherlands, Hungary, and Italy and using the HE gene of Bovine torovirus isolates Niigata1 (AB661456) and Niigata3 (AB661458) as outgroups. The HE gene sequence data segregated all the PToV isolates into two well-supported monophyletic groups; however, various isolates from Spain, Italy, and South Korea did not segregate geographically suggesting very recent translocation of the viruses to these localities. Evidence of recombination was observed between two South Korean isolates that partitioned into two distinct subclades. Data further suggest that most of the nucleotides in the HE gene are under negative selection; however, changes within codon 237 showed an evidence of positive selection.