Animal noroviruses

Among enteric caliciviruses, noroviruses belong to the genus Norovirus, one of the four accepted genera in the family Caliciviridae. These single-stranded, positive-sense RNA viruses are highly variable both genetically and antigenically. Several animal enteric caliciviruses that are morphologically indistinguishable and genetically closely related to human noroviruses have been identified. The first bovine enteric noroviruses were described in Great Britain and are known as Newbury Agent 2. At least three genetic clusters of porcine noroviruses join together within genogroup II noroviruses. Human noroviruses are the most important cause of acute gastroenteritis illness in people of all ages. In the USA, they are associated with approximately 30-50% of all food-borne outbreaks. Until now, noroviruses have not been associated with gastroenteritis outbreaks in immunocompetent animals. Neither bovine nor porcine noroviruses can replicate in cell culture, although human norovirus can grow in a complex 3D culture system. However, the recently discovered murine noroviruses can replicate in cell culture and are therefore used as model viruses to study human noroviruses. This review focusses on virus classification, virion structure, pathogenesis, epidemiology, immune response and diagnosis of animal noroviruses in comparison with human noroviruses. The classification of animal enteric caliciviruses within the Norovirus genus raises the question of whether transmission from an animal reservoir to humans could occur. Answering this question is important in determining the risk of cross-species infections affecting the epidemiology and evolution of these viruses and so complicating the control of human norovirus infections.     

First detection of porcine norovirus GII.18 in Latin America

Human (Hu) noroviruses (NoVs) circulate worldwide infecting people of all ages in developing and developed countries. Animal NoVs present some antigenic and genetic relationship to HuNoVs, although their zoonotic potential has not been established yet. Among animal NoVs, porcine (Po) NoVs are the most genetically related to HuNoVs. PoNoVs have only been detected in healthy finisher pigs in a few developed countries. Information about them lacks in developing countries. In this study 96 fecal samples from pigs of different ages from five farms in Rio de Janeiro State, Brazil were tested for NoVs. We report detection and genotyping by RT-PCR, nucleotide sequencing and phylogenetic analysis of partial polymerase and capsid regions of viral genome PoNoV genogroup II genotype 18 (GII.18) in one stool sample from a healthy finisher pig. This is the first report of PoNoV detection in Latin America and it supports the assumption that PoNoVs present a worldwide distribution.     

Binding of recombinant norovirus like particle to histo-blood group antigen on cells in the lumen of pig duodenum

Histo-blood group antigens (HBGA) expressed on cells in the human GI tract have been shown to function as receptors for noroviruses. In concordance with earlier reports (Backer et al., 1997; Yamamoto and Yamamoto, 2001), this study found that individual pigs are either HBGA type A positive or type H1 (type O) positive. Recombinant norovirus like particles from a genogroup I (rNVLP) or three genogroup II (rMOH, rVA207, and rVA387) strains bound to plates coated with pig gastro-intestinal washings with similar binding patterns to humans. The binding of human norovirus like particles was inhibited by pre-incubating the wells with MAbs specific for either type A or type H1 HBGA, or by the presence of free HBGAs from human saliva. Co-localization of rNVLP and corresponding HBGA on epithelial cells of pig gastro-intestinal tissue (PGIT) was also observed. These findings suggest that rNVLP binds to HBGAs expressed on PGIT epithelial cells. This is the first report of the specific binding of human rNVLP to HBGAs in epithelial cells of pig gastrointestinal tissue. It highlights the importance of further study of human norovirus incidence and potential infection and residence in non-human animal hosts and suggests the possibility that norovirus may be a zoonotic pathogen.     

Binding of recombinant norovirus like particle to histo-blood group antigen on cells in the lumen of pig duodenum

Histo-blood group antigens (HBGA) expressed on cells in the human GI tract have been shown to function as receptors for noroviruses. In concordance with earlier reports (Backer et al., 1997, Yamamoto and Yamamoto, 2001), this study found that individual pigs are either HBGA type A positive or type H1 (type O) positive. Recombinant norovirus like particles from a genogroup I (rNVLP) or three genogroup II (rMOH, rVA207, and rVA387) strains bound to plates coated with pig gastro-intestinal washings with similar binding patterns to humans. The binding of human norovirus like particles was inhibited by pre-incubating the wells with MAbs specific for either type A or type H1 HBGA, or by the presence of free HBGAs from human saliva. Co-localization of rNVLP and corresponding HBGA on epithelial cells of pig gastro-intestinal tissue (PGIT) was also observed. These findings suggest that rNVLP binds to HBGAs expressed on PGIT epithelial cells. This is the first report of the specific binding of human rNVLP to HBGAs in epithelial cells of pig gastrointestinal tissue. It highlights the importance of further study of human norovirus incidence and potential infection and residence in non-human animal hosts and suggests the possibility that norovirus may be a zoonotic pathogen.     

RT-PCR test for detecting porcine sapovirus in weanling piglets in Hunan Province, China

The prevalence of porcine sapovirus infection in weanling pigs was investigated in Hunan Province, China, between August 2006 and October 2007. A total of 153 diarrheic fecal samples from ten intensive pig farms from ten representative administrative regions in Hunan province were examined for porcine sapoviruses using RT-PCR. Twenty-two of 153 (14.37?%) samples were found to contain porcine sapoviruses. Phylogenetic analysis showed that all the porcine sapovirus isolates in Hunan Province belonged to the porcine sapovirus genogroup III. The results of the present investigation have implications for the control of porcine sapovirus infection in pigs in Hunan Province, China.     

Seroprevalence and molecular detection of porcine sapovirus in symptomatic suckling piglets in Guangdong Province,China

The seroprevalence and genetic identification of sapovirus (SaV) in symptomatic suckling piglets were investigated in Guangdong Province, China, between November 2011 and April 2013. Serum (n?=?960) and diarrheic fecal (n?=?101) samples collected from symptomatic suckling piglets in Guangdong Province were evaluated for antibodies against SaV using indirect enzyme-linked immunosorbent assay (iELISA). The overall seroprevalence of SaV in symptomatic suckling piglets was 61.9 % (594/960). Positive animals were found in all regions with seroprevalence ranging from 52 to 67.8 %, but the difference was not statistically significant (P?>?0.05). In addition, RNA of SaV was extracted from diarrheic fecal samples, and the partial polymerase gene was amplified by RT-PCR and then sequenced. Seven of 101 (6.9 %) samples were found to contain porcine SaV. Phylogenetic analysis showed that all the porcine SaV isolates belong to the porcine SaV genogroup III (GIII). This is the first report of SaV seroprevalence in symptomatic pigs in China.     

Serological investigation of aborted sheep and pigs for infection by Neospora caninum

Serology for Neospora caninum was undertaken using direct ELISAs on sera from 660 aborted sheep and 454 breeding sows, which had aborted or were considered infertile. All ovine sera were further tested by indirect fluorescent antibody test (IFAT) for N. caninum, and a latex agglutination test (LAT) for Toxoplasma gondii was performed on 423 of the samples, including all those positive by ELISA. ELISA-positive porcine sera were tested by IFAT and an inhibition ELISA for antibodies to N. caninum and by LAT for T. gondii. Only 3 (0.45%) of the ovine sera were seropositive for N. caninum by both ELISA and IFAT whereas although 40 porcine sera were seropositive by ELISA all were negative by IFAT. The results suggest that environmental exposure to N. caninum occurs rarely in sheep and pigs.     

Seroprevalence in Household Raised Pigs Indicate High Exposure to GII Noroviruses in Rural Nicaragua

Information about porcine norovirus (PoNoV), genetically similar to human NoV (HuNoV), is limited from rural areas where household‐raised pigs are heavily exposed to faecal material which could facilitate transmission. Histo‐blood group antigens (HBGAs) are known susceptibility factors to NoV in humans and in a germfree piglet model but their role in susceptibility in the porcine population remains unknown. This study reports: (i) the seroprevalence and antibody titres to human norovirus (NoV) VLPs in household raised pigs; (ii) the distribution of HBGAs in relation to NoV IgG antibody titres and further characterization by blocking of GII.4 VLP binding to pig gastric mucins (PGM). The majority of pigs were seropositive to all three VLPs tested (58–70%) with seropositivity and cross‐reactivity increasing significantly with age. However, pig sera could not block the binding of NoV GII.4 VLPs (Dijon) to PGM suggesting no previous infection with this genotype. The majority of the pigs were H‐positive (84%), a susceptibility factor for human infections. IgG antibody titres were however higher in H‐negative (GMT = 247) as compared with H‐positive (GMT = 57) pigs, but after age stratification, this difference in antibody titres was only observed in pigs ≤1 month of age. In conclusion, serological data show that the porcine population of Nicaragua is highly exposed to NoV infections, and the association to HBGAs warrants further investigation.     

Genetic characterisation of antibiotic resistance and virulence factors in vanA-containing enterococci from cattle, sheep and pigs subsequent to the discontinuation of the use of avoparcin

The prevalence of vancomycin resistant-enterococci (VRE) in faecal samples from cattle, sheep and pigs slaughtered for human consumption was evaluated. Enterococci containing the vanA gene were detected in 25.3% and 2.7% of the porcine and ovine samples, respectively, and were identified as Enterococcus faecium. No vanA-containing enterococcal strains were detected in bovine samples. Enterococcal strains with intrinsic vancomycin resistance were detected in seven (9.9%) faecal samples from pigs and in two samples from both cattle and sheep (3.7% and 2.7%, respectively). All vanA-positive isolates from pigs were resistant to tetracycline and erythromycin, and the mobile element Tn916/Tn1545-like transposon was detected in 90.5% of the tetracycline-resistant isolates that contained the tet(M) gene. Although gelatinase and haemolytic activity were not detected, the hyl and cylB virulence genes were found within the VRE strains isolated.     

Experimental induction of malignant catarrhal fever in pigs with ovine herpesvirus 2 by intranasal nebulization

Malignant catarrhal fever (MCF), a frequently fatal herpesviral disease primarily of ruminant species, has been sporadically reported in pigs. All cases of naturally occurring porcine MCF reported to date have been linked to ovine herpesvirus 2 (OvHV-2), a gammaherpesvirus in the genus Macavirus carried by sheep. Experimental induction of MCF by aerosolization of the virus in nasal secretions collected from infected sheep has been successful in bison, cattle and rabbits. The goals of this study were to determine the susceptibility of pigs to MCF following experimental intranasal inoculation of OvHV-2, and to characterize the disease. Twelve pigs in four groups were nebulized with 10(5), 10(6), 10(7), or 10(8) DNA copies of OvHV-2 from sheep nasal secretions. Three control pigs were nebulized with nasal secretions from uninfected sheep. Three additional pigs were inoculated intravenously with 10(7) DNA copies of OvHV-2 to evaluate this route of infection with cell-free virus. Seven of twelve intranasally challenged pigs became infected with OvHV-2. Five of these seven, all in higher dose groups, developed MCF. Lesions resembled those reported in natural cases of porcine MCF. The most striking and consistent histological lesions were in trachea, lung, kidney and brain. These comprised mucopurulent tracheitis, interstitial pneumonia, necrotizing arteritis-periarteritis, and nonpurulent meningoencephalitis. No infection was established in the intravenously challenged or control groups. The study showed that MCF can be experimentally induced in pigs by aerosol challenge using sheep nasal secretions containing OvHV-2. Domestic pigs are a natural clinically susceptible host for sheep-associated MCF. They represent a useful, cost-effective model for MCF research.     

Detection of multiple genotypes of calicivirus infection in asymptomatic swine in Taiwan

Noroviruses (NoVs) and sapoviruses (SaVs) of the family Caliciviridae are emerging enteric pathogens in humans and animals. Recent detection of genogroup II norovirus (GII NoV) RNA from swine raises public health concerns about zoonotic transmission of porcine NoVs to humans. However, few papers reported genotype distributions and epidemiological features in swine farms and their genetic relationship to human strains, which was the objective of our study. This study investigated the epidemiological features and genotypes of caliciviruses in swine farms using 533 pig faecal samples from six farms in central and southern Taiwan, tested for viral RNA using RT-PCR targeting the conserved polymerase gene. NoVs and SaVs were detected with a positive rate of 7.1% and 0.6%, respectively. To confirm the positive rate of NoVs, 255 pig faecal samples from two farms in central Taiwan were tested with primer pairs targeting the partial capsid gene of GII, and 32.3% of the positive rate was found. Furthermore, the results from the capsid region suggested a higher positive rate of 41.7% in winter than 26.4% in summer with statistical significance (P < 0.05). Sequence analysis showed 29 strains belonging to GII.4 (human) and nine strains belonging to GII.11 (swine) identified based on the partial polymerase gene. Additional genotypes clustered with GII.2 (human) and GII.18 (swine) were also characterized based on the partial capsid gene. SaVs detected in porcine faecal samples belonged to genogroup III (GIII), which clustered with the PEC-Cowden strain. Our study demonstrated the presence of multiple genotypes of both human and porcine NoVs infecting swine of various ages asymptomatically. Although the zoonotic potential of detected human NoVs in swine was not conclusive owing to the lack of local human faecal samples, our study revealed the importance of monitoring emerging strains in swine to mitigate the potential impact of recombinant NoVs infecting the human population.     

Detection and characterization of potentially zoonotic viruses in faeces of pigs at slaughter in Germany

Pigs can harbour a variety of viruses in their gastrointestinal tract. Some of them are closely related to human viruses and are therefore suspected to have a zoonotic potential. Only little is known about the presence of those viruses in pigs at slaughter. However, by contamination of meat with zoonotic viruses during the slaughtering process, food-borne transmission to humans may be possible. Here we analyzed 120 faecal samples of pigs at slaughter from 3 different geographical regions of Germany for the presence of astrovirus (AstV), encephalomyocarditis virus (EMCV), hepatitis E virus (HEV), norovirus genogroup II (NoV GII) and group A rotavirus (GARV). Using real-time RT-PCR, the most frequently detected virus was AstV, which was present in 20.8% of the samples, followed by NoV GII with a detection rate of 14.2%. EMCV, HEV and GARV were found only occasionally with detection rates of 4.2%, 2.5% and 0.8%, respectively. Analyses of partial genome sequences of the viruses indicated that the detected AstV and NoV GII mainly represented typical pig virus strains, which have not been detected in humans so far. However, the GARV and HEV strains were more closely related to human strains. The results indicate that enteric viruses, some of them with zoonotic potential, are present in pig faeces at slaughter. Application of good hygiene practice is necessary to minimize the risk of introducing these viruses into the food and to prevent virus transmission to highly exposed persons such as slaughterers and veterinarians.     

Genetic diversity of porcine sapoviruses

Sapoviruses (SaVs) within the Caliciviridae family are an important cause of gastroenteritis in both humans and animals. Although the widespread occurrence of divergent human SaV strains has been reported, there have only been a few studies of porcine SaVs examining their genetic diversity. The aim of this study was to assess the genetic diversity of porcine SaVs in piglets with diarrhea in South Korea. Two hundred and thirty-seven fecal specimens from piglets with diarrhea were examined from 78 farms over a 2-year period from six provinces in South Korea. Overall, 69 (29.1%) of the samples from five provinces tested positive for porcine SaVs by either RT-PCR or nested PCR with the primer pairs specific to porcine SaVs. An analysis of the partial capsid gene (757bp) of 12 porcine SaVs detected from fecal samples showed genetic divergence, not only among the Korean porcine SaVs (67.7-99.1%), but also between Korean and American porcine SaVs (69.1-90.2%). Interestingly, one strain (Po/SaV/JN-MA113/05/Korea), formed a second porcine SaV/GIII genotype, and is tentatively called GIII/2. This strain had a 0.236-0.405 inter-cluster distance with the other strains in the same genogroup, which is comparable to the distances between the established GI and GII SaVs. Furthermore, two potential recombinant porcine SaVs were identified. In conclusion, porcine SaV infections are common in diarrheic piglets in South Korea. The infecting strains are genetically diverse, and include a newly recognized genotype and recombinant viruses within genogroup III.     

Norovirus outbreak in a natural playground: A One Health approach

Norovirus constitutes the most frequently identified infectious cause of disease outbreaks associated with untreated recreational water. When investigating outbreaks related to surface water, a One Health approach is insightful. Historically, there has been a focus on potential contamination of recreational water by bird droppings and a recent publication demonstrating human noroviruses in bird faeces suggested this should be investigated in future water‐related norovirus outbreaks. Here, we describe a One Health approach investigating a norovirus outbreak in a natural playground. On social media, a large amount of waterfowl were reported to defecate near these playground premises leading to speculations about their potential involvement. Surface water, as well as human and bird faecal specimens, was tested for human noroviruses. Norovirus was found to be the most likely cause of the outbreak but there was no evidence for transmission via waterfowl. Cases had become known on social media prior to notification to the public health service underscoring the potential of online media as an early warning system. In view of known risk factors, advice was given for future outbreak investigations and natural playground design.     

Effects of Escherichia coli heat-stable enterotoxin b on small intestinal villi in pigs, rabbits, and lambs

Culture supernates from two strains of E. coli were placed into different ligated intestinal sections (loops) of each animal. The two bacterial strains were identical except that one contained a plasmid carrying the heat-stable toxin b (STb) gene, while the other did not. Morphometric techniques were used to assess villous epithelial surface areas and mucosal volumes in both intestinal segments exposed to STb-positive (test) and to STb-negative (control) supernates. In pigs whose intestines were exposed to STb-positive supernatants for 2 hours, both villous epithelial surface area and mucosal volume were significantly smaller in test loops than in control loops (P less than 0.02). In test loops of pigs incubated for 1 hour, and in test loops of lambs incubated for 2 hours, there was a decrease in villous epithelial surface area which approached the test for significance but did not meet it (0.05 less than P less than 0.10). Rabbit test loops did not differ from rabbit control loops in either villous epithelial surface area or mucosal volume. Histological examination of the tissues from all three species revealed epithelial changes in porcine and ovine tissues only. In porcine and ovine tissues, epithelium at villous tips was seen to be cuboidal or squamous, or even to be absent. Villi with similarly altered epithelium were seen in control loops, but were seen much more frequently in test loops. These epithelial changes were seen as early as 30 minutes of incubation in pigs. Intestinal tissues from these pigs were examined by transmission electron microscopy, but no difference between test and control tissues was seen.(ABSTRACT TRUNCATED AT 250 WORDS)     

检测绵羊源爱知病毒D型荧光RT-PCR方法的建立和应用

绵羊源爱知病毒D型(ovine Aichivirus D)是在国内绵羊中新发现的病毒,本研究根据绵羊源Aichivirus D 3D基因序列设计检测引物,通过反应体系和条件优化,成功建立了检测绵羊源Aichivirus D的TB Green染料法荧光RT-PCR方法,该方法特异性和稳定性良好,灵敏度高。对2020年4月―2021年5月采集自四川6个场253份绵羊粪便样本(健康羊的133份,腹泻羊的120份)进行检测,结果该病毒的平均检出率为8.7％,场阳性率为66.7％。其中,腹泻粪便样本中绵羊源Aichivirus D阳性率(17.5%)显著高于非腹泻粪便样本中绵羊源Aichivirus D阳性率(0.75%,P<0.001)。表明绵羊源Aichivirus D可能是引起以上地区绵羊腹泻的病原。从绵羊源Aichivirus D的阳性样本中成功克隆出大小为1 422 bp的13个完整的绵羊源Aichivirus D 3D基因,其核苷酸相似性为99.4％～100.0％。遗传演化分析发现这13株绵羊源Aichivirus D 3D基因与本实验室前期研究上传的绵羊源Aichivirus D 3D基因共同聚为单独的一个大支。本研究为绵羊源Aichivirus D的分子检测提供了一种新的方法和基础流行病学数据。     

华南地区猪圆环病毒和猪繁殖与呼吸综合征病毒检测分析

为了解华南地区猪圆环病毒及猪繁殖与呼吸综合征病毒的最新流行情况,采集了华南地区11个规模猪场各饲养阶段猪血清807份,用套式PCR(nPCR)检测猪圆环病毒1型(PCV-1)和猪圆环病毒2型(PCV-2);采集了若干猪场2010年1月至2011年8月期间有咳嗽、喘气、消瘦及疑似PDNS等临床症状的猪血清312份,以及无临床症状猪血清104份,以nPCR检测PCV-2,以一步法反转录PCR(RT-PCR)检测猪繁殖与呼吸综合征病毒。结果发现,所调查的11个规模猪场中只有5个检出PCV-1,所有猪场均检出PCV-2。PCV-2阳性率为30.61%,而PCV-1阳性率仅为4.21%;经产母猪和7周龄以上保育猪PCV阳性率最高;有临床症状的猪血清PCV-2阳性率为58.65%,PRRSV阳性率为37.82%,PCV-2阳性猪群中有39.89%的猪同时感染PRRSV;有症状猪群7月～9月的PCV-2感染率最高,而1月～3月最低;无临床症状猪血清PCV-2阳性率为27.9%,PRRSV阳性率为0.96%,PCV-2与PRRSV无混合感染。证明PCV尤其是PCV-2在华南地区仍广泛传播并流行,而且PCV-2与PRRSV混合感染致病情况较多。PCV-2的感染率与季节有一定的相关性,种猪带毒情况严重。