Complete genome sequence of a new-genotype porcine norovirus isolated from piglets with diarrhea

Noroviruses (NoVs) are members of the family Caliciviridae and are emerging enteric pathogens of humans and animals. So far, porcine NoVs have been detected exclusively in fecal samples from adult swine without clinical signs. Here we report the genome sequence of a NoV strain isolated from piglets with diarrhea. Experimental infection of miniature pigs with this porcine NoV-positive fecal sample confirmed that this strain can cause diarrhea in piglets. A phylogenetic tree based on the predicted amino acid sequence of the complete capsid region showed that this strain is separate from known porcine GII strains (GII-11, GII-18, and GII-19), constituting the sole member of a new branch.     

Rapid detection of porcine circovirus type 2 using a TaqMan-based real-time PCR

Porcine circovirus type 2 (PCV2) and the associated disease postweaning multisystemic wasting syndrome (PMWS) have caused heavy losses in global agriculture in recent decades. Rapid detection of PCV2 is very important for the effective prophylaxis and treatment of PMWS. To establish a sensitive, specific assay for the detection and quantitation of PCV2, we designed and synthesized specific primers and a probe in the open reading frame 2. The assay had a wide dynamic range with excellent linearity and reliable reproducibility, and detected between 102 and 1010 copies of the genomic DNA per reaction. The coefficient of variation for Ct values varied from 0.59% to 1.05% in the same assay and from 1.9% to 4.2% in 10 different assays. The assay did not cross-react with porcine circovirus type 1, porcine reproductive and respiratory, porcine epidemic diarrhea, transmissible gastroenteritis of pigs and rotavirus. The limits of detection and quantitation were 10 and 100 copies, respectively. Using the established real-time PCR system, 39 of the 40 samples we tested were detected as positive.     

Isolation and oral immunogenicity assessment of porcine epidemic diarrhea virus NH-TA2020 strain: One of the predominant strains circulating in China from 2017 to 2021

Porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV) is one of the most devastating diseases in the global pig industry due to its high mortality rate in piglets. Maternal vaccines can effectively enhance the gut-mammary gland-secretory IgA axis to boost lactogenic immunity and passive protection of nursing piglets against PEDV challenge. From 2017 to 2021, we collected 882 diarrhea samples from 303 farms in China to investigate the epidemiology of PEDV. The result showed that about 52.15% (158/303) of the farms were positive for PEDV with an overall detection rate of 63.95% (564/882) of the samples. The S1 fragments of S gene from 104 strains were sequenced for the phylogenetic analysis. A total of 71 PEDV strains (68.27%) sequenced in this study were clustered into the predominant G2c subgroup, while the newly-defined G2d strains (9.62%) were identified in three provinces of China. The NH-TA2020 strain of G2c subgroup was isolated and cultured, and its infection to piglets caused watery diarrhea within 24 h, indicating its strong pathogenicity. Oral administration of NH-TA2020 strain to pregnant gilts stimulated high levels of IgA antibody in colostrum. The piglets fed by the gilts above were challenged with NH-TA2020 strain or CH–HeB-RY-2020 strain from G2d subgroup, and the clinical symptoms and virus shedding were significantly reduced compared to the mock group. Our findings suggest that G2c subgroup is the predominant branch circulating in China from 2017 to 2021. Oral administration of NH-TA2020 enhances maternal IgA and lactogenic immune responses, which confer protection against the homologous and emerging G2d PEDV strains challenges in neonates.     

猪细小病毒LDR-PCR检测方法的建立和应用

为准确特异灵敏地检测猪细小病毒(PPV),建立一种新的LDR-PCR方法。首先在病毒的保守区内设计一对LDR探针,LDR探针两端各连有一段引物对应序列,以连接产物为模板进行PCR,琼脂糖凝胶电泳检测结果。以标准质粒为模板,通过对LDR反应的退火温度、连接酶浓度及探针浓度等反应条件进行优化,确定了LDR最佳的反应体系,并建立了LDR-PCR方法。结果表明,可以特异地检测PPV,与猪繁殖与呼吸障碍综合征病毒(PRRSV)、猪瘟病毒(CSFV)、伪狂犬病毒(PRV)、猪圆环病毒(PCV)、猪传染性胃肠炎病毒(TGEV)、猪流行性腹泻病毒(PEDV)无交叉反应;最低检测限为102个拷贝。利用建立的方法对41例临床样本进行检测,14份样品PPV阳性,与普通PCR检测结果符合率为97.6%。     

GII.3型诺如病毒GZ31597株变异情况及与受体组织血型抗原结合模式分析

诺如病毒(Noroviruses,NoVs)是引起非菌型胃肠炎暴发流行的主要病原体之一。为了解我国GII.3型NoVs毒株的变异以及受体结合模式,本研究对来自2015年一起中国广州NoVs胃肠炎暴发的GII.3型毒株GZ31597株进行聚合酶区和完整VP1区基因扩增、序列测定和序列分析,并表达VP1突出区蛋白(P蛋白),通过P蛋白与不同血型唾液样本的酶免疫分析法(EIA)测定实验确定其组织血型抗原(Histo-blood group antigens,HBGAs)结合模式。GZ31597株聚合酶和VP1基因系统进化分析表明,GZ31597株为GII.P12/GII.3-SubD基因型(聚合酶/衣壳区),该毒株较先前的GII.3毒株相比,在既是抗原表位又是HBGAs受体结合位点的氨基酸385残基发生了氨基酸转换。根据Western Blotting结果,证实P蛋白成功表达。唾液结合分析结果显示,该毒株P蛋白与A、B、AB、O型分泌型以及O型非分泌型唾液均可以结合,但结合值相对低。本研究表明该GII.P12/GII.3-SubD亚型的GII.3毒株在长期的流行过程中,通过氨基酸的转换,改变抗原性和受体结合活性,使GII.3型毒株在人群中继续流行。通过探索GII.3型NoVs在人群中长期广泛流行的原因,为GII.3型诺如病毒性胃肠炎的预防和控制提供重要依据。     

Complete Genome Sequence of a Variant Porcine Epidemic Diarrhea Virus Strain Isolated in China

Since October 2010, an outbreak of porcine epidemic diarrhea (PED) has been observed in some provinces of China. Here we report the complete genome sequence of porcine epidemic diarrhea virus (PEDV) strain LC, which was recently isolated from sucking piglets that suffered from severe watery diarrhea in Guangdong. It will help in understanding the epidemiological and molecular characteristics of PEDV in China.     

猪和牛轮状病毒的分离与鉴定

应用MA-104细胞、胰蛋白酶处理培养物和旋转培养技术,成功地从患腹泻仔猪和犊牛的粪便样品中,分离到6株猪轮状病毒和4株牛轮状病毒。经形态学、血清学和病毒RNA电泳分析等鉴定,确为典型轮状病毒。经MA-104细胞连续传代,这10株轮状病毒均成为细胞适应毒株,并在该细胞上产生明显的细胞病变。     

安徽省部分地区2016–2018年猪圆环病毒2型遗传进化分析

【背景】猪圆环病毒(Porcinecircovirus,PCV)可以引起断奶仔猪多系统衰竭综合症(Postweaningmultisystemicwastingsyndrome,PMWS)。【目的】了解安徽省部分地区猪圆环病毒2型(PCV2)的遗传变异情况。【方法】采用PCR技术,对2016-2018年间安徽省部分地区猪场疑似PMWS感染的组织样品共计31份进行PCV2检测和全基因扩增,并通过DNAStar等生物信息学软件对所得到的PCV2毒株基因序列进行遗传进化分析。【结果】所得的8株PCV2安徽株与GenBank上已发表的国内外参考毒株相比较,核苷酸相似性为92.8%-99.0%,ORF2及其推导的氨基酸序列相似性分别为85.8%-99.6%和82.5%-100%。遗传进化树分析结果显示8株安徽株中有1株PCV2a,2株PCV2b,5株PCV2d,未发现PCV2c、PCV2e和PCV2f基因型。此外,通过对ORF2基因编码的氨基酸序列分析,发现各基因型Cap蛋白氨基酸序列上的位点具有其独特性。【结论】近年来PCV2在安徽地区的猪群中感染较为普遍,其中PCV2d基因型的感染病例增多,逐渐成为安徽地区的优势流行株。本研究为安徽地区的PCV2防控提供了一定的参考依据。     

Identification of a novel Cryptosporidium genotype in pigs

Over a 3-year period, a total of 646 fecal samples from pigs in 22 indoor and outdoor herds from Western Australia were screened for Cryptosporidium spp. by microscopy. Results revealed that 39 of 646 samples (6.03%) were positive for Cryptosporidium. Cryptosporidium was much more common in outdoor herds (17.2%) than in indoor herds (0.5%) and was more common in animals between the ages of 5 and 8 weeks (69.2%) than in younger animals (P < 0.0001). Molecular characterization of the positive samples at the 18S ribosomal DNA locus identified two distinct genotypes of Cryptosporidium: the previously identified pig genotype I and a novel pig genotype (pig genotype II), both of which warrant species status.     

猪流行性腹泻病毒功能性受体的鉴定

为研究猪氨基肽酶(Porcine Aminopeptidase N,pAPN)是否作为猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)的细胞感染受体,通过转染技术,使PEDV非容许性细胞MDCK表达pAPN,并用PEDV感染转染细胞。结果发现转染的MDCK细胞可以感染PEDV,并且该病毒可以在转染细胞中连续传代。免疫荧光法鉴定存在病毒抗原。进一步实验证实,抗pAPN血清可以抑制PEDV感染转染的MDCK细胞。这些结果展示转染的MDCK细胞、pAPN表达及PEDV病毒复制之间存在直接联系,证明pAPN是PEDV的细胞感染受体之一。     

Development and Application of an ELISA for the Detection of Porcine Deltacoronavirus IgG Antibodies

Porcine deltacoronavirus (PDCoV), also known as porcine coronavirus HKU15, was first detected in North America in early 2014 and associated with enteric disease in pigs, resulting in an urgent need to further investigate the ecology of this virus. While assays detecting nucleic acids were implemented quickly, assays to detect anti-PDCoV antibodies have not been available. In this study, an indirect anti-PDCoV IgG enzyme-linked immunosorbent assay (ELISA) based on the putative S1 portion of the spike protein was developed and utilized to determine the prevalence of anti-PDCoV IgG in U.S. pigs. The diagnostic sensitivity of the PDCoV ELISA was 91% with a diagnostic specificity of 95%. A total of 968 serum samples were tested including samples with confirmed infection with PDCoV, porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus or porcine respiratory coronavirus. There was no cross-reactivity with any of the other coronaviruses. Among 355 arbitrarily selected serum samples collected in 2014 and originating from 51 farms across 18 U.S. states, anti-PDCoV IgG antibodies were detected in 8.7% of the samples and in 25.5% of the farms whereas anti-PEDV IgG was detected in 22.8% of the samples and in 54.9% of the farms. In addition, anti-PDCoV IgG antibodies were detected in archived samples collected in 2010, perhaps indicating an earlier undetected introduction into the U.S. pig population. Overall, the obtained data suggest that PDCoV seroprevalence in U.S. pigs is lower compared to PEDV and PDCoV may have been introduced to the U.S. prior to PEDV.     

Complete genome sequences of a Korean virulent porcine epidemic diarrhea virus and its attenuated counterpart

A virulent porcine epidemic diarrhea virus (PEDV) strain, DR13, was obtained from suckling pigs suspected of having porcine epidemic diarrhea in 1999 in Korea, and its attenuated counterpart was derived from virulent strain DR13 by serial propagation in Vero cells. This report describes the first complete genome sequences of virulent PEDV and its attenuated counterpart, which will provide important insights into the molecular basis of the attenuation of PEDV.     

猪和牛轮状病毒抗原和抗体的检测

应用ELISA直接双抗体夹心法检查轮状病毒抗原,24份仔猪和29份犊牛的腹泻粪样,分别有12和16份阳性。用病毒RNA电泳分析检查阳性粪样,各出现两种病毒RNA电泳型,用中和试验检查17份成年牛和16份成年猪血清,分别有16和15份病毒抗体阳性。将其与ELISA间接法和结合法进行了比较。     

Complete genome sequence of a novel porcine epidemic diarrhea virus in South china

Since early 2010, outbreaks of porcine epidemic diarrhea (PED) have been observed frequently in immunized swine herds in southern China. The suckling piglets are particularly susceptible to porcine epidemic diarrhea virus (PEDV), with a high mortality rate (90%). Recently, a virulent PEDV strain, GD-A, was isolated from an immunized-swine breeding farm in Guangdong, China. This report describes the complete genome sequence of GD-A, and the data will provide important insights into the variation of PEDV field isolates in southern China.     

Impact of Porcine Epidemic Diarrhea on Performance of Growing Pigs

The impact of porcine epidemic diarrhea virus (PEDv) infection on the US pork industry has mainly been attributed to the mortality that it causes in suckling piglets, and, consequently, much effort has been invested in the quantification of its effect in sow farms. However, no information on the performance of surviving pigs that were exposed to the PEDv as piglets is available. Here, a retrospective cohort study to evaluate the impact of porcine epidemic diarrhea virus (PEDv) infection on growing pigs’ performance, as indicated by mortality, average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR) was performed using production records from weaned pigs in nursery and wean-to-finish sites from sow farms that became PEDv-infected between May 2013 and June 2014. Production records from the first batch of growing pigs weaned in infected flows after the PEDv outbreak (“infected batches”) were compared with those from pigs weaned within the previous 14 to 120 days (“control batches”). Performance records from infected and control batches, paired by flow, were compared using non-parametric paired tests. Mortality, ADG and FCR were significantly different in PEDv-positive (infected) compared with PEDv-negative (control) batches, with a mean increase of mortality and FCR of 11% and 0.5, respectively, and a decrease of ADG of 0.16 lb/day. Our results demonstrate a poorer performance of growing pigs weaned after a PEDv outbreak compared with those weaned within the previous 14-120 days, suggesting that in addition to the mortality induced by PEDv in suckling pigs, the disease also impairs the performance of surviving pig. These findings help to quantify the impact of PEDv infection in the US and, ultimately, contribute to efforts to quantify the cost-effectiveness of disease prevention and control measures.     

A Unique Human Norovirus Lineage with a Distinct HBGA Binding Interface

Norovirus (NoV) causes epidemic acute gastroenteritis in humans, whereby histo-blood group antigens (HBGAs) play an important role in host susceptibility. Each of the two major genogroups (GI and GII) of human NoVs recognizes a unique set of HBGAs through a distinct binding interface that is conserved within a genogroup, indicating a distinct evolutionary path for each genogroup. Here, we characterize a Lewis a (Le^a) antigen binding strain (OIF virus) in the GII.21 genotype that does not share the conserved GII binding interface, revealing a new evolution lineage with a distinct HBGA binding interface. Sequence alignment showed that the major residues contributing to the new HBGA binding interface are conserved among most members of the GII.21, as well as a closely related GII.13 genotype. In addition, we found that glycerol inhibits OIF binding to HBGAs, potentially allowing production of cheap antivirals against human NoVs. Taken together, our results reveal a new evolutionary lineage of NoVs selected by HBGAs, a finding that is important for understanding the diversity and widespread nature of NoVs.     

Aminopeptidase N Knockout Pigs Are Not Resistant to Porcine Epidemic Diarrhea Virus Infection

<正>Dear Editor,Porcine epidemic diarrhea virus(PEDV) is the etiologic agent of porcine epidemic diarrhea(PED), which is an acute, highly contagious, and devastating enteric viral disease in pigs(Lee 2015). PEDV is a coronavirus that mainly infects and replicates in villous enterocytes of the small intestine in pigs(Li et al. 2016). PEDV can infect     

Clinical Characteristics and Molecular Epidemiology of Noroviruses in Outpatient Children with Acute Gastroenteritis in Huzhou of China

BackgroundNoroviruses (NoVs) are considered major causative pathogens associated with the morbidity and mortality of young children with acute gastroenteritis. However, few studies have examined NoVs causing acute diarrhea among outpatient children worldwide. This study was conducted to investigate the clinical features and molecular epidemiology of NoVs in outpatient children with acute gastroenteritis in Huzhou, China, between April 2013 and April 2014.MethodsStool specimens from 1346 outpatient children enrolled (under 5 years of age) with acute gastroenteritis were examined for NoVs by multiplex RT-PCR, and sequences of the partial capsids of NoVs were analyzed phylogenetically, while the relevant clinical data were analyzed statistically.ResultsOf 1346 specimens, 383 (28.5%, 383/1346) were positive for NoVs. The proportion of GII genotypes (26.9%) was significantly higher than that of GI genotypes (1.6%). The GII.4 genotype was the most prevalent of GII genotypes and was clustered into GII.4/Sydney (37.8%) and GII.4/2006b (62.2%), whereas GI strains were clustered into GI.1. Additionally, the younger children (12 to <24 months of age) were more susceptible to NoVs than children in other age groups, and the highest percentage of NoV infections occurred in April 2013. The diarrheal frequency (times/d) and WBC counts of the infected outpatient group with NoVs were significantly higher than were those of the uninfected outpatient group.ConclusionNoVs were confirmed to be the major viral agents responsible for acute gastroenteritis in outpatient children in Huzhou, China, and GII.4/Sydney and GII.4/2006b variants were identified as the predominant strains in this study.     

The epidemiology of clostridium perfringens type a on Ontario swine farms, with special reference to cpb2-positive isolates

ABSTRACT: BACKGROUND: There is poor understanding of most aspects of Clostridium perfringens type A as a possible cause of neonatal diarrhea in piglets, and the prevalence and types of C. perfringens present on Ontario swine farms is unknown. To study the prevalence of fecal C. perfringens and selected toxin genes, 48 Ontario swine farms were visited between August 2010 and May 2011, and 354 fecal samples were collected from suckling pigs, lactating sows, weanling pigs, grower-finisher pigs, and gestating sows, as well as from manure pits. The fecal samples were cultured quantitatively, and toxin genes were detected by real-time multiplex polymerase chain reaction (PCR). RESULTS: In mixed multivariable linear analysis, log10 C. perfringens in fecal samples from suckling pigs were higher than that of weanling pigs, grower-finisher pigs, and manure pit samples (P <0.05). In mixed multivariable logistic analysis, the C. perfringens isolates recovered from lactating sows (OR = 0.069, P <0.001), gestating sows (OR = 0.020, P <0.001), grower-finishers (OR = 0.017, P <0.001), and manure pits (OR = 0.11, P <0.001) were less likely to be positive for the consensus beta2 toxin gene cpb2 compared to the isolates from suckling pigs. The prevalence of cpb2 in the isolates recovered from weanlings did not differ significantly from suckling pigs. C. perfringens isolates that were positive for cpb2 were more likely to carry the atypical cpb2 gene (atyp-cpb2) (OR = 19, P <0.001) compared to isolates that were negative for cpb2. Multivariable analysis did not identify farm factors affecting the presence of consensus cpb2 and atyp-cpb2 genes. CONCLUSIONS: This study provides baseline data on the prevalence of C. perfringens and associated toxin genes in healthy pigs at different stages of production on Ontario swine farms. The study suggests that if C. perfringens type A are involved in neonatal enteritis, there may be strains with specific characteristics that cannot be identified by the existing genotyping system.