Detection and molecular characterization of porcine type 3 orthoreoviruses circulating in South Korea

Orthoreoviruses infect virtually all mammalian species, causing systemic infections including mild gastrointestinal and respiratory illnesses. However, little is known about the prevalence or genetic diversity of porcine orthoreoviruses in South Korea. We examined 237 diarrheic fecal samples collected from 78 pig farms around the country. RT-PCR utilizing primers specific for the L1 gene of mammalian orthoreoviruses showed that 45 (19.0%) samples were positive. The 10 strains isolated from orthoreovirus-positive samples formed typical perinuclear cytoplasmic inclusion bodies and had an atypical hemagglutination pattern; these are characteristics of type 3 orthoreovirus. Phylogenetic analysis of the S1 gene in these 10 Korean and other strains showed that type 3 orthoreoviruses could be divided into four lineages; the 10 Korean strains were included in porcine lineage IV, along with T3/porcine/Sichuan/2006. Sequence analysis showed that strains in lineage IV had nucleotide identities of 97.0-98.1% and deduced amino acid identities of 96.4-98.2%. Sequence analysis of the σ1 protein, a viral attachment protein, revealed that the amino acid sequences associated with neurotropism (amino acids 198-204, 249I, 350D, and 419E) were highly conserved among the Korean strains, confirming that neural tropism was present. In conclusion, our findings suggest that porcine orthoreovirus infections are endemic in pig farms in South Korea and that the 10 novel Korean porcine orthoreoviruses belong to porcine lineage IV of type 3 orthoreovirus. In addition, sequence analysis of S1 genes encoding the σ1 protein showed that the 9 of 10 Korean porcine orthoreoviruses exhibited neural tropism.     

Detection and genetic characterization of porcine group A rotaviruses in asymptomatic pigs in smallholder farms in East Africa: Predominance of P[8] genotype resembling human strains

Viral enteritis is a serious problem accounting for deaths in neonatal animals and humans worldwide. The absence of surveillance programs and diagnostic laboratory facilities have resulted in a lack of data on rotavirus associated diarrheas in pigs in East Africa. Here we describe the incidence of group A rotavirus (RVA) infections in asymptomatic young pigs in East Africa. Of the 446 samples examined, 26.2% (117/446) were positive for RVA. More nursing piglets (78.7%) shed RVA than weaned (32.9%) and grower (5.8%) pigs. RVA incidence was higher in pigs that were either housed_free-range (77.8%) or tethered_free-range (29.0%) than those that were free-range or housed or housed-tethered pigs. The farms with larger herd size (>10 pigs) had higher RVA prevalence (56.5%) than farms with smaller herd size (24.1–29.7%). This study revealed that age, management system and pig density significantly (p < 0.01) influenced the incidence of RVA infections, with housed_free-range management system and larger herd size showing higher risks for RVA infection. Partial (811–1604 nt region) sequence of the VP4 gene of selected positive samples revealed that different genotypes (P[6], P[8] and P[13]) are circulating in the study area with P[8] being predominant. The P[6] strain shared nucleotide (nt) and amino acid (aa) sequence identity of 84.4–91.3% and 95.1–96.9%, respectively, with known porcine and human P[6] strains. The P[8] strains shared high nt and aa sequence identity with known human P[8] strains ranging from 95.6–100% to 92–100%, respectively. The P[13] strains shared nt and aa sequence identity of 83.6–91.7% and 89.3–96.4%, respectively, only with known porcine P[13] strains. No P[8] strains yielded RNA of sufficient quality/quantity for full genome sequencing. However analysis of the full genome constellation of the P[6], two P[13] and one untypeable strains revealed that the P[6] strain (Ke-003-5) genome constellation was G26-P[6]-I5-R1-C1-M1-A8-N1-T1-E1-H1, P[13] strains (Ug-049 and Ug-453) had G5-P[13]-I5-R1-C1-M1-A8-N1-T7-E1-H1 while the untypeable strain (Ug-218) had G5-P[?]-I5-R1-C1-M1-A8-N1-T1-E1-H? In conclusion, P[6] and P[8] genotypes detected were genetically closely related to human strains suggesting the possibility of interspecies transmission. Further studies are required to determine the role of RVA in swine enteric disease burden and to determine the genetic/antigenic heterogeneity of the circulating strains for development of accurate diagnostic tools and to implement appropriate prophylaxis programs.     

猪轮状病毒的分离鉴定及部分特性研究

从吉林省某猪场发生腹泻的猪群粪便中,用MA-104细胞培养,分离到一株猪轮状病毒,盲传至4代,感染细胞出现明显病变,经电镜观察,见有典型的轮状病毒颗粒.其RNA电泳型为4:2:3:2型,属A群轮状病毒,同时对其蚀斑特性和血凝特性进行了初步研究,这株病毒被命名为JL94株.     

Detection and molecular characterization of calf diarrhoea bovine coronaviruses circulating in South Korea during 2004-2005

Although the widespread occurrence of calf diarrhoea (CD) bovine coronavirus (BCoV) infections have been reported in most cattle producing countries, only the genetic differences in the BCoVs from American and Canadian isolates and/or strains have been identified and compared. Hence, it is unclear if the BCoVs circulating in the other countries have distinct genetic characteristics. The aim of this study was to determine the prevalence and genetic diversity of CD BCoVs based on the deduced amino acid (aa) sequences of the spike (S) and haemagglutinin/esterase (HE) proteins in South Korea. RT-PCR and nested PCR using the primer pairs specific to the nucleocapsid gene, BCoVs detected the BCoVs in 56 (15.6%) of 359 diarrhoeic faecal samples. Phylogenetic analysis of the entire S gene indicated that 10 Korean CD BCoV strains clustered with other Korean BCoV strains with different clinical forms but were different from the American and Canadian BCoV strains. Moreover, the phylogenetic data of the aa sequences of the HE gene revealed all the Korean CD strains to be distinct from the other Korean BCoV strains with different clinical forms. These results suggest that the Korean BCoVs cause endemic infections in diarrhoeic calves in Jeonnam province and have taken a different evolutionary pathway from the BCoVs in other countries. Moreover, the different BCoV strains are circulating in the different clinical forms in South Korea. These results also suggest that vaccines against the BCoVs can be developed with each Korean BCoV in different clinical forms.     

Serological update and molecular characterization of Dirofilaria immitis in dogs,South Korea

Eighty-one dogs in the Chungnam province were tested for heartworm (Dirofilaria immitis) infection by ELISA (SNAP test, IDEXX Laboratories, Maine, USA). Seventeen (20.9%) of the 81 samples were found to be positive and further analyzed by 16S rRNA sequencing. In this study, all dogs tested lived outdoors. Using the χ² test and Fisher’s exact test, no significant differences in the prevalence of dirofilariasis were observed among different gender and age groups, although the prevalence of this disease among dogs 2–4 yrs of age remains highest. Sequence analysis revealed that the species prevalent in Chungnam province were genetically distinct from the type strain of D. immitis based on the nucleotide deletion found at position nt 276 (cytosine) and nucleotide substitution at position 428 (G to A) of the partial 16S rRNA sequence of the type strain. Furthermore, phylogenetic analysis suggests at least 2 groups of D. immitis circulating in the Chungnam area between the year 2007 and 2008.     

Detection of porcine rotavirus by EM, ELISA and CIET

Feces samples from swine-herds with severe problems of neonatal diarrhoea, 3 weeks scours or early weaning diarrhoea were examined for porcine rotavirus using three techniques: EM, ELISA and CIET. Infection with rotavirus was found in about one third of the feces samples, representing more than half of the examined swine-herds. EM and ELISA revealed nearly all the samples found to be positive, while CIET as used here detected only half of them.     

A群猪轮状病毒JS株vp7基因序列分析

根据已发表的猪轮状病毒OSU毒株vp7基因核苷酸序列ORF两端保守区序列,设计一对特异引物,以猪轮状病毒JS毒株反转录cDNA为模板,通过PCR方法扩增出长约1000 bp目的片段。将其进行T-A克隆、序列测定和分析。结果表明,vp7基因全长1062 bp,含有一个981 bp的开放阅读框,编码326个氨基酸。与已知的15个毒株vp7全长基因的核苷酸及推导的氨基酸序列比较,同源性分别为74.5%～78.5%和75.2%～83.1%,核苷酸系统发育进化树结果表明,JS毒株与轮状病毒G9型参考毒株ICB2185、O-1亲缘关系较近,分为一个群,表明JS毒株血清型为G9型。目前,我国尚未见猪及其它动物轮状病毒G9型流行株的报道。     

Impact of porcine group A rotavirus co-infection on porcine epidemic diarrhea virus pathogenicity in piglets

Porcine epidemic diarrhea virus (PEDV) and porcine group A rotavirus (PGAR) are the main causative agents of acute diarrhea in piglets. In South Korea, PGAR is prevalent in piglets naturally infected with PEDV. Piglets naturally co-infected with PEDV and PGAR appeared to have severe and prolonged diarrhea that was distinct from that commonly observed. The aim of this study was to determine the impact of PGAR co-infection on PEDV pathogenicity in piglets. Thirty-six colostrum-deprived, one-day old, Large White-Duroc crossbred pigs were randomly divided into four equal groups: PEDV, PEDV/PGAR, PGAR, and control groups. The piglets were euthanized at 1, 2, or 3 days post-inoculation (DPI) to measure the villous height:crypt depth (VH:CD) ratio and to collect fecal samples for RT-PCR and virus isolation. No significant differences in mean VH:CD ratio and clinical symptoms (diarrhea, vomiting, dehydration, and anorexia) were observed between the PEDV/PGAR-infected and PEDV-infected groups of piglets at 1, 2 and 3 DPI; however, at 2 and 3 DPI, PGAR was detected in all fecal samples by RT-PCR and virus isolation. These findings failed to detect any interaction between PEDV and porcine rotavirus in the small intestines of piglets, suggesting that concurrent infection of PGAR may not synergistically enhance intestinal villous atrophy of piglets with PEDV disease. We propose that the severe diarrhea exhibited in PEDV and PGAR co-infected piglets may be more associated with the immunity level of the host rather than to any synergistic effect of PGAR on PEDV enteritis.     

猪A组轮状病毒VP4全基因在大肠杆菌中的表达与检测

以猪轮状病毒mRNA为模板,应用反转录聚合酶链式反应(RT-PCR)技术,扩增了2331bp的VP4全基因,通过T-A克隆技术,将PCR产物克隆至pGEM-T Vector中,成功地构建出克隆质粒pGEM-T-VP4.以BamHⅠ和SalⅠ双酶切pGEM-T-VP4和pGEX-6P-1,并将纯化的VP4基因亚克隆至融合型表达载体pGEX-6P-1中,构建出原核表达质粒pGEX-6P-VP4.将pGEX-6P-VP4转化至感受态E.coli BL21(DE3)中,经IPTG诱导和SDS-PAGE分析,可见约111.47 ku融合蛋白带.Western blot分析发现,该蛋白具有轮状病毒抗原性,从而为进一步研究轮状病毒的亚单位疫苗及DNA疫苗奠定基础.     

Unexpected detection of porcine rotavirus C strains carrying human origin VP6 gene

Background: Rotavirus C (RVC), a known etiological agent of diarrheal outbreaks, mainly inflicts swine population globally with sporadic incidence in human, cattle, ferret, mink and dog.

Objective: To demonstrate the presence of RVC in Indian swine population and characterization of its selected structural (VP6) and non-structural (NSP4 and NSP5) genes.

Methods: A total of 108 diarrheic samples from different regions of India were used. Isolated RNA was loaded onto polyacrylamide gel to screen for the presence of RVs through the identification of specific electrophoretic genomic migration pattern. To characterize the RVC strains, VP6 gene and NSP4 and NSP5 genes were amplified, sequenced and analyzed.

Results: Based on VP6 gene specific diagnostic RT-PCR, the presence of RVC was confirmed in 12.0% (13/108) piglet fecal specimens. The nucleotide sequence analysis of VP6 gene, encoding inner capsid protein, from selected porcine RVC (PoRVC) strains revealed more than 93% homologies to human RVC strains (HuRVC) of Eurasian origin. These strains were distant from hitherto reported PoRVCs and clustered with HuRVCs, owning I2 genotype. However, the two non-structural genes, i.e. NSP4 and NSP5, of these strains were found to be of swine type, signifying a re-assortment event that has occurred in the Indian swine population.

Conclusion: The findings indicate the presence of human-like RVC in Indian pigs and division of RVC clade with I2 genotype into further sub-clades. To the best of our knowledge, this appears to be the first report of RVC in Indian swine population. Incidence of human-like RVC VP6 gene in swine supports its subsequent zoonotic prospective.     

Full-length genome analysis of G2, G9 and G11 porcine group A rotaviruses

Group A rotaviruses with G2 and G9 VP7 specificity are common in humans, while G11 strains have been detected only sporadically. G2, G9 and G11 rotaviruses also circulate in pigs and swine rotaviruses have been suspected of interspecies and zoonotic transmissions in numerous studies. However, the complete gene constellation of G2 and G9 porcine rotaviruses has not yet been determined. In order to start filling this gap, the genomic make up of two G2, one G9 and one G11 porcine rotavirus strains, detected in Canada in 2005–2007, was determined. With the exception of a G2P[34] strain, with E9 NSP4 type and mixed I5 + I14 VP6 type, the constellation of genomic segments was rather conserved and were closely related to prototype porcine strains in the four viruses characterized (I5-R1-C1-M1-A8-N1-T7-E1-H1). Most notably, all the viruses displayed a rare NSP3 genotype, T7, which has also been identified in rare human reassortant strains and in the reference strain RVA/Cow-tc/GBR/UK/1973/G6P[5]. This study provides crucial genetic data on these complex viruses and will help understand the origin and ecological niche of gene segments and the role played by pigs in their evolution.     

Genetic characterization of porcine circovirus type 2 in Republic of Korea

Porcine circovirus type 2 (PCV2) is a major causative agent of postweaning multisystemic wasting syndrome (PMWS). Sequence and phylogenetic analyses based on the ORF2 capsid protein gene fragment showed that field isolate in Republic of Korea (ROK), PCV2 YJK 0703, was closely related with the PCV2 Fh18 isolate. PCV2 YJK 0703 was genetically distinct and not related to previously reported ROK isolates. Therefore, genotypic variation exists among prevailing PCV2 in ROK. This result suggests that several PCV2 genotypes exist in Korean pig farms.     

Evolution of rotavirus C in humans and several domestic animal species

Rotavirus C (RVC) causes enteric disease in multiple species, including humans, swine, bovines, and canines. To date, the evolutionary relationships of RVC populations circulating in different host species are poorly understood, owing to the low availability of genetic sequence data. To address this gap, we sequenced 45 RVC complete genomes from swine samples collected in the United States and Mexico. A phylogenetic analysis of each genome segment indicates that RVC populations have been evolving independently in human, swine, canine, and bovine hosts for at least the last century, with inter‐species transmission events occurring deep in the phylogenetic tree, and none in the last 100 years. Bovine and canine RVC populations clustered together nine of the 11 gene segments, indicating a shared common ancestor centuries ago. The evolutionary relationships of RVC in humans and swine were more complex, due to the extensive genetic diversity and multiple RVC clades identified in pigs, which were not structured geographically. Topological differences between trees inferred for different genome segments occurred frequently, including at nodes deep in the tree, indicating that RVC's evolutionary history includes multiple reassortment events that occurred a long time ago. Overall, we find that RVC is evolving within host‐defined lineages, but the evolutionary history of RVC is more complex than previously recognized due to the high genetic diversity of RVC in swine, with a common ancestor dating back centuries. Pigs may act as a reservoir host for RVC, and a source of the lineages identified in other species, including humans, but additional sequencing is needed to understand the full diversity of this understudied pathogen across multiple host species.     

Comparison of blood electrolyte and biochemical parameters between single infections of rotavirus and Cryptosporidium parvum in diarrheic Hanwoo calves

BackgroundNeonatal calf diarrhea is a major problem in the cattle industry worldwide. Rotavirus and Cryptosporidium parvum are the primary causative agents, especially during the first three weeks of the calf’s life.ObjectivesThis study investigated the differences in acid-base, electrolytes, and biochemical parameters of diarrheic calves with infection of either rotavirus or C. parvum.MethodsA total of 61 Korean native calves (≤ 20 days old) were divided into two groups based on rotavirus or C. parvum infections: rotavirus infection (n = 44) and C. parvum infection (n = 17). The calves with at a specific blood pH range (pH 6.92–7.25) were chosen for comparison. The acid-base, electrolyte, chemistry, and serum proteins were analyzed, Further, fecal examinations were performed.ResultsCompared to C. parvum-infected calves, the rotavirus-infected calves showed lower levels of total carbon dioxide, bicarbonate (HCO₃ ⁻), anion gap, total protein, and albumin/globulin ratio, and significantly lower levels of potassium, globulin, and α2-globulin (p < 0.05). The C. parvum-infected calves (r = 0.749) had stronger correlations between pH and HCO₃ ⁻ than the rotavirus-infected calves (r = 0.598). Compared to rotavirus-infected calves, strong correlations between globulin and α2-globulin, α2-globulin and haptoglobin were identified in C. parvum-infected calves.ConclusionsThis study is the first to investigate acid-base, electrolyte, and biochemical parameters in calves in response to infections of rotavirus and C. parvum. Although rotavirus and C. parvum cause malabsorptive and secretory diarrhea in similar-aged calves, blood parameters were different. This would help establish the diagnostic and treatment strategies.     

Biologic and molecular characterization of Toxoplasma gondii isolates from pigs from Portugal

Little is known of Toxoplasma gondii infections in animals in Portugal. In the present paper, we report the first isolation of viable T. gondii from pigs in Portugal. Antibodies to T. gondii were found in 52 (15.6%) of 333 pigs prior to slaughter using the modified agglutination test (MAT) at a serum dilution of 1:20. Attempts were made to isolate T. gondii from 37 seropositive pigs. Samples of brain and/or heart from each pig were digested in acid pepsin, and bioassayed into mice. Viable T. gondii was isolated from 15 pigs. Restriction fragment length polymorphism on products of SAG2 locus amplified by PCR and microsatellite analysis revealed that 11 isolates were Type II and four were Type III. The results indicate that phenotypically and genetically T. gondii are similar to isolates from pigs from the U.S.     

Molecular detection and characterization of unclassified bovine enteric caliciviruses in South Korea

The unclassified bovine enteric calicivirus (BEC) is a new bovine enteric calicivirus that is different from bovine norovirus, and causes diarrhea and pathologies in the small intestine of calves. This virus includes Nebraska (NB)- and Newbury agent 1 (NA1)-like strains. The prevalence of this BEC and its genetic characterization has only been reported in the UK and the USA. This study examined the prevalence and genetic diversity of these BECs in diarrheic calves in South Korea. Among a total of 645 diarrheic fecal specimens obtained from 629 cattle herds, these unclassified BECs were detected in 59 (9.1%) diarrheic fecal samples from 57 herds (9.3%) by either RT-PCR or nested PCR. Sequence and phylogenetic analyses of the partial RdRp gene showed that all the Korean BECs clustered together and were closely related to the NB-like viruses (80.9-88.1% nucleotide and 84.5-98.4% amino acid) but not to the NA1-like viruses (75.8-78.4% nucleotide and 79.7-82.8% amino acid). Although these viruses could not be classified into NA1- and NB-like viruses from the sequence and phylogenetic data of the entire capsid gene, all the Korean BECs clustered together on a branch separate from the other known BECs. These results show that these BEC infections are endemic in diarrheic calves in South Korea. The infecting strains are genetically closer to the NB-like viruses but have a distinct evolutionary pathway.     

Descriptive summary of an outbreak of porcine post-weaning multisystemic wasting syndrome (PMWS) in New Zealand

CASE HISTORY: Investigations were conducted to determine the cause of an acute, multi-farm outbreak of porcine respiratory disease that included diarrhoea and subsequent loss of body condition in affected pigs. A definition for post-weaning multisystemic wasting syndrome (PMWS) including both clinical and pathological features, previously developed for the pig industry in New Zealand, was applied to the current outbreak. In addition to self-reporting by owners of affected farms, local veterinarians, disease and epidemiology consultants, and animal health officials from the Ministry of Agriculture and Forestry (MAF) were involved in conducting farm visits and submission of diagnostic specimens.

CLINICAL FINDINGS AND DIAGNOSIS: Pathogens known to be endemic in the pig industry in New Zealand as well as likely exotic diseases were excluded as causative agents of the outbreak. Clinical signs including dyspnoea, diarrhoea, and rapid loss of body condition were consistent with the New Zealand case definition for PMWS. Interstitial pneumonia, pulmonary oedema, generalised lymph-node enlargement, and presence of porcine circovirus type 2 (PCV2) inclusion bodies were consistently identified in affected pigs. Classical swine fever virus (CSFv), Porcine reproductive and respiratory syndrome virus (PRRSv), and Influenza virus were ruled out, using molecular and traditional virological techniques. Spread of the disease between farms was hypothesised to be facilitated by locally migrating flocks of black-backed seagulls. The original source of the disease incursion was not identified.

DIAGNOSIS: Based on the consistent presence of circovirusassociated lesions in lymphoid tissues in combination with generalised enlargement of lymph nodes, histiocytic interstitial pneumonia, clinical wasting, and poor response to antibiotic therapy, a diagnosis of PMWS was made.

CLINICAL RELEVANCE: PMWS should be considered in the differential diagnoses of sudden onset of respiratory dyspnoea, diarrhoea, and rapid loss of body condition in young pigs in New Zealand pig herds.     

猪MEF2C基因慢病毒表达载体构建及C2C12细胞转染条件的优化

MEF2C基因能够控制肌细胞分化过程中的基因转录,特别在骨骼肌、心肌和平滑肌中介导细胞的分化。本研究将猪 MEF2C 基因化学合成后经 Bam HI 和 Asc I 双酶切后克隆到慢病毒表达载体 pLen-ti6.3_MCS_IRES2-EGFP中,获得的重组质粒用限制性内切酶酶切分析和测序鉴定;利用慢病毒阴性对照侵染靶细胞C2C12来确定最佳MOI值以及靶细胞最适抗生素Blasticidin剂量。结果显示猪MEF2C基因成功克隆到慢病毒表达载体中;该重组质粒侵染靶细胞C2C12的最佳MOI值为300;靶细胞抗生素的筛选剂量为4μg/mL,维持剂量为3μg/mL上述试验为建立MEF2C稳定过表达细胞系提供了前期工作基础。