NSP4 Genes Research Articles

Whole Genome Classification and Phylogenetic Analyses of Rotavirus B strains from the United States

Rotaviruses (RVs) are a major etiological agent of acute viral gastroenteritis in humans and young animals, with rotavirus B (RVB) often detected in suckling and weaned pigs. Group A rotavirus classification is currently based on the two outer capsid proteins, VP7 and VP4, and the middle layer protein, VP6. Using RVB strains generated in this study and reference sequences from GenBank, pairwise identity frequency graphs and phylogenetic trees were constructed for the eleven gene segments of RVB to estimate the nucleotide identity cutoff values for different genotypes and determine the genotype diversity per gene segment. Phylogenetic analysis of VP7, VP4, VP6, VP1–VP3, and NSP1–NSP5 identified 26G, 5P, 13I, 5R, 5C, 5M, 8A, 10N, 6T, 4E, and 7H genotypes, respectively. The analysis supports the previously proposed cutoff values for the VP7, VP6, NSP1, and NSP3 gene segments (80%, 81%, 76% and 78%, respectively) and suggests new cutoff values for the VP4, VP1, VP2, VP3, NSP2, NSP4, and NSP5 (80%, 78%, 79%, 77% 83%, 76%, and 79%, respectively). Reassortment events were detected between the porcine RVB strains from our study. This research describes the genome constellations for the complete genome of Group B rotaviruses in different host species.

Emergence of Human G2P[4] Rotaviruses in the Post-vaccination Era in South Korea: Footprints of Multiple Interspecies Re-assortment Events

After the introduction of two global rotavirus vaccines, RotaTeq in 2007 and Rotarix in 2008 in South Korea, G1[P8] rotavirus was the major rotavirus genotype in the country until 2012. However, in this study, an emergence of G2P[4] as the dominant genotype during the 2013 to 2015 season has been reported. Genetic analysis revealed that these viruses had typical DS-1-like genotype constellation and showed evidence of re-assortment in one or more genome segments, including the incorporation of NSP4 genes from strains B-47/2008 from a cow and R4/Haryana/2007 from a buffalo in India, and the VP1 and VP3 genes from strain GO34/1999 from a goat in Bangladesh. Compared to the G2 RotaTeq vaccine strain, 17–24 amino acid changes, specifically A87T, D96N, S213D, and S242N substitutions in G2 epitopes, were observed. These results suggest that multiple interspecies re-assortment events might have contributed to the emergence of G2P[4] rotaviruses in the post-vaccination era in South Korea.

Detection and molecular characterization of two rare G8P[14] and G3P[3] rotavirus strains collected from children with acute gastroenteritis in Japan

This study describes the detection and molecular characterization of two rare G8P[14] and G3P[3] rotavirus strains, which were collected from children with acute gastroenteritis in 2014 in Japan. Among 247 rotaviruses, one G8P[14] (strain 12,597) and one G3P[3] (strain 12,638) rotaviruses were detected. The genotypes of 11 gene segments of these two rotavirus strains (RVA/Human-wt/JPN/12597/2014/G8P[14] and RVA/Human-wt/JPN/12638/2014/G3P[3]) were characterized. The genotype constellation of strain 12,597 was assigned to G8-P[14]-I2-R2-C2-M2-A3-N2-T9-E2-H3, and this strain possessed a rare T9 genotype of NSP3 gene which has never been reported previously in combination with G8 genotype of VP7 gene. Molecular characterization and phylogenetic analysis suggested that the strain 12,597 had the consensus G8P[14] backbone that originated from the rotaviruses of animal origins such as cows, deer, dogs, and cats. The genotype constellation of strain 12,638 was identified as G3-P[3]-I3-R3-C3-M3-A9-N2-T3-E3-H6. The VP7 and VP4 genotypes of strain 12,638 was similar to those of the Cat97-like strains, but the VP1, VP2, and VP3 were closely related to those of the AU-1-like strain. Interestingly, the NSP1 to NSP3 genes shared highest identities with those of a bat rotavirus (RVA/Bat-wt/ZMB/LUS12–14/2012/G3P[3] strain). These findings indicated that the strain 12,638 was an intra-genotype reassortant strain among the AU-1-like strains, the Cat97-like strains and the bat strain. Interestingly, the strains 12,597 and 12,638 possessed the same N2 genotype of NSP2 gene. The results of this study support the possible roles of interspecies transmission and multiple reassortment events for generating the genetic diversity of rotavirus in human.

Equine-like G3 rotavirus strains as predominant strains among children in Indonesia in 2015–2016

Rotavirus A (RVA) is a major cause of acute gastroenteritis in humans and animals worldwide. As a result of the segmented nature of the rotavirus genome, genetic reassortment commonly occurs. This study aims to clarify the genetic characteristics of RVAs circulating in Indonesia. From June 2015 through August 2016, stool samples were collected from 134 children aged <5 years (71 male and 63 female) with acute gastroenteritis who were inpatients at a private hospital in Surabaya, Indonesia. All stool samples were screened for RVA antigen using immunochromatography. Forty-two samples (31.3%, 42/134) were RVA antigen-positive. All RVA positive samples tested showed the unusual combinations of G3P[8] (n = 36) and G3P[6] (n = 3) with a short RNA pattern by G/P typing and polyacrylamide gel electrophoresis (PAGE). Whole genome analysis by next-generation sequencing (NGS) was performed for 11 strains to determine the RVA genotypes. Eleven rotavirus strains were found to carry a DS-like genetic backbone; nine strains showed a G3-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 genome constellation, which was recently reported in Australia, Hungary, Spain and Brazil; as well, two strains showed a G3-P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H2 genome constellation. The phylogenetic tree based on the VP7 gene showed that all 11 strains were classified as equine-like G3, which is genetically distinct and different in origin from typical human G3 strains. The phylogenetic tree based on the NSP4 gene showed that six strains were classified as bovine-like strain and the remaining five were classified as human strain. In conclusion, we identified the strains which are intergenogroup reassortants containing an equine-like G3 VP7, a P[8])/P[6] VP4, with a DS-1-like genetic backbone. These findings suggest that equine-like G3P[8] and P[6] RVA strains have been circulating in the Indonesian population for at least 1 year and probably longer, indicating a diversity of RVAs in this area.

A novel HRM assay for differentiating classical strains and highly pathogenic strains of type 2 porcine reproductive and respiratory syndrome virus

Differentiation of classical strains and highly pathogenic strains of porcine reproductive and respiratory syndrome virus is crucial for effective vaccination programs and epidemiological studies. We used nested PCR and high resolution melting curve analysis with unlabeled probe to distinguish between the classical and the highly pathogenic strains of this virus. Two sets of primers and a 20 bp unlabeled probe were designed from the NSP3 gene. The unlabeled probe included two mutations specific for the classical and highly pathogenic strains of the virus. An additional primer set from the NSP2 gene of the highly pathogenic vaccine strain JXA1-R was used to detect its exclusive single nucleotide polymorphism. We tested 107 clinical samples, 21 clinical samples were positive for PRRSV (consistent with conventional PCR assay), among them four were positive for the classical strain with the remainder 17 for the highly pathogenic strain. Around 10 °C difference between probe melting temperatures showed the high discriminatory power of this method. Among highly pathogenic positive samples, three samples were determined as positive for JXA1-R vaccine-related strain with a 95% genotype confidence percentage. All these genotyping results using the high resolution melting curve assay were confirmed with DNA sequencing. This unlabeled probe method provides an alternative means to differentiate the classical strains from the highly pathogenic porcine reproductive and respiratory syndrome virus strains rapidly and accurately.

Complete genome characterization of a rotavirus B (RVB) strain identified in Alpine goat kids with enteritis reveals inter-species transmission with RVB bovine strains.

Rotavirus B (RVB) has been associated with enteric disease in many animal species. An RVB strain was identified in pooled intestinal samples from Alpine caprine kids (between 2 and 3 days of age) experiencing high (>90 %) morbidity, and the complete caprine RVB genome was characterized. Histology revealed villus atrophy, the samples tested positive for RVB by real-time RT-PCR and metagenomic next-generation sequencing identified only RVB and orf virus. In the VP4 gene segment, the caprine RVB strain had a higher percentage nucleotide identity to the Indian bovine RVB strains than to the Japanese bovine RVB strains, but the VP7, VP6, VP2, NSP1, NSP2 and NSP5 gene segments of the American caprine RVB strain were genetically related to the Japanese bovine RVB strains. The results indicate a lack of RVB sequences to understand reassortment or the evolutionary relationship of RVB strains from cattle and goats.

Species C Rotaviruses in Children with Diarrhea in India, 2010-2013: A Potentially Neglected Cause of Acute Gastroenteritis.

All over the world, children and adults are severely affected by acute gastroenteritis, caused by one of the emerging enteric pathogens, rotavirus C (RVC). At present, no extensive surveillance program is running for RVC in India, and its prevalence is largely unknown except cases of local outbreaks. Here, we intended to detect the presence of RVC in diarrheic children visiting or admitted to hospitals in Haldwani (state of Uttarakhand, India), a city located in the foothills of the Himalayas. During 2010–2013, we screened 119 samples for RVC by an RVC VP6 gene-specific RT-PCR. Of these, 38 (31.93%) were found positive, which is higher than the incidence rates reported so far from India. The phylogenetic analysis of the derived nucleotide sequences from one of the human RVC (HuRVC) isolates, designated as HuRVC/H28/2013/India, showed that the study isolate belongs to genotype I2, P2 and E2 for RVC structural genes 6 and 4 (VP6, and VP4) and non-structural gene 4 (NSP4), respectively. Furthermore, the VP6 gene of HuRVC/H28/2013/India shows the highest similarity to a recently-reported human-like porcine RVC (PoRVC/ASM140/2013/India, KT932963) from India suggesting zoonotic transmission. We also report a full-length NSP4 gene sequence of human RVC from India. Under the One-health platforms there is a need to launch combined human and animal RVC surveillance programs for a better understanding of the epidemiology of RVC infections and for implementing control strategies.

Genetic analysis of rotavirus G2P[4] strains in Pune, Western India: circulation of a novel reassortant bearing E6 NSP4 genotype.

In India, G2P[4] strains are known to be the second most predominant group A rotaviruses causing acute gastroenteritis among children. This study was performed to determine the diversity within VP7(G), VP4(P), VP6(I) and NSP4(E) genes of 16 G2P[4] rotavirus strains detected in children hospitalized for acute gastroenteritis in Pune, Western India during 2009-2013. Fourteen strains showed G2-P[4]-I2-E2 and two strains showed G2-P[4]-I2-E6 genotype constellation. Phylogenetic analysis showed their clustering into G2-IV-a3, P[4]-5bi/ii, I2-3ii and E2-4i/ii or E6 genotypes/lineages. These data reveal inter- and/or intra-genotypic variations in a genogroup-2 constellation of G2P[4] rotavirus strains circulating in Pune, Western India, providing evidence of a novel G2P[4] reassortant bearing a rare NSP4 genotype, E6 during 2009-2013.

Human bocavirus in hospitalized children under 5 years with acute respiratory infection, São Paulo, Brazil, 2010.

The aims of this study were to investigate the human bocavirus (HBoV) frequency and genotypes in hospitalized children <5 years presenting acute respiratory infections (ARI) within the São Paulo metropolitan area. Nasopharyngeal samples from 300 patients, previously screened for common respiratory viruses, were tested by qPCR for the NSP1 and NP-1 genes. The VP1/2 gene in positive samples was then amplified by PCR and sequenced. A total of 49 positive HBoV cases (16.3%; mean Ct value of 34.41) were detected with the mean age being 18.1 months (range 1 month to 5 years) and the median age being 1 year of age. Children aged between 0 and 12 months had higher detection rates of HBoV (69.4%; 34/49; mean Ct = 34.45) than children from other age groups (30.6%; 15/49; mean Ct = 34.34). No significant differences were observed between HBoV Ct levels and clinical illness. The occurrence was more frequently associated with fall (38.8%; 19/49) and spring (36.7%; 18/49). All 12 sequenced isolates were identified as HBoV-1, displaying minor genetic variation compared to the Swedish reference strains ST1 and ST2 (99.1–99.7% nt). The sole identification of HBoV-1 supports the hypothesis that this particular genotype is strongly related to ARI, and contributes to the role of this virus in the aetiology of respiratory diseases.

Analysis of structure-function relationship in porcine rotavirus A enterotoxin gene.

Rotavirus (RV)-infected piglets are presumed to be latent sources of heterologous RV infection in humans and other animals. In RVs, non-structural protein 4 (NSP4) is the major virulence factor with pleiotropic properties. In this study, we analyzed the nsp4 gene from porcine RVs isolated from diarrheic and non-diarrheic cases at different levels of protein folding to explore correlations to diarrhea-inducing capabilities and evolution of nsp4 in the porcine population. Full-length nsp4 genes were amplified, cloned, sequenced, and then analyzed for antigenic epitopes, RotaC classification, homology, genetic relationship, modeling of NSP4 protein, and prediction of post-translational modification. RV presence was observed in both diarrheic and non-diarrheic piglets. All nsp4 genes possessed the E1 genotype. Comparison of primary, secondary, and tertiary structure and the prediction of post-translational modifications of NSP4 from diarrheic and non-diarrheic piglets revealed no apparent differences. Sequence analysis indicated that nsp4 genes have a multi-phyletic evolutionary origin and exhibit species independent genetic diversity. The results emphasize the evolution of the E9 nsp4 genotype from the E1 genotype and suggest that the diarrhea-inducing capability of porcine RVs may not be exclusively linked to its enterotoxin gene.

Phylogenetic comparison of the VP7, VP4, VP6, and NSP4 genes of rotaviruses isolated from children in Nizhny Novgorod, Russia, 2015-2016, with cogent genes of the Rotarix and RotaTeq vaccine strains.

Group A rotaviruses (RVA) are one of the leading causes of gastroenteritis in young children worldwide. The introduction of universal mass vaccination around the world has contributed to a reduction in hospitalizations and outpatient visits associated with rotavirus infection. Continued surveillance of RVA strains is needed to determine long-term effects of vaccine introduction. In the present work, we carried out the analysis of the genotypic diversity of RVA strains isolated in Nizhny Novgorod (Russia) during the 2015-2016 epidemic season. Also we conducted a comparative analysis of the amino acid sequences of T-cell epitopes of wild-type and vaccine (RotaTeq and Rotarix) strains. In total, 1461 samples were examined. RVAs were detected in 30.4% of cases. Rotaviruses with genotype G9P[8] (40.5%) dominated in the 2015-16 epidemic season. Additionally, RVAs with the following genotypes were detected: G4P[8] (25.4%), G1P[8] (13%), G2P[4] (3.2%). Rotaviruses with genotypes G3P[9], G6P[9], and G1P[9] totaled 3%. The number of partially typed and untyped RVA samples was 66 (14.9%). The findings of a RVA of G6P[9] genotype in Russia were an original observation. Our analysis of VP6 and NSP4 T-cell epitopes showed highly conserved amino acid sequences. The found differences seem not to be caused by the immune pressure but were rather related to the genotypic affiliations of the proteins. Vaccination against rotavirus infection is not included in the national vaccination schedule in Russia. Monitoring of the genotypic and antigenic diversity of contemporary RVA will allow providing a comparative analysis of wild-type strains in areas with and without vaccine campaign.

Molecular characterization of a human G20P[28] rotavirus a strain with multiple genes related to bat rotaviruses

Group A rotaviruses are the major cause of severe gastroenteritis in the young of mammals and birds. This report describes characterization of an unusual G20P[28] rotavirus strain detected in a 24month old child from Suriname. Genomic sequence analyses revealed that the genotype constellation of the Suriname strain RVA/Human-wt/SUR/2014735512/2013/G20P[28] was G20-P[28]-I13-R13-C13-M12-A23-N13-T15-E20-H15. Genes VP1, VP2, VP3, NSP1, NSP2, NSP3, NSP4 and NSP5 were recently assigned novel genotypes by the Rotavirus Classification Working Group (RCWG). Three of the 11 gene segments (VP7, VP4, VP6) were similar to cognate gene sequences of bat-like human rotavirus strain Ecu534 from Ecuador and the VP7, NSP3 and NSP5 gene segments of strain RVA/Human-wt/SUR/2014735512/2013/G20P[28] were found to be closely related to gene sequences of bat rotavirus strain 3081/BRA detected in Brazil. Although distantly related, the VP1 gene of the study strain and bat strain BatLi09 detected in Cameroon in 2014 are monophyletic. The NSP1 gene was found to be most closely related to human strain QUI-35-F5 from Brazil. These findings suggest that strain RVA/Human-wt/SUR/2014735512/2013/G20P[28] represents a zoonotic infection from a bat host.

Real-time RT-PCR for Mayaro virus detection in plasma and urine

BackgroundMayaro virus (MAYV) causes an acute febrile illness which can be difficult to differentiate from dengue or chikungunya. MAYV RNA can be detected in plasma during the first 3–5days of illness, but only a single rRT-PCR has been fully evaluated in the literature. ObjectivesTo develop an rRT-PCR for MAYV and evaluate assay performance using human plasma and urine samples spiked with different MAYV strains. Study designA MAYV rRT-PCR targeting a region of the 5′UTR and nsp1 gene was designed from the alignment of all complete-genome MAYV sequences to be compatible with existing laboratory protocols. The assay was evaluated using human samples spiked with six MAYV strains, including strains from each of the three genotypes. ResultsThe linear range of the MAYV rRT-PCR extended from 1.0 to 8.0 log10copies/μL, and the lower limit of 95% detection was 8.2copies/μL. No detection was observed when the MAYV rRT-PCR was tested with genomic RNA from related arboviruses. The assay demonstrated linear amplification of all 6 MAYV strains when spiked into human plasma samples as well as 2 strains spiked into urine. ConclusionsWe report the design and evaluation of an rRT-PCR for MAYV. Given the concern for MAYV emergence in the Americas and the few molecular tests that have been evaluated in the literature, this assay should provide a useful diagnostic for patients with an acute febrile illness.

Disruption of the Opal Stop Codon Attenuates Chikungunya Virus-Induced Arthritis and Pathology.

ABSTRACTChikungunya virus (CHIKV) is a mosquito-borne alphavirus responsible for several significant outbreaks of debilitating acute and chronic arthritis and arthralgia over the past decade. These include a recent outbreak in the Caribbean islands and the Americas that caused more than 1 million cases of viral arthralgia. Despite the major impact of CHIKV on global health, viral determinants that promote CHIKV-induced disease are incompletely understood. Most CHIKV strains contain a conserved opal stop codon at the end of the viral nsP3 gene. However, CHIKV strains that encode an arginine codon in place of the opal stop codon have been described, and deep-sequencing analysis of a CHIKV isolate from the Caribbean identified both arginine and opal variants within this strain. Therefore, we hypothesized that the introduction of the arginine mutation in place of the opal termination codon may influence CHIKV virulence. We tested this by introducing the arginine mutation into a well-characterized infectious clone of a CHIKV strain from Sri Lanka and designated this virus Opal524R. This mutation did not impair viral replication kinetics in vitro or in vivo. Despite this, the Opal524R virus induced significantly less swelling, inflammation, and damage within the feet and ankles of infected mice. Further, we observed delayed induction of proinflammatory cytokines and chemokines, as well as reduced CD4+ T cell and NK cell recruitment compared to those in the parental strain. Therefore, the opal termination codon plays an important role in CHIKV pathogenesis, independently of effects on viral replication.

Optimization and application of a DNA-launched infectious clone of equine arteritis virus.

Reverse genetics is one of the most powerful tools in modern virology. Equine arteritis virus (EAV) is the prototype member of the Equartevirus. In this study, a new reverse genetics system for the recovery of equine arteritis virus from a cDNA plasmid, which contains viral cDNA sequence flanked by hammerhead ribozyme (HamRz) and hepatitis delta virus ribozyme (HdvRz) sequences in both terminals of the viral genome, was developed by optimization of the promoter and terminator regions. Cellular RNA polymerase II drove the transcription of the viral genome. The results showed that the rescued virus (ic-EAV) shared similar morphological and growth characteristics with the wild-type (WT) virus, and could be distinguished from the WT virus via an engineered BspEI restriction site in the nsp3 gene. By using the reverse genetics method established in this study, a G-to-C silent mutation at site 12642 resulted in a significant change in the plaque size of the rescued virus. Moreover, an eGFP-labeled EAV was constructed by introducing the eGFP gene into the infectious clone of EAV, which facilitated the observation of the infection of EAV in target cells. Hence, the newly reverse genetics method of EAV established in this study can be easily manipulated and would be helpful for studying the pathogenic mechanism of EAV.

Molecular characterization of different equine-like G3 rotavirus strains from Germany

The genetic heterogeneity of rotaviruses constitutes a substantial burden to human and animal health. Occasional interspecies transmissions can generate novel virus strains in the human population. We detected equine-like G3P[8] strains in feces sampled from three children in Germany in 2015 and 2016, respectively. Thereof two showed a DS-1-like backbone. In one strain the NSP2 gene segment was of distinct genotype (G3-P[8]-I2-R2-C2-M2-A2-N1-T2-E2-H2). Phylogenetic analyses of the German strains showed a relation to other equine-like G3 rotaviruses circulating in different countries. The reconstruction of reassortment events in the evolution of novel equine-like G3 rotaviruses suggests an independent introduction of the three strains into the local human rotavirus population.

Use of Salmon Cardiac Primary Cultures (SCPCs) of different genotypes for comparative kinetics of mx expression, viral load and ultrastructure pathology, after infection with Salmon Pancreas Disease Virus (SPDV)

In vitro fish based models have been extensively applied in human biomedical research but, paradoxically, less frequently in the research of fish health issues. Farmed Atlantic salmon can suffer from several viral conditions affecting the heart. Therefore, species-specific, cardiac in vitro models may represent a useful tool to help further understanding and management of these diseases. The mechanisms underlying genotype based resistance are complex and usually rely on a combined effect of elements from both the innate and adaptive immune response, which are further complicated by external environmental factors. Here we propose that Salmon Cardiac Primary Cultures (SCPCs) are a useful tool to investigate these mechanisms as the basis for genotypic differences between Atlantic salmon families in susceptibility to cardiotropic viral disease.Using SCPCs produced from two different commercially available Atlantic salmon embryonated ova (Atlantic Ova IPN sensitive” (S) and “Atlantic QTL-innOva® IPN/PD” (R)), the influence of host genotype on the viral load and mx expression following Salmon Pancreas Disease Virus infection was assessed over a 15 day period. Both R and S SCPCs groups were successfully infected. A measurable difference between groups of viral nsP1 and host antiviral mx gene expression was observed (i.e. a later, but larger onset of mx expression in the R group). Mx expression peaks were followed by a decrease in viral nsP1 in both groups. Additionally, ultrastructural examination of infected SCPCs allowed the description of degenerative changes at the individual cell level. The SCPC model presents some advantages, over current fish cell culture monolayers and in vivo material, such as the presence of different cell components normally present in the target organ, as well as the removal of a layer of functional complexity (acquired immunity), making it possible to focus on tissue specific, early innate immune mechanisms. These preliminary results highlight the importance of considering genetic origin when selecting the fish source for the production of SCPCs, as well as their usefulness as screening tools for assessment of genotypic differences in disease resistance.

Rotavirus-encoded virus-like small RNA triggers autophagy by targeting IGF1R via the PI3K/Akt/mTOR pathway

Rotaviruses are double-stranded RNA viruses that are a major cause of viral diarrhea in infants. Examining virus–host cell interaction is important for elucidating mechanisms of virus proliferation in host cells. Viruses can create an environment that promotes their survival and self-proliferation by encoding miRNAs or miRNA-like molecules that target various host cell. However, it remains unclear whether RNA viruses encode viral miRNAs, and their regulation mechanisms are largely unknown. We previously performed deep sequencing analysis to investigate rotavirus-encoded miRNAs, and identified the small RNA molecule Chr17_1755, which we named RV-vsRNA1755. In our present study, we determined that RV-vsRNA1755 is encoded by the rotavirus NSP4 gene and that it targets the host cell IGF1R, which is part of the PI3K/Akt pathway. We further explored the biological characteristics and functions of RV-vsRNA1755.Our results suggest that rotavirus adapts to manipulate PI3K/Akt signaling at early phases of infection. RV-vsRNA1755 targets IGF1R, blockading the PI3K/Akt pathway and triggering autophagy, but it ultimately inhibits autophagy maturation. A mechanism through which rotavirus encodes a virus-like small RNA (RV-vsRNA1755) that triggers autophagy by targeting the host cell IGF1R gene was revealed. These data provide a theoretical basis for therapeutic drug screening targeting RV-vsRNA1755.

Identification of two novel Rotavirus A genotypes, G35 and P[50], from Peruvian alpaca faeces

Rotavirus A (RVA) Alp11B was detected from a neonatal Peruvian alpaca presenting with diarrhea, and the Alp11B VP7, VP4, VP6, NSP4, and NSP5 genes were sequenced. The partial genotype constellation of this strain, RVA/Alpaca-wt/PER/Alp11B/2010, was determined to be G35-P[50]-I13-E16-H6.

Preparation and identification of polyclonal antibodies specific for nsp4 protein of porcine reproductive and respiratory syndrome virus

To obtain specific antibodies against nsp4 protein of porcine reproductive and respiratory syndrome virus (PRRSV), nsp4 gene was amplified by RT-PCR and cloned into pET-28a(+) vector, designated pET28a-nsp4. pET28a-nsp4 was transformed into Escherichia coli Trasseta (DE3) cells and expressed after induction of IPTG. SDS-PAGE analysis showed that the recombinant protein was expressed in soluble form with the molecular weight of 26 kDa. The soluble fusion protein in the supernatant was purified using Ni+-NTA affinity chromatography. New Zealand rabbits were immunized by the purified nsp4 and anti-sera against nsp4 were obtained. The titer of polyclonal antibodies was about 106 and showed good specificity and sensitivity in the immunofluorescence assay and Western blotting analysis. The polyclonal antibodies also recognized native nsp4 form PRRSV infected Marc-145 cells, providing a useful tool in PRRSV replication mechanism study.