Prevalence and genomic characterization of rotavirus group A genotypes in piglets from southern highlands and eastern Tanzania.

Animals have been identified as the potential reservoirs of rotavirus group A (RVA) for human infection. However, very little is known regarding the genotype and genomic profiles of circulating RVA in Tanzanian piglets. The rotavirus genetic diversity and genome analysis was assessed among piglets from Southern highlands and Eastern Tanzania. A total of 241 faecal samples were collected from piglets in the regions of Mbeya, Iringa, and Morogoro. RVA was detected and genotyped using reverse transcription polymerase chain reaction (RT-PCR). Sanger dideoxynucleotide cycle sequencing of the viral protein (VP) 4 and VP7 genes was afterwards performed to confirm the RT-PCR results. Selected genotypes were subjected to whole genome sequencing. The overall prevalence of RVA was 35.26% (85/241) in piglets (30.58% in Mbeya, 43.75% in Iringa and 31.16% in Morogoro). Upon genotyping, the G genotypes were G4 (26), G9 (10), G3 (6), G5 (3) and the remaining 40 were untypeable, while the P genotype, were P[6] (35), P[13] (3) and the remaining 47 were untypeable. The G4P[6] were the predominant genotype followed by G3P[6], G3P[13], G4P[13] and G5P[13] were most common genotypes combinations. On phylogenetic analysis, G4 was grouped to lineage V, sublineages VIIa and VIIc, G9 to lineage I, G5 to lineage II, G3 to lineage IV, P[6] to lineage V and sublineage Ic and the P[13] to lineage IV. We revealed amino acid differences between the circulating G4 and the G4 in the ProSystems RCE vaccine used in pigs. The whole genome reveals genomic constellation of G4-P[6]-I1-R1-C1-M1-A8-N1-T1-E1-H1, G5-P[x]-I5-R1-C1-M1-A8-N1-Tx-E1-H1, G3/G4-P[13]/P[6]-Ix-R1-C1-M1-A8-N1-T1-E1-H1, G3-P[6]-Ix-R1-C1-M1-A8-N1-Tx-E1-H1 and G9-P[x]-Ix-R1-C1-M1-Ax-N1-Tx-E1-H1. The VP7 gene of G9, the VP4 gene of P[6] and NSP4 (E1) gene of some genotypes clustered together and closely related to humans origin or porcine-human reassortant strains with nucleotide similarities ranging from 97.90% to 99.74% from neighboring countries, implying possibility intragenogroup reassortment and interspecies transmission. The higher strain diversity observed within the gene segments highlight the importance of genomic analysis and continuous monitoring of RVA genotypes. Further research is needed to determine the risk factors associated with RVA infection in Tanzanian pigs in order to properly design a control program.

Phylogenetic analysis of VP7 and VP4 genes of the most predominant human group A rotavirus G12 identified in children with acute gastroenteritis in Himachal Pradesh, India during 2013-2016.

G12 strains are now considered to be the sixth most prevalent human rotaviruses globally. India has introduced rotavirus vaccine Rotavac® into the national immunization program in 2016 and Himachal Pradesh (HP) is the first state to launch it. During epidemiological rotavirus surveillance in HP, predominance of G12 rotaviruses was observed. This study investigated the genetic variability and evolution of HP G12 strains (n = 15) associated with P-genotypes P[6], P[4], and P[8] identified between 2013 and 2016. Phylogenetic analysis of VP7 gene revealed that all characterized G12 strains clustered in lineage-III and diversified into three subclusters indicating that these strains may have originated from three different ancestral G12 strains. The comparative sequence analysis of HP strains with Rotavac® and Rotarix® vaccine strains revealed various amino acid substitutions in epitope regions of VP7 and VP4 proteins especially at the antibody neutralization sites. Only 12/29 VP7 epitope residues and 2/25 VP4 epitope residues were found to be conserved between HP rotavirus strains and vaccine strains. Both long and short electropherotypes were observed in G12P[4] strains, while a single long electropherotype was observed in G12P[6] strains. Children of ≤11 months were significantly infected with G12 rotaviruses. The frequency of vomiting episodes (≥5/day) was significantly higher in children infected with G12 rotavirus strains as compared to non-G12 rotaviruses (p = 0.0405). Our study provides the comprehensive data on clinical characteristics and evolutionary pattern of the G12 rotavirus, the most prevalent strain in HP and emphasizes the need to monitor these strains for inclusion in future vaccine.

Recombinant Lactobacillus plantarum NC8 strain expressing porcine rotavirus VP7 induces specific antibodies in BALB/c mice.

The major etiologic agent that causes acute gastroenteritis worldwide in young animals and children is Group A rotavirus. Currently, commercially available vaccines do not often prevent porcine rotavirus (PRV) infection. In this study, we evaluated the efficacy of oral recombinant Lactobacillus vaccine against PRV in a mouse model. Lactobacillus plantarum NC8 was used as the host strain, and bacterial vectors were constructed, because the NC8 isolated has shown the capability to survive gastric transit and to colonize the intestinal tract of humans and other mammals. To explore the immunological mechanisms, lactic acid bacterial vectors were used to express VP7 antigen from PRV. We constructed an L. plantarum strain with surface-displayed VP7, named NC8-pSIP409-pgsA-VP7-DCpep. The expressed recombinant protein had a molecular weight of ∼37 kDa. The strain was used to immunize BALB/c mice to evaluate their immunomodulatory characteristics. Mice were orally immunized with recombinant L. plantarum NC8-pSIP409-pgsA-VP7-DCpep at a dose of 2 × 109 colony forming units/200 µl. The results showed that NC8-pSIP409-pgsA-VP7-DCpep significantly stimulated the differentiation of dendritic cells (DCs) in Peyer's patches (PPs) and increased the serum levels of IL-4 and IFN-γ, as measured by enzyme-linked immunosorbent assay in mice treated with NC8-pSIP409-pgsA-VP7-DCpep. Compared to the empty vector group, NC8-pSIP409-pgsA-VP7-DCpep significantly increased the production of B220+ B cells in mesenteric lymph nodes (MLNs) and PPs and also increased the titer levels of the VP7-specific antibodies, including IgG and sIgA. The administration of NC8-pSIP409-pgsA-VP7-DCpep mediated relatively broad cellular responses. This study reveals that clear alternatives exist for PRV control strategies and provides information on PRV infection.

Genetic diversity of G9 rotavirus strains circulating among diarrheic children in North India: A comparison with 116E rotavirus vaccine strain.

The parental rotavirus strain 116E (G9P[11]) used to generate Rotavac® vaccine was isolated in 1986 in New Delhi. Thenceforward, there is no comprehensive report on diversity of G9 rotavirus strains from 116E; therefore, the present study evaluates the VP7 gene sequence diversity of G9 strains (retrieved from GenBank) from different geographical regions (1987-2016). Additionally, 22 recently collected G9 strains from Himachal Pradesh and Delhi (2013-2016) were included in the phylogenetic analysis. Interestingly, unlike 116E which belong to lineage-II all other G9 rotavirus including these 22 samples clustered together in a separate lineage (III). Further, six amino acid substitutions including one novel, K143M (epitope 7-2) different from 116E were detected mostly in the neutralization epitopes of VP7 protein (neutralization escape mutants). Overall, the accumulation of identified substitutions in VP7 epitopes and evolution of G9 strains in India may have impact on Rotavac® efficacy.

Detection and molecular characterization of rotavirus of pigs in Kerala, India.

Group A rotaviruses (GAR) are an important cause of diarrhoea in infants and newborn animals especially pigs. In this paper, we report the detection, G and P typing and phylogenetic analysis of GAR of pigs in Kerala. A total of 100 fecal samples from diarrhoeic piglets were collected from organized farms in Wayanad, Ernakulam, Thrissur, and Palakkad districts of Kerala. The samples were tested for the presence of GAR employing reverse transcriptase polymerase chain reaction (RT-PCR) targeting VP6 gene. Positive samples were tested by G and P genotyping primers and representative amplicons were sequenced. Of the 100 samples, 12 were positive for GAR. The G and P types detected were G2, G4, G5, G6, G9, P[6] and P[19]. An untypable P type (P21-5 like) was also detected. In some of the samples more than one G type was detected. The nucleotide sequences of G2, G4 and G5 types were similar to those seen in pigs and that of G6 was similar to bovine sequences. G9, P[6] and P[19] sequences showed similarity to human rotavirus sequences. The findings of this study provide the first information on the G and P genotypes of GAR of pigs in Kerala.

Genetic Characterizations and Molecular Evolution of VP7 Gene in Human Group A Rotavirus G1.

Rotavirus group A (RVA) G1 is one leading genotype circulating in humans worldwide, and related molecular information from a global perspective is still limited. Here, we present a comprehensive description of the genetic characterizations and molecular evolution of the RVA G1 VP7 gene. Our results show that RVA G1 can be divided into two lineages and multiple sub-lineages with a relatively high genetic diversity. Vaccine strains are phylogenetic, closer to lineage I. The evolutionary rate of the RVA G1 VP7 gene is 8.869 × 10-4 substitutions/site/year, and its most recent common ancestor was in 1933. The RVA G1 VP7 gene shows a linear evolution at the nucleotide level and a linear accumulation of difference at the amino acid level. Sub-lineage replacement of G1 VP7 gene is also observed and the effective population size of the G1 VP7 gene has had great change in the past decades and has remained stable in recent years. Altogether, the RVA G1 VP7 gene constantly evolves and there is no clear evidence that the evolution of the RVA G1 VP7 gene was influenced by vaccines. Continuous surveillance is still indispensable to evaluate the molecular epidemiology of RVA, especially in the post-vaccination era.

G and P genotype profiles of rotavirus A field strains circulating in beef and dairy cattle herds in Brazil, 2006-2015.

The aim of this retrospective study was to use RT-PCR and nucleotide sequencing analysis to determine the G (VP7 gene) and P (VP4 gene) genotypes of 155 Brazilian bovine rotavirus A (RVA) wild-type strains detected in diarrheic calves from all Brazilian geographical regions from 2006 to 2015. The RVA strains evaluated belonged to the G6, G10, P[5], and P[11] genotypes. The G6P[5] genotype was prevalent (65.5%; P < 0.05) in beef, and the G10P[11] (38.4%) and G6P[11] (30.8%) genotypes were more prevalent in dairy cattle herds. The Midwest was the region with the highest number of genotyped RVA strains, where the genotypes G6, P[5], and P[11] were identified. Genotype combination G6-IV/P[5]-IX, prevalent in beef herds, and G6-III/P[11]-III or G10-IV/P[11]-III, prevalent in dairy herds, were detected. In addition, for the first time in Brazil, we detected the P[5] and P[11] genotype RVA strains that belong to lineage II and VII, respectively.

Detection of mutations in the VP7 gene of vaccine-derived strains shed by monovalent rotavirus vaccine recipients.

Strains of Rotarix, a live attenuated monovalent oral rotavirus vaccine, replicate in the intestine and are shed for about one month in immunocompetent recipients. The current study aimed to identify genetic changes of shed strains to reveal any significant mutations and their clinical impact on recipients. Stool samples of recipients of the first dose of Rotarix were sequentially collected for one month from the day of administration. Sequence analyses of the VP7 gene in eight recipients revealed five amino acid substitutions. Among them, two were observed in aa123, which is located in antigenic region 7-1a. Since there were no associated clinical symptoms, the genetic changes were unlikely to have caused reversion of pathogenicity of vaccine strain. Of interest, the virus in one case became closer to wild-type rotavirus via an amino acid change at aa123 occurring 14 days after administration, which might have resulted from multiple replications and long-term shedding of the vaccine strain.

Identification and In Silico Characterization of a Genetically Distinct Avian Rotavirus D Capsid Gene, VP7.

Rotavirus D (RV-D) is gaining importance as a cause of gastroenteritis and runting and stunting syndrome (RSS) in poultry. To date, information is scarce on the molecular analysis of RV-D isolates worldwide. In this study, the VP7 gene, a major constituent of outer capsid structural protein, from a RV-D isolate (UKD48) obtained from Uttarakhand state was analyzed. Phylogenetically, the RV-D isolate was found to be closely related to a South Korean strain, and the nucleotide percent identity varied from 80.4–84.2% with other RV-D strains available globally. Furthermore, domain investigation within 21 aligned amino acid sequences of the VP7 gene affirmed that this gene has several domains: a conserved glycosylation site (N–I–T) having an important role in protein folding; a N-terminal signal peptide (“ITG”) for endoplasmic reticulum retention; and two hydrophobic sites for elucidating transmembrane portions, antigenic structures, and so forth. The findings suggest that the VP7 gene of the Indian RV-D isolate is genetically distinct from those of other avian RV-Ds. Although biological evidence is still needed to prove the functional characteristics of these domains in outer capsid structural proteins, the present study adds new knowledge and derives the need for further investigation.

Longitudinal study of bovine rotavirus group A in newborn calves from vaccinated and unvaccinated dairy herds.

Reports of rotavirus excretion in calves usually result from cross-sectional studies, and in face of the conflicting results regarding protection of calves born to vaccinated dams against diarrhea, the aim of the present study was to evaluate rotavirus excretion in dairy calves born to vaccinated or unvaccinated dams, to identify the genotypes of bovine rotavirus group A (RVA) strains isolated from these animals as well as to investigate characteristics of the disease in naturally occurring circumstances throughout the first month of life. Five hundred fifty-two fecal samples were taken from 56 calves, 28 from each farm and, in the vaccinated herd, 11/281 samples (3.91%) taken from six different calves tested positive for RVA while in the unvaccinated herd, 3/271 samples (1.11%) taken from 3 different calves tested positive. The genotyping of the VP7 genes showed 91.2% nucleotide sequence identity to G6 genotype (NCDV strain), and for the VP4 gene, strains from the vaccinated herd were 96.6% related to B223 strain, while strains from the unvaccinated herd were 88% related to P[5] genotype (UK strain). Genotypes found in this study were G6P[11] in the vaccinated herd and G6P[5] in the unvaccinated herd. All calves infected with rotavirus presented an episode of diarrhea in the first month of life, and the discrepancy between the genotypes found in the commercial vaccine (G6P[1] and G10P[11]) and the rotavirus strains circulating in both vaccinated and unvaccinated herds show the importance of keeping constant surveillance in order to avoid potential causes of vaccination failure.

Zooanthroponotic transmission of rotavirus in Haryana State of Northern India.

Rotaviruses are the major cause of severe gastroenteritis and mortality in young children and animals. Due to segmented nature of dsRNA genome and wide host range, vast genetic and antigenic diversity exists amongst different isolates of rotaviruses. A total of 230 fecal ovine and caprine samples collected from organized farms and villages in Haryana were screened for rotavirus detection. Samples were screened by latex agglutination test and RNA-PAGE followed by RT-PCR and nucleic acid sequencing. The latex agglutination test showed 25 newborn lamb and 4 kid fecal samples positive for rotavirus. However, RNA-PAGE showed only 9 lamb fecal samples positive for rotavirus. All the samples were subjected to RT-PCR employing vp4 and vp7 gene specific primers of group A rotavirus of ovine, bovine and human origin. Only two samples from lamb (Sheep18/Hisar/2013 and Sheep22/Hisar/2013) showed vp4 and vp7 gene specific amplification with human group A rotavirus (GAR) specific primer. However, they did not show any amplification with ovine and bovine rotavirus specific primers. The nucleotide as well as deduced amino acid sequence analysis of vp4 gene of these isolates showed >98/97% and vp7 gene >95/94% nt/aa identity with human GAR from different regions of the world. Based on nucleotide similarity search, Sheep18/Hisar/2013 and Sheep22/Hisar/2013 isolates were genotyped as G1P[8] and G1P[4]. Phylogenetic analysis also confirmed that these isolates were clustered closely with human rotaviruses from different regions of the world. Earlier, higher prevalence of human rotaviruses was reported from the sample collecting area. The amplification of ovine samples with human rotavirus gene specific primers, sequence identity and phylogenetic analysis strongly suggests the zoonotic transmission of human GAR to sheep.

Baculovirus-mediated GCRV vp7 and vp6 genes expression in silkworm and grass carp.

Grass carp hemorrhagic disease is a common fish disease and often results in significant economic losses in grass carp aquaculture in China. This study was aimed to develop a novel oral vaccine against grass carp reovirus (GCRV). GCRV vp6 and vp7 genes with ß-actin promoter of Megalobrama amblycephala and polyhedrin promoter (Ph10) of baculovirus, respectively, were cloned into plasmid pFast™-Dual to construct a vector pFast-PHVP7-AVP6, which was used to generate a recombinant baculovirus BacFish-vp6/vp7 via Bac-to-Bac system. The VP7 expression was analyzed from freeze-dried powder of the BacFish-vp6/vp7-infected silkworm pupae by western blotting, and VP6 expression was analyzed from orally vaccinated fish with the freeze-dried powder by RT-PCR. The VP6 expression was also analyzed from both CIK cells transduced with BacFish-vp6/vp7 and tissues of vaccinated fish by immunofluorescence analysis. Recombinant VP7 could be detected from the BacFish-vp6/vp7-infected silkworm pupae. Pathological changes were not observed in CIK cells transduced with BacFish-vp6/vp7, and VP6 expression was found in CIK cells. When the grass carps were orally administrated with the freeze-dried powder, vp6 gene transcription was found in blood of the vaccinated fishes and VP6 protein was observed in liver and kidney of the vaccinated fish by immunofluorescence analysis. These results indicated that vp7 gene was expressed in the BacFish-vp6/vp7-infected silkworm and vp6 gene was expressed in orally vaccinated fish with freeze-dried powder of the BacFish-vp6/vp7-infected silkworm pupae, suggesting the possibility to use the powder as an orally administrated vaccine.

Phylogenetic inference of the porcine Rotavirus A origin of the human G1 VP7 gene.

Rotavirus A (RVA) is an important cause of acute gastroenteritis in children worldwide. The most common VP7 genotype of human RVA is G1, but G1 is rarely detected in porcine strains. To understand the evolutionary relationships between human and porcine G1 VP7 genes, we sequenced the VP7 genes of three Japanese G1 porcine strains; the first two (PRV2, S80B) were isolated in 1980 and the third (Kyusyu-14) was isolated in 2001. Then, we performed phylogenetic and in-silico structural analyses. All three VP7 sequences clustered into lineage VI, and the mean nucleotide sequence identity between any pair of porcine G1 VP7 sequences belonging to lineage VI was 91.9%. In contrast, the mean nucleotide sequence identity between any pair of human G1 VP7 sequences belonging to lineages I-V was 95.5%. While the mean nucleotide sequence identity between any pair of porcine lineage VI strain and human lineage I-V strain was 85.4%, the VP7 genes of PRV2 and a rare porcine-like human G1P[6] strain (AU19) were 98% identical, strengthening the porcine RVA origin of AU19. The phylogenetic tree suggests that human G1 VP7 genes originated from porcine G1 VP7 genes. The time of their most recent common ancestor was estimated to be 1948, and human and porcine RVA strains evolved along independent pathways. In-silico structural analyses identified 7 amino acid residues within the known neutralisation epitopes that show differences in electric charges and shape between different porcine and human G1 strains. When compared with much divergent porcine G1 VP7 lineages, monophyletic, less divergent human G1 VP7 lineages support the hypothesis that all human G1 VP7 genes included in this study originated from a rare event of a porcine RVA transmitting to humans that was followed by successful adaptation to the human host. By contrast, AU19 represents interspecies transmission that terminated in dead-end infection.

Molecular characterization of group B rotavirus circulating in pigs from India: identification of a strain bearing a novel VP7 genotype, G21.

The occurrence of group B rotavirus (RVB) infections in pigs has been reported from different parts of world. However, such infection in the pig population maintained in Indian farms has not been investigated as yet. A total of 187 faecal specimens were collected from pigs reared in different pig farms/pigsties located in western and northern regions of India and tested for the presence of porcine RVB by amplification of the NSP2 gene using conventional RT-PCR. Nine specimens (4.8%) were shown to contain RVB RNA. N2 and N4 genotypes of NSP2 gene were detected in three and six RVB strains respectively. VP7 (G-type) and NSP5 (H-type) genes of selected six RVB strains were characterized to identify the genotypes. Multiple G (G7, G19 and G20) and H (H4 and H5) genotypes detected in the RVB strains indicated circulation of heterogeneous population of RVB strains in pigs of India. Additionally, one strain was proposed to belong to a novel RVB genotype designated as G21 on account of <80% identity of VP7 gene sequence with its counterpart in RVB strains from 20 established genotypes. Deduced amino acid sequence of VP7 gene also displayed the presence of seven unique substitutions in the strain. The study reports for the first time the occurrence of RVB infections in Indian pig herds and provides important epidemiological data useful for better understanding of ecology and evolution of porcine RVBs.

Severe diarrhea outbreak in beef calves (Bos indicus) caused by G6P[11], an emergent genotype of bovine rotavirus group A / Surto de diarreia neonatal em bezerros de corte (Bos indicus) ocasionado por um genotipo emergente G6P[11] de rotavírus bovino grupo A

The episodes of diarrhea caused by neonatal bovine rotavirus group A (BoRVA) constitute one of the major health problems in the calf rearing worldwide. The main G (VP7) and P (VP4) genotypes of BoRVA strains involved in the etiology of diarrhea in calves are G6P[1], G10P[11], G6P[5], and G8P[1]. However, less frequently, other G and P genotypes have been described in BoRVA strains identified in diarrheic fecal samples of calves. This study describes the identification and molecular characterization of an emerging genotype (G6P[11]) in BoRVA strains involved in the etiology of a diarrhea outbreak in beef calves in a cattle herd of high production in extensive management system. The diarrhea outbreak, which showed high morbidity (60%) and lethality (7%) rates, occurred in calves (n= 384) Nelore (Bos indicus) up to 30-day-old from the State of Mato Grosso do Sul, Brazil. BoRVA was identified in 80% (16/20) of the fecal samples analyzed by polyacrylamide gel electrophoresis (PAGE) technique. In all PAGE-positive fecal samples were amplified products with 1,062-bp and 876-bp in the RT-PCR assays for VP7 (G type) and VP4 (VP8*) (P type) of BoRVA, respectively. The nucleotide sequence analysis of VP7 and VP4 genes of four wild-type BoRVA strains showed G6-III P[11]-III genotype/lineage. The G6P[11] genotype has been described in RVA strains of human and animal hosts, however, in calves this genotype was only identified in some cross-sectional studies and not as a single cause of diarrhea outbreaks in calves with high morbidity and lethality rates as described in this study...

Os episódios de diarreia neonatal ocasionados pelo rotavírus bovino grupo A (BoRVA) constituem-se em um dos principais problemas sanitários na criação de bezerros em todo o mundo. Os principais genotipos G (VP7) e P (VP4) de cepas de BoRVA envolvidos na etiologia da diarreia em bezerros são G6P[1], G10P[11], G6P[5] e G8P[1]. No entanto, com menor frequência, outros genotipos G e P têm sido descritos em cepas de BoRVA identificadas em amostras de fezes diarreicas de bezerros. Este estudo descreve a identificação e caracterização molecular de um genotipo emergente (G6P[11]) em cepas de BoRVA envolvidas na etiologia de um surto de diarreia em bezerros de um rebanho bovino de corte de alta produção em sistema de manejo extensivo. O surto, que apresentou altas taxas de morbidade (60%) e de letalidade (7%), ocorreu em bezerros (n=384) da raça Nelore (Bos indicus) com até 30 dias de idade, provenientes do estado do Mato Grosso do Sul, Brasil. O BoRVA foi identificado em 80% (16/20) das amostras fecais analisadas pela técnica de eletroforese em gel de poliacrilamida (PAGE). Em todas as amostras fecais PAGE-positivas foi possível a amplificação por RT-PCR de produtos com 1.062 pb e 876 pb referentes aos genes VP7 (G tipo) e VP4 (VP8*) (P tipo), respectivamente, de BoRVA. A análise da sequência de nucleotídeos dos genes VP7 e VP4 de quatro cepas de BoRVA demonstrou a presença do genotipo/linhagem G6-III P[11]-III. O genotipo G6P[11] tem sido descrito em cepas de RVA de hospedeiros humanos e animais. Contudo, em bezerros, este genotipo foi apenas identificado em alguns estudos transversais e não como a única causa de surtos de diarreia em bezerros com altas taxas de morbidade e...