Rotavirus Group Research Articles

904. Epidemiology of Rotavirus Group A Mixed Genotypes Isolated from Children with Acute Gastroenteritis over a 15-Year Period

Abstract Background Limited data exist worldwide regarding the genetic epidemiology of mixed G and/or P Rotavirus group A (RVA) strains and their association with demographic and clinical characteristics in children with acute gastroenteritis (AGE). Methods Positive fecal samples for mixed RVA previously G (VP7) and P (VP4) genotyped from children aged ≤16 years with AGE between 2007-2021 (15 years) in Greece were further genotyped for I (VP6) and E (NSP4) using RT-PCR and Sanger sequencing. Demographic, clinical and laboratory data of children infected with a mixed RVA strain were also collected. Results Over the 15-year period, 129/4427 (2.9%) mixed RVA strains were detected. Higher detection rate was observed in 2016 (7.5%) and in 2021 (5.8%) (Figure 1). Children infected with a mixed RVA strain had a median age of 21.1 months (IQR: 56.2) and were predominantly males (85/129, 65.9%). Mixed G genotype were 73/129 (56.6%), mixed P type 54/129 (41.1%) and both G and P mixed genotypes 3/129 (2.3%). In total, 27 different G/P combinations were detected with most prevalent the: G1G4P[8] (47/129, 36.4%), G1P[4]P[8] (18/129, 14%) and G4P[4]P[8] (18/129, 14%) (Figure 2). Higher seasonal peaks were observed for G mixed types in spring (68.8%) and for P mixed types in summer (66.7%). Infection with a G mixed type had a higher relative frequency in children aged 0-12 months, while infection with P mixed type had a higher relative frequency in children aged 13-48 months (p=0.043). I and E typing was successful in 49.6% (64/129) of mixed samples from 2013-2021. Two different I (I1, I2) and three different E (E1, E2, E3) genotypes were detected, with I1 (42.2%, 27/64) and E1 (39.1%, 25/64) being the most common I and E genotypes, respectively. In 31.3% (20/64) of samples, mixed I (I1I1, I1I2) and/or E (E1E1, E1E2) genotypes were also detected. No association has been detected between mixed genotypes and clinical characteristics of the patients. Annual distribution of total and mixed RVA cases in a 15-year period G-P combinations of RVA mixed strains. Other combinations found in a smaller proportion (<2.3%) were: G3P[4]P[8] (1.6%), G4P[8]P[9] (1.6%), G6P[4]P[9] (1.6%), GUDP[4]P[8] (1.6%), G2G4P[4]P[8] (1.6%), G1G2P[4] (0.8 %), G1G3P[9] (0.8%), G1G8P[4]P[8] (0.8%), G10P[8]P[10] (0.8%), G12P[4]P[8] (0.8%), G2P[8]P[10] (0.8%), G2G4P[8] (0.8%), G2G9P[8] (0.8%), G4G12P[4] (0.8%), G8G9P[8] (0.8%), G9P[4]P[8] (0.8%), G9P[6]P[8] (0.8%), G9G12P[8](0.8%). Conclusion These results indicate the great genetic diversity of RVA strains. Significant differences were found in the seasonality of the mixed G and P strains as well as in the age group of children infected with mixed G and P strains. Disclosures All Authors: No reported disclosures

Metatranscriptomic assessment of diarrhoeic faeces reveals diverse RNA viruses in rotavirus group A infected piglets and calves from India.

RNA viruses are a major group contributing to emerging infectious diseases and neonatal diarrhoea, causing morbidity and mortality in humans and animals. Hence, the present study investigated the metatranscriptomic-derived faecal RNA virome in rotavirus group A (RVA)-infected diarrheic piglets and calves from India. The viral genomes retrieved belonged to Astroviridae in both species, while Reoviridae and Picornaviridae were found only in piglets. The nearly complete genomes of porcine RVA (2), astrovirus (AstV) (6), enterovirus G (EVG) (2), porcine sapelovirus (PSV) (2), Aichivirus C (1), and porcine teschovirus (PTV) (1) were identified and characterised. In the piglet, AstVs of PAstV2 (MAstV-26) and PAstV4 (MAstV-31) lineages were predominant, followed by porcine RVA, EVG, PSV, Aichivirus C, teschovirus (PTV-17) in decreasing order of sequence reads. In contrast, AstV accounted for the majority of reads in bovines and belonged to MAstV-28 and a proposed MAstV-35. Both RVA G4P[6] strains exhibited prototype Gottfried strains like a genotypic constellation of G4-P[6]-I1-R1-C1-M1-A8-N1-T1-E1-H1. Ten out of eleven genes were of porcine origin, while the VP7 gene clustered with G4-lineage-1, consisting of human strains, suggesting a natural porcine-human reassortant. In the recombination analysis, multiple recombination events were detected in the PAstV4 and PAstV2 genomes, pointing out that these viruses were potential recombinants. Finally, the study finds diverse RNA virome in Indian piglets and calves for the first time, which may have contributed to diarrhoea. In the future, the investigation of RNA virome in animals will help in revealing pathogen diversity in multifactorial diseases, disease outbreaks, monitoring circulating viruses, viral discovery, and evaluation of their zoonotic potential.

Coinfection of porcine astrovirus and other porcine viruses in diarrheic pigs in Haryana, India.

In this study, 306 rectal swabs from diarrheal pigs of various ages (0-3 weeks, 3-6 weeks, and >6 weeks) were collected from 54 piggery units in different climatic zones in Haryana state, India. These samples were tested for the presence of porcine astrovirus (PAstV), porcine rotavirus group A (PRV-A), and classical swine fever virus (CSFV) by reverse transcription polymerase chain reaction (RT-PCR), and porcine circovirus 2 (PCV-2) by polymerase chain reaction (PCR). Out of the 306 samples tested, 153 (50%), 108 (35.3%), 32 (10.6%), and three (0.9%) tested positive for PAstV, PCV-2, PRV-A, and CSFV, respectively. A single infection was detected in 135 samples, while mixed infections were found in 77 samples: 70 with two viruses and seven samples with more than two. PAstV was detected most frequently (55.31%) in pigs aged 3-6 weeks. PCV-2 wasmore predominant in pigs aged 0-3 weeks (36.53%), whereas PRV-A was more common in pigs aged 3-6 weeks (11.3%). CSFV was observed in the age group of 0-3 weeks (1.92%). Phylogenetic analysis revealed the circulation of lineages 2 and 4 of PAstV in this region. Thus, it can be concluded that one or more than one virus is circulating in piggery units in Haryana, India.

Genome analyses ofspecies A rotavirus isolated from various mammalian hosts inNorthern Ireland during 2013-2016.

Rotavirus group A (RVA) is the most important cause of acute diarrhoea and severe dehydration in young mammals. Infection in livestock is associated with significant mortality and economic losses and, together with wildlife reservoirs, acts as a potential source of zoonotic transmission. Therefore, molecular surveillance of circulating RVA strains in animal species is necessary to assess the risks posed to humans and their livestock. An RVA molecular epidemiological surveillance study on clinically diseased livestock species revealed high prevalence in cattle and pigs (31 per cent and 18 per cent, respectively) with significant phylogenetic diversity including a novel and divergent ovine artiodactyl DS-1-like constellation G10-P[15]-I2-R2-C2-M2-A11-N2-T6-E2-H3. An RVA gene reassortment occurred in an RVA asymptomatic pig and identified as a G5-P[13] strain, and a non-structural protein (NSP)2 gene had intergenomically reassorted with a human RVA strain (reverse zoonosis) and possessed a novel NSP4 enterotoxin E9 which may relate to the asymptomatic RVA infection. Analysis of a novel sheep G10-P[15] strain viral protein 4 gene imparts a putative homologous intergenic and interspecies recombination event, subsequently creating the new P[15] divergent lineage. While surveillance across a wider range of wildlife and exotic species identified generally negative or low prevalence, a novel RVA interspecies transmission in a non-indigenous pudu deer (zoo origin) with the constellation of G6-P[11]12-R2-C2-M2-A3-N2-T6-E2-H3 was detected at a viral load of 11.1 log10 copies/gram. The detection of novel emerging strains, interspecies reassortment, interspecies infection, and recombination of RVA circulating in animal livestock and wildlife reservoirs is of paramount importance to the RVA epidemiology and evolution for the One Health approach and post-human vaccine introduction era where highly virulent animal RVA genotypes have the potential to be zoonotically transmitted.

Genotypic shift in rotavirus associated with neonatal outbreaks in Seoul, Korea.

Rotavirus group A (RVA) is a causative agent of acute gastroenteritis among young children worldwide, despite the global expansion of rotavirus vaccination. In Korea, although the prevalence of RVA has been reduced among young children owing to vaccination, nosocomial infections still occur among neonates. The aim of this study was to investigate the molecular epidemiology of RVA strains associated with several neonatal outbreaks in Seoul from 2017 to 2020. Clinical and environmental samples were collected and screened for the presence of RVA using ELISA and PCR targeting VP6, respectively. RVA-positive strains were genotyped via RT-PCR and subsequent sequencing of VP4 and VP7 and were phylogenetically compared with RVA strains from other countries. During 2017-2020, a total of 15 RVA outbreaks occurred at neonatal facilities (six in hospital neonatal wards and nine in postpartum care centers) in Seoul, and only two RVA genotypes were detected: G4P[6] and G8P[6]. G8P[6] emerged in Seoul November 2018 and immediately became the predominant genotype among neonates, at least up to 2020. Phylogenetic analysis revealed that the G8P[6] genotype in this study was closely related to G8P[6] strains first identified in Korea in 2017, but differed from G8P[6] strains detected in Africa. A novel G8P[6] genotype of RVA strains has emerged and caused outbreaks among neonates in Seoul. Continued surveillance for circulating RVA genotypes is imperative to monitor genotype changes and their potential risks to public health.

English

Rotavirus group A (RVA) causes neonatal calves diarrhoea, causing massive economic losses in many countries due to deaths, treatment costs, and stunted growth. However, little is known about Tanzanian cattle's RVA burden and genotypes. The prevalence and genetic diversity of rotaviruses circulating in cattle in Southern and Eastern Tanzania regions were investigated. A total of 272 faecal samples collected from calves (102 from Mbeya, 89 from Iringa, and 81 from Morogoro) were tested for RVA. 30 (11.03%) of the 272 samples tested positive for RVA, 7 (6.8%) of the 30 RVA positive samples were from Mbeya, 23 (28.39%) from Morogoro, and no positive samples were found in the Iringa region. Using RT-PCR, 20 samples were identified as G10 genotypes, while the remaining 10 samples were untypeable with the primers used, whereas all P genotypes were untypeable. To confirm the RT-PCR results, representative samples were chosen and sequenced in the ABI 3130XL using a Big-dye Terminator kit with Sanger dideoxy sequencing method.  Phylogenetic analysis and nucleotide comparison revealed that G10 was closely related to and clustered with human G10 strains from neighboring Kenya, implying that circulating G10 strains may have the potential for zoonotic transmission. When circulating G10 amino acid alignment was compared to bovine vaccine strain B223, our G10 samples had various nucleotide substitutions at antigenic epitopes, which resulted in the acquisition of glycosylation sites suggesting that G10 has the potential to evade neutralization antibodies induced by a vaccine. Our study provided preliminary data on RVA in cattle in Tanzania, paving the way for further research into the rotavirus epidemiology and risk factors associated with RVA in cattle in Tanzania.   Key words: Cattle, rotavirus groups A, G10 genotype, Tanzania.

Genetic and phylogenetic relationship of an American caprine rotavirus B strain with equine rotavirus B

Rotaviruses (RVs) are significant enteric pathogens of humans and animals. In March 2021, the University of Kentucky Veterinary Diagnostic Laboratory (UKVDL) received a fecal sample from a 1-week-old goat kid with diarrhea from a farm with 5 additional diarrheic kids. The fecal sample was tested negative for Bovine coronavirus, Bovine rotavirus Group A, E. coli K99+, Cryptosporidium parvum and Salmonella spp by multiplex real-time PCR assays. Interestingly, a novel Equine Rotavirus B (ERVB) in Kentucky was identified from neonatal foals also with watery diarrhea in the Spring of 2021. Once the ERVB-specific real-time PCR assay became available, the fecal sample from the goat kid was tested and found positive for RVB. Genome sequence of the caprine RVB from fecal sample was obtained using shotgun metagenomic sequencing by Illumina MiSeq. All of the eleven viral segments of caprine RVB were sequenced either completely or partially. Genetic and phylogenetic analysis of VP7, VP4, VP6, VP1–VP3, and NSP1- NSP5 genes indicated the caprine RVB strain had the genotype constellation of G3-P[3]-I3- R3-C3-M3-A4-N3-T3-E3-H3. In addition, the caprine RVB sequence showed the highest nucleotide identity and evolutionary relationship to the ERVB and previously sequenced caprine RVB strains. Given the similar geographical location of the equine and caprine strains from our study, these findings suggest a possible common source of infection.

Association among the Presence of Rotavirus Group A and Types of Sources Located in Rural Communities

Among the etiological agents of acute gastroenterocolitis, group A Rotavirus stands out. Proper hygiene and basic sanitation are the prophylactic means. The present research aimed to carry out the tracking of Rotavirus in waters of rural communities. The collections were carried out in 86 residences, distributed in 16 communities, considering the types of sources: tubular wells, excavated wells, surface springs, cistern with rainwater and springs. The elution of membranes with negative charges was used to obtain viral particles. Extraction of the genetic material and cDNA synthesis were performed according to the rules of the kit used, then qPCR was performed with an Applied Biosystems StepOne® thermal cycler. Statistical analysis was performed using Stata® 16.0, BioEstat® 5.3 and Minitab® 1. Rotavirus was detected in 47 samples (54.6%), however, there was no significant association among municipalities and the presence of Rotavirus. There was a strong association with shallow excavated well OR = 3.59 [1.05–12.28, p = 0.04]. The virus to infect humans and non-human animals, it can infect poultry, cattle and pigs. The presence of Rotavirus in surface water qualifies it as a biological marker of water quality and would occur through fecal contamination in water surface and underground.

Epidemiological study of unusual rotavirus strains and molecular characterization of emerging P[14] strains isolated from children with acute gastroenteritis during a 15-year period

Rotavirus group A (RVA) is characterized by molecular and epidemiological diversity. To date, 42 G and 58 P RVA genotypes have been identified, some of which, like P[14], have a zoonotic origin. In this study, we describe the epidemiology of unusual RVA genotypes and the molecular characteristics of P[14] strains. Fecal samples from children ≤ 16 years of age with acute gastroenteritis (AGE) who were hospitalized during 2007–2021 in Greece were tested for RVA by immunochromatography. Positive RVA samples were G and P genotyped, and part of the VP7 and VP4 genes were sequenced by the Sanger method. Epidemiological data were also recorded. Phylogenetic analysis of P[14] was performed using MEGA 11 software. Sixty-two (1.4%) out of 4427 children with RVA AGE were infected with an unusual G (G6/G8/G10) or P (P[6]/P[9]/P[10]/P[11]/P[14]) genotype. Their median (IQR) age was 18.7 (37.3) months, and 67.7% (42/62) were males. None of the children were vaccinated against RVA. P[9] (28/62; 45.2%) was the most common unusual genotype, followed by P[14] (12/62; 19.4%). In the last two years, during the period of the COVID-19 pandemic, an emergence of P[14] was observed (5/12, 41.6%) after an 8-year absence. The highest prevalence of P[14] infection was seen in the spring (91.7%). The combinations G8P[14] (41.7%), G6P[14] (41.7%), and G4P[14] (16.6%) were also detected. Phylogenetic analysis showed a potential evolutionary relationship of three human RVA P[14] strains to a fox strain from Croatia. These findings suggest a possible zoonotic origin of P[14] and interspecies transmission between nondomestic animals and humans, which may lead to new RVA genotypes with unknown severity.

Genomic epidemiology of the rotavirus G2P[4] strains in coastal Kenya pre- and post-rotavirus vaccine introduction, 2012-8.

The introduction of rotavirus vaccines into the national immunization programme in many countries has led to a decline in childhood diarrhoea disease burden. Coincidentally, the incidence of some rotavirus group A (RVA) genotypes has increased, which may result from non-vaccine-type replacement. Here, we investigate the evolutionary genomics of rotavirus G2P[4] which has shown an increase in countries that introduced the monovalent Rotarix® vaccine. We examined sixty-three RVA G2P[4] strains sampled from children (aged below 13 years) admitted to Kilifi County Hospital, coastal Kenya, pre- (2012 to June 2014) and post-(July 2014 to 2018) rotavirus vaccine introduction. All the sixty-three genome sequences showed a typical DS-1-like genome constellation (G2-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2). Pre-vaccine G2 sequences predominantly classified as sub-lineage IVa-3 and co-circulated with low numbers of sub-lineage IVa-1 strains, whereas post-vaccine G2 sequences mainly classified into sub-lineage IVa-3. In addition, in the pre-vaccine period, P[4] sub-lineage IVa strains co-circulated with low numbers of P[4] lineage II strains, but P[4] sub-lineage IVa strains predominated in the post-vaccine period. On the global phylogeny, the Kenyan pre- and post-vaccine G2P[4] strains clustered separately, suggesting that different virus populations circulated in the two periods. However, the strains from both periods exhibited conserved amino acid changes in the known antigenic epitopes, suggesting that replacement of the predominant G2P[4] cluster was unlikely a result of immune escape. Our findings demonstrate that the pre- and post-vaccine G2P[4] strains circulating in Kilifi, coastal Kenya, differed genetically but likely were antigenically similar. This information informs the discussion on the consequences of rotavirus vaccination on rotavirus diversity.

Genetic diversity and genomic analysis of G3P[6] and equine-like G3P[8] in children under-five from Southern Highlands and Eastern Tanzania

Rotavirus group A genomic characterization is critical for understanding the mechanisms of rotavirus diversity, such as reassortment events and possible interspecies transmission. However, little is known about the genetic diversity and genomic relationship of the rotavirus group A strains circulating in Tanzania. The genetic and genomic relationship of RVA genotypes was investigated in children under the age of five. A total of 169 Fecal samples were collected from under-five with diarrhea in Mbeya, Iringa and Morogoro regions of Tanzania. The RVA were screened in children under five with diarrhea using reverse transcription PCR for VP7 and VP4, and the G and P genotypes were determined using Sanger dideoxynucleotide cycle sequencing. Whole-genome sequencing was performed on selected genotypes. The overall RVA rate was 4.7% (8/169). The G genotypes were G3 (7/8) and G6 (1/8) among the 8 RVA positives, while the P genotypes were P[6] (4/8) and P[8] (2), and the other two were untypeable. G3P[6] and G3P[8] were the identified genotype combinations. The genomic analysis reveals that the circulating G3P[8] and G3P[6] isolates from children under the age of five with diarrhea had a DS-1-like genome configuration (I2-R2-C2-M2-axe-N2-T2-E2-H2). The phylogenic analysis revealed that all 11 segments of G3P[6] were closely related to human G3P[6] identified in neighboring countries such as Uganda, Kenya, and other African countries, implying that G3P[6] strains descended from a common ancestor. Whereas, G3P[8] were closely related to previously identified equine-like G3P[P8] from Kenya, Japan, Thailand, Brazil, and Taiwan, implying that this strain was introduced rather than reassortment events. We discovered amino acid differences at antigenic epitopes and N-linked glycosylation sites between the wild type G3 and P[8] compared to vaccine strains, implying that further research into the impact of these differences on vaccine effectiveness is warranted. The phylogenic analysis of VP7 also identified a bovine-like G6. For the first time in Tanzania, we report the emergence of novel equine-like G3 and bovine-like G6 RVA strains, highlighting the importance of rotavirus genotype monitoring and genomic analysis of representative genotypes.

A G3P[9] rotavirus strain with an unusual genome constellation in a diarrheic cat in Thailand.

Rotavirus infection can cause diarrhea in many animal species. A 2-year-old indoor female Siamese cat with bloody mucoid diarrhea tested positive for rotavirus (RV) group A by real-time reverse transcription polymerase chain reaction (RT-PCR). Subsequent conventional RT-PCR amplification of the 11 RV segments and sequencing revealed a G3-P[9]-I2-R2-C2-M2-A3-N2-T3-E3-H3 genome constellation. Phylogenetic analysis showed that the VP4, VP7, NSP1, NSP3, NSP4, and NSP5 genes were closely related to those of human feline-like rotaviruses, while the VP1, VP2, VP3, VP6, and NSP2 genes were genetically closest to those of human bovine-like rotaviruses, suggesting that genetic reassortment had occurred. The uniqueness of this G3P[9] feline rotavirus strain expands our knowledge about feline rotaviruses.

Lack of molecular evidence of fecal-borne viruses in capybaras from São Paulo state, Brazil, 2018-2020: a minor public health issue.

Capybara (Hydrochoerus hydrochaeris) is the world's largest rodent species distributed throughout South America. These animals are incredibly tolerant to anthropogenic environments and are occupying large urban centers. Capybaras are known to carry potentially zoonotic agents, including R. rickettsia, Leishmania spp., Leptospira spp., Trypanosoma spp., Salmonella spp., Toxoplasma gondii, and rabies virus. Focusing on the importance of monitoring potential sources of emerging zoonotic viruses and new viral reservoirs, the aim of the present study was to assess the presence of fecal-borne viruses in the feces of capybaras living in urban parks in São Paulo state, Brazil. A total of 337 fecal samples were collected between 2018 and 2020 and screened for the following: (i) Rotavirus group A (RVA) by ELISA; (ii) non-RVA species and Picobirnavirus (PBV) using PAGE; (iii) Human Bocaparvovirus (HBoV), Bufavirus (BuV), Tusavirus (TuV), and Cutavirus (CuV) qPCR; (iv) Human Enterovirus (EV), Norovirus GII (NoV), and Hantavirus by in houses RT-qPCR; (v) SARS-CoV-2 via commercial RT-qPCR kit assay; and (vi) Astrovirus (AstV) and Adenovirus (AdV) using conventional nested (RT)-PCRs. All fecal samples tested were negative for fecal-borne viruses. This study adds further evidence that the fecal-borne viruses is a minor public health issue in Brazilian capybaras, at least during the surveillance period and surveyed areas. Continuous monitoring of sylvatic animals is essential to prevent and control the emergence or re-emergence of newly discovered virus as well as viruses with known zoonotic potential.

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.

Genome Analysis of the G6P6 Genotype of Porcine Group C Rotavirus in China.

Swine enteric disease is the predominant cause of morbidity and mortality, and viral species involved in swine enteric disease include rotaviruses and coronaviruses, among others. Awareness of the circulating porcine rotavirus group C (PoRVC) in pig herds is critical to evaluate the potential impact of infection. At present, due to the lack of disease awareness and molecular diagnostic means, the research on RVC infection in China is not well-studied. In this study, diarrhea samples collected from pig farms were detected positive for RVC by PCR, and the full-length RVC was not previously reported for Chinese pig farms. This rotavirus strain was designated as RVC/Pig/CHN/JS02/2018/G6P6. A natural recombination event was observed with breakpoints at nucleotides (nt) 2509 to 2748 of the VP2 gene. Phylogenetic analysis based on nsp1 revealed that a new branch A10 formed. Collectively, our data suggest a potentially novel gene recombination event of RVC in the VP2 gene. These findings provide a new insight into the evolution of the rotavirus.

Production and Evaluation of Chicken Egg Yolk Immunoglobulin (IgY) against Human and Simian Rotaviruses.

Producing specific antibodies in chickens is an attractive approach for diagnosis or therapeutic applications. Besides the high immunoglobulin Y (IgY) yield transferred to the egg yolk and its suitability for large-scale production, such an approach is more bioethical for animal maintenance. The IgY technology offers new possibilities for application in human and veterinary diagnostics and therapeutics, including strategies for treating severe intestinal diseases in children, particularly in emerging countries. Herein, we describe the production and purification of polyclonal antibodies against rotavirus group A (RVA) in immunised hens aiming at its application in prophylaxis and treatment of rotavirus-induced diarrhoea. For this purpose, we inoculated Rhodia laying chickens (Gallus gallus domesticus) with two or three doses of RVA combined with adjuvants or only adjuvants (control group). As the egg-laying period began, the yolk protein purification processes yielded a high concentration of specific IgY, the highest titre resulting from the group of hens that received three doses of the immunogen. The purified IgY blocked the functional activity of RVA in MA-104 cells, thus confirming the neutralisation ability. Therefore, anti-RVA IgY could be a promising candidate for pre- and post-exposure prevention or treatment of rotavirus-induced diarrhoea.

Estimates the Evolution of G8P[8] Rotaviruses in Thailand during 2012 - 2014

Point mutation and abrupt changes of segmented genomes of rotavirus group A (RVAs) from mixed infection are critical factors for generating genetically divergent strains. Our preceding study of rotavirus infections in 2012 - 2014 in Thailand indicated the high prevalence of G8P[8] strains. This study aimed to identify the origin of each segment of the genome of these G8P[8] RVA strains by using phylogenetic analysis. All gene segments from the 5 selected G8P[8] were sequenced partially and aligned with locally circulating rotavirus strains to estimate the phylogenic relationship. Based on the genome's constellation of the genome, these 5 strains’ genetic backgrounds possessed genotype constellation 2 or called the DS-1-like genotype. Ten genes, except for VP6, were clustered within the same phylogenic tree of the cognate genes of Thai circulating G8P[8]strains isolated between 2013 - 2014. Their 5 genes VP2, VP3, NSP1, NSP3, and NSP5, also exhibited high nucleotide similarities with the first unusual Japanese DS-1-like strains G1P[8] NT004. Besides, the VP4 shared a high nucleotide sequence similarity with the cognate gene of circulating Thai human G1P[8] Wa-like and G1P[8] NT004. However, the remaining 4 gene segments (VP1, VP7, NSP2, and NSP4) were clustered with other human or animal RVA strains within the same monophyletic. All of these G8P[8] strains shown to be an intragenogroup reassortment. Thus, characterization of the genomes is crucial to monitor the evolutionary dynamics and relationships of co-circulating RVAs in predicting the effectiveness of current vaccines and future vaccine development strategies.
 HIGHLIGHTS
 
 G8P[8] strains’ genetic backgrounds possessed genotype constellation 2 ( DS-1-like genotype)
 G8P[8] strains might originate from a sequential process of multiple reassortment events involving intra-genogroups from humans and animal species
 Ten genes, except for VP6, were clustered within the same phylogenic tree of the cognate genes of Thai circulating G8P[8]strains isolated between 2013 - 2014
 
 GRAPHICAL ABSTRACT

Differences in epidemiology of enteropathogens in children pre- and post-rotavirus vaccine introduction in Kilifi, coastal Kenya

BackgroundKenya introduced Rotarix® (GlaxoSmithKline Biologicals, Rixensart, Belgium) vaccination into its national immunization programme beginning July 2014. The impact of this vaccination program on the local epidemiology of various known enteropathogens is not fully understood.MethodsWe used a custom TaqMan Array Card (TAC) to screen for 28 different enteropathogens in 718 stools from children aged less than 13 years admitted to Kilifi County Hospital, coastal Kenya, following presentation with diarrhea in 2013 (before vaccine introduction) and in 2016–2018 (after vaccine introduction). Pathogen positivity rate differences between pre- and post-Rotarix® vaccination introduction were examined using both univariate and multivariable logistic regression models.ResultsIn 665 specimens (92.6%), one or more enteropathogen was detected, while in 323 specimens (48.6%) three or more enteropathogens were detected. The top six detected enteropathogens were: enteroaggregative Escherichia coli (EAggEC; 42.1%), enteropathogenic Escherichia coli (EPEC; 30.2%), enterovirus (26.9%), rotavirus group A (RVA; 24.8%), parechovirus (16.6%) and norovirus GI/GII (14.4%). Post-rotavirus vaccine introduction, there was a significant increase in the proportion of samples testing positive for EAggEC (35.7% vs. 45.3%, p = 0.014), cytomegalovirus (4.2% vs. 9.9%, p = 0.008), Vibrio cholerae (0.0% vs. 2.3%, p = 0.019), Strongyloides species (0.8% vs. 3.6%, p = 0.048) and Dientamoeba fragilis (2.1% vs. 7.8%, p = 0.004). Although not reaching statistical significance, the positivity rate of adenovirus 40/41 (5.8% vs. 7.3%, p = 0.444), norovirus GI/GII (11.2% vs. 15.9%, p = 0.089), Shigella species (8.7% vs. 13.0%, p = 0.092) and Cryptosporidium spp. (11.6% vs. 14.7%, p = 0.261) appeared to increase post-vaccine introduction. Conversely, the positivity rate of sapovirus decreased significantly post-vaccine introduction (7.8% vs. 4.0%, p = 0.030) while that of RVA appeared not to change (27.4% vs. 23.5%, p = 0.253). More enteropathogen coinfections were detected per child post-vaccine introduction compared to before (mean: 2.7 vs. 2.3; p = 0.0025).ConclusionsIn this rural Coastal Kenya setting, childhood enteropathogen infection burden was high both pre- and post-rotavirus vaccination introduction. Children who had diarrheal admissions post-vaccination showed an increase in coinfections and changes in specific enteropathogen positivity rates. This study highlights the utility of multipathogen detection platforms such as TAC in understanding etiology of childhood acute gastroenteritis in resource-limited regions.

Oncolytic and protective effects of strain 100 of a new group of non-classified rotavirus of reoviridae family on B16/F10 melanoma growth in experiment.

e21587 Background: The purpose of this study was to assess the effect of a strain 100 of a new group of rotavirus of Reoviridae family on the growth of transplantable B16/F10 melanoma in mice and the survival of the animals. Methods: We studied the strain 100 of a new group of rotavirus of Reoviridae family (RVK) ( http://jbks.ru/archive/issue-10/article-6 ). RVK 100 is a live attenuated non-pathogenic virus growing on pig embryo kidney cell culture with concentration 5·109 per 1 ml. The dynamics of melanoma growth and the survival of tumor-bearing animals receiving RVK were studied in 25 male mice divided into 5 equal groups, each n = 5. RVK was administered intramuscularly and orally once a week 0.3 mL and 25 µL, respectively: to groups 1-2 21 days prior to the tumor inoculation, a total of 4 injections (“vaccination” regimen); to groups 3-4 – 7 days after tumor inoculation with the onset of tumor nodes, a total of 3 injections (“treatment” regimen); group 5 included controls receiving 0.85% NaCl solution. Groups 1 and 3 received live RVK, groups 2 and 4 - RVK inactivated by UV radiation. Results: Survival of mice in groups 1-2 receiving both live and inactivated RVK in the “vaccination” regimen was increased statistically significantly: in group 1, Me = 25 days after melanoma transplantation (LQ 24; UQ 30), in group 2 Me = 26 days (LQ 25; UQ 30), in controls Me = 14 days (LQ 10; UQ 22); p = 0.032 and p = 0.0079, respectively. RVK administration in the “treatment” regimen did not cause such changes. While mice of group 3 receiving the live strain 100 showed a tendency to higher survival, compared to controls (Me = 23 days, LQ 22; UQ 24, p = 0.056), the administration of inactivated RVK (group 4) did not significantly improve the survival of animals, compared to controls (Me = 22 days (LQ 21; UQ 33, p = 0.151). Conclusions: Improved survival of animals with B16/F10 melanoma after the RVK administration indicates the virus ability to slow down tumor growth and confirms our previously published data on the RVK strain 228, although, in contrast to it, the strain 100 is able to increase the survival rate of tumor-bearing mice only when used in the “vaccination” regimen, which refers mainly to the inactivated virus. Since the inactivation of the RVK strain 100 eliminates its effect when used in the “treatment”, but not “vaccination” regimen, it seems to be associated with an immunostimulatory effect but not with an oncolytic one.

Diverse human and bat-like rotavirus G3 strains circulating in suburban Bangkok.

Although rotavirus vaccines are available in many parts of the world and are effective in reducing the overall incidence of rotavirus infection, it remains a major cause of diarrhea in less-developed countries. Among various rotavirus group A (RVA) strains, the increasingly common genotype G3 (defined by the VP7 gene) has been identified in both humans and animals. Our previous epidemiological surveillance in Bangkok found several unusual non-vaccine-like G3 strains in patients with diarrhea. In this study, we sequenced and characterized the genomes of seven of these G3 strains, which formed combinations with genotypes P[4], P[6], P[9], and P[10] (defined by the VP4 gene). Interestingly, we identified a bat-like RVA strain with the genome constellation G3-P[10]-I3-R3-C3-M3-A9-N3-T3-E3-H6, which has not been previously reported in the literature. The amino acid residues deduced from the nucleotide sequences of our G3 strains differed at the antigenic epitopes to those of the VP7 capsid protein of the G3 strain in RotaTeq vaccine. Although it is not unusual for the segmented genomes of RVA to reassort and give rise to emerging novel strains, the atypical G3 strains identified in this study suggest possible animal-to-human RVA zoonotic spillover even in urban areas.