Equine-like G3 Research Articles

Genetic diversity of G9, G3, G8 and G1 rotavirus group A strains circulating among children with acute gastroenteritis in Vietnam from 2016 to 2021

Rotavirus group A (RVA) is the most common cause of severe childhood diarrhea worldwide. The introduction of rotavirus vaccination programs has contributed to a reduction in hospitalizations and mortality caused by RVA. From 2016 to 2021, we conducted surveillance to monitor RVA prevalence and genotype distribution in Nam Dinh and Thua Thien Hue (TT Hue) provinces where a pilot Rotavin-M1 vaccine (Vietnam) implementation took place from 2017 to 2020. Out of 6626 stool samples, RVA was detected in 2164 (32.6%) by ELISA. RT-PCR using type-specific primers were used to determine the G and P genotypes of RVA-positive specimens. Whole genome sequences of a subset of 52 specimens randomly selected from 2016 to 2021 were mapped using next-generation sequencing. From 2016 to 2021, the G9, G3 and G8 strains dominated, with detected frequencies of 39%, 23%, and 19%, respectively; of which, the most common genotypes identified were G9P[8], G3P[8] and G8P[8]. G1 strains re-emerged in Nam Dinh and TT Hue (29.5% and 11.9%, respectively) from 2020 to 2021. G3 prevalence decreased from 74% to 20% in TT Hue and from 21% to 13% in Nam Dinh province between 2017 and 2021. The G3 strains consisted of 52% human typical G3 (hG3) and 47% equine-like G3 (eG3). Full genome analysis showed substantial diversity among the circulating G3 strains with different backgrounds relating to equine and feline viruses. G9 prevalence decreased sharply from 2016 to 2021 in both provinces. G8 strains peaked during 2019–2020 in Nam Dinh and TT Hue provinces (68% and 46%, respectively). Most G8 and G9 strains had no genetic differences over the surveillance period with very high nucleotide similarities of 99.2–99.9% and 99.1–99.7%, respectively. The G1 strains were not derived from the RVA vaccine. Changes in the genotype distribution and substantial diversity among circulating strains were detected throughout the surveillance period and differed between the two provinces. Determining vaccine effectiveness against circulating strains over time will be important to ensure that observed changes are due to natural secular variation and not from vaccine pressure.

Equine Rotavirus A under the One Health Lens: Potential Impacts on Public Health.

Group A rotaviruses are a well-known cause of viral gastroenteritis in infants and children, as well as in many mammalian species and birds, affecting them at a young age. This group of viruses has a double-stranded, segmented RNA genome with high genetic diversity linked to point mutations, recombination, and, importantly, reassortment. While initial molecular investigations undertaken in the 1900s suggested host range restriction among group A rotaviruses based on the fact that different gene segments were distributed among different animal species, recent molecular surveillance and genome constellation genotyping studies conducted by the Rotavirus Classification Working Group (RCWG) have shown that animal rotaviruses serve as a source of diversification of human rotavirus A, highlighting their zoonotic potential. Rotaviruses occurring in various animal species have been linked with contributing genetic material to human rotaviruses, including horses, with the most recent identification of equine-like G3 rotavirus A infecting children. The goal of this article is to review relevant information related to rotavirus structure/genomic organization, epidemiology (with a focus on human and equine rotavirus A), evolution, inter-species transmission, and the potential zoonotic role of equine and other animal rotaviruses. Diagnostics, surveillance and the current status of human and livestock vaccines against RVA are also reviewed.

Emergence of equine-like G3 and porcine-like G9 rotavirus strains in Sarawak, Malaysia: 2019-2021.

Rotavirus is the leading causative viral agent of pediatric acute gastroenteritis globally, infecting mostly children 5 years old and below. Data on rotavirus prevalence in Malaysia is scarce, despite the WHO's recommendation for continuous rotavirus surveillance, and has underestimated the need for national rotavirus vaccination. Characteristics of the current rotavirus strains in Malaysia have to be determined to understand the rotavirus epidemiology and vaccine compatibility. This study sought to determine the genetic relatedness of Sarawak rotavirus strains with global strains and to determine the antigenic coverage and epitope compatibility of Rotarix and RotaTeq vaccines with the Sarawak rotavirus strains via in silico analysis. A total of 89 stool samples were collected from pediatric patients (<5 years old) with acute gastroenteritis at private hospitals in Kuching, Sarawak. Rotavirus was detected using reverse transcription-polymerase chain reaction. Positive amplicons were analyzed using nucleotide sequencing before phylogenetic analyses and assessment of epitope compatibility. Genotyping revealed G1P[8] (1/13; 7.7%), G3P[8] (3/13; 23%), G9P[4] (1/13; 7.7%), and G9P[8] (3/13; 23%), G9P[X] (1/13; 7.7%), GXP[4] (1/13; 7.7%), and GXP[8] (3/13; 23%) in samples. All wild-type Sarawak rotavirus strains, with the exception of G1, showed variations in their phylogenetic and antigenic epitope characteristics.

Increase in rotavirus prevalence with the emergence of genotype G9P[8] in replacement of genotype G12P[6] in Sabah, Malaysia.

Rotaviruses are major causative agents of acute diarrhea in children under 5 years of age in Malaysia. However, a rotavirus vaccine has not been included in the national vaccination program. To date, only two studies have been carried out in the state of Sabah, Malaysia, although children in this state are at risk of diarrheal diseases. Previous studies showed that 16%-17% of cases of diarrhea were caused by rotaviruses and that equine-like G3 rotavirus strains are predominant. Because the prevalence of rotaviruses and their genotype distribution vary over time, this study was conducted at four government healthcare facilities from September 2019 through February 2020. Our study revealed that the proportion of rotavirus diarrhea increased significantly to 37.2% (51/137) after the emergence of the G9P[8] genotype in replacement of the G12P[8] genotype. Although equine-like G3P[8] strains remain the predominant rotaviruses circulating among children, the Sabahan G9P[8] strain belonged to lineage VI and was phylogenetically related to strains from other countries. A comparison of the Sabahan G9 strains with the G9 vaccine strains used in the RotaSiil and Rotavac vaccines revealed several mismatches in neutralizing epitopes, indicating that these vaccines might not be effective in Sabahan children. However, a vaccine trial may be necessary to understand the precise effects of vaccination.

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.

Genetic diversity of G3 rotavirus strains circulating among children with acute gastroenteritis in Nam Dinh and Thua Thien Hue provinces, Vietnam, 2016 - 2021

Rotavirus (RV) is the most common cause of severe childhood diarrhea worldwide. Since 2006, the WHO has approved the use of two vaccines against Rotavirus (RotaTeq and Rotarix), which has contributed to a significant reduction in morbidity and mortality from this disease in children under 5 years of age. Beside of that, the presence of vaccines could also cause selective pressure among circulating RV strains. Diarrhea samples were a part of the study of the project “Evaluation of rotavirus vaccine effectiveness in Vietnam” and some diarrhea samples were randomly selected from 2010 - 2015 and were stored in our laboratory. Rotavirus antigen in diarrhea samples was detected using the Premier Rotaclone kit. G and P genotypes were determined by RT-PCR using type specific primers. The nucleotide sequencewas determined by next generation sequencing and phylogenetic analysis was performed by the neighbor-joining method. 375/1715 (21.9%) samples detected G3 strain include equine-like G3 (eG3) and human G3 (hG3). The prevalence of G3 strains decreased from 2016 to 2021. All eleven gene segments were typed among 34 samples and substantial diversity among the circulating strains was observed. The circulating G3 strains were also phylogenetically diverse and related to strains from several different countries, different origins such as human (20 samples), pig (1 sample), cat (3 samples) and equine (10 samples). Comparing with the vaccine strains, the antigenic epitopes present on VP7 ofG3 in Vietnam differed considerably, especially the K238N. With the genetic characteristics of the G3 strain, it could contribute to the vaccine effectiveness in Vietnam.

Changing distribution of rotavirus A genotypes circulating in Japanese children with acute gastroenteritis in outpatient clinic, 2014–2020

BackgroundRotavirus A (RVA) is a major cause of severe acute gastroenteritis (AGE) in infants and children worldwide. In Japan, two kinds of rotavirus vaccines have been introduced as voluntary vaccines in 2011 and 2012, respectively, and launched into the national vaccine program in October 2020. MethodsIn this study, we investigated prevalence of RVA and their molecular characterization in the stool samples collected from infants and children with AGE who visited one outpatient clinic in Japan, from July 2014 to June 2020, during voluntary vaccination with two kinds of rotavirus vaccines. ResultsThe RVA detection rates decreased from 44.7 % in 2014–2015 to 35.4 % in 2018–2019, whereas in 2019–2020 the numbers of samples collected were dramatically decreased and none of RVA was detected. During this study period, rotavirus vaccination rates in this area increased from 32.4 % to 62.2 %. Distribution of RVA VP7 (G), VP4 (P), and VP6 (I) genotypes in this area had changed year by year; the major genotype combinations were G1P[8]I1 and G1P[8]I2 in 2014–2015, G2P[4]I2 and G9P[8]I1 in 2015-2016, G1P[8]I1 and G8P[8]I2 in 2017–2018, and G8P[8]I2 in 2018–2019. Phylogenetic analysis demonstrated that VP7 nucleotide sequences of G1 were genetically diverse compared with those of other G genotypes in this study. Meanwhile, predominance of unusual G2P[8]I1, G2P[8]I2 and mixed P genotypes were observed only in 2016–2017, but did not carry on in 2017–2019. The equine-like G3 was detected only in 2016–2017. ConclusionsThe results revealed diversity of RVA genotypes and the genotype combinations have changed year by year in Japan, during the study period of 2016–2020.

Development of a Real-Time Reverse Transcription-PCR Assay To Detect and Quantify Group A Rotavirus Equine-Like G3 Strains.

Since 2013, group A rotavirus strains characterized as novel DS-1-like intergenogroup reassortant "equine-like G3" strains have emerged and spread across 5 continents among human populations in at least 14 countries. Here, we report a novel one-step TaqMan quantitative real-time reverse transcription-PCR assay developed to genotype and quantify the viral load for samples containing rotavirus equine-like G3 strains. Using a universal G forward primer and a newly designed reverse primer and TaqMan probe, we developed and validated an assay with a linear dynamic range of 227 to 2.3 × 109 copies per reaction and a limit of detection of 227 copies. The percent positive agreement, percent negative agreement, and precision of our assay were 100.00%, 99.63%, and 100.00%, respectively. This assay can simultaneously detect and quantify the viral load for samples containing DS-1-like intergenogroup reassortant equine-like G3 strains with high sensitivity and specificity, faster turnaround time, and decreased cost. It will be valuable for high-throughput screening of stool samples collected to monitor equine-like G3 strain prevalence and circulation among human populations throughout the world.

Emergence of equine-like G3 strains as the dominant rotavirus among children under five with diarrhea in Sabah, Malaysia during 2018-2019.

Rotavirus infection is a dilemma for developing countries, including Malaysia. Although commercial rotavirus vaccines are available, these are not included in Malaysia’s national immunization program. A scarcity of data about rotavirus genotype distribution could be partially to blame for this policy decision, because there are no data for rotavirus genotype distribution in Malaysia over the past 20 years. From January 2018 to March 2019, we conducted a study to elucidate the rotavirus burden and genotype distribution in the Kota Kinabalu and Kunak districts of the state of Sabah. Stool specimens were collected from children under 5 years of age, and rotavirus antigen in these samples was detected using commercially available kit. Electropherotypes were determined by polyacrylamide gel electrophoresis of genomic RNA. G and P genotypes were determined by RT-PCR using type specific primers. The nucleotide sequence of the amplicons was determined by Sanger sequencing and phylogenetic analysis was performed by neighbor-joining method. Rotavirus was identified in 43 (15.1%) children with watery diarrhea. The male:female ratio (1.9:1) of the rotavirus-infected children clearly showed that it affected predominantly boys, and children 12–23 months of age. The genotypes identified were G3P[8] (74% n = 31), followed by G1P[8] (14% n = 6), G12P[6](7% n = 3), G8P[8](3% n = 1), and GxP[8] (3% n = 1). The predominant rotavirus circulating among the children was the equine-like G3P[8] (59.5% n = 25) with a short electropherotype. Eleven electropherotypes were identified among 34 strains, indicating substantial diversity among the circulating strains. The circulating genotypes were also phylogenetically diverse and related to strains from several different countries. The antigenic epitopes present on VP7 and VP4 of Sabahan G3 and equine-like G3 differed considerably from that of the RotaTeq vaccine strain. Our results also indicate that considerable genetic exchange is occurring in Sabahan strains. Sabah is home to a number of different ethnic groups, some of which culturally are in close contact with animals, which might contribute to the evolution of diverse rotavirus strains. Sabah is also a popular tourist destination, and a large number of tourists from different countries possibly contributes to the diversity of circulating rotavirus genotypes. Considering all these factors which are contributing rotavirus genotype diversity, continuous surveillance of rotavirus strains is of utmost importance to monitor the pre- and post-vaccination efficacy of rotavirus vaccines in Sabah.

Prevalence and Distribution of Rotavirus Genotypes Among Children With Acute Gastroenteritis in Areas Other Than Java Island, Indonesia, 2016-2018.

Group A rotaviruses (RVAs) are the leading cause of acute gastroenteritis, which is often associated with severe symptoms in children under 5 years old. Genetic reassortments and interspecies transmission commonly occur, resulting in a great diversity of RVA circulating in the world. The aim of this study is to determine the prevalence and distribution of RVA genotypes among children in Indonesia over the years 2016–2018 across representative areas of the country. Stool samples were collected from 202 pediatric patients with acute gastroenteritis in three regions of Indonesia (West Nusa Tenggara, South Sumatra, and West Papua) in 2016–2018. Rotavirus G and P genotypes were determined by reverse transcription PCR (RT-PCR) and direct sequencing analysis. The prevalences of RVA in South Sumatra (55.4%) and West Papua (54.0%) were significantly higher than that in East Java (31.7%) as determined in our previous study. The prevalence in West Nusa Tenggara (42.6%) was the lowest among three regions, but higher than that in East Java. Interestingly, equine-like G3 rotavirus strains were found as predominant strains in South Sumatra in 2016 and in West Papua in 2017–2018. Moreover, the equine-like G3 strains in South Sumatra detected in 2016 were completely replaced by human G1 and G2 in 2018. In conclusion, RVA infection in South Sumatra and West Papua was highly endemic. Equine-like G3 strains were also spread to South Sumatra (West Indonesia) and West Papua (East Indonesia), as well as Java Island. Dynamic change in rotavirus genotypes from equine-like G3 to human genotypes was also observed. Continuous monitoring may be warranted in isolated areas in Indonesia.

Emergence in 2017-2019 of novel reassortant equine-like G3 rotavirus strains in Palermo, Sicily.

Rotavirus A (RVA) is a major etiologic agent of gastroenteritis in children worldwide. Hospital-based surveillance of viral gastroenteritis in paediatric population in Palermo (Italy) from 2017 onwards revealed a sharp increase in G3P[8] RVAs, accounting for 71% of all the RVAs detected in 2019. This pattern had not been observed before in Italy, with G3 RVA usually being detected at rates lower than 3%. In order to investigate this unique epidemiological pattern, the genetic diversity of G3 RVAs identified during a 16-year long surveillance (2004-2019) was explored by systematic sequencing of the VP7 and VP4 genes and by whole genome sequencing of selected G3 strains, representative of the various RVA seasons. Sequence and phylogenetic analyses of the VP7 and VP4 genes revealed the emergence, in 2017 of reassortant equine-like G3P[8], which gradually replaced former G3P[8] strains. The G3P[8] circulating before 2017 showed a Wa-like constellation of genome segments while the G3P[8] that emerged in 2017 had a DS-1-like backbone. On direct inspection of the VP7 and VP4 antigenic epitopes, the equine-like G3P[8] strains possessed several amino acid variations in neutralizing regions compared with vaccine strains. The equine-like G3P[8] RVAs are a further example of the zoonotic impact of animal viruses on human health.

Molecular and clinical characterization of the equine-like G3 rotavirus that caused the first outbreak in Japan, 2016.

Since 2013, equine-like G3 rotavirus (eG3) strains have been detected throughout the world, including in Japan, and the strains were found to be dominant in some countries. In 2016, the first eG3 outbreak in Japan occurred in Tomakomai, Hokkaido prefecture, and the strains became dominant in other Hokkaido areas the following year. There were no significant differences in the clinical characteristics of eG3 and non-eG3 rotavirus infections. The eG3 strains detected in Hokkaido across 2 years from 2016 to 2017 had DS-1-like constellations (i.e. G3-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2), and the genes were highly conserved (97.5-100 %). One strain, designated as To16-12 was selected as the representative strain for these strains, and all 11 genes of this strain (To16-12) exhibited the closest identity to one foreign eG3 strain (STM050) seen in Indonesia in 2015 and two eG3 strains (IS1090 and MI1125) in another Japanese prefecture in 2016, suggesting that this strain might be introduced into Japan from Indonesia. Sequence analyses of VP7 genes from animal and human G3 strains found worldwide did not identify any with close identity (>92 %) to eG3 strains, including equine RV Erv105. Analysis of another ten genes indicated that the eG3 strain had low similarity to G2P[4] strains, which are considered traditional DS-1-like strains, but high similarity to DS-1-like G1P[8] strains, which first appeared in Asia in 2012. These data suggest that eG3 strains were recently generated in Asia as mono-reassortant strain between DS-1-like G1P[8] strains and unspecified animal G3 strains. Our results indicate that rotavirus surveillance in the postvaccine era requires whole-genome analyses.

Multiple Introductions and Predominance of Rotavirus Group A Genotype G3P[8] in Kilifi, Coastal Kenya, 4 Years after Nationwide Vaccine Introduction.

Globally, rotavirus group A (RVA) remains a major cause of severe childhood diarrhea, despite the use of vaccines in more than 100 countries. RVA sequencing for local outbreaks facilitates investigation into strain composition, origins, spread, and vaccine failure. In 2018, we collected 248 stool samples from children aged less than 13 years admitted with diarrheal illness to Kilifi County Hospital, coastal Kenya. Antigen screening detected RVA in 55 samples (22.2%). Of these, VP7 (G) and VP4 (P) segments were successfully sequenced in 48 (87.3%) and phylogenetic analysis based on the VP7 sequences identified seven genetic clusters with six different GP combinations: G3P[8], G1P[8], G2P[4], G2P[8], G9P[8] and G12P[8]. The G3P[8] strains predominated the season (n = 37, 67.2%) and comprised three distinct G3 genetic clusters that fell within Lineage I and IX (the latter also known as equine-like G3 Lineage). Both the two G3 lineages have been recently detected in several countries. Our study is the first to document African children infected with G3 Lineage IX. These data highlight the global nature of RVA transmission and the importance of increasing global rotavirus vaccine coverage.

Group A Rotavirus Detection and Genotype Distribution before and after Introduction of a National Immunisation Programme in Ireland: 2015-2019.

Immunisation against rotavirus infection was introduced into Ireland in December 2016. We report on the viruses causing gastroenteritis before (2015–2016) and after (2017–2019) implementation of the Rotarix vaccine, as well as changes in the diversity of circulating rotavirus genotypes. Samples from patients aged ≤ 5 years (n = 11,800) were received at the National Virus Reference Laboratory, Dublin, and tested by real-time RT-PCR for rotavirus, Rotarix, norovirus, sapovirus, astrovirus, and enteric adenovirus. Rotavirus genotyping was performed either by multiplex or hemi-nested RT-PCR, and a subset was characterised by sequence analysis. Rotavirus detection decreased by 91% in children aged 0–12 months between 2015/16 and 2018/19. Rotarix was detected in 10% of those eligible for the vaccine and was not found in those aged >7 months. Rotavirus typically peaks in March–May, but following vaccination, the seasonality became less defined. In 2015–16, G1P[8] was the most common genotype circulating; however, in 2019 G2P[4] was detected more often. Following the introduction of Rotarix, a reduction in numbers of rotavirus infections occurred, coinciding with an increase in genotype diversity, along with the first recorded detection of an equine-like G3 strain in Ireland.

Whole-gene analysis of inter-genogroup reassortant rotaviruses from the Dominican Republic: Emergence of equine-like G3 strains and evidence of their reassortment with locally-circulating strains

Inter-genogroup reassortant group A rotavirus (RVA) strains possessing a G3 VP7 gene of putative equine origin (EQL-G3) have been detected in humans since 2013. Here we report detection of EQL-G3P[8] RVA strains from the Dominican Republic collected in 2014–16. Whole-gene analysis of RVA in stool specimens revealed 16 EQL-G3P[8] strains, 3 of which appear to have acquired an N1 NSP1 gene from locally-circulating G9P[8] strains and a novel G2P[8] reassortant possessing 7 EQL-G3-associated genes and 3 genes from a locally-circulating G2P[4] strain. Phylogenetic/genetic analyses of VP7 gene sequences revealed nine G3 lineages (I–IX) with newly-assigned lineage IX encompassing all reported human EQL-G3 strains along with the ancestral equine strain. VP1 and NSP2 gene phylogenies suggest that EQL-G3P[8] strains were introduced into the Dominican Republic from Thailand. The emergence of EQL-G3P[8] strains in the Dominican Republic and their reassortment with locally-circulating RVA could have implications for current vaccination strategies.

First detection of a reassortant G3P[8] rotavirus A\xa0strain in Italy: a case report in an 8-year-old child

BackgroundAcute gastroenteritis (AGE) due to group A rotavirus (RVA) agent is one of the major causes of hospitalization in paediatric age. The G3P[8] RVA genotype has been usually considered as one of the major human genotypes, largely circulating in Asia, but showing low detection rates in the European countries. In recent years, the G3P[8] RVAs emerged also in Europe as a predominant genotype and the viral strains detected revealed high similarities with equine-like G3P[8] RVA strains, resulting in a new variant circulating in humans and able to cause AGE in the paediatric population.Case presentationAn 8-year-old boy was admitted to the Emergency Room because he had suffered from severe diarrhoea, vomiting, and high fever over the previous two days. Severe dehydration was evident based on low serum concentrations of potassium and sodium, low glycaemia, and pre-renal failure (creatinine 2.48 mg/dL, urea 133 mg/dL). Immunological tests were within normal range. Enzyme immunoassay for the detection of RV was positive, and a sample of faeces was collected in order to perform the molecular characterization of the viral strain. The phylogenetic trees revealed relatedness between the VP7 and VP4 genes of the G3P[8] RVA Italian strain (namely PG2) and those belonging to recent G3P[8] RVAs detected worldwide. The G3 VP7 belonged to the G3-I lineage and shared the highest nucleotide sequence identity (99.8%) with the equine-like G3 previously identified in other countries. The P [8] VP4 revealed a similar clustering pattern to that observed for the VP7. In addition, the molecular characterization of the 11 gene segments of strain PG2 revealed a G3-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 genomic constellation.ConclusionsThis case shows the first detection in Italy of a reassortant G3P[8] RVA associated with a severe AGE, which is unusual in a school-age child without any known severe underlying problems. The findings reported in this paper highlight the importance of continuously monitoring the RVA strains circulating in paediatric age in order to detect novel viral variants able to spread in the general population.

Molecular Epidemiology and Clinical Features of Rotavirus Infection Among Pediatric Patients in East Java, Indonesia During 2015-2018: Dynamic Changes in Rotavirus Genotypes From Equine-Like G3 to Typical Human G1/G3.

Group A rotavirus (RVA) is the most important cause of severe gastroenteritis among children worldwide, and effective RVA vaccines have been introduced in many countries. Here we performed a molecular epidemiological analysis of RVA infection among pediatric patients in East Java, Indonesia, during 2015–2018. A total of 432 stool samples were collected from hospitalized pediatric patients with acute gastroenteritis. None of the patients in this cohort had been immunized with an RVA vaccine. The overall prevalence of RVA infection was 31.7% (137/432), and RVA infection was significantly more prevalent in the 6- to 11-month age group than in the other age groups (P < 0.05). Multiplex reverse transcription-PCR (RT-PCR) revealed that the most common G-P combination was equine-like G3P[8] (70.8%), followed by equine-like G3P[6] (12.4%), human G1P[8] (8.8%), G3P[6] (1.5%), and G1P[6] (0.7%). Interestingly, the equine-like strains were exclusively detected until May 2017, but in July 2017 they were completely replaced by a typical human genotype (G1 and G3), suggesting that the dynamic changes in RVA genotypes from equine-like G3 to typical human G1/G3 in Indonesia can occur even in the country with low RVA vaccine coverage rate. The mechanism of the dynamic changes in RVA genotypes needs to be explored. Infants and children with RVA-associated gastroenteritis presented more frequently with some dehydration, vomiting, and watery diarrhea, indicating a greater severity of RVA infection compared to those with non-RVA gastroenteritis. In conclusion, a dynamic change was found in the RVA genotype from equine-like G3 to a typical human genotype. Since severe cases of RVA infection were prevalent, especially in children aged 6 to 11 months or more generally in those less than 2 years old, RVA vaccination should be included in Indonesia’s national immunization program.

Improvement of Rotavirus Genotyping Method by Using the Semi-Nested Multiplex-PCR With New Primer Set.

Rotavirus A (RVA) is a major cause of gastroenteritis in infants and young children. After vaccine introduction, RVA surveillance has become more important for monitoring changes in genotype distribution, and the semi-nested multiplex-PCR is a popular method for RVA genotyping. In particular, the VP7 primer set reported by Gouvea and colleagues in 1990 is still widely used worldwide as the recommended WHO primer set in regional and national reference RVA surveillance laboratories. However, this primer set yielded some mistakes with recent epidemic strains. The newly emerged equine-like G3 strains were mistyped as G1, G8 strains were mistyped as G3, the G9 lineage 3 strains showed very weak band, and the G9 lineage 6 strains showed a G9-specific band and a non-specific band. Gouvea’s standard protocol has become relatively unreliable for identifying genotypes correctly. To overcome this limitation, we redesigned the primer set to include recent epidemic strains. Our new primer set enabled us to correctly identify the VP7 genotypes of representative epidemic strains by agarose gel electrophoresis (G1, G2, human typical G3, equine-like G3, G4, G8, G9, and G12). We believe that the multiplex-PCR method with our new primer set is a useful and valuable tool for surveillance of RVA epidemics.

Molecular characterisation of rotavirus strains detected during a clinical trial of the human neonatal rotavirus vaccine (RV3-BB) in Indonesia

BackgroundThe RV3-BB human neonatal rotavirus vaccine aims to provide protection from severe rotavirus disease from birth. The aim of the current study was to characterise the rotavirus strains causing gastroenteritis during the Indonesian Phase IIb efficacy trial. MethodsA randomized, double-blind placebo-controlled trial involving 1649 participants was conducted from January 2013 to July 2016 in Central Java and Yogyakarta, Indonesia. Participants received three doses of oral RV3-BB vaccine with the first dose given at 0–5 days after birth (neonatal schedule), or the first dose given at ∼8 weeks after birth (infant schedule), or placebo (placebo schedule). Stool samples from episodes of gastroenteritis were tested for rotavirus using EIA testing, positive samples were genotyped by RT-PCR. Full genome sequencing was performed on two representative rotavirus strains. ResultsThere were 1110 episodes of acute gastroenteritis of any severity, 105 episodes were confirmed as rotavirus gastroenteritis by EIA testing. The most common genotype identified was G3P[8] (90/105), the majority (52/56) of severe (Vesikari score ≥11) rotavirus gastroenteritis episodes were due to the G3P[8] strain. Full genome analysis of two representative G3P[8] samples demonstrated the strain was an inter-genogroup reassortant, containing an equine-like G3 VP7, P[8] VP4 and a genogroup 2 backbone I2-R2-C2-M2-A2-N2-T2-E2-H2. The complete genome of the Indonesian equine-like G3P[8] strain demonstrated highest genetic identity to G3P[8] strains circulating in Hungary and Spain. ConclusionsThe dominant circulating strain during the Indonesian Phase IIb efficacy trial of the RV3-BB vaccine was an equine-like G3P[8] strain. The equine-like G3P[8] strain is an emerging cause of severe gastroenteritis in Indonesia and in other regions.

Spread of the emerging equine-like G3P[8] DS-1-like genetic backbone rotavirus strain in Brazil

Background: In 2013, a novel equine-like G3P[8] DS-1 like rotavirus (RVA) emerged as dominant strain worldwide. In 2016, this strain was reported in Brazil. The aims of the present study were to conduct a genetic retrospective investigation since 2013 in order to identify the possible entry of this novel G3P[8] strain in Brazil, describe its distribution across the country, help the understanding of evolutionary dynamics and potential implications in RVA vaccine programs. Methods & Materials: Between January/2013 and October/2017, a total of 4025 fecal specimens were collected from Southern, Southeastern and Midwest regions, and likely to be representative of Brazilian population. Specimens were screened for RVA using ELISA, and genotyped by RT-PCR. VP7 amplicons of positive G3P[8] samples were submitted to sequencing in order to identify the equine-like G3P[8] strains by nucleotide similarity search. Six equine-like G3P[8] representative samples were selected for investigation of the whole genome. Results: G3P[8] represented 16.7% (123/736) of all RVA-positive samples, and further divided as equine-like G3 (6.4%; 47/736) and wild-type G3 (11.5%; 85/736). During 2013-2014, wild-type G3P[8] was the dominant strain across Brazil, and no equine-like G3P[8] was detected. Equine-like G3P[8] strain was first identified in March/2015 in Paraná State, suggesting that the strain stepped into Brazil through Southern region. The equine-like G3P[8] circulated only in Paraná till February/2016, but rapidly spread across the country after that, reaching Amazonas in March, Goiás in July and the Federal District in September. Equine-like G3P[8] strain reached São Paulo in August/2017. No wild-type G3 was detected since March/2015, therefore, it could be speculated that the atypical G3P[8] genotype was able to completely replace wild-type G3P[8] strains. Whole genome analysis revealed a DS-1 like genotype constellation (G3-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2), and segments shared the highest nucleotide sequence identity with strains isolated in Brazil and Spain. Conclusion: This study highlights the emergence of the atypical equine-like G3P[8] genotype circulating in Brazil, and reinforces the potential for novel human/animal reassortant to arise within human population. Equine-like G3P[8] DS-1-like strains pose a challenge for the RVA surveillance, once G/P typing characterization by RT-PCR is unable to identify this intergenogroup reassortant without inclusion of sequencing analysis.