Surveillance and molecular characterization of rotavirus strains circulating in Manipur,North-Eastern India: Increasing prevalence of emerging G12 strains

To determine the frequency and genotypes of rotavirus strains, samples were collected from children hospitalized with acute diarrhea at the Regional Institute of Medical Sciences, Manipur. The globally common genotypes G1P[8] and G2P[4] constituted 58% of the total positive strains, while 3% and 8% strains were emerging genotypes, G9P[6] and G12P[6]. This is the first report of genotype G12 in Manipur. The G12 strains clustered with lineage III strains and had >98% identity with corresponding rotaviruses from Bangladesh, Thailand and the USA. Other uncommon G–P combinations including G4P[4], G4P[6], G10P[6] and G9P[19], along with a few strains that could not be typed were also found. The VP7 genes of G4 and G10 strains clustered with porcine and bovine strains, indicating possible zoonotic transmission. High frequency (36–62%) of rotavirus infection and predominance of G1P[8] and G2P[4] among children with acute diarrhea emphasized the need for implementation of currently available vaccines to reduce the burden of rotavirus induced diarrhea in India.     

Distribution of rotavirus genotypes in Dhaka,Bangladesh, 2012–2016: Re-emergence of G3P[8] after over a decade of interval

Group A rotavirus causes a substantial proportion of diarrhoea related deaths worldwide among children under five years. We analyzed rotavirus prevalence and genotypes distribution among patients admitted with diarrhoea at icddr,b hospital in Dhaka during 2012–16. Stool specimens (n = 1110) were collected from diarrhoea patients and tested for RVA antigen using enzyme immunoassay. Rotavirus positive samples were G (VP7) and P (VP4) genotyped by RT-PCR and sanger sequencing. Data on clinical manifestations were collected from icddr,b hospital surveillance system. A total of 351 (32%) patients were positive for rotavirus antigen, about half of those were children under two years old. During the study period, G1P[8] (27%) was the most prevalent strain, followed by G12P[8] (15%) and G9[P4] (9%). Mixed G or P genotypes were identified in a substantial proportion (23%) with few strains of rare combinations such as G1P[4], G1P[6], G2P[6], G2P[8], G9P[6]. The genotypic fluctuation was noteworthy; G12P[8] was the major strain in 2012–14 but sharply decreased in 2015–16 when G1P[8] became the most common strain. G3P[8] re-emerged (17%) in 2016 after 11 years. Since the Government of Bangladesh has planned to include rotavirus vaccine in national immunization programme from 2018, our data will provide baseline information on rotavirus genotypes in the pre-vaccination era to observe the selection pressure on genotypes in the post vaccination epoch.     

The prevalence and genotype diversity of Human Rotavirus A circulating in Thailand, 2011–2014

Human rotavirus A (RVA) is the major infectious virus causing acute watery diarrhea in children, especially those younger than 5 years of age, and is a major public health problem in Thailand. Outbreaks of this virus have been reported worldwide. Besides the common genotypes, unusual genotypes providing evidence of inter-species transmission have also been described. Therefore, the aim of this study was to investigate the prevalence and genotypes of RVA in Thailand. A total of 688 samples were collected from children who were hospitalized with acute diarrhea in Chumphae Hospital in Khon Kaen and Chulalongkorn Hospital in Bangkok. RVA was detected using one-step RT-PCR and the genotypes were evaluated by sequencing. Overall, 204 of the 688 samples (30%) were positive for RVA. Nine genotypes were identified: three common in humans (G1P[8] [53%], G2P[4] [18%], G3P[8] [12%]), one feline-like (G3P[9] [1%]), four porcine-like (G4P[6] [0.5%], G5P[6] [0.5%], G9P[8] [0.5%], G12P[6] [1.5%]), and one bovine-like (G8P[8] [13%]). The variation in virus genotypes and the animal-like genotypes detected in this study suggested that a high diversity of RVA types is circulating in the Thai population. Therefore, continuous molecular epidemiological monitoring of RVA is essential and has implications for the national vaccination program.     

Characterization of human rotaviruses circulating in Iraq in 2008: Atypical G8 and high prevalence of P[6] strains

Fecal samples from 976 children with gastroenteritis were collected and analyzed for group A rotavirus (RVA), in three different cities in Iraq between January 2008 and December 2008. RVA antigen was detected in 394 (40%) of the samples, and 98 samples were available for further genotype analyses using multiplex RT-PCR and sequence analyses for untypeable strains. The G/P-genotype combination was determined for 69 samples, and 19, 2 and 8 samples remained P-untypeable, G-untypeable and G/P-untypeable (UT), respectively. The most prevalent genotype was G2 (40%, 39/98) most often associated with P[6]. G1 was the second most common genotype (16%, 16/98) mainly associated with P[8] and P[UT]. G3, G4 and G9 were detected at a lower prevalence (3%, 11%, 3%, respectively), mainly associated with P[6]. Surprisingly, five G8P[6], and seven G12 RVA strains in combination with P[6] and P[8] were also detected for the first time in Iraq. Overall, a striking high prevalence of 47% of the analyzed samples possessed the P[6] genotype (65% of the P-typed RVA strains). Atypical genotype combinations such as G1P[4], G1P[6], G2P[8] or strains with mixed G-types were detected sporadically. The detection of unusual G8P[6] RVA strains prompted us to further analyze the NSP2, NSP3, NSP4 and NSP5 gene segments of three selected G8P[6] strains, resulting in their designation to the N2, T2, E2 and H2 genotypes, respectively. The VP7, VP4, NSP2, NSP3 and NSP5 gene segments clustered closely with common human RVA strains, whereas the NSP4 gene sequences were found to cluster with animal derived RVA strains, suggesting a potential reassortment event. The high prevalence of RVA strains with the G8, G12 and P[6] genotypes in combination with a DS-1-like genotype constellation in Iraq, needs to be monitored closely as these RVA strains might challenge the effectiveness of current RVA vaccines.     

Molecular characterization of rotaviruses circulating in the population in Turkey

Of a total of 508 stool specimens from children with acute diarrhoea, mostly under the age of 5 years, collected in nine cities in the western and southeastern regions of Turkey between May 2000 and October 2002, 119 (23.4%) were found positive for rotaviruses (RV) by ELISA. Positive samples were characterized by electropherotyping and G and P genotyping. A subset of G and P types were confirmed by nucleic acid sequencing. The most prevalent types found in this collection included G4P[8], accounting for 27/64 (42.2%) of the fully characterized strains. G1P[8], G2P[4] and G3P[8] were found in 17 (26.6%), 2 (3.1%) and one (1.5%) samples respectively. Less common strains such as G9P[8] were found in two (3.2%) samples and G2P[8], G1P[6], G2P[6] and G4P[6], possible reassortant viruses, were found in five (7.8%), 2 (3.1%), one (1.5%) and four (6.3%) samples respectively. Mixed infections were found in six (7.3%) samples and were associated with combinations of G1 + G2, G1 + G4, G1 + G9 and G4 + G9 strains. This is the first molecular epidemiology study of its kind to be carried out in Turkey and suggests a significant diversity of co-circulating rotavirus strains.     

Genetic diversity of species A rotaviruses detected in clinical and environmental samples,including porcine-like rotaviruses from hospitalized children in the Philippines

Rotaviruses are the major cause of severe acute diarrhea in infants and young children. Rotaviruses exhibit zoonosis and thereby infect both humans and animals. Viruses detected in urban rivers possibly reflect the presence of circulating viruses in the catchment. The present study investigates the genetic diversity of species A rotaviruses detected from river water and stool of hospitalized children with acute diarrhea in Tacloban City, the Philippines. Species A rotaviruses were detected by real-time RT-PCR and their genotypes were identified by multiplex PCR and sequencing of partial regions of VP7 and VP4. Rotaviruses were detected in 85.7% (30/35) of the river water samples and 62.7% (151/241) of the clinical samples. Genotypes of VP7 in the river water samples were G1, G2, G3, G4, G5, and G9, and those of VP4 were P[3], P[4], P[6], P[8], and P[13]. Genotypes of viruses from the clinical samples were G2P[4], G1P[8], G3P[8], G4P[6], G5P[6], and G9P[8]. Among those, G2P[4] in clinical samples (77.9%, 81/104) and P[4] of VP4 in river water samples (67.5%, 56/83)) were the most frequently detected rotavirus genotypes. However, G5 was the more frequently detected than G2 in the river water samples (42% vs. 13%) which may be originated from porcine rotavirus. Sequence analyses of eleven gene segments revealed one G5P[6] and two G4P[6] rotaviruses in the clinical samples, wherein, several gene segments were closely related to porcine rotaviruses. The constellation of these rotavirus genes suggests the emergence of reassortment between human and porcine rotavirus due to interspecies transmission.Although two commercial rotavirus vaccines are available now, these vaccines are designed to confer immunity against the major human rotaviruses. Constant monitoring of viral variety in populated areas where humans and domestic animals live in close proximity provides vital information related to the diversity of rotaviruses in a human population.     

Rotavirus strain diversity in Eastern and Southern African countries before and after vaccine introduction

BackgroundThe African Rotavirus Surveillance Network has been detecting and documenting rotavirus genotypes in the African sub-continent since 1998 in anticipation of the rollout of rotavirus vaccination in routine Expanded Programme on Immunisation. This paper reports distribution of the rotavirus strains circulating in 15 Eastern and Southern African (ESA) countries from 2010-2015 as part of active World Health Organization (WHO) rotavirus surveillance, and investigates possibility of emergence of non-vaccine or unusual strains in six selected countries post-vaccine introduction.Material and methodsStool samples were collected from children <5 years of age presenting with acute gastroenteritis at sentinel hospitals pre- and post-rotavirus vaccine introduction. Samples were tested for group A rotavirus using an enzyme immunoassay by the national and sentinel laboratories. At the WHO Rotavirus Regional Reference Laboratory in South Africa, molecular characterisation was determined by PAGE (n = 4186), G and P genotyping (n = 6447) and DNA sequencing for both G and P types (n = 400).ResultsThe six-year surveillance period demonstrated that 23.8% of the strains were G1P[8], followed by G2P[4] (11.8%), G9P[8] (10.4%), G12P[8] (4.9%), G2P[6] (4.2%) and G3P[6] (3.7%) in 15 ESA countries. There was no difference in circulating strains pre- and post-rotavirus vaccine introduction with yearly fluctuation of strains observed over time. Atypical rotavirus G and P combinations (such as G1P[4], G2P[8], G9P[4] and G12P[4]) that might have arisen through inter-genogroup or inter-genotypes reassortment were detected at low frequency (2%). Close genetic relationship of African strains were reflected on the phylogenetic analysis, strains segregated together to form an African cluster in the same lineages/sub-lineage or monophyletic branch.ConclusionThere has been considerable concern about strain replacement post-vaccine introduction, it was not clear at this early stage whether observed cyclical changes of rotavirus strains were due to vaccine pressure or this was just part of natural annual fluctuations in the six ESA countries, long-term surveillance is required.     

Molecular and epidemiological trend of rotavirus infection among infants and children in Japan

This study investigated the prevalence of group A, B, and C rotavirus (RAV, RBV, RCV) and adenovirus (AdV) infections in infants and children with acute gastroenteritis in Japan from July 2006 to June 2007. A total of 628 fecal specimens collected from infants and children with acute gastroenteritis in five different places (Maizuru, Tokyo, Sapporo, Saga and Osaka) in Japan during the period of July 2006–June 2007 were examined for RAV, RBV, RCV and AdV by RT-multiplex PCR. RAV was further characterized to G-typing and P-typing by RT-multiplex PCR and sequencing method. It was found that 123 (19.6%) fecal specimens were positive for RAV followed by AdV of 4.5%. RBV and RCV could not be detected in this study. Serotype G1 (58.5%) was identified at high levels followed by G9 (20.3%), G2 (11.4%), and G3 (7.3%). P genotyping revealed P[8] as predominant (84.6%) followed by P[4] (13.8%) and P[6] (1.6%). During the 2006/2007 rotavirus season, G1P[8] strains were most common with G9P[8], G2P[4], G3P[8], G1P[4] and G9P[6] also detected. It is clear from this study that RAV is still the cause of diseases in Japan. To our knowledge, this is the first report of RV P[6] strain in humans in Japan.     

Epidemiological features and genetic characterization of virus strains in rotavirus associated gastroenteritis in children of Odisha in Eastern India

We have studied the clinical characteristics, severity and seasonality of rotavirus infection and prevalent genotypes in 652 non-rota vaccinated children in Odisha in eastern India. P genotypes were analysed for their association with host blood group antigens. P type of the virus is determined by the VP8* gene, and specific recognition of A - type of Histo - blood group antigen by P[14]VP8* has been reported. VP4, VP7 and VP6 genes of commonly identified G1P[8] strain were compared with genes of the same strain isolated from other parts of India, elsewhere and strains used for Rotarix and Rotateq vaccines.In 54.75% of children with gastroenteritis, rota virus was found. 9.65% of children had moderate, 78.07% severe, and 12.28% very severe disease as assessed using the Vesikari scoring system. The incidence of infection was highest during winter months. There was no association between any blood group and specific P genotypes. G1P[8] was the commonest cause of gastroenteritis, followed by G1P[11], G3P[8], G9P[8], G2P[4], G2P[6], G9P[4], G9P[11] and G1P[6]. Predominant G genotypes identified were G1 (72.9%), G9 (10.81%), G2 (8.10%) and G3 (8.10%). Sequence analysis of the VP7 gene, placed the G1P[8] strain in lineage 1 and of VP6 gene placed nine G1P[8] strains in subgroup II and one in subgroup I. The VP7 gene segment of two Odisha G1P[8] strains were found to cluster relatively close to the VP7 sequences of Rotarix vaccine. Antigenic differences were found with vaccine strains. Ten G1P[8] strains sequenced for the VP4 gene had 91–93% nucleotide and 92–96% amino acid identity with Rotateq vaccine P[8]). Rotarix vaccine VP4 had 89–91% nucleotide and 90–92% amino acid identity. Our findings indicate genetic variability of rotavirus strains circulating in the region and are significant, given the introduction of rota vaccination in the State.     

Prevalence and phylogenetic analysis of rotavirus genotypes in Thailand between 2007 and 2009

Rotaviruses are the most common cause of severe diarrhea among infants and young children worldwide, especially in developing countries. In Thailand, rotavirus has presented a major public health problem causing severe diarrhea in infants and young children. It was responsible for about one-third of diarrheal diseases in hospitalized patients. In this study, we have analyzed the distribution and performed molecular characterization of rotaviruses circulating in infants and young children with diarrhea admitted to the city and rural hospitals in Thailand between July 2007 and May 2009. Group A human rotavirus was detected in 158 (28.4%) of 557 fecal specimens by RT-PCR. The peak incidence of infection was found in the winter months between December and March. The G1P[8] strain was identified as the most prevalent (49.4%) followed by G9P[8] (22.2%), G2P[4] (20.2%) and G3P[8] (0.6%). The uncommon strains G12P[8], G12P[6] and G3P[9] were also detected. Phylogenetic analysis of selected G and P genotypes isolated in this study was performed to compare with the reference strains from different countries. Emergence of G12 in the northern part of Thailand was observed and phylogenetic analysis demonstrated close relation between Thai isolates and strains from India. The present study reveals the recurring changing genotypes of rotavirus circulating in Thailand. The genetic association between isolates from Thailand and other countries ought to be considered with regard to local and global dissemination of rotavirus as it is crucial for prevention especially, with respect to vaccine implementation.     

Analysis of rotavirus genotypes in Korea during 2013: An increase in the G2P[4] genotype after the introduction of rotavirus vaccines

Background

Group A rotavirus is the leading cause of acute gastroenteritis in children worldwide. We investigated G and P genotypes of group A rotavirus strains isolated from patients during 2013 and investigated which genotypes were identified from vaccinated patients.

Methods

From January to December 2013, 2235 fecal specimens were tested for rotavirus antigen, of which 374 specimens (16.7%) showed positive results. Strains from 288 rotavirus-positive specimens were genotyped using PCR and sequencing, and individual patients’ corresponding vaccine histories were investigated through the Korean Center for Disease Control website.

Results

G2 (22.6%) and P[4] (24.0%) were the most frequently identified G and P genotypes, respectively; accordingly, G2P[4] (19.8%) was the most prevalent G/P genotype observed in this period. G4P[6] (10.1%) was the second most prevalent G/P genotype and was mostly detected in neonates. Other genotypes, G1P[8], G9P[8], G1P[6], and G3P[6], were also detected. Of 288 rotavirus-positive specimens, 48 specimens were obtained from previously vaccinated patients. G2P[4] was also the genotype most frequently isolated from vaccinated patients. VP7 epitope analysis of G1P[8] and G2P[4] strains showed at least one amino acid differences in comparison with Rotarix and RotaTeq vaccine strains. The genotypic distribution of rotavirus strains in Korea has been shown temporal and geographical differences.

Conclusion

This study showed that G2P[4] was the genotype most frequently isolated from both vaccinated and unvaccinated patients in Korea during 2013. However, it is unclear whether the change of predominant genotype is due to the effect of vaccination or due to natural variation.     

A decade of G3P[8] and G9P[8] rotaviruses in Brazil: Epidemiology and evolutionary analyses

This study aims to estimate the frequency of group A rotaviruses (RVA) infection with genotypes G3P[8] and G9P[8] in children that suffered from diarrheal disease (DD) between 2001 and 2011 in different Brazilian regions. In addition, the genetic diversity of G3P[8] and G9P[8] RVA strains recovered from vaccinated and non-vaccinated children was assessed. Laboratory-based RVA surveillance included 15,115 cases of DD, and RVA was detected by enzyme immune-assay and/or polyacrylamide gel electrophoresis in 3357 (22%) samples. RVA was genotyped by the semi-nested RT-PCR and among RVA-positive samples, 100 (2.9%) were G3 (63 G3P[8], 32 G3P not typed [NT], and 5 G3P[6]) and 378 (16.2%) were G9 (318 G9P[8], 59 G9P[NT], and 1 G9P[6]). From the G3 and G9 positive samples, 16 and 12, respectively, were obtained from children aged 4–48 months vaccinated with the monovalent vaccine (Rotarix®, RV1). Phylogenetic analyses of the VP7 and VP8¹ encoding genes were performed for 26 G3P[8] and 48 G9P[8] strains. VP8¹ phylogenetic analysis revealed that all strains analyzed belonged to P[8] lineage III, whereas RV1 belongs to P[8]-I lineage. VP7 analysis revealed that all G3 and G9 strains belonged to G3-lineage III and G9-lineage III. The comparison of the VP7 and VP8¹ antigenic epitopes regions of Brazilian strains with RV1 strain revealed several amino acid changes. However, no particular differences among Brazilian strains detected before and after vaccine introduction were observed, or among strains detected from vaccinated and non-vaccinated children. Complete genome characterization of four G3P[8] and seven G9P[8] strains revealed a typical conserved human Wa-like genomic constellation. Changes in the genetic diversity of G3P[8] and G9P[8] RVA detected from 2001 to 2011 in Brazil seemed not be related to RV1 introduction in Brazil.     

Rotavirus strain distribution in Ghana pre- and post- rotavirus vaccine introduction

BackgroundGhana introduced the monovalent rotavirus vaccine (Rotarix) into its national paediatric vaccination programme in May2012. Vaccine introduction was initiated nationwide and achieved >85% coverage within a few months. Rotavirus strain distribution pre- and post-RV vaccine introduction is reported.MethodsStool samples were collected from diarrhoeic children <5 years of age hospitalized between 2009 and 2016 at sentinel sites across Ghana and analyzed for the presence of group A rotavirus by enzyme immunoassay. Rotavirus strains were characterized by RT-PCR and sequencing.ResultsA total of 1363 rotavirus EIA-positive samples were subjected to molecular characterization. These were made up of 823 (60.4%) and 540 (39.6%) samples from the pre- and post-vaccine periods respectively. Rotavirus VP7 genotypes G1, G2 and G3, and VP4 genotypes P[6] and P[8] constituted more than 65% of circulating G and P types in the pre–vaccine period. The common strains detected were G1P[8] (20%), G3P[6] (9.2%) and G2P[6] (4.9%).During the post-vaccine period, G12, G1 and G10 genotypes, constituted more than 65% of the VP7 genotypes whilst P[6] and P[8] made up more than 75% of the VP4 genotypes. The predominant circulating strains were G12P[8] (26%), G10P[6] (10%) G3P[6] (8.1%) and G1P[8] (8.0%). We also observed the emergence of the unusual rotavirus strain G9P[4] during this period.ConclusionRotavirus G1P[8], the major strain in circulation during the pre-vaccination era, was replaced by G12P[8] as the most predominant strain after vaccine introduction. This strain replacement could be temporary and unrelated to vaccine introduction since an increase in G12 was observed in countries yet to introduce the rotavirus vaccine in West Africa. A continuous surveillance programme in the post-vaccine era is necessary for the monitoring of circulating rotavirus strains and the detection of unusual/emerging genotypes.     

Impact of rotavirus vaccine on all-cause diarrhea and rotavirus hospitalizations in Madagascar

BackgroundRotavirus vaccine was introduced into the Extended Program on Immunization in Madagascar in May 2014. We analyzed trends in prevalence of all cause diarrhea and rotavirus hospitalization in children <5 years of age before and after vaccine introduction and assessed trend of circulating rotavirus genotypes at Centre Hospitalier Universitaire Mère Enfant Tsaralalàna (CHU MET).MethodsFrom January 2010 to December 2016, we reviewed the admission logbook to observe the rate of hospitalization caused by gastroenteritis among 19619 children <5 years of age admitted at the hospital. In June 2013–December 2016, active rotavirus surveillance was also conducted at CHUMET with support from WHO. Rotavirus antigen was detected by EIA from stool specimen of children who are eligible for rotavirus gastroenteritis surveillance at sentinel site laboratory and rotavirus positive specimens were further genotyped at Regional Reference Laboratory by RT-PCR.ResultsDiarrhea hospitalizations decreased after rotavirus vaccine introduction. The median proportion of annual hospitalizations due to diarrhea was 26% (range: 31–22%) before vaccine introduction; the proportion was 25% the year of vaccine introduction, 17% in 2015 and 16% in 2016. Rotavirus positivity paralleled patterns observed in diarrhea. Before vaccine introduction, 56% of stool specimens tested positive for rotavirus; the percent positive was 13% in 2015, 12% in 2016. Diverse genotypes were detected in the pre-vaccine period; the most common were G3P[8] (n = 53; 66%), G2P[4] (n = 12; 15%), and G1P[8] (n = 11; 14%). 6 distinct genotypes were found in 2015; the most common genotype was G2P[4] (n = 10; 67%), the remaining, 5, G12[P8], G3[P8], G1G3[P4], G3G12[P4][P8] and G1G3[NT] had one positive specimen each.ConclusionsFollowing rotavirus vaccine introduction all-cause diarrhea and rotavirus-specific hospitalizations declined dramatically. The most common genotypes detected in the pre-vaccine period were G3P[8] and G2P[4] in 2015, the post vaccine period.     

Emergence of unusual G6P[6] rotaviruses in children, Burkina Faso, 2009-2010

To obtain more information about rotavirus (ROTAV) genotypes in Burkina Faso, we characterized 100 ROTAVs isolated from fecal samples of children with acute gastroenteritis in the capital city of Ouagadougou, during December 2009-March 2010. Of note, 13% of the ROTAV-positive samples, including those with mixed infections, were positive for the unusual G6 genotype ROTAV strain. The genotypes identified were G9P[8], G6P[6], G1P[6], G3P[6], G1P[8], and G2P[4]. G9P[8] subgroup (SG)II strains dominated during the beginning of the ROTAV season, but later in the season, other G types associated with P[6] and SGI specificity emerged. This emergence was related to a shift in the overall age of infected children; ROTAV SGII infected younger children and induced more severe symptoms. The finding of a high incidence of G6P[6] strains highlights the need for long-term surveillance of ROTAV strains in Burkina Faso, especially when ROTAV vaccination is being considered in several African countries.