RVA Vaccines Research Articles

Epidemiology and Genetic Diversity of Rotavirus in Kunming, China, in 2015

Group A rotavirus (RVA) is a serious public problem in China, with a prevalence of 29.7% in diarrhea cases under 5 years of age. A total of 1,224 fecal specimens were collected in 2015 from children under 5 years of age with acute gastroenteritis in Kunming, China, for detection and characterization of rotavirus. The prevalence of RVA was 28.5%. The frequency of RVA detection was greatest (52%) among children aged 7–12 months. The following strains were the most common types: G9P(8) (58.2%), G3P(8) (14.9%), and G1P(8) (6.9%). The following strains were uncommon types: G3P(4), G8P(9), and G9P(4) (1.1%); G1P(4), G2P(8), and G4P(8) (0.9%); G2P(6), G3P(9), G4P(6), and G9P(6) (0.6%); and G4P(4) (0.3%). G3G1P(8) (5.2%), G1G2P(4) (0.9%), G1G9P(8) (0.6%), and G2P(4)P(8) (0.3%) were mixed types. This study documents the molecular epidemiology, genetics, and diversity of rotavirus strains in children under 5 years of age in Kunming, China, and suggests that it may be important to offer RVA vaccination and scientific evidence to stop and control RVA-related diarrhea.

Molecular analysis of group A rotaviruses detected in hospitalized children from Rawalpindi, Pakistan during 2014

As a part of strategy to control diarrheal diseases, World Health Organization (WHO) recommends to include rotavirus vaccines in national immunization programs. Sentinel surveillance networks have been established to monitor rotavirus disease burden and genotype distribution in both pre and post vaccine era in many countries. Unfortunately, due to lack of proper surveillance programs, data on rotavirus disease burden and genotype distribution from Pakistan is scarce. We investigated 502 stool samples from children (<5years) hospitalized due to gastroenteritis in Rawalpindi, Pakistan during 2014 for the presence of group A rotavirus (RVA) and its genotypic diversity. Among 147 ELISA positive samples, 131 were successfully genotyped for RVA. Common G types detected were G1 (23.6%), followed by G3 (22.9%), G12 (19.8%), G2 (19.08%) and G9 (9.9%). The most common P-type was P[8] (41.2%), followed by P[6] (29%) and P[4] (28.24%). G3P[8] (17.55%) was the most prevalent genotype combination followed by G12P[6] (16.7%), G2P[4] (15.2%) and G1P[8] (14.5%). Mixed infection of rotavirus G-P types was also observed in 6% of samples. Phylogenetic analysis of VP7 and VP4 genes of Pakistani strains showed that G1, G2, G9 and P[4], P[6], P[8] were closely related to strains circulating worldwide as well as previously reported strains from Pakistan. Pakistani G12P[8] strains NIH-BBH-3981 and NIH-BBH-4003 belonged to lineage 3 cluster 3a along with strains from USA and Italy whereas G12P[6] strains NIH-BBH-3978, NIH-BBH-4052 and NIH-BBH-4444 were closely related to strains from Italy, Thailand, United Kingdom and with previously reported G12 strains from Pakistan within lineage 3 cluster 3b. This pre-vaccination data supports the need for RVA vaccine inclusion at our national level and will be helpful in assessing the effect of vaccination on RVA genotype diversity due to vaccine selection pressure once post-vaccination data becomes available.

Putative vaccine breakthrough event associated with heterotypic rotavirus infection in newborn calves, Turkey, 2015

Group A rotaviruses (RVA) are regarded as major enteric pathogens of large ruminants, including cattle. Rotavirus vaccines administered to pregnant cows are commonly used to provide passive immunity that protects newborn calves from the clinical disease. In this study we report the detection of RVA from calves with severe diarrhea in a herd regularly vaccinated to prevent enteric infections including RVA. Diarrheic disease was observed in newborn calves aged 4–15days, with high morbidity and mortality rates, but no diarrhea was seen in adult animals. Rotavirus antigen was detected by enzyme-immunoassay in the intestinal content or the fecal samples of all examined animals. Besides RVA, bovine coronavirus and bovine enteric calicivirus were detected in some samples. Selected RVA strains were characterized by whole genome sequencing. Two strains, RVA/Cow-wt/TUR/Amasya-1/2015/G8P[5] and RVA/Cow-wt/TUR/Amasya-2/2015/G8P[5] were genotyped as G8-P[5]-I2-R2-C2-M2-A3-N2-T6-E2-H3 and showed >99% nucleotide sequence identity among themselves. This genomic constellation is fairly common among bovine RVA strains; however, phylogenetic analysis of the G8 VP7 gene showed close genetic relationship to some European human RVA strains (up to 98.4% nt identity). Our findings is the first indication regarding the circulation of G8 RVA strains in Turkey. Given that the administered RVA vaccines contained type G6 and G10 VP7 antigens some concerns raised with regard to the level of heterotypic protection elicited by the vaccine strains against circulating bovine G8 RVA strains. Enhancement of surveillance of circulating RVA strains in calves across Turkey is needed to support ongoing vaccination programs.

Outbreak of G2P[4] rotavirus gastroenteritis in a retirement community, Brazil, 2015: An important public health risk?

The present study described a group A rotavirus (RVA) outbreak in an age-care facility in Brazil, using epidemiologic and molecular diagnostic methods. A descriptive clinical, epidemiological and environmental investigation was conducted. Stool samples were collected and screened for RVA, Norovirus (NoV), Enteric Adenovirus 40/41 (AdV 40/41) and Astrovirus (AstV) using ELISA, RT-PCR, qRT-PCR, electron microscopy and sequencing methods. Outbreak occurred during 26th–29th October, 2015; 28 individuals affected (22 residents; 6 staff). The attack rate was 25.9% and 8.5% among residents (median-age: 85.5 years) and staff (median-age: 28 years), respectively. Female staff was identified as the index case. RVA G2P[4] genotype was detected in 87.5% (7/8). Genetic analysis demonstrated that the outbreak involved one single strain, suggesting a common-source infection. RVA should be considered during outbreaks investigations in residential facilities, and raise the question if the current licensed RVA vaccines for children could also be helpful for the elderly.

Analysis of a genotype G3P[9] rotavirus a strain that shows evidence of multiple reassortment events between animal and human rotaviruses.

The species A rotaviruses (RVA) are important gastroenteric pathogens that infect humans and animals. RVA genotype G3P[9] has been described in human-animal reassortment events, and the complexity of its hosts motivates the genetic investigation of this strain. Therefore, the aim of this study is to analyse a G3P[9] sample that was detected in a child with acute gastroenteritis. The 1A3739 sample featured the constellation G3P[9]-I18-R3-C3-Mx-A19-N3-T3-E3-H6. The sequence for VP3 gene was not obtained. The phylogeny showed a closer relationship among genes VP7, VP1, NSP3, NSP4, and NSP5 with genes of animal origin, such as chiropter, alpaca, equine, and simian. In addition, the genes VP6 and NSP1 belong to the new genotypes I18 and A19, respectively. The emergence of strains such as these can interfere with the effectiveness of the RVA vaccine, and continuous monitoring is therefore important. Additional studies are needed to determine the evolutionary source and to identify a possible reservoir of RVA in nature.

VP7 and VP8* genetic characterization of group A rotavirus genotype G12P[8]: Emergence and spreading in the Eastern Brazilian coast in 2014.

Group A rotavirus (RVA) genotype G12 is habitually associated with diarrhea disease (DD) in African children and recently its detection has increased worldwide. A total of 970 stool samples collected from individuals with DD in the Northeastern, Southeastern, and Southern Brazilian regions, Eastern coast, were analyzed and 321 (33%) were positive for RVA and of these, 241 (75%) genotyped as G12P[8]. The rate of RVA positivity was higher among children aged 5-10 years old (60%). All RVA infections observed in adults aged >21 years were G12P[8] (n = 27) showing that this genotype affected older age groups during the year of 2014 in Brazil. Phylogenetic analysis of VP7 and VP8* G12P[8] strains demonstrated an elevated similarity among Brazilian and G12-III prototypes strains circulating worldwide recently, suggesting that this lineage is associated with the global spread of the G12 genotype, considered as the 6th most prevalent human RVA genotype nowadays; while other G12 lineages remain sporadically detected and usually detected in association with other P genotypes. VP8* analysis revealed that Brazilian strains belong to P[8]-3 lineage, the single P[8] lineage presently detected in the country. No major nucleotide/amino acid disparities were observed among strains recovered from children and adults for VP7 and VP8* genes. These data are essential to support the surveillance studies, particularly in countries where the RVA vaccine was introduced in their National Immunization Program enabling identification of potential alterations in the epidemiological profile that can impact its efficacy in vaccination programs. J. Med. Virol. 89:64-70, 2017. © 2016 Wiley Periodicals, Inc.

Epidemiology and genetic diversity of group A rotavirus in acute diarrhea patients in pre-vaccination era in southwest China.

Group A rotavirus (RVA) is one of the leading cause of acute diarrhea worldwide, the RVA-related disease burden and the genotypes of RVA is important reference to introduce RVA variance to national immunisation programmes, 1,121 diarrhea cases and 319 healthy controls were recruited from four sentinel hospital outpatient from July 2014 to June 2015. The prevalence of RVA was 244 (21.8%) in gastroenteritis cases and in 12 (3.8%) in healthy controls across all age group (OR = 7.12, 95%CI = 3.93-12.89); the detection rate of RVA in diarrhea patients under 5 years was more higher than in diarrhea cases over 5 years (26.1%, 222/850; 8.1%, 22/271, respectively, P = 0.000). Of 244 RVA strains isolated from acute diarrhea cases, G9 (66.4%) was predominant G genotype, followed by G3 (18.7%), G1 (8.9%), and G1G3 (3.8%); P[8] was the overwhelming prevalence P genotype, followed by P[4] (4.7%); G9P[8] (54.9%) was most common G and P Combination, followed by G3P[8] (17.6%) and G1[8] (8.6%). The conclusion of the study was important to provide reference for introducing the RVA vaccine to prevent and control RVA-associated disease burden. J. Med. Virol. 89:71-78, 2017. © 2016 Wiley Periodicals, Inc.

A non-enteric adenovirus A12 gastroenteritis outbreak in Rio de Janeiro, Brazil.

A gastroenteritis outbreak that occurred in 2013 in a low-income community in Rio de Janeiro was investigated for the presence of enteric viruses, including species A rotavirus (RVA), norovirus (NoV), astrovirus (HAstV), bocavirus (HBoV), aichivirus (AiV), and adenovirus (HAdV). Five of nine stool samples (83%) from patients were positive for HAdV, and no other enteric viruses were detected. Polymerase chain reaction products were sequenced and subjected to phylogenetic analysis, which revealed four strains and one strain of non-enteric HAdV-A12 and HAdV-F41, respectively. The HAdV-A12 nucleotide sequences shared 100% nucleotide similarity. Viral load was assessed using a TaqMan real-time PCR assay. Stool samples that were positive for HAdV-A12 had high viral loads (mean 1.9 X 107 DNA copies/g stool). All four patients with HAdV-A12 were < 25 months of age and had symptoms of fever and diarrhoea. Evaluation of enteric virus outbreaks allows the characterisation of novel or unique diarrhoea-associated viruses in regions where RVA vaccination is routinely performed.

Detection of the emerging rotavirus G12P[8] genotype at high frequency in brazil in 2014: Successive replacement of predominant strains after vaccine introduction

The continuum characterization of rotavirus (RVA) genotypes is essential to understand how vaccine introduction could impact virus epidemiology. In the present study, an unexpected rapid changing pattern of RVA genotypes distribution in Brazilian population during three followed seasons is described. From January/2012 to December/2014, a total of 3441 fecal specimens were collected from collaborating centers across Southern, Southeastern and Midwest of Brazil. All specimens were screened for RVA using ELISA, and genotyped by RT-PCR. Differences in proportions were tested using Chi-Squares. A p-value of less than 0.05 was considered statistically significant. RVA was detected in 19.7% (677/3441). Among RVA positive cases (n=677), a total of 652 (96.3%) samples were successfully amplified by RT-PCR. G3P[8] remained prevalent in 2012 (37.6%, 69/185) and 2013 (40.1%, 74/186) (χ2=0.107, p=0.743), but declined markedly in 2014 (3.5%, 10/281) (χ2=71.770, p=0.000). G12P[8] was second highest strain in 2012 (22.7%, 42/185), decrease rapidly in 2013 (2.7%, 5/186) (χ2=26.224, p=0.000) and re-emerged as the predominant genotype in 2014 (86.6%, 243/281) (χ2=118.299, p=0.000). From July/2014, G12P[8] was the single genotype detected in all regions studied. The sudden emergence, spread and predominance of G12P[8] strain in Brazil, raised the hypothesis of a possible G12 outbreak being in progress. Nationally, the long term decline in gastroenteritis hospitalization observed in the country after RVA vaccine introduction was confirmed. Nevertheless, the sharp increase in diarrhea hospitalization prevalence from 2013 to 2014 observed in Southern and Southeastern regions is consistent with what appears to be an outbreak of G12P[8]. Continued surveillance is needed to verify the effectiveness of the RotarixTM vaccine in Brazil together with potential emergence of unusual genotypes.

Rotavirus genotypes and the indigenous children of Brazilian midwest in the vaccine era, 2008-2012: Footprints of animal genome.

World group A rotavirus (RVA) surveillance data provides useful estimates of the disease burden, however, indigenous population might require special consideration. The aim of this study was to describe the results of G- and P-types from Brazilian native children ≤ 3 years. Furthermore, selected strains have been analyzed for the VP7, VP6, VP4, and NSP4 encoding genes in order to gain insight into genetic variability of Brazilian strains. A total of 149 samples, collected during 2008-2012, were tested for RVA using ELISA and PAGE, following by RT-PCR and sequencing. RVA infection was detected in 8.7% of samples (13/149). Genotype G2P[4] was detected in 2008 and 2010, G8P[6] in 2009, and G3P[8] in 2011. The phylogenetic analysis of the VP7 and VP4 genes grouped the Brazilian G2P[4] and G3P[8] strains within the lineages currently circulating in humans worldwide. However, the phylogenetic analysis of the VP6 and NSP4 from the Brazilian G2P[4] strains, and the VP7 and NSP4 from the Brazilian G3P[8] strains suggest a distant common ancestor with different animal strains (bovine, caprine, and porcine). The epidemiological and genetic information obtained in the present study is expected to provide an updated understanding of RVA genotypes circulating in the native infant population, and to formulate policies for the use of RVA vaccines in indigenous Brazilian people. Moreover, these results highlight the great diversity of human RVA strains circulating in Brazil, and an in-depth surveillance of human and animal RVA will lead to a better understanding of the complex dynamics of RVA evolution.

Rotavirus vaccine effectiveness in Latin American and Caribbean countries: A systematic review and meta-analysis

IntroductionThere are two group A rotavirus (RVA) vaccines available worldwide since 2006: monovalent (Rotarix®, RV1) and pentavalent (RotaTeq®, RV5). Currently, 16 countries and 1 territory in Latin America and the Caribbean (LAC) have introduced RVA vaccines and since their introduction several impact and effectiveness studies have been conducted in different countries. The purpose of this study was to assess RVA vaccine effectiveness in LAC countries. MethodologyWe conducted a systematic review and meta-analysis of studies in children under-five who were admitted with laboratory-confirmed RVA diarrhea. We searched Medline, WOS, LILACS, Scopus, and other sources from 2006 to October 2013. Two independent evaluators identified the studies that met predefined selection criteria and extracted relevant information according to a protocol. Pooled estimates were obtained with fixed and random-effects models and stratified according to selected effect modifiers. ResultsOf the 806 articles meeting the initial criteria, 8 case-control studies which involved 27,713 participants (6265 cases and 21,448 controls) were included in the final analyses. The pooled estimates were calculated using different types of controls, leading to different degrees of effectiveness. The effectiveness of two doses of RV1 against rotavirus-related hospitalizations ranged from 63.5% (95% CI: 39.2–78.0) to 72.2% (95%CI: 60.9–80.2). Effectiveness ranged from 75.4% (95%CI: 64.6–82.9) to 81.8% (CI 95%:72.3–88.1) among infants <12 months for RV1, and from 56.5% (95%CI: 26.2–74.3) to 66.4% (95%CI: 54.1–75.5) for infants >12 months. The RV5 effectiveness for diarrhea with a Vesikari score >11 in infants 6 to 11 months old ranged from 76.1% (95%CI: 57.6–86.6) to 88.8% (95%CI: 78.3–94.3). Also, it showed 63.5% (95%CI: 29.4–82.6) of effectiveness against G2P [4]. ConclusionRVA vaccines consistently showed protection against diarrhea-related hospitalizations in LAC. Results were more robust for RV1. Effectiveness was shown with different types of controls, but appeared somewhat higher with community controls. Effectiveness was higher among infants <12 months and lower in older children.

Unusual rotavirus genotypes among children with acute diarrhea in Saudi Arabia.

BackgroundHuman rotavirus A (human RV-A) is the most common cause of viral gastroenteritis in infants. The objective of the study was to characterize the G and P genotypes among clinical rotavirus isolates from children with acute diarrhea admitted to a tertiary care hospital in Riyadh, Saudi Arabia.MethodsFrom 2011 to 2012, 541 pediatric patients with acute diarrhea were tested for rotavirus infection. RNA extractions from the fecal specimens were done by commercial kit. RT-PCR and sequencing techniques were used to detect the prevalent genotypes. Phylogenetic analysis by Maximum Likelihood method was used to study the clustering of the circulating genotypes.ResultsThe data showed that 171/541 (31.6%) faecal samples were positive for human RVA and majority were children aged below 2 years. From the G and P [types] detected it was seen that (a) 171 minus 43 ie. 128 rotavirus positives were G typed successfully (b) 171 minus 20 ie. 151 rotavirus positives were P typed successfully; (c) overall G [P] nature was determined for 113 rotavirus positives out of 171. VP4 genotyping showed that majority of the positives 146/151 (96.7%) were P [8]; 4/151 (2.6%) were P [4]; 1/151 (0.66%) was P [6]. The dominant strains included G1P [8] 70/113 (61.9%); G9P [8] 19/113 (16.8%); G12P [8] 7/113 (6.2%) and G3P [8] 5/113 (4.4%) while the uncommon strains detected from Saudi Arabia during the study were G1P [4] 1/113 (0.88%) and G12P [6] 1/113 (0.88%). Phylogenetic tree, based on VP4/VP7 sequence analysis, revealed that G1P [8] was distinctly related to homologous strains included in human RV-A vaccine strains. Nevertheless, the uncommon genotypes G1P [4] and G12P [6] were clustered with isolates from other countries such as Bangladesh, China, Japan, Thailand and Philippines.ConclusionsMore studies will be required to further focus on newly emerging genotypes in our region together with the seasonality of rotavirus infection in the region, especially after January 2013 when the rotavirus vaccination has become part of routine childhood immunizations.

Differences in lineage replacement dynamics of G1 and G2 rotavirus strains versus G9 strain over a period of 22 years in Bangladesh

Group A rotaviruses (RVAs) have been a major cause of severe gastroenteritis in Bangladesh, mainly in children below the age of five. At the icddr,b, RVA strains collection and characterization dates back for more than 20years. This sample collection was used to study the molecular evolution of the VP7 gene of G1, G2 and G9 RVA strains, which have been circulating in Bangladesh for most of this study period. The evolutionary rates (95% HPD) for G1, G2 and G9 were calculated to be 0.93×10−3 (0.68–1.18), 1.45×10−3 (1.12–1.78) and 1.07×10−3 (0.78–1.39), respectively, which is in line with previous data for the RVA VP7 outer capsid protein, which is under strong negative selective pressure. Bayesian analyses revealed that for the G1 and G2 genotypes, one or multiple lineages co-circulated for one or a few seasons, frequently followed by replacement with genetically different lineages. This can be explained by the existence of a large variety of G1 and G2 RVA lineages and the rapid dissemination of different lineages across the globe. In contrast, circulating G9 lineages were rather closely related to each other across the study period and they were usually derived from variants circulating in the previous season(s). This is consistent with the fact that G9 RVAs have circulated in the human population for less than 20years, and therefore their genetic diversity is much smaller, not resulting in the replacement of circulating G9 strains by highly divergent G9 lineages from abroad. Such different evolutionary dynamics for different RVA genotypes may alter their response to the selective pressure that might be exerted by the introduction of RVA vaccines and therefore a continued close monitoring is warranted.

Rotavirus genotypes in Belarus, 2008–2012

This study describes group A rotavirus (RVA) genotype prevalence in Belarus from 2008 to 2012. In 2008, data from 3 sites in Belarus (Brest, Mogilev, Minsk) indicated that G4P[8] was the predominant genotype. Data from Minsk (2008–2012) showed that G4P[8] was the predominant RVA genotype in all years except in 2011 when G3P[8] was most frequently detected. Other RVA genotypes common in Europe (G1P[8], G2P[4]) were detected each year of the study. This study reveals the dominance of genotype G4P[8] in Belarus and helps to establish the baseline genotype prevalence prior to RVA vaccine introduction in the country.

Rotavirus surveillance in Kisangani, the Democratic Republic of the Congo, reveals a high number of unusual genotypes and gene segments of animal origin in non-vaccinated symptomatic children.

Group A rotavirus (RVA) infections form a major public health problem, especially in low-income countries like the Democratic Republic of the Congo (COD). However, limited data on RVA diversity is available from sub-Saharan Africa in general and the COD in particular. Therefore, the first aim of this study was to determine the genetic diversity of 99 RVAs detected during 2007–2010 in Kisangani, COD. The predominant G-type was G1 (39%) and the most predominant P-type was P[6] (53%). A total of eight different G/P-combinations were found: G1P[8] (28%), G8P[6] (26%), G2P[4] (14%), G12P[6] (13%), G1P[6] (11%), G9P[8] (4%), G4P[6] (2%) and G8P[4] (1%). The second aim of this study was to gain insight into the diversity of P[6] RVA strains in the COD. Therefore, we selected five P[6] RVA strains in combination with the G1, G4, G8 (2x) or G12 genotype for complete genome analysis. Complete genome analysis showed that the genetic background of the G1P[6] and G12P[6] strains was entirely composed of genotype 1 (Wa-like), while the segments of the two G8P[6] strains were identified as genotype 2 (DS-1-like). Interestingly, all four strains possessed a NSP4 gene of animal origin. The analyzed G4P[6] RVA strain was found to possess the unusual G4-P[6]-I1-R1-C1-M1-A1-N1-T7-E1-H1 constellation. Although the majority of its genes (if not all), were presumably of porcine origin, this strain was able to cause gastro-enteritis in humans. The high prevalence of unusual RVA strains in the COD highlights the need for continued surveillance of RVA diversity in the COD. These results also underline the importance of complete genetic characterization of RVA strains and indicate that reassortments and interspecies transmission among human and animal RVAs strains occur regularly. Based on these data, RVA vaccines will be challenged with a wide variety of different RVA strain types in the COD.

Surveillance of group A Rotavirus in Buenos Aires 2008–2011, long lasting circulation of G2P[4] strains possibly linked to massive monovalent vaccination in the region

BackgroundGroup A rotaviruses (RVA) are the most frequent single etiological agents of severe diarrhea in infants. Since 2006 RVA vaccines have been introduced in national schedules of middle and high income countries with substantial declines in rotavirus associated disease burden. However, surveillance must be maintained to, eventually, detect emerging types or variants selected by the new pressure imposed by vaccination. ObjectivesTo analyze the molecular epidemiology of group A rotavirus after vaccine introduction in the region in the context of data from more than 15 years of continuous surveillance in Buenos Aires. Study designRVA positive diarrhea samples collected in Buenos Aires from 2008 to 2011 were genotyped by RT-PCR. Selected samples were sequenced to gain insight on evolution of common and globally emerging human RVA strains. ResultsLineage III G12P[8] strain emerged in 2008 in Buenos Aires and shared co-dominancy with G3 strains during 2009. An atypical long lasting circulation of G2P[4] strains since 2004 reached rates around 80% in 2011 in Buenos Aires. Sequencing of the VP7 and VP4 genes of representative G2P[4] isolates suggests Brazil as the origin of the 2010–2011 strains. ConclusionsGlobally emergent G12 lineage III strains could be established as dominant strains in a very populated area in two years since emergence. In this work it was also shown that the persistence of G2P[4] strains during 8 years could be related to massive immunization with the monovalent vaccine in the region.

Real-time RT-PCR assays to differentiate wild-type group A rotavirus strains from Rotarix® and RotaTeq® vaccine strains in stool samples

Group A rotaviruses (RVA) are the leading cause of severe diarrhea in young children worldwide. Two live-attenuated RVA vaccines, Rotarix® and RotaTeq® are recommended by World Health Organization (WHO) for routine immunization of all infants. Rotarix® and RotaTeq® vaccines have substantially reduced RVA associated mortality but occasionally have been associated with acute gastroenteritis (AGE) cases identified in vaccinees and their contacts. High-throughput assays are needed to monitor the prevalence of vaccine strains in AGE cases and emergence of new vaccine-derived strains following RVA vaccine introduction. In this study, we have developed quantitative real-time RT-PCR (qRT-PCR) assays for detection of Rotarix® and RotaTeq® vaccine components in stool samples. Real-time RT-PCR assays were designed for vaccine specific targets in the genomes of Rotarix® (NSP2, VP4) and RotaTeq® (VP6, VP3-WC3, VP3-human) and validated on sequence confirmed stool samples containing vaccine strains, wild-type RVA strains, and RVA-negative stools. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Rotarix® NSP2 and VP4 qRT-PCR assays exhibited 92–100% sensitivity, 99–100% specificity, 94–105% efficiency, and a limit of detection of 2–3 copies per reaction. RotaTeq® VP6, VP3-WC3, and VP3-human qRT-PCR assays displayed 100% sensitivity, 94–100% specificity, 91–102% efficiency and limits of detection of 1 copy, 2 copies, and 140 copies, respectively. These assays permit rapid identification of Rotarix® and RotaTeq® vaccine components in stool samples from clinical and surveillance studies and will be helpful in determining the frequency of vaccine strain-associated AGE.

Diarrhoea-related hospitalizations in children before and after implementation of monovalent rotavirus vaccination in Mexico

To assess, by socioeconomic setting, the effect of nationwide vaccination against species A rotavirus (RVA) on childhood diarrhoea-related hospitalizations in Mexico. Data on children younger than 5 years who were hospitalized for diarrhoea in health ministry hospitals between 1 January 2003 and 31 December 2011 were collected from monthly discharge reports. Human development indexes were used to categorize the states where hospitals were located as having generally high, intermediate or low socioeconomic status. Annual rates of hospitalization for diarrhoea--per 10,000 hospitalizations for any cause--were calculated. Administrative data were used to estimate vaccine coverage. In the states with high, intermediate and low socioeconomic status, coverage with a two-dose monovalent RVA vaccine--among children younger than 5 years--had reached 93%, 86% and 71%, respectively, by 2010. The corresponding median annual rates of hospitalization for diarrhoea--per 10,000 admissions--fell from 1001, 834 and 1033 in the "prevaccine" period of 2003-2006, to 597, 497 and 705 in the "postvaccine" period from 2008 to 2011, respectively. These decreases correspond to rate reductions of 40% (95% confidence interval, CI: 38-43), 41% (95% CI: 38-43) and 32% (95% CI: 29-34), respectively. Nationwide, RVA vaccination appeared to have averted approximately 16,500 hospitalizations for childhood diarrhoea in each year of the postvaccine period. Monovalent RVA vaccination has substantially reduced childhood diarrhoea-related hospitalizations for four continuous years in discretely different socioeconomic populations across Mexico.

Recombinant Monovalent Llama-Derived Antibody Fragments (VHH) to Rotavirus VP6 Protect Neonatal Gnotobiotic Piglets against Human Rotavirus-Induced Diarrhea

Group A Rotavirus (RVA) is the leading cause of severe diarrhea in children. The aims of the present study were to determine the neutralizing activity of VP6-specific llama-derived single domain nanoantibodies (VHH nanoAbs) against different RVA strains in vitro and to evaluate the ability of G6P[1] VP6-specific llama-derived single domain nanoantibodies (VHH) to protect against human rotavirus in gnotobiotic (Gn) piglets experimentally inoculated with virulent Wa G1P[8] rotavirus. Supplementation of the daily milk diet with 3B2 VHH clone produced using a baculovirus vector expression system (final ELISA antibody -Ab- titer of 4096; virus neutralization -VN- titer of 256) for 9 days conferred full protection against rotavirus associated diarrhea and significantly reduced virus shedding. The administration of comparable levels of porcine IgG Abs only protected 4 out of 6 of the animals from human RVA diarrhea but significantly reduced virus shedding. In contrast, G6P[1]-VP6 rotavirus-specific IgY Abs purified from eggs of hyperimmunized hens failed to protect piglets against human RVA-induced diarrhea or virus shedding when administering similar quantities of Abs. The oral administration of VHH nanoAb neither interfered with the host's isotype profiles of the Ab secreting cell responses to rotavirus, nor induced detectable host Ab responses to the treatment in serum or intestinal contents. This study shows that the oral administration of rotavirus VP6-VHH nanoAb is a broadly reactive and effective treatment against rotavirus-induced diarrhea in neonatal pigs. Our findings highlight the potential value of a broad neutralizing VP6-specific VHH nanoAb as a treatment that can complement or be used as an alternative to the current strain-specific RVA vaccines. Nanobodies could also be scaled-up to develop pediatric medication or functional food like infant milk formulas that might help treat RVA diarrhea.

VP8∗P[8] lineages of group A rotaviruses circulating over 20 years in Brazil: Proposal of six different sub-lineages for P[8]-3 clade

Group A rotaviruses (RVA) is the most important cause of severe gastroenteritis among children worldwide. Vaccination is considered the best alternative among public health measures to reduce and prevent the global burden caused by RVA infections. Rotarix™, a monovalent vaccine based on a human strain with a G1P[8]-1 specificity, was introduced in the National Brazilian Immunization Programs (NIP) in March, 2006. RVA P[8] is the most prevalent P genotype worldwide and four distinct phylogenetic lineages: P[8]-1, -2, -3, and -4 have been described. In the current study phylogenetic analysis of the VP8∗ gene of 135 RVA P[8] Brazilian strains, in combination with G1, G3, G5 or G9 VP7 genotype, collected from 1986 to 2011 were carried out for a better understanding of the evolution of this viral genotype in Brazil. Lineages P[8]-1, P[8]-2, and P[8]-3 were observed circulating in Brazil. In 2001 these three P[8] lineages co-circulated simultaneously and this is the first report in South America to date. Considering the P[8] lineage and the G genotype, all G3 strains were related to lineage P[8]-3, whereas the G9 strains were related to P[8]-2 and P[8]-3 and G1 and G5 were related to P[8]-1, P[8]-2, and P[8]-3. In addition, the phylogenetic analysis based on estimate of genetic distances between P[8]-3 strains and the definition of a 1.5% cutoff value (with relevant statistical support) it was possible to propose a new classification for the P[8]-3 lineage into six different sub-lineages: P[8]-3.1 to P[8]-3.6. These findings reinforce the notion of the existence of constraints within specific RVA strains populations. The results obtained in this study reinforce the importance of a continuous RVA surveillance of circulating strains in order to predict the possible variants that will circulate in a country, assess the effects of vaccination on RVA circulating strains, and ultimately help in the design, challenges, and prospects of RVA vaccines.