Rotavirus genotypes in children under five years hospitalized with diarrhea in low and middle-income countries: Results from the WHO-coordinated Global Rotavirus Surveillance Network.

Rotavirus is the most common pathogen causing pediatric diarrhea and an important cause of morbidity and mortality in low- and middle-income countries. Previous evidence suggests that the introduction of rotavirus vaccines in national immunization schedules resulted in dramatic declines in disease burden but may also be changing the rotavirus genetic landscape and driving the emergence of new genotypes. We report genotype data of more than 16,000 rotavirus isolates from 40 countries participating in the Global Rotavirus Surveillance Network. Data from a convenience sample of children under five years of age hospitalized with acute watery diarrhea who tested positive for rotavirus were included. Country results were weighted by their estimated rotavirus disease burden to estimate regional genotype distributions. Globally, the most frequent genotypes identified after weighting were G1P[8] (31%), G1P[6] (8%) and G3P[8] (8%). Genotypes varied across WHO Regions and between countries that had and had not introduced rotavirus vaccine. G1P[8] was less frequent among African (36 vs 20%) and European (33 vs 8%) countries that had introduced rotavirus vaccines as compared to countries that had not introduced. Our results describe differences in the distribution of the most common rotavirus genotypes in children with diarrhea in low- and middle-income countries. G1P[8] was less frequent in countries that had introduced the rotavirus vaccine while different strains are emerging or re-emerging in different regions.

Antiviral activity of 4-oxoquinoline-3-carboxamide derivatives against bovine herpesvirus type 5.

BACKGROUND: Bovine herpesvirus type 5 is an important agent of meningoencephalitis in cattle and has been identified in outbreaks of bovine neurological disease in several Brazilian states. In recent years, oxoquinoline derivatives have become an important focus in antiviral drug research. METHODS: The cytotoxicity and anti BoHV-5RJ42/01 activity of a set of synthetic 4-oxoquinoline derivatives 4a-k were assayed on Madin-Darby Bovine Kidney cell and antiviral activity by plaque reduction assay. RESULTS: The most promising substance (4h) exhibited CC50 and EC50 values of 1,239 µM ±5.5 and 6.0 µM ±1.5, respectively, with an SI =206. Two other compounds 4j (CC50 = 35 µM ±2 and EC50 = 24 µM ±7.0) and 4k (CC50= 55 µM ±2 and EC50 = 24 µM ±5.1) presented similar inhibitory profile and selectivity indexes of 1.4 and 2.9, respectively. The results of the time-of-addition studies revealed expressive reduction of virus production (≥80%) in different stages of virus replication cycle except for compound 4h that slightly inhibited virus yield in the first 2 h post infection, but it showed expressive virus inhibition after this time. CONCLUSIONS: All three compounds slightly interact with the virus on the virucidal assay and they are not able to block virus attachment and penetration. Antiviral effect of oxoquinoline 4h was more prominent than acyclovir which leads us to suggest compound 4h as a promising molecule for further anti-BoHV-5 drug design.

The evolving epidemiology of rotavirus A infection in Brazil a decade after the introduction of universal vaccination with Rotarix®.

BACKGROUND: Brazil introduced the monovalent rotavirus vaccine (Rotarix®) in 2006. This study aimed to assess the epidemiology and genotype distribution of species-A rotavirus (RVA) in Brazil, comparing the pre- and post-vaccination periods. METHODS: Laboratory-based RVA surveillance included 866 municipalities in 22 Brazilian states, over a 21-year period. A total of 16,185 children with diarrheal diseases (DD) aged up to 12 years between 1996 and 2005 (pre-vaccination period, n = 7030) and from 2006 to 2017 (post-vaccination period, n = 9155) were enrolled. RVA was detected using ELISA immune assay and/or polyacrylamide gel electrophoresis and genotyped using nested PCR and/or nucleotide sequencing. RVA-positivity and genotypes detection rates were compared in distinct periods and age groups and Rotarix vaccination status. RESULTS: RVA-positivity in pre- and post-vaccination periods was, respectively: 4-11 months bracket, 33.3% (668/2006) and 16.3% (415/2547) (p <  0.001); 12-24 months, 28.2% (607/2154) and 22.2% (680/3068) (p <  0.001); 25-48 months, 17.4% (215/1235) and 29.4% (505/1720) (p <  0.001). Genotypes distribution in the pre- and post-vaccination periods was, respectively: G1P [8]/G1P[Not Typed], 417/855 (48.8%) and 118/1835 (6.4%) (p <  0.001); G2P [4]/G2P[NT], 47/855 (5.5%) and 838/1835 (45.7%) (p <  0.001); G3P [8]/G3P[NT], 55/855 (6.4%) and 253/1835 (13.8%) (p <  0.001); G9P [8]/G9P[NT], 238/855 (27.8%) and 152/1835 (8.3%) (p <  0.001); G12P [8]/G129P[NT], 0/871 (0%) and 249/1835(13.6%) (p <  0.001). Concerning infants aged 4-11 months, RVA frequency in fully vaccinated and non-vaccinated individuals was 11.9% (125/1052) and 24.5% (58/237) (p <  0.001), respectively. In children aged 12-24 months, RVA detection rate was 18.1% (253/1395) and 29.6% (77/260) (p <  0.001), for the vaccinated and non-vaccinated individuals, respectively (p <  0.001). CONCLUSIONS: RVA infection was significantly less frequent in children aged ≤2 years with DD after implementing vaccination, mainly among vaccinated children. It was also observed a decrease of P [8] circulation and emergence of G2P[4] in 2005, and afterwards in the post-vaccine era, with spreading of G12P[8] in 2014-2015 and of G3P[8] in 2017. Continuous RVA surveillance must be carried out in this scenario.

G26P[19] rotavirus A strain causing acute gastroenteritis in the American continent

In Brazil, the rotavirus A genotype G26 was first identified in suckling piglets, while the P[19] genotype has not been identified in any animal species so far. This report details the genetic characterisation of a G26P[19] RVA strain detected from an eight year-old child, vaccinated with Rotarix®, hospitalised with acute diarrhoeal disease in Rio de Janeiro in 2015. Most likely, the genome constellation (I5-R1-C1-M1-A8-N1-T1-E1-H1) observed in the G26P[19] Brazilian strain was a result of interspecies transmission events between humans and pigs. In addition, a rearrangement in the NSP5 gene was observed downstream of the 3' non-coding region.

Etiology of Severe Acute Watery Diarrhea in Children in the Global Rotavirus Surveillance Network Using Quantitative Polymerase Chain Reaction.

Background: The etiology of acute watery diarrhea remains poorly characterized, particularly after rotavirus vaccine introduction. Methods: We performed quantitative polymerase chain reaction for multiple enteropathogens on 878 acute watery diarrheal stools sampled from 14643 episodes captured by surveillance of children <5 years of age during 2013-2014 from 16 countries. We used previously developed models of the association between pathogen quantity and diarrhea to calculate pathogen-specific weighted attributable fractions (AFs). Results: Rotavirus remained the leading etiology (overall weighted AF, 40.3% [95% confidence interval {CI}, 37.6%-44.3%]), though the AF was substantially lower in the Americas (AF, 12.2 [95% CI, 8.9-15.6]), based on samples from a country with universal rotavirus vaccination. Norovirus GII (AF, 6.2 [95% CI, 2.8-9.2]), Cryptosporidium (AF, 5.8 [95% CI, 4.0-7.6]), Shigella (AF, 4.7 [95% CI, 2.8-6.9]), heat-stable enterotoxin-producing Escherichia coli (ST-ETEC) (AF, 4.2 [95% CI, 2.0-6.1]), and adenovirus 40/41 (AF, 4.2 [95% CI, 2.9-5.5]) were also important. In the Africa Region, the rotavirus AF declined from 54.8% (95% CI, 48.3%-61.5%) in rotavirus vaccine age-ineligible children to 20.0% (95% CI, 12.4%-30.4%) in age-eligible children. Conclusions: Rotavirus remained the leading etiology of acute watery diarrhea despite a clear impact of rotavirus vaccine introduction. Norovirus GII, Cryptosporidium, Shigella, ST-ETEC, and adenovirus 40/41 were also important. Prospective surveillance can help identify priorities for further reducing the burden of diarrhea.

Detection and molecular characterization of emergent GII.P17/GII.17 Norovirus in Brazil, 2015.

A newly GII.17 Kawazaki_2014 variant strain was detected recently in Brazil. Phylogenetic analysis reveals at least four independent introduction events of this lineage into this country that took place throughout 2014, coinciding with FIFA World Cup in Brazil, 2014, and Hong Kong has been identified as the most likely source of introduction. This variant emerged in Asia causing outbreaks and replacing prevalent GII.4. Emergence of GII.P17/GII.17 variant emphasizes the need for active laboratory surveillance for NoV including molecular epidemiology and studies on virus evolution.

Dissemination of human adenoviruses and rotavirus species A on fomites of hospital pediatric units.

Rotavirus A and human adenovirus dissemination were demonstrated both in a pediatric ward and in a neonatal intensive care unit (NICU) of the same pediatric hospital. Virus detection from fomites samples were higher in the pediatric ward (42.3% [137 out of 324]) than in the NICU (4.5% [7 out of 156]), revealing that cleaning processes used in our NICU are effective in reducing viral contamination, suggesting human adenovirus as a potential biomarker of contamination of hospital fomites.

Performance of a one-step quantitative duplex RT-PCR for detection of rotavirus A and noroviruses GII during two periods of high viral circulation.

Rotavirus A (RVA) and noroviruses (NoV) are the major viral agents of acute gastroenteritis (AGE) worldwide. In the present study, we aimed to evaluate the performance of a one-step duplex quantitative RT-PCR (dRT-qPCR) assay, established for detection and quantification of RVA and NoV genogroup II (GII) using a single DNA standard curve (SC), as well as to investigate the association between fecal viral load and optical density (OD) values, and viruses' genotyping. The results obtained by dRT-qPCR in 530 fecal samples from AGE cases were compared with methods employed for the diagnosis of those viruses as follows: enzyme immunoassay (EIA) and polyacrylamide gel electrophoresis (PAGE) for RVA; and qualitative PCR for NoV. By using dRT-qPCR, we detected RVA and NoV in 353 (66%), increasing the positivity rate by 22.5% for RVA and 11.5% NoV, comparing the number of positive samples. RVA and NoV GII were detected in a range of 5.17 × 10(3) to 6.56 × 10(9) and 3.76 × 10(3) to 9.13 × 10(10) genome copies per gram of feces, respectively. We observed a significant direct correlation between genome copies values and optical density, using dRT-qPCR and EIA assays, respectively (Spearman ρ=0.41; p<0.0001). Viruses characterization demonstrated a predominance of NoV GII.4 Sidney 2012 variant during October 2013 to February 2014, followed by the emergence of RVA genotype G12P[8] in 2014. The established assay using a single SC provides an early feedback concerning detection and quantification, with the advantage of detecting simultaneously RVA and NoV GII, reducing time and reagent costs.

Detection and molecular characterization of caliciviruses (vesivirus and norovirus) in an outbreak of acute diarrhea in kittens from Brazil.

Feline caliciviruses (FCVs) have occasionally been described in cats in association with enteric disease, but an etiological role for these viruses in acute gastroenteritis is still unclear. In this study, molecular characterization of FCV and feline norovirus (FNoV) was undertaken using real-time PCR (RT-PCR) and sequence analysis of the ORF1 region in fecal specimens from 29 diarrheic cats. The specimens were also screened for parvovirus, coronavirus, astrovirus and group A rotavirus. A quantitative one step RT-PCR was also performed to detect and quantitate NoV genogroup IV and the role of these animal caliciviruses in feline gastroenteritis was investigated. This is the first description of enteric FCV and FNoV in South America.

Group a rotavirus and norovirus genotypes circulating in the northeastern Brazil in the post-monovalent vaccination era.

Group A rotaviruses (RVA) and noroviruses (NoV) are the leading cause of acute gastroenteritis (AGE) worldwide. Childhood diarrhea deaths and hospital admissions have declined since the introduction of the monovalent (G1P[8]) vaccine (Rotarix(®) [RV1]) in the National Immunization Program in Brazil in 2006. This study aims to investigate the epidemiological profile of NoV and RVA infections from children with AGE in the Northeastern region of Brazil in the post vaccine season. Two-hundred fecal samples collected from children up to 10 years old in Fortaleza, Ceará between 2008-2009 were screened for the presence of RVA and NoV. Positive samples were genotyped and sequenced. The RVA screening revealed 12% prevalence and all RVA strains belonged to G2P[4] genotype. Phylogenetic analysis based on the 11 RVA genome segments sequenced from eight samples revealed a DS-1-like genotype constellation: I2-R2-C2-M2-A2-N2-T2-E2-H2. For NoV screening, the prevalence observed was 17% and the following genotypes were detected: GII.4 (59%), GII.12 (17%), GII.6 (9%), GII.3 (6%), and GII.? (9%). At least four different NoVs genotypes and two RVA G2P[4] variants were identified circulating in the Northeastern region of Brazil. RVA phylogenetic analysis suggests that the RVA G2P[4] strains might have originated from intragenogroup reassortment events. Whether the genetic modifications observed in these contemporary G2P[4] RVA strains may impact the long-term effectiveness of the current vaccination programs remains to be explored. These data reinforce the importance of surveillance for monitoring the emergence of new strains of RVA and NoV and their impact on cases of acute gastroenteritis.

Phylogenetic analyses of Norovirus strains detected in Uruguay reveal the circulation of the novel GII.P7/GII.6 recombinant variant.

Noroviruses (NoV) are one of the major etiological agent of acute gastroenteritis (AGE) outbreaks worldwide. Distinct NoV genotypes have been associated with different transmission patterns and disease severity in humans. Therefore, it is important to identify genetically different NoV genotypes circulating in a particular region. However, genotyping has become a challenge due to recombination events occurring mainly nearby ORF1/ORF2 junction of NoV genome, leading to distinct genotypes with polymerase and capsid regions derived from parenteral strains. Taking this into account, ORF1/ORF2 sequences were obtained from NoV strains collected from patients with AGE in Uruguay. This study reveals in silico evidences of recombination events taking place in four out of six strains analyzed for which its polymerase gene and its capsid region correspond to GII.P7 and to GII.6 genotype, respectively. These results also reveal the circulation of a GII.P7/GII.6 recombinant variant in the natural populations of NoV strains in the northwestern region of Uruguay. As far as we know this is the first report about the circulation of a NoV GII.P7/GII.6 recombinant variant in the Americas.

Viability of human adenovirus from hospital fomites.

The monitoring of environmental microbial contamination in healthcare facilities may be a valuable tool to determine pathogens transmission in those settings; however, such procedure is limited to bacterial indicators. Viruses are found commonly in those environments and are rarely used for these procedures. The aim of this study was to assess distribution and viability of a human DNA virus on fomites in an Adult Intensive Care Unit of a private hospital in Rio de Janeiro, Brazil. Human adenoviruses (HAdV) were investigated in 141 fomites by scraping the surface area and screening by quantitative PCR (qPCR) using TaqMan® System (Carlsbad, CA). Ten positive samples were selected for virus isolation in A549 and/or HEp2c cell lines. A total of 63 samples (44.7%) were positive and presented viral load ranging from 2.48 × 10(1) to 2.1 × 10(3) genomic copies per millilitre (gc/ml). The viability was demonstrated by integrated cell culture/nested-PCR in 5 out of 10 samples. Nucleotide sequencing confirmed all samples as HAdV and characterized one of them as specie B, serotype 3 (HAdV-3). The results indicate the risk of nosocomial transmission via contaminated fomites and point out the use of HAdV as biomarkers of environmental contamination.

Synthetic aminomethylnaphthoquinones inhibit the in vitro replication of bovine herpesvirus 5.

Bovine herpesvirus type 5 (BoHV-5) is an etiologic agent of meningoencephalitis in cattle. The aim of this study was to evaluate the antiviral potential of a series of synthetic Mannich bases derived from lawsone and to investigate at which stage of the BoHV-5 replicative cycle the compounds might be acting. The most potent and selective inhibitor exhibited CC50 and EC50 values of 1867 µM ± 8.3 and 3.8 µM ± 1.2, respectively (ACV: 989 µM ± 2 and 166 µM ± 2, respectively).

Epidemiologia das rotaviroses antes e após a introdução da vacina / Rotavirus epidemiology before and after vaccine introduction

OBJETIVOS: Avaliar a prevalência e a circulação dos genótipos de rotavírus, antes e após a introdução da vacina oral contra rotavírus humano, bem como verificar uma possível mudança na faixa etária de ocorrência da infecção pelo RV-A. MÉTODOS: Trata-se de um estudo transversal realizado no período de 2002 a 2011, em Juiz de Fora, MG. Foram avaliados 1.144 espécimes fecais diarreicos, obtidos de crianças de 0 a cinco anos não hospitalizadas, que foram analisadas por PAGE e RT-PCR. Os dados relativos à prevalência e distribuição etária dos casos de rotavirose foram analisados pelo teste χ2 (p < 0,05), utilizando-se o programa SPSS, versão 13.0. RESULTADOS: Infecções por rotavírus foram detectadas em 9,35% (107/1.144) das amostras, com prevalências variando de 11,12% (90/809) no período pré-vacinal a 5,07% (17/335) no pós-vacinal (p = 0,001). Dentre as amostras caracterizadas, os genótipos mais frequentemente detectados foram G1P[6] (6/33 = 18,2%) no período 2002-2005 e G2P[4] no ano de 2006 (11/33 = 33,3%) e no período 2007-2011 (5/33 = 15,2%). Observou-se, ainda, uma redução significativa no número de casos de rotavirose em crianças de 0 a 36 meses, após a introdução da vacina. CONCLUSÕES: O estudo revelou queda significativa na prevalência de rotavírus, principalmente na faixa etária de 0 a 36 meses, no período 2007-2011, bem como redução na circulação do genótipo G1.

OBJECTIVES: To evaluate the prevalence and circulation of rotavirus genotypes before and after the introduction of oral vaccine against human rotavirus (OVHR), and to check for a possible change in the age of occurence of the infection by RV-A. METHODS: This was a cross-sectional study conducted between 2002-2011, in the city of Juiz de Fora, state of Minas Gerais, Brazil. A total of 1,144 diarrheal stool specimens were obtained from nonhospitalized children aged between 0 and 5 years, and analyzed by polyacrylamide gel electrophoresis and reverse-transcription polymerase chain reaction for genotype characterization. Data on prevalence and age distribution of rotavirus cases were analyzed through the chi-squared test (p < 0.05), using SPSS, release 13.0. RESULTS: Rotavirus infection was detected in 9.35% (107/1,144) samples, with prevalence rates ranging from 11.12% (90/809) in the pre-vaccine to 5.07% (17/335) in the post-vaccine period (p = 0.001). Among the samples tested, the most frequently detected genotypes were G1P[6] (6/33 = 18.2%) in the period between 2002 and 2005 and G2P[4] in 2006 (11/33 = 33.3%) and in the period between 2007 and 2011 (5/33 = 15.2%). There was also a significant reduction in the number of cases of rotavirus disease in children aged between 0 and 36 months after the vaccine introduction. CONCLUSIONS: The study evidenced a significant decrease in the prevalence of rotavirus, mainly in children aged between 0 and 36 months in the 2007-2011 period, as well as a reduction in G1 genotype circulation.

Rotavirus epidemiology before and after vaccine introduction.

OBJECTIVES: To evaluate the prevalence and circulation of rotavirus genotypes before and after the introduction of oral vaccine against human rotavirus, and to check for a possible change in the age of occurence of the infection by RV-A. METHODS: This was a cross-sectional study conducted between 2002-2011, in the city of Juiz de Fora, state of Minas Gerais, Brazil. A total of 1,144 diarrheal stool specimens were obtained from nonhospitalized children aged between 0 and 5 years, and analyzed by polyacrylamide gel electrophoresis and reverse-transcription polymerase chain reaction for genotype characterization. Data on prevalence and age distribution of rotavirus cases were analyzed through the chi-squared test (p < 0.05), using SPSS, release 13.0. RESULTS: Rotavirus infection was detected in 9.35% (107/1,144) samples, with prevalence rates ranging from 11.12% (90/809) in the pre-vaccine to 5.07% (17/335) in the post-vaccine period (p = 0.001). Among the samples tested, the most frequently detected genotypes were G1P[6] (6/33 = 18.2%) in the period between 2002 and 2005 and G2P[4] in 2006 (11/33 = 33.3%) and in the period between 2007 and 2011 (5/33 = 15.2%). There was also a significant reduction in the number of cases of rotavirus disease in children aged between 0 and 36 months after the vaccine introduction. CONCLUSIONS: The study evidenced a significant decrease in the prevalence of rotavirus, mainly in children aged between 0 and 36 months in the 2007-2011 period, as well as a reduction in G1 genotype circulation.

Marine natural seaweed products as potential antiviral drugs against Bovine viral diarrhea virus

Bovine viral diarrhea virus (BVDV) is an etiologic agent that causes important economic losses in the world. It is endemic in cattle herds in most parts of the world. The purpose of this study was to evaluate the in vitro cytotoxic effect and antiviral properties of several marine natural products obtained from seaweeds: the indole alkaloid caulerpin (CAV, 1) and three diterpenes: 6-hydroxydichotoma-3,14-diene-1,17-dial (DA, 2), 10,18-diacetoxy-8-hydroxy-2,6-dolabelladiene (DB1, 3) and 8,10,18-trihydroxy-2,6-dolabelladiene (DB3, 4). The screening to evaluate the cytotoxicity of compounds did not show toxic effects to MDBK cells. The antiviral activity of the compounds was measured by the inhibition of the cytopathic effect on infected cells by plaque assay (PA) and EC50 values were calculated for CAV (EC=2,0± 5.8), DA (EC 2,8± 7.7), DB1 (EC 2,0±9.7), and DB3 (EC 2,3±7.4). Acyclovir (EC50 322± 5.9) was used in all experiments as the control standard. Although the results of the antiviral activity suggest that all compounds are promising as antiviral agents against BVDV, the Selectivity Index suggests that DB1 is the safest of the compounds tested.

Factors associated with rotavirus diarrhoea in children living in a socially diverse urban centre in Brazil.

A case-control study, aimed at identifying factors associated with rotavirus diarrhoea cases presenting to health facilities, was conducted in children from low-income and middle-low-income families in Brazil. Cases were 390 children with diarrhoea and rotavirus in stools; controls were 1674 children without diarrhoea presenting to the same facilities. Data were collected by questionnaire and observations during home visits. Explanatory variables were grouped according to a conceptual model of causation. The ORs by non-conditional logistic regression and population-attributable fractions were calculated. Socioeconomic factors contributed a third of cases, followed by contact with diarrhoea cases and by not being breast fed. In cases aged <1 year, not being breast fed was the main determinant, followed by socioeconomic factors, and crowding and contact outside the home; in older children, socioeconomic factors followed by contact inside and outside the home were the main determinants. Environmental and sanitation variables were not associated with diarrhoea in the final model, and socioeconomic factors were only partly mediated by proximal variables. Transmission of rotavirus appears to be mostly by person-to-person contact, and shows marked social differentials not explained by the biological factors studied. The rotavirus vaccine is unlikely to protect against the full range of circulating genotypes of rotavirus, and understanding rotavirus epidemiology remains essential to the development of control policies.

Group A rotavirus detection on environmental surfaces in a hospital intensive care unit.

BACKGROUND: Environmental surfaces can play a role in the spread of pathogens, such as enteric viruses, within a hospital. This study assessed the level of contamination of group A rotavirus (RV-A) on environmental surfaces samples from an adult intensive care unit in a hospital in Rio de Janeiro, Brazil. METHODS: A total of 504 environmental surface samples were obtained from multiple sites in the intensive care unit, including flushing buttons, telephones, and alcohol gel supports. Nested and quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) were used to detect and quantify RV-A levels through partial amplification of VP6 and NSP3 genes, respectively, and the viability of the viruses detected was assessed by MA-104 cell integrated cell culture/RT-PCR. RESULTS: RV-A was detected by nested RT-PCR in 14% of the samples (73 of 504), with viral loads ranging from 3.4 genomic copies/mL to 2.9 × 10(3) genomic copies/mL. The nucleotide sequence of the amplicons obtained from nested RT-PCR confirmed that the positive samples were RV-A. Moreover, 3 of 10 strains investigated demonstrated viability by integrated cell culture/RT-PCR. CONCLUSION: The detection of RV-A on environmental surface samples indicates a need for improvements to hospital cleaning procedures to reduce viral contamination, and suggests, as reported previously, that RV-A can be used as a biomarker to assess contamination in hospitals.

Phylogenetic analysis of VP1, VP2, and VP3 gene segments of genotype G5 group A rotavirus strains circulating in Brazil between 1986 and 2005.

Genotype G5 group A rotavirus (RV-A), which is common in pigs and also detected in horses and cattle, circulated as endemic genotype in the 1980s and early 1990s in Brazil. After 1996, G5 RV-A has been replaced by G9 RV-A, becoming only sporadically detected. Recently, G5 has been reported in children with severe diarrhea in Argentina, Cameroon, Paraguay, People's Republic of China, and Vietnam, suggesting that, although uncommon in humans, it has a worldwide distribution. In a previous study, Brazilian G5 RV-A VP7 gene analysis demonstrated the existence of three main lineages: I, II, and III; all Brazilian strains and three porcine strains from Thailand grouped inside Lineage I. The VP8(*) subunit of VP4 gene showed that all P[8] strains fell into three major genetic lineages: P[8]-1; P[8]-2; and P[8]-3. Partial sequencing and phylogenetic analysis of VP1, VP2 and VP3 genes of P[8]G5 human RV-A strains were determined from 28 Brazilian strains collected from 1986 to 2005. The VP1-VP3 partial sequences analysis showed that the Brazilian strains have high amino acid identity with the human RV-A prototype IAL28 and other Wa-like genogroup strains. It was also shown that G5 RV-A Brazilian VP1-VP3 and VP7 sequences have a similar pattern of gouping: The study strains and the G5 prototype strain IAL-28 grouped together, while other prototypes, like OSU grouped separately. These results suggest that the core protein genes (VP1-VP3) of the G5 RV-A Brazilian strains might have originated from porcine and human strains. Phylogenetic analyses of VP7, VP4, VP1, VP2, and VP3 genes of P[8]G5 strains revealed a conserved genomic constellation (G5-P[8]-R1-C1-M1) with sequence similarity to Wa-like strains: IAL28, Wa, BE00048, CK00032, CK00033, DC4772 and DC1898, suggesting that despite the differences in genotypes (i.e., G5, G1 and G3) these viruses are genetically similar. The results presented here are fundamental to understand the epidemiology and evolution of genotype G5 RV-A and demonstrate the importance of continuous monitoring and molecular characterization of RV-A strains circulating in human and animal populations.

Analysis of human P[4]G2 rotavirus strains isolated in Brazil reveals codon usage bias and strong compositional constraints.

The Rotavirus genus belongs to the family Reoviridae and its genome consist of 11 segments of double-stranded RNA. Group A rotaviruses (RV-A) are the main etiological agent of acute viral gastroenteritis in infants and young children worldwide. Understanding the extent and causes of biases in codon usage is essential to the understanding of viral evolution. However, the factors shaping synonymous codon usage bias and nucleotide composition in human RV-A are currently unknown. In order to gain insight into these matters, we analyzed the codon usage and base composition constraints on the two genes that codify the two outer capsid proteins (VP4 [VP8*] and VP7) of 58 P[4]G2 RV-A strains isolated in Brazil and investigated the possible key evolutionary determinants of codon usage bias. The results of these studies revealed that the frequencies of codon usage in both RV-A proteins studied are significantly different than the ones used by human cells. In order to observe if similar trends of codon usage are found when RV-A complete genomes are considered, we compare these results with results found using a dataset of 10 reference strains for whom the complete codes of the 11 segments are known. Similar results were obtained using capsid proteins or complete genomes. The general correlations found between the position of each sequence on the first axis generated by correspondence analysis and the relative dinucleotide abundances indicate that codon usage in RV-A can also be strongly influenced by underlying biases in dinucleotide frequencies. CpG and GpC containing codons are markedly suppressed. Thus, the results of this study suggest that RV-A genomic biases are the result of the evolution of genome composition in relation to host adaptation and the ability to escape antiviral cell responses.