Diversity of Rotavirus Strains Circulating in Botswana before and after introduction of the Monovalent Rotavirus Vaccine.

BACKGROUND: Globally, rotavirus is the leading cause of acute gastroenteritis (AGE) in children aged <5 years. Botswana introduced the monovalent rotavirus vaccine (Rotarix) in July 2012. To study the impact of this vaccine on rotavirus genotypes circulating in Botswana, a comparison of the genotypes pre-vaccination (2011-2012) and post-vaccination (2013-2018) periods was conducted. SUBJECTS AND METHODS: Residual samples from 284 children <5 years of age that tested positive for rotavirus by enzyme immunoassay were genotyped. One hundred and five samples were from the pre-vaccination period and 179 were from the post-vaccination period. Genotyping was performed using two multiplexed one-step reverse transcription polymerase chain reaction (RT-PCR) assays for the amplification and genotyping of rotavirus VP7 (G) and VP4 (P) genes. RESULTS: Prior to vaccine introduction, the predominant rotavirus circulating genotypes were G9P[8] (n = 63, 60%) and G1P[8] (n = 22, 21%). During the vaccine period, G2P[4] was the predominant genotype (n = 49, 28%), followed by G9P[8] (n = 40, 22%) and G1P[8] (n = 33, 18.5%). There was a significant decline in the prevalence of G9P[8] (p = 0.001) in the post-vaccination period. There was also a notable decline in G1P[8]. A spike in G2P[4] was observed in 2013, one year post-vaccine introduction. Rotavirus strain G3P[4] (n = 8) was only detected in the post-vaccine introduction period. In 2018 there was a marked increase in genotype G3P[8] (p = 0.0003). CONCLUSIONS: The distribution of circulating rotavirus genotypes in Botswana changed after vaccine implementation. Further studies are needed to examine whether these changes are related to vaccination or simply represent natural secular variation.

Prevalence of rotavirus among children under five years of age with diarrhea in Kaduna State, Nigeria

Rotavirus (RV) is a major etiological agent of acute infantile gastroenteritis and is associated with 20%-25% of diarrhea cases in infants. Nigeria continues to be among the first five countries with greatest number of RV disease associated deaths per year. The objective was to determine some demographic factors that might be associated with rotavirus diarrhea among children in Kaduna State. From September 2013-August 2014, 401 diarrheic stool samples were collected from children under 5 years of age in Kaduna State, Nigeria and analyzed for RV antigen using ELISA. An overall RV prevalence of 32.2% (129/401) was obtained with the infection occurring throughout the study period. The infection was higher in males (33.0%:63/111) compared to females (31.4%:66/210). The highest burden was detected in children 25 -36 months of age (37.3%:22/59). Highest prevalence was detected in children whose parents had primary education (35.8%:19/53) and those whose parents were civil servants (35.6%:36/101). There was no statistically significant association between breast feeding and RV infection (P> 0.05). The study has revealed that rotavirus remains an important cause of acute diarrhea in children under five years in Kaduna State, Nigeria. Hence the need to introduce the vaccines into the childhood immunization program in the country

Human G9P[8] rotavirus strains circulating in Cameroon, 1999-2000: Genetic relationships with other G9 strains and detection of a new G9 subtype.

Group A rotaviruses (RV-A) are the leading cause of viral gastroenteritis in children worldwide and genotype G9P[8] is one of the five most common genotypes detected in humans. In order to gain insight into the degree of genetic variability of G9P[8] strains circulating in Cameroon, stool samples were collected during the 1999-2000 rotavirus season in two different geographic regions in Cameroon (Southwest and Western Regions). By RT-PCR, 15 G9P[8] strains (15/89=16.8%) were identified whose genomic configurations was subsequently determined by complete or partial gene sequencing. In general, all Cameroonian G9 strains clustered into current globally-spread sublineages of the VP7 gene and displayed 86.6-100% nucleotide identity amongst themselves and 81.2-99.5% nucleotide identity with global G9 strains. The full genome classification of all Cameroonian strains was G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 but phylogenetic analysis of each gene revealed that the strains were spread across 4 or more distinct lineages. An unusual strain, RVA/Human-wt/CMR/6788/1999/G9P[8], which shared the genomic constellation of other Cameroonian G9P[8] strains, contained a novel G9 subtype which diverged significantly (18.8% nucleotide and 19% amino acid distance) from previously described G9 strains. Nucleotide and amino acid alignments revealed that the 3' end of this gene is highly divergent from other G9 VP7 genes suggesting that it arose through extensive accumulation of point mutations. The results of this study demonstrate that diverse G9 strains circulated in Cameroon during 1999-2000.

Sequencing and phylogenetic analysis of the coding region of six common rotavirus strains: evidence for intragenogroup reassortment among co-circulating G1P[8] and G2P[4] strains from the United States.

The segmented genome of rotaviruses provides an opportunity for rotavirus strains to generate a large genetic diversity through reassortment; however, this mechanism is considered to play little role in the generation of mosaic gene constellations between Wa-like and DS-1-like strains in genes other than the neutralization antigens. A pilot study was undertaken to analyze these two epidemiologically important strains at the genomic level in order to (i) identify intergenogroup reassortment and (ii) to make available additional reference genome sequences of G1P[8] and G2P[4] for future genomics analyses. The full or nearly complete coding region of all 11 genes for 3 G1P[8] (LB2719, LB2758, and LB2771) and 3 G2P[4] (LB2744, LB2764, and LB2772) strains isolated from children hospitalized with severe diarrhea in Long Beach, California, where these strains were circulating at comparable rates during 2005-2006 are described in this study. Based on the full-genome classification system, all G1P[8] strains had a conserved genomic constellation: G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-E1-H1 and were mostly identical to the few Wa-like strains whose genome sequences have already been determined. Similarly, the genome sequences of the 3 G2P[4] strains were highly conserved: G2-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-E2-H2 and displayed an overall lesser genetic divergence with reference DS-1-like strains. While intergenogroup reassortment was not seen between the G1P[8] and G2P[4] strains studied here, evidence for intragenogroup reassortment events was identified. Similar studies in the post-rotavirus genomic era will help uncover whether intergenogroup reassortment affecting the backbone genes could play a significant role in any potential vaccine breakthrough events by evading immunity of vaccinated children.

Genomic characterization of human rotavirus G10 strains from the African Rotavirus Network: relationship to animal rotaviruses.

Global rotavirus surveillance has led to the detection of many unusual human rotavirus (HRV) genotypes. The aim of this study was to elucidate the genetic and evolutionary relationships of short fragments of all 11 gene segments of G10 HRV strains identified in West Africa through the African Rotavirus Network (ARN) system. During 1998-2004 surveillance within the ARN, we identified 5 G10 P[8] HRV strains. Fragments of all 11 gene segments of these G10 strains were sequenced. Phylogenetic and sequence analyses of each gene segment revealed high nucleotide similarities amongst the ARN strains (97-100%) except in the case of the VP1(85-96%) and NSP2 genes (87.8-99.7%) where some strains were divergent. All genes of the ARN strains were classified as Wa-like (genotype 1) with the exception of their VP7 gene of all strains (genotype G10) and the VP6 gene of a single strain, 6755/2002/ARN (DS-1 like, genotype 2). While classified as Wa-like, the NSP2 genes of four of the ARN strains occupied a distinct sub-lineage related to simian strain Tuch, while the NSP2 of strain 6755/2002/ARN and NSP5 genes of all strains were closely related to the cognate genes of both human and animal strains belonging to the Wa-like genogroup. Although these findings help to elucidate the evolution of ARN G10 strains, additional sequence studies of cognate animal rotavirus genes are needed to determine irrefutably the specific origin of those genes relative to both human and animal rotavirus strains.

Genome sequence based molecular epidemiology of unusual US Rotavirus A G9 strains isolated from Omaha, USA between 1997 and 2000.

After discovery in the early 1980s, Rotavirus A serotype G9 was detected infrequently for almost a decade. Since the mid-1990s, however, serotype G9 has emerged to become a globally common strain linked to the introduction of a single, new genetic variant of G9 VP7 gene. Studies have demonstrated that genetically divergent G9 strains co-circulated at low frequency with the emerging variants. Examples include unique U.S. G9 strains Om46/Hu/USA/1998 and Om67/Hu/USA/1998, isolated in Omaha during the 1997-1998 rotavirus season, that are more closely related phylogenetically to reference strains from the 1980s than to most emerging G9 strains from the U.S. and globally. Here, we sequenced the VP7 full open reading frame for all available G9 strains (n=12) identified in Omaha during 1996-2000 seasons to investigate their epidemiology and evolution. In addition, the full or partial length open reading frames of the remaining 10 genes for five divergent Om46-like strains and one modern G9 variant were sequenced to evaluate their potential origin. Our findings suggest that Om46-like G9 strains may have been introduced into humans recently, perhaps in 1997-1998 when it was first detected, and the presumed original host of this VP7 gene variant may have been an animal species based on the unexpected detection of porcine rotavirus related NSP2 gene in the genome. The relatively high fitness of Om46-like strains during the 1997-1998 rotavirus season, 1 year after the globally important G9 variant was documented to be already spreading in the study area and other sites of the United States, appears to parallel findings on seasonal replacement of various genetic and antigenic variants of other common human rotavirus antigen specificities.

Quantification of human immunodeficiency virus-1 viral load using nucleic acid sequence-based amplification (NASBA) in north central Nigeria.

BACKGROUND: Viral load (VL) quantification is considered an integral part of the standard care in human immunodeficiency virus (HIV) infected individuals but in Nigeria as in most of sub-Saharan Africa, this has not reached the majority ofpatients. METHODS: We report the first field application of the NucliSens EasyQ HIV-1 platform for the real time quantification of HIV-1 VL combining NASBA amplification and real time detection with molecular beacons among HIV-1 infected individuals in north central Nigeria where the predominant HIV-1 subtypes are CRF02_AG and G. CD4+ counts were enumerated using a fluorescence-activated cell sorter system. RESULTS: Of one hundred and forty nine (n=149) plasma sample from patients with mean age of 32 years and made up of 77 males and 72 females, fifty {n=50 (37.9%); 28 males and 22 females} had VLs below the lower detection limit (LDL=25 IU/ml) set by the assay while eighty-two {n=82 (62.1%); 39 males and 43 females} had VL levels above the LDL. Furthermore, 13 of 82 (15.9%) patients with viral loads above the LDL had VLs between 26-1000 IU/ml while 69 (84.1%) had VLs of 1001-2,400,000 IU/ml. 17 (11.4%) of the samples could not be analyzed due to poor viral amplification. Among individuals with both CD4+ and VL results (n=56), those with CD4+ of 1-418 cell/microl presented with higher VL usually above 45,000 IU/ml when compared with those with CD4+ of over 500 cell/microl. CONCLUSION: Our findings highlight the pattern, usefulness and feasibility ofVL quantification by NucliSens EasyQ in monitoring HIV-1 patients in Nigeria.

Characterization of human rotavirus recovered from children with acute diarrhea in Kinshasa, Democratic Republic Of Congo.

Between July and October of 2003, 2004, and 2005, outbreaks of acute gastroenteritis occurred among children <5 years of age in Kinshasa, Democratic Republic of the Congo. Stool specimens (67 in 2003, 108 in 2004, and 116 in 2005) were collected and screened for rotaviruses using either latex agglutination (Diarlex LAA; Orion Diagnostics) or enzyme immunoassay (IDEIA; DakoCytomation). The molecular characteristics of the rotavirus strains were then determined. Group A rotavirus was detected in 195 (76%) of 258 stool specimens. Polyacrylamide gel electrophoresis was used to observe the 11 rotavirus double-stranded RNA segments in 83% of the 195 rotavirus-positive specimens. Six rotavirus group A electropherotypic patterns were noted, predominantly within the short classic pattern (111 [69%]) and the long pattern (37 [23%]). Mixed patterns were noted in the 14 remaining specimens (9%). Of the 29 samples subjected to subgrouping VP6 enzyme immunoassay, subgroup I predominated. Some of the specimens collected in 2003 (n = 26), 2004 (n = 38), and 2005 (n = 52) were analyzed by reverse-transcription polymerase chain reaction, which showed that t G8P[6] and G8P[8] strains predominated in 2003, and G1P[6] strains with short electropherotypic patterns predominated in 2004 and 2005. The emergence in Kinshasa of G8 serotypes, unusually associated with the P[6] genotype, as well as uncommon G1 rotavirus strains showing a short RNA pattern, is significant in relation to the introduction of a rotavirus vaccine and underscores the need for continued rotavirus serotype surveillance in the Democratic Republic of the Congo.

Molecular detection of novel adenoviruses in fecal specimens of captive monkeys with diarrhea in China.

Adenovirus (AdV) has been recently detected among monkeys with diarrhea in a major research primate colony in China. To better assess disease burden and epidemiology of adenoviruses in the colony, we examined the prevalence of this virus in fecal specimens by PCR using broadly reactive hexon gene-specific primers. Of the 29 strains that were characterized by sequence and phylogenetic analysis, we identified a broad spectrum of simian AdV (SAdV) types, including species SAdV-A (n=14) and HAdV-G (n=9). Six additional strains represented two genetic clusters distantly related to other known SAdVs. A better understanding of the epidemiology of SAdVs and their potential role in gastroenteritis is critical to the implementation of advanced prevention strategies against AdV infection in captive primates.

Genomic characterization of human rotavirus G8 strains from the African rotavirus network: relationship to animal rotaviruses.

Global rotavirus surveillance has led to the detection of many unusual human rotavirus (HRV) genotypes. During 1996-2004 surveillance within the African Rotavirus Network (ARN), six P[8],G8 and two P[6],G8 human rotavirus strains were identified. Gene fragments (RT-PCR amplicons) of all 11-gene segments of these G8 strains were sequenced in order to elucidate their genetic and evolutionary relationships. Phylogenetic and sequence analyses of each gene segment revealed high similarities (88-100% nt and 91-100% aa) for all segments except for gene 4 encoding VP4 proteins P[8] and P[6]. For most strains, almost all of the genes of the ARN strains other than neutralizing antigens are related to typical human strains of Wa genogroup. The VP7, NSP2, and NSP5 genes were closely related to cognate genes of animal strains (83-99% and 97-99% aa identity). This study suggests that the ARN G8 strains might have arisen through VP7 or VP4 gene reassortment events since most of the other gene segments resemble those of common human rotaviruses. However, VP7, NSP2 (likely), and NSP5 (likely) genes are derived potentially from animals consistent with a zoonotic introduction. Although these findings help elucidate rotavirus evolution, sequence studies of cognate animal rotavirus genes are needed to conclusively determine the specific origin of those genes relative to both human and animal rotavirus strains.

Role of astrovirus in intussusception in Nigerian infants.

Intussusception (IS) is a form of intestinal obstruction in which a segment of the bowel prolapses into a more distal segment. Viral infections, mostly adenovirus, enteroviruses, human herpesvirus and Epstein-Barr virus are reported in 20-50% of childhood cases of IS. Between January and July 2004, six stool specimens collected from infants 0- to 8-months old diagnosed and admitted for IS were investigated for the presence of rotavirus, astrovirus and adenovirus antigens. Astrovirus antigen was detected in three of the six stool specimens by enzyme immune assay (EIA) and confirmed in two specimens by reverse transcription-polymerase chain reaction (RT-PCR). Rotavirus, non-enteric adenovirus and astrovirus were detected by EIA, as mixed infections in a single specimen. The rotavirus strain revealed a SGI+II, mixed G1G2G8P[6] genotype and had no visible electrophoretic profile. A larger study is needed to determine the extent of involvement of astroviruses in IS in infants and the virus should be included in studies investigating the aetiology of IS.

Epidemiology of rotavirus infection in north-western Nigeria.

Rotaviruses (RV) are associated with approximately 33 000 deaths in children <5 years of age annually in Nigeria. However, limited data exit on RV infection in north-western Nigeria. During July 2002 to July 2004, 1063 (869 diarrhoeic and 194 control) stool samples were collected from children <5 years of age presenting with diarrhoea in north-western Nigeria. The stools were analysed for RV antigen and further characterized by antigenic and genomic methods. RV was detected in 18% of children with diarrhoea and 7.2% of the age-matched case controls. The highest RV burden was detected in children <6-months-old. Long electropherotypes and VP6 subgroup I + II specificity predominated.

Epidemiology of rotavirus and astrovirus infections in children in northwestern Nigeria.

BACKGROUND: Recent estimates attribute 527,000 deaths in children less than five years of age to rotavirus diarrhea annually, with 145,000 occurring in sub-Saharan Africa. Human astroviruses have been identified as one of the most frequent causes of infantile diarrhea, second in incidence only to rotavirus. This study was conducted to determine the prevalence of rotavirus and astrovirus and also to establish the circulating strains of rotavirus in a community in Nigeria where most diarrheic patients do not visit clinics or health care centers. METHODS: A total of 154 stool samples (134 diarrheic and 20 non-diarrheic) were collected from infants and young children less than 5 years of age from January-March 2002. Samples were obtained by house-to-house visit in randomly selected districts in Zaria, Northwestern Nigeria. The samples were screened for rotavirus and astrovirus antigens using commercially available Enzyme Linked Immunosorbent Assay (ELISA) kits. All positive group A rotavirus samples were further subjected to VP6 sub-group ELISA, Polyacrylamide gel electrophoresis (PAGE) to determine their RNA electropherotypes and Reverse transcription polymerase chain reaction (RT-PCR) to determine their VP7 and VP4 genotypes. RESULTS: Rotavirus and astrovirus antigens were detected in 9% (12) and 5% (7) of the 134 diarrheic stool samples respectively. No viral antigen was detected in the non-diarrheic stools. Rotavirus infection was more common in younger children than astrovirus infection. VP6 sub-group II specificity (58.3%), long RNA electropherotypes (41.6%), VP7 genotype G1 (33.3%) and VP4 genotype P [6] (33.3%) were the most common strains in circulation at that time in the community. Of significance is the fact that a large proportion of the rotavirus strains in circulation could not be assigned either a VP6 subgroup or RNA electrophoretic pattern probably as a result of low viral load. CONCLUSION: In this community-based study, rotavirus and astrovirus were significantly associated with diarrhea. However, the prevalence of rotavirus infection among children appears to be low while that of astrovirus falls in the range seen in hospital-based studies around the continent.

Epidemiology of rotavirus and astrovirus infections in children in Northwestern Nigeria

Background: Recent estimates attribute 527 000 deaths in children less than five years of age to rotavirus diarrhea annually, with 145 000 occurring in sub-Saharan Africa. Human astroviruses have been identified as one of the most frequent causes of infantile diarrhea, second in incidence only to rotavirus. This study was conducted to determine the prevalence of rotavirus and astrovirus and also to establish the circulating strains of rotavirus in a community in Nigeria where most diarrheic patients do not visit clinics or health care centers. Methods: A total of 154 stool samples (134 diarrheic and 20 non-diarrheic) were collected from infants and young children less than 5 years of age from January-March 2002. Samples were obtained by house-to-house visit in randomly selected districts in Zaria, Northwestern Nigeria. The samples were screened for rotavirus and astrovirus antigens using commercially available Enzyme Linked Immunosorbent Assay (ELISA) kits. All positive group A rotavirus samples were further subjected to VP6 sub-group ELISA, Polyacrylamide gel electrophoresis (PAGE) to determine their RNA electropherotypes and Reverse transcription polymerase chain reaction (RT-PCR) to determine their VP7 and VP4 genotypes. Results: Rotavirus and astrovirus antigens were detected in 9% (12) and 5% (7) of the 134 diarrheic stool samples respectively. No viral antigen was detected in the non-diarrheic stools. Rotavirus infection was more common in younger children than astrovirus infection. VP6 sub-group II specificity (58.3%), long RNA electropherotypes (41.6%), VP7 genotype G1 (33.3%) and VP4 genotype P [6] (33.3%) were the most common strains in circulation at that time in the community. Of significance is the fact that a large proportion of the rotavirus strains in circulation could not be assigned either a VP6 subgroup or RNA electrophoretic pattern probably as a result of low viral load. Conclusion: In this community-based study, rotavirus and astrovirus were significantly associated with diarrhea. However, the prevalence of rotavirus infection among children appears to be low while that of astrovirus falls in the range seen in hospital-based studies around the continent

Adenovirus infection in children with diarrhea disease in Northwestern Nigeria.

BACKGROUND: Adenoviruses, particularly enteric adenoviruses (EAds) type 40 (Ad40) and type 41(Ad41), can cause acute and severe diarrhea in young children worldwide. This study was conducted to delineate the epidemiological features of adenoviruses identified in children with gastroenteritis in Northwestern Nigeria. METHODS: All 282 specimens comprising 248 diarrheic and 34 non-diarrheic stools were randomly selected from 1063 stools previously analyzed for rotaviruses. These specimens were collected between July 2002 and July 2004 from children < 5 years of age. The specimens were screened for the presence of adenoviruses using monoclonal antibody-based Enzyme Immuno Assay (EIA), (Adenovirus RIDASCREEN r-Biopharm, UK) and the positive specimens were further examined for Ad40 and Ad41 using Premier Adenoclone -Type 40/41 EIA (Meridian Biosciences, USA). Negative staining electron microscopy was performed on selected specimens to confirm the presence of adenovirus particles. RESULTS: Adenovirus antigen was detected in 63/282 (23%) of the diarrheic diarrheic and in 6/34 (17.6%) of the non-diarrheic specimens. Adenoviruses were detected throughout the study period with most patients infected in the age group 25-36 months. The male-to-female ratio was 2.2:1 (43/20). Clinical features included fever (60%: 38/63), vomiting (56%: 35/63), mild dehydration (49%: 31/63), symptoms of upper respiratory tract infection (13%: 8/63) and abdominal pain (5%: 3/63). Analysis of stool specimen in adenovirus infected patients showed watery diarrhea in 87% (55/63), diarrhea with mucus in 19% (12/63) and diarrhea with mucus and blood in 3% (2/63). Ten (10) percent of the children were hospitalized due to gastroenteritis while 9 patients (14.3%) had co-infections with rotavirus. Human EAds were detected in 8% of specimens mainly in the dry season and among children older than 2 years. The principal symptoms were diarrhea (100%), dehydration (80%) and fever (80%). CONCLUSION: The findings of this study suggest that adenoviruses are important etiologic agents of gastroenteritis in Northwestern Nigerian children.