The Association between Symptomatic Rotavirus Infection and Histo-Blood Group Antigens in Young Children with Diarrhea in Pretoria, South Africa.

OBJECTIVES: Recently, histo-blood group antigens (HBGAs) have been identified as receptors or attachment factors of several viral pathogens. Among rotaviruses, HBGAs interact with the outer viral protein, VP4, which has been identified as a potential susceptibility factor, although the findings are inconsistent throughout populations due to HBGA polymorphisms. We investigated the association between HBGA phenotypes and rotavirus infection in children with acute gastroenteritis in northern Pretoria, South Africa. METHODS: Paired diarrheal stool and saliva samples were collected from children aged ≤ 59 months (n = 342) with acute moderate to severe diarrhea, attending two health care facilities. Rotaviruses in the stool samples were detected by commercial EIA and the rotavirus strains were characterized by RT-PCR targeting the outer capsid VP7 (G-type) and VP4 (P-type) antigens for genotyping. Saliva-based ELISAs were performed to determine A, B, H, and Lewis antigens for blood group typing. RESULTS: Blood type O was the most common blood group (62.5%) in this population, followed by groups A (26.0%), B (9.3%), and AB (2.2%). The H1-based secretors were common (82.7%) compared to the non-secretors (17.3%), and the Lewis antigen positive phenotypes (Le(a+b+)) were predominant (54.5%). Blood type A children were more likely to be infected by rotavirus (38.8%) than any other blood types. P[4] rotaviruses (21/49; 42.9%) infected only secretor individuals, whereas P[6] rotaviruses (3/49; 6.1%) only infected Le(a-b-), although the numbers were very low. On the contrary, P[8] rotaviruses infected children with a wide range of blood group phenotypes, including Le(a-b-) and non-secretors. CONCLUSIONS: Our findings demonstrated that Lewis antigens, or the lack thereof, may serve as susceptibility factors to rotaviral infection by specific VP4 genotypes as observed elsewhere. Potentially, the P[8] strains remain the predominant human VP4 genotype due to their ability to bind to a variety of HBGA phenotypes.

Genetic characterisation of south african and mozambican bovine rotaviruses reveals a typical bovine-like artiodactyl constellation derived through multiple reassortment events

This study presents whole genomes of seven bovine rotavirus strains from South Africa and Mozambique. Double-stranded RNA, extracted from stool samples without prior adaptation to cell culture, was used to synthesise cDNA using a self-annealing anchor primer ligated to dsRNA and random hexamers. The cDNA was subsequently sequenced using an Illumina MiSeq platform without prior genome amplification. All strains exhibited bovine-like artiodactyl genome constellations (G10/G6-P[11]/P[5]-I2-R2-C2-M2-A3/A11/A13-N2-T6-E2-H3). Phylogenetic analysis revealed relatively homogenous strains, which were mostly related to other South African animal strains or to each other. It appears that these study strains represent a specific bo-vine rotavirus population endemic to Southern Africa that was derived through multiple reassortment events. While one Mozambican strain, MPT307, was similar to the South African strains, the second strain, MPT93, was divergent from the other study strains, exhibiting evi-dence of interspecies transmission of the VP1 and NSP2 genes. The data presented in this study not only contribute to the knowledge of circulating African bovine rotavirus strains, but also em-phasise the need for expanded surveillance of animal rotaviruses in African countries in order to improve our understanding of rotavirus strain diversity

Genetic Characterisation of South African and Mozambican Bovine Rotaviruses Reveals a Typical Bovine-like Artiodactyl Constellation Derived through Multiple Reassortment Events.

This study presents whole genomes of seven bovine rotavirus strains from South Africa and Mozambique. Double-stranded RNA, extracted from stool samples without prior adaptation to cell culture, was used to synthesise cDNA using a self-annealing anchor primer ligated to dsRNA and random hexamers. The cDNA was subsequently sequenced using an Illumina MiSeq platform without prior genome amplification. All strains exhibited bovine-like artiodactyl genome constellations (G10/G6-P[11]/P[5]-I2-R2-C2-M2-A3/A11/A13-N2-T6-E2-H3). Phylogenetic analysis revealed relatively homogenous strains, which were mostly related to other South African animal strains or to each other. It appears that these study strains represent a specific bovine rotavirus population endemic to Southern Africa that was derived through multiple reassortment events. While one Mozambican strain, MPT307, was similar to the South African strains, the second strain, MPT93, was divergent from the other study strains, exhibiting evidence of interspecies transmission of the VP1 and NSP2 genes. The data presented in this study not only contribute to the knowledge of circulating African bovine rotavirus strains, but also emphasise the need for expanded surveillance of animal rotaviruses in African countries in order to improve our understanding of rotavirus strain diversity.

Whole Genome In-Silico Analysis of South African G1P[8] Rotavirus Strains Before and After Vaccine Introduction Over A Period of 14 Years.

Rotavirus G1P[8] strains account for more than half of the group A rotavirus (RVA) infections in children under five years of age, globally. A total of 103 stool samples previously characterized as G1P[8] and collected seven years before and seven years after introducing the Rotarix® vaccine in South Africa were processed for whole-genome sequencing. All the strains analyzed had a Wa-like constellation (G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1). South African pre- and post-vaccine G1 strains were clustered in G1 lineage-I and II while the majority (84.2%) of the P[8] strains were grouped in P[8] lineage-III. Several amino acid sites across ten gene segments with the exception of VP7 were under positive selective pressure. Except for the N147D substitution in the antigenic site of eight post-vaccine G1 strains when compared to both Rotarix® and pre-vaccine strains, most of the amino acid substitutions in the antigenic regions of post-vaccine G1P[8] strains were already present during the pre-vaccine period. Therefore, Rotarix® did not appear to have an impact on the amino acid differences in the antigenic regions of South African post-vaccine G1P[8] strains. However, continued whole-genome surveillance of RVA strains to decipher genetic changes in the post-vaccine period remains imperative.

Molecular Characterisation of a Rare Reassortant Porcine-Like G5P[6] Rotavirus Strain Detected in an Unvaccinated Child in Kasama, Zambia.

A human-porcine reassortant strain, RVA/Human-wt/ZMB/UFS-NGS-MRC-DPRU4723/2014/G5P[6], was identified in a sample collected in 2014 from an unvaccinated 12 month old male hospitalised for gastroenteritis in Zambia. We sequenced and characterised the complete genome of this strain which presented the constellation: G5-P[6]-I1-R1-C1-M1-A8-N1-T1-E1-H1. The genotype A8 is often observed in porcine strains. Phylogenetic analyses showed that VP6, VP7, NSP2, NSP4, and NSP5 genes were closely related to cognate gene sequences of porcine strains (e.g., RVA/Pig-wt/CHN/DZ-2/2013/G5P[X] for VP7) from the NCBI database, while VP1, VP3, VP4, and NSP3 were closely related to porcine-like human strains (e.g., RVA/Human-wt/CHN/E931/2008/G4P[6] for VP1, and VP3). On the other hand, the origin of the VP2 was not clear from our analyses, as it was not only close to both porcine (e.g., RVA/Pig-tc/CHN/SWU-1C/2018/G9P[13]) and porcine-like human strains (e.g., RVA/Human-wt/LKA/R1207/2009/G4P[6]) but also to three human strains (e.g., RVA/Human-wt/USA/1476/1974/G1P[8]). The VP7 gene was located in lineage II that comprised only porcine strains, which suggests the occurrence of independent porcine-to-human reassortment events. The study strain may have collectively been derived through interspecies transmission, or through reassortment event(s) involving strains of porcine and porcine-like human origin. The results of this study underline the importance of whole-genome characterisation of rotavirus strains and provide insights into interspecies transmissions from porcine to humans.

Genetic characterisation of novel G29P[14] and G10P[11] rotavirus strains from African buffalo.

We report the first description of rotavirus A strains in African buffalo (Syncerus caffer). Following RNA extraction from stool samples, cDNA was prepared, followed either by sequence-independent amplification and 454 pyrosequencing or direct sequencing on an Illumina MiSeq platform. RVA/Buffalo-wt/ZAF/4426/2002/G29P[14] exhibited a novel G29P[14] combination and an artiodactyl backbone: I2-R2-C2-M2-A11-N2-T6-E2-H3. RVA/Buffalo-wt/ZAF/1442/2007/G10P[11] also exhibited an artiodactyl backbone: I2-R2-C2-M2-A13-N2-T6-E2-H3. Characterisation of these genome constellations indicate that the two buffalo strains are moderately diverse from each other and related to South African bovine RVA strains. The detection of RVA in buffalo contribute to our understanding of the host range of rotavirus in animals.

Whole Genome Analysis of African G12P[6] and G12P[8] Rotaviruses Provides Evidence of Porcine-Human Reassortment at NSP2, NSP3, and NSP4.

Group A rotaviruses (RVA) represent the most common cause of pediatric gastroenteritis in children <5 years, worldwide. There has been an increase in global detection and reported cases of acute gastroenteritis caused by RVA genotype G12 strains, particularly in Africa. This study sought to characterize the genomic relationship between African G12 strains and determine the possible origin of these strains. Whole genome sequencing of 34 RVA G12P[6] and G12P[8] strains detected from the continent including southern (South Africa, Zambia, Zimbabwe), eastern (Ethiopia, Uganda), central (Cameroon), and western (Togo) African regions, were sequenced using the Ion Torrent PGM method. The majority of the strains possessed a Wa-like backbone with consensus genotype constellation of G12-P[6]/P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1, while a single strain from Ethiopia displayed a DS-1-like genetic constellation of G12-P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H2. In addition, three Ethiopian and one South African strains exhibited a genotype 2 reassortment of the NSP3 gene, with genetic constellation of G12-P[8]-I1-R1-C1-M1-A1-N1-T2-E1-H1. Overall, 10 gene segments (VP1-VP4, VP6, and NSP1-NSP5) of African G12 strains were determined to be genetically related to cognate gene sequences from globally circulating human Wa-like G12, G9, and G1 strains with nucleotide (amino acid) identities in the range of 94.1-99.9% (96.5-100%), 88.5-98.5% (93-99.1%), and 89.8-99.0% (88.7-100%), respectively. Phylogenetic analysis showed that the Ethiopian G12P[6] possessing a DS-1-like backbone consistently clustered with G2P[4] strains from Senegal and G3P[6] from Ethiopia with the VP1, VP2, VP6, and NSP1-NSP4 genes. Notably, the NSP2, NSP3, and NSP4 of most of the study strains exhibited the closest relationship with porcine strains suggesting the occurrence of reassortment between human and porcine strains. Our results add to the understanding of potential roles that interspecies transmission play in generating human rotavirus diversity through reassortment events and provide insights into the evolutionary dynamics of G12 strains spreading across selected sub-Saharan Africa regions.

Diversity of rotavirus strains circulating in children under five years of age who presented with acute gastroenteritis before and after rotavirus vaccine introduction, University Teaching Hospital, Lusaka, Zambia, 2008-2015.

BACKGROUND: Following the introduction of rotavirus vaccine into the routine immunization schedule, the burden of rotavirus disease has significantly reduced in Zambia. Although rotavirus vaccines appear to confer good cross-protection against both vaccine and non-vaccine strains, concerns about strain replacement following vaccine implementation remain. We describe the diversity of the circulating rotavirus strains before and after the Rotarix® vaccine was introduced in Lusaka from January 2012. METHODS: Under five children were enrolled through active surveillance at University Teaching Hospital using a standardized WHO case investigation form. Stool samples were collected from children who presented with ≥3 loose stool in 24 h and were admitted to the hospital for acute gastroenteritis as a primary illness. Samples were tested for group A rotavirus antigen enzyme-linked immunosorbent assay. Randomly selected rotavirus positive samples were analysed by reverse transcription polymerase chain reaction for G and P genotyping and and Nucleotide sequencing was used to confirm some mixed infections. RESULTS: A total of 4150 cases were enrolled and stool samples were collected from 4066 (98%) children between 2008 and 2011, before the vaccine was introduced. Rotavirus antigen was detected in 1561/4066 (38%). After vaccine introduction (2012 to 2015), 3168 cases were enrolled, 3092 (98%) samples were collected, and 977/3092 (32%) were positive for rotavirus. The most common G and P genotype combinations before vaccine introduction were G1P[8] (49%) in 2008; G12P[6] (24%) and G9P[8] (22%) in 2009; mixed rotavirus infections (32%) and G9P[8] (20%) in 2010, and G1P[6] (46%), G9P[6] (16%) and mixed infections (20%) in 2011. The predominant strains after vaccine introduction were G1P[8] (25%), G2P[4] (28%) and G2P[6] (23%) in 2012; G2P[4] (36%) and G2P[6] (44%) in 2013; G1P[8] (43%), G2P[4] (9%), and G2P[6] (24%) in 2014, while G2P[4] (54%) and G2P[6] (20%) continued to circulate in 2015. CONCLUSION: These continual changes in the predominant strains suggest natural secular variation in circulating rotavirus strains post-vaccine introduction. These findings highlight the need for ongoing surveillance to continue monitoring how vaccine use affects strain evolution over a longer period of time and assess any normal seasonal fluctuations of the rotavirus strains.

Complete genome analyses of the first porcine rotavirus group H identified from a South African pig does not provide evidence for recent interspecies transmission events.

Rotaviruses (RVs) are classified into eight species/groups (RVA-RVH) according to the migration patterns of their 11 genome segments, as well as by serological and molecular properties of Viral Protein 6 (VP6). In 1997 a new unclassified RV was reported infecting adults in Bangladesh and China. This virus was initially named novel adult diarrhoea rotavirus (ADRV-N), but later renamed as RVH. Since then, RVH has been detected in humans only very sporadically. However, RVH is increasingly being detected in pig populations in the USA, Brazil and Japan, but not yet in Africa. Unfortunately, whole genome sequence data of porcine RVH strains in GenBank is currently restricted to a single strain (SKA-1) from Japan. Porcine diarrhoeic samples were collected in South Africa and analysed for rotavirus using an RVA ELISA and electropherotyping by PAGE. One sample displayed a 4:2:1:1:1:1:1 migration pattern, typical for RVH. In order to further investigate this strain, sequence-independent amplification followed by random sequencing using the 454/Roche GS FLX Sequencer was performed, resulting in the second complete porcine RVH strain (MRC-DPRU1575) available in databases. Phylogenetically, all segments of MRC-DPRU1575 clustered closely with the SKA-1 strain and in some segments with known porcine RVH strains from Brazil and the USA. In contrast, the porcine RVH strains were only distantly related to human RVH strains from Asia and a partial RVH-like strain recently detected in bats from Cameroon. Overall, strain MRC-DPRU1575 is the first complete genome of a porcine RVH from Africa and allows for the development of improved RVH screening methods. Our analyses indicate that RVH strains cluster according to their host species, not suggesting any evidence of recent interspecies transmission events. However, more RVH genomes from a wider host range are needed to better understand their evolutionary pathways and zoonotic potential.

Complete genomic sequence for an avian group G rotavirus from South Africa.

We report the first complete sequence for an avian group G rotavirus (RVG) genome from Africa, which is the third publically available RVG genome. These RVG genomes are highly diverse, especially in their VP4, VP7, NSP4, and NSP3 segments, indicating that RVG diversity is comparable to that of rotavirus A.

Whole genome detection of rotavirus mixed infections in human, porcine and bovine samples co-infected with various rotavirus strains collected from sub-Saharan Africa.

Group A rotaviruses (RVA) are among the main global causes of severe diarrhea in children under the age of 5years. Strain diversity, mixed infections and untypeable RVA strains are frequently reported in Africa. We analysed rotavirus-positive human stool samples (n=13) obtained from hospitalised children under the age of 5years who presented with acute gastroenteritis at sentinel hospital sites in six African countries, as well as bovine and porcine stool samples (n=1 each), to gain insights into rotavirus diversity and evolution. Polyacrylamide gel electrophoresis (PAGE) analysis and genotyping with G-(VP7) and P-specific (VP4) typing primers suggested that 13 of the 15 samples contained more than 11 segments and/or mixed G/P genotypes. Full-length amplicons for each segment were generated using RVA-specific primers and sequenced using the Ion Torrent and/or Illumina MiSeq next-generation sequencing platforms. Sequencing detected at least one segment in each sample for which duplicate sequences, often having distinct genotypes, existed. This supported and extended the PAGE and RT-PCR genotyping findings that suggested these samples were collected from individuals that had mixed rotavirus infections. The study reports the first porcine (MRC-DPRU1567) and bovine (MRC-DPRU3010) mixed infections. We also report a unique genome segment 9 (VP7), whose G9 genotype belongs to lineage VI and clusters with porcine reference strains. Previously, African G9 strains have all been in lineage III. Furthermore, additional RVA segments isolated from humans have a clear evolutionary relationship with porcine, bovine and ovine rotavirus sequences, indicating relatively recent interspecies transmission and reassortment. Thus, multiple RVA strains from sub-Saharan Africa are infecting mammalian hosts with unpredictable variations in their gene segment combinations. Whole-genome sequence analyses of mixed RVA strains underscore the considerable diversity of rotavirus sequences and genome segment combinations that result from a complex evolutionary history involving multiple host species.

Genetic diversity of rotavirus genome segment 6 (encoding VP6) in Pretoria, South Africa.

BACKGROUND: Rotavirus viral protein 6 (VP6), encoded by genome segment (GS) 6, is the primary target for rotavirus diagnosis by serological and some molecular techniques. Selected full length nucleotide sequences of GS 6 of rotavirus strains from South Africa were sequenced and analysed to determine genetic diversity and variations within the circulating rotaviruses. FINDINGS: The VP6 amplicons were sequenced using the Sanger ABI 3130xl. Phylogenetic and pairwise analysis revealed that the VP6 genes of the study strains belonged to two different VP6 [I] genotypes. Five sequences were assigned genotype I1 and seven as genotype I2. Comparison of the group specific antigenic regions of the South African strains to the reference strains, shows that the South African VP6 sequences belonging to the VP6 genotype I2 were highly conserved, with only two amino acids changes at positions 239 (T>N) and 261(I>V). On the other hand, South African VP6 sequences belonging to I1 genotypes revealed several amino acid variations mostly within the antigenic region III. CONCLUSIONS: Rotavirus strains with I1 and I2 genotype are predominantly circulating within the South African communities of which the later seems to be more conserved within the antigenic regions. The observed genetic variations observed within GS 6 of rotaviruses analysed in the current study are unlikely to impact negatively on the performance of the current VP6-based detection methods. Nevertheless, investigators should continually consider this diversity and adapt the primer design for the detection and characterization of the VP6 gene accordingly.

Novel NSP1 genotype characterised in an African camel G8P[11] rotavirus strain.

Animal-human interspecies transmission is thought to play a significant role in influencing rotavirus strain diversity in humans. Proving this concept requires a better understanding of the complete genetic constellation of rotaviruses circulating in various animal species. However, very few whole genomes of animal rotaviruses, especially in developing countries, are available. In this study, complete genetic configuration of the first African camel rotavirus strain (RVA/Camel-wt/SDN/MRC-DPRU447/2002/G8P[11]) was assigned a unique G8-P[11]-I2-R2-C2-M2-A18-N2-T6-E2-H3 genotype constellation that has not been reported in other ruminants. It contained a novel NSP1 genotype (genotype A18). The evolutionary dynamics of the genome segments of strain MRC-DPRU447 were rather complex compared to those found in other camelids. Its genome segments 1, 3, 7-10 were closely related (>93% nucleotide identity) to those of human-animal reassortant strains like RVA/Human-tc/ITA/PA169/1988/G6P[14] and RVA/Human-wt/HUN/Hun5/1997/G6P[14], segments 4, 6 and 11 shared common ancestry (>95% nucleotide identity) with bovine rotaviruses like strains RVA/Cow-wt/CHN/DQ-75/2008/G10P[11] and RVA/Cow-wt/KOR/KJ19-2/XXXX/G6P[7], whereas segment 2 was closely related (94% nucleotide identity) to guanaco rotavirus strain RVA/Guanaco-wt/ARG/Rio_Negro/1998/G8P[1]. Its genetic backbone consisted of DS-1-like, AU-1-like, artiodactyl-like and a novel A18 genotype. This suggests that strain MRC-DPRU447 potentially emerged through multiple reassortment events between several mammalian rotaviruses of at least two genogroups or simply strain MRC-DPRU447 display a unique progenitor genotypes. Close relationship between some of the genome segments of strain MRC-DPRU447 to human rotaviruses suggests previous occurrence of reassortment processes combined with interspecies transmission between humans and camels. The whole genome data for strain MRC-DPRU447 adds to the much needed animal rotavirus data from Africa which is limited at the moment.

Epidemiologic and genotypic characteristics of rotavirus strains detected in children less than 5 years of age with gastroenteritis treated at 3 pediatric hospitals in Zimbabwe during 2008-2011.

BACKGROUND: In anticipation of rotavirus vaccine introduction, the Zimbabwe Ministry of Health initiated rotavirus surveillance in 2008 to describe the rotavirus epidemiological trends and circulating genotypes among children <5 years of age. METHODS: Active hospital-based surveillance for diarrhea was conducted at 3 sentinel sites from January 2008 to December 2011. Children aged <5 years, who presented with acute gastroenteritis as a primary illness and who were admitted to a hospital ward or treated at the emergency unit, were enrolled in the surveillance program and had a stool specimen collected and tested for rotavirus by enzyme immunoassay. Genotyping of a sample of positive specimens was performed using reverse-transcription polymerase chain reaction. RESULTS: A total of 3728 faecal samples were collected and tested during the 4 year surveillance period and 1804 (48.5%) tested rotavirus positive. The highest prevalence of rotavirus diarrhea was found during the dry, cool season. Rotavirus positivity peaked in children 3-17 months of age with almost 80% of cases. Compared with rotavirus-negative cases, rotavirus-positive cases were more likely to be dehydrated (26% vs. 14%, P ≤ 0.001) and have vomiting (77% vs. 57%, P ≤ 0.001) and less likely to have fever (17% vs. 24%, P = 0.03). G9P[8] (43.3%), G1P[8] (11.8%), G2P[4] (8.7%), G2P[6] (8.7%) and G12P[6] (8.7%) were the most common genotypes detected. DISCUSSION: Rotavirus causes a significant disease burden among children <5 years of age in Zimbabwe. This active surveillance system can serve as a platform to monitor the impact of rotavirus vaccine on disease burden following vaccine introduction.

Update of rotavirus strains circulating in Africa from 2007 through 2011.

BACKGROUND: The African Rotavirus Surveillance Network has been detecting and documenting rotavirus genotypes in the subcontinent since 1998, largely based on intercountry workshops conducted at Rotavirus Regional Reference Laboratories. This article reports on rotavirus genotypes generated at Regional Reference Laboratories, South Africa between 2007 and 2011 from 16 African countries. METHODS: Stool samples were collected from <5-year-old children with diarrhea following World Health Organization criteria of hospital-based rotavirus surveillance. Enzyme immunoassay (EIA) was performed by National Laboratories. Regional Reference Laboratories retested 10% of randomly selected EIA positives and 10% of EIA negatives from each country as part of quality control. At least 50 rotavirus EIA positives from each country per year were subjected to reverse transcriptase polymerase chain reaction based on G-/P-types. Sequencing was conducted in 5-10% of each representative G or P genotype to confirm the genotype, as well as to type some of the samples that could not be genotyped with reverse transcriptase polymerase chain reaction-based methods. RESULTS: A total of 2555 of rotavirus EIA positives were genotyped. G1 was the most predominant (28.8%), followed by G9 (17.3%), G2 (16.8%), G8 (8.2%), G12 (6.2%) and G3 (5.9%). Similarly, the P[8] strain was the most prevalent (40.6%), followed by P[6] (30.9%) and P[4] (13.9%). The top G/P combinations detected were G1P[8] (18.4%), G9P[8] (11.7%), G2P[4] (8.6%), G2P[6] (6.2%), G1P[6] (4.9%), G3P[6] (4.3%), G8P[6] (3.8%) and G12P[8] (3.1%). CONCLUSIONS: There is high genetic diversity of rotavirus strains circulating in the subcontinent. Understanding the strain diversity pre- and postvaccine introduction are important in Africa to understand the broader impact of the rotavirus vaccines on regionally circulating strains.

Sequence analysis of the whole genomes of five African human G9 rotavirus strains.

The G9 rotaviruses are amongst the most common global rotavirus strains causing severe childhood diarrhoea. However, the whole genomes of only a few G9 rotaviruses have been fully sequenced and characterised of which only one G9P[6] and one G9P[8] are from Africa. We determined the consensus sequence of the whole genomes of five African human group A G9 rotavirus strains, four G9P[8] strains and one G9P[6] strain collected in Cameroon (central Africa), Kenya (eastern Africa), South Africa and Zimbabwe (southern Africa) in 1999, 2009 and 2010. Strain RVA/Human-wt/ZWE/MRC-DPRU1723/2009/G9P[8] from Zimbabwe, RVA/Human-wt/ZAF/MRC-DPRU4677/2010/G9P[8] from South Africa, RVA/Human-wt/CMR/1424/2009/G9P[8] from Cameroon and RVA/Human-wt/KEN/MRC-DPRU2427/2010/G9P[8] from Kenya were on a Wa-like genetic backbone and were genotyped as G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. Strain RVA/Human-wt/ZAF/MRC-DPRU9317/1999/G9P[6] from South Africa was genotyped as G9-P[6]-I2-R2-C2-M2-A2-N1-T2-E2-H2. Rotavirus A strain MRC-DPRU9317 is the second G9 strain to be reported on a DS-1-like genetic backbone, the other being RVA/Human-wt/ZAF/GR10924/1999/G9P[6]. MRC-DPRU9317 was found to be a reassortant between DS-1-like (I2, R2, C2, M2, A2, T2, E2 and H2) and Wa-like (N1) genome segments. All the genome segments of the five strains grouped strictly according to their genotype Wa- or DS-1-like clusters. Within their respective genotypes, the genome segments of the three G9 study strains from southern Africa clustered most closely with rotaviruses from the same geographical origin and with those with the same G and P types. The highest nucleotide identity of genome segments of the study strains from eastern and central Africa regions on a Wa-like backbone was not limited to rotaviruses with G9P[8] genotypes only, they were also closely related to G12P[6], G8P[8], G1P[8] and G11P[25] rotaviruses, indicating a close inter-genotype relationship between the G9 and other rotavirus genotypes. Rotavirus strain MRC-DPRU9317 is the first G9P[6] to be characterised on a DS-1-like genetic backbone with a reassortant segment 8 (NSP2) and fourth G9P[6] to be fully sequenced globally.

Whole genome sequence analyses of three African bovine rotaviruses reveal that they emerged through multiple reassortment events between rotaviruses from different mammalian species.

Animal-to-human interspecies transmission is one of the evolutionary mechanisms driving rotavirus strain diversity in humans. Although quite a few studies emanating from Africa revealed evidence of bovine-to-human rotavirus interspecies transmission, whole genome data of African bovine rotavirus strains are not yet available. To gain insight into the complete genome constellation of African bovine rotaviruses, the full genomes of three bovine rotavirus strains were extracted from stool samples collected from calves, amplified using a sequence-independent procedure, followed by 454(®) pyrosequencing. Strains RVA/Cow-wt/ZAF/1603/2007/G6P[5] and RVA/Cow-wt/ZAF/1605/2007/G6P[5] were both genotyped as G6-P[5]-I2-R2-C2-M2-A3-N2-T6-E2-H3 and were probably two variants of the same rotavirus due to their close nucleotide sequence similarity. The genotype constellation of strain RVA/Cow-wt/ZAF/1604/2007/G8P[1] was G8-P[1]-I2-R2-C2-M2-A3-N2-T6-E2-H3. The genetic relationships and phylogenetic analyses suggested that these three bovine rotavirus strains may have emerged through multiple reassortment events between bovine, giraffe and antelope rotaviruses. Due to the close relatedness of genome segments 1 (encoding VP1), 7 (NSP2), 9 (VP7) and 10 (NSP4) of strain RVA/Cow-wt/ZAF/1604/2007/G8P[1] to those of the corresponding segments of human rotaviruses, RVA strain 1604 may represent bovine strains that were transmitted to humans and possibly reassorted with human rotaviruses previously. The complete nucleotide sequences of the bovine rotavirus strains reported in this study represent the first whole genome data of bovine rotaviruses from Africa.

Rotavirus genetic diversity, disease association, and temporal change in hospitalized rural Kenyan children.

BACKGROUND: The effectiveness of rotavirus vaccines will be dependent on the immunity conferred against prevalent and emergent variants causing severe diarrheal disease. Longitudinal surveillance of disease-causing strains is a prerequisite to intervention. METHODS: Molecular characterization was conducted on rotavirus-positive stool samples from children admitted with diarrhea to a rural district hospital during 2002-2004. Extracted viral RNA was separated by polyacrylamide gel electrophoresis, and rotavirus VP4 (P types) and VP7 (G types) specificities were determined. RESULTS: Among 558 investigated cases, the predominant genotype was P[8]G1 (42%), followed by P[8]G9 (15%), P[4]G8 (7%), P[6]G8 (6%), and P[8]G8 (4%), with 10% mixed strains. Overall, there were 6 different P types and 7 G types. No association was identified between genotype and child age, sex, or severity of diarrhea. The P and G genotypes and polyacrylamide gel electropherotypes showed significant temporal variation in frequency: P[8]G1 decreased from 51% (95% confidence interval [CI], 43%-58%) in 2002 to 30% (95% CI, 24%-37%) in 2004, and P[4]G8 increased from 2% (95% CI, 0%-5%) in 2002 to 13% (95% CI, 9%-19%). Quarterly data revealed seasonally endemic and emergence and/or decay patterns. CONCLUSIONS: Our study of rotavirus strains causing severe diarrhea in rural Kenyan children showed a predominance of P[8]G1 and confirms the importance of G8 and G9 strains in sub-Saharan Africa. Considerable genetic diversity of rotavirus strains was observed, including substantial mixed and unusual types, coupled with significant temporal strain variation and emergence. These results warn of variable vaccine efficacy and the need for long-term surveillance of circulating rotavirus genotypes.

Characterization of human rotavirus strains from children with diarrhea in Nairobi and Kisumu, Kenya, between 2000 and 2002.

Rotavirus infection is a major cause of diarrheal illness and hospitalization in children <5 years old in Kenya and has been described in various settings and locations across the country and for different time points. In this study, we expand on the molecular characterization of rotavirus strains collected in Nairobi and Kisumu, Kenya, between 2000 and 2002. Rotavirus strains were typed by reverse-transcription polymerase chain reaction and characterized using VP6 monoclonal antibodies and RNA electrophoresis of the viral genome. A large proportion of specimens could not be genotyped; 41% did not produce a G type result, and 43% did not produce a P type result. Of the strains that could be genotyped, G1P[8] strains were predominant, followed by G2P[4] strains. In addition, G8 and G9 strains were seen in similar proportions Interestingly, the G and P combinations were more diverse among G8 and G9 rotavirus strains, suggesting the recent introduction of these strains into the human population. These observations are a link between the occasional observation of G8 and G9 strains at the turn of the century and the high predominance of G9P[8] strains observed in Kenya in 2005.

Molecular characterization of rotavirus strains circulating in Oman in 2005.

Limited genotyping data are available for rotavirus strains in the Middle East. In this study, we investigated the molecular epidemiology of human rotavirus strains circulating in the Sultanate of Oman during 2005. Rotavirus was detected in 178 (57.4%) of 310 of the diarrheal stools of young children <5 years admitted to hospitals and outpatients clinics. Polyacrylamide gel electrophoresis demonstrated the cocirculation of 8 strains, although 2 strains predominated across the Sultanate. Genotyping revealed the presence of human rotavirus strains of types G1P[8], G2P[4], and G3P[8]. Several strains exhibited unusual combinations of G and P genotypes and RNA electropherotypes, indicating the likelihood of natural reassortment events occurring with a high frequency. In addition, the unusual P[10] genotype was identified among the rotavirus strains, in combination with the G1 type.