Rotaviruses are the most common cause of severe diarrhea requiring hospitalization in young children and it is estimated to cause approximately 453,000 deaths in children <5 years of age annually.1 More than 50% of these 453,000 deaths occur in Africa. Surveillance studies in 9 African countries (Cameroon, Ethiopia, Ghana, Kenya, Tanzania, Togo, Uganda, Zambia and Zimbabwe) have shown 41% median detection (range: 16–57%) rate of rotavirus in hospitalized children <5 years of age.2,3 Previous data from African countries have also estimated that rotavirus infections cause approximately 25–48% of all diarrheal hospital admissions.4–6
Rotavirus genotypes are classified according to 2 outer capsid proteins: VP7 and VP4. The VP7 specifies the G (glycoprotein) and VP4 specifies the P (protease-sensitive) genotype.7 These proteins segregate independently and elicit neutralizing antibodies, and thus are crucial components of rotavirus vaccine development. More recently, rotavirus classification based on 11 genome segments has been established, and this system has shown 27G and 35P genotypes.8 Among these genotypes, 12G and 15P are commonly associated with human infections. The G1P, G2P, G3P, G4P and G9P represent >90% genotypes circulating in developed countries of North America, Europe and Australia.9–11 Rotavirus strains in Africa are known to be very diverse compared with many regions and several unusual strains have been reported from the continent.12,13
The African Rotavirus Surveillance Network (AFR RSN) was established in 1998 as a regional partnership between African countries, the World Health Organization (WHO) and its partners. The AFR RSN initiative successfully grew over the years and has been instrumental in creating awareness of the burden of rotavirus disease in Africa, and it has facilitated collection of data, skills development and training of regional experts. The network is actively participating in activities to determine the burden of rotavirus disease in hospital settings, define rotavirus epidemiology and identify rotavirus strains circulating in Africa. Based on annual regional intercountry workshops, rotavirus surveillance studies were conducted in many African countries including South Africa, Zimbabwe, Zambia, Malawi, Tanzania, Uganda, Kenya, Ethiopia, Democratic Republic of Congo (DRC), Ghana, Togo, Senegal, Guinea Bissau, Nigeria and Burkina Faso.2,5,14–17 This article describes the rotavirus genotypes generated by 16 countries from Southern Africa, East Africa, West Africa and Central Africa, between 2007 and 2011, before rotavirus vaccines were introduced.
MATERIAL AND METHODS
Participating Countries of the African Rotavirus Surveillance Network
The genotyping workshops were conducted at the WHO Rotavirus Regional Reference Laboratory (RRL), located at the South African Medical Research Council Diarrheal Pathogens Research Unit, based at the University of Limpopo, Medunsa Campus, Pretoria, South Africa (hereinafter referred to as RRL South Africa). Between 2007 and 2011, a total of 30 participants (some participants attended more than once) from 16 African countries representing over 32 sentinel sites attended the workshops to generate regional data on rotavirus burden and circulating genotypes (Table 1).
Sample Collection and Transfer to RRL
Samples were collected from children <5 years of age with diarrhea who fulfilled the criteria for hospital-based rotavirus surveillance using the WHO generic protocol. For every workshop, each country was requested to bring a minimum of 50 enzyme immunoassay (EIA) positives spanning January to December of the year of collection for genotyping purpose and 10% of EIA negatives for quality control purpose. In total, 3412 rotavirus EIA-positive and 1223 EIA-negative stool samples were brought to RRL between 2007 and 2011. Genotyping was performed in 2555 randomly selected rotavirus EIA-positive samples. The hospital-based surveillance studies in Africa follow the WHO generic protocol and WHO Regional Office for Africa. Standard Operating Procedures for stool collection, storage and laboratory testing.
Detection of Rotavirus Antigen by EIA
The initial testing of group A rotavirus infection was performed by participating National Laboratories or Sentinel Laboratories in the respective countries using ProSpecT Rotavirus kit (Oxoid Ltd, United Kingdom). At the RRL South Africa, 10% of randomly selected EIA positives and 10% of EIA negatives from each country were validated by retesting with ProSpecT Rotavirus kit.
Reverse Transcriptase Polymerase Chain Reaction (PCR) and Genotyping Assays for Gene Segments 4 and 9
Viral dsRNA was extracted from 140 μL of 10% fecal suspensions using the QIAamp viral RNA extraction method ( QIAGEN, Hilden, Germany). The extracted RNA was reverse transcribed and amplified in the presence of avian myloblastosis virus and Taq DNA polymerase using consensus primer pairs Con2/Con3 and sBeg/end9 as described by Gentsch et al and Gouvea et al,18,19 respectively. The resulting cDNA template was used for G and P typing using seminested PCR amplification of the genes encoding the VP7 and VP4.18–22
Sequence Analyses of the Genomic Segments 4 and 9
Partial gene sequencing was conducted in 5–10% of each representative G or P genotype to confirm the genotype, as well as to identify some of the strains that could not be genotyped with reverse transcriptase PCR-based methods. The VP4 and VP7 PCR amplicons were sequenced with the forward and reverse primers using the dideoxynucleotide chain termination method (ABI 3130XL sequencer, Inqaba Biotechnological Industry, Pretoria, South Africa). The chromatogram sequencing data were edited and aligned with the corresponding reference strains from GenBank using ChromasPro (www.technelysium.com.au) and BioEdit (www.mbio.ncsu.edu/bioEdit/bioedit.html) software packages. The sequences were verified by Nucleotide Basic Local Alignment Search Tool (BLASTN) analysis on the National Centre for Biotechnology Information website. Genotype data generated through sequencing were included in the overall genotyping results.
Quality Control of EIA-positive and EIA-negative Samples
Quality control was performed on 1823 samples (711 positive stool samples and 1112 negative samples). Quality control results on negative stool samples showed 80–100% concordance (with the majority being at least 90%) between the National Laboratories/Sentinel Laboratories and the RRL, while the quality control for rotavirus-positive samples showed 60–100% concordance (with the majority being at least 90%) between the National Laboratories/Sentinel Laboratories and the RRL. Countries that did not achieve 100% correlation during validation process were given feedback with possible solutions to avoid false positive or negative results during EIA testing and were able to improve their national surveillance efforts.
Distribution of Rotavirus Genotypes in Africa
Single G and P Genotypes
From a total of 2555 rotavirus positives, G1 was the most predominant strain widely distributed in all regions (n = 736; 28.8%) during the 5-year period, with the exception of central Africa where G1 was detected less frequently (Table 2). The second most predominant strain circulating in the region was G9 (17.3%), with the majority found in Southern Africa (Zimbabwe and Zambia), East Africa (Kenya) and Central Africa (Cameroon). The rotavirus G2 strain represented the third most prevalent strain, detected at 16.8% in the subcontinent. The temporal distribution of G2 rotavirus strains varied seasonally, year to year and country to country. In some African countries (Cameroon, Tanzania, Uganda and Cote d’Ivoire), G2 strains were sporadically identified while it was prevalent in other countries such as Guinea Bissau, DRC, The Gambia and South Africa. G8 rotavirus strains (8.2%) were also 1 of the major genotypes circulating in most African countries. G8 represented the fourth most prevalent strain and was detected in Kenya, Uganda, Cameroon, Zimbabwe and Zambia. G12 represented the fifth most prevalent strain (6.2%). G12s have been detected in many Africa countries including Ethiopia, Togo, DRC, Zambia, Zimbabwe, Cameroon, Uganda and Kenya. G3 genotypes were the sixth most prevalent strains, detected in 5.9% of samples. G3 strains were mainly circulating in Ethiopia, Togo, Nigeria and Cameroon. G3 strains with long RNA pattern were only detected in Mauritius.
The P genotypes circulating in the region showed that P strains were the most prevalent, detected at 40.6% (Table 3). This was followed by P (30.9%) and P (13.9%) strains. The P genotype was found in combination with G1, G3, G4, G9, G8 and G12. The P genotype was also detected in association with a wide variety of G types (G1, G2, G3, G9, G8, G12 and G6), while P was found in combination with limited G types (G2, G9 and G8).
G and P Combinations
The G and P combinations were determined in 2555 of 3412 (74.8%) rotavirus EIA positives (Table 4). The genotyping results revealed a high diversity of rotavirus strains circulating in the subcontinent. Overall, the top 8 predominating G/P combinations were G1P (18.4%), G9P (11.7%), G2P (8.6%), G2P (6.2%), G1P (4.9%), G3P (4.3%), G8P (3.8%) and G12P (3.1%). These strains were unevenly distributed during the 5-year period. The circulating strains showed considerable geographic and temporal variation, while the prevalence of individual strains showed yearly changes or steady increase year on year. Mixed genotype infections (multiple G or P genotypes identified in 1 sample) and partially typed samples (either G or P) were detected frequently in many African countries representing 17.5% and 7.2%, respectively, of samples. Atypical rotavirus G and P combinations were detected at low frequency including G2P, G9P, G8P and G12P. Furthermore, the presence of possible human-animal G and P strains in Mauritius, Tanzania, Kenya, Guinea Bissau, Senegal and Nigeria was also detected during the surveillance period. These included G8P, G6P, G4P, G9P and G10P that are commonly found in either cattle or porcine.
G and P Combinations in Southern Africa
In Southern Africa (Zimbabwe and Zambia), 6 G/P combinations most prevalent included G9P (20%), G1P (14%), G1P (6%), G12P (6%), G8P (5%) and G9P (5%) (Fig. 1A). In Zimbabwe, G9P has been predominant since 2009.
G and P Combinations in East Africa
The East African region was represented by Ethiopia, Kenya, Mauritius, Tanzania and Uganda. G1P was the most predominant strain detected at 23%, followed by G9P (12%), G2P (8%), G8P (5%) and 5 different genotypes (G8P, G9P, G3P, G12P and G1P) each at 4% (Fig. 1B). The G1P strain was detected in relatively high incidence in all the participating countries, with the exception of Kenya in 2010 and 2011. Instead, G9P and G8P strains were the most prevalent genotypes in Kenya during 2010–2011. There was also high prevalence of G3P and G12P strains in Ethiopia.
G and P Combinations in West Africa
The West African region consisted of Burkina Faso, Cote d’Ivoire, Guinea Bissau, The Gambia, Nigeria, Senegal and Togo. Overall, G1P was the most frequent strain observed (17%), followed by G2P (13%), G2P (9%), G3P (9%), G9P (7%), G1P (5%) and G12P (3%; Fig. 1C). Partially typed P or G and mixed G and P types were also identified. The distribution of G1P/G1P and G2P/G2P genotypes was nearly similar during the study period. The G2P and G2P strains were shown to be important strains circulating in Guinea Bissau in 2011 and The Gambia in 2010. G3P rotavirus strains were mainly detected in Togo (2009–2011) and Nigeria (2011). G6P strains were sporadically identified in Guinea Bissau, Nigeria and Senegal during 2011.
G and P Combinations in Central Africa
This region comprised Cameroon and DRC. The prevalent strains were G2P (17%), G2P (14%), G9P (11%), G1P (9%), G1P (6%) and G8P (5%) (Fig. 1D). G9P was found to be the predominant strain in Cameroon during 2008 and 2009.
The findings of our analysis confirm that the worldwide common strains (G1 to G4, G9) are not present at the same proportions in Africa as in industrialized countries. The 5-year (2007–2011) analysis in Africa, demonstrated that while G1 strain was the most predominant (28.8%) and G2 (16.8%), G3 (5.9%) and G9 (17,3%) strains occurred commonly, there are still a large proportion of strains that are novel, such as G8 (8.2%) and G12 (6.2%), and large numbers that are mixed or untypables using current primers sets. Similarly, the P genotypes detected in the continent showed that while P (40.6%) and P (13.9%) strains were prevalent, the P genotype strains were very common (30.9%) in this study as reported previously for African rotaviruses. This seems to be a peculiar distribution of rotavirus strains circulating in Africa compared with other regions.
The predominant strains identified reflect the great diversity and unusual nature of rotavirus strains in Africa. G/P combinations included G1P (18.4%), G9P (11.7%) and G2P (8.6%) which are found globally, and G2P (6.2%), G1P (4.9%), G3P (4.3%), G8P (3.8%) and G12P (3.1%) which are unusual (Table 4). In contrast, G1P, G2P, G3P, G4P and G9P remain the most important strains globally and represent >90% in industrialized countries of North America, Europe and Australia.9,11 The frequencies of these globally prevalent strains have been examined since the beginning of the AFR RSN and represent <50% of strains in Africa. The greater strain diversity among rotavirus strains circulating in Africa remains consistently high as documented in the last 5 years.
Consistent with the findings from numerous published data from Africa, rotavirus G1 strains were widely distributed in many African countries during this study period. Between 1996 and 1999 during the 3 African rotavirus network workshops, G1 serotypes were found to be the most significant strains in Cote I’voire, Nigeria, Tunisia, Kenya, Tanzania, Cameroon, Zambia, Botswana, South Africa, Namibia and Zimbabwe,16 highlighting the likely biological competitive advantage of this strain in human infants. Since the global emergence of G9 rotavirus strains in mid 1990s, and when they were first detected in Africa in 1995 in Malawi, they have become widely distributed in the continent. Our results showed the continued and widespread distribution of these strains across the continent as G9 strains were detected in Zimbabwe, Zambia, Kenya and Cameroon to corroborate documentation of G9 strains previously in Libya, Kenya, Ghana, Malawi, Nigeria, Guinea Bissau and South Africa.2,15–17,23,24 Interestingly, G9 strains are associated with both P and P VP4 genotypes in southern and eastern Africa.
G2P strains remain a constant strain identified in many African countries, and the strains have been found at increasing frequencies in many countries including South Africa, Ghana, Guinea Bissau, Burkina Faso, Mauritius, Ivory Coast, Kenya, Tunisia and Zambia.16,25–27 In South Africa, at Dr George Mukhari Hospital, the incidence of rotavirus G2 strains has been documented to occur after every 3–4 years since 1984,5,26,27 indicating a classical “cyclic” nature of occurrence, which has been documented in other settings worldwide. It is of possible concern, that the 2010 surveillance data from Blantyre, Malawi has documented a 10-year absence of G2 strains,28 which may mean that a major G2P outbreak could occur in this naïve population.
The presence of G8 rotavirus strains have been reported in many African countries previously including Kenya, Malawi, Nigeria, South Africa, DRC, Egypt, Guinea Bissau and Ghana.14,17,29–31 This report shows the consistent and steady dominance of the G8 strains in Africa, with high prevalence detected in Kenya, Zimbabwe, Zambia and Cameroon. This unusual distribution and detection of G8 strains in Africa was believed linked to possible zoönotic transmission, but the consistency of the observed rates implies that there is person-to-person transmission occurring, albeit at a low level.
Interestingly, during 1996 and 1999, the AFR RSN reported more cases of G3 strains, with subgroup II and a long RNA migration patterns in Zimbabwe, Ghana, Nigeria and Cameroon.16,32 G3 strains became uncommon in many African countries for nearly a decade; only reported in a few countries such as South Africa in 2005.27 However, between 2009 and 2011, the G3 strains seem to have reemerged in Africa, detected in Mauritius, Togo, Ethiopia, Cameroon and Nigeria (this study).
Second, in the last 5 years, the distribution of rotavirus genotypes varied geographically from region to region and from country to country both temporally and spatially, confirming that the molecular epidemiology of rotavirus strains is complex; the strains likely to circulate each year cannot be entirely predicted. This has important implications for the introduction of rotavirus vaccines and for monitoring strain diversity after introduction. It is crucial to recognize that the annual circulating strains will fluctuate over time and in different regions of the continent and to integrate this information with interpretation of the postvaccine introduction surveillance. For example, G9 rotavirus strains were common across all regions of the continent during 2007–2011 including South Africa, Zambia, Zimbabwe, Cameroon, Kenya and Senegal. However, G2 strains were commonly detected in West Africa (Guinea Bissau and The Gambia) and DRC. Thus, as we study postvaccine introduction impact on the strain diversity, we need to recognize the temporal variations in strain diversity.
Also, as the G12 continue to emerge as an important strain in most regions of the world including Asia, Europe, South America, North America and Africa, it may well become a predominant strain in coming years—with or without rotavirus vaccine introduction.33–38 In Africa, the G12 strains were detected for the first time in 2004 in South Africa, and since then it has been also detected in other regions of Africa, including Zambia, Zimbabwe, Ethiopia, Kenya, DRC and Cameroon,2 as is the case in this study. G12 strains will likely continue to appear as vaccines are introduced into some of these countries in 2012 and 2013 and may lead to false assumptions that G12 strains have emerged because of vaccine introduction.
Third, the potential for reassortment between human and animal rotaviruses in Africa is high, taking into account that many of the population live in close proximity with domesticated animals such as cattle, goats, donkeys, pigs, dogs and chickens. The driving force for rotavirus evolution is essentially through either “antigenic shift,” including gene reassortment when there is direct interspecies transmission, gene rearrangements, or through an accumulation of point mutations. This latter may lead to development of untypables rotavirus strains that cannot be typed by the currently available primers.9 In the last 5-year period, mixed genotype infections, either P or G types and partially typed G or P were commonly identified. Possible human-animal G and P strains including G8P, G6P, G4P, G9P and G10P were observed widely in Mauritius, Tanzania, Guinea Bissau, Senegal, Kenya and Nigeria. Group A rotavirus strains carrying G10P, G10P G11P, G5P, G6P, G8P, G3P, G4P, G9P, G12P and G9P are commonly found in animals and these strains have been reported sporadically to cause acute gastroenteritis in young children suggesting animal-human reassortment or direct animal to human rotavirus transmission. Although most of these genotypes are reported in Africa,14,17,29,39 they are also detected in other parts of the world.40–43
A recent study in Burkina Faso reported the unusual G6P that was found to be the second most predominant strain (detected at 23%) after G9P (32%),44 supporting the concept of interspecies transmission. Also, identification of G5 (a porcine rotavirus) first in Cameroon39 and now in several other African countries suggests that this genotype is likely to spread in Africa. G8 rotavirus strains which are commonly found to infect cattle are of epidemiological importance in Africa, where they are frequently detected in children with rotavirus diarrhea. The presence of G8 rotavirus strains have been reported across the continent from Egypt to South Africa and Kenya to Guinea Bissau including countries in between.14,17,29,30 The observation of G8 strains raises the possibility of direct zoönotic transmission or the possibility of a reassortant strain between human and animal rotaviruses. In addition, atypical rotavirus G/P combinations naturally includes intergenogroup or intergenotypes reassortment representing G1P, G2P, G9P, G12P, G8P and G8P have been reported worldwide that might be the results of mixed genotype infections.30,45 These strains are commonly detected in Asia (14%), South America (11%) and Africa (27%).9,10,12,13
Our study has several limitations. Notable were: (1) EIA results may be affected by stool storage, transportation, uneven distribution of the virus in solid stool samples and incorrect interpretation of low optical density values; (2) selection of samples for genotyping was based on the date of collection and not on the age distribution or gender and (3) countries participating in the AFR RSN may not be generalizable to other countries that are not participating in the network.
The annual regional intercountry surveillance workshops conducted between 2007 and 2011 afforded the opportunity to examine the rotavirus strains recently circulating in Africa. Taken together, the results clearly support the need for continuous assessment and monitoring of genotypes circulating in Africa. The anticipated large scale introduction of the rotavirus vaccines in many African countries in the coming years will represent the most significant public health intervention to reduce mortality and morbidity associated with rotavirus infections in Africa. Although Rotarix (GlaxoSmithKline, Rixensart, Belgium) and RotaTeq (Merck, Blue Bell, PA) have shown to be safe and efficacious in protecting severe rotavirus diarrhea in young children in both low- and high-income settings, the impact of regionally prevalent and mixed genotypes on vaccine effectiveness in Africa is not clear, and this needs to be monitored. Thus, postvaccine introduction surveillance studies are crucial in Africa to understand the broader impact of the vaccine on regionally circulating strains. The data generated across the continent has been crucial to guide evidence-based decisions on introduction of rotavirus vaccine in the African continent. Currently 7 African countries (South Africa, Sudan, Ghana, Rwanda, Malawi, Botswana and Zambia) have introduced rotavirus vaccine and 9 others (Ethiopia, Tanzania, Cameroon, Angola, Burundi, DRC, Djibouti, Madagascar, Niger) are approved for support through Global Alliance for Vaccines and Immunization over the next 2 years. The AFR RSN is now well-placed to study the impact of the rotavirus vaccine on disease and monitor strain replacement postvaccine introduction.
The authors thank representatives from Ministries of Health who participated in the African Rotavirus Surveillance Network training workshops (2007–2011): I. Bonkoungou (Burkina Faso); A. Boula and N.K.M. Ntoto (Cameroon); C. Boni Epse Cessi (Cote d ‘Ivoire); J.-P. Wandje, G. Kitambala, E. Pukuta and A. Nkongolo (DRC); B. Asfaw, A. Abebe and F. Tassew (Ethiopia), M. Betts and A. Martin (The Gambia); S. Monteiro (Guinea Bissau); B. Mwinyi, N. Kiulia and I. Amina (Kenya); V. Pursem ( Mauritius); C. Chukwubike (Nigeria); D. Amadou and K. Ndiaye (Senegal); J. Daffi, K. Omari, A. Mohamed and A. Machaga ( Tanzania); T. Segla-Dangloba and E. Tsolenyanu (Togo); P. Namuwulya, A. Mulindwa, A. Odiit and A. Kisakye (Uganda); J.S. Chibumbya and E.M. Mpabalwani (Zambia); A. Shonai, A. Mukaratirwa and P. Nziramasanga (Zimbabwe). We are grateful to workshop facilitators J. Nyangao from Kenya Medical Research Institute, RRL in East Africa; G. Armah from Noguchi Memorial Research Institute, RRL in West Africa; N. Page from National Institute for Communicable Diseases, NHLS; M. Esona, M. Bowen and J. Gentsch from Centers for Disease Control and Prevention, United States; D. Makinita, P. Moche, B. Dooka, L. Mapuroma and P. Bos from RRL South Africa.
1. Tate JE, Burton AH, Boschi-Pinto C, et al.WHO-coordinated Global Rotavirus Surveillance Network. 2008 estimate of worldwide rotavirus-associated mortality in children younger than 5 years before the introduction of universal rotavirus vaccination programmes: a systematic review and meta-analysis. Lancet Infect Dis. 2012;12:136–141
2. Mwenda JM, Ntoto KM, Abebe A, et al. Burden and epidemiology of rotavirus diarrhea in selected African countries: preliminary results from the African Rotavirus Surveillance Network. J Infect Dis. 2010;202(suppl):S5–S11
3. Centers for Disease Control and Prevention (CDC). . Rotavirus surveillance-worldwide 2009. MMWR Morb Mortal Wkly Rep. 2011;60:514–516
4. Cunliffe NA, Kilgore PE, Bresee JS, et al. Epidemiology of rotavirus diarrhoea in Africa: a review to assess the need for rotavirus immunization. Bull World Health Organ. 1998;76:525–537
5. Steele AD, Peenze I, de Beer MC, et al. Anticipating rotavirus vaccines: epidemiology and surveillance of rotavirus in South Africa. Vaccine. 2003;21:354–360
6. Communicable Diseases Surveillance. Bulletin, Rotavirus Surveillance in South Africa 2009. NICD, NHLS. 2010;8:p11–p14
7. Estes MK, Kapikian AZFields BN, Knipe DM, Howley PM. Rotaviruses. Fields Virology. 20075th ed Philadelphia, PA Lippincott Williams & Wilkins:1917–1974
8. Matthijnssens J, Ciarlet M, McDonald SM, et al. Uniformity of rotavirus strain nomenclature proposed by the Rotavirus Classification Working Group (RCWG). Arch Virol. 2011;156:1397–1413
9. Santos N, Hoshino Y. Global distribution of rotavirus serotypes/genotypes and its implication for the development and implementation of an effective rotavirus vaccine. Rev Med Virol. 2005;15:29–56
10. Gentsch JR, Laird AR, Bielfelt B, et al. Serotype diversity and reassortment between human and animal rotavirus strains: implications for rotavirus vaccine programs. J Infect Dis. 2005;192(suppl 1):S146–S159
11. Bányai K, László B, Duque J, et al. Systematic review of regional and temporal trends in global rotavirus strain diversity in the pre rotavirus vaccine era: insights for understanding the impact of rotavirus vaccination programs. Vaccine. 2012;30(suppl 1):A122–A130
12. Sanchez-Padillla E, Grais FR, Guerin PJ, et al. Burden of disease and circulating serotypes of rotavirus infection in sub-Saharan Africa: systematic review and meta-analysis. Lancet Infect Dis. 2009;9:567–576
13. Todd S, Page NA, Duncan Steele A, et al. Rotavirus strain types circulating in Africa: Review of studies published during 1997–2006. J Infect Dis. 2010;202(suppl):S34–S42
14. Adah MI, Nagashima S, Wakuda M, et al. Close relationship between G8-serotype bovine and human rotaviruses isolated in Nigeria. J Clin Microbiol. 2003;41:3945–3950
15. Armah GE, Steele AD, Binka FN, et al. Changing patterns of rotavirus genotypes in ghana: emergence of human rotavirus G9 as a major cause of diarrhea in children. J Clin Microbiol. 2003;41:2317–2322
16. Steele AD, Ivanoff B. Rotavirus strains circulating in Africa during 1996-1999: emergence of G9 strains and P strains. Vaccine. 2003;21:361–367
17. Nielsen NM, Eugen-Olsen J, Aaby P, et al. Characterization of rotavirus strains among hospitalised and non hospitalised children in Guinea-Bissau, 2002 a high frequency of mixed infections with serotype G8 J Clin Virol. 2005;35:13–21
18. Gentsch JR, Glass RI, Woods P, et al. Identification of group A rotavirus gene 4 types by polymerase chain reaction. J Clin Microbiol. 1992;30:1365–1373
19. Gouvea V, Glass RI, Woods P, et al. Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens. J Clin Microbiol. 1990;28:276–282
20. Iturriza-Gómara M, Green J, Brown DW, et al. Diversity within the VP4 gene of rotavirus P strains: implications for reverse transcription-PCR genotyping. J Clin Microbiol. 2000;38:898–901
21. Iturriza-Gómara M, Kang G, Gray J. Rotavirus genotyping: keeping up with an evolving population of human rotaviruses. J Clin Virol. 2004;31:259–265
22. Aladin F, Nawaz S, Iturriza-Gómara M, et al. Identification of G8 rotavirus strains determined as G12 by rotavirus genotyping PCR: updating the current genotyping methods. J Clin Virol. 2010;47:340–344
23. Cunliffe NA, Dove W, Bunn JEG, et al. Expanding global distribution of rotavirus serotype G9: detection in Libya, Kenya and Cuba. Emerg Infect Dis. 2001;7:890–892
24. Page N, Esona M, Armah G, et al. Emergence and characterization of serotype G9 rotavirus strains from Africa. J Infect Dis. 2010;202(suppl):S55–S63
25. Fischer TK, Steinsland H, Molbak K, et al. Genotype profiles of rotavirus strains from children in a suburban community in Guinea-Bissau, Western Africa. J Clin Microbiol. 2000;38:264–267
26. Page NA, Steele AD. Antigenic and genetic characterization of serotype G2 human rotavirus strains from the African continent. J Clin Microbiol. 2004;42:595–600
27. Seheri LM, Page N, Dewar JB, et al. Characterization and molecular epidemiology of rotavirus strains recovered in Northern Pretoria, South Africa during 2003-2006. J Infect Dis. 2010;202(suppl):S139–S147
28. Cunliffe NA, Ngwira BM, Dove W, et al. Epidemiology of rotavirus infection in children in Blantyre, Malawi, 1997-2007. J Infect Dis. 2010;202(suppl):S168–S174
29. Cunliffe NA, Dove W, Bunn JE, et al. Expanding global distribution of rotavirus serotype G9: detection in Libya, Kenya, and Cuba. Emerg Infect Dis. 2001;7:890–892
30. Matthijnssens J, Rahman M, Yang X, et al. G8 rotavirus strains isolated in the Democratic Republic of Congo belong to the DS-1-like genogroup. J Clin Microbiol. 2006;44:1801–1809
31. Page N, Esona M, Seheri M, et al. Characterization of genotype G8 strains from Malawi, Kenya, and South Africa. J Med Virol. 2010;82:2073–2081
32. Asmah RH, Green J, Armah GE, et al. Rotavirus G and P genotypes in rural Ghana. J Clin Microbiol. 2001;39:1981–1984
33. Griffin DD, Nakagomi T, Hoshino Y, et al.National Rotavirus Surveillance System. Characterization of nontypeable rotavirus strains from the United States: identification of a new rotavirus reassortant (P2A,G12) and rare P3 strains related to bovine rotaviruses. Virology. 2002;294:256–269
34. Das S, Varghese V, Chaudhury S, et al. Emergence of novel human group A rotavirus G12 strains in India. J Clin Microbiol. 2003;41:2760–2762
35. Castello AA, Argüelles MH, Rota RP, et al. Molecular epidemiology of group A rotavirus diarrhea among children in Buenos Aires, Argentina, from 1999 to 2003 and emergence of the infrequent genotype G12. J Clin Microbiol. 2006;44:2046–2050
36. Bányai K, Bogdán A, Kisfali P, et al. Emergence of serotype G12 rotaviruses, Hungary. Emerg Infect Dis. 2007;13:916–919
37. Rahman M, Matthijnssens J, Yang X, et al. Evolutionary history and global spread of the emerging g12 human rotaviruses. J Virol. 2007;81:2382–2390
38. Page NA, de Beer MC, Seheri LM, et al. The detection and molecular characterization of human G12 genotypes in South Africa. J Med Virol. 2009;81:106–113
39. Esona MD, Armah GE, Geyer A, et al. Detection of an unusual human rotavirus strain with G5P specificity in a Cameroonian child with diarrhea. J Clin Microbiol. 2004;42:441–444
40. Santos N, Lima RC, Pereira CF, et al. Detection of rotavirus types G8 and G10 among Brazilian children with diarrhea. J Clin Microbiol. 1998;36:2727–2729
41. Cooney MA, Gorrell RJ, Palombo EA. Characterisation and phylogenetic analysis of the VP7 proteins of serotype G6 and G8 human rotaviruses. J Med Microbiol. 2001;50:462–467
42. Iturriza Gómara M, Kang G, Mammen A, et al. Characterization of G10P rotaviruses causing acute gastroenteritis in neonates and infants in Vellore, India. J Clin Microbiol. 2004;42:2541–2547
43. Uchida R, Pandey BD, Sherchand JB, et al. Molecular epidemiology of rotavirus diarrhea among children and adults in Nepal: detection of G12 strains with P or P and a G11P strain. J Clin Microbiol. 2006;44:3499–3505
44. Nordgren J, Bonkoungou IJ, Nitiema LW, et al. Rotavirus in diarrheal children in rural Burkina Faso: high prevalence of genotype G6P. Infect Genet Evol. 2012;12:1892–1898
45. Iturriza-Gómara M, Isherwood B, Desselberger U, et al. Reassortment in vivo: driving force for diversity of human rotavirus strains isolated in the United Kingdom between 1995 and 1999. J Virol. 2001;75:3696–3705