Detection of the first G6P[14] human rotavirus strain in an infant with diarrhoea in Ghana

Background Rotaviruses with G6P[14] specificity are mostly isolated in cattle and have been established as a rare cause of gastroenteritis in humans. This study reports the first detection of G6P[14] rotavirus strain in Ghana from the stool of an infant during a hospital-based rotavirus surveillance study in 2010. Methods Viral RNA was extracted and rotavirus VP7 and VP4 genes amplified by one step RT-PCR using gene-specific primers. The DNA was purified, sequenced and genotypes determined using BLAST and RotaC v2.0. Phylogenetic tree was constructed using maximum likelihood method in MEGA v6.06 software and statistically supported by bootstrapping with 1000 replicates. Phylogenetic distances were calculated using the Kimura-2 parameter model. Results The study strain, GHA-M0084/2010 was characterised as G6P[14]. The VP7 gene of the Ghanaian strain clustered in G6 lineage-III together with artiodactyl and human rotavirus (HRV) strains. It exhibited the highest nucleotide (88.1 %) and amino acid (86.9 %) sequence identity with Belgian HRV strain, B10925. The VP8* fragment of the VP4 gene was closely related to HRV strains detected in France, Italy, Spain and Belgium. It exhibited the strongest nucleotide sequence identity (87.9 %) with HRV strains, PA169 and PR/1300 (Italy) and the strongest amino acid sequence identity (89.3 %) with HRV strain R2775/FRA/07 (France). Conclusion The study reports the first detection of G6P[14] HRV strain in an infant in Ghana. The detection of G6P[14], an unusual strain pre-vaccine introduction in Ghana, suggests a potential compromise of vaccine effectiveness and indicates the necessity for continuous surveillance in the post vaccine era. Electronic supplementary material The online version of this article (doi:10.1186/s12985-016-0643-y) contains supplementary material, which is available to authorized users.


Background
Group A rotaviruses (RVAs) are the leading cause of severe, dehydrating, acute diarrhoea in children <5 years of age worldwide and are estimated to be responsible for 453,000 deaths annually, with a disproportionate number of these deaths occurring in sub-Saharan Africa and South East Asia [1]. The World Health organization (WHO) recommended global use of rotavirus vaccines as one of the effective interventions to reduce RVAassociated diseases [2]. Currently, two RVA vaccines, RotaTeq (Merck & Co., Inc., United States) and Rotarix (GlaxoSmithKline Biologicals, Belgium) have been licensed and introduced into routine childhood immunisation programmes.
Rotaviruses with G6 specificity are recognised as common serotype in cattle and other ruminants and have been found to be an infrequent cause of human disease. Bovine-like human G6 rotavirus strains PA151 and PA169 were first identified in 1987 and 1988 respectively from two Italian children hospitalised with acute gastroenteritis [9]. These two strains were characterised as G6P [9] (strain PA151) and G6P [14] (strain PA169). Subsequently, an increasing number of G6 HRV strains have been detected in combination with other P genotypes across the globe (Hungary, Australia, United States and India) [10][11][12][13]. G6 HRV strains are reported to be found mainly in combination with P [14] rotaviruses (commonly detected in antelope, cattle, rabbits, goats, sheep and guanacos) [14].
Previous reports in Africa, documented the isolation of G6 rotavirus strains only in animals [15]. The first detection of G6P [14] HRV strain in Africa was in 2004 from the stool of a child from Egypt [7].
In our previous study, rotavirus was detected in the stool of a four month old child hospitalised with severe, acute dehydrating gastroenteritis at Maamobi Polyclinic in Accra, Ghana in August 2010. This sample exhibited the classical human rotavirus group A migration pattern (4-2-3-2) with a long electrophoretype by Polyacrylamide Gel Electrophoresis (PAGE) (data not shown). However, the amplified VP7 and VP8* fragment of the VP4 gene could not be genotyped using the routine HRV genotype specific primers. In this study, the G and P genotypes of strain GHA-M0084/2010 were identified by sequencing.

Discussion
G6 rotaviruses have been found to be an infrequent cause of human disease. Their detection in the human population was suggestive of zoonotic transmission as they have been detected almost exclusively in artiodactyls such as cattle [8,14]. We report the detection of a G6P [14] rotavirus strain in Ghana from the stool of an infant participating in a hospital-based diarrhea surveillance study conducted in 2010 (pre-vaccine era). This strain was detected in a locality where the human population live in close proximity with their cattle, goats, sheep and pigs. This setting and proximity provide an ideal environment for dual RV infections from human and animal sources which can lead to reassortment of genes and eventual generation of novel strains.
Phylogenetic analysis of the VP7 gene of the study strain (GHA-M0084/2010) showed clustering with artiodactyl and human G6 strains in lineage III (Fig. 1). The Ghanaian strain displayed the strongest nucleotide and amino acid sequence identity (74.9-88.1 % and 80.0-86.9 % respectively) to randomly selected non-African reference strains (Table 1). It exhibited the lowest nucleotide identity (74.9 %) to African HRV strains, BA356 (Cameroon) and 48-BF (Burkina Faso) both isolated in 2010 ( Table 1). The VP8* fragment of the VP4 gene of the study strain clustered together with HRV strains detected in France (2007)   Belgium (1997) (Fig. 2). It displayed the strongest aa (89.3 %) sequence identity to HRV strain R2775 isolated in France and nt (87.9 %) sequence identity to HRV strains PA169 and PR/1300 isolated in Italy (Table 2). Two paediatric rotavirus vaccines (Rotarix and RotaTeq) are available and have been introduced into national immunization programmes in many countries [16,17]. The monovalent vaccine, Rotarix, comprises a human G1P [8] rotavirus strain, whereas the pentavalent vaccine, RotaTeq, (a human-bovine reassortant vaccine) comprises human serotypes G1, G2, G3, G4, and P[8] on a bovine strain background. Both vaccines demonstrated broad protection against the most common RVA genotypes [18]. However, their efficacy is low especially on the African continent with diverse pool of strain types [19,20]. G6P [14] strain detected in this study shares neither Gnor P-genotype with either of the two current vaccines. Though, other rotavirus proteins such as VP6 and NSP4 have been identified to play a role in the protective immunity against rotavirus infection, it is unknown whether Rotarix will provide protection against completely heterotypic G6P [14], an animal-derived human rotavirus strain.

Conclusion
In this study, we determined the VP7 and VP4 genotypes of previously non-typeable rotavirus strain. The study reports for the first time the detection of G6P [14] rotavirus strain in a child in West Africa. Data on the impact of rotavirus vaccination globally has shown that two rotavirus vaccines, RotaTeq™ and Rotarix™, provide good homotypic and heterotypic protection. However, there are concerns about the degree of protection these vaccines provide against rare/unusual rotavirus strains such as G6P [14] that bears G/P genotype specificities not included in the vaccines. The detection of a G6P [14] strain pre-vaccine introduction in Ghana, suggests a potential compromise of vaccine effectiveness and indicates the necessity for continuous surveillance in the post vaccine era.

Viral RNA extraction and reverse transcription
Viral RNA was extracted from 10 % faecal suspension in phosphate buffered saline using the QIAamp viral RNA Mini kit (Qiagen/Westburg, Leusden, The Netherlands). PCR primer pairs, 9Con1/9Con2 and VP4-F/VP4-R (Additional file 1: Table S1) were used in one step RT-PCR to target the VP7 gene and the VP8* fragment of the VP4 gene to generate 903 and 663 bp respectively [21,22]. PCR products were purified using the Exo-SAP-IT clean up kit (Affymetrix, Miles Rd Cleveland, OH, USA). Products were directly sequenced for VP7 and VP4 genes using the Bigdye terminator sequencing kit v3.1 (Perkin-Elmer Applied Biosystems, Foster City, CA, USA) on an automated Genetic analyzer ABI PRISM 3130 (Applied Biosystems).

Nucleotide sequence accession number
The nucleotide sequences of the strain reported in this study (GHA-M0084/2010) have been deposited in the GenBank database under accession numbers KJ616414 and KJ616415 respectively.

Additional file
Additional file 1: Table S1. List of primers used in this study.