Browsing by Author "Damanka, S.A."
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Item Genetic analysis of Ghanaian G1P[8] and G9P [8] rotavirus A strains reveals the impact of P [8] VP4 gene polymorphism on P-genotyping(PLOS ONE, 2019-06-10) Damanka, S.A.; Agbemabiese, C.A.; Dennis, F.E.; Lartey, B.L.; Adiku, T.K.; Enweronu-Laryea, C.C.; Armah, G.E.The World Health Organisation rotavirus surveillance networks have documented and shown eclectic geographic and temporal diversity in circulating G- and P- genotypes identified in children <5 years of age. To effectively monitor vaccine performance and effectiveness, robust molecular and phylogenetic techniques are essential to detect novel strain variants that might emerge due to vaccine pressure. This study inferred the phylogenetic history of the VP7 and VP4 genes of previously non-typeable strains and provided insight into the diversity of P[8] VP4 sequences which impacted the outcome of our routine VP4 genotyping method. Near-full-length VP7 gene and the VP8* fragment of the VP4 gene were obtained by Sanger sequencing and genotypes were determined using RotaC v2.0 web-based genotyping tool. The genotypes of the 57 rotavirus-positive samples with sufficient stool was determined. Forty-eight of the 57 (84.2%) had the P[8] specificity, of which 43 (89.6%) were characterized as P[8]a subtype and 5 (10.4%) as the rare OP354-like subtype. The VP7 gene of 27 samples were successfully sequenced and their G-genotypes confirmed as G1 (18/27) and G9 (9/27). Phylogenetic analysis of the P[8]a sequences placed them in subcluster IIIc within lineage III together with contemporary G1P[8], G3P[8], G8P[8], and G9P[8] strains detected globally from 2006–2016. The G1 VP7 sequences of the study strains formed a monophyletic cluster with African G1P[8] strains, previously detected in Ghana and Mali during the RotaTeq vaccine trial as well as Togo. The G9 VP7 sequences of the study strains formed a monophyletic cluster with contemporary African G9 sequences from neighbouring Burkina Faso within the major sub-cluster of lineage III. Mutations identified in the primer binding region of the VP8* sequence of the Ghanaian P[8]a strains may have resulted in the genotyping failure since the newly designed primer successfully genotyped the previously non-typeable P[8] strains. In summary, the G1, G9, and P[8]a sequences were highly similar to contemporary African strains at the lineage level. The study also resolved the methodological challenges of the standard genotyping techniques and highlighted the need for regular evaluation of the multiplex PCR-typing method especially in the post-vaccination era. The study further highlights the need for regions to start using sequencing data from local rotavirus strains to design and update genotyping primers.Item Genetic analysis of Ghanaian G1P[8] and G9P [8] rotavirus A strains reveals the impact of P [8] VP4 gene polymorphism on P-genotyping(PLOS ONE, 2019-06-10) Damanka, S.A.; Agbemabiese, C.A.; Dennis, F.E.; Lartey, 'B.L.; Adiku, T.K.; Enweronu-Laryea, C.C.; Armah, G.E.The World Health Organisation rotavirus surveillance networks have documented and shown eclectic geographic and temporal diversity in circulating G- and P- genotypes identified in children <5 years of age. To effectively monitor vaccine performance and effectiveness, robust molecular and phylogenetic techniques are essential to detect novel strain variants that might emerge due to vaccine pressure. This study inferred the phylogenetic history of the VP7 and VP4 genes of previously non-typeable strains and provided insight into the diversity of P[8] VP4 sequences which impacted the outcome of our routine VP4 genotyping method. Near-full-length VP7 gene and the VP8* fragment of the VP4 gene were obtained by Sanger sequencing and genotypes were determined using RotaC v2.0 web-based genotyping tool. The genotypes of the 57 rotavirus-positive samples with sufficient stool was determined. Forty-eight of the 57 (84.2%) had the P[8] specificity, of which 43 (89.6%) were characterized as P[8]a subtype and 5 (10.4%) as the rare OP354-like subtype. The VP7 gene of 27 samples were successfully sequenced and their G-genotypes confirmed as G1 (18/27) and G9 (9/27). Phylogenetic analysis of the P[8]a sequences placed them in subcluster IIIc within lineage III together with contemporary G1P[8], G3P[8], G8P[8], and G9P[8] strains detected globally from 2006–2016. The G1 VP7 sequences of the study strains formed a monophyletic cluster with African G1P[8] strains, previously detected in Ghana and Mali during the RotaTeq vaccine trial as well as Togo. The G9 VP7 sequences of the study strains formed a monophyletic cluster with contemporary African G9 sequences from neighbouring Burkina Faso within the major sub-cluster of lineage III. Mutations identified in the primer binding region of the VP8* sequence of the Ghanaian P[8]a strains may have resulted in the genotyping failure since the newly designed primer successfully genotyped the previously non-typeable P[8] strains. In summary, the G1, G9, and P[8]a sequences were highly similar to contemporary African strains at the lineage level. The study also resolved the methodological challenges of the standard genotyping techniques and highlighted the need for regular evaluation of the multiplex PCR-typing method especially in the post-vaccination era. The study further highlights the need for regions to start using sequencing data from local rotavirus strains to design and update genotyping primers.Item Identification of Amino Acid Substitutions Within the VP7 Genes of G2 Rotavirus Strains in Ghana(The Pediatric infectious disease journal, 2018-11) Damanka, S.A.; Agbemabiese, C.A.; Lartey, B.L.; Dennis, F.E.; Asamoah, F.K.; Adiku, T.K.; Enweronu-Laryea, C.C.; Sagoe, K.W.; Ofori, M.F.; Armah, G.E.We used the dideoxynucleotide chain termination method to determine the strains of nine non-typeable rotavirus enzyme immunoassay–positive samples, which were identified as G2. We detected nucleotide changes in the primer-binding region and amino acid substitutions within the VP7 protein of the G2 rotavirus strains. Genotyping primers need to be updated regularly.Item Molecular Characterization Of Previously Non-Typeable Rotavirus Strains In Ghana(University of Ghana, 2015-10) Damanka, S.A.Background Rotavirus is associated with severe infantile diarrhoea requiring hospitalization. In order to control severe disease caused by rotavirus infection, vaccination is considered an essential strategy. Currently, two rotavirus vaccines (Rotarix™ and RotaTeq™) have been included in the Expanded Program on Immunization of most African countries to help reduce the high disease indections. Ghana has over the past 20 years been involved in the surveillance of rotavirus diseases by isolating rotaviruses from diarrhoeic stools and determining the strain types as part of the build up to vaccine introduction. During rotavirus burden of disease surveillance between 2007 and 2011, three hundred and one rotavirus samples could not genotype with the pool of primers recommended for use within the WHO Regional Reference Laboratory network. Samples with sufficient stools were characterized. Aim The study sought to characterize and identify primer mismatches for nontypeable rotavirus strains at the Regional Rotavirus Reference laboratory in Ghana. Methodology One hundred and eleven G and 89 P-types had sufficient stool and were characterized by sequencing methods. To confirm previous EIA results, 10% of randomly selected nontypeable samples were reanalyzed using the commercially available DAKO IDEIA kit (Dako Diagnostics, Cambridgeshire, UK). To assess the integrity of RV dsRNA, Polyacrylamide Gel Electrophoresis (PAGE) was carried out. Viral ribonucleic acids were extracted by the phenol-chloroform-guanidine-isothiocyanate method and one step RT-PCR targeting the VP7 and VP4 genes of RVA was carried out using PCR primer pairs, Beg9/End9 and 9Con1/9Con2 for VP7 xv University of Ghana http://ugspace.ug.edu.gh genes and Con2/Con3 and VP4F/VP4R for VP4 genes. The RT-PCR products were purified with the QIAquick PCR purification kit (Qiagen/Westburg), sequenced with the ABI PRISM® BigDye Terminator v.3.1 Ready Reaction mix. Amplicons were purified using ethanol-sodium acetate precipitation method. Sequences were subjected to BLAST (Basic Local Alignment Search Tool) and subsequently characterized using the web-based genotyping tool, RotaC v2.0. Results The G-types were determined for 74 out of the 111 samples that had sufficient stool to work with. The G-types detected were as follows: G1; 31 (41.8%), G2; 9 (12.1%), G3; 11 (14.8%), G6; 1 (1.4%), G8; 2 (2.7%), G9; 14 (18.9%) and G12; 6 (8.1%). Sequence analyses of the Ghanaian VP7 isolates and cognate genes of known ancestral and contemporary reference strains revealed the accumulation of point mutations in the antigenic regions. The P-types were successfully characterized for 57 of the 89 samples that had sufficient stool. Forty-eight (84.2%) of these were identified as P[8]a strains of which 5 (10.4%) were characterized as the rare OP354-like human rotavirus P[8]b subtype. Other strains detected in the study were P[4], 1 (1.8%), P[6], 7 (12.3%) and P[14], 1 (1.8%). P[14] was found in combination with G6. In Silico PCR performed with in-house primers failed to genotype Ghanaian P[8] isolates. However, In Silico PCR with published primers, Rev compl 1T-1D and P8b-MMC38 successfully genotyped the Ghanaian P[8]a and P[8]b isolates respectively. Discussion, conclusion and recommendation The study determined the identity of previously nontypeable rotavirus strains in Ghana and established their genetic relatedness to globally circulating strains. The study also showed that the VP7 and VP4 genes of Ghanaian rotavirus strains have evolved as a result of accumulated point mutations over the years which might have led to genotyping failure. The amino acid xvi University of Ghana http://ugspace.ug.edu.gh differences detected in the antigenic regions of the Ghanaian isolates may lead to conformational changes that may influence the effectiveness of the vaccine. The study revealed for the first time the detection of OP354-like ([P[8]b) genotype in Ghana. It also identified for the first time G6P[14] rotavirus strain in Ghana. The study highlights the importance of including sequencing in the characterization of rotavirus strains as well as regularly updating the primer sequences employed for molecular typing of rotaviruses. Continuous surveillance of circulating rotavirus strains is important for the detection of new rotavirus strains as well as for the evaluation of the rotavirus vaccination program in Ghana.Item Next-generation sequencing of a human-animal reassortant G6P[14] rotavirus A strain from a child hospitalized with diarrhoea(Archives of Virology, 2020-02-10) Damanka, S.A.; Dennis, F.E.; Lartey, B.L.; Nyarko, K.M.; Agbemabiese, C.A.; Armah, G.E.We previously reported the VP4 and the VP7 genotypes of the first G6P[14] rotavirus strain (RVA/Human-wt/GHA/M0084/2010/G6P[14]) from the stool of an infant with diarrhoea in Ghana. In the current study, we obtained the complete genome sequences using Illumina MiSeq next-generation sequencing to enable us to determine the host species origin of the genes by phylogenetic analysis. The genotype constellation was G6-P[14]-I2-R2-C2-M2-A11-N2-T6-E2-H3. Phylogenetic analysis showed that M0084 was a reassortant strain from RVAs of both artiodactyl and human host species origin. The level of sequence identity of the individual genes of M0084 to other sequences in the GenBank ranged from 95.2 to 99.5%; however, there was no single strain from the GenBank database with a complete genome sequence that was highly similar to that of M0084. To help trace the source of such unique gene pools being introduced into human RVAs, it will be useful to examine RVA sequences from potential reservoirs such as sheep and goats, which are common domestic animals in this locality.Item Sub-genotype phylogeny of the non-G, non-P genes of genotype 2 Rotavirus A strains(PLoS ONE, 2019-05-10) Agbemabiese, C.A.; Nakagom, T.; Damanka, S.A.; Dennis, F.E.; Lartey, B.L.; Armah, G.E.; Nakagomi, O.Recent increase in the detection of unusual G1P[8], G3P[8], G8P[8], and G9P[4] Rotavirus A (RVA) strains bearing the DS-1-like constellation of the non-G, non-P genes (hereafter referred to as the genotype 2 backbone) requires better understanding of their evolutionary relationship. However, within a genotype, there is lack of a consensus lineage designation framework and a set of common sequences that can serve as references. Phylogenetic analyses were carried out on over 8,500 RVA genotype 2 genes systematically retrieved from the rotavirus database within the NCBI Virus Variation Resource. In line with previous designations, using pairwise comparison of cogent nucleotide sequences and stringent bootstrap support, reference lineages were defined. This study proposes a lineage framework and provides a dataset ranging from 34 to 145 sequences for each genotype 2 gene for orderly lineage designation of global genotype 2 genes of RVAs detected in human and animals. The framework identified five to 31 lineages depending on the gene. The least number of lineages (five to seven) were observed in genotypes A2 (NSP1), T2 (NSP3) and H2 (NSP5) which are limited to human RVA whereas the most number of lineages (31) was observed in genotype E2 (NSP4). Sharing of the same lineage constellations of the genotype 2 backbone genes between recently-emerging, unusual G1P[8], G3P[8], G8P[8] and G9P[4] reassortants and many contemporary G2P[4] strains provided strong support to the hypothesis that unusual genotype 2 strains originated primarily from reassortment events in the recent past involving contemporary G2P[4] strains as one parent and ordinary genotype 1 strains or animal RVA strains as the other. The lineage framework with selected reference sequences will help researchers to identify the lineage to which a given genotype 2 strain belongs, and trace the evolutionary history of common and unusual genotype 2 strains in circulation.Item Whole genome characterization and evolutionary analysis of OP354-like P[8] Rotavirus A strains isolated from Ghanaian children with diarrhoea(PLOS ONE, 2019-05-30) Damanka, S.A.; Kwofie, S.; Dennis, F.E.; Lartey, B.L.; Agbemabiese, C.A.; Doan, Y.H.; Adiku, T.K.; Katayama, K.; Enweronu-Laryea, C.C.; Armah, G.E.In 2010, the rare OP354-like P[8]b rotavirus subtype was detected in children less than 2 years old in Ghana. In this follow-up study, to provide insight into the evolutionary history of the genome of Ghanaian P[8]b strains RVA/Human-wt/GHA/GHDC949/2010/G9P[8] and RVA/Human-wt/GHA/GHM0094/2010/G9P[8] detected in an infant and a 7-month old child hospitalised for acute gastroenteritis, we sequenced the complete genome using both Sanger sequencing and Illumina MiSeq technology followed by phylogenetic analysis of the near-full length sequences. Both strains possessed the Wa-like/genotype 1 constellation G9P[8]b-I1-R1-C1-M1-A1-N1-T1-E1-H1. Sequence comparison and phylogenetic inference showed that both strains were identical at the lineage level throughout the 11 genome segments. Their VP7 sequences belonged to the major sub-lineage of the G9-lineage III whereas their VP4 sequences belonged to P[8]b cluster I. The VP7 and VP4 genes of the study strains were closely related to a Senegalese G9P[8]b strain detected in 2009. In the remaining nine genome segments, both strains consistently clustered together with Wa-like RVA strains possessing either P[8]a or P[8]b mostly of African RVA origin. The introduction of a P[8]b subtype VP4 gene into the stable Wa-like strain backbone may result in strains that might propagate easily in the human population, with a potential to become an important public health concern, especially because it is not certain if the monovalent rotavirus vaccine (Rotarix) used in Ghana will be efficacious against such strains. Our analysis of the full genomes of GHM0094 and GHDC949 adds to knowledge of the genetic make-up and evolutionary dynamics of P[8]b rotavirus strains