http://hdl.handle.net/123456789/60 2026-04-13T10:38:42Z http://hdl.handle.net/123456789/715 GENETIC DIVERSITY OF HEPATITIS C VIRUS AMONG BLOOD DONORS AND SYMPTOMATIC PATIENTS IN SOUTHWESTERN NIGERIA SHENGE, JULIET ADAMMA Hepatitis C virus (HCV) infection results in liver cirrhosis, hepatic failure or hepatocellular carcinoma (HCC). The virus exhibits extreme sequence variability, which has resulted in emergence of variants that are distributed differently. There is dearth of information on HCV diversity and its association with disease progression. This study was designed to investigate HCV strains and disease outcomes in blood donors and patients in Nigeria. Three hundred and one anti-HCV positive blood samples were collected from 99 symptomatic hepatitis patients, 125 HIV-infected individuals and 77 asymptomatic blood donors, over a period of three years (2014-2017) in Ibadan, Nigeria. Viral RNA was extracted from samples and reverse–transcribed to complimentary DNA and HCV nonstructural gene 5b (NS5B) amplified by PCR, using specific primers. The amplified products were sequenced by Sanger’s method and edited on CLC workbench and BioEdit programs. Basic Local Alignment Search Tool (BLAST) was used to compare gene sequences with those from GenBank and HCV databases. Sequence alignment was done on MEGA 7.0 and phylogenetic trees constructed using Neighbor-Joining method with bootstrap of 1000 replicates. Viral proteins were analysed on Netphos 3.1 software and data analysis was done using Fisher’s Exact test at Chiᴧ2 = 0.05. A total of 301 samples from individuals aged 3 months to 81 years were analysed. The NS5B genes were amplified by PCR in 60 (20%) of the samples, of which the gene was successfully sequenced in 42 with 97- 99% homology to HCV prototype H77 and sequences from around the world. Of the 42 isolates, 12 (28.6%), 5 (11.9%), 4 (9.5%), 1 (2.4%), 1 (2.4%) and 19 (45.2%) were subtypes 1a, 1b, 2b, 2c, 3a and 5a respectively, with 5a and 1a as predominant strains. Subtypes 1b and 2b were found only in patients with hepatitis, while subtypes 1a and 5a were predominant in blood donors and individuals with HIV respectively (p=0.0004). A 10% nucleotide variation was observed within isolates and 20% when compared with H77 prototype, while amino acid substitution varied among isolates with highest substitutions observed in symptomatic patients. Major clinically-important resistance mutations detected were S15G in 28 isolates, Q47H (35 isolates), T7N (24 isolates), S54L (22 isolates), G61R (23 isolates), and T79M (12 isolates). Analyses of viral proteins for conformational modifications revealed Serine/Threonine phosphorylated residues at positions 52 and 73, which are viral markers for disease progression. Four (30.8%) phosphorylated residues were found in hepatitis patients; 2 (15.4%) in blood donors and 7 (53.8%) in HIV-infected individuals (p=0.0400). Multiple hepatitis C virus genotypes circulate in Nigeria with predominance of genotype 5, found for the first time in West Africa. The observed variations in hepatitis C virus NS5B gene have implications for therapy. Routine survey of hepatitis C virus circulating in Nigeria is recommended. 2019-03-01T00:00:00Z http://hdl.handle.net/123456789/713 GENETIC DIVERSITY AND DRUG RESISTANCE PATTERNS OF HIV-1 ISOLATES FROM DELTA, ANAMBRA AND IMO STATES, NIGERIA UDEZE, AUGUSTINE OKECHUKWU, Human Immunodeficiency Virus (HIV) is characterized by high genetic diversity which affects diagnosis, transmission, disease progression, vaccine design, co-receptor usage, response to antiretroviral therapy and development of drug resistance. Previous studies in Northern and Southwestern parts of Nigeria indicate the presence of multiple HIV strains, with different dominant strains and increasing resistance to some antiretroviral drugs. There is however limited information on strains circulating in the Southeastern part of the country. This study was undertaken to determine the diversity of circulating HIV-1 strains, their co-receptor usage potential and resistance patterns to Protease Inhibitors (PIs) in Delta, Anambra and Imo States. Blood samples were collected from 246 consenting HIV-1 infected individuals attending antiretroviral treatment centers in Delta, Anambra and Imo States. Genomic DNA was extracted from the samples using phenol/chloroform extraction procedure, and P24 gag, protease and C2-V3 envgenes amplified by nested PCR using specific primers for each of the genes. Thirty isolates with amplified products in all 3 genomic regions were sequenced using Sanger’s method. The nucleotide sequences were assembled, edited, blasted in genbank and phylogenetically analysed using ClustalW and Neighbour Joining method based on their nucleotide and amino acid sequences. Co-receptor usage and resistance patterns were determined using Geno2Pheno method and Stanford algorithm respectively. Data were analysed using descriptive statistics and Chi-square at α0.05. A total of 17 gag, 28 protease and 14 env sequences were obtained. Phylogenetic analysis showed that 17.7, 29.4 and 41.2% of the gag sequences were subtypes A, G and CRF02_AG respectively(p=0.00001) while 35.7 and 57.1% of protease sequences were identified as subtypes G and CRF02_AG respectively (p=0.00001). Also, 11.8 of gag and 7.1% of protease sequences were unclassifiable (U). Based on the env C2-V3 sequences, 7.1, 35.7, 50.0 and 7.1% were identified as subtypes A, G, CRF02_AG and J, respectively (p=0.00001). Mosaic structure was found among 71.4 and 26.7% of isolates in which 3 and 2 genomic regions respectively were successfully sequenced. The V3 loop of the 14 env sequences displayed total amino acid diversity of 82.9%. The CXCR4-tropic and CCR5-tropic viruses accounted for 42.9 and 57.1%, respectively (p=0.047715) among the study population. Maraviroc associated resistant mutations occurred in 12.5% of the CCR5-tropic viruses. From the 28 protease sequences, amino acid substitution occurred in 41.4% out of the 99 amino acid positions. Major PI resistance-associated mutations were found at two protease sites (M46L and V82L) in 3 (10.7%) of the sequences. Minor PI mutations identified include; L10V/I (25.0%), K20I (100%) and N88D (3.6%). Other polymorphisms identified include; I13V/A, E35Q, M36I/L, N37D/S/E/H, R57K/G, L63T/P/S/Q, C67E/S, H69K/R, K70R, V82I and L89M in the range of 28.6% to 100% among the different subtypes. The predominant HIV-1 subtypes circulating in Delta, Anambra and Imo States, Nigeria are CRF02_AG and G. Identification of mosaic strains including unclassifiable sequences highlights the complexity of HIV epidemic in Nigeria. Significant proportion of the isolates uses CXCR4 co-receptor with the potential of undermining success of Maraviroc in treatment. 2019-03-01T00:00:00Z http://hdl.handle.net/123456789/711 GENOTYPIC CHARACTERISATION OF HUMAN RESPIRATORY SYNCYTIAL VIRUS IN CHILDREN PRESENTING WITH RESPIRATORY TRACT INFECTIONS IN IBADAN, NIGERIA OGUNSEMOWO, OLUKUNLE SEUN, Human Respiratory Syncytial Virus (HRSV) is the most common viral cause of acute lower respiratory tract infections in infants and young children,however, effective vaccine is yet to be licensed for human use till date. Palivizumab, a Monoclonal Antibody (MAb) against the Fusion protein of the virus is used for prophylactic treatment in developed countries but not yet in Nigeria. Mutation at the binding site of this MAb may lead to drug resistance. Information on the circulating genotypes of HRSV from regions with high-burden of infection is vital in the global efforts towards the development of protective vaccine. This study was designed to determine the genotypes of HRSV circulating among children in Ibadan, and the presence of resistance mutation in the F gene of the strains identified. Nasopharyngeal and oropharyngeal swab samples were collected between March and October, 2015, from 231 children (aged 1 to 72 months) with symptoms of respiratory infections. The samples were collected at Oranyan and Omi-Adio primary health centres as well as Our Lady of Apostles Catholic Hospital, Oluyoro (a secondary health facility) in Ibadan. Viral RNA was extracted from the swab specimens and used to identify HRSV using primers targeting the conserved region of the matrix gene. The HRSV-positive samples were subtyped by PCR using subtype-specific primers targeting the second Hypervariable Region (HVR2) of the glycoprotein gene and then sequenced to determine the genotype(s). Antigenic site II of the F gene of HRSV-positive samples were also amplified, sequenced and analysed for amino acid substitutions. Demographic information and medical history were obtained using structured questionnaire. Data were analysed using Chi square at α0.05. The HRSV was detected in 41 specimens (17.8%), out of which 22 (53.7%) were subtype A and 11 (26.8%) were subtype B. Eight (19.5%) of the HRSV-positive samples could not be subtyped with primers used. Phylogenetic analysis showed that genotype ON1 with 72 nucleotide duplications was the major subgroup A virus detected alongside genotype NA2. All the detected HRSV subtype B belong to the BA genotype with characteristic 60 nucleotide duplications. Amino acid substitutions were found in the ON1 genotypes including threonine substituted with isoleucine at position 292 (T292I) in isolate NGR/OL76/15-RSVA. The NA2 genotype have mutation on all antigenic sites in the HVR2. Substitution of asparagine with serine at position 276 (N276S), previously reported to confer resistance against Palivizumab was detected in all the F gene analysed. Symptoms like cough (p=0.007), wheezing (p=0.001) and nasal congestion (p=0.001) were associated with HRSV positivity. Three genotypes of human respiratory syncytial viruswere detected among children in Ibadan, Nigeria. Substitution T292I found in one of the isolates has not been reported elsewhere although its significance is not yet known. The presence of the Palivizumab-resistance mutation on the circulating virus suggest the likelihood of treatment failure if the monoclonal antibody is introduced for use in Nigeria. Studies to determine the effect of the N276S mutation on Palivizumab is recommended to inform policy on the monoclonal antibody use in Nigeria. 2019-04-01T00:00:00Z http://hdl.handle.net/123456789/709 MOLECULAR EPIDEMIOLOGY AND SOME FACTORS ASSOCIATED WITH GENITAL HUMAN PAPILLOMAVIRUS INFECTION AMONG WOMEN IN OYO STATE, NIGERIA NEJO, YEWANDE TOLULOPE, Genital Human Papillomavirus (HPV) infection is a well-established causative agent of cervical cancer; a major cancer in most developing countries.Persistent infection with high-risk HPV, especially types 16 and 18 have been associated with cervical cancer. Two HPV vaccines (Cervarix and Gardasil) are currently available in Nigeria, targeting two (16 and 18) and four (6, 11, 16 and 18) types, respectively. However, the distribution of HPV types varies greatly across geographical regions with little information on the circulating types among Nigerian women, thus raising concerns about the effectiveness of the available vaccines in the country. This study was therefore designed to determine the circulating HPV types among women in Oyo State, Nigeria and identify some factors associated with the infection. A total of 295 endocervical swab samples were collected from consenting women attending routine cervical cancer screening, STI clinics and community-based outreach programme. The participants were enrolled from University College Hospital, Ibadan; Baptist Medical Centre, Saki and during an outreach in Molete community, Ibadan.Structured questionnaire was used to capture demographic, medical information and sexual history.Genomic DNA was extracted from samples using commercial extraction reagents. The presence of HPV was detected by PCR using two sets of consensus primers targeting the L1 and E6/E7 genes. Six pairs of HPV type-specific primers (16, 18, 31, 33, 35 and 6/11) were then used to genotype the HPV isolates in a nested PCR. Samples not identified by the primers used were sequenced and typed by phylogenetic analysis. Data were analysed using Chi-square at α0.05. Fifty five (18.6%) individuals were positive for HPV infection. Primers targeting E6/E7 region detected more HPV infections (17.3%) than those targeting L1 region (9.2%). Five HPV types were detected using type-specific primers (HPV 16, 18, 31, 33 and 35), while 14 HPV types (HPV 6, 16, 18, 31, 35, 42, 43, 44, 52, 58, 66, 74, 81, 86) were identified by sequencing. In all, 15 HPV types were detected with HPV 31 being the most predominant (32.8%), followed by HPV 35 (17.2%) and HPV 16 (15.5%). About 21.0% of individuals had dual infections while high-risk HPV genotypes were found among 86.2% of HPV-positive individuals. Highest nucleotide substitutions (n=32) were found in HPV 44 genotype while the only HPV 74 isolate had three nucleotide (CCT) insertionsat E7 gene that translated into amino acid proline. Some factors including divorce (p=0.019), illiteracy (p=0.003), polygamy(p=0.027), unemployment (p=0.023), low income earnings (p=0.018), younger age (<18years)at sexual debut (p=0.039) and passive smoking (p=0.017) were associated with HPV infection. Multiple Human Papillomavirus types co-circulated in Oyo State, Nigeria. Most of the circulating Human Papillomavirus are high-risk type with type 31 being the most predominant. Although the implication of the rare HPV 74 with proline insertion detected for the first time in Nigeria is unknown, it may have effect on the transformation potential of the virus. Primers targeting E6/E7 region may be more appropriate for the detection of Human Papillomavirus circulating in Oyo State, Nigeria. 2019-03-01T00:00:00Z