http://adhlui.com.ui.edu.ng/jspui/handle/123456789/23 Theses in Virology 2026-02-24T09:26:04Z http://adhlui.com.ui.edu.ng/jspui/handle/123456789/702 Title: CHARACTERISATION AND SERO-EPIDEMIOLOGY OF POTISKUM VIRUS Authors: OMILABU, S. A. Abstract: Some aspects of biochemical and biophysical characteristics of Potiskum (POT) virus (a flavivirus isolated from Cricetomys gambianus) were studied in experimental animals, mosquitoes and tissue culture systems. The laboratory and domestic animals infected included Swiss albino mice, rabbits, chicks and goats. The tissue culture systems used were BHK-21, Vero, HEP-2, LLC-MK₂, E6, MA 104 MDCK, MDBK, and MEC. Aedes aegypti was the only mosquito species tested. The Nero-epidemiological pattern of the virus were also studied in both human and animal populations. POT virus passed freely through 200 and 450 nm diameter sized membrane filters. The virus titre was reduced by 2.0 logs when supsended in a diluent supplemented with serum collected from local cattle. The virus titre in tissue culture was not as high as observed in Swipe albino mice. Potiskum virus lost its infectivity to ether and chloroform. The virus lose was between 3.3 and 4.5 lost of virus particles to Ether and Chloroform respectively. POT virus also lost appreciable virus titre when suspended in glycerin buffer. The virus was also shown to be highly heat labile as more than 5.0 logs of POT virus was lost to heat (56°C) under 30 minutes. However, the virus loss at 4°C was minimal ( 2.0 logs) when stored for 120 hours. Potiskum virus haemagglutinated goose, sheep, pig and chick erythrocytes, optimally at either room, +4°C or 37°C using pH values ranging from 6.4 to 6.8; the POT haemagglutinating antigens were only detectable in sucrose-acetone extracted mouse brain materials. The enzyme-linked immunosorbent assay (ELISA) was shown to be sensitive but it could not detect POT virus antigen beyond 10⁻⁴ dilutions. The results of complement fixation and haemaglutination inhibiting antibodies assayed in either mouse or rabbit sera showed that the titres increased with POT virus dose/number of virus inocura. It was was observed that POT virus cross-reacted extensively with Uganda S, West Nile, Wesselebron and Yellow fever; antibodies to POT virus cross-reacted broadly with theme flaviviruses while their antibodies selectively reacted with POT virus. It was observed that POT virus was related to Uganda S, West Nile, Wesselsbron and Yellow fever viruses in descending order. The results of experimental infections of both laboratory and domestic animals indicated that mice, goats and chicks tested were susceptible to POT virus infection. The infection resulted in clinical disease; the animals developed viraemia of variable duration (4 - 8 days). Their susceptibility was however age and dose- dependent. The Swiss albino mice aced 2 - 15 days succumbed to IOLD₅₀ of the virus while the 7 - 15 days old chicks were refractory to infection with 1OLD₅₀ and 100LD₅₀ of POT virus. Variations in susceptibility were also observed in different routes of infection. The virus was however shown to be highly neurotropic in most of the POT virus-infected animals. High virus titres ( 6.5 logs) and marked histopathological lesions were confined to the brains of infected animals. There was 50-100% mortalities in chicks infected with 1000LD₅₀ or more of POT virus using subcutaneous (s.c.) or oral routes. The s. c. inoculated birds succumbed faster than the orally inoculated chicks. Striking pathological lesions and infective virus particles were detected in most of the organs assayed. Similarly the West African Dwarf goats showed 50% mortality when infected s.c. Their response was also dependent on age, the younger the more susceptible. The infected animals developed fever, starry hair coats, leucopaenia, nervousness, hindlimb paralysis and lateral recumbency among other signs. Infective virus particles and striking lesions were detected in the brain, lungs, liver and small intestine. Generally, complement fixing antigens were detected in the organs of infected mice and goats, however, CF antigen was not detected in the organs of POT virus infected chicks. The birds also failed to circulate CF antibodies. Potlskum virus multiplied in four of the 9 cell culture systems tested namely, BHK-21, E6, Vero and HEP-2. The virus produced cell rounding cytopathology and also formed plaques under Carboxy-methyl Cellulose (CMC) overlay medium. CF antigens were also detected in these four cell cultures. The cytological examinations of two of the susceptible cultures (BHK-21 and E6) suggested the replication of the virus both in the cytoplasm and the nucleus of these cells. The experimentally infected mosquitoes (Aedes aegypti) successfully transmitted POT virus from day 5 extrinsic incubation period (EIP). Transmission of the virus was delayed till day 10 in mosquito that received low virus dose. POT virus was circulated in the mosquitoes for 35 days when the last batch of the mosquitoes was harvested and in the suckling mice exposed to these infected mosquitoes. The sero-epidemiological studies showed that HI antibodies to flaviviruses were prevalent in both the human and animal populations tested. However the prevalence varied with different ecological zones. In humans, high POT virus activity (70%) was observed alongside yellow fever, Wesselebron, Uganda S, Dengues 1 & 2, West Nile and Banzi viruses. Few monotypic HI reaction, were found with POT virus despite its high cross-reactivity with other flaviviruses tested. The result of the plaque reduction test performed on some sera showed that 22%, of these sera reduced 50% or more of the POT virus plaques. The prevalence of flavivirus infection in animals were also moderate and POT virus activities were highest in camels, pigs and dogs as against the low response observed in goats, sheep, cattle and fowls. Generally, the prevalence observed in this study in both human and animal populations in 5 different ecological zones further accentuates the hyperendemicity of flavivirus Infections in this environments. Description: A Thesis in the Department of Virology submitted to the Faculty of Basic Medical Sciences, College of Medicine, in partial fulfillment of the requirement for the degree of Doctor of Philosophy, University of Ibadan 1992-11-01T00:00:00Z http://adhlui.com.ui.edu.ng/jspui/handle/123456789/696 Title: GENETIC DIVERSITY AND CORECEPTOR USAGE OF HIV-1 STRAINS AMONG INFECTED INDIVIDUALS PRESENTING AT A VOLUNTARY COUNSELLING AND TESTING CENTRE IN IBADAN, NIGERIA Authors: DONBRAYE, EMMANUEL Abstract: Molecular analyses of Human Immunodeficiency Virus (HIV) have shown great genetic diversity among strains of the virus. The diversity has implications on efficiency of its transmission, pathogenesis, diagnosis, vaccine design, coreceptor usage, response to anti-retroviral therapy and development of drug resistance. The HIV epidemic in Nigeria is characterised by the presence of multiple strains of the virus and increasing resistance to some anti-retroviral drugs. This study was carried out to determine the circulating HIV-1 strains and their coreceptor usage patterns at a voluntary counseling and testing centre in Nigeria. Blood samples were collected from 85 consenting HIV-1 infected (40 seroconcondant couples and partners of 5 serodiscordant couples) anti-retroviral therapy-naive patients. They include 42 males and 43 females with median age of 37 years (range 18-58 years) who presented at a voluntary counselling and testing centre in the University College Hospital, lbadan. Genomic DNA was extracted from whole blood using a commercial DNA extraction kit. The env C2-V3 region of HIV-1 from the genomic DNA extract was amplified by nested PCR using primers WT1and WT2, and KK₄₀ and KK₃₀ for the first and second rounds, respectively. Amplified DNA was directly sequenced in both forward and reverse directions, edited and analysed for phylogenetic relationships by ClustaW and Maximum Likelihood methods in a specialized software based on their nucleotide and amino acid sequences. The target region was successfully sequenced from 64 of the 85 patterns comprising, 23 of the 40 seroconcordant couples, 13 single partners of the remaining 17 seroconcordant couples and the 5 serodiscordant couples. Overall, 3.1%, 53.1%, 28.1%, 14.1% and 1.6% of the HIV-1 env C2-V3 nucleotide sequences were identified as subtype A, G, CRFO2_AG, CRF06_cpx, and CRF35_AD, respectively, with variation in subtype distribution. Eighteen of the 23 positive seroconcordant couples were infected with the same HIV.1 strain while the other five HIV-1 seroconcordant couples were infected with different HIV-1 strains indicating independent sources of infection of each spouse .The V3 loop region of the viruses had amino acid sequence conservation in the base positions 1-8 and 26-35 and tip regions and sequence variability, including mutations and deletions at positions 9-25. Most (76.5%) of the sequences lead the GPGQ crown motif while the GPGQ/L/R/K substitution was observed in 18.8%. The number of N-linked glycosylation sites ranged from 0 to 4 per env C2-V3 amino acid sequence with only 37.5% of the sequences having all four N-Iinked glycosylation sites. The CXCR4- tropic viruses known to be associated with the presence of multiple ammo acid mutations were predominant (68.8%) among the studied population. Ten percent of the CCR5-tropic viruses showed Maraviroc associated resistant mutations. There was evidence of circulation of HIV-1 CRF35_AD in Nigeria, and multiple circulation of HIV-1 subtypes, with the predominance of HIV-1G. The predominance of CXCR4 -tropic viruses and presence of primary resistance to Maraviroc in 10% of CCR5-tropic viruses suggest the need for further investigation prior to introduction of coreceptor inhibitors like Maraviroc for management of HIV infection in Nigeria. Description: A Doctoral Degree Thesis in the Department of Virology submitted to the Faculty of Basic Medical Sciences in partial fulfillment of the requirement for the degree of Doctor of Philosophy of the University of Ibadan, Ibadan, Nigeria 2013-01-01T00:00:00Z http://adhlui.com.ui.edu.ng/jspui/handle/123456789/689 Title: GENETICS AND PATHOGENICITY OF THREE H5N1 AVIAN INFLUENZA A VIRUS ISOLATES OF CHICKEN IN SOUTHWESTERN NIGERIA Authors: AIKI-RAJI, C.O. Abstract: Highly Pathogenic Avian influenza A (HPAI) H₅NI outbreak has been reported in several African countries including Nigeria. The outbreak continues to pose a risk to human health and livelihood. The genetics and pathogenicity of three Nigerian H₅NI avian influenza virus isolates were therefore investigated. Carcasses of chicken were obtained from Ogun State for post mortem examination. Nasopharyngeal and cloacal swabs, lung, brain, intestine, heart and tracheal tissues were collected for virus isolation in embryonated chicken eggs. Three virus isolates were obtained and identified serologically with haemagglutination-inhibition assay. Viral RNA was extracted with phenol and guanidinium isothiocyanate and the genes were amplified using polymerase chain reaction, cloned into pGEM-T vector for mini preps, sequenced and analysed phylogenetically. The non-structural genes were transfected into 293T cells to assess their effects on interferon production in mammalian cells. Intravenous and intranasal pathogenicity teas were carried out in eight 4-week old specific pathogen free White Leghorn chickens. Tissue samples were collected for virus isolation and titration. Gross and histopathological examinations and immunohistochemical staining were also carried out. The isolates were designated influenza chicken/Nigeria/228-5/2006, A/chicken/Nigeria/228-6/2006 and A/chicken/Nigeria/228-10/2006. Sequence analyses confirmed the presence of multibasic amino acids in the haemagglutinin cleavage site as PQGERRKK which has been associated with high virulence. The neuraminidase did not have histidine substitution for tyrosine at position 274 (H274Y) which is associated with antiviral drug (oseltamivir) resistance. The non-structural protein 1 (NSI) open reading frame encoded a five-amino acid deletion at positions 80 to 84 in the three isolates while A/chicken/Nigeria/228-10/2006 had a 7 amino acid C-terminal extension. Intravenous and intranasal pathogenicity tests revealed virulence and high pathogenicity with mean death time of 1.4 and 2.2 days respectively. Gross lesions included congestion of the lungs, petechiae in epicardial fat, in the gizzard and swollen kidneys. Histologically, the most severe lesions were severe interstitial pneumonia with edema, myocyte degeneration and necrosis in the heart and moderate nonsuppurative encephalitis. Tissues with lesions coincided with sites of virus replication and indicated a severe systemic infection. This was confirmed by immunohistochemical staining of the viral antigen. The sequence changes did not affect the ability of NSI to block interferon induction when expressed transiently in 293T cells. The PB2 protein also had a lysine residue at position 627, which has been implicated in mammalian adaptation of H₅NI avian viruses. The sequence changes observed in NSI did not influence the ability of the virus to establish in mammalian cells. Based on the H₅NI Evolution Working Group classification, the three Nigerian H₅NI isolates are highly pathogenic and belong to clade 2.2.2 as other viruses from Africa, Asia, Middle East and Europe. Description: A Thesis in the Department of Virology submitted to the Faculty of Basic Medical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy of the University of Ibadan 2011-09-01T00:00:00Z http://adhlui.com.ui.edu.ng/jspui/handle/123456789/641 Title: RIFT VALLEY FEVER VIRUS IN NIGERIA: COMPARATIVE EXPERIMENTAL INFECTIONS OF THREE BREEDS OF INDIGENOUE SHEEP AND SERO-SURVEY OF HUMAN AND ANIMAL POPULATIONS Authors: OLALEYE, D. O. Abstract: Electron microscopic examination by negative staining and antigen controlled immunodiagnosis techniques on the Zinga virus used for this study showed that it is a strain of Rift Valley fever (RVF) virus. This is based on its morphology and specific reaction with reference to Rift valley fever virus antiserum. Retrospective and prospective immunity surveys in human and animal populations in different ecological zones of Nigeria showed endemic presence of Rift valley fever virus in the country. Out of 3,121 human sera tested for haemagglutination inhibition (HI) antibodies to RVF, 463 (14.8%) were positive of which 390 (85.4%) or 12.8% of total sera tested positive by plaque reduction neutralization test. The highest prevalence of neutralizing (NT) anti-bodies was found in the Sudan Savannah (17.9%) followed by derived savannah (17.7%). The prevalence rate in other zones included swamp forest (17.4%), Plateau (11.3%), rain forest (11.1%) and Guinea Savannah (4.5%). Antibody prevalence was significantly higher among livestock and forest related workers than hospital workers and the general population in different locations of all the ecological zones. Sex distribution analysis of all positive sera in the six ecological zones showed no significant difference between the male and female sex groups tested. In a longitudinal serological survey conducted at University College Hospital, Ibadan, a 5.5 RVF virus infection rate was demonstrated in the human population. There was a significantly higher infection rate during the wet than dry season of the same year. Human sera tested for specific RVF IgM in different ecological zones showed that 107 of 380 (27.4%) contained specific RVF IgM indicating recent exposure to the virus. Similar to results of HI and plaque reduction neutralization test (PRNT) , the highest incidence of RVF virus infection was found in the Sudan savannah. Results also showed significantly higher prevalence of specific RVF among livestock and forest-related workers than the general population and hospital workers. The highest prevalence of individuals with RVF virus specific IgM was found among cattle rearers at Maiduguri with over 50% positivity. Retrospective animal serological survey for RVF immunity showed that 261 of 2365 (11.0%) animal sera tested had HI and NT antibodies. The highest prevalence was found in sheep (17.8%) followed by goats (10.4%), cattle (10.2%) and horse (9.8%). Other animals included camel (3.3%), free range domestic fowls (3.4%) and pigs (0%). Closely related to findings in the human population, the highest prevalence of RVF antibody was found in the Guinea savannah (16.5%), followed by Sudan savannah (15.1%), swamp forest (11.4%), rain forest (4.8%) and derived savannah (4.0). A prevalence of 14.7% and derived savannah 14.7% was found among trade animals. Results showed that animals aged 3 years and above had higher prevalence of RVF antibodies than younger animals. Prospective animal serological survey using 10 sentinel flocks in 4 farms at Ibadan and Ile-Ife showed evidence of active circulation of the virus. This was demonstrated by seroconversions in 10 animals from 4 flocks in 2 farms, namely: University of Ibadan cattle, sheep and goats flocks and Iwo Roadairy cattle. One of the seroconversions took place during the early wet season (April), 6 occurred at the peak of rains, while the others sero converted towards the end of the wet season in the area. Experimental infection of three indigenous breeds of sheep in Nigeria, namely: West African dwarf (WAD), Yankasa and Ouda resulted in fatal disease with Zinga RVF virus. Infected sheep of the three breeds responded by pyrexia within 24 hours of infection. Fever peaked between the 2nd and 4th day p.i. and lasted 6 to 7 days. Viraemia coincided with pyrexia and peaked (10 9 PFU/ml) 3 days p.i. in two Yankasa and one West African dwarf sheep. Peak titre of 10 7.5 PFU/ml and above was detected in all other infected sheep. Infected sheep showed characteristic clinical changes which included hyperactivity, watery and mucoid nasal discharges, watery projectile and bloody diarrhoea, external haemorrhage and manifestations of nervous disorders. Viraemia was followed by mild antibody development in all the infected sheep. There was severe external haemorrhage in the Yankasa sheep but mild to moderate in the other breeds. Haematological changes included a sharp fall in the packed cell volume, haemoglobin concentration and total red blood cell count during the course of the disease. These changes were most severe in Yankasa with a drop of 10.3%, 40.0% and 13.3% in the PCV in WAD, Yankasa and Ouda breeds respectively on day 3 p.i. There were thrombocytopaemia, prolongation of the prothrombin and clotting times of all the infected sheep. Infected animals also showed progressive leucopaenia which was associated with severe lymphopaenia. Total protein dropped sharply during the early phase of the disease but rose gradually from day 5 p.i. The rise was associated with gradual increase in the globulin level from day 5. p.i. On the other hand, albumin level decreased progressively until the animals died. Evaluation of serum biochemical constituents of infected sheep showed sharp and progressive increase in the level of two liver enzymes, alanine aminotransferase and aspartate aminotransferase. The sodium level decreased gradually while potassium was initially stable but later increased in the level of blood urea nitrogen of all the infected animals from day 3 p.i. and this continued terminally. Gross and microscopic examinations of the carcasses of all the infected animals showed significant lesions in many organs which showed that Zinga RVF virus is pantropic for sheep and can also cause disseminated intravascular coagulation in this species. Description: A Thesis in the Department of Virology, submitted to the Faculty of Basic Medical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy of the University of Ibadan, Ibadan, Nigeria. 1990-09-01T00:00:00Z