Normalization of Gene Expression by Quantitative RT-PCR in Human Cell Line: comparison of 12 Endogenous Reference Genes

BACKGROUND: Polymerase Chain Reaction (PCR) has become an important diagnostic and research tool of modern molecular biology globally. Real-time PCR allows for rapid and reliable quantification of mRNA transcription. Reference genes are used as internal reaction control to normalise mRNA levels between different samples in order to allow for an exact comparison of mRNA transcription level.METHODS: In this study, twelve commonly used human reference genes were investigated in Human Embryonic Kidney Cell Lines (HEK293) using real-time qPCR with SYBR green. The genes included beta-2-microglobulin (B2M), glyceraldehyde-3- phosphate dehydrogenase (GAPDH), succinate dehydrogenase complex subunit A (SDHA), and tyrosine 3- monooxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ). The stability of these reference genes was investigated using the geNorm application.RESULTS: The range of expression stability in the genes analysed was (from the most stable to the least stable): UBC, TOP1, ATP5B, CYC1, GAPDH, SDHA, YWHAZ, CTB, 18S, EIFA-2, B2M and RPL13A. The optimal number of reference targets in the experiment was calculated to be 2 (geNorm V<0.15) when comparing a normalization factor based on the 2 or 3 most stable targets).CONCLUSION: The expression stability varied greatly between the 12 candidate reference genes. UBC, TOP1, ATP5B, CYC1 and GAPDH respectively showed the highest stability in HEK293 cells based on both expression stability and expression level. Overall, our data suggest that UBC and TOP1show the least variation and the highest expression stability. This report validates the need for rational selection of reference genes for data normalization to ensure accuracy of quantitative PCR assays.

Human Papillomavirus (HPV) Infection in Males: A Need for More Awareness

Globally, human papillomavirus (HPV) is the most common viral sexually transmitted pathogen, which is significantly associated with high morbidity and mortality in both sexes. Except those vaccinated, virtually all sexually active individuals will be infected with HPV in their lifetime. Although most HPV infections are transient, association with anogenital warts, cervical, penile, and other malignancies have been reported. HPV can be transmitted from one person to another through contact especially during sexual contact including anal, vaginal, or oral. Although HPV infection affects both males and females, its causal association with cervical cancer has made most literature to be mainly on females. In view of its sexual transmissibility and the increasing prevalence of HPV-related malignancies among males worldwide, there is need for more awareness on the infection in males. Most developed countries offer HPV vaccination for girls, but vaccine recommendations for boys are still relatively uncommon especially in developing countries where the burden of HPV-related malignancies is still very high. The current discourse highlights the need for increased awareness on HPV vaccination among this neglected gender group

Assessment of hepatitis B surface antigen negative blood units for HBV DNA among replacement blood donors in a hospital based blood bank in Nigeria

Background: Hepatitis B virus infection is one of the greatest threats to blood safety all over the world. The laboratory algorithm based on only the detection of hepatitis B surface antigen (HBsAg) leaves a gap for infected HBsAg negative donors to donate blood during the \u201cwindow period\u201d (WP) and late stages of infection. Objective: To estimate the frequency of the presence of HBV deoxyribonucleic acid (DNA) in HBsAg negative blood units screened using two different assays for HBsAg in a high endemic region. Methods: Frozen serum aliquot of 100 replacement blood donors who donated blood units that were HBsAg negative were retrieved and tested for HBV DNA. Sample positive for HBV DNA was sequenced by Sanger\u2019s method, genotyped and the viral load was determined. Results: One sample (1%) was positive for HBV DNA. The HBV viral load of the sample was 768,000 IU/ml. The partial S-gene of the Hepatitis B virus isolated was genotype E using the NCBI viral genotyping tool. Conclusions: There is still a risk of HBV infected blood unit escaping detection when donor testing is limited to HBsAg screening. The use of NAT which can substantially reduce HBV infected blood donors from the donor pool should be considered

Seroprevalence of hepatitis E among restaurant food handlers in Ibadan, Nigeria

 Background: Hepatitis E virus (HEV) is one of the causative agent of acute viral hepatitis in humans. HEV is an important public health disease in many parts of the world because it is transmitted faeco-orally.  Majority of the documented studies on hepatitis E virus in Nigeria have focused on pregnant women and animal handlers with limited data among food handlers. Thus the current study aimed at investigating the prevalence of HEV infection among food handlers operating within the premises of a tertiary care facility.Methods: One hundred and seventy seven (177) food handlers were screened using commercial Enzyme-Linked Immuno-Sorbent Assay (ELISA) to detect IgM antibodies to Hepatitis E. A semi-structured questionnaire was used to assess risk factors for HEV infection.Results: HEV IgM antibodies were detected in 16 (9.0%) of the participants. Age-specific HEV IgM seroprevalence appeared to decrease with age, however there were no significant differences in HEV IgM seropositivity regarding age (P=0.251), gender (P = 0.231), marital status (P=0.735) and religion (P = 0.906). Significant risk factors for HEV IgM seropositivity included source of water for drinking (P=0.03) and the use of soap for hand washing (P=0.02).Conclusion: Our findings suggest that HEV remains a public health problem, as the virus circulates at low but considerable levels especially among food handlers; thus posing a threat to potential contacts. Proper hand washing practices as well as provision of portable water are important factors for the control of Hepatitis E

Emergence and spread of two SARS-CoV-2 variants of interest in Nigeria.

Identifying the dissemination patterns and impacts of a virus of economic or health importance during a pandemic is crucial, as it informs the public on policies for containment in order to reduce the spread of the virus. In this study, we integrated genomic and travel data to investigate the emergence and spread of the SARS-CoV-2 B.1.1.318 and B.1.525 (Eta) variants of interest in Nigeria and the wider Africa region. By integrating travel data and phylogeographic reconstructions, we find that these two variants that arose during the second wave in Nigeria emerged from within Africa, with the B.1.525 from Nigeria, and then spread to other parts of the world. Data from this study show how regional connectivity of Nigeria drove the spread of these variants of interest to surrounding countries and those connected by air-traffic. Our findings demonstrate the power of genomic analysis when combined with mobility and epidemiological data to identify the drivers of transmission, as bidirectional transmission within and between African nations are grossly underestimated as seen in our import risk index estimates

A year of genomic surveillance reveals how the SARS-CoV-2 pandemic unfolded in Africa.

The progression of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic in Africa has so far been heterogeneous, and the full impact is not yet well understood. In this study, we describe the genomic epidemiology using a dataset of 8746 genomes from 33 African countries and two overseas territories. We show that the epidemics in most countries were initiated by importations predominantly from Europe, which diminished after the early introduction of international travel restrictions. As the pandemic progressed, ongoing transmission in many countries and increasing mobility led to the emergence and spread within the continent of many variants of concern and interest, such as B.1.351, B.1.525, A.23.1, and C.1.1. Although distorted by low sampling numbers and blind spots, the findings highlight that Africa must not be left behind in the global pandemic response, otherwise it could become a source for new variants

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance.

Investment in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing in Africa over the past year has led to a major increase in the number of sequences that have been generated and used to track the pandemic on the continent, a number that now exceeds 100,000 genomes. Our results show an increase in the number of African countries that are able to sequence domestically and highlight that local sequencing enables faster turnaround times and more-regular routine surveillance. Despite limitations of low testing proportions, findings from this genomic surveillance study underscore the heterogeneous nature of the pandemic and illuminate the distinct dispersal dynamics of variants of concern-particularly Alpha, Beta, Delta, and Omicron-on the continent. Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve while the continent faces many emerging and reemerging infectious disease threats. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Molecular virology of KSHV : elucidating vIRF2 and vIRF4 function.

The innate type I interferon antiviral response is the first line of defence invoked to limit the spread of viral infections. Hence a number of viruses including Kaposi's sarcoma-associated herpesvirus (KSHV) have evolved defence strategies against this antiviral response. KSHV is the aetiologic agent of KS and almost one quarter of the KSHV genes specify either demonstrated or potential immunomodulatory activity including the four viral interferon regulatory factors (vIRFs). vIRFs 1, 2 and 3 have previously been shown to inhibit type I IFN signalling, whereas the inhibitory role of vIRF4 is yet to be reported. A previous stable isotope labelling of amino acids in cell culture (SILAC) study coupled to LC-MS/MS identified USP7 and ribosomal proteins as binding partners of both vIRF2 and vIRF4. The aim of the present study was to investigate the role of these binding partners in type I IFN signalling and to determine the regulatory mechanisms behind the effects of these partner proteins on the functions of the vIRF2 and vIRF4 proteins. Polysome profiling and microarray studies were carried out on vIRF4 expressing cells and suggested a novel regulatory role for vIRF4 in translation. USP7 was also characterised as a positive regulator of IFN signalling and the mechanism behind this effect was explored

Asymptomatic Bacteriuria in Antenatal Patients in Ilorin, Nigeria

 Objective: To determine the prevalence of asymptomaticbacteriuria, bacteriology and sensitivity pattern in Ilorin using thegold standard of urine culture.Methods: A prospective study was carried out from 1st Julyto 31st October 2007, at the University of Ilorin TeachingHospital (UITH) on 125 consenting asymptomatic pregnantwomen. A structured proforma was used to collect informationfrom the women and a midstream urine specimen collected forbacteriological culture.Results: Of the 125 pregnant women, 50 had bacteriuria on urineculture giving a prevalence of 40�20The mean age of the womenwas 28.5 years with a standard deviation of 4.95. The age rangedbetween 14 and 40 years. Staphylococcus aureus was the commonestpathogen isolated (72� followed by Proteus spp (14� Most ofthe organisms showed good sensitivity to Nitrofurantoin andgentamicin.Conclusion: The prevalence of asymptomatic bacteriuria in Ilorinis high and routine urine culture is advocated for all pregnantwomen at booking