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Isolation and Characterization of SARS-CoV-2 in Kenya

American Journal of Molecular Biology 2024年第2期14卷 66-83页

作者： Albina Makio robinson mugasiali irekw. Matthew Mutinda Munyao Caroline Wangui Njoroge Peter Kipkemboi Rotich Tonny Teya Nyandwaro Joanne Jepkemei Yego Anne Wanjiru Mwangi James Hungo Kimotho Ronald Tanui Vincent Rutto Samson Muuo Nzou Center for Traditional Medicine and Drug Research Kenya Medical Research Institute Nairobi Kenya Center for Microbiology Research Kenya Medical Research Institute Nairobi Kenya Production Department Kenya Medical Research Institute Nairobi Kenya Department of Biochemistry and Microbiology Rhodes University Grahamstown South Africa Center for Virus Research Kenya Medical Research Institute Nairobi Kenya

The discovery of Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) in Wuhan, Hubei province, China, in December 2019 raised global health warnings. Quickly, in 2020, the virus crossed borders and infected individuals across the world, evolving into the COVID-19 pandemic. Notably, early signs of the virus’s existence were observed in various countries before the initial outbreak in Wuhan. As of 12th of April, the respiratory disease had infected over 762 million people worldwide, with over 6.8 million deaths recorded. This has led scientists to focus their efforts on understanding the virus to develop effective means to diagnose, treat, prevent, and control this pandemic. One of the areas of focus is the isolation of this virus, which plays a crucial role in understanding the viral dynamics in the laboratory. In this study, we report the isolation and detection of locally circulating SARS-CoV-2 in Kenya. The isolates were cultured on Vero Cercopithecus cell line (CCL-81) cells, RNA extraction was conducted from the supernatants, and reverse transcriptase-polymerase chain reaction (RT-PCR). Genome sequencing was done to profile the strains phylogenetically and identify novel and previously reported mutations. Vero CCL-81 cells were able to support the growth of SARS-CoV-2 in vitro, and mutations were detected from the two isolates sequenced (001 and 002). Genome sequencing revealed the circulation of two isolates that share a close relationship with the Benin isolate with the D614G common mutation identified along the S protein. These virus isolates will be expanded and made available to the Kenya Ministry of Health and other research institutions to advance SARS-CoV-2 research in Kenya and the region.

关键词： SARS-CoV-2 COVID-19 Whole Genome Sequencing Phylogenetic Analysis Nucleotide Substitutions Amino Acid Changes

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Identification and Characterization of Hepatitis B Virus Immune Escape Mutants in Kenya

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American Journal of Molecular Biology 2023年第1期13卷 1-17页

作者： Rhoda Elizabeth King James Kimotho Rosaline Macharia Faith Njoki Ndung’u Samson Muuo Nzou robinson mugasiali irekw. Department of Molecular Biology and Biotechnology Institute for Basic Sciences Technology and Innovation Pan African University Nairobi Kenya Department of Innovation Technology and Transfer Kenya Medical Research Institute Nairobi Kenya Department of Biochemistry University of Nairobi Nairobi Kenya Department for Microbiology Research Kenya Medical Research Institute Nairobi Kenya

Hepatitis B Virus (HBV) infections affect about 400 million people globally and cause about 1.4 million deaths annually. The virus displays high levels of genetic variations/mutations, some of which are immune escape mutants. The prevalence of HBV infection in Kenya is high at about 8%. This study aimed at identifying and characterizing HBV immune escape mutants in Kenya. From 547 HBV sequences available in Kenya in NCBI, and HBVdb databases in July 2021, 120 full sequences were retrieved. The S gene sequences at position 1-225, which included the “a” determinant region of the gene were analyzed using various bioinformatics tools such as Bioedit software, and Emboss Cons. The clinical significance was flagged from the search of peer-reviewed journals. Forty-six HBV-positive blood donor samples were obtained from the Kenya National Blood Transfusion Services without personal identifiers, DNA extracted, and sequenced targeting positions 1 to 520 of S genes. Mutations were similarly identified from seventeen sequences after cleaning and analysis. Out of 120 sequences that were extracted from databases and analyzed, 79 different mutations were identified. Fifteen of them were of clinical importance with an occurrence frequency of at least 5% were obtained. The majority (64.6%, n = 51), with S207N and A194V being most dominant, could result in immune escape and reduced HBsAg detection signals while 24.1% (n = 19) could result in immune escape/reduced HBsAg detection signals and high probability of hepatocellular carcinoma. Most likely to occur on the amino acids Alanine, Lysine, Serine, Asparagine, and Valine in decreasing order. The most dominant genotype was found to be Genotype A (N = 10), while four sequences were Genotype D. In contrast to the in-silico studies, the sequences from HBV samples from blood donors did not demonstrate the presence of S207N and A194V mutations and all the genotypes were type A1. Only two (13.3%) samples showed the same mutations of sK122R

关键词： Hepatitis B Virus Mutations Hepatitis B Surface Antigen Gene Genotype

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Investigation of Plasmodium falciparum Resistance Biomarkers among Primary School Children in Western Kenya

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American Journal of Molecular Biology 2022年第2期12卷 43-53页

作者： Otilmoi Poul Stephen Tonny Teya Nyandwaro robinson mugasiali irekw. Rebecca Wanjiku Waihenya Matthew Mutinda Munyao Peter Kipkemboi Rotich Caroline Wangui Njoroge Anne Wanjiru Mwangi Joanne Jepkemei Yego Nicole Sian Tanchu Dawala Koromtili Oumar Grace Ngendo Kanyita Primrose Muthoni Ndungu Samson Muuo Nzou Department of Molecular Biology and Biotechnology Pan-African University Institute of Basic Sciences Technology and Innovation Nairobi Kenya Innovation Technology Transfer Department Kenya Medical Research Institute (ITTD-KEMRI) Nairobi Kenya Zoology Department School of Biological Sciences College of Pure and Applied Sciences Jomo Kenyatta University of Agriculture and Technology Nairobi Kenya Production Department KEMRI Nairobi Kenya Centre for Microbiology Research Kenya Medical Research Institute Nairobi Kenya Nagasaki University Institute of Tropical Medicine Kenya Medical Research Institute (NUITM-KEMRI) Nairobi Kenya Department of Biochemistry Microbiology and Biotechnology Kenyatta University Nairobi Kenya Department of Pharmacology and Pharmacognosy University of Nairobi Nairobi Kenya

Background: A marked decrease in malaria-related deaths worldwide has been attributed to the administration of effective antimalarials against Plasmodium falciparum. However, the continuous spread of P. falciparum resistance to anti-malarial drugs is raising a serious problem in controlling Malaria to the vulnerable children’s immune system. In recent studies, Plasmodium falciparum Kelch 13 propeller gene (Pfk13) has been reported to develop resistance to artemisinin in South Asia. In this study, we checked Plasmodium falciparum chloroquine resistance transporter gene (Pfcrt) involved in chloroquine (CQ) resistance. Method: In this study, archived 280 samples were collected from Alupe primary school children in Busia, Western Kenya from May, 2016 to November, 2016. Genomic DNA was extracted using the MightyPrep reagent. The samples were investigated for P. falciparum positivity out of which 67 of them tested positive giving a prevalence rate of 24%. The sixty-seven were subjected to PCR amplification for the molecular marker resistance to Pfcrt. After PCR amplification, the amplicons were purified and sequenced using Sanger Sequencing. The sequence data were analyzed using BioEdit software to identify point mutations. Results: 14 samples sequences were analyzed on Bioedit software giving the following amino acid changes F76C, Y66H, L70A, Y58C, T59V, V65I, P67L, T81L, Y60S, Y66S, P67T and I71F). New mutations have been reported at position 76 leading to an amino acid change, one of Pfcrt gold standard biomarkers. However, amino acid changes Y66H, L70A, Y58C, T59V, V65I, P67L, T81L, Y60S, Y66S, P67T and I71F are newly reported giving an increase in Pfcrt prevalence of concern from zero to 5.0%. A phylogenetic evolutionary relationship was constructed as shown below. Generally, the results showed a continuous resistance of *** to Pfcrt which calls for robust continuous monitoring and surveillance. Conclusion: Due to the increase of the resistant Pfcrt gene pre

关键词： P. falciparum Chloroquine Drug Resistance Malaria P. falciparum Chloroquine Resistance Transporter Gene Mutations

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Expression and Evaluation of Wb-SXP-1 and Wb-123 Recombinant Antigens as Potential Diagnostic Biomarkers for Lymphatic Filariasis

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American Journal of Molecular Biology 2023年第2期13卷 95-112页

作者： Sharlene Kerubo Mageto Rebecca Wanjiku Waihenya Anne Wanjiru Mwangi Peter Kipkemboi Rotich Matthew Mutinda Munyao Tonny Teya robinson mugasiali irekw. Joanne Jepkemei Yego Caroline Wangui Njoroge Grace Ng’endo Kanyita Nicole Sian Tanchu Dawala Koromtili Oumar Primrose Muthoni Ndungu Samson Muuo Nzou Department of Molecular Biology and Biotechnology Pan-African University Institute of Basic Sciences Technology and Innovation Nairobi Kenya Zoology Department Jomo Kenyatta University for Agriculture and Technology Nairobi Kenya Nagasaki University Institute of Tropical Medicine Kenya Medical Research Institute (NUITM-KEMRI) Nairobi Kenya Production Department Kenya Medical Research Institute Nairobi Kenya Centre for Microbiology Research Kenya Medical Research Institute Nairobi Kenya Department of Biochemistry Microbiology and Biotechnology Kenyatta University Nairobi Kenya Department of Pharmacology Clinical Pharmacy and Pharmacy Practice University of Nairobi Nairobi Kenya

Lymphatic filariasis (LF) remains a public health concern as it can cause permanent morbidity and disability to those infected. While the global elimination of LF in these endemic areas is ongoing through mass drug administration, there is the need to develop diagnostic tools that would be utilized to track the progress of total global eradication as well as perform surveillance for the recurrence of lymphatic filariasis transmission. Currently, approved LF diagnosis tools are faced with lack of specificity, low sensitivity, and periodicity dependence. Recombinant filarial antigen-based assays can address these drawbacks and offer practical instruments for LF diagnosis and surveillance. This present study, evaluated rWb-SXP-1 and rWb-123 antigens as potential diagnostic biomarker tools for Wuchereria banchrofti in human sera using microspheres-based multiplex serological assay. Based on statistical analysis using XLSTAT 2019 (Addinsoft) on data generated from multiplex technology assay, generated ROC curves for both rWb-SXP-1 and rWb-123 demonstrated 87.1% sensitivity to Wuchereria banchrofti human sera with rWb-SXP-1 antigens having the highest specificity of 96%. Indication that rWb-SXP-1 and rWb-123 antigens are capable of detecting immunoglobulin G4 (IgG4) antibodies in human sera synthesized specifically against W. banchrofti infections. Therefore, rWb-SXP-1 and rWb-123 antigens can be utilized to detect W. banchrofti infections by antibody profiling with excellent diagnostic sensitivity and specificity using microsphere-based multiplex serological tests. This method can be particularly practical for screening a large number of sera samples and/or for quick, extensive field-testing due to the high-throughput and quick formats applied.

关键词： Lymphatic Filariasis Recombinant Antigens Diagnosis

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Use of Polyclonal Antibody for the Diagnosis of Human African Trypanosomiasis

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American Journal of Molecular Biology 2023年第2期13卷 127-139页

作者： Dawala Koromtili Oumar Matthew Mutinda Munyao Anne Wanjiru Mwangi Rebecca Wanjiku Waihenya Peter Kipkemboi Rotich robinson mugasiali irekw. Tonny Teya Nyandwaro Caroline Wangui Njoroge Joanne Jepkemei Yego Primrose Muthoni Ndungu Sharlene Kerubo Mageto Damaris Mutethya Kilei Otilmoi Poul Stephen Nicole Sian Tanchu Grace Ngendo Kanyita Shingo Inoue Lucy Gitau Samson Muuo Nzou Department of Molecular Biology and Biotechnology Pan-African University Institute of Basic Sciences Technology and Innovation Nairobi Kenya Centre for Microbiology Research Kenya Medical Research Institute Nairobi Kenya Nagasaki University Institute of Tropical Medicine-Kenya Medical Research Institute Project Nairobi Kenya Production Department Kenya Medical Research Institute Nairobi Kenya Zoology Department Jomo Kenyatta University for Agriculture and Technology Nairobi Kenya Department of Pharmacology and Pharmacognosy University of Nairobi Nairobi Kenya Department of Microbiology Jomo Kenyatta University for Agriculture and Technology Nairobi Kenya Department of Biochemistry Microbiology and Biotechnology Kenyatta University Nairobi Kenya Center for Biotechnology Research and Development Kenya Medical Research Institute Nairobi Kenya

Human African trypanosomiasis (HAT), commonly known as sleeping sickness is one of the neglected tropical diseases (NTDs), which is fatal if left untreated. Its diagnosis is a challenge since the signs and symptoms of the primary phase are not specific, the existing diagnostic methods have low sensitivity and specificity, and the available drugs have some toxicity. New, robust, and cost-effective techniques are needed for the early identification of parasites. This study aimed to assess the sensitivity and specificity of two different types of polyclonal antibodies against T. b. gambiense using antigen detection ELISA. Polyclonal antibodies against the expressed proteins Tbg I2 and Tbg I17 were produced using New Zealand white rabbits. The antibody titer measured was greater than 32 g/L after the 3rd immunization for the expressed protein Tbg I2. For the expressed protein Tbg I17, the antibody titer measured was greater than 32 g/L after the 4th immunization. The sensitivity and specificity of the Tbg I2 polyclonal antibody confirmed with Polymerase Chain Reaction (PCR) as gold standard were respectively 89.5% and 80.6%, while for the Tbg I17 polyclonal antibody, the sensitivity and specificity were respectively 92.1% and 88.9%. The area under the curve for the Tbg I2 polyclonal antibody was 0.90 ± 0.032, while for the Tbg I17 polyclonal antibody, the area under the curve was 0.92 ± 0.0. The Tbg I17 polyclonal antibody produced in New Zealand white rabbits has good sensitivity and good specificity;it can be successfully used in the diagnosis of HAT.

关键词： Human African Trypanosomiasis Polyclonal Antibody Tbg I2 Expressed Protein Tbg I17 Expressed Protein Sensitivity Specificity

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