Molecular Surveillance of Adamantane Resistance among Human Influenza A Viruses Isolated in Four Epidemic Seasons in Kenya

Background: Adamantanes impede influenza A virus replication and are important in the treatment and prophylaxis of disease caused by these viruses. Genotypic characterization of influenza A viruses for mutations associated with resistance to adamantanes has not been fully investigated in Kenya. Objective: To characterize susceptibility of influenza A virus subtypes that circulated in Kenya from 2008-2011 to adamantanes. Methods: Archived influenza A virus strains obtained from 2008 to 2011 were propagated in MDCK cells prior to sequencing of the matrix and hemagglutinin gene segments, followed by bioinformatics analyses. Results: Ninety two virus strains consisting of 21 A/H3N2, 18 A/H1N1 and 53 A/H1N1pdm09 were analyzed.  All A/H3N2 and A/H1N1pdm09 viruses displayed resistance to adamantanes due to the S31N/S31D amino acid substitution. All A/H1N1pdm09 virus strains belonged to the N-lineage characterized by S203T amino acid substitution in the HA1. All A/H1N1 viruses were sensitive to adamantane and were characterized by K140E amino acid substitution in the HA1. Conclusion: All Kenyan influenza A/H3N2 and A/H1N1pdm09 virus strains were resistant to adamantanes while seasonal A/H1N1 strains were sensitive to these drugs. During the study period, Amantadine and Rimantadine were inappropriate for prophylaxis and treatment of influenza disease caused by A/H3N2 and A/H1N1pdm09 virus subtypes in Kenya. Key words: Kenya, influenza A/H3N2, A/H1N1pdm09, A/H1N1, adamantane

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

Genotypic Characterization of Resistance to Neuraminidase Inhibitors amongst Influenza A viruses that circulated in Kenya from 2008 to 2011

Background: Vaccines and antivirals are the mainstay for mitigation and clinical management of influenza infections. However, due to the ever changing antigenic profile, vaccine formulations are revised every year to keep them efficacious. Neuraminidase (NA) inhibitors, mainly oseltamivir and zanamivir, function both as prophylactic and treatment agents. In neuraminidase inhibition, inhibitor molecules mimic NA’s natural substrate and bind to the active site, preventing NA from cleaving host cell receptors and releasing new virus. Currently there exists no data on antiviral susceptibility profile of influenza A isolates circulating within the Eastern African region. Here we characterized the antiviral susceptibility of the 2008-2011 influenza A viruses circulating in Kenya. Methodology: Nasopharyngeal swab specimen from consenting outpatients of ages greater than or equal to two months were obtained and transported under the cold chain to the National Influenza Center (NIC) and screened by real-time RT-PCR using primers targeted at the matrix, and hemagglutinin genes of influenza A subtypes. Positive specimens were inoculated onto MDCK monolayers to isolate virus. RNA was extracted from virus isolates followed by PCR amplification of NA gene segments using gene-specific primers. Nucleotide sequencing of the NA amplicons was carried out using the BigDye chemistry prior to analyses using a suite of bioinformatics tools. Results: 836 influenza A viruses were isolated. 108 (13%) isolates were analyzed for susceptibility to NA inhibitors. 64% (7/11) of the 2008 seasonal influenza A/H1N1 isolates depicted oseltamivir resistant marker H275Y while all 33 influenza A/H3N2 isolates had H at position 275 hence were sensitive to oseltamivir. Similarly, genetic analysis of the A(H1N1)pdm09 strains in 2009 showed that all had H275 hence sensitive to oseltamivir. The same pattern was duplicated in 2 of the pandemic influenza A/ H1N1 isolates analyzed in the year 2010. Thus all A(H1N1)pdm09 isolated were sensitive to oseltamivir. In 2011 we isolated 14 isolates belonging to influenza A/H3N2 subtype. All these had H 275 in the NA protein implying sensitivity to oseltamivir. Overall, our genotypic data demonstrate that there was oseltamivir resistance in seasonal influenza A (H1N1) viruses isolated in Kenya in 2008-2009. Conclusion: Our study shows that seasonal influenza A/H1N1 was displaced in 2010 and 2011 after introduction influenza A(H1N1)pdm09 which has since replaced the previous seasonal influenza A/H1N1

Surveillance of Human Parainfluenza viruses in Kenya during the 2007-2011 Period

Background: Human parainfluenza viruses (HPIVs) belong to the paramyxoviridae family and are classified into four types. These viruses account for a large percentage of pediatric respiratory disease, including syndromes such as upper respiratory tract infections (URTIs), laryngotracheobronchitis (croup), bronchiolitis, and pneumonia. HPIV is the major cause of croup in which type 1 is most frequent cause, followed by type 3 and type 2 respectively. In January 2007, through an existing influenza surveillance network, the Kenyan National Influenza center started screening for parainfluenza and other non-influenza respiratory viruses within the designated Influenza surveillance network made up of eight sentinel sites spread throughout the country. Objective: The objective of this study was to monitor and document circulation of Human parainfluenza viruses in Kenya in the period 2007-2011. Methodology: Specimens were collected from the nasopharynx using a flocked swab from consenting patients meeting the WHO influenza-like-illness (ILI) case definition. Specimens were transported to the NIC while observing the cold chain and inoculated into LLCMK2 cell line. After incubation and observation for cytopathic effect, all samples were screened by direct immunofluorescence assay (IFA) using the Respiratory Panel I Viral Screening and Identification kit (Chemicon International, Inc). Results: 14,990 nasopharyngeal swab samples were collected between January 2007-October 2011. HPIV were detected in 801 (5.3%) cases. 361 (45%) of the detections were HPIV-3 followed by HPIV-1 in 296 (37%) and 144 (18%) for HPIV-2 respectively. This confirms what has been observed elsewhere that HPIV1 and HPIV3 are the most frequently detected types. Analyses of co-infections involving HPIVs showed that HPIV1/HPIV2 (16cases) were the most frequent followed by HPIV1/HPIV3 (15) cases and HPIV2/HPIV3 (15 cases). There were 30 cases of triple infections of HPIV1/HPIV2/HPIV3. Generally, parainfluenza viruses circulated throughout the period under study. Parainfluenza virus infections were observed throughout the year with no distinct seasonal patterns. Conclusion: This study shows that parainfluenza viruses contributed to a significant level to the respiratory disease burden in Kenya in 2007-2011. Furthermore, our study has shown that parainfluenza viruses circulated in the human population in Kenya throughout the study period and did not show any distinct seasonality

Analyses of selection pressure on the Hemagglutinin gene of influenza A/H3N2 Viruses circulating in Kenya 2007-2011

Background: The hemagglutinin (HA) gene of Influenza viruses, especially the HA1 portion, exhibits a rapid rate of change, largely in response to human immune surveillance in a partially immune human population. Mutations in influenza viral genes accumulate over time and are under selection pressure during epidemics or pandemics. Objective: To determine whether the Kenyan influenza A/H3N2 viruses are undergoing adaptive evolution to become epidemic threats. Methods: Nasopharyngeal samples from patients meeting the WHO ILI case definition were collected between 2007 and 2010 from across Kenya. The detection of H3N2 virus was carried out using real-time RT-PCR. Positive samples were then cultured in MDCK cells and confirmed using the HAI assay. 156 isolates from this period were selected for amplification of the HA1 portion of the HA gene and the resulting amplicons sequenced. Global estimates, ω, of dN and dS, averaged over the entire alignment, were compared to calculate the overall strength of selection using the HyPhy 2.0 software package implemented in datamonkey. Results: Analysis of neutrality using, Kumar’s method showed that ω varied from 0.50 in 2007, 0.36 in 2008, 0.32 in 2009, 0.61 in 2010 and 0.41 in 2011. Further site by site analysis identified amino acid positions 46, 158 and 173 to be under positive selection. Analysis of differential selection showed 7 sites that harbored two or more amino acid substitutions. Amino acid positions 158, 160 and 189 had the highest number of amino acid polymorphisms. Conclusions: Overall, this study shows that local Influenza A(H3N2) viruses have been evolving via a series of ‘adaptive bursts’ characterized by positive selection occurring largely in immunological epitopes B and D. In between these bursts there is little evidence for positive selection and newly-emergent strains slowly replace the existent strains. Based on this study alone, we propose that these bursts occur after every two years. Since in a single population, dN/dS <1does not follow monotonic function, it is difficult to infer selection pressure in these results. However, by focusing on the antigenic sites we were able to observe an evolutionary pattern in the Kenyan samples. Thus evolution of Kenyan Influenza A(H3N2) is characterized by non synonymous changes followed by a period of stasis that is then followed by another period of non synonymous changes which is followed by purification selection

Respiratory Adenovirus Species Circulating In Kenya from 2007-2010

Background: Human adenoviruses [HAdvs] are causative agents of several diseases including acute respiratory disease (ARD), keratoconjunctivitis, gastroenteritis, acute hemorrhagic cystitis, opportunistic infection in immuno - compromised patients and severe and potentially fatal pneumonia. Adenoviruses have been shown to affect mainly pediatric populations, military recruits and congested institutions such as hospitals and schools where they are a major cause of morbidity and mortality. In this study we examined respiratory adenovirus species and types associated with respiratory infection circulating at eight study sites in Kenya from 2007-2010. Methods: Nasopharyngeal swab samples collected in viral transport media were transported to the National Influenza Centre in dry shippers while maintaining the cold chain. Samples originated from patients with Influenza like Illness (ILI) participating in the United States Army Medical Research unit in Kenya (USAMRU-K) sentinel surveillance network across the country. The samples were inoculated into Hep2 cells and incubated at 37 °C with 5% CO2 for 14 days or until cytopathic effect was observed. Presence of HAdv in the supernatants was determined by immunofluorescence assays. To begin to analyze these Kenyan HAdvs, 10% were molecularly genotyped using HAdv type-specific primers followed by nucleotide sequencing and analysis using a suite of bioinformatics software. This work received ethical approval under the KEMRI ERC-approved protocol SSC#981. Results: A total of 12,959 samples were screened during the period. 385 (3%) of these were positive for HAdv by cell culture. Molecular characterization of ~10% of the viruses using PCR yielded 20 (45%) HAdvs of the B species and 24 (55%) HAdvs species C. We did not detect any HAdv species E during the period. Analysis of the nucleotide sequences differentiated the species into types B3 [n=5], B7 [n=15], C1 [n=6], C2 [n=13] and C5 [n=4]. Although cell culture is not the most sensitive method for screening respiratory viruses, our results showed that respiratory HAdvs contributed at least 3% of the respiratory disease burden in Kenya during this period. Furthermore, the results showed that type B7 was the most prevalent HAdv followed by type C2. HAdv type B7 has been associated with the most severe forms of respiratory infections and fatalities globally, and our results showed that a substantial burden of serious respiratory disease was due to this HAdv type. These results suggest that if a respiratory HAdv vaccine were to be introduced in Kenya, it ought to contain the HAdv types B7 and C2 components. Conclusion: We have for the first time molecularly characterized HAdv types isolated from the respiratory tract of humans in Kenya and showed that overall, types B7 and C2 substantially contribute to severe respiratory disease in Kenya. The Kenyan Ministry of Health should consider introducing a vaccine containing these two components to mitigate against respiratory illness