2020 Ebola virus disease outbreak in Équateur Province, Democratic Republic of the Congo: a retrospective genomic characterisation.

BACKGROUND: The Democratic Republic of the Congo has had 15 Ebola virus disease (EVD) outbreaks, from 1976 to 2023. On June 1, 2020, the Democratic Republic of the Congo declared an outbreak of EVD in the western Équateur Province (11th outbreak), proximal to the 2018 Tumba and Bikoro outbreak and concurrent with an outbreak in the eastern Nord Kivu Province. In this Article, we assessed whether the 11th outbreak was genetically related to previous or concurrent EVD outbreaks and connected available epidemiological and genetic data to identify sources of possible zoonotic spillover, uncover additional unreported cases of nosocomial transmission, and provide a deeper investigation into the 11th outbreak. METHODS: We analysed epidemiological factors from the 11th EVD outbreak to identify patient characteristics, epidemiological links, and transmission modes to explore virus spread through space, time, and age groups in the Équateur Province, Democratic Republic of the Congo. Trained field investigators and health professionals recorded data on suspected, probable, and confirmed cases, including demographic characteristics, possible exposures, symptom onset and signs and symptoms, and potentially exposed contacts. We used blood samples from individuals who were live suspected cases and oral swabs from individuals who were deceased to diagnose EVD. We applied whole-genome sequencing of 87 available Ebola virus genomes (from 130 individuals with EVD between May 19 and Sept 16, 2020), phylogenetic divergence versus time, and Bayesian reconstruction of phylogenetic trees to calculate viral substitution rates and study viral evolution. We linked the available epidemiological and genetic datasets to conduct a genomic and epidemiological study of the 11th EVD outbreak. FINDINGS: Between May 19 and Sept 16, 2020, 130 EVD (119 confirmed and 11 probable) cases were reported across 13 Équateur Province health zones. The individual identified as the index case reported frequent consumption of bat meat, suggesting the outbreak started due to zoonotic spillover. Sequencing revealed two circulating Ebola virus variants associated with this outbreak-a Mbandaka variant associated with the majority (97%) of cases and a Tumba-like variant with similarity to the ninth EVD outbreak in 2018. The Tumba-like variant exhibited a reduced substitution rate, suggesting transmission from a previous survivor of EVD. INTERPRETATION: Integrating genetic and epidemiological data allowed for investigative fact-checking and verified patient-reported sources of possible zoonotic spillover. These results demonstrate that rapid genetic sequencing combined with epidemiological data can inform responders of the mechanisms of viral spread, uncover novel transmission modes, and provide a deeper understanding of the outbreak, which is ultimately needed for infection prevention and control during outbreaks. FUNDING: WHO and US Centers for Disease Control and Prevention.

Whole genome sequencing of outbreak strains from 2017 to 2018 reveals an endemic clade of dengue 1 virus in Cameroon.

Dengue fever is expanding as a global public health threat including countries within Africa. For the past few decades, Cameroon has experienced sporadic cases of arboviral infections including dengue fever. Here, we conducted genomic analyses to investigate the origin and phylogenetic profile of Cameroon DENV-1 outbreak strains and predict the impact of emerging therapeutics on these strains. Bayesian and maximum-likelihood phylogenetic inference approaches were employed in virus evolutionary analyses. An in silico analysis was performed to assess the divergence in immunotherapeutic and vaccine targets in the new genomes. Six complete DENV-1 genomes were generated from 50 samples that met a clinical definition for DENV infection. Phylogenetic analyses revealed that the strains from the current study belong to a sub-lineage of DENV-1 genotype V and form a monophyletic taxon with a 2012 strain from Gabon. The most recent common ancestor (TMRCA) of the Cameroon and Gabon strains was estimated to have existed around 2008. Comparing our sequences to the vaccine strains, 19 and 15 amino acid (aa) substitutions were observed in the immuno-protective prM-E protein segments of the Dengvaxia® and TetraVax-DV-TV003 vaccines, respectively. Epitope mapping revealed mismatches in aa residues at positions E155 and E161 located in the epitope of the human anti-DENV-1 monoclonal antibody HMAb 1F4. The new DENV strains constitute a conserved genomic pool of viruses endemic to the Central African region that needs prospective monitoring to track local viral evolution. Further work is needed to ascertain the performance of emerging therapeutics in DENV strains from the African region.

Whole genome analysis and antimicrobial resistance of Neisseria gonorrhoeae isolates from Ghana.

Introduction: Gonorrhoea is a major public health concern. With the global emergence and spread of resistance to last-line antibiotic treatment options, gonorrhoea threatens to be untreatable in the future. Therefore, this study performed whole genome characterization of Neisseria gonorrhoeae collected in Ghana to identify lineages of circulating strains as well as their phenotypic and genotypic antimicrobial resistance (AMR) profiles. Methods: Whole genome sequencing (WGS) was performed on 56 isolates using both the Oxford Nanopore MinION and Illumina MiSeq sequencing platforms. The Comprehensive Antimicrobial Resistance Database (CARD) and PUBMLST.org/neisseria databases were used to catalogue chromosomal and plasmid genes implicated in AMR. The core genome multi-locus sequence typing (cgMLST) approach was used for comparative genomics analysis. Results and Discussion: In vitro resistance measured by the E-test method revealed 100%, 91.0% and 85.7% resistance to tetracycline, penicillin and ciprofloxacin, respectively. A total of 22 sequence types (STs) were identified by multilocus sequence typing (MLST), with ST-14422 (n = 10), ST-1927 (n = 8) and ST-11210 (n = 7) being the most prevalent. Six novel STs were also identified (ST-15634, 15636-15639 and 15641). All isolates harboured chromosomal AMR determinants that confer resistance to beta-lactam antimicrobials and tetracycline. A single cefixime-resistant strain, that belongs to N. gonorrhoeae multiantigen sequence type (NG-MAST) ST1407, a type associated with widespread cephalosporin resistance was identified. Neisseria gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR), identified 29 unique sequence types, with ST-464 (n = 8) and the novel ST-3366 (n = 8) being the most prevalent. Notably, 20 of the 29 STs were novel, indicative of the unique nature of molecular AMR determinants in the Ghanaian strains. Plasmids were highly prevalent: pTetM and pblaTEM were found in 96% and 92% of isolates, respectively. The TEM-135 allele, which is an amino acid change away from producing a stable extended-spectrum ß-lactamase that could result in complete cephalosporin resistance, was identified in 28.5% of the isolates. Using WGS, we characterized N. gonorrhoeae strains from Ghana, giving a snapshot of the current state of gonococcal AMR in the country and highlighting the need for constant genomic surveillance.

Molecular characterization of haemagglutinin genes of influenza B viruses circulating in Ghana during 2016 and 2017.

Recent reports of haemagglutinin antigen (HA) mismatch between vaccine composition strains and circulating strains, have led to renewed interest in influenza B viruses. Additionally, there are concerns about resistance to neuraminidase inhibitors in new influenza B isolates. To assess the potential impact in Ghana, we characterized the lineages of influenza B viruses that circulated in Ghana between 2016 and 2017 from different regions of the country: Southern, Northern and Central Ghana. Eight representative specimens from the three regions that were positive for influenza B virus by real-time RT-PCR were sequenced and compared to reference genomes from each lineage. A total of eleven amino acids substitutions were detected in the B/Victoria lineage and six in the B/Yamagata lineage. The strains of influenza B viruses were closely related to influenza B/Brisbane/60/2008 and influenza B/Phuket/3073/2013 for the Victoria and Yamagata lineages, respectively. Three main amino acid substitutions (P31S, I117V and R151K) were found in B/Victoria lineages circulating between 2016 and 2017, while one strain of B/Victoria possessed a unique glycosylation site at amino acid position 51 in the HA2 subunit. Two main substitutions (L172Q and M251V) were detected in the HA gene of the B/Yamagata lineage. The U.S. CDC recently reported a deletion sub-group in influenza B virus, but this was not identified among the Ghanaian specimens. Close monitoring of the patterns of influenza B evolution is necessary for the efficient selection of representative viruses for the design and formulation of effective influenza vaccines.

Retrospective Genomic Characterization of a 2017 Dengue Virus Outbreak, Burkina Faso.

Knowledge of contemporary genetic composition of dengue virus (DENV) in Africa is lacking. By using next-generation sequencing of samples from the 2017 DENV outbreak in Burkina Faso, we isolated 29 DENV genomes (5 serotype 1, 16 serotype 2 [DENV-2], and 8 serotype 3). Phylogenetic analysis demonstrated the endemic nature of DENV-2 in Burkina Faso. We noted discordant diagnostic results, probably related to genetic divergence between these genomes and the Trioplex PCR. Forward and reverse1 primers had a single mismatch when mapped to the DENV-2 genomes, probably explaining the insensitivity of the molecular test. Although we observed considerable homogeneity between the Dengvaxia and TetraVax-DV-TV003 vaccine strains as well as B cell epitopes compared with these genomes, we noted unique divergence. Continual surveillance of dengue virus in Africa is needed to clarify the ongoing novel evolutionary dynamics of circulating virus populations and support the development of effective diagnostic, therapeutic, and preventive countermeasures.

Phylogenetic and genetic characterization of influenza A H9N2 viruses isolated from backyard poultry in selected farms in Ghana.

INTRODUCTION: Avian influenza viruses (AIV) cause significant economic losses to poultry farmers worldwide. These viruses have the ability to spread rapidly, infect entire poultry flocks, and can pose a threat to human health. The National Influenza Centre (NIC) at the Noguchi Memorial Institute for Medical Research in collaboration with the Ghana Armed forces (GAF) and the U.S. Naval Medical Research Unit No. 3, Ghana Detachment (NAMRU-3) performs biannual surveillance for influenza viruses among poultry at military barracks throughout Ghana. This study presents poultry surveillance data from the years 2017 to 2019. METHODOLOGY: Tracheal and cloacal swabs from sick and healthy poultry were collected from the backyards of GAF personnel living quarters and transported at 4°C to the NIC. Viral ribonucleic acid (RNA) was isolated and analyzed for the presence of influenza viruses using real-time polymerase chain reaction (PCR) assays. Viral nucleic acids extracted from influenza A-positive specimens were sequenced using universal influenza A-specific primers. RESULTS: Influenza A H9N2 virus was detected in 11 avian species out of 2000 samples tested. Phylogenetic analysis of viral haemagglutinin (HA) protein confirms the possibility of importation of viruses from North Africa and Burkina Faso. Although the detected viruses possess molecular markers of virulence and mammalian host adaptation, the HA cleavage site anlaysis confirmed low pathogenicity of the viruses. CONCLUSIONS: These findings confirm the ongoing spread of H9 viruses among poultry in Ghana. Poultry farmers need to be vigilant for sick birds and take the appropriate public health steps to limit the spread to other animals and spillover to humans.

Resurgence of Ebola virus in 2021 in Guinea suggests a new paradigm for outbreaks.

Seven years after the declaration of the first epidemic of Ebola virus disease in Guinea, the country faced a new outbreak-between 14 February and 19 June 2021-near the epicentre of the previous epidemic1,2. Here we use next-generation sequencing to generate complete or near-complete genomes of Zaire ebolavirus from samples obtained from 12 different patients. These genomes form a well-supported phylogenetic cluster with genomes from the previous outbreak, which indicates that the new outbreak was not the result of a new spillover event from an animal reservoir. The 2021 lineage shows considerably lower divergence than would be expected during sustained human-to-human transmission, which suggests a persistent infection with reduced replication or a period of latency. The resurgence of Zaire ebolavirus from humans five years after the end of the previous outbreak of Ebola virus disease reinforces the need for long-term medical and social care for patients who survive the disease, to reduce the risk of re-emergence and to prevent further stigmatization.

Chikungunya viruses containing the A226V mutation detected retrospectively in Cameroon form a new geographical subclade.

BACKGROUND: Chikungunya virus (CHIKV) is a re-emerging arbovirus associated with sporadic outbreaks in Cameroon since 2006. Viral whole genomes were generated to analyze the origins of evolutionary lineages, the potential of emergence/re-emergence, and to infer transmission dynamics of recent Cameroon CHIKV outbreak strains. METHODS: Samples collected between 2016 and 2019 during CHIKV outbreaks in Cameroon were screened for CHIKV using reverse transcription PCR (RT-PCR), followed by whole genome sequencing of positive samples. RESULTS: Three coding-complete CHIKV genomes were obtained from samples, which belong to an emerging sub-lineage of the East/Central/South African genotype and formed a monophyletic taxon with previous Central African strains. This clade, which we have named the new Central African clade, appears to be evolving at 3.0 × 10-4 nucleotide substitutions per site per year (95% highest posterior density (HPD) interval of 1.94 × 10-4 to 4.1 × 10-4). Notably, mutations in the envelope proteins (E1-A226V, E2-L210Q, and E2-I211T), which are known to enhance CHIKV adaptability and infectious potential in Aedes albopictus, were present in all strains and mapped to established high-density Ae. albopictus populations. CONCLUSIONS: These new CHIKV strains constitute a conserved genomic pool of an emerging sub-lineage, reflecting a putative vector host adaptation to Ae. albopictus, which has practically displaced Aedes aegypti from select regions of Cameroon.

Novel viruses in hard ticks collected in the Republic of Korea unveiled by metagenomic high-throughput sequencing analysis.

Ticks are vectors of a wide range of zoonotic viruses of medical and veterinary importance. Recently, metagenomics studies demonstrated that they are also the source of potentially pathogenic novel viruses. During the period from 2015 to 2017, questing ticks were collected by dragging the vegetation from geographically distant locations in the Republic of Korea (ROK) and a target-independent high-throughput sequencing method was utilized to study their virome. A total of seven viruses, including six putative novel viral entities, were identified. Genomic analysis showed that the novel viruses were most closely related to members in the orders Jingchuvirales and Bunyavirales. Phylogenetic reconstruction showed that the Bunyavirales-like viruses grouped in the same clade with other viruses within the Nairovirus and Phlebovirus genera, while the novel Jingchuvirales-like virus grouped together with other viruses within the family Chuviridae. Real-time RT-PCR was used to determine the geographic distribution and prevalence of these viruses in adult ticks. These novel viruses have a wide geographic distribution in the ROK with prevalences ranging from 2% to 18%. Our study expands the knowledge about the composition of the tick virome and highlights the wide diversity of viruses they harbor in the ROK. The discovery of novel viruses associated with ticks in the ROK highlights the need for an active tick-borne disease surveillance program to identify possible reservoirs of putative novel human pathogens.

Integration of genomic sequencing into the response to the Ebola virus outbreak in Nord Kivu, Democratic Republic of the Congo.

On 1 August 2018, the Democratic Republic of the Congo (DRC) declared its tenth Ebola virus disease (EVD) outbreak. To aid the epidemiologic response, the Institut National de Recherche Biomédicale (INRB) implemented an end-to-end genomic surveillance system, including sequencing, bioinformatic analysis and dissemination of genomic epidemiologic results to frontline public health workers. We report 744 new genomes sampled between 27 July 2018 and 27 April 2020 generated by this surveillance effort. Together with previously available sequence data (n = 48 genomes), these data represent almost 24% of all laboratory-confirmed Ebola virus (EBOV) infections in DRC in the period analyzed. We inferred spatiotemporal transmission dynamics from the genomic data as new sequences were generated, and disseminated the results to support epidemiologic response efforts. Here we provide an overview of how this genomic surveillance system functioned, present a full phylodynamic analysis of 792 Ebola genomes from the Nord Kivu outbreak and discuss how the genomic surveillance data informed response efforts and public health decision making.

Ebola Virus Transmission Initiated by Relapse of Systemic Ebola Virus Disease.

During the 2018-2020 Ebola virus disease (EVD) outbreak in North Kivu province in the Democratic Republic of Congo, EVD was diagnosed in a patient who had received the recombinant vesicular stomatitis virus-based vaccine expressing a ZEBOV glycoprotein (rVSV-ZEBOV) (Merck). His treatment included an Ebola virus (EBOV)-specific monoclonal antibody (mAb114), and he recovered within 14 days. However, 6 months later, he presented again with severe EVD-like illness and EBOV viremia, and he died. We initiated epidemiologic and genomic investigations that showed that the patient had had a relapse of acute EVD that led to a transmission chain resulting in 91 cases across six health zones over 4 months. (Funded by the Bill and Melinda Gates Foundation and others.).

Shedding of Marburg Virus in Naturally Infected Egyptian Rousette Bats, South Africa, 2017.

We detected Marburg virus RNA in rectal swab samples from Egyptian rousette bats in South Africa in 2017. This finding signifies that fecal contamination of natural bat habitats is a potential source of infection for humans. Identified genetic sequences are closely related to Ravn virus, implying wider distribution of Marburg virus in Africa.

Selective Phosphodiesterase 1 Inhibitor BTTQ Reduces Blood Pressure in Spontaneously Hypertensive and Dahl Salt Sensitive Rats: Role of Peripheral Vasodilation.

Regulation of the peripheral vascular resistance via modulating the vessel diameter has been considered as a main determinant of the arterial blood pressure. Phosphodiesterase enzymes (PDE1-11) hydrolyse cyclic nucleotides, which are key players controlling the vessel diameter and, thus, peripheral resistance. Here, we have tested and reported the effects of a novel selective PDE1 inhibitor (BTTQ) on the cardiovascular system. Normal Sprague Dawley, spontaneously hypertensive (SHR), and Dahl salt-sensitive rats were used to test in vivo the efficacy of the compound. Phosphodiesterase radiometric enzyme assay revealed that BTTQ inhibited all three isoforms of PDE1 in nanomolar concentration, while micromolar concentrations were needed to induce effective inhibition for other PDEs. The myography study conducted on mesenteric arteries revealed a potent vasodilatory effect of the drug, which was confirmed in vivo by an increase in the blood flow in the rat ear arteriols reflected by the rise in the temperature. Furthermore, BTTQ proved a high efficacy in lowering the blood pressure about 9, 36, and 24 mmHg in normal Sprague Dawley, SHR and, Dahl salt-sensitive rats, respectively, compared to the vehicle-treated group. Moreover, additional blood pressure lowering of about 22 mmHg could be achieved when BTTQ was administered on top of ACE inhibitor lisinopril, a current standard of care in the treatment of hypertension. Therefore, PDE1 inhibition induced efficient vasodilation that was accompanied by a significant reduction of blood pressure in different hypertensive rat models. Administration of BTTQ was also associated with increased heart rate in both models of hypertension as well as in the normotensive rats. Thus, PDE1 appears to be an attractive therapeutic target for the treatment of resistant hypertension, while tachycardia needs to be addressed by further compound structural optimization.

Modeling mosquito-borne and sexual transmission of Zika virus in an enzootic host, the African green monkey.

Mosquito-borne and sexual transmission of Zika virus (ZIKV), a TORCH pathogen, recently initiated a series of large epidemics throughout the Tropics. Animal models are necessary to determine transmission risk and study pathogenesis, as well screen antivirals and vaccine candidates. In this study, we modeled mosquito and sexual transmission of ZIKV in the African green monkey (AGM). Following subcutaneous, intravaginal or intrarectal inoculation of AGMs with ZIKV, we determined the transmission potential and infection dynamics of the virus. AGMs inoculated by all three transmission routes exhibited viremia and viral shedding followed by strong virus neutralizing antibody responses, in the absence of clinical illness. All four of the subcutaneously inoculated AGMs became infected (mean peak viremia: 2.9 log10 PFU/mL, mean duration: 4.3 days) and vRNA was detected in their oral swabs, with infectious virus being detected in a subset of these specimens. Although all four of the intravaginally inoculated AGMs developed virus neutralizing antibody responses, only three had detectable viremia (mean peak viremia: 4.0 log10 PFU/mL, mean duration: 3.0 days). These three AGMs also had vRNA and infectious virus detected in both oral and vaginal swabs. Two of the four intrarectally inoculated AGMs became infected (mean peak viremia: 3.8 log10 PFU/mL, mean duration: 3.5 days). vRNA was detected in oral swabs collected from both of these infected AGMs, and infectious virus was detected in an oral swab from one of these AGMs. Notably, vRNA and infectious virus were detected in vaginal swabs collected from the infected female AGM (peak viral load: 7.5 log10 copies/mL, peak titer: 3.8 log10 PFU/mL, range of detection: 5-21 days post infection). Abnormal clinical chemistry and hematology results were detected and acute lymphadenopathy was observed in some AGMs. Infection dynamics in all three AGM ZIKV models are similar to those reported in the majority of human ZIKV infections. Our results indicate that the AGM can be used as a surrogate to model mosquito or sexual ZIKV transmission and infection. Furthermore, our results suggest that AGMs are likely involved in the enzootic maintenance and amplification cycle of ZIKV.

Complete genomic sequences of Venezuelan equine encephalitis virus subtype IIID isolates from mosquitoes.

Venezuelan equine encephalitis virus (VEEV) is an important pathogen of medical and veterinary importance in the Americas. In this report, we present the complete genome sequences of five VEEV isolates obtained from pools of Culex (Melanoconion) gnomatos (4) or Culex (Melanoconion) pedroi (1) from Iquitos, Peru. Genetic and phylogenetic analyses showed that all five isolates grouped within the VEEV complex sister to VEEV IIIC and are members of subtype IIID. This is the first report of full-length genomic sequences of VEEV IIID.

Complete Coding Sequence of Western Equine Encephalitis Virus Strain Fleming, Isolated from a Human Case.

We sequenced the complete coding genome of the western equine encephalitis virus (WEEV) strain Fleming. This strain was originally isolated in 1938 from a human WEEV case.

Active Targeted Surveillance to Identify Sites of Emergence of Hantavirus.

BACKGROUND: Endemic outbreaks of hantaviruses pose a critical public health threat worldwide. Hantaan orthohantavirus (HTNV) causes hemorrhagic fever with renal syndrome (HFRS) in humans. Using comparative genomic analyses of partial and nearly complete sequences of HTNV from humans and rodents, we were able to localize, with limitations, the putative infection locations for HFRS patients. Partial sequences might not reflect precise phylogenetic positions over the whole-genome sequences; finer granularity of rodent sampling reflects more precisely the circulation of strains. METHODS: Five HFRS specimens were collected. Epidemiological surveys were conducted with the patients during hospitalization. We conducted active surveillance at suspected HFRS outbreak areas. We performed multiplex polymerase chain reaction-based next-generation sequencing to obtain the genomic sequence of HTNV from patients and rodents. The phylogeny of human- and rodent-derived HTNV was generated using the maximum likelihood method. For phylogeographic analyses, the tracing of HTNV genomes from HFRS patients was defined on the bases of epidemiological interviews, phylogenetic patterns of the viruses, and geographic locations of HTNV-positive rodents. RESULTS: The phylogeographic analyses demonstrated genetic clusters of HTNV strains from clinical specimens, with HTNV circulating in rodents at suspected sites of patient infections. CONCLUSIONS: This study demonstrates a major shift in molecular epidemiological surveillance of HTNV. Active targeted surveillance was performed at sites of suspected infections, allowing the high-resolution phylogeographic analysis to reveal the site of emergence of HTNV. We posit that this novel approach will make it possible to identify infectious sources, perform disease risk assessment, and implement preparedness against vector-borne viruses.

Comparison of targeted next-generation sequencing for whole-genome sequencing of Hantaan orthohantavirus in Apodemus agrarius lung tissues.

Orthohantaviruses, negative-sense single-strand tripartite RNA viruses, are a global public health threat. In humans, orthohantavirus infection causes hemorrhagic fever with renal syndrome or hantavirus cardiopulmonary syndrome. Whole-genome sequencing of the virus helps in identification and characterization of emerging or re-emerging viruses. Next-generation sequencing (NGS) is a potent method to sequence the viral genome, using molecular enrichment methods, from clinical specimens containing low virus titers. Hence, a comparative study on the target enrichment NGS methods is required for whole-genome sequencing of orthohantavirus in clinical samples. In this study, we used the sequence-independent, single-primer amplification, target capture, and amplicon NGS for whole-genome sequencing of Hantaan orthohantavirus (HTNV) from rodent specimens. We analyzed the coverage of the HTNV genome based on the viral RNA copy number, which is quantified by real-time quantitative PCR. Target capture and amplicon NGS demonstrated a high coverage rate of HTNV in Apodemus agrarius lung tissues containing up to 103-104 copies/µL of HTNV RNA. Furthermore, the amplicon NGS showed a 10-fold (102 copies/µL) higher sensitivity than the target capture NGS. This report provides useful insights into target enrichment NGS for whole-genome sequencing of orthohantaviruses without cultivating the viruses.