Long-term Sudan virus Ebola survivors maintain multiple antiviral defense mechanisms.

BACKGROUND: The critical issues of sustained memory immunity following ebolavirus disease among long-term survivors (EVD) are still unclear. METHODS: Here, we examine virus-specific immune and inflammatory responses in 12 Sudan virus (SUDV) long-term survivors from Uganda's 2000-1 Gulu outbreak, 15 years after recovery following in vitro challenge. Total RNA from isolated SUDV-stimulated and unstimulated PBMCs was extracted and analyzed. Matched serum samples were also collected to determine SUDV IgG levels and functionality. RESULTS: We detected persistent humoral (58%, 7 of 12) and cellular (33%, 4 of 12) immune responses in SUDV long-term survivors and identified critical molecular mechanisms of innate and adaptive immunity. Gene expression in immune pathways, the IFN signaling system, antiviral defense response, and activation and regulation of T- and B-cell responses were observed. SUDV long-term survivors also maintained robust virus-specific IgG antibodies capable of polyfunctional responses, including neutralizing and innate Fc effector functions. CONCLUSIONS: Data integration identified significant correlations among humoral and cellular immune responses and pinpointed a specific innate and adaptive gene expression signature associated with long-lasting immunity. This could help identify natural and vaccine correlates of protection against ebolavirus disease.

Confirmation of Zika virus infection through hospital-based sentinel surveillance of acute febrile illness in Uganda, 2014-2017.

Zika virus (ZIKV), transmitted by Aedes species mosquitoes, was first isolated in Uganda in 1947. From February 2014 to October 2017, the Uganda Virus Research Institute, in collaboration with the US Centers for Diseases Control and Prevention, conducted arbovirus surveillance in acute febrile illness (AFI) patients at St Francis hospital in Nkonkonjeru. Three hundred and eighty-four serum samples were collected and tested for IgM antibodies to yellow fever virus (YFV), West Nile virus (WNV), dengue virus (DENV), chikungunya virus (CHIKV) and ZIKV. Of the 384 samples, 5 were positive for ZIKV IgM. Of these five, three were confirmed by plaque reduction neutralization test (PRNT) to be ZIKV infections. Of the remaining two, one was determined to be a non-specific flavivirus infection and one was confirmed to be alphavirus-positive by reverse transcriptase polymerase chain reaction (RT-PCR). This study provides the first evidence of laboratory-confirmed ZIKV infection in Uganda in five decades, and emphasizes the need to enhance sentinel surveillance.

Profile and persistence of the virus-specific neutralizing humoral immune response in human survivors of Sudan ebolavirus (Gulu).

To better understand humoral immunity following ebolavirus infection, a serological study of the humoral immune response against the individual viral proteins of Sudan ebolavirus (Gulu) in human survivors was performed. An enzyme-linked immunosorbent assay specific for full-length recombinant viral proteins NP, VP30, VP40, and GP1-649 (GP lacking the transmembrane domain) of Sudan ebolavirus (Gulu) was used as well as a plaque reduction neutralization test. Serum samples from human survivors, which were collected up to 10 years following recovery, were screened and analyzed. Results demonstrate that samples obtained 10 years following infection contain virus-specific antibodies that can neutralize virus. Neutralization correlates well with immunoreactivity against the viral proteins NP, VP30, and GP1-649. Sera from individuals who died or those with no documented infection but immunoreactive to ebolavirus did not neutralize. This work provides insight into the duration, profile of immunoreactivity, and neutralization capacity of the humoral immune response in ebolavirus survivors.

Detection of two alphaviruses: Middelburg virus and Sindbis virus from enzootic amplification cycles in southwestern Uganda.

Our knowledge of alphavirus genetic diversity is mainly based on viruses isolated from anthropophilic mosquito species, humans, and livestock during outbreaks. Studies on alphaviruses from sylvatic amplification cycles in sub-Saharan Africa have been conducted less often than from epizootic environments. To gain insight into alphavirus diversity in enzootic transmission cycles, we collected over 23,000 mosquitoes in lowland rainforest and savannah gallery forest in southwestern Uganda and tested them for alphavirus infections. We detected Sindbis virus (SINV) in a Culex Culex sp. mosquito and Middelburg virus (MIDV) in Eretmapodites intermedius and Mansonia africana. MIDV is a mosquito-borne alphavirus that causes febrile illness in sheep, goats, and horses and was previously not known to occur in Uganda. SINV, also a mosquito-borne alphavirus, causes mild infections in humans. Full genomes of SINV and MIDV were sequenced, showing a nucleotide identity of 99% to related strains. Both isolates replicated to high titres in a wide variety of vertebrate cells. Our data suggest endemic circulation of SINV and MIDV in Uganda.

Influenza surveillance in 15 countries in Africa, 2006-2010.

BACKGROUND: In response to the potential threat of an influenza pandemic, several international institutions and governments, in partnership with African countries, invested in the development of epidemiologic and laboratory influenza surveillance capacity in Africa and the African Network of Influenza Surveillance and Epidemiology (ANISE) was formed. METHODS: We used a standardized form to collect information on influenza surveillance system characteristics, the number and percent of influenza-positive patients with influenza-like illness (ILI), or severe acute respiratory infection (SARI) and virologic data from countries participating in ANISE. RESULTS: Between 2006 and 2010, the number of ILI and SARI sites in 15 African countries increased from 21 to 127 and from 2 to 98, respectively. Children 0-4 years accounted for 48% of all ILI and SARI cases of which 22% and 10%, respectively, were positive for influenza. Influenza peaks were generally discernible in North and South Africa. Substantial cocirculation of influenza A and B occurred most years. CONCLUSIONS: Influenza is a major cause of respiratory illness in Africa, especially in children. Further strengthening influenza surveillance, along with conducting special studies on influenza burden, cost of illness, and role of other respiratory pathogens will help detect novel influenza viruses and inform and develop targeted influenza prevention policy decisions in the region.

Whole-genome analysis to determine the rate and patterns of intra-subtype reassortment among influenza type-A viruses in Africa.

Influenza type-A viruses (IAVs) present a global burden of human respiratory infections and mortality. Genome reassortment is an important mechanism through which epidemiologically novel influenza viruses emerge and a core step in the safe reassortment-incompetent live-attenuated influenza vaccine development. Currently, there are no data on the rate, spatial and temporal distribution, and role of reassortment in the evolution and diversification of IAVs circulating in Africa. We aimed to detect intra-subtype reassortment among Africa pandemic H1N1pdm09 (2009-10), seasonal H1N1pdm09 (2011-20), and seasonal H3N2 viruses and characterize the genomic architecture and temporal and spatial distribution patterns of the resulting reassortants. Our study was nested within the Uganda National Influenza Surveillance Programme. Next-generation sequencing was used to generate whole genomes (WGs) from 234 H1N1pdm09 (n = 116) and H3N2 (n = 118) viruses sampled between 2010 and 2018 from seven districts in Uganda. We combined our newly generated WGs with 658 H1N1pdm09 and 1131 H3N2 WGs sampled between 1994 and 2020 across Africa and identified reassortants using an automated Graph Incompatibility Based Reassortment Finder software. Viral reassortment rates were estimated using a coalescent reassortant constant population model. Phylogenetic analysis was used to assess the effect of reassortment on viral genetic evolution. We observed a high frequency of intra-subtype reassortment events, 12 · 4 per cent (94/758) and 20 · 9 per cent (256/1,224), and reassortants, 13 · 3 per cent (101/758) and 38 · 6 per cent (472/1,224), among Africa H1N1pdm09 and H3N2 viruses, respectively. H1N1pdm09 reassorted at higher rates (0.1237-0.4255) than H3N2 viruses (0 · 00912-0.0355 events/lineage/year), a case unique to Uganda. Viral reassortants were sampled in 2009 through 2020, except in 2012. 78 · 2 per cent (79/101) of H1N1pdm09 reassortants acquired new non-structural, while 57 · 8 per cent (273/472) of the H3N2 reassortants had new hemagglutinin (H3) genes. Africa H3N2 viruses underwent more reassortment events involving larger reassortant sets than H1N1pdm09 viruses. Viruses with a specific reassortment architecture circulated for up to five consecutive years in specific countries and regions. The Eastern (Uganda and Kenya) and Western Africa harboured 84 · 2 per cent (85/101) and 55 · 9 per cent (264/472) of the continent's H1N1pdm09 and H3N2 reassortants, respectively. The frequent reassortment involving multi-genes observed among Africa IAVs showed the intracontinental viral evolution and diversification possibly sustained by viral importation from outside Africa and/or local viral genomic mixing and transmission. Novel reassortant viruses emerged every year, and some persisted in different countries and regions, thereby presenting a risk of influenza outbreaks in Africa. Our findings highlight Africa as part of the global influenza ecology and the advantage of implementing routine whole-over partial genome sequencing and analyses to monitor circulating and detect emerging viruses. Furthermore, this study provides evidence and heightens our knowledge on IAV evolution, which is integral in directing vaccine strain selection and the update of master donor viruses used in recombinant vaccine development.

Retrospective in silico mutation profiling of SARS-CoV-2 structural proteins circulating in Uganda by July 2021: Towards refinement of COVID-19 disease vaccines, diagnostics, and therapeutics.

The SARS-CoV-2 virus, the agent of COVID-19, caused unprecedented loss of lives and economic decline worldwide. Although the introduction of public health measures, vaccines, diagnostics, and therapeutics disrupted the spread of the SARS-CoV-2, the emergence of variants poses substantial threat. This study traced SARS-CoV-2 variants circulating in Uganda by July 2021 to inform the necessity for refinement of the intervention medical products. A comprehensive in silico analysis of the SARS-CoV-2 genomes detected in clinical samples collected from COVID-19 patients in Uganda revealed occurrence of structural protein variants with potential of escaping detection, resisting antibody therapy, or increased infectivity. The genome sequence dataset was retrieved from the GISAID database and the open reading frame encoding the spike, envelope, membrane, or nucleocapsid proteins was translated. The obtained protein sequences were aligned and inspected for existence of variants. The variant positions on each of the four alignment sets were mapped on predicted epitopes as well as the 3D structures. Additionally, sequences within each of the sets were clustered by family. A phylogenetic tree was constructed to assess relationship between the encountered spike protein sequences and Wuhan-Hu-1 wild-type, or the Alpha, Beta, Delta and Gamma variants of concern. Strikingly, the frequency of each of the spike protein point mutations F157L/Del, D614G and P681H/R was over 50%. The furin and the transmembrane serine protease 2 cleavage sites were unaffected by mutation. Whereas the Delta dominated the spike sequences (16.5%, 91/550), Gamma was not detected. The envelope protein was the most conserved with 96.3% (525/545) sequences being wild-type followed by membrane at 68.4% (397/580). Although the nucleocapsid protein sequences varied, the variant residue positions were less concentrated at the RNA binding domains. The dominant nucleocapsid sequence variant was S202N (34.5%, 205/595). These findings offer baseline information required for refining the existing COVID-19 vaccines, diagnostics, and therapeutics.

Sporadic outbreaks of crimean-congo haemorrhagic fever in Uganda, July 2018-January 2019.

INTRODUCTION: Crimean-Congo haemorrhagic fever (CCHF) is a tick-borne, zoonotic viral disease that causes haemorrhagic symptoms. Despite having eight confirmed outbreaks between 2013 and 2017, all within Uganda's 'cattle corridor', no targeted tick control programs exist in Uganda to prevent disease. During a seven-month-period from July 2018-January 2019, the Ministry of Health confirmed multiple independent CCHF outbreaks. We investigated to identify risk factors and recommend interventions to prevent future outbreaks. METHODS: We defined a confirmed case as sudden onset of fever (≥37.5°C) with ≥4 of the following signs and symptoms: anorexia, vomiting, diarrhoea, headache, abdominal pain, joint pain, or sudden unexplained bleeding in a resident of the affected districts who tested positive for Crimean-Congo haemorrhagic fever virus (CCHFv) by RT-PCR from 1 July 2018-30 January 2019. We reviewed medical records and performed active case-finding. We conducted a case-control study and compared exposures of case-patients with age-, sex-, and sub-county-matched control-persons (1:4). RESULTS: We identified 14 confirmed cases (64% males) with five deaths (case-fatality rate: 36%) from 11 districts in western and central region. Of these, eight (73%) case-patients resided in Uganda's 'cattle corridor'. One outbreak involved two case-patients and the remainder involved one. All case-patients had fever and 93% had unexplained bleeding. Case-patients were aged 6-36 years, with persons aged 20-44 years more affected (AR: 7.2/1,000,000) than persons ≤19 years (2.0/1,000,000), p = 0.015. Most (93%) case-patients had contact with livestock ≤2 weeks before symptom onset. Twelve (86%) lived <1 km from grazing fields compared with 27 (48%) controls (ORM-H = 18, 95% CI = 3.2-∞) and 10 (71%) of 14 case-patients found ticks attached to their bodies ≤2 weeks before symptom onset, compared to 15 (27%) of 56 control-persons (ORM-H = 9.3, 95%CI = 1.9-46). CONCLUSIONS: CCHF outbreaks occurred sporadically during 2018-2019, both within and outside 'cattle corridor' districts of Uganda. Most cases were associated with tick exposure. The Ministry of Health should partner with the Ministry of Agriculture, Animal Industry and Fisheries to develop joint nationwide tick control programs and strategies with shared responsibilities through a One Health approach.

High insecticide resistances levels in Anopheles gambiaes s.l. in northern Uganda and its relevance for future malaria control.

OBJECTIVE: The aim of the study was to determine the level of insecticide resistance and diversity in Anopheles mosquitoes in northern Uganda. Standard WHO insecticide susceptibility test assays were used to test for susceptibility to 0.5% malathion, 0.1% bendiocarb, 0.05% deltamethrin and 0.75% permethrin on 3-5 day old generation one progeny. We also screened for species diversity and knockdown resistance using PCR assay. RESULTS: Anopheles gambiae s.s. is the predominant malaria vector in northern Uganda followed by An. arabiensis. An. gambiae s.s. was susceptible to malathion and bendiocarb with the observed mortality rate of 100% and 98-100% observed respectively while very high resistance was observed with deltamethrin and permethrin. Minimal KDR-eastern variant homozygous forms of 8.3% in An. gambiae s.s. were detected in Oyam district. In conclusion, this study confirms that An. gambiae s.s. females are susceptible to malathion and bendiocarb while high intensity of resistance was observed with deltamethrin and permethrin in the same area. Use of carbamate and organophosphate insecticides bendiocarb and malathion for indoor residual spraying activities in northern Uganda is highly recommended since high levels of pyrethroids resistance (deltamethrin and permethrin) was detected in the area.

Multiple viral proteins and immune response pathways act to generate robust long-term immunity in Sudan virus survivors.

BACKGROUND: Profiles of immunity developed in filovirus patients and survivors have begun to shed light on antigen-specific cellular immune responses that had been previously under-studied. However, our knowledge of the breadth and length of those responses and the viral targets which mediate long-term memory immunity still lags significantly behind. METHODS: We characterized antigen-specific immune responses in whole blood samples of fifteen years post-infected survivors of the Sudan virus (SUDV) outbreak in Gulu, Uganda (2000-2001). We examined T cell and IgG responses against SUDV complete antigen and four SUDV proteins; glycoprotein (GP), nucleoprotein (NP), and viral protein 30 (VP30), and 40 (VP40). FINDINGS: We found survivors-maintained antigen-specific CD4+ T cell memory immune responses mediated mainly by the viral protein NP. In contrast, activated CD8+ T cell responses were nearly absent in SUDV survivors, regardless of the stimulating antigen used. Analysis of anti-viral humoral immunity revealed antigen-specific IgG antibodies against SUDV and SUDV proteins. Survivor IgGs mediated live SUDV neutralization in vitro and FcγRI and FcγRIII antibody Fc-dependent responses, mainly via antibodies to the viral proteins GP and VP40. INTERPRETATION: We highlight the key role of several proteins, i.e., GP, NP, and VP40, to act as mediators of distinctive and sustained cellular memory immune responses in long-term SUDV survivors. We suggest that the inclusion of these viral proteins in vaccine development may best mimic survivor native memory immune responses with the potential of protecting against viral infection. FUNDS: This research was funded by the Defense Threat Reduction Agency (CB4088) and by the National Institute Of Allergy And Infectious Diseases of the National Institutes of Health under Award Number R01AI111516. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

The Development and Validation of a Novel Nanobody-Based Competitive ELISA for the Detection of Foot and Mouth Disease 3ABC Antibodies in Cattle.

Effective management of foot and mouth disease (FMD) requires diagnostic tests to distinguish between infected and vaccinated animals (DIVA). To address this need, several enzyme-linked immunosorbent assay (ELISA) platforms have been developed, however, these tests vary in their sensitivity and specificity and are very expensive for developing countries. Camelid-derived single-domain antibodies fragments so-called Nanobodies, have demonstrated great efficacy for the development of serological diagnostics. This study describes the development of a novel Nanobody-based FMD 3ABC competitive ELISA, for the serological detection of antibodies against FMD Non-Structural Proteins (NSP) in Uganda cattle herds. This in-house ELISA was validated using more than 600 sera from different Uganda districts, and virus serotype specificities. The evaluation of the performance of the assay demonstrated high diagnostic sensitivity and specificity of 94 % (95 % CI: 88.9-97.2), and 97.67 % (95 % CI: 94.15-99.36) respectively, as well as the capability to detect NSP-specific antibodies against multiple FMD serotype infections. In comparison with the commercial PrioCHECK FMDV NSP-FMD test, there was a strong concordance and high correlation and agreement in the performance of the two tests. This new developed Nanobody based FMD 3ABC competitive ELISA could clearly benefit routine disease diagnosis, the establishment of disease-free zones, and the improvement of FMD management and control in endemically complex environments, such as those found in Africa.

Marburg virus survivor immune responses are Th1 skewed with limited neutralizing antibody responses.

Until recently, immune responses in filovirus survivors remained poorly understood. Early studies revealed IgM and IgG responses to infection with various filoviruses, but recent outbreaks have greatly expanded our understanding of filovirus immune responses. Immune responses in survivors of Ebola virus (EBOV) and Sudan virus (SUDV) infections have provided the most insight, with T cell responses as well as detailed antibody responses having been characterized. Immune responses to Marburg virus (MARV), however, remain almost entirely uncharacterized. We report that immune responses in MARV survivors share characteristics with EBOV and SUDV infections but have some distinct differences. MARV survivors developed multivariate CD4+ T cell responses but limited CD8+ T cell responses, more in keeping with SUDV survivors than EBOV survivors. In stark contrast to SUDV survivors, rare neutralizing antibody responses in MARV survivors diminished rapidly after the outbreak. These results warrant serious consideration for any vaccine or therapeutic that seeks to be broadly protective, as different filoviruses may require different immune responses to achieve immunity.

Human Survivors of Disease Outbreaks Caused by Ebola or Marburg Virus Exhibit Cross-Reactive and Long-Lived Antibody Responses.

A detailed understanding of serological immune responses to Ebola and Marburg virus infections will facilitate the development of effective diagnostic methods, therapeutics, and vaccines. We examined antibodies from Ebola or Marburg survivors 1 to 14 years after recovery from disease, by using a microarray that displayed recombinant nucleoprotein (NP), viral protein 40 (VP40), envelope glycoprotein (GP), and inactivated whole virions from six species of filoviruses. All three outbreak cohorts exhibited significant antibody responses to antigens from the original infecting species and a pattern of additional filoviruses that varied by outbreak. NP was the most cross-reactive antigen, while GP was the most specific. Antibodies from survivors of infections by Marburg marburgvirus (MARV) species were least cross-reactive, while those from survivors of infections by Sudan virus (SUDV) species exhibited the highest cross-reactivity. Based on results revealed by the protein microarray, persistent levels of antibodies to GP, NP, and VP40 were maintained for up to 14 years after infection, and survival of infection caused by one species imparted cross-reactive antibody responses to other filoviruses.

Correspondence of Neutralizing Humoral Immunity and CD4 T Cell Responses in Long Recovered Sudan Virus Survivors.

Robust humoral and cellular immunity are critical for survival in humans during an ebolavirus infection. However, the interplay between these two arms of immunity is poorly understood. To address this, we examined residual immune responses in survivors of the Sudan virus (SUDV) outbreak in Gulu, Uganda (2000-2001). Cytokine and chemokine expression levels in SUDV stimulated whole blood cultures were assessed by multiplex ELISA and flow cytometry. Antibody and corresponding neutralization titers were also determined. Flow cytometry and multiplex ELISA results demonstrated significantly higher levels of cytokine and chemokine responses in survivors with serological neutralizing activity. This correspondence was not detected in survivors with serum reactivity to SUDV but without neutralization activity. This previously undefined relationship between memory CD4 T cell responses and serological neutralizing capacity in SUDV survivors is key for understanding long lasting immunity in survivors of filovirus infections.

Immune memory to Sudan virus: comparison between two separate disease outbreaks.

Recovery from ebolavirus infection in humans is associated with the development of both cell-mediated and humoral immune responses. According to recent studies, individuals that did not survive infection with ebolaviruses appear to have lacked a robust adaptive immune response and the expression of several early innate response markers. However, a comprehensive protective immune profile has yet to be described. Here, we examine cellular memory immune responses among survivors of two separate Ebolavirus outbreaks (EVDs) due to Sudan virus (SUDV) infection in Uganda-Gulu 2000-2001 and Kibaale 2012. Freshly collected blood samples were stimulated with inactivated SUDV, as well as with recombinant SUDV or Ebola virus (EBOV) GP (GP1-649). In addition, ELISA and plaque reduction neutralization assays were performed to determine anti-SUDV IgG titers and neutralization capacity. Cytokine expression was measured in whole blood cultures in response to SUDV and SUDV GP stimulation in both survivor pools, demonstrating recall responses that indicate immune memory. Cytokine responses between groups were similar but had distinct differences. Neutralizing, SUDV-specific IgG activity against irradiated SUDV and SUDV recombinant proteins were detected in both survivor cohorts. Furthermore, humoral and cell-mediated crossreactivity to EBOV and EBOV recombinant GP1-649 was observed in both cohorts. In conclusion, immune responses in both groups of survivors demonstrate persistent recognition of relevant antigens, albeit larger cohorts are required in order to reach greater statistical significance. The differing cytokine responses between Gulu and Kibaale outbreak survivors suggests that each outbreak may not yield identical memory responses and promotes the merits of studying the immune responses among outbreaks of the same virus. Finally, our demonstration of cross-reactive immune recognition suggests that there is potential for developing cross-protective vaccines for ebolaviruses.

Profiling the native specific human humoral immune response to Sudan Ebola virus strain Gulu by chemiluminescence enzyme-linked immunosorbent assay.

Ebolavirus, a member of the family Filoviridae, causes high lethality in humans and nonhuman primates. Research focused on protection and therapy for Ebola virus infection has investigated the potential role of antibodies. Recent evidence suggests that antibodies can be effective in protection from lethal challenge with Ebola virus in nonhuman primates. However, despite these encouraging results, studies have not yet determined the optimal antibodies and composition of an antibody cocktail, if required, which might serve as a highly effective and efficient prophylactic. To better understand optimal antibodies and their targets, which might be important for protection from Ebola virus infection, we sought to determine the profile of viral protein-specific antibodies generated during a natural cycle of infection in humans. To this end, we characterized the profile of antibodies against individual viral proteins of Sudan Ebola virus (Gulu) in human survivors and nonsurvivors of the outbreak in Gulu, Uganda, in 2000-2001. We developed a unique chemiluminescence enzyme-linked immunosorbent assay (ELISA) for this purpose based on the full-length recombinant viral proteins NP, VP30, and VP40 and two recombinant forms of the viral glycoprotein (GP(1-294) and GP(1-649)) of Sudan Ebola virus (Gulu). Screening results revealed that the greatest immunoreactivity was directed to the viral proteins NP and GP(1-649), followed by VP40. Comparison of positive immunoreactivity between the viral proteins NP, GP(1-649), and VP40 demonstrated a high correlation of immunoreactivity between these viral proteins, which is also linked with survival. Overall, our studies of the profile of immunorecognition of antibodies against four viral proteins of Sudan Ebola virus in human survivors may facilitate development of effective monoclonal antibody cocktails in the future.