Using Multiplex Amplicon PCR Technology to Efficiently and Timely Generate Rift Valley Fever Virus Sequence Data for Genomic Surveillance.

Rift Valley fever (RVF) is a febrile vector-borne disease endemic in Africa and continues to spread in new territories. It is a climate-sensitive disease mostly triggered by abnormal rainfall patterns. The disease is associated with high mortality and morbidity in both humans and livestock. RVF is caused by the Rift Valley fever virus (RVFV) of the genus Phlebovirus in the family Phenuiviridae. It is a tripartite RNA virus with three genomic segments: small (S), medium (M) and large (L). Pathogen genomic sequencing is becoming a routine procedure and a powerful tool for understanding the evolutionary dynamics of infectious organisms, including viruses. Inspired by the utility of amplicon-based sequencing demonstrated in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and Ebola, Zika and West Nile viruses, we report an RVFV sample preparation based on amplicon multiplex polymerase chain reaction (amPCR) for template enrichment and reduction of background host contamination. The technology can be implemented rapidly to characterize and genotype RVFV during outbreaks in a near-real-time manner. To achieve this, we designed 74 multiplex primer sets covering the entire RVFV genome to specifically amplify the nucleic acid of RVFV in clinical samples from an animal tissue. Using this approach, we demonstrate achieving complete RVFV genome coverage even from samples containing a relatively low viral load. We report the first primer scheme approach of generating multiplex primer sets for a tripartite virus which can be replicated for other segmented viruses.

Congenital Rift Valley fever in Sprague Dawley rats is associated with diffuse infection and pathology of the placenta.

Rift Valley fever (RVF) is a disease of animals and humans associated with abortions in ruminants and late-gestation miscarriages in women. Here, we use a rat model of congenital RVF to identify tropisms, pathologies, and immune responses in the placenta during vertical transmission. Infection of late-gestation pregnant rats resulted in vertical transmission to the placenta and widespread infection throughout the decidua, basal zone, and labyrinth zone. Some pups from infected dams appeared normal while others had gross signs of teratogenicity including death. Histopathological lesions were detected in placenta from pups regardless of teratogenicity, while teratogenic pups had widespread hemorrhage throughout multiple placenta layers. Teratogenic events were associated with significant increases in placental pro-inflammatory cytokines, type I interferons, and chemokines. RVFV displays a high degree of tropism for all placental tissue layers and the degree of hemorrhage and inflammatory mediator production is highest in placenta from pups with adverse outcomes. Given the potential for RVFV to emerge in new locations and the recent evidence of emerging viruses, like Zika and SARS-CoV-2, to undergo vertical transmission, this study provides essential understanding regarding the mechanisms by which RVFV crosses the placenta barrier.

Rift Valley Fever Outbreak during COVID-19 Surge, Uganda, 2021.

Rift Valley fever, endemic or emerging throughout most of Africa, causes considerable risk to human and animal health. We report 7 confirmed Rift Valley fever cases, 1 fatal, in Kiruhura District, Uganda, during 2021. Our findings highlight the importance of continued viral hemorrhagic fever surveillance, despite challenges associated with the COVID-19 pandemic.

Vector and Host C-Type Lectin Receptor (CLR)-Fc Fusion Proteins as a Cross-Species Comparative Approach to Screen for CLR-Rift Valley Fever Virus Interactions.

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus endemic to Africa and the Arabian Peninsula, which causes diseases in humans and livestock. C-type lectin receptors (CLRs) represent a superfamily of pattern recognition receptors that were reported to interact with diverse viruses and contribute to antiviral immune responses but may also act as attachment factors or entry receptors in diverse species. Human DC-SIGN and L-SIGN are known to interact with RVFV and to facilitate viral host cell entry, but the roles of further host and vector CLRs are still unknown. In this study, we present a CLR-Fc fusion protein library to screen RVFV-CLR interaction in a cross-species approach and identified novel murine, ovine, and Aedes aegypti RVFV candidate receptors. Furthermore, cross-species CLR binding studies enabled observations of the differences and similarities in binding preferences of RVFV between mammalian CLR homologues, as well as more distant vector/host CLRs.

Low Density Lipoprotein Receptor-Related Protein 1 (LRP1) is a host factor for RNA viruses including SARS-CoV-2 (preprint)

Viruses with an RNA genome are the main causes of zoonotic infections. In order to identify novel pro-viral host cell factors, we screened a haploid insertion-mutagenized mouse embryonic cell library for clones that rendered them resistant to the zoonotic Rift Valley fever virus (RVFV; family Phleboviridae, order Bunyavirales). This screen returned the Low Density Lipoprotein Receptor-Related protein 1 (LRP1, or CD91) as top hit, a 600 kDa plasma membrane protein known to be involved in a wide variety of cell activities. Inactivation of LRP1 expression in human cells reduced RVFV infection at the early stages of infection, including the particle attachment to the cell. In the highly LRP1-positive human HuH-7 cell line, LRP1 was required for the early infection stages also of Sandfly fever Sicilian virus (SFSV; family Phleboviridae, order Bunyavirales), vesicular stomatitis (VSV; family Rhabdoviridae, order Mononegavirales), Encephalomyocarditis virus (EMCV, family Picornaviridae), and the coronaviruses MERS-CoV, SARS-CoV-1, and SARS-CoV-2. While for RVFV, EMCV, and MERS-CoV the replication cycle could eventually catch up, LRP1 requirement for the late infection stage in HuH-7 cells was observed for SFSV, La Crosse virus (LACV; family Peribunyaviridae, order Bunyavirales), VSV, SARS-CoV-1, and SARS-CoV-2. For SARS-CoV-2, the absence of LRP1 stably reduced viral RNA levels in human lung Calu-3 cells, and both RNA levels and particle production in the hepatic HuH-7 cells. Thus, we identified LRP1 as a host factor that supports various infection cycle stages of a broad spectrum of RNA viruses.

Sensitivity Analysis in an Immuno-Epidemiological Vector-Host Model.

Sensitivity Analysis (SA) is a useful tool to measure the impact of changes in model parameters on the infection dynamics, particularly to quantify the expected efficacy of disease control strategies. SA has only been applied to epidemic models at the population level, ignoring the effect of within-host virus-with-immune-system interactions on the disease spread. Connecting the scales from individual to population can help inform drug and vaccine development. Thus the value of understanding the impact of immunological parameters on epidemiological quantities. Here we consider an age-since-infection structured vector-host model, in which epidemiological parameters are formulated as functions of within-host virus and antibody densities, governed by an ODE system. We then use SA for these immuno-epidemiological models to investigate the impact of immunological parameters on population-level disease dynamics such as basic reproduction number, final size of the epidemic or the infectiousness at different phases of an outbreak. As a case study, we consider Rift Valley Fever Disease utilizing parameter estimations from prior studies. SA indicates that [Formula: see text] increase in within-host pathogen growth rate can lead up to [Formula: see text] increase in [Formula: see text] up to [Formula: see text] increase in steady-state infected host abundance, and up to [Formula: see text] increase in infectiousness of hosts when the reproduction number [Formula: see text] is larger than one. These significant increases in population-scale disease quantities suggest that control strategies that reduce the within-host pathogen growth can be important in reducing disease prevalence.

Seropositivity rates of zoonotic pathogens in small ruminants and associated public health risks at informal urban markets in Zambia.

Informal livestock markets are an important source of animal-derived proteins for growing urban populations in countries such as Zambia. In parallel, they can also constitute pathways of zoonotic pathogen transmission to humans. This risk is aggravated by limited disease monitoring and poor control systems with regards to biosecurity and public health. The aim of this study was to investigate the risks for spread of zoonotic diseases in Zambia's two largest informal small ruminant markets, located in Lusaka and Kasumbalesa, through combining seroepidemiology with interviews and observations. In April, May and September 2018, serum samples (n = 237) were collected and analysed for antibodies for the zoonotic pathogens Brucella spp., Coxiella (C.) burnetii and Rift Valley fever virus (RVFV), using commercially available enzyme linked immunosorbent assays (ELISA). In addition, slaughterhouse activities were observed and semi-structured interviews and focus group discussions held with slaughterhouse workers and small ruminant traders, focusing on the handling of animals and meat, and the perceptions of zoonotic disease risks at slaughter and consumption. The study found seropositivity rates of 10.1% (95% confidence interval [CI] 6.60-14.7) for Brucella spp., 5.9% (95% CI 3.27-9.71) for C. burnetii, and 0.8% (95% CI 0.10-3.01) for RVFV. Interviews with value chain members and observations at the slaughterhouse revealed unsanitary procedures and multiple occupational hazards for slaughterhouse workers. This study showed that the Zambian informal small ruminant trade system poses risks to public health, and that these risks are exacerbated by a lack of information about food-borne diseases and how associated risks can be mitigated amongst value chain actors. The results of this study can be used to formulate preventive measures to improve informal meat markets and reduce the risks to public health.

Cross-Reactivity of SARS-CoV-2 Laboratory Diagnostics to Endemic Diseases in Africa: A Diagnostic Accuracy Study (preprint)

Background: Serology is a great tool to assess the level of immunity against SARS-CoV-2 in settings with limited access to molecular diagnostics. However, African populations displays a particular immunological profile with massive circulation of infectious agents from different aetiologies that can affect assays performance.Methods: We evaluated the OMEGA Diagnostics COVID-19 ELISA-IgG and the ID Screen® SARS-CoV-2-N IgG Indirect in Senegal using a panel of 636 blood samples covering several African-endemic diseases and healthy donors to determine test sensitivity and specificity. The sensitivity panel of sera includes 461 serum samples collected from 91 patients hospitalized for COVID-19 disease. COVID-19 cases were confirmed by qRT-PCR and samples were collected on an interval of three days until viral clearance. In addition, 272 sera obtained from COVID-19 negative individuals were selected from a well-documented biobank of sera collected before the COVID-19 outbreak.Finding: High-cross reactivity have been found in individuals with a history of exposure to Chikungunya, HIV, malaria (Plasmodium falciparum), rheumatoid factor as well as healthy donors with respective specificities of 55%, 41.8%, 70%, 70% and 75%. ELISA experiments with commercial assays targeting either SARS-CoV-2 Nucleocapsid protein and Spike 2 protein or nucleocapsid protein only suggest that cross-reactivity might be directed against Spike 2 protein and not Nucleocapsid protein. Further samples characterisation reveals that anti-malaria IgG is the leading cause of such poor specificities, but exposure to other diseases contributed as well.Interpretation: We anticipate that COVID-19 seroprevalence can be biased if assays are not contextualized. Since malaria is endemic in African settings, we propose that a particular attention must be given in serological surveillance of COVID-19 or anti-SARS-CoV-2 antibodies quantification as vaccines are being rolled out.FundingUK Foreign, Commonwealth and Development Office/Wellcome Trust Joint Initiative for Research in Epidemic Preparedness and Response (JIREP grant number 220764/Z/20/Z).Funding Information: UK Foreign, Commonwealth and Development Office/Wellcome Trust Joint Initiative for Research in Epidemic Preparedness and Response (JIREP grant number 220764/Z/20/Z).Declaration of Interests: JRAF was an employee of Mologic Ltd, which was the development partner of one of the ELISAs adopted in this study. The remaining authors declare no competing interest.Ethics Approval Statement: Pre-COVID-19 samples for malaria (PCR), dengue, yellow fever, Zika, Chikungunya, Influenza A/B, HIV, rheumatoid arthritis and samples tested negative for the same diseases and also Crimean Congo haemorrhagic fever, West Nile fever encephalitis and Rift Valley fever were part of national public health surveillance program of the Senegalese Ministry of Social Action and Health performed in collaboration with Institut Pasteur de Dakar. Therefore, consultation with ethics committee was not required. Pre-COVID-19 samples for malaria endemic areas were from a longitudinal cohort survey performed in Dielmo village and approved by the Senegalese National Ethics Committee for Research in Health (reference number 00000007/MSAS/CNERS/Sec 26 January 2021). Samples from COVID-19 RT-PCR positive patients were obtained from a multicentre cohort survey approved by the Senegalese National Ethics Committee for Research in Health (reference number 00000068/MSAS/CNERS/Sec, 10 April 2020).

Safety of COVID-19 vaccines, their components or their platforms for pregnant women: A rapid review (preprint)

ABSTRACT Background Pregnant women with COVID-19 are at an increased risk of severe COVID-19 illness as well as adverse pregnancy and birth outcomes. Many countries are vaccinating or considering vaccinating pregnant women with limited available data about the safety of this strategy. Early identification of safety concerns of COVID-19 vaccines, including their components, or their technological platforms is therefore urgently needed. Methods We conducted a rapid systematic review, as the first phase of an ongoing full systematic review, to evaluate the safety of COVID-19 vaccines in pregnant women, including their components, and their technological platforms (whole virus, protein, viral vector or nucleic acid) used in other vaccines, following the Cochrane methods and the PRISMA statement for reporting (PROSPERO-CRD42021234185). We searched literature databases, COVID-19 and pregnancy registries from inception February 2021 without time or language restriction and explored the reference lists of relevant systematic reviews retrieved. We selected studies of any methodological design that included at least 50 pregnant women or pregnant animals exposed to the vaccines that were selected for review by the COVAX MIWG in August 2020 or their components or platforms included in the COVID-19 vaccines, and evaluated adverse events during pregnancy and the neonatal period. Pairs of reviewers independently selected studies through the COVIDENCE web software and performed the data extraction through a previously piloted online extraction form. Discrepancies were resolved by consensus. Results We identified 6768 records, 256 potentially eligible studies were assessed by full-text, and 37 clinical and non-clinical studies (38 reports, involving 2,397,715 pregnant women and 56 pregnant animals) and 12 pregnancy registries were included. Most studies (89%) were conducted in high-income countries. The most frequent study design was cohort studies (n=21), followed by surveillance studies, randomized controlled trials, and registry analyses. Most studies (76%) allowed comparisons between vaccinated and unvaccinated pregnant women (n=25) or animals (n=3) and reported exposures during the three trimesters of pregnancy. The most frequent exposure was to AS03 adjuvant in the context of A/H1N1 pandemic influenza vaccines (n=24), followed by aluminum-based adjuvants (n=11). Aluminum phosphate was used in Respiratory Syncytial Virus Fusion candidate vaccines (n=3) and Tdap vaccines (n=3). Different aluminum-based adjuvants were used in hepatitis vaccines. The replication-deficient simian adenovirus ChAdOx1 was used for a Rift Valley fever vaccine. Only one study reported exposure to messenger RNA (mRNA) COVID-19 vaccines that also used lipid nanoparticles. Except for one preliminary report about A/H1N1 influenza vaccination (adjuvant AS03) - corrected by the authors in a more thorough analysis, all studies concluded that there were no safety concerns. Conclusion This rapid review found no evidence of pregnancy-associated safety concerns of COVID-19 vaccines that were selected for review by the COVAX MIWG or of their components or platforms when used in other vaccines. However, the need for further data on several vaccine platforms and components is warranted given their novelty. Our findings support current WHO guidelines recommending that pregnant women may consider receiving COVID-19 vaccines, particularly if they are at high risk of exposure or have comorbidities that enhance the risk of severe disease.

In vitro screening of anti-viral and virucidal effects against SARS-CoV-2 by Hypericum perforatum and Echinacea. (preprint)

Special Infectious Agent Unit in King Fahd Medical Research Center at King Abdulaziz University, Jeddah, Saudi Arabia, has pursed the anti-viral project field to optimize the group of medicinal plants for human-infectious diseases. We have begun virtually in this field since COVID-19 pandemic, besides our divergence in the infectious agents. In this study and based on the previous review, Hypericum perforatum (St. Johns Wort) and Echinacea (gaia HERBS(R)) were tested in vitro using Vero E6 cells for their anti-viral effects against the newly identified Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) through its infectious cycle from 0 to 48 hours post infection. The hypericin (0.9 mg) of H. perforatum and the different parts (roots, seeds, aerial) of two types of Echinacea species (Echinacea purpurea and Echinacea angustifolia) were examined their efficacy in certain concentration and under light-dependent anti-viral activities to measure the inhibition of the SARS-CoV-2 mRNA expression of RNA-dependent RNA polymerase (RdRP) gene and the viral load with quantitative real-time polymerase chain reaction (qRT-PCR), and to assess the neutralization of the SARS-CoV-2 spike receptor binding on cell culture assay. Interestingly, the mixture (H.E.) of 100 mg/mL of H. perforatum and Echinacea was tested too on SARS-CoV-2 and showed crucial anti-viral activity competing H. perforatum then Echinacea effects as anti-viral treatment. Therefore, the results of gaia HERBS(R) products, H. perforatum and Echinacea species, applied in this study showed significant anti-viral and virucidal effects in the following order of potency: H. perforatum, H.E., and Echinacea on SARS-CoV-2 infectious cycle; and will definitely required a set up of clinical trial with specific therapeutic protocol based on the outcome of this study. Author SummaryAfter an outbreak of Rift Valley Fever in the Southern region of Saudi Arabia, particularly in May 2003, Special Infectious Agents Unit (SIAU) was established and founded by Prof. Esam Ibraheem Azhar. This unit contains a full range of facilities including Biosafety Level 3, allows him and his research groups to ambulate and culture risk group 3 viruses in Saudi Arabia & Gulf States for the first time. Since that time, SIAU and our international collaboration have been extended to implement a standard protocols in the infectious agents diagnostics procedure through different mode of collaboration including exchange of expertise, joint research program and more recently a technology transfer agreements with number of international institute sharing same interests. Furthermore, we have been engaged in number of researches related to Hajj & Umrah plus number of national services with the Ministry of Health (MOH) through which, we utilize our Mobile biosafety level 3 Lab to enhance the diagnostics of MERS CoV in the Holly sites during Hajj since 2014. In our SIAU and with a powerful team, we have excellent researches made valuable contributions through in vivo and in vitro animal and human studies, and several human viral pathogens which are a threat to global health security due to millions of pilgrims visiting Saudi Arabia every year from 182 countries: with particular areas of interests in: Alkhurma Viral Hemorrhagic Fever, Dengue Hemorrhagic Fever Viruses, Rift Valley Fever Virus, MERS-CoV and more recently the new global infectious diseases threat, Sever Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2).

In Search for the Hotspots of Disease X: A Biogeographic Approach to Mapping the Predictive Risk of WHO's Blueprint Priority Diseases (preprint)

Background: Anthropization of natural habitats including climate change along with overpopulation and global travel have been contributing to EIDs outbreaks. The ongoing COVID-19 outbreak highlights the threats to human health, social stability and global trade and economy. The Blueprint Priority Diseases (BPDs) is a list established by the World Health Organization, which are in urgent need of research. We proposed mapping the predictive risk of the BPDs, identifying the environmental drivers and validating the use of disease biogeography as a tool for identifying the potential hotspots for Disease X.Methods: We used environmental predictor variables from satellite imagery such as landscape, deforestation, climatic covariates, and species distribution modelling by classical regression and Support Vector Machine (SVM) to model BPD occurrences from 2000 to 2019. The models were evaluated using AUC (>0.9) and a weighed mean was used to map the predictive risk of the BPDs and identify potential sites of Disease X.Results: We generated predictive risk maps for Ebola, Marburg, Nipah, Rift Valley Fever and coronavirus diseases using glm and SVM models. Elevation, tree cover loss and climatic covariates were found to significant triggers for disease emergence of BPD outbreaks. We identified regions in China and Uganda to be potential hotspots for Disease X.Conclusions: Using a biogeographic approach, existing disease databases and freely accessible environmental data, we were able to identify Wuhan as a potential hotspot of disease emergence in the absence of COVID-19 data. We confirm that distribution of disease emergence in humans is spatially dependent on environmental factors.Funding Statement: None to declare Declaration of Interests: None to declareEthical Approval: Not needed

Novel ionophores active against La Crosse virus identified through rapid antiviral screening (preprint)

Bunyaviruses are significant human pathogens, causing diseases ranging from hemorrhagic fevers to encephalitis. Among these viruses, La Crosse virus (LACV), a member of the California serogroup, circulates in the eastern and midwestern United States. While LACV infection is often asymptomatic, dozens of cases of encephalitis are reported yearly. Unfortunately, no antivirals have been approved to treat LACV infection. Here, we developed a method to rapidly test potential antivirals against LACV infection. From this screen, we identified several potential antiviral molecules, including known antivirals. Additionally, we identified many novel antivirals that exhibited antiviral activity without affecting cellular viability. Valinomycin, a potassium ionophore, was among our top targets. We found that valinomycin exhibited potent anti-LACV activity in multiple cell types in a dose-dependent manner. Valinomycin did not affect particle stability or infectivity, suggesting that it may preclude virus replication by altering cellular potassium ions, a known determinant of LACV entry. We extended these results to other ionophores and found that the antiviral activity of valinomycin extended to other viral families including bunyaviruses (Rift Valley fever virus, Keystone virus), enteroviruses (Coxsackievirus, rhinovirus), flavirivuses (Zika), and coronaviruses (229E and MERS-CoV). In all viral infections, we observed significant reductions in virus titer in valinomycin-treated cells. In sum, we demonstrate the importance of potassium ions to virus infection, suggesting a potential therapeutic target to disrupt virus replication. ImportanceNo antivirals are approved for the treatment of bunyavirus infection. The ability to rapidly screen compounds and identify novel antivirals is one means to accelerate drug discovery for viruses with no approved treatments. We used this approach to screen hundreds of compounds against La Crosse virus, an emerging bunyavirus that causes significant disease, including encephalitis. We identified several known and previously unidentified antivirals. We focused on a potassium ionophore, valinomycin, due to its promising in vitro antiviral activity. We demonstrate that valinomycin, as well as a selection of other ionophores, exhibits activity against La Crosse virus as well as several other distantly related bunyaviruses. We finally observe that valinomycin has activity against a wide array of human viral pathogens, suggesting that disrupting potassium ion homeostasis with valinomycin may be a potent host pathway to target to quell virus infection.