Genome-scale CRISPR screening reveals that C3aR signaling is critical for rapid capture of fungi by macrophages

The fungal pathogen Histoplasma capsulatum (Hc) invades, replicates within, and destroys macrophages. To interrogate the molecular mechanisms underlying this interaction, we conducted a host-directed CRISPR-Cas9 screen and identified 361 genes that modify macrophage susceptibility to Hc infection, greatly expanding our understanding of host gene networks targeted by Hc. We identified pathways that have not been previously implicated in Hc interaction with macrophages, including the ragulator complex (involved in nutrient stress sensing), glycosylation enzymes, protein degradation machinery, mitochondrial respiration genes, solute transporters, and the ER membrane complex (EMC). The highest scoring protective hits included the complement C3a receptor (C3aR), a G-protein coupled receptor (GPCR) that recognizes the complement fragment C3a. Although it is known that the complement system reacts with the fungal surface, leading to opsonization and release of small peptide fragments such as C3a, a role for C3aR in macrophage susceptibility to fungi has not been elucidated. We demonstrated that whereas C3aR is dispensable for macrophage phagocytosis of bacteria and latex beads, it is critical for optimal macrophage capture of pathogenic fungi, including Hc,the ubiquitous fungal pathogen Candida albicans, and the causative agent of Valley Fever Coccidioides posadasii. We showed that C3aR localizes to the early phagosome during H. capsulatum infection where it coordinates the formation of actin-rich membrane protrusions that promote Hc capture. We also showed that the EMC promotes surface expression of C3aR, likely explaining its identification in our screen. Taken together, our results provide new insight into host processes that affect Hc-macrophage interactions and uncover a novel and specific role for C3aR in macrophage recognition of fungi.

Tilorone-Dihydrochloride Protects against Rift Valley Fever Virus Infection and Disease in the Mouse Model

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to Africa and the Middle East that can affect humans and ruminant livestock. Currently, there are no approved vaccines or therapeutics for the treatment of severe RVF disease in humans. Tilorone-dihydrochloride (Tilorone) is a broad-spectrum antiviral candidate that has previously shown efficacy against a wide range of DNA and RNA viruses, and which is clinically utilized for the treatment of respiratory infections in Russia and other Eastern European countries. Here, we evaluated the antiviral activity of Tilorone against Rift Valley fever virus (RVFV). In vitro, Tilorone inhibited both vaccine (MP-12) and virulent (ZH501) strains of RVFV at low micromolar concentrations. In the mouse model, treatment with Tilorone significantly improved survival outcomes in BALB/c mice challenged with a lethal dose of RVFV ZH501. Treatment with 30 mg/kg/day resulted in 80% survival when administered immediately after infection. In post-exposure prophylaxis, Tilorone resulted in 30% survival at one day after infection when administered at 45 mg/kg/day. These findings demonstrate that Tilorone has potent antiviral efficacy against RVFV infection in vitro and in vivo and supports further development of Tilorone as a potential antiviral therapeutic for treatment of RVFV infection.

Detection of Rift Valley Fever Virus Lineage H from South Africa through Syndromic Sentinel Surveillance Network in Senegal

Background Rift Valley fever virus (RVFV) is an arbovirus that causes epizootics and epidemics among livestock population and humans. Our surveillance system has revealed multiple emergences and re-emergences of RVFV in West Africa over the last decade. Methods In Senegal a sentinel syndromic surveillance network (4S) has been implemented since 2011. Samples from human suspected arbovirus infection in 4S sentinel sites were sent to Institut Pasteur de Dakar (IPD) where arbovirus diagnosis by enzyme-linked immunosorbent assay (ELISA), real-time reverse transcription polymerase chain reaction (RT-PCR), and virus isolation were performed. Overall, IPD has received a total of 1,149 samples from arboviral suspected patients through the 4S network from January to December 2020. These samples were screened for seven arboviruses including RVFV. Whole genome sequencing of positive RVFV samples by RT-PCR were performed using Illumina Miseq platform followed by genome assembly. Phylogenetic analysis were performed using MEGA X. Results Out of the 1,149 arbovirus suspected cases, four RVFV positive samples were detected with RT-PCR while five RVFV positive samples were detected by ELISA. Complete genome sequences were obtained for three strains among the four positive samples by RT-PCR. Phylogenetic analyses indicated an emergence of a virus first described in South Africa during a major outbreak. Conclusion Strong surveillance system allowed the detection of RVFV outbreak in Senegal in 2020. The obtained genomes clustered with strains from South Africa belonging to lineage H. This calls for an implementation of a strong surveillance system in wild animals, humans, and livestock simultaneously in all African Countrries.

Viral Haemorrhagic Fevers and Malaria Co-Infections Among Febrile Patients Seeking Health Care in Tanzania

Background: In recent years there have been reports of viral haemorrhagic fever (VHF) epidemics in Sub-Saharan Africa where malaria is endemic. VHF and malaria have overlapping clinical presentations making differential diagnosis a challenge. The objective of this study was to determine the prevalence of selected zoonotic VHFs and malaria co-infections among febrile patients seeking health care in Tanzania. Methods: This facility-based cross-section study was carried out in Buhigwe, Kalambo, Kyela, Kilindi, Kinondoni, Kondoa, Mvomero, and Ukerewe districts in Tanzania. The study involved febrile patients seeking health care from primary healthcare facilities. Blood samples were collected and tested for infections due to malaria, Crimean-Congo haemorrhagic fever (CCHF), Ebola virus disease (EVD), Marburg virus disease (MVD), Rift Valley fever (RVF) and yellow fever (YF). Malaria infections were tested using rapid diagnostics tests while exposure to VHFs was determined by screening for immunoglobulin M antibodies using commercial enzyme-linked immunosorbent assays. Results: A total of 308 participants (mean age=35±18.9 years) were involved in the study. Of these, 54 (17.5%) had malaria infection and 15 (4.8%) were positive for IgM antibodies against VHFs (RVF=8; CCHF=2; EBV=3; MBV=1; YF=1). Six (1.9%) individuals had both VHF (RVF=2; CCHF=1; EVD=2; MVD=1) and malaria infections. The highest co-infection prevalence (0.6%), was observed among individuals aged 46-60 years (p<0.05). District was significantly associated with co-infection (p<0.05) with the highest prevalence recorded in Buhigwe (1.2%) followed by Kinondoni (0.9%) districts. Headache (100%) and muscle, bone, back and joint pains (83.3%) were the most significant complaints among those infected with both VHFs and malaria (p=0.001). Conclusions: Co-infections of VHF and malaria are prevalent in Tanzania and affect more the older than the younger population. Since the overlapping symptoms in co-infected individuals may challenge accurate diagnosis, adequate laboratory diagnosis should be emphasized in the management of febrile illnesses.

Co-circulation of Orthobunyaviruses and Rift Valley Fever Virus in Mauritania, 2015

Ngari virus (NRIV) has been mostly detected during concurrent outbreaks of Rift Valley fever virus (RVFV). NRIV is grouped in the genus Orthobunyavirus within the Bunyaviridae family and RVFV in the genus Phlebovirus in the family Phenuiviridae. Both are zoonotic arboviruses and can induce hemorrhagic fever displaying the same clinical picture in humans and small ruminants. To investigate if NRIV and its parental viruses, Bunyamwera virus (BUNV) and Batai virus (BATV), played a role during the Mauritanian RVF outbreak in 2015/16, we analyzed serum samples of sheep and goats from central and southern regions in Mauritania by quantitative real-time RT-PCR, serum neutralization test (SNT) and ELISA. 41 of 458 samples exhibited neutralizing reactivity against NRIV, nine against BATV and three against BUNV. Moreover, complete virus genomes from BUNV could be recovered from two sheep as well as two NRIV isolates from a goat and a sheep. No RVFV-derived viral RNA was detected, but 81 seropositive animals including 22 IgM-positive individuals were found. Of these specimens, 61 samples revealed antibodies against RVFV and at least against one of the three orthobunyaviruses. An indirect ELISA based on NRIV/BATV and BUNV derived Gc protein was established as complement to SNT, which showed high performance regarding NRIV, but decreased sensitivity and specificity regarding BATV and BUNV. Moreover, we observed high cross-reactivity among NRIV and BATV serological assays. Taken together, the data indicate the co-circulation of at least BUNV and NRIV in the Mauritanian sheep and goat populations.

MAVS mediates a protective immune response in the brain to Rift Valley fever virus

Rift Valley fever virus (RVFV) is a highly pathogenic mosquito-borne virus capable of causing hepatitis, encephalitis, blindness, hemorrhagic syndrome, and death in humans and livestock. Upon aerosol infection with RVFV, the brain is a major site of viral replication and tissue damage, yet pathogenesis in this organ has been understudied. Here, we investigated the immune response in the brain of RVFV infected mice. In response to infection, microglia initiate robust transcriptional upregulation of antiviral immune genes, as well as increased levels of activation markers and cytokine secretion that is dependent on mitochondrial antiviral-signaling protein (MAVS) and independent of toll-like receptors 3 and 7. In vivo, Mavs-/- mice displayed enhanced susceptibility to RVFV as determined by increased brain viral burden and higher mortality. Single-cell RNA sequence analysis identified microglia-specific defects in type I interferon and interferon responsive gene expression in Mavs-/- mice, as well as dysregulated lymphocyte infiltration. The results of this study provide a crucial step towards understanding the precise molecular mechanisms by which RVFV infection is controlled in the brain and will help inform the development of vaccines and antiviral therapies that are effective in preventing encephalitis.

Safety, Immunogenicity and Antibody Persistence of Rift Valley Fever Virus Clone 13 Vaccine in Sheep, Goats and Cattle in Tanzania

Background: Vaccination is considered to be the best approach to control Rift Valley fever (RVF) in animals and consequently in humans. This study assessed the efficacy and safety of the RVF virus (RVFV) Clone 13 vaccine under field conditions.Methodology: A vaccine trial was conducted in sheep (230), goats (230), and cattle (140) in Ngorongoro district, Tanzania. Half of each of the animal species were vaccinated and the other half received the placebo. Animals were clinically monitored and bled before vaccination and at days 15, 30, 60, 180 and 360 (+/– 10) post-vaccination to measure Immunoglobulin M (IgM) and IgG antibody responses to RVFV. Survival analysis was conducted using cox-proportional hazard regression model to measure the time until an event of interest had occurred and to compare the cumulative proportion of events over time.Results: Of 600 animals included in the study, 120 animals were lost during the study, leaving a total of 480 (243 in the vaccinated group and 237 in the control group) for complete follow-up sampling. There was no adverse reaction reported at the injection site of the vaccine/placebo in all animals. Abortions, deaths, or body temperature variations were not associated with vaccination (p &gt; 0.05). By day 15 post-inoculation, the IgG seroconversion in vaccinated goats, cattle and sheep was 27.0% (n = 115), 20.0% (n = 70) and 10.4% (n = 115), respectively. By day 30 post-inoculation, it was 75.0% (n = 113), 74.1% (n = 112) and 57.1% (n = 70) in vaccinated sheep, goats and cattle, respectively. By day 60 post-inoculation, IgG seroconversion in sheep, goats and cattle was 88.1% (n = 109), 84.3% (n = 108) and 64.60% (n = 65), respectively. By day 180, the IgG seroconversion in sheep, goats and cattle was 88.0% (n = 108), 83.8% (n = 105) and 66.1% (n = 62), respectively. By day 360, the IgG seroconversion in sheep, goats and cattle was 87.2% (n = 94), 85.6% (n = 90) and 66.1% (n = 59), respectively. Only five animals from the vaccinated group were RVFV IgM positive, which included four sheep and a goat.Conclusion: RVFV Clone 13 vaccine was well tolerated by sheep, goats, and cattle. The vaccine induced detectable, but variable levels of IgG responses, and of different duration. The vaccine is considered safe, with high immunogenicity in sheep and goats and moderate in cattle.