Molecular characterization of an avian rotavirus a strain detected from a large-billed crow (Corvus macrorhynchos) in Japan.

Avian rotaviruses A (RVAs) are occasionally transmitted to animals other than the original hosts across species barriers. Information on RVAs carried by various bird species is important for identifying the origin of such interspecies transmission. In this study, to facilitate an understanding of the ecology of RVAs from wild birds, we characterized all of the genes of an RVA strain, JC-105, that was detected in a fecal sample of a large-billed crow (Corvus macrorhynchos) in Japan. All of the genes of this strain except for the VP4 and VP7 genes, which were classified as novel genotypes (P[56] and G40, respectively), were closely related to those of the avian-like RVA strain detected from a raccoon, indicating the possibility that crows had been involved in the transmission of avian RVAs to raccoons. Our findings highlight the need for further viral investigations in wild birds and mammals to understand the mechanisms of avian-to-mammal RVA transmission.

Dopamine-Derived Guanidine Alkaloids from a Didemnidae Tunicate: Isolation, Synthesis, and Biological Activities.

Mellpaladines A-C (1-3) and dopargimine (4) are dopamine-derived guanidine alkaloids isolated from a specimen of Palauan Didemnidae tunicate as possible modulators of neuronal receptors. In this study, we isolated the dopargimine derivative 1-carboxydopargimine (5), three additional mellpaladines D-F (6-8), and serotodopalgimine (9), along with a dimer of serotonin, 5,5'-dihydroxy-4,4'-bistryptamine (10). The structures of these compounds were determined based on spectrometric and spectroscopic analyses. Compound 4 and its congeners dopargine (11), nordopargimine (15), and 2-(6,7-dimethoxy-3,4-dihydroisoquinolin-1-yl)ethan-1-amine (16) were synthetically prepared for biological evaluations. The biological activities of all isolated compounds were evaluated in comparison with those of 1-4 using a mouse behavioral assay upon intracerebroventricular injection, revealing key functional groups in the dopargimines and mellpaladines for in vivo behavioral toxicity. Interestingly, these alkaloids also emerged during a screen of our marine natural product library aimed at identifying antiviral activities against dengue virus, SARS-CoV-2, and vesicular stomatitis Indiana virus (VSV) pseudotyped with Ebola virus glycoprotein (VSV-ZGP).

Seroprevalence of Bas-Congo virus in Mangala, Democratic Republic of the Congo: a population-based cross-sectional study.

BACKGROUND: Bas-Congo virus (BASV), an emerging tibrovirus, was associated with an outbreak of acute haemorrhagic fever in Mangala, Democratic Republic of the Congo, in 2009. In 2012, neutralising antibodies to BASV were detected in the lone survivor and one of his close contacts. However, subsequent serological and molecular surveys were unsuccessful as neither BASV antibodies nor its RNA were detected. In this study, we determined the seroprevalence of BASV infection in Mangala 13 years after the initial outbreak. METHODS: We conducted a population-based serological survey from Jan 17 to Jan 23, 2022. Consenting individuals at least 5 years of age, living in Mangala for at least 4 weeks, and who had no contraindication to venepuncture were enrolled. Participants were interviewed using a pre-tested questionnaire for sociodemographic and clinical characteristics. We supplemented the collected serum samples with 284 archived samples from Matadi and Kinshasa. All samples were tested for antibodies to BASV and other tibroviruses using a pseudovirus-based neutralisation test. FINDINGS: Among the 267 individuals from Mangala, the prevalence of BASV antibodies was 55% (95% CI 49-61; n=147). BASV seropositivity odds significantly increased with age (5·2 [95% CI 2·1-12·9] to 83·9 [20·8-337·7] times higher in participants aged 20 years or older than participants aged 5-19 years). Some occupational categories (eg, farmer or public servant) were associated with seropositivity. Only nine (6%) of 160 samples from Matadi and one (<1%) of 124 samples from Kinshasa had neutralising antibodies to BASV. Moreover, we also detected neutralising antibodies to other tibroviruses-Ekpoma virus 1, Ekpoma virus 2, and Mundri virus-in 84 (31%), 251 (94%), and 219 (82%) of 267 Mangala samples; 14 (9%), 62 (39%), and 120 (75%) of 160 Matadi samples; and six (5%), five (4%), and 33 (27%) of 124 Kinshasa samples, respectively. INTERPRETATION: Human infection with BASV and other tibroviruses seems common in Mangala, although no deadly outbreak has been reported since 2009. Exposure to BASV might be highly restricted to Mangala and the increasing prevalence of neutralising antibodies with age suggests regular contact with the virus in this city. Altogether, our findings suggest that human infection with tibroviruses could be common in the study areas and not associated with deadly haemorrhagic or debilitating syndromes. FUNDING: Japan Agency for Medical Research and Development (AMED) and Japan International Cooperation Agency (JICA) under the Science and Technology Research Partnership for Sustainable Development (SATREPS) and Japan Program for Infectious Diseases Research and Infrastructure from AMED.

ICTV Virus Taxonomy Profile: Filoviridae 2024.

Filoviridae is a family of negative-sense RNA viruses with genomes of about 13.1-20.9 kb that infect fish, mammals and reptiles. The filovirid genome is a linear, non-segmented RNA with five canonical open reading frames (ORFs) that encode a nucleoprotein (NP), a polymerase cofactor (VP35), a glycoprotein (GP1,2), a transcriptional activator (VP30) and a large protein (L) containing an RNA-directed RNA polymerase (RdRP) domain. All filovirid genomes encode additional proteins that vary among genera. Several filovirids (e.g., Ebola virus, Marburg virus) are pathogenic for humans and highly virulent. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Filoviridae, which is available at www.ictv.global/report/filoviridae.

Phylogenetic Analysis of Newcastle Disease Virus Isolated from Poultry in Live Bird Markets and Wild Waterfowl in Zambia.

Poultry production is essential to the economy and livelihood of many rural Zambian households. However, the industry is threatened by infectious diseases, particularly Newcastle disease virus (NDV) infection. Therefore, this study employed next-generation sequencing to characterise six NDV isolates from poultry in Zambia's live bird markets (LBMs) and wild waterfowl. Four NDV isolates were detected from 410 faecal samples collected from chickens in LBMs in Lusaka and two from 2851 wild birds from Lochinvar National Park. Phylogenetic analysis revealed that the four NDVs from LBM clustered in genotype VII and sub-genotype VII.2 were closely related to viruses previously isolated in Zambia and other Southern African countries, suggesting possible local and regional transboundary circulation of the virus. In contrast, the two isolates from wild birds belonged to class I viruses, genotype 1, and were closely related to isolates from Europe and Asia, suggesting the possible introduction of these viruses from Eurasia, likely through wild bird migration. The fusion gene cleavage site motif for all LBM-associated isolates was 112RRQKR|F117, indicating that the viruses are virulent, while the isolates from wild waterfowl had the typical 112ERQER|L117 avirulent motif. This study demonstrates the circulation of virulent NDV strains in LBMs and has, for the first time, characterised NDV from wild birds in Zambia. The study further provides the first whole genomes of NDV sub-genotype VII.2 and genotype 1 from Zambia and stresses the importance of surveillance and molecular analysis for monitoring the circulation of NDV genotypes and viral evolution.

Development of an Immunochromatography Assay to Detect Marburg Virus and Ravn Virus.

The recent outbreaks of Marburg virus disease (MVD) in Guinea, Ghana, Equatorial Guinea, and Tanzania, none of which had reported previous outbreaks, imply increasing risks of spillover of the causative viruses, Marburg virus (MARV) and Ravn virus (RAVV), from their natural host animals. These outbreaks have emphasized the need for the development of rapid diagnostic tests for this disease. Using monoclonal antibodies specific to the viral nucleoprotein, we developed an immunochromatography (IC) assay for the rapid diagnosis of MVD. The IC assay was found to be capable of detecting approximately 102-4 50% tissue culture infectious dose (TCID50)/test of MARV and RAVV in the infected culture supernatants. We further confirmed that the IC assay could detect the MARV and RAVV antigens in the serum samples from experimentally infected nonhuman primates. These results indicate that the IC assay to detect MARV can be a useful tool for the rapid point-of-care diagnosis of MVD.

The evolution and determinants of neutralization of potent head-binding antibodies against Ebola virus.

Monoclonal antibodies against the Ebola virus (EBOV) surface glycoprotein are effective treatments for EBOV disease. Antibodies targeting the EBOV glycoprotein (GP) head epitope have potent neutralization and Fc effector function activity and thus are of high interest as therapeutics and for vaccine design. Here we focus on the head-binding antibodies 1A2 and 1D5, which have been identified previously in a longitudinal study of survivors of EBOV infection. 1A2 and 1D5 have the same heavy- and light-chain germlines despite being isolated from different individuals and at different time points after recovery from infection. Cryoelectron microscopy analysis of each antibody in complex with the EBOV surface GP reveals key amino acid substitutions in 1A2 that contribute to greater affinity, improved neutralization potency, and enhanced breadth as well as two strategies for antibody evolution from a common site.

Prevalence and Genomic Characterization of Rotavirus A from Domestic Pigs in Zambia: Evidence for Possible Porcine-Human Interspecies Transmission.

Rotavirus is a major cause of diarrhea globally in animals and young children under 5 years old. Here, molecular detection and genetic characterization of porcine rotavirus in smallholder and commercial pig farms in the Lusaka Province of Zambia were conducted. Screening of 148 stool samples by RT-PCR targeting the VP6 gene revealed a prevalence of 22.9% (34/148). Further testing of VP6-positive samples with VP7-specific primers produced 12 positives, which were then Sanger-sequenced. BLASTn of the VP7 positives showed sequence similarity to porcine and human rotavirus strains with identities ranging from 87.5% to 97.1%. By next-generation sequencing, the full-length genetic constellation of the representative strains RVA/pig-wt/ZMB/LSK0137 and RVA/pig-wt/ZMB/LSK0147 were determined. Genotyping of these strains revealed a known Wa-like genetic backbone, and their genetic constellations were G4-P[6]-I5-R1-C1-M1-A8-N1-T1-E1-H1 and G9-P[13]-I5-R1-C1-M1-A8-N1-T1-E1-H1, respectively. Phylogenetic analysis revealed that these two viruses might have their ancestral origin from pigs, though some of their gene segments were related to human strains. The study shows evidence of reassortment and possible interspecies transmission between pigs and humans in Zambia. Therefore, the "One Health" surveillance approach for rotavirus A in animals and humans is recommended to inform the design of effective control measures.

Renaming of genera Ebolavirus and Marburgvirus to Orthoebolavirus and Orthomarburgvirus, respectively, and introduction of binomial species names within family Filoviridae.

The International Committee on Taxonomy of Viruses (ICTV) Filoviridae Study Group continues to prospectively refine the established nomenclature for taxa included in family Filoviridae in an effort to decrease confusion of genus, species, and virus names and to adhere to amended stipulations of the International Code of Virus Classification and Nomenclature (ICVCN). Recently, the genus names Ebolavirus and Marburgvirus were changed to Orthoebolavirus and Orthomarburgvirus, respectively. Additionally, all established species names in family Filoviridae now adhere to the ICTV-mandated binomial format. Virus names remain unchanged and valid. Here, we outline the revised taxonomy of family Filoviridae as approved by the ICTV in April 2023.

Susceptibility of common dabbling and diving duck species to clade 2.3.2.1 H5N1 high pathogenicity avian influenza virus: an experimental infection study.

In the winter of 2010-2011, Japan experienced a large outbreak of infections caused by clade 2.3.2.1 H5N1 high pathogenicity avian influenza viruses (HPAIVs) in wild birds. Interestingly, many tufted ducks (Aythya fuligula), which are migratory diving ducks, succumbed to the infection, whereas only one infection case was reported in migratory dabbling duck species, the major natural hosts of the influenza A virus, during the outbreak. To assess whether the susceptibility of each duck species to HPAIVs was correlated with the number of cases, tufted duck and dabbling duck species (Eurasian wigeon, Mareca penelope; mallard, Anas platyrhynchos; Northern pintail, Anas acuta) were intranasally inoculated with A/Mandarin duck/Miyazaki/22M807-1/2011 (H5N1), an index clade 2.3.2.1 virus previously used for experimental infection studies in various bird species. All ducks observed for 10 days post-inoculation (dpi) mostly shed the virus via the oral route and survived. The tufted ducks shed a higher titer of the virus than the other dabbling duck species, and one of them showed apparent neurological symptoms after 7 dpi, which were accompanied by eye lesions. No clinical symptoms were observed in the dabbling ducks, although systemic infection and viremia were observed in some of them sacrificed at 3 dpi. These results suggest that the susceptibility of clade 2.3.2.1 HPAIVs might differ by duck species.

Surveillance, Isolation, and Genetic Characterization of Bat Herpesviruses in Zambia.

Bats are of significant interest as reservoirs for various zoonotic viruses with high diversity. During the past two decades, many herpesviruses have been identified in various bats worldwide by genetic approaches, whereas there have been few reports on the isolation of infectious herpesviruses. Herein, we report the prevalence of herpesvirus infection of bats captured in Zambia and genetic characterization of novel gammaherpesviruses isolated from striped leaf-nosed bats (Macronycteris vittatus). By our PCR screening, herpesvirus DNA polymerase (DPOL) genes were detected in 29.2% (7/24) of Egyptian fruit bats (Rousettus aegyptiacus), 78.1% (82/105) of Macronycteris vittatus, and one Sundevall's roundleaf bat (Hipposideros caffer) in Zambia. Phylogenetic analyses of the detected partial DPOL genes revealed that the Zambian bat herpesviruses were divided into seven betaherpesvirus groups and five gammaherpesvirus groups. Two infectious strains of a novel gammaherpesvirus, tentatively named Macronycteris gammaherpesvirus 1 (MaGHV1), were successfully isolated from Macronycteris vittatus bats, and their complete genomes were sequenced. The genome of MaGHV1 encoded 79 open reading frames, and phylogenic analyses of the DNA polymerase and glycoprotein B demonstrated that MaGHV1 formed an independent lineage sharing a common origin with other bat-derived gammaherpesviruses. Our findings provide new information regarding the genetic diversity of herpesviruses maintained in African bats.

Pathogenicity of Lloviu and Bombali Viruses in Type I Interferon Receptor Knockout Mice.

Type I interferon receptor knockout (IFNAR-/-) mice are not able to generate a complete innate immune response; therefore, these mice are often considered to assess the pathogenicity of emerging viruses. We infected IFNAR-/- mice with a low or high dose of Lloviu virus (LLOV) or Bombali virus (BOMV) by the intranasal (IN) or intraperitoneal (IP) route and compared virus loads at early and late time points after infection. No signs of disease and no viral RNA were detected after IN infection regardless of LLOV dose. In contrast, IP infections resulted in increased viral loads in the high-dose LLOV and BOMV groups at the early time point. The low-dose LLOV and BOMV groups achieved higher viral loads at the late time point. However, there was 100% survival in all groups and no signs of disease. In conclusion, our results indicate a limited value of the IFNAR-/- mouse model for investigation of the pathogenicity of LLOV and BOMV.

RNA Editing as a General Trait of Ebolaviruses.

RNA editing has been discovered as an essential mechanism for the transcription of the glycoprotein (GP) gene of Ebola virus but not Marburg virus. We developed a rapid transcript quantification assay (RTQA) to analyze RNA transcripts generated through RNA editing and used immunoblotting with a pan-ebolavirus monoclonal antibody to confirm different GP gene-derived products. RTQA successfully quantified GP gene transcripts during infection with representative members of 5 ebolavirus species. Immunoblotting verified expression of the soluble GP and the transmembrane GP. Our results defined RNA editing as a general trait of ebolaviruses. The degree of editing, however, varies among ebolaviruses with Reston virus showing the lowest and Bundibugyo virus the highest degree of editing.

Field-deployable multiplex detection method of SARS-CoV-2 and influenza virus using loop-mediated isothermal amplification and DNA chromatography.

A novel multiplex loop-mediated isothermal amplification (LAMP) method combined with DNA chromatography was developed for the simultaneous detection of three important respiratory disease-causing viruses: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus, and influenza B virus. Amplification was performed at a constant temperature, and a positive result was confirmed by a visible colored band. An in-house drying protocol with trehalose was used to prepare the dried format multiplex LAMP test. Using this dried multiplex LAMP test, the analytical sensitivity was determined to be 100 copies for each viral target and 100-1000 copies for the simultaneous detection of mixed targets. The multiplex LAMP system was validated using clinical COVID-19 specimens and compared with the real-time qRT-PCR method as a reference test. The determined sensitivity of the multiplex LAMP system for SARS-CoV-2 was 71% (95% CI: 0.62-0.79) for cycle threshold (Ct) ≤ 35 samples and 61% (95% CI: 0.53-0.69) for Ct ≤40 samples. The specificity was 99% (95%CI: 0.92-1.00) for Ct ≤35 samples and 100% (95%CI: 0.92-1.00) for the Ct ≤40 samples. The developed simple, rapid, low-cost, and laboratory-free multiplex LAMP system for the two major important respiratory viral diseases, COVID-19 and influenza, is a promising field-deployable diagnosis tool for the possible future 'twindemic, ' especially in resource-limited settings.

Head-to-head comparison of diagnostic accuracy of four Ebola virus disease rapid diagnostic tests versus GeneXpert® in eastern Democratic Republic of the Congo outbreaks: a prospective observational study.

BACKGROUND: Ebola virus disease (EVD) outbreaks have emerged in Central and West Africa. EVD diagnosis relies principally on RT-PCR testing with GeneXpert®, which has logistical and cost restrictions at the peripheral level of the health system. Rapid diagnostic tests (RDTs) would offer a valuable alternative at the point-of-care to reduce the turn-around time, if they show good performance characteristics. We evaluated the performance of four EVD RDTs against the reference standard GeneXpert® on stored EVD positive and negative blood samples collected between 2018 and 2021 from outbreaks in eastern Democratic Republic of the Congo (DRC). METHODS: We conducted a prospective and observational study in the laboratory on QuickNavi-Ebola™, OraQuick® Ebola Rapid Antigen, Coris® EBOLA Ag K-SeT, and Standard® Q Ebola Zaïre Ag RDTs using left-over archived frozen EDTA whole blood samples. We randomly selected 450 positive and 450 negative samples from the EVD biorepositories in DRC, across a range of GeneXpert® cycle threshold values (Ct-values). RDT results were read by three persons and we considered an RDT result as "positive", when it was flagged as positive by at least two out of the three readers. We estimated the sensitivity and specificity through two independent generalized (logistic) linear mixed models (GLMM). FINDINGS: 476 (53%) of 900 samples had a positive GeneXpert Ebola result when retested. The QuickNavi-Ebola™ showed a sensitivity of 56.8% (95% CI 53.6-60.0) and a specificity of 97.5% (95% CI 96.2-98.4), the OraQuick® Ebola Rapid Antigen test displayed 61.6% (95% CI 57.0-65.9) sensitivity and 98.1% (95% CI 96.2-99.1) specificity, the Coris® EBOLA Ag K-SeT showed 25.0% (95% CI 22.3-27.9) sensitivity and 95.9% (95% CI 94.2-97.1) specificity, and the Standard® Q Ebola Zaïre Ag displayed 21.6% (95% CI 18.1-25.7) sensitivity and 99.1% (95% CI 97.4-99.7) specificity. INTERPRETATION: None of the RDTs evaluated approached the "desired or acceptable levels" for sensitivity set out in the WHO target product profile, while all of the tests met the "desired level" for specificity. Nevertheless, the QuickNavi-Ebola™ and OraQuick® Ebola Rapid Antigen Test demonstrated the most favorable profiles, and may be used as frontline tests for triage of suspected-cases while waiting for RT-qPCR confirmatory testing. FUNDING: Institute of Tropical Medicine Antwerp/EDCTP PEAU-EBOV-RDC project.

Morphogenesis of Bullet-Shaped Rabies Virus Particles Regulated by TSG101.

Viral protein assembly and virion budding are tightly regulated to enable the proper formation of progeny virions. At this late stage in the virus life cycle, some enveloped viruses take advantage of the host endosomal sorting complex required for transport (ESCRT) machinery, which contributes to the physiological functions of membrane modulation and abscission. Bullet-shaped viral particles are unique morphological characteristics of rhabdoviruses; however, the involvement of host factors in rhabdovirus infection and, specifically, the molecular mechanisms underlying virion formation are not fully understood. In the present study, we used a small interfering RNA (siRNA) screening approach and found that the ESCRT-I component TSG101 contributes to the propagation of rabies virus (RABV). We demonstrated that the matrix protein (M) of RABV interacts with TSG101 via the late domain containing the PY and YL motifs, which are conserved in various viral proteins. Loss of the YL motif in the RABV M or the downregulation of host TSG101 expression resulted in the intracellular aggregation of viral proteins and abnormal virus particle formation, indicating a defect in the RABV assembly and budding processes. These results indicate that the interaction of the RABV M and TSG101 is pivotal for not only the efficient budding of progeny RABV from infected cells but also for the bullet-shaped virion morphology. IMPORTANCE Enveloped viruses bud from cells with the host lipid bilayer. Generally, the membrane modulation and abscission are mediated by host ESCRT complexes. Some enveloped viruses utilize their late (L-) domain to interact with ESCRTs, which promotes viral budding. Rhabdoviruses form characteristic bullet-shaped enveloped virions, but the underlying molecular mechanisms involved remain elusive. Here, we showed that TSG101, one of the ESCRT components, supports rabies virus (RABV) budding and proliferation. TSG101 interacted with RABV matrix protein via the L-domain, and the absence of this interaction resulted in intracellular virion accumulation and distortion of the morphology of progeny virions. Our study reveals that virion formation of RABV is highly regulated by TSG101 and the virus matrix protein.

Structural and Energetic Basis for Differential Binding of Ebola and Marburg Virus Glycoproteins to a Bat-Derived Niemann-Pick C1 Protein.

BACKGROUND: Our previous study demonstrated that the fruit bat (Yaeyama flying fox)-derived cell line FBKT1 showed preferential susceptibility to Ebola virus (EBOV), whereas the human cell line HEK293T was similarly susceptible to EBOV and Marburg virus (MARV). This was due to 3 amino acid differences of the endosomal receptor Niemann-Pick C1 (NPC1) between FBKT1 and HEK293T (ie, TET and SGA, respectively, at positions 425-427), as well as 2 amino acid differences at positions 87 and 142 of the viral glycoprotein (GP) between EBOV and MARV. METHODS/RESULTS: To understand the contribution of these amino acid differences to interactions between NPC1 and GP, we performed molecular dynamics simulations and binding free energy calculations. The average binding free energies of human NPC1 (hNPC1) and its mutant having TET at positions 425-427 (hNPC1/TET) were similar for the interaction with EBOV GP. In contrast, hNPC1/TET had a weaker interaction with MARV GP than wild-type hNPC1. As expected, substitutions of amino acid residues at 87 or 142 in EBOV and MARV GPs converted the binding affinity to hNPC1/TET. CONCLUSIONS: Our data provide structural and energetic insights for understanding potential differences in the GP-NPC1 interaction, which could influence the host tropism of EBOV and MARV.

Isolation and Characterization of Distinct Rotavirus A in Bat and Rodent Hosts.

Rotavirus A (RVA) causes diarrheal disease in humans and various animals. Recent studies have identified bat and rodent RVAs with evidence of zoonotic transmission and genome reassortment. However, the virological properties of bat and rodent RVAs with currently identified genotypes still need to be better clarified. Here, we performed virus isolation-based screening for RVA in animal specimens and isolated RVAs (representative strains: 16-06 and MpR12) from Egyptian fruit bat and Natal multimammate mouse collected in Zambia. Whole-genome sequencing and phylogenetic analysis revealed that the genotypes of bat RVA 16-06 were identical to that of RVA BATp39 strain from the Kenyan fruit bat, which has not yet been characterized. Moreover, all segments of rodent RVA MpR12 were highly divergent and assigned to novel genotypes, but RVA MpR12 was phylogenetically closer to bat RVAs than to other rodent RVAs, indicating a unique evolutionary history. We further investigated the virological properties of the isolated RVAs. In brief, we found that 16-06 entered cells by binding to sialic acids on the cell surface, while MpR12 entered in a sialic acid-independent manner. Experimental inoculation of suckling mice with 16-06 and MpR12 revealed that these RVAs are causative agents of diarrhea. Moreover, 16-06 and MpR12 demonstrated an ability to infect and replicate in a 3D-reconstructed primary human intestinal epithelium with comparable efficiency to the human RVA. Taken together, our results detail the unique genetic and virological features of bat and rodent RVAs and demonstrate the need for further investigation of their zoonotic potential. IMPORTANCE Recent advances in nucleotide sequence detection methods have enabled the detection of RVA genomes from various animals. These studies have discovered multiple divergent RVAs and have resulted in proposals for the genetic classification of novel genotypes. However, most of these RVAs have been identified via dsRNA viral genomes and not from infectious viruses, and their virological properties, such as cell/host tropisms, transmissibility, and pathogenicity, are unclear and remain to be clarified. Here, we successfully isolated RVAs with novel genome constellations from three bats and one rodent in Zambia. In addition to whole-genome sequencing, the isolated RVAs were characterized by glycan-binding affinity, pathogenicity in mice, and infectivity to the human gut using a 3D culture of primary intestinal epithelium. Our study reveals the first virological properties of bat and rodent RVAs with high genetic diversity and unique evolutional history and provides basic knowledge to begin estimating the potential of zoonotic transmission.

Current status and molecular epidemiology of rabies virus from different hosts and regions in Malawi.

Although rabies is endemic in Malawi, there have been no studies in which rabies virus was systematically investigated and characterized in multiple animal hosts in that country. In order to provide molecular epidemiological data on rabies virus in Malawi, 683 suspected rabies case reports from 2008 to 2021 were examined, and 46 (dog = 40, cow = 5, and cat = 1) viable rabies-positive brain samples archived at the Central Veterinary Laboratory (CVL), Lilongwe, Malawi, were analyzed genetically. The results showed an increase in the submission of brain samples from 2008 to 2010, with the highest number of submissions observed in 2020. Of the 683 case reports analyzed for the period under review, 38.1% (260/683) (CI: 34.44 - 41.84) were confirmed by direct fluorescent antibody test. Among the confirmed cases, 65.4% (170/260) (CI: 59.23 - 71.09) were canine rabies. Further, phylogenetic analysis revealed that sequences from different animal hosts clustered together within the Africa 1b lineage, suggesting that the strains circulating in livestock are similar to those in domestic dogs. This finding supports the hypothesis that canine rabies is spilling over to livestock and emphasizes the need for further studies to provide data for effective control of rabies in Malawi.

Detection of Old and New World Relapsing Fever Borreliae in Ornithodoros Ticks Collected from Warthog Burrows in Zambia.

Relapsing fever (RF) is an arthropod-borne disease caused by Borrelia spirochete, which is one of the major public health concerns in endemic regions including Africa. However, information on Borrelia spirochetes is limited in Zambia. Here, we investigate the Borrelia spirochetes harbored by Ornithodoros ticks in Zambian National Parks. We analyzed 182 DNA samples pooled from 886 Ornithodoros ticks. Of these, 43 tested positive, and their sequence revealed that the ticks harbored both Old and New World RF borreliae. This research presents the first evidence of Old-World RF borreliae in Zambia. The New World RF borreliae detected herein differed from the Candidatus Borrelia fainii previously reported in Zambia and were closely related to the pathogenic Borrelia sp. VS4 identified in Tanzania. Additionally, Borrelia theileri was recently reported in Zambia. Hence, at least four different Borrelia species occur in Zambia, and the organisms causing relapsing fever there might be more complex than previously thought. We empirically confirmed that real-time PCR with TaqMan minor groove binder probes accurately and simultaneously detected both Old and New World RF. In this manner, they could facilitate quantitative analyses of both types of RF borreliae. Subsequent investigations should endeavor to isolate the aforementioned Borrelia spp. and perform serosurveys on patients with RF.