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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.

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.

While evidence suggests presence of HEV infection in humans in Zambia, currently, there is no information on its occurrence in domestic pigs. Here, we investigated the presence of HEV antibodies and genome in domestic pigs in Zambia. Sera (n = 484) from domestic pigs were screened for antibodies against HEV by ELISA while genome detection in fecal (n = 25) and liver (n = 100) samples from slaughter pigs was conducted using nested RT–PCR assay. Overall, seroprevalence was 47.7% (231/484) while zoonotic genotype 3 HEV RNA was detected in 16.0% (20/125) of slaughtered pigs. This is the first report to highlight occurrence of HEV infection in domestic pigs in Zambia. This finding suggests possible contamination of the pork supply chain. Moreover, there is a potential risk of zoonotic transmission of HEV to abattoir workers, pig farmers and handlers.

Nelson Bay orthoreovirus (NBV) is an emerging bat-borne virus and causes respiratory tract infections in humans sporadically. Over the last two decades, several strains genetically related to NBV were isolated from humans and various bat species, predominantly in Southeast Asia (SEA), suggesting a high prevalence of the NBV species in this region. In this study, an orthoreovirus (ORV) belonging to the NBV species was isolated from Indonesian fruit bats' feces, tentatively named Paguyaman orthoreovirus (PgORV). Serological studies revealed that 81.2% (108/133) of Indonesian fruit bats sera had neutralizing antibodies against PgORV. Whole-genome sequencing and phylogenetic analysis of PgORV suggested the occurrence of past reassortments with other NBV strains isolated in SEA, indicating the dispersal and circulation of NBV species among bats in this region. Intranasal PgORV inoculation of laboratory mice caused severe pneumonia. Our study characterized PgORV's unique genetic background and highlighted the potential risk of PgORV-related diseases in Indonesia.

Mammalian orthoreovirus (MRV) has been identified in humans, livestock and wild animals; this wide host range allows individual MRV to transmit into multiple species. Although several interspecies transmission and genetic reassortment events of MRVs among humans, livestock and wildlife have been reported, the genetic diversity and geographic distribution of MRVs in Africa are poorly understood. In this study, we report the first isolation and characterization of MRVs circulating in a pig population in Zambia. In our screening, MRV genomes were detected in 19.7 % (29/147) of faecal samples collected from pigs by reverse transcription PCR. Three infectious MRV strains (MRV-85, MRV-96 and MRV-117) were successfully isolated, and their complete genomes were sequenced. Recombination analyses based on the complete genome sequences of the isolated MRVs demonstrated that MRV-96 shared the S3 segment with a different MRV isolated from bats, and that the L1 and M3 segments of MRV-117 originated from bat and human MRVs, respectively. Our results suggest that the isolated MRVs emerged through genetic reassortment events with interspecies transmission. Given the lack of information regarding MRVs in Africa, further surveillance of MRVs circulating among humans, domestic animals and wildlife is required to assess potential risk for humans and animals.

Orthoreoviruses have been indentified in several mammals, however, there is no information about orthoreoviruses in shrews. In this study, we screened wild animals in Zambia, including shrews, rodents, and bats for the detection of orthoreoviruses. Two orthoreovirus RNA genomes were detected from a shrew intestinal-contents (1/24) and a bat colon (1/96) sample by reverse-transcription (RT)-PCR targeting the RNA-dependent RNA polymerase gene of orthoreoviruses. Phylogenetic analyses revealed that each of the identified orthoreoviruses formed a distinct branch among members of the Orthoreovirus genus. This is the first report that shrews are susceptible to orthoreovirus infection. Our results suggest the existence of undiscovered orthoreoviruses in shrews and provide important information about the genetic diversity of orthoreoviruses.

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.

Bat trypanosomes consist of more than 30 trypanosome species from over 70 species of bats. Recent studies suggest that bats play a role in disseminating trypanosomes from African continent to the terrestrial mammals both in the Afrotropic-Palearctic Ecozones and Nearctic Ecozone. However, the diversity, distribution, and evolution of bat trypanosomes are still unclear. To better understand their evolution, more genetic data of bat trypanosomes from a variety of locations are required. During a survey of Borrelia spp. of bats inhabiting a cave in Zambia, we observed flagellate parasites from 5 of 43 hemocultures. Sequence and phylogenetic analyses of the glycosomal glyceraldehyde 3-phosphate dehydrogenase gene (gGAPDH; 572 bp) and the 18S ribosomal RNA gene (18S rRNA gene; 1,079–1,091 bp) revealed that all were Trypanosoma spp. belonged to the Trypanosoma cruzi clade. Three and two of them exhibited the similarity with T. conorhini and T. dionisii, respectively. The present study provides the first genetic data on Trypanosoma spp. of bats inhabiting Zambia., Graphical abstract Image 1, Highlights • Two putative species of trypanosomes were detected from bats. • First molecular characterization of bat trypanosome in Zambia. • First record of trypanosome in Hipposideros vittatus.

Background Pteropine orthoreovirus (PRV) is an emerging bat-borne zoonotic virus that causes severe respiratory illness in humans. Although PRVs have been identified in fruit bats and humans in Australia and Asia, little is known about the prevalence of PRV infection in Africa. Therefore, this study performed an PRV surveillance in fruit bats in Zambia. Methods Egyptian fruit bats (Rousettus aegyptiacus, n = 47) and straw-colored fruit bats (Eidolon helvum, n = 33) captured in Zambia in 2017–2018 were screened for PRV infection using RT-PCR and serum neutralization tests. The complete genome sequence of an isolated PRV strain was determined by next generation sequencing and subjected to BLAST and phylogenetic analyses. Replication capacity and pathogenicity of the strain were investigated using Vero E6 cell cultures and BALB/c mice, respectively. Results An PRV strain, tentatively named Nachunsulwe-57, was isolated from one Egyptian fruit bat. Serological assays demonstrated that 98% of sera (69/70) collected from Egyptian fruit bats (n = 37) and straw-colored fruit bats (n = 33) had neutralizing antibodies against PRV. Genetic analyses revealed that all 10 genome segments of Nachunsulwe-57 were closely related to a bat-derived Kasama strain found in Uganda. Nachunsulwe-57 showed less efficiency in viral growth and lower pathogenicity in mice than another PRV strain, Miyazaki-Bali/2007, isolated from a patient. Conclusions A high proportion of Egyptian fruit bats and straw-colored fruit bats were found to be seropositive to PRV in Zambia. Importantly, a new PRV strain (Nachunsulwe-57) was isolated from an Egyptian fruit bat in Zambia, which had relatively weak pathogenicity in mice. Taken together, our findings provide new epidemiological insights about PRV infection in bats and indicate the first isolation of an PRV strain that may have low pathogenicity to humans., Author summary Pteropine orthoreovirus (PRV) is a causative agent of acute respiratory illness in humans in tropical and sub-tropical regions in Southeast Asia. PRVs have been originally isolated from fruit bats, and it is assumed that PRVs spread to humans by both bat-to-human and human-to-human transmission. Recently, an PRV was also detected from a fruit bat in the Afrotropical region and might potentially cause an emerging infection of the bat-borne zoonotic virus in Africa. However, little is known about the prevalence of PRV infection in Africa. In this study, we demonstrated the high prevalence of PRV infection in bat populations in Zambia and isolated a new strain of PRV from Egyptian fruit bats. In addition, we found that the bat-derived PRV strain had lower pathogenicity in mice than a human-derived PRV strain isolated from a patient in Southeast Asia. Our findings provide new epidemiological information about PRV in fruit bats in the Afrotropical region and indicate the first isolation of an PRV strain that may cause attenuated infection in humans.

Tick-borne flaviviruses (TBFVs) infect mammalian hosts through tick bites and can cause various serious illnesses, such as encephalitis and hemorrhagic fevers, both in humans and animals. Despite their importance to public health, there is limited epidemiological information on TBFV infection in Africa is limited. Herein, we report that a novel flavivirus, Mpulungu flavivirus (MPFV), was discovered in a Rhipicephalus muhsamae tick in Zambia. MPFV was found to be genetically related to Ngoye virus detected in ticks in Senegal, and these viruses formed a unique lineage in the genus Flavivirus. Analyses of dinucleotide contents of flaviviruses indicated that MPFV was similar to those of other TBFVs with a typical vertebrate genome signature, suggesting that MPFV may infect vertebrate hosts leading to significant public health problems. Bioinformatic analyses of the secondary structures in the 3′-untranslated regions (UTRs) revealed that MPFV exhibits unique exoribonuclease-resistant RNA (xrRNA) structures. Utilizing biochemical approaches, we clarified that two xrRNA structures of MPFV in the 3′-UTR could prevent exoribonuclease activity. In summary, our findings provide new information regarding the geographical distribution of TBFV and xrRNA structures in the 3′-UTR of flaviviruses.

Tick-borne flaviviruses (TBFVs) infect mammalian hosts through tick bites and can cause various serious illnesses, such as encephalitis and hemorrhagic fevers, both in humans and animals. Despite their importance to public health, there is limited epidemiological information on TBFV infection in Africa. Herein, we report that a novel flavivirus, Mpulungu flavivirus (MPFV), was discovered in a Rhipicephalus muhsamae tick in Zambia. MPFV was found to be genetically related to Ngoye virus detected in ticks in Senegal, and these viruses formed a unique lineage in the genus Flavivirus. Analyses of dinucleotide contents of flaviviruses indicated that MPFV was similar to those of other TBFVs with a typical vertebrate genome signature, suggesting that MPFV may infect vertebrate hosts. Bioinformatic analyses of the secondary structures in the 3′-untranslated regions (UTRs) revealed that MPFV exhibited unique exoribonuclease-resistant RNA (xrRNA) structures. Utilizing biochemical approaches, we clarified that two xrRNA structures of MPFV in the 3′-UTR could prevent exoribonuclease activity. In summary, our findings provide new information regarding the geographical distribution of TBFV and xrRNA structures in the 3′-UTR of flaviviruses.

Mosquito-borne alphaviruses are disseminated globally and cause febrile illness in humans and animals. Since the prevalence and diversity of alphaviruses has not been previously investigated in Zambia, reverse transcription PCR was employed as a broad-spectrum approach for the detection of alphaviruses in mosquitoes. From 552 mosquito pools, a novel alphavirus, tentatively named Mwinilunga alphavirus (MWAV), was discovered from a single Culex quinquefasciatus mosquito pool. The full genome of MWAV was subsequently determined, and pairwise comparisons demonstrated that MWAV represented a new alphavirus species. Phylogenetic analyses and a linear discriminant analysis based on the dinucleotide ratios in various virus sequences indicated that MWAV is related to a mosquito-specific alphavirus distinct from other known mosquito-borne alphaviruses due to its inability to replicate in vertebrate cell lines. Further analyses of these novel alphaviruses will help to facilitate a greater understanding of the molecular determinants of host range restriction and the evolutionary relationships of alphaviruses.

Ticks (Ixodidae and Argasidae) are important arthropod vectors of various pathogens that cause human and animal infectious diseases. Many previously published studies on tick-borne pathogens focused on those transmitted by ixodid ticks. Although there are increasing reports of viral pathogens associated with argasid ticks, information on bacterial pathogens they transmit is scarce. The aim of this molecular study was to detect and characterize Rickettsia and Anaplasmataceae in three different argasid tick species, Ornithodoros faini, Ornithodoros moubata, and Argas walkerae collected in Zambia. Rickettsia hoogstraalii and Rickettsia lusitaniae were detected in 77 % (77/100) of Ar. walkerae and 10 % (5/50) of O. faini, respectively. All O. moubata pool samples (n = 124) were negative for rickettsial infections. Anaplasmataceae were detected in 63 % (63/100) of Ar. walkerae and in 82.2 % (102/124) of O. moubata pools, but not in O. faini. Phylogenetic analysis based on the concatenated sequences of 16S rRNA and groEL genes revealed that Anaplasma spp. detected in the present study were distinct from previously validated Anaplasma species, indicating that the current knowledge on the diversity and vector range of Anaplasma spp. is incomplete. Our findings highlight new geographical records of R. lusitaniae and R. hoogstraalii and confirm that the wide geographic distribution of these species includes the African continent. The data presented here increase our knowledge on argasid tick-borne bacteria and contribute toward understanding their epidemiology.

To monitor the arthropod-borne virus transmission in mosquitoes, we have attempted both to detect and isolate viruses from 3304 wild-caught female mosquitoes in the Livingstone (Southern Province) and Mongu (Western Province) regions in Zambia in 2017. A pan-flavivirus RT-PCR assay was performed to identify flavivirus genomes in total RNA extracted from mosquito lysates, followed by virus isolation and full genome sequence analysis using next-generation sequencing and rapid amplification of cDNA ends. We isolated a newly identified Barkedji virus (BJV Zambia) (10,899 nt) and a novel flavivirus, tentatively termed Barkedji-like virus (BJLV) (10,885 nt) from Culex spp. mosquitoes which shared 96% and 75% nucleotide identity with BJV which has been isolated in Israel, respectively. These viruses could replicate in C6/36 cells but not in mammalian and avian cell lines. In parallel, a comparative genomics screening was conducted to study evolutionary traits of the 5&prime, and 3&prime, untranslated regions (UTRs) of isolated viruses. Bioinformatic analyses of the secondary structures in the UTRs of both viruses revealed that the 5&prime, UTRs exhibit canonical stem-loop structures, while the 3&prime, UTRs contain structural homologs to exoribonuclease-resistant RNAs (xrRNAs), SL-III, dumbbell, and terminal stem-loop (3&prime, SL) structures. The function of predicted xrRNA structures to stop RNA degradation by Xrn1 exoribonuclease was further proved by the in vitro Xrn1 resistance assay.

Bat-associated bartonellae, including Bartonella mayotimonensis and Candidatus Bartonella rousetti, were recently identified as emerging and potential zoonotic agents, respectively. However, there is no report of bat-associated bartonellae in Zambia. Thus, we aimed to isolate and characterize Bartonella spp. from bats and bat flies captured in Zambia by culturing and PCR. Overall, Bartonella spp. were isolated from six out of 36 bats (16.7%), while Bartonella DNA was detected in nine out of 19 bat flies (47.3%). Subsequent characterization using a sequence of five different genes revealed that three isolates obtained from Egyptian fruit bats (Rousettus aegyptiacus) were Ca. B. rousetti. The isolates obtained from insectivorous bats (Macronycteris vittatus) were divided into two previously unclassified bat-associated bartonellae. A phylogenetic analysis of the six genotypes of Bartonella gltA sequences from nine pathogen-positive bat flies revealed that three genotypes belonged to the same clades as bat-associated bartonellae, including Ca. B. rousetti. The other three genotypes represented arthropod-associated bartonellae, which have previously been isolated only from ectoparasites. We demonstrated that Ca. B. rousetti is maintained between bats (R. aegyptiacus) and bat flies in Zambia. Continuous surveillance of Bartonella spp. in bats and serological surveys in humans in Africa are warranted to evaluate the public health importance of bat-associated bartonellae.

Bats are suspected to play important roles in the ecology of filoviruses, including ebolaviruses and marburgviruses. A cave-dwelling fruit bat, Rousettus aegyptiacus, has been shown to be a reservoir of marburgviruses. Using an enzyme-linked immunosorbent assay with the viral glycoprotein antigen, we detected immunoglobulin G antibodies specific to multiple filoviruses in 158 of 290 serum samples of R aegyptiacus bats captured in Zambia during the years 2014-2017. In particular, 43.8% of the bats were seropositive to marburgvirus, supporting the notion that this bat species continuously maintains marburgviruses as a reservoir. Of note, distinct peaks of seropositive rates were repeatedly observed at the beginning of rainy seasons, suggesting seasonality of the presence of newly infected individuals in this bat population. These data highlight the need for continued monitoring of filovirus infection in this bat species even in countries where filovirus diseases have not been reported.

Bacillus anthracis is a Gram-positive endospore-forming bacterial species that causes anthrax in both humans and animals. In Zambia, anthrax cases are frequently reported in both livestock and wildlife, with occasional transmission to humans, causing serious public health problems in the country. To understand the genetic diversity of B. anthracis strains in Zambia, we sequenced and compared the genomic DNA of B. anthracis strains isolated across the country. Single nucleotide polymorphisms clustered these strains into three groups. Genome sequence comparisons revealed a large deletion in strains belonging to one of the groups, possibly due to unequal crossing over between a pair of rRNA operons. The deleted genomic region included genes conferring resistance to bacitracin, and the strains with the deletion were confirmed with loss of bacitracin resistance. Similar deletions between rRNA operons were also observed in a few B. anthracis strains phylogenetically distant from Zambian strains. The structure of bacitracin resistance genes flanked by rRNA operons was conserved only in members of the Bacillus cereus group. The diversity and genomic characteristics of B. anthracis strains determined in this study would help in the development of genetic markers and treatment of anthrax in Zambia. IMPORTANCE Anthrax is caused by Bacillus anthracis, an endospore-forming soil bacterium. The genetic diversity of B. anthracis is known to be low compared with that of Bacillus species. In this study, we performed whole-genome sequencing of Zambian isolates of B. anthracis to understand the genetic diversity between closely related strains. Comparison of genomic sequences revealed that closely related strains were separated into three groups based on single nucleotide polymorphisms distributed throughout the genome. A large genomic deletion was detected in the region containing a bacitracin resistance gene cluster flanked by rRNA operons, resulting in the loss of bacitracin resistance. The structure of the deleted region, which was also conserved among species of the Bacillus cereus group, has the potential for both deletion and amplification and thus might be enabling the species to flexibly control the level of bacitracin resistance for adaptive evolution.

Feline calicivirus (FCV) is a pathogenic microorganism that causes upper respiratory diseases in cats. Recently, an FCV infection with a high mortality rate has been confirmed, and there is need to develop a treatment for cases of acute infection. We evaluated whether the replication of FCV could be prevented by RNA interference. For this study, we designed an siRNA targeted to the polymerase region of the strain FCV-B isolated from a cat that died after exhibiting neurological symptoms. Cells transfected with siR-pol dose-dependently suppressed the replication of FCV-B. siR-pol suppressed its replication by suppressing the target viral RNA.

The JM1/1 strain of fowl adenovirus (FAV) serotype 1 isolated from gizzard erosion was used to investigate the biology of FAV in homologous (susceptible) and heterologous cells. The FAV JM1/1 strain is capable of efficient multiplication in primary chicken kidney (CK) cells, but not in Crandell-Rees feline kidney (CRFK) cells or Vero cells. FAV adsorption in heterologous cells was slightly higher than in CK cells. An early gene encoding a DNA-binding protein and a late gene encoding the hexon protein were expressed in CK cells. Only the early gene was expressed in Vero cells. Neither of these genes was expressed in CRFK cells. These results suggest that the virus was unable to multiply effectively due to suppression of viral gene expression in the heterologous cells used in this study.