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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.

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.

Rabies remains endemic in Zambia. Despite conducting canine vaccinations in Lusaka district, the vaccination coverage and actual seropositivity in the dog population in Lusaka district are rarely evaluated. This study estimated the seropositivity-based immunization coverage in the owned dog population in Lusaka district using the expanded program on immunization cluster survey method. The time-series trend of neutralizing antibodies against rabies in vaccinated dogs was also evaluated. Of 366 dogs in 200 dog-owning households in Lusaka district, blood samples were collected successfully from 251 dogs. In the sampled dogs, 42.2% (106/251) had an antibody titer ≥0.5 IU/mL. When the 115 dogs whose blood was not collected were assumed to be seronegative, the minimum immunization coverage in Lusaka district’s owned dog population was estimated at 29.0% (95% confidence interval: 22.4–35.5). It was also found that a single vaccination with certified vaccines is capable of inducing protective levels of antibodies. In contrast, higher antibody titers were observed in multiple-vaccinated dogs than in single-vaccinated dogs, coupled with the observation of a decline in antibody titer over time. These results suggest the importance of continuous booster immunization to maintain herd immunity and provide useful information to plan mass vaccination against rabies in Zambia.

Whilst bovine leukemia virus (BLV) causes considerable economic losses to the dairy industry worldwide, information on its molecular epidemiology and economic impact in beef cattle is limited. Here, blood from 880 animals from Zambia's major cattle-rearing provinces was screened for BLV by nested PCR. Positive pools were sequenced and phylogenetically analyzed. The estimated pooled prevalence was 2.1%. All strains belonged to genotype 1 and formed a distinct phylogenetic cluster. The study suggests circulation of genotype 1 BLV in beef cattle in these regions. This is the first report on molecular detection and characterization of BLV from beef cattle in Africa.

Rabies is endemic in Zambia and Zimbabwe. The previously investigated strains of rabies virus in central Zambia belong to the Africa 1b lineage, with similar circulating virus strains found in the various tested hosts and regions. However, prior work assessed only limited regions and host species. Thus, this study aimed to more comprehensively determine the genetic diversity of rabies virus across regions of Zambia and Zimbabwe. RNA (n = 76) was extracted from positive direct fluorescent antibody test brain tissues from dog, cow, goat, cat, pig, human, and jackal collected from Zambia and Zimbabwe. The amplicons of the nucleoprotein and glycoprotein genes were obtained from all examined samples by nested RT-PCR and subsequently sequenced. A phylogenetic analysis of the N gene confirmed that all the endemic strains of rabies virus in Zambia and Zimbabwe belong to the Africa 1b lineage. The obtained viral gene sequences were phylogenetically divided into two clusters. Cluster II comprised only Zambian strains. In contrast, cluster I comprised both Zambia and Zimbabwe strains, with strains from Zimbabwe forming a distinct lineage from Zambian strains, implying viral genetic divergence due to geographical barriers. However, no evidence of clustering based on host or region was observed, implying the circulation of similar virus strains occurs in different hosts and regions of Zambia and Zimbabwe. The clustering of rabies virus strains from jackals with those from domestic animals provides evidence of similar virus strains circulating in both wildlife and domestic animals, and that the jackal might be one of the potential reservoirs of rabies virus infection. In this study, no strains circulating in Zimbabwe were detected in Zambia.

Zika virus (ZIKV) circulation occurs between non-human primates (NHPs) in a sylvatic transmission cycle. To investigate evidence of flavivirus infection in NHPs in Zambia, we performed a plaque reduction neutralization test (PRNT) to quantify neutralizing antibodies. PRNT revealed that sera from NHPs (African green monkeys and baboons) exhibited neutralizing activity against ZIKV (34.4%; 33/96), whereas a PRNT for yellow fever virus using NHP sera showed no neutralization activity. ZIKV genomic RNA was not detected in splenic tissues from NHPs, suggesting that the presence of anti-ZIKV neutralizing antibodies represented resolved infections. Our evidence suggests that ZIKV is maintained in NHP reservoirs in Zambia.

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.

Giardia duodenalis is one of the most common causes of diarrhea in humans with about 250–300 million cases per year. It is considered to be a species complex comprising of eight genetic assemblages (A to H), with assemblages A and B being the major causes of human infections. In this study we carried out genotypic characterization of G. duodenalis isolates detected in asymptomatic school-going children aged 3–16 years. Between May and September 2017, a total of 329 fecal samples were collected from school-going children from Chawama compound of Lusaka City and were screened for Giardia by microscopic examination. All microscopically positive fecal samples were analyzed by semi-nested polymerase chain reaction (PCR) targeting the glutamate dehydrogenase (gdh) gene. Genotyping of amplified PCR products was conducted by restriction fragment length polymorphism (RFLP) and DNA sequence analysis. Microscopically, Giardia was found in 10% (33/329) of fecal samples. The PCR-RFLP analysis of the gdh gene revealed assemblages A and B in 27.3% (9/33) and 72.7% (24/33), respectively. Furthermore, analysis with restriction enzymes identified sub-assemblages AII (27.3%, 9/33), BIII (12.1%, 4/33), BIV (51.5%, 17/33) and mixed infections of BIII and BIV (9.1%, 3/33). Phylogenetic analysis showed the clustering of 27.6% (8/29) and 72.4% (21/29) of Zambian Giardia gdh gene sequences into assemblages A and B, respectively. This study has revealed the presence of both assemblage A and B and that spread of G. duodenalis in school-going children appears to be mostly through anthroponotic transmission. To our knowledge, this is the first report of genotypic characterization of G. duodenalis identified in Zambia.

Group A rotavirus (RVA) is a major cause of diarrhea in children worldwide. Although RVA infects many animals, little is known about RVA in bats. The present study investigated the genetic diversity of RVA in Zambian bats. We identified RVA from two straw-colored fruit bats (Eidolon helvum) and an Egyptian fruit bat (Rousettus aegyptiacus), and analyzed the genome sequences of these strains. Genome segments of the RVA strains from Zambian E. helvum showed 97%–99% nucleotide sequence identity with those of other RVA strains from E. helvum in Cameroon, which is 2800 km from the sampling locations. These findings suggest that migratory straw-colored fruit bat species, distributed across sub-Saharan Africa, have the potential to disseminate RVA across long distances. By contrast, the RVA strain from Zambian R. aegyptiacus carried highly divergent NSP2 and NSP4 genes, leading us to propose novel genotypes N21 and E27, respectively. Notably, this RVA strain also shared the same genotype for VP6 and NSP3 with the RVA strains from Zambian E. helvum, suggesting interspecies transmission and genetic reassortment may have occurred between these two bat species in the past. Our study has important implications for RVA dispersal in bat populations, and expands our knowledge of the ecology, diversity and evolutionary relationships of RVA., Highlights • Detection of group A rotavirus from Zambian fruit bats. • Some viral genes were almost identical to those of rotavirus from Cameroonian bats. • The findings provide evidence for long-distance dispersal events of rotavirus. • First report of novel N21 and E27 genotypes.

Mosquito surveillance studies to identify mosquito-borne flaviviruses have identified West Nile Virus (WNV) for the first time in Zambia. The Zambian WNV isolate from Culex quinquefasciatus mosquitoes collected in the Western Province was closely related genetically to WNV lineage 2 South African strains which have been previously shown to be highly neuroinvasive. These data provide the first evidence of the circulation of WNV in Zambia and suggest there should be an increased awareness of possible associated human and animal diseases in that country.

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.

Infectious bursal disease (IBD) is a highly contagious, immunosuppressive disease of chickens and causes substantial economic losses to the poultry industry globally. This study investigated the genetic characteristics and pathological lesions induced by IBD viruses (IBDVs) that were associated with 60 suspected outbreaks in chickens during 2015-2016 in Lusaka Province, Zambia. Nucleotide sequences of VP2 hypervariable region (VP2-HVR) (n = 38) and part of VP1 (n = 37) of Zambian IBDVs were phylogenetically analysed. Phylogenetic analysis of the VP2-HVR and VP1 revealed that most viruses (n = 31 of each genome segment) clustered with the very virulent (vv) strains. The rest of the viruses clustered with the classical strains, with two of the viruses being closely related to attenuated vaccine isolates. Two of the viruses that belonged to the vv genotype had a unique amino acid (aa) substitution Q324L whereas one virus had two unique changes, N280S and E300A in the VP2-HVR aa sequence. Although Zambian strains with a vv genotype possessed virulence marker aa within VP1 at 145T, 146D and 147N, two viruses showed unique substitutions, with one virus having 147T while the other had 147H. Pathologically, it was noted that only viruses with a vv genotype appeared to be associated with inducing pathological lesions in non-lymphoid organs (proventriculus and gizzard). Whilst documenting for the first time the presence of classical virulent IBDVs, this study demonstrates the involvement of multiple genotypes, with predominance of vvIBDVs in the epidemiology of IBD in Zambia.

Although tick-borne pathogens, Anaplasma platys and Anaplasma phagocytophilum are recognized as zoonotic agents associated with appreciable morbidity and mortality in dogs and humans worldwide, there is limited information on these infections in many African countries, including Zambia. The purpose of this study was to detect, identify and phylogenetically characterize Anaplasma species from dogs in Chilanga District in Lusaka Province, Zambia. A total of 301 blood samples were collected from apparently healthy and semi-confined dogs. Initial screening by polymerase chain reaction with specific primers targeting the 16S rRNA gene of Anaplasma species revealed that 9% (27/301) of our samples were positive. Subsequent sequence and phylogenetic analysis of a longer fragment of the 16S rRNA and citrate synthase (gltA) genes of four positive samples showed the presence of A. platys and an Anaplasma species, which was closely related to those detected in dogs in South Africa. This is the first report on molecular identification and characterization of canine-associated zoonotic Anaplasma species in Zambia.

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne zoonosis with a high case fatality rate in humans. Although the disease is widely found in Africa, Europe, and Asia, the distribution and genetic diversity of CCHF virus (CCHFV) are poorly understood in African countries. To assess the risks of CCHF in Zambia, where CCHF has never been reported, epidemiologic studies in cattle and ticks were conducted. Through an indirect immunofluorescence assay, CCHFV nucleoprotein-specific serum IgG was detected in 8.4% (88/1,047) of cattle. Among 290 Hyalomma ticks, the principal vector of CCHFV, the viral genome was detected in 11 ticks. Phylogenetic analyses of the CCHFV S and M genome segments revealed that one of the detected viruses was a genetic reassortant between African and Asian strains. This study provides compelling evidence for the presence of CCHFV in Zambia and its transmission to vertebrate hosts., Author summary Crimean-Congo hemorrhagic fever (CCHF) is a severe viral disease mainly transmitted by ticks. Effective prophylactics and therapeutics have not been established for this disease yet. While CCHF is endemic in Africa, information on the distribution and genetic diversity of CCHF virus (CCHFV) is quite limited in many Sub-Saharan African countries. In this study, we conducted serologic and molecular epidemiologic investigations for CCHFV infection in cattle and ticks in Zambia. Serologic screening revealed that 8.4% of cattle were tested positive for CCHFV-specific IgG. Hyalomma ticks infected with CCHFV were also identified by genetic screening. Phylogenetic analyses showed that one of the CCHFVs detected in Zambia was a genetic reassortant between African and Asian CCHFV strains. Currently, Zambia is considered CCHF-free country because CCHF cases have never been reported. However, the findings in this study indicate that CCHFV is maintained in Hyalomma ticks and occasionally transmitted to vertebrate hosts such as cattle in Zambia. Further epidemiologic studies and continuous monitoring of CCHFV infection should be implemented in the southern African region.

Orf or contagious ecthyma is a neglected and economically important zoonotic disease caused by a dermatotropic parapoxvirus that commonly affects domestic small ruminants. Although orf is globally distributed, there is a paucity of information on the disease in many African countries. Here, a suspected severe outbreak of orf in goats at a farm in Lusaka was investigated. Orf virus (ORFV) infection was confirmed by PCR amplification of viral DNA (RNA polymerase, B2L and virus interferon-resistance genes) in clinical samples. Some detected genes were sequenced and phylogenetically analyzed. This is the first report on molecular characterization of ORFV in goats in Zambia.

Bat species represent natural reservoirs for a number of high-consequence human pathogens. The present study investigated the diversity of polyomaviruses (PyVs) in Zambian insectivorous and fruit bat species. We describe the complete genomes from four newly proposed African bat PyV species employing the recently recommended criteria provided by the Polyomaviridae Study Group of the International Committee on Taxonomy of Viruses. A comprehensive phylogenetic and recombination analysis was performed to determine genetic relationships and the distribution of recombination events in PyV from mammalian and avian species. The novel species of PyV from Zambian bats segregated with members of the genera Alphapolyomavirus and Betapolyomavirus, forming monophyletic clades with bat and non-human primate PyVs. Miniopterus schreibersii polyomavirus 1 and 2 segregated in a clade with South American bat PyV species, Old World monkey and chimpanzee PyVs and Human polyomavirus 13 (New Jersey PyV). Interestingly, the newly described Egyptian fruit bat PyV, tentatively named Rousettus aegyptiacus polyomavirus 1, had the highest nucleotide sequence identity to species of PyV from Indonesian fruit bats, and Rhinolophus hildebrandtii polyomavirus 1 was most closely related to New World monkey PyVs. The distribution of recombination events in PyV genomes was non-random: recombination boundaries existed in the intergene region between VP1 and LTAg and also at the 3' end of VP2/3 in the structural genes, whereas infrequent recombination was present within the LTAg gene. These findings indicate that recombination within the LTAg gene has been negatively selected against during polyomaviral evolution and support the recent proposal for taxonomic classification based on LTAg to define novel PyV species.

During 2013-2015, several and severe outbreaks of African swine fever (ASF) affected domestic pigs in six provinces of Zambia. Genetic characterization of ASF viruses (ASFVs) using standardized genotyping procedures revealed that genotypes I, II and XIV were associated with these outbreaks. Molecular and epidemiological data suggest that genotype II ASFV (Georgia 2007/1-like) detected in Northern Province of Zambia may have been introduced from neighbouring Tanzania. Also, a genotype II virus detected in Eastern Province of Zambia showed a p54 phylogenetic relationship that was inconsistent with that of p72, underscoring the genetic variability of ASFVs. While it appears genotype II viruses detected in Zambia arose from a domestic pig cycle, genotypes I and XIV possibly emerged from a sylvatic cycle. Overall, this study demonstrates the co-circulation of multiple genotypes of ASFVs, involvement of both the sylvatic and domestic pig cycle in ASF outbreaks in Zambia and possible trans-boundary spread of the disease in south-eastern Africa. Indeed, while there is need for regional or international concerted efforts in the control of ASF, understanding pig marketing practices, pig population dynamics, pig housing and rearing systems and community engagement will be important considerations when designing future prevention and control strategies of this disease in Zambia.

To investigate the diversity of simian immunodeficiency virus (SIV) among nonhuman primates (NHPs) in Zambia, next-generation sequencing was performed to determine the complete genome sequence of a novel SIV recovered by co-culturing African green monkey (AGM) peripheral blood lymphocytes with human CD4(+) T-cell lines. We report the first described SIV (SIVagmMAL-ZMB) from a malbrouck (Chlorocebus cynosuros). SIVagmMAL-ZMB was detected by real-time PCR analysis of splenic RNA in 3.2% (3/94) of AGMs and was undetectable in baboons (0/105). SIVagmMAL-ZMB possessed < 80% nucleotide sequence identity to known SIV isolates and was located basally to vervet monkey SIV strains in all phylogenies.

Giardia duodenalis is one of the most common causes of diarrhea in humans with about 250–300 million cases per year. It is considered to be a species complex comprising of eight genetic assemblages (A to H), with assemblages A and B being the major causes of human infections. In this study we carried out genotypic characterization of G. duodenalis isolates detected in asymptomatic school-going children aged 3–16 years. Between May and September 2017, a total of 329 fecal samples were collected from school-going children from Chawama compound of Lusaka City and were screened for Giardia by microscopic examination. All microscopically positive fecal samples were analyzed by semi-nested polymerase chain reaction (PCR) targeting the glutamate dehydrogenase (gdh) gene. Genotyping of amplified PCR products was conducted by restriction fragment length polymorphism (RFLP) and DNA sequence analysis. Microscopically, Giardia was found in 10% (33/329) of fecal samples. The PCR-RFLP analysis of the gdh gene revealed assemblages A and B in 27.3% (9/33) and 72.7% (24/33), respectively. Furthermore, analysis with restriction enzymes identified sub-assemblages AII (27.3%, 9/33), BIII (12.1%, 4/33), BIV (51.5%, 17/33) and mixed infections of BIII and BIV (9.1%, 3/33). Phylogenetic analysis showed the clustering of 27.6% (8/29) and 72.4% (21/29) of Zambian Giardia gdh gene sequences into assemblages A and B, respectively. This study has revealed the presence of both assemblage A and B and that spread of G. duodenalis in school-going children appears to be mostly through anthroponotic transmission. To our knowledge, this is the first report of genotypic characterization of G. duodenalis identified in Zambia., Graphical abstract Unlabelled Image, Highlights • Giardia duodenalis infection observed in asymptomatic children • First report of genotypes circulating in Zambia • Presence of sub-assemblages AII, BIII and BIV • Co-infection with sub-assemblages BIII and BIV reported

The Smacoviridae has recently been classified as a family of small circular single-stranded DNA viruses. An increasing number of smacovirus genomes have been identified exclusively in faecal matter of various vertebrate species and from insect body parts. However, the genetic diversity and host range of smacoviruses remains to be fully elucidated. Herein, we report the genetic characterization of eleven circular replication-associated protein (Rep) encoding single-stranded (CRESS) DNA viruses detected in the faeces of Zambian non-human primates. Based on pairwise genome-wide and amino acid identities with reference smacovirus species, ten of the identified CRESS DNA viruses are assigned to the genera Porprismacovirus and Huchismacovirus of the family Smacoviridae, which bidirectionally encode two major open reading frames (ORFs): Rep and capsid protein (CP) characteristic of a type IV genome organization. The remaining unclassified CRESS DNA virus was related to smacoviruses but possessed a genome harbouring a unidirectionally oriented CP and Rep, assigned as a type V genome organization. Moreover, phylogenetic and recombination analyses provided evidence for recombination events encompassing the 3'-end of the Rep ORF in the unclassified CRESS DNA virus. Our findings increase the knowledge of the known genetic diversity of smacoviruses and highlight African non-human primates as carrier animals.

The Smacoviridae has recently been classified as a family of small circular single-stranded DNA viruses. An increasing number of smacovirus genomes have been identified exclusively in faecal matter of various vertebrate species and from insect body parts. However, the genetic diversity and host range of smacoviruses remains to be fully elucidated. Herein, we report the genetic characterization of eleven circular replication-associated protein (Rep) encoding single-stranded (CRESS) DNA viruses detected in the faeces of Zambian non-human primates. Based on pairwise genome-wide and amino acid identities with reference smacovirus species, ten of the identified CRESS DNA viruses are assigned to the genera Porprismacovirus and Huchismacovirus of the family Smacoviridae, which bidirectionally encode two major open reading frames (ORFs): Rep and capsid protein (CP) characteristic of a type IV genome organization. The remaining unclassified CRESS DNA virus was related to smacoviruses but possessed a genome harbouring a unidirectionally oriented CP and Rep, assigned as a type V genome organization. Moreover, phylogenetic and recombination analyses provided evidence for recombination events encompassing the 3′-end of the Rep ORF in the unclassified CRESS DNA virus. Our findings increase the knowledge of the known genetic diversity of smacoviruses and highlight African non-human primates as carrier animals.

Rift Valley fever (RVF) is a zoonotic mosquito-borne disease caused by RVF virus (RVFV) that causes abortions and high mortalities in livestock and is also associated with acute and fatal disease in humans. In the Democratic Republic of Congo (DRC), information on the epidemiology of RVF is limited, particularly among cattle reared by smallholder farmers. This cross-sectional study was conducted to investigate the seroprevalence of RVF in cattle raised by smallholder farmers in Kwilu Province of DRC, which has not yet reported an RVF epidemic. A total of 677 cattle sera were collected from four territories and tested for anti-RVFV antibodies using immunofluorescent assay and enzyme-linked immunosorbent assay. The overall seroprevalence of anti-RVFV IgG was 6.5% (44/677) (95% CI 4.81–8.7). There was a statistically significant difference in the seroprevalence among the territories (χ2 = 28.79, p 3 years. Forest area (18.92% (95% CI 12.35–27.7)) had higher seropositivity than savannah area (4.06% (95% CI 2.65–6.12)). Sex difference was not significant (χ2 = 0.14, p = 0.704). These findings indicate that cattle in Kwilu Province had been exposed to RVFV, which represents a significant risk for both livestock and human health.

Lujo virus is a novel Old World arenavirus identified in Southern Africa in 2008 as the cause of a viral hemorrhagic fever (VHF) characterized by nosocomial transmission with a high case fatality rate of 80% (4/5 cases). Whereas this outbreak was limited, the unprecedented Ebola virus disease outbreak in West Africa, and recent Zika virus disease epidemic in the Americas, has brought into acute focus the need for preparedness to respond to rare but potentially highly pathogenic outbreaks of zoonotic or arthropod-borne viral infections. A key determinant for effective control of a VHF outbreak is the time between primary infection and diagnosis of the index case. Here, we review the Lujo VHF outbreak of 2008 and discuss how preparatory measures with respect to developing diagnostic capacity might be effectively embedded into existing national disease control networks, such as those for human immunodeficiency virus, tuberculosis, and malaria.

The latest outbreak of Ebola virus disease (EVD) in West Africa has highlighted the urgent need for the development of rapid and reliable diagnostic assays. We used monoclonal antibodies specific to the ebolavirus nucleoprotein to develop an immunochromatography (IC) assay (QuickNavi-Ebola) for rapid diagnosis of EVD. The IC assay was first evaluated with tissue culture supernatants of infected Vero E6 cells and found to be capable of detecting 10(3)-10(4) focus-forming units/mL of ebolaviruses. Using serum samples from experimentally infected nonhuman primates, we confirmed that the assay could detect the viral antigen shortly after disease onset. It was also noted that multiple species of ebolaviruses could be detected by the IC assay. Owing to the simplicity of the assay procedure and absence of requirements for special equipment and training, QuickNavi-Ebola is expected to be a useful tool for rapid diagnosis of EVD.

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.

Although canine parvovirus (CPV) causes severe gastroenteritis in dogs globally, information on the molecular epidemiology of the virus is lacking in many African countries. Here, 32 fecal samples collected from diarrheic dogs in Zambia were tested for CPV infection using molecular assays. CPV was detected in 23 samples (71.9%). Genetic characterization revealed the predominance of CPV-2c (91.3%). This finding differs from previous reports in Africa, which indicated that CPV-2a and CPV-2b were most prevalent. Phylogenetically, most Zambian CPVs formed a distinct cluster. This is the first report on the molecular characterization of CPV in Zambia.

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.

The open reading frame of the nucleocapsid protein (NP) of Rift Valley fever virus (RVFV) strain MP12 was cloned and expressed in Vero E6 cells. The recombinant NP (rNP)-expressing cells were used as antigens for an indirect immunofluorescent antibody assay (IFA). The rNP-based IFA and RVFV-infected Vero E6 cell (authentic antigen)-based IFA showed similar IFA profiles with immune rabbit serum, which was prepared by immunization with rNP expressed using a baculovirus vector. A total of 942 traditional cattle sera obtained in five districts in Central, Southern, and Western provinces of Zambia were screened for anti-RVFV antibodies by the authentic antigen-based and rNP-based IFAs. Significant agreement was obtained between the two IFAs. The findings show that the rNP-based IFA is a safe and useful diagnostic tool as an alternative to the authentic antigen-based IFA. The antibody titers given by the rNP-based IFA were higher than those by the authentic antigen-based IFA. Therefore, the rNP-based IFA might be useful for serosurveillance of RVFV infection among cattle. Antibody prevalence rates in the five districts were 1.3% to 13.5% in the authentic antigen-based IFA and 6.0% to 21.4% in the rNP-based IFA. The results indicated that despite no reports of active cases of RVF in these provinces of Zambia, the virus is circulating among cattle herds.

Coxiella burnetii, the causative agent of Q fever, is a zoonotic pathogen associated with sylvatic or domestic transmission cycles, with rodents being suspected to link the two transmission cycles. Infection and subsequent disease in humans has historically been associated with contact with infected livestock, especially sheep. However, recently there have been reports of Q fever outbreaks associated with contact with infected rodents and dogs. Studies exploring the potential role of these animal hosts in the epidemiology of Q fever in many developing countries in Africa are very limited. This study aimed to determine the potential role of rodents and dogs in the epidemiological cycle of C. burnetti in Zambia. Using pathogen-specific polymerase chain reaction assays targeting the 16S rRNA gene, C. burnetii was detected for the first time in 45% of rodents (9/20), in one shrew and in 10% of domestic dogs (15/150) screened in Zambia. Phylogenetic characterization of six samples based on the isocitrate synthase gene revealed that the strains were similar to a group of isolates from chronic human Q fever patients, goats and rodents reported in multiple continents. Considering the close proximity of domestic dogs and rodents to humans, especially in resource-limited communities, the presence of C. burnetii in these animals could be of significant public health importance. It is thus important to determine the burden of Q fever in humans in such resource-limited communities where there is close contact between humans, rodents and dogs.

Bats are important reservoirs for emerging zoonotic viruses. For extensive surveys of potential pathogens in straw-colored fruit bats (Eidolon helvum) in Zambia, a total of 107 spleen samples of E. helvum in 2006 were inoculated onto Vero E6 cells. The cell culture inoculated with one of the samples (ZFB06-106) exhibited remarkable cytopathic changes. Based on the ultrastructural property in negative staining and cross-reactivity in immunofluorescence assays, the virus was suspected to be an adenovirus, and tentatively named E. helvum adenovirus 06-106 (EhAdV 06-106). Analysis of the full-length genome of 30,134 bp, determined by next-generation sequencing, showed the presence of 28 open reading frames. Phylogenetic analyses confirmed that EhAdV 06-106 represented a novel bat adenovirus species in the genus Mastadenovirus. The virus shared similar characteristics of low G + C contents with recently isolated members of species Bat mastadenoviruses E, F and G, from which EhAdV 06-106 diverged by more than 15% based on the distance matrix analysis of DNA polymerase amino acid sequences. According to the taxonomic criteria, we propose the tentative new species name “Bat mastadenovirus H”. Because EhAdV 06-106 exhibited a wide in vitro cell tropism, the virus might have a potential risk as an emerging virus through cross-species transmission.

Fruit bats are suspected to be a natural reservoir of filoviruses, including Ebola and Marburg viruses. Using an enzyme-linked immunosorbent assay based on the viral glycoprotein antigens, we detected filovirus-specific immunoglobulin G antibodies in 71 of 748 serum samples collected from migratory fruit bats (Eidolon helvum) in Zambia during 2006-2013. Although antibodies to African filoviruses (eg, Zaire ebolavirus) were most prevalent, some serum samples showed distinct specificity for Reston ebolavirus, which that has thus far been found only in Asia. Interestingly, the transition of filovirus species causing outbreaks in Central and West Africa during 2005-2014 seemed to be synchronized with the change of the serologically dominant virus species in these bats. These data suggest the introduction of multiple species of filoviruses in the migratory bat population and point to the need for continued surveillance of filovirus infection of wild animals in sub-Saharan Africa, including hitherto nonendemic countries.

Viral metagenomic analysis identified a new parvovirus genome in the intestinal contents of wild shrews in Zambia. Related viruses were detected in spleen tissues from wild shrews and nonhuman primates. Phylogenetic analyses showed that these viruses are related to human bufaviruses, highlighting the presence and genetic diversity of bufaviruses in wildlife.

Human monkeypox is a viral zoonosis caused by monkeypox virus, an orthopoxvirus (OPXV). The majority of human monkeypox cases have been reported in moist forested regions in West and Central Africa, particularly in the Democratic Republic of the Congo (DRC). In this study we investigated zoonotic OPXV infection among wild animals in Zambia, which shares a border with DRC, to assess the geographical distribution of OPXV. We screened for OPXV antibodies in sera from non-human primates (NHPs), rodents and shrews by ELISA, and performed real-time PCR to detect OPXV DNA in spleen samples. Serological analysis indicated that 38 of 259 (14.7 %) rodents, 14 of 42 (33.3 %) shrews and 4 of 188 (2.1 %) NHPs had antibodies against OPXV. The OPXV DNA could not be detected in spleens from any animals tested. Our results indicated that wild animals living in rural human habitation areas of Zambia have been infected with OPXV.

A study was conducted to determine the serotypes of foot-and-mouth disease viruses (FMDV) circulating in African buffaloes (Syncerus caffer) from selected areas in Zambia. Sera and probang samples were collected between 2011 and 2012 and analysed for presence of antibodies against FMDV while probang samples were used to isolate the FMDV by observing cytopathic effect (CPE). Samples with CPE were further analysed using antigen ELISA. High FMD seroprevalence was observed and antibodies to all the three Southern African Territories (SAT) serotypes were detected in four study areas represented as follows: SAT2 was 72.7 percent; SAT1 was 62.6 percent; and SAT3 was 26.2 percent. Mixed infections accounted for 68.6 percent of those that were tested positive. For probang samples, CPE were observed in three of the samples, while the antigen ELISA results showed positivity and for SAT1 (n=1) and SAT2 (n=2). It is concluded that FMDV is highly prevalent in Zambian buffaloes which could play an important role in the epidemiology of the disease. Therefore livestock reared at interface with the game parks should be included in all routine FMDV vaccination programmes.

Childhood lead poisoning is a serious public health concern worldwide. Blood lead levels exceeding 5 mu g dL(-1) are considered elevated. In Kabwe, the capital of Zambia's Central Province, extensive Pb contamination of township soils in the vicinity of a Pb-Zn mine and posing serious health risk to children has been reported. We investigated BLLs in children under the age of 7 years in townships around the mine; where blood samples were collected and analyzed using an ICP-MS. All of the sampled children had BLLs exceeding 5 mu g dL(-1). Children in these areas could be at serious risk of Pb toxicity as 18% of the sampled children in Chowa, 57% (Kasanda) and 25% (Makululu) had BLLs exceeding 65 mu g dL(-1). Eight children had BLLs exceeding 150 mu g dL(-1) with the maximum being 427.8 mu g dL(-1). We recommend that medical intervention be commenced in the children with BLL exceeding 45 mu g dL(-1). (C) 2015 Elsevier Ltd. All rights reserved.