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.

Epidemiology of epizootic ulcerative syndrome (EUS) in fish in the main water bodies of the Kavango-Zambezi and Great Limpopo transfrontier conservation areas of Zimbabwe.

A three-year study investigated the epidemiology of epizootic ulcerative syndrome (EUS) in fish from Kavango-Zambezi (KAZA) and Great Limpopo (GL) transfrontier conservation areas of Zimbabwe. A total of 38 sites comprising 27 wild fisheries and 11 aquacultures, from Mashonaland West, Matabeleland North and South, and Midlands were selected. Of the 27 wild fisheries, EUS-positive fish were detected from 9 (33.3%) and none from the 11 aquacultures. No positive cases were detected from Nile tilapia (Oreochromis niloticus) from both aquacultures and wild fisheries. A total of 9.9% (239/2423) fish from the nine positive fisheries had typical EUS lesions, and infection was confirmed in 15 species. Prevalence was significantly higher (p < 0.05) in KAZA (11.5%; 95% CI: 9.6-13.4) compared with GL (8.6%; 95% CI: 7.1-10.1). The most affected were Clarias, followed by Barbus and Oreochromis species. Most cases (>80%) were reported in winter when ambient temperature was low. Further studies are required to determine water parameters associated with EUS outbreaks. These results suggested that the African sharptooth catfish (Clarias gariepinus) could be used potentially as an indicator species for EUS surveillance programmes. Thus, implementation of surveillance and biosecurity programmes that take into consideration the epidemiology of EUS will be beneficial.

Isolation, discrimination, and molecular detection of Listeria species from slaughtered cattle in Namwala District, Zambia.

BACKGROUND: The food industry is increasingly becoming more scrutinized, given the frequency and intensity with which zoonotic diseases are being reported. Pathogen tracking has become more applicable with regards food safety. It is in this regard that the present study was formulated to track Listeria species. in freshly slaughtered cattle carcasses by utilizing standard and molecular biological techniques. METHODS: A cross-sectional study design was conducted from March to December 2020 with 200 samples being equally collected in the rainy and dry seasons. A total of 180 and 20 swabs were aseptically collected from carcasses and the environment respectively. Samples were first subjected to pre-enrichment in half-strength Fraser broth followed by enrichment in full strength Fraser broth and subsequent plating on Listeria agar. Listeria growth characteristics were identified up to species level based on their morphological and biochemical characteristics. Further, molecular detection and phylogenetic analysis was conducted. Quantitative proportionate survey data were analyzed using Stata Version 15 software to estimate crude prevalence taking into account complex design at abattoir level. Factors associated with contamination were characterized using logistic regression. Sequences were analyzed using, Genetyyx version 12 and phylogenetic Mega. RESULTS: Of the 200 samples, 19 were positive for Listeria species identified as L.innocua 14/19 (73.7%) and L. monocytogenes 5/19 (26.3%). All isolates were from freshly slaughtered carcasses, and none from environment. Siginificant differences in contamination levels were observed based on season: rainy season yielded 14 (73.6%) whilst the dry season 5 (26.3%). The L. monocytogenes strains showed a high degree of homogeneity on phylogenetic analysis and clustered based on abattoir. Seasonality was identified as a major determinant influencing contamination based on the final logistic regression model. CONCLUSION: This study found evidence of L. monocytogenes contamination on traditionally raised beef carcasses across various abattoirs surveyed. The failure to find Listeria contamination on the abattoir environment may to a greater extent intimate cattle carccases as primary sources of contamination. However, a more comprerehnsive study incorporating different geographical regions is needed to conclusively ascertain these present findings.

Mastomys natalensis is a possible natural rodent reservoir for encephalomyocarditis virus.

Encephalomyocarditis virus (EMCV) infects a wide range of hosts and can cause encephalitis, myocarditis, reproductive disorders and diabetes mellitus in selected mammalian species. As for humans, EMCV infection seems to occur by the contact with animals and can cause febrile illnesses in some infected patients. Here we isolated EMCV strain ZM12/14 from a natal multimammate mouse (Mastomys natalensis: M. natalensis) in Zambia. Pairwise sequence similarity of the ZM12/14 P1 region consisting of antigenic capsid proteins showed the highest similarity of nucleotide (80.7 %) and amino acid (96.2%) sequence with EMCV serotype 1 (EMCV-1). Phylogenetic analysis revealed that ZM12/14 clustered into EMCV-1 at the P1 and P3 regions but segregated from known EMCV strains at the P2 region, suggesting a unique evolutionary history. Reverse transcription PCR (RT-PCR) screening and neutralizing antibody assays for EMCV were performed using collected tissues and serum from various rodents (n=179) captured in different areas in Zambia. We detected the EMCV genome in 19 M. natalensis (19/179=10.6 %) and neutralizing antibody for EMCV in 33 M. natalensis (33/179=18.4 %). However, we did not detect either the genome or neutralizing antibody in other rodent species. High neutralizing antibody litres (≧320) were observed in both RT-PCR-negative and -positive animals. Inoculation of ZM12/14 caused asymptomatic persistent infection in BALB/c mice with high antibody titres and high viral loads in some organs, consistent with the above epidemiological results. This study is the first report of the isolation of EMCV in Zambia, suggesting that M. natalensis may play a role as a natural reservoir of infection.

Lactococcus garvieae isolated from Lake Kariba (Zambia) has low invasive potential in Nile tilapia (Oreochromis niloticus).

The pathogenesis of Lactococcus garvieae (L. garvieae) was assessed in Nile tilapia (Oreochromis niloticus) following administration by two different routes of infection (intraperitoneal versus immersion), using 180 fish divided into three groups. The first group of fish was injected intraperitoneally (IP) with 3 × 105 colony-forming units (cfu) of L. garvieae; the second group was infected by immersion (IMM) into water containing 9.6 × 105  cfu/ml L. garvieae, and in group 3 (Control), the fish were injected IP with sterile normal saline. Mortalities were recorded daily, and on 3, 5, 7, and 13 days post-infection (dpi), liver, kidney, spleen, brain and eyes were sampled. The level of infection between groups was assessed by number of mortalities that occurred, pathology/histopathology of internal organs, bacterial re-isolation and presence of bacteria in situ determined using immunohistochemistry. A significant difference (p < .0001) was observed between L. garvieae re-isolation from tilapia following administration by IP injection and IMM. Similarly, more clinical signs and mortalities (p < .001) were observed in the IP group compared to the IMM group where no mortalities were observed. These findings suggest that L. garvieae has a low invasive potential in Nile tilapia with intact skin/external barriers and highlights the importance of maintaining fish without cuts or abrasions under field conditions.

West Nile Virus in Farmed Crocodiles, Zambia, 2019.

We detected West Nile virus (WNV) nucleic acid in crocodiles (Crocodylus niloticus) in Zambia. Phylogenetically, the virus belonged to lineage 1a, which is predominant in the Northern Hemisphere. These data provide evidence that WNV is circulating in crocodiles in Africa and increases the risk for animal and human transmission.

Characterization of mammalian orthoreoviruses isolated from faeces of pigs in Zambia.

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.

Human Borreliosis Caused by a New World Relapsing Fever Borrelia-like Organism in the Old World.

BACKGROUND: Relapsing fever is an infectious disease previously neglected in Africa, which imposes a large public health burden in the country. We aimed to investigate and report on a case of relapsing fever borreliosis in Zambia. METHODS: A previously unknown Borrelia species was isolated from the blood of a febrile patient. Investigations of the presumptive vector ticks and natural hosts for the Borrelia species were conducted by culture isolation and/or DNA detection by Borrelia-specific polymerase chain reaction. Using culture isolates from the patient and bat specimens, genetic characterization was performed by multilocus sequence analysis based on the draft genome sequences. RESULTS: The febrile patient was diagnosed with relapsing fever. The isolated Borrelia species was frequently detected in Ornithodoros faini (n = 20/50 [40%]) and bats (n = 64/237 [27%]). Multilocus sequence analysis based on a draft genome sequence revealed that the Borrelia species isolates from the patient and presumptive reservoir host (bats) formed a monophyletic lineage that clustered with relapsing fever borreliae found in the United States. CONCLUSIONS: A febrile illness caused by a Borrelia species that was treatable with erythromycin was identified in Zambia. This is the first study to report on relapsing fever Borrelia in Zambia and suggesting the likely natural reservoir hosts of the isolated Borrelia species. Interestingly, the isolated Borrelia species was more closely related to New World relapsing fever borreliae, despite being detected in the Afrotropic ecozone.

Marburgvirus in Egyptian Fruit Bats, Zambia.

We detected Marburg virus genome in Egyptian fruit bats (Rousettus aegyptiacus) captured in Zambia in September 2018. The virus was closely related phylogenetically to the viruses that previously caused Marburg outbreaks in the Democratic Republic of the Congo. This finding demonstrates that Zambia is at risk for Marburg virus disease.

Serological evidence of Zika virus infection in non-human primates 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.

Seroprevalence of Filovirus Infection of Rousettus aegyptiacus Bats 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.

Molecular epidemiological investigations of plague in Eastern Province of Zambia.

BACKGROUND: Plague is a flea-borne zoonotic and invasive disease caused by a gram negative coccobacillus bacterium called Yersinia pestis. Plague has caused three devastating pandemics globally namely: the Justinian, Black Death and Oriental plague. The disease in the Eastern Province of Zambia has been reported in Nyimba and Sinda Districts in the past 15 years. The aim of this study was to investigate the molecular epidemiology of plague in the two affected districts. Polymerase Chain Reaction (PCR), targeting Plasminogen activator gene (pla gene) of Y. pestis, was performed on suspected human bubo aspirates (n = 7), rodents (n = 216), shrews (n = 27) and fleas (n = 1494). Of these, one positive sample from each source or host was subjected to sequencing followed by phylogenetic analysis. RESULTS: The plasminogen activator gene (pla gene) of Y. pestis was detected in 42.8% bubo aspirates, 6.9% rodents, 3.7% shrew and 0.8% fleas. The fleas were from pigs (n = 4), goats (n = 5) and rodents (n = 3). The sequencing and phylogenetic analysis suggested that the pla gene of Y. pestis in Nyimba and Sinda was similar and the isolates demonstrated a high degree of evolutionary relationship with Antiqua strains from the Republic of Congo and Kenya. CONCLUSION: It can be concluded that pla gene of Y. pestis was present in various hosts in the two districts and the strains circulating in each district were similar and resembles those in the Republic of Congo and Kenya.

Characterization of field infectious bursal disease viruses in Zambia: evidence of co-circulation of multiple genotypes with predominance of very virulent strains.

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.

Discovery of African bat polyomaviruses and infrequent recombination in the large T antigen in the Polyomaviridae.

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.

Identification of the same polyomavirus species in different African horseshoe bat species is indicative of short-range host-switching events.

Polyomaviruses (PyVs) are considered to be highly host-specific in different mammalian species, with no well-supported evidence for host-switching events. We examined the species diversity and host specificity of PyVs in horseshoe bats (Rhinolophus spp.), a broadly distributed and highly speciose mammalian genus. We annotated six PyV genomes, comprising four new PyV species, based on pairwise identity within the large T antigen (LTAg) coding region. Phylogenetic comparisons revealed two instances of highly related PyV species, one in each of the Alphapolyomavirus and Betapolyomavirus genera, present in different horseshoe bat host species (Rhinolophus blasii and R. simulator), suggestive of short-range host-switching events. The two pairs of Rhinolophus PyVs in different horseshoe bat host species were 99.9 and 88.8 % identical with each other over their respective LTAg coding sequences and thus constitute the same virus species. To corroborate the species identification of the bat hosts, we analysed mitochondrial cytb and a large nuclear intron dataset derived from six independent and neutrally evolving loci for bat taxa of interest. Bayesian estimates of the ages of the most recent common ancestors suggested that the near-identical and more distantly related PyV species diverged approximately 9.1E4 (5E3-2.8E5) and 9.9E6 (4E6-18E6) years before the present, respectively, in contrast to the divergence times of the bat host species: 12.4E6 (10.4E6-15.4E6). Our findings provide evidence that short-range host-switching of PyVs is possible in horseshoe bats, suggesting that PyV transmission between closely related mammalian species can occur.

Isolation of a simian immunodeficiency virus from a malbrouck (Chlorocebus cynosuros).

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.

Multi-reassortant G3P[3] group A rotavirus in a horseshoe bat in Zambia.

Group A rotavirus is a major cause of diarrhoea in humans, especially in young children. Bats also harbour group A rotaviruses, but the genetic backgrounds of bat rotavirus strains are usually distinct from those of human rotavirus strains. We identified a new strain of group A rotavirus in the intestinal contents of a horseshoe bat in Zambia. Whole genome sequencing revealed that the identified virus, named RVA/Bat-wt/ZMB/LUS12-14/2012/G3P[3], possessed the genotype constellation G3-P[3]-I3-R2-C2-M3-A9-N2-T3-E2-H3. Several genome segments of LUS12-14 were highly similar to those of group A rotaviruses identified from humans, cows and antelopes, indicating interspecies transmission of rotaviruses between bats and other mammals with possible multiple genomic reassortment events.

Molecular characterization of infectious bursal disease viruses detected in vaccinated commercial broiler flocks in Lusaka, Zambia.

Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive viral disease of young chickens and remains one of the economically most important diseases threatening the poultry industry worldwide. In this study, 16 and 11 nucleotide sequences of the VP2 hypervariable region (VP2-HVR) and part of VP1, respectively, of IBD virus (IBDV) detected in vaccinated broiler chickens in Lusaka in 2012 were determined. Phylogenetic analysis revealed that these Zambian IBDVs separated into three genotypes of very virulent (VV) IBDVs. Although the majority of these viruses belonged to the African VV type (VV1), which consisted of viruses from West Africa, South Africa and Zambia, one virus belonged to the East African VV type (VV2). Interestingly, a Zambian IBDV belonging to the VV3 genotype (composed of viruses from several continents) clustered with attenuated vaccine strains. Although sequence analysis of VP2-HVR showed that all detected Zambian IBDVs had conserved putative virulence marker amino acids (i.e., 222A, 242I, 256I, 294I and 299S), one virus had two unique amino acid substitutions, N280S and E300A. This study demonstrates the diversity of Zambian IBDVs and documents for the first time the possible involvement of attenuated vaccine strains in the epidemiology of IBD in Zambia. Strict biosecurity of poultry farms, monitoring of live vaccine use in the field, surveillance and characterization of IBDV in poultry and development of a vaccine from local or regional IBDV field strains are recommended for improved IBD control in Zambia.

Seroepidemiological Prevalence of Multiple Species of Filoviruses in Fruit Bats (Eidolon helvum) Migrating in Africa.

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.

Distinct Lineages of Bufavirus in Wild Shrews and Nonhuman Primates.

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.