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

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

Viruses 2023, 15, 1369 3 of 22 determine their complete or nearly complete genome sequences. The bat herpesviruses for which complete genome sequences are available have been identified for bats in Oceania (Australia), Asia (Vietnam, Indonesia, and Japan), and North America (United States of America and Canada), but no complete genome sequence of herpesviruses detected in African bats has been reported.
As part of the surveillance program of zoonotic virus infection in bats in Zambia, we screened frugivorous bats (Egyptian fruit bats, Rousettus aegyptiacus) and insectivorous bats (striped leaf-nosed bats, Macronycteris vittatus; Sundevall's roundleaf bats, Hipposideros caffer) for herpesviruses, followed by phylogenetic analysis of the detected sequences. Here, we also report the isolation of a novel gammaherpesvirus named Macronycteris gammaherpesvirus 1 (MaGHV1) from Macronycteris vittatus bats in Zambia. We performed complete genome sequencing and genetically characterized the isolates.

Sample Collection
In 2018, 24 cave-dwelling Egyptian fruit bats (Rousettus aegyptiacus), 105 striped leafnosed bats (Macronycteris vittatus), and one Sundevall's roundleaf bat (Hipposideros caffer) were captured in Chongwe (15.6 • S, 28.7 • E) with approval from the Department of National Parks and Wildlife, Ministry of Tourism and Arts, Zambia (DNPW8/27/1) [21]. Bat species were identified based on morphological characteristics and nucleotide sequence analysis of the mitochondrial 16S ribosomal RNA and the cytochrome b genes. We collected oral and rectal swabs from each captured bat and promptly placed them in Eagle's minimum essential medium (MEM) supplemented with 1000 units/mL penicillin, 1000 µg/mL streptomycin, 25 µg/mL amphotericin B, 0.01 M HEPES, and 0.5% bovine serum albumin (BSA). After brief centrifugation, the supernatants were stored at −80 • C.

Amplification of the Herpesvirus DNA Polymerase Gene by PCR
Screening for the herpesvirus detection was conducted using pooled swab specimens. Oral and rectal swabs were pooled for each bat and nucleic acids were individually extracted from 130 pooled swab specimens using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Nucleic acids extracted by the kit include not only RNA but also DNA as previously described [22]. The herpesvirus DNA genome was detected by semi-nested PCR using Tks Gflex DNA Polymerase (TaKaRa, Shiga, Japan) with pan-herpesvirus primer sets targeting the DNA polymerase (DPOL) gene (Table S1) [23]. PCR products were subjected to direct sequencing using the Big Dye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA). The 5 -and 3 -ends of the sequences derived from primers were trimmed and obtained sequences were analyzed through a BLAST search (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 23 May 2023). The determined partial DPOL nucleotide sequences were deposited into the DNA Data Bank of Japan (DDBJ) under accession numbers LC762209-LC762247.

Virus Isolation
African green monkey kidney (Vero E6) cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM Lglutamine, 100 units/mL penicillin, 100 µg/mL streptomycin, 3.5 mg/mL D-glucose, and 1.0 mg/mL NaHCO 3 at 37 • C with 5% CO 2 . The media from the swabs that tested positive for herpesvirus detection by genetic screening were inoculated onto Vero E6 cell cultures, followed by 1 h incubation at 37 • C in 5% CO 2 for virus adsorption. After the inocula were removed, the cells were washed twice with PBS and maintained in DMEM containing 5 µg/mL trypsin, 0.3% BSA, 2 mM L-glutamine, 4% antibiotic-antimycotic solution (Gibco, Waltham, MA, USA), and 1.0 mg/mL NaHCO 3 at 37 • C in 5% CO 2 for 2 weeks. The supernatant of the inoculated cells was blindly passaged to fresh Vero E6 cells. Subsequently, the supernatant of the passaged culture from oral and rectal swabs was pooled for each bat and subjected to DNA extraction and NGS analysis as described below. Total DNA was extracted from the pool by using a DNeasy Blood & Tissue Kit (QIAGEN), and isolation of herpesviruses was confirmed by PCR.

Genome Sequencing, Construction, and Annotation
The complete genome sequences of isolated herpesviruses were determined by NGS analyses. The sequencing library was synthesized from total DNA of isolated viruses using a Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA, USA) according to the manufacturer's instructions, and was then sequenced on a MiSeq instrument with a MiSeq Reagent Kit v3 (600 cycles) to generate 300 bp paired-end reads. Sequence reads were trimmed and analyzed by de novo assembly using CLC Genomics Workbench software (CLC bio, Hilden, Germany). Consensus sequences with coverage of over 20 reads were obtained from the herpesvirus genome contigs.
For filling the remaining gap, PCR was performed using a specific primer designed based on the upstream and downstream sequences of the gaps (Table S1). The obtained amplicons were sequenced using a Frongle and Ligation Sequencing Kit (SQK-Q20EA) (Oxford Nanopore Technologies, Oxford, United Kingdom). Base calling was performed using Guppy version 5.0.16+b9fcd7b. Extra sequences were trimmed using Porechop [24]. Then, consensus sequences were generated using Canu [25]. To compliment the terminal repeat sequences at both ends of the genome, the genome DNA was amplified using an illustra GenomiPhi V2 DNA Amplification Kit (GE Healthcare, Chicago, IL, USA), and their terminals were repaired using T7 Endonuclease I (New England Biolabs, Ipswich, MA, USA). Following sequencing, base calling and trimming were performed as described above. To select reads including the terminal sequence, they were aligned with one kb regions flanking each terminal repeat using BWA [26]. The selected reads for each terminal were assembled using Canu, and the most representative contig was manually selected. The obtained gap-filling and terminal repeat sequences were concatenated with the genome assembly and polished with Pilon [27]. The determined genome sequences were deposited in the DDBJ database under accession numbers LC763829-LC763830.
ORFs were predicted by the ORF finder tool provided by the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/orffinder/, accessed on 23 May 2023). ORFs were identified using the following criteria: (i) sequence similarity to known viral or cellular genes, (ii) the presence of an initiating methionine and a stop codon, and/or (iii) length greater than 100 amino acids. ORFs with more than two of these criteria were considered to be genes encoding viral proteins. The translated amino acid sequences of the ORFs were subjected to BLAST search to determine whether they matched known viral or cellular proteins. The nomenclature used for MaGHV1 ORFs is based on a similar strategy used for other gammaherpesviruses such as equid herpesvirus 2 [28]. The unified name of the ORF for viral genes conserved across the subfamily Gammaherpesvirinae is used in this study. Additional ORFs are numbered in order of the appearance in the genome from 5 to 3 . The complete sequence of the isolated virus was analyzed in a tandem repeat finder program [29] to identify the repeat regions.

Genetic Comparison and Phylogenetic Analysis
Bioinformatic analyses were performed using herpesvirus sequences deposited in the DDBJ/EMBL-Bank/GenBank databases. Identity comparison analyses were conducted between isolated herpesviruses using GENETYX version 15 (GENETYX Corporation, Tokyo, Japan). Phylogenetic analyses based on the amino acid sequences of DPOL and glycoprotein B (gB) genes were performed using MEGA X software [30]. The MUSCLE protocol was used to align the sequences. Phylogenetic trees were constructed using the maximum likelihood method based on the model of LG+G+I for complete and partial DPOL genes and the model of LG+G+I+F for the gB gene as the best-fit models, with 1000 bootstrap replicates. The viruses and their accession numbers of the sequences used in the analysis are listed in Table S2 in the Supplementary Materials. BLAST analyses revealed that 26 of the 90 obtained sequences (181 or 184 bp in length) originated from betaherpesviruses and that 60 of the sequences (172 bp in length) originated from gammaherpesviruses. These sequences showed 45.6-100% and 68.4-100% amino acid identities with known betaherpesviruses and gammaherpesviruses, respectively (Table S3). Exceptionally, 4 sequences (172 bp in length of sample ID 3, 6, 114, and 126) were related to various alpha-, beta-, or gammaherpesviruses with 40-60% amino acid identities through BLAST analyses. Since these four sequences showed the highest identities with a bat-derived betaherpesvirus (Plecotus austriacus/Spain/psc-23/2004or2007), we tentatively classified them into the subfamily Betaherpesvirinae (Table 2).

Phylogenetic Analyses Based on the Partial DPOL Gene
Phylogenetic trees of the partial DPOL gene were constructed using the amino acid sequences of the detected herpesviruses with other alpha-, beta-, and gammaherpesviruses identified in bats worldwide ( Figure 1). The Zambian bat betaherpesviruses were phylogenetically divided into seven groups (BHV-G1 to -G7) as shown in Figure 1B. BHV-G1, -G2, and -G7 each consisted of a single virus identified in Macronycteris vittatus bats. BHV-G3, -G4, and -G7 were phylogenetically related to bat betaherpesviruses previously identified in various countries. BHV-G3 formed a cluster near the branches of BHV-G2 and a betaherpesvirus detected in Molossus molossus bats in Uruguay. BHV-G4 formed a cluster with betaherpesviruses from Rousettus aegyptiacus bats in South Africa and Hungary, and BHV-G7 was closely related to betaherpesviruses in Rhinolophus ferrumequinum bats in China and Spain. BHV-G5 formed a unique lineage near the branch of murid betaherpesvirus 8. Of note, BHV-G6 contained betaherpesviruses identified in Rousettus aegyptiacus and Macronycteris vittatus bats and formed an independent cluster under the subfamily Betaherpsvirinae. The Rousettus aegyptiacus/Zambia/3/2018 virus shared 100% nucleotide identity with the Macronycteris vittatus/Zambia/114/2018 virus, suggesting that the viruses were distributed in both frugivorous and insectivorous bats. On the other hand, the Zambian bat gammaherpesviruses were phylogenetically divided into five groups (GHV-G1 to -G5) as shown in Figure 1C. GHV-G1, -G2, -G3, and -G4 each formed independent clusters or branches. GHV-G1 were monophyletic along with a gammaherpesvirus detected in Rhinophus blythi bats in China. GHV-G2 belonged to a large clade including GHV-G1 and gammaherpesviruses identified in various insectivorous bats in China and Japan. GHV-G3 and G4 formed distinct lineages within the Betaherpsvirinae clade. GHV-G5 were phylogenetically related to previously described gammaherpesviruses detected in frugivorous bats in Hungary and Bangladesh. These results indicated that genetically diverse herpesviruses were maintained in bats in the cave in Zambia.

Isolation of a Novel Gammaherpesvirus from Macronycteris Vittatus Bats
To isolate the viruses detected by genetic screening, the herpesvirus-positive swab samples were inoculated onto Vero E6 cells. Gammaherpesviruses were isolated from seven Macronycteris vittatus bats (sample IDs 59, 80, 82, 86, 88, 89, and 106). After several passages, a cytopathic effect (CPE) was observed in Vero E6 cells infected with the isolates (Figure 2). The isolated viruses were genetically analyzed by PCR and sequencing targeting the partial DPOL gene which were used for screening. According to the sequencing analyses, the seven isolates were divided into two closely related groups (i.e., sample IDs 59/80/86/89/106 and IDs 82/88). The two sequences showed 98.3% similarity (169/172 bp). These newly isolated herpesvirus strains were tentatively named Macronycteris gammaherpesvirus 1 (MaGHV1). The two representative MaGHV1 strains from sample IDs 80 and 82 (strains 80 and 82, respectively) were used for further analyses.

Complete Genome of MaGHV1 and Genetic Comparison between Strains
To determine the complete genome sequences of the MaGHV1 strains, we performed whole genome sequencing. The genome of MaGHV1 strain 82 was 116,972 bp in length with a GC content of 37.3% ( Figure 3). The genome structure comprised a unique region that was flanked by tandem terminal repeats at each end ( Table 3). The size of the consensus repeat sequences at both termini was 284 bp and the copy numbers were 7 and 3.8 at 5 and 3 termini, respectively. In the unique region, two repeat regions at coordinates 25,665 to 26,556 (repeat region 1) and 105,538 to 106,545 (repeat region 2) were found. A total of 79 ORFs were identified and most of the ORFs showed the highest identities to viral genes of rhinolophus gammaherpesvirus 1 (RGHV1) isolated from a greater horseshoe bat (Rhinolophus ferrumequinum) in Japan [20], followed by myotis gammaherpesvirus 8 isolated from a microbat (Myotis velifer incautus) cell line [18]. Notably, at least 67 ORFs showed homology with viral genes of equid herpesvirus 2, which is a well-known gammaherpesvirus, and one of them (ORF38) shared the highest identity (43.3%) with the myristylated tegument protein of equid gammaherpesvirus 2 rather than RGHV1. The BM1, BM4, and BM6 genes showed sequence similarity to cellular genes, CASP8 and FADD-like apoptosis regulator (32.5% identity), E3 ubiquitin-protein ligase (38.9% identity), and bcl-2 like protein (34.0% identity), respectively, and the BM4 and BM6 genes also had homologies to ORF 4 and ORF13 of RGHV1. Six ORFs (BM2, BM10, and BM13-16) showed no significant similarity to any known genes. In addition, ORF73, which encoded the homolog of nuclear antigen LANA-1, was mapped to a direct repeat (repeat region 2). To identify herpesviruses genetically related to MaGHV1, BLAST analyses were performed using the deduced amino acid sequences of viral proteins. The complete DPOL sequence (ORF9) of MaGHV1 strain 82 shared the highest identity (78.4%) with RGHV1. Among other ORFs, the deduced amino acid sequences of glycoprotein B (gB; ORF8), helicase-primase helicase subunit (ORF44), Uracil-DNA glycosylase (ORF46), deoxyribonuclease (ORF37), and major capsid protein (ORF25) showed 72.0, 78.6, 75.8, 68.0, and 78.1% identities to RGHV1 homologs, respectively (Table S4).     ( Figure 2). The isolated viruses were genetically analyzed by PCR and sequencing targeting the partial DPOL gene which were used for screening. According to the sequencing analyses, the seven isolates were divided into two closely related groups (i.e., sample IDs 59/80/86/89/106 and IDs 82/88). The two sequences showed 98.3% similarity (169/172 bp). These newly isolated herpesvirus strains were tentatively named Macronycteris gammaherpesvirus 1 (MaGHV1). The two representative MaGHV1 strains from sample IDs 80 and 82 (strains 80 and 82, respectively) were used for further analyses.

Complete Genome of MaGHV1 and Genetic Comparison between Strains
To determine the complete genome sequences of the MaGHV1 strains, we performed whole genome sequencing. The genome of MaGHV1 strain 82 was 116,972 bp in length with a GC content of 37.3% (Figure 3). The genome structure comprised a unique region that was flanked by tandem terminal repeats at each end ( Table 3). The size of the consensus repeat sequences at both termini was 284 bp and the copy numbers were 7 and 3.8 at 5′ and 3′ termini, respectively. In the unique region, two repeat regions at coordinates 25,665 to 26,556 (repeat region 1) and 105,538 to 106,545 (repeat region 2) were found. A total of 79 ORFs were identified and most of the ORFs showed the highest identities to viral genes of rhinolophus gammaherpesvirus 1 (RGHV1) isolated from a greater horseshoe bat (Rhinolophus ferrumequinum) in Japan [20], followed by myotis gammaherpesvirus 8 isolated from a microbat (Myotis velifer incautus) cell line [18]. Notably, at least 67 ORFs showed homology with viral genes of equid herpesvirus 2, which is a well-known gammaherpesvirus, and one of them (ORF38) shared the highest identity (43.3%) with the myristylated tegument protein of equid gammaherpesvirus 2 rather than RGHV1. The BM1, BM4, and BM6 genes showed sequence similarity to cellular genes, CASP8 and FADD-like apoptosis regulator (32.5% identity), E3 ubiquitin-protein ligase (38.9% identity), and bcl-2 like protein (34.0% identity), respectively, and the BM4 and BM6 genes also had homologies to ORF 4 and ORF13 of RGHV1. Six ORFs (BM2, BM10, and BM13- 16) showed no significant similarity to any known genes. In addition, ORF73, which encoded the homolog of nuclear antigen LANA-1, was mapped to a direct repeat (repeat region 2). To identify herpesviruses genetically related to MaGHV1, BLAST analyses were performed using the deduced amino acid sequences of viral proteins. The complete DPOL        On the other hand, most of the 79 ORFs of MaGHV1 described above were also identified in the genome of strain 80: BM3-7 and ORF6-11 encoded in contig 1 (21,962 bp), BM8-13, ORF17-40, ORF42-50, and ORF52-70 in contig 2 (79,285 bp), and ORF73-75, BM14-16 in contig 3 (8979 bp) (Table S5). Identity comparison of each viral protein between the MaGHV1 strains demonstrated > 90% amino acid identity of 70 ORFs. Relatively low identities (52.6-89.5%) were found in the remaining viral proteins, including three membrane proteins (BM3, ORF47, and BM11), one tegument protein (ORF45), and two hypothetical proteins (BM12 and BM13).

Phylogenetic Characterization of MaGHV1
Phylogenetic trees were constructed on the basis of DPOL and gB amino acid sequences. The phylogenetic analysis of DPOL demonstrated that two MaGHV1 strains were in the subfamily Gammaherpesvirinae and formed an independent lineage sharing a common origin with other bat-derived gammaherpesviruses such as RGHV1, myotis gammaherpesvirus 8, and myotis ricketti herpesvirus 2 detected by virome analyses in China [31] ( Figure 4A). Similar results were obtained in the phylogenetic tree of gB ( Figure 4B). In both the DPOL and gB phylogenetic trees, MaGHV1 strains were located near the branches of equid gammaherpesvirus 2 and equid gammaherpesvirus 5. were in the subfamily Gammaherpesvirinae and formed an independent lineage sharing a common origin with other bat-derived gammaherpesviruses such as RGHV1, myotis gammaherpesvirus 8, and myotis ricketti herpesvirus 2 detected by virome analyses in China [31] (Figure 4A). Similar results were obtained in the phylogenetic tree of gB ( Figure  4B). In both the DPOL and gB phylogenetic trees, MaGHV1 strains were located near the branches of equid gammaherpesvirus 2 and equid gammaherpesvirus 5.

Discussion
In this study, we isolated a novel gammaherpesvirus, tentatively named MaGHV1, from Macronycteris vittatus bats in Zambia. We determined its complete genome sequence including the repeat sequences, and genetically characterized the encoded sequences. During the past two decades, many partial herpesvirus genes have been detected in various bats worldwide using NGS or PCR-based approaches [8,13,[31][32][33]. However, few investigations isolated infectious herpesviruses. Isolated infectious viruses are often needed for downstream studies aiming at biological characterization of novel viruses, such as their host tropisms, transmissibility, and pathogenicity.
Herpesviruses are highly disseminated in nature and most animal species have yielded at least one herpesvirus and frequently, several distinct herpesviruses [5]. By our PCR screening, at least seven betaherpesviruses and five gammaherpesviruses were detected in three Zambian bat species: Rousettus aegyptiacus, Macronycteris vittatus, and Hipposideros caffer. Our phylogenetic analyses indicate that diverse herpesviruses are distributed in the bat population in Zambia. It is generally believed that mammalian herpesviruses are mainly host-specific and have coevolved with their specific host species [34]. In a previous study, host-virus cophylogenetic analyses demonstrated that the phylogenetic tree of bat betaherpesviruses showed congruence with features in the phylogeny of corresponding host organisms, providing evidence for coevolutionary development of virus and host lineages [35]. Zambian bat herpesviruses were phylogenetically divided into several groups depending on their host species, except for BHV-G6. Interestingly, the nucleotide sequence of one betaherpesvirus from Rousettus aegyptiacus frugivorous bats was identical to that from Macronycteris vittatus insectivorous bats. Although evolutionary analyses of bat gammaherpesviruses have shown that host-switching events occur frequently [36], little information about the cross-species transmission of bat betaherpesviruses is available. In an epidemiological survey of herpesviruses in bats in Peru, betaherpesviruses detected in an Artibeus lituratus fruit bat and Desmodus rotundus vampire bats phylogenetically formed a cluster, suggesting a possible cross-species infection within the family Phyllostomidae (between the genera Artibeus and Desmodus) [35]. Our Zambian case may also show possible cross-species infection within order Chiroptera (between the families Hipposideridae and Pteropodidae), suggesting a unique and rare instance of cross-species transmission of betaherpesviruses. In our study, bats were captured using a harp trap, and trapped bats closely contacted each other in the collection chamber for a few hours. Considering this situation, bat saliva might be transferred to the oral cavities of other bats, implying possible contamination of the virus in an improper host without actual infection. To clarify the true frequency of betaherpesvirus transmission between the families Hipposideridae and Pteropodidae bat species, further investigation with virus isolation is required.
It is important to characterize herpesviruses in various host species for better understanding of the evolutionary history and host specificity of herpesviruses. Although partial herpesvirus genes have been detected in bats in various African countries [9][10][11][12][13], Macronycteris vittatus and Hipposideros caffer bats, which live in Africa, had not been analyzed for the detection of herpesviruses. In this study, we carried out the first genetic characterization of herpesviruses from these insectivorous bats living in Africa. Having a high Chiroptera (bat) diversity, there are currently 18 families (including 5 extinct), 84 genera (25 extinct), and 403 species (54 extinct) recognized as occurring in Africa [37]. Some of the bat species, including Macronycteris vittatus and Hipposideros caffer, were distributed only in Africa and the southwestern Arabian Peninsula, including Saudi Arabia and Yemen [37,38]. Therefore, surveillance of herpesviruses in bats in each country or region is necessary to fully understand the evolutionary host-virus relationship.
The prototype of gammaherpesvirus, Kaposi's sarcoma-associated herpesvirus, is estimated to encode over 85 genes, of which about 58 are conserved in all gammaherpesvirus genomes, including 40 core genes for all herpesviruses [6,7,39]. Overall, the structure of the MaGHV1 genome is similar to other gammaherpesviruses such as RGHV1, equid herpesvirus 2, and equid herpesvirus 5, and MaGHV1 carries all the conserved ORFs among the subfamily Gammaherpesvirinae. The conserved genes in the MaGHV1 genome are arranged colinearly in direction and position, mostly as in other gammaherpesvirus genomes. We found unique putative ORFs of MaGHV1, some of which were defined as hypothetical proteins with no homology with any known proteins, and it would be of interest to analyze the functions of these unique viral proteins in future studies. The MaGHV1 genome was flanked by the terminal repeat region consisting of multiple copies of tandemly repeated sequences. According to the ICTV criteria for the classification of the herpesvirus genome [6], MaGHV1 could be classified into the class 2 genome structure, which is common in members of the subfamily Gammaherpesvirinae [6]. The genome structure of RGHV1, which is genetically related to MaGHV1, could also be classified into the class 2 genome [20]. On the other hand, myotis gammaherpesvirus 8, which might share a common origin with MaGHV1, is currently unclassified because its terminal repeat sequences were not determined. Therefore, complete genome sequences of herpesviruses, including the repeat regions, are necessary to clarify the genetic relationships among diverse herpesviruses.
In the present study, we showed the presence of multiple bat-derived herpesviruses in Zambia. Our findings provide new information regarding the genetic diversity of bat herpesviruses. Similar prevalence patterns were also observed in bats in other countries such as Indonesia and South Africa [11,32]. As the first investigation on bat-derived herpesviruses in Zambia, we only screened three bat species in the present study. However, since there are 65 known bat species in Zambia [40], more diverse herpesviruses might be associated with various bat species in the same manner as other bat herpesviruses that have been previously recognized. It would be of interest to further investigate the prevalence of herpesvirus infection of bats in Zambia and other African countries.
Supplementary Materials: The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/v15061369/s1, Table S1: Primers used in this study; Table S2: Reference sequences used in this study; Table S3: Highest amino acid identities for partial DPOL genes detected by screening; Table S4: Identity comparison of each viral protein among Macronycteris gammaherpesvirus 1 strains and rhinolophus gammaherpesvirus 1; Table S5: Predicted protein coding regions in the genome of Macronycteris gammaherpesvirus 1 strain 80.