Characterization of a Novel Alphaherpesvirus Isolated from the Fruit Bat Pteropus lylei in Vietnam

A novel bat alphaherpesvirus, Pteropus lylei-associated alphaherpesvirus (PLAHV), was isolated from urine of the fruit bat Pteropus lylei in Vietnam. The whole-genome sequence was determined and was predicted to include 72 open reading frames in the 144,008-bp genome. PLAHV is circulating in a species of fruit bats, Pteropus lylei, in Asia. This study expands the knowledge on bat-associated alphaherpesvirology.

ABSTRACT

Herpesviruses exist in nature within each host animal. Ten herpesviruses have been isolated from bats and their biological properties reported. A novel bat alphaherpesvirus, which we propose to name “Pteropus lylei-associated alphaherpesvirus (PLAHV),” was isolated from urine of the fruit bat Pteropus lylei in Vietnam and characterized. The entire genome sequence was determined to be 144,008 bp in length and predicted to include 72 genes. PLAHV was assigned to genus Simplexvirus with other bat alphaherpesviruses isolated from pteropodid bats in Southeast Asia and Africa. The replication capacity of PLAHV in several cells was evaluated in comparison with that of herpes simplex virus 1 (HSV-1). PLAHV replicated better in the bat-originated cell line and less in human embryonic lung fibroblasts than HSV-1 did. PLAHV was serologically related to another bat alphaherpesvirus, Pteropodid alphaherpesvirus 1 (PtAHV1), isolated from a Pteropus hypomelanus-related bat captured in Indonesia, but not with HSV-1. PLAHV caused lethal infection in mice. PLAHV was as susceptible to acyclovir as HSV-1 was. Characterization of this new member of bat alphaherpesviruses, PLAHV, expands the knowledge on bat-associated alphaherpesvirology.

IMPORTANCE A novel bat alphaherpesvirus, Pteropus lylei-associated alphaherpesvirus (PLAHV), was isolated from urine of the fruit bat Pteropus lylei in Vietnam. The whole-genome sequence was determined and was predicted to include 72 open reading frames in the 144,008-bp genome. PLAHV is circulating in a species of fruit bats, Pteropus lylei, in Asia. This study expands the knowledge on bat-associated alphaherpesvirology.

INTRODUCTION

Herpesviruses are enveloped viruses with a double-stranded linear DNA genome ranging from 124 to 295 kbp in length (1, 2). The viral genome is encapsulated in an icosahedral protein cage called a capsid, which is surrounded by a protein layer called the tegument, and a lipid bilayer, the envelope, derived from host membrane with viral membrane glycoproteins (1, 2). Herpesviruses are widely disseminated in nature and coexist with each of their hosts, with more than 100 species making up the large order Herpesvirales (International Committee on Taxonomy of Viruses [https://talk.ictvonline.org/taxonomy/]) (1, 2). Mammalian herpesviruses are assigned to the family Herpesviridae, which is subdivided into 3 subfamilies, Alphaherpesvirinae, Betaherpesvirinae, and Gammaherpesvirinae, based on biological properties and genome sequence similarities (1, 2).

Numerous species of viruses belonging to a variety of families are hosted in bats, some of which show high pathogenicity to humans and livestock and threaten global public health (3). Lyssaviruses, including rabies virus, are maintained in and geographically distributed with some species of bats and cause diseases in humans (3–5). Henipaviruses are harbored in some species of pteropodid bats and have repeatedly spilled over to livestock and humans (3–6). Other species of viruses, including filoviruses, coronaviruses, and reoviruses, have sporadically spilled over from bats and caused human infection (3–5).

Herpesviruses have been isolated from several species of bats and their characteristics have been elucidated. To date, 5 kinds of alphaherpesviruses have been isolated from 6 species of frugivorous bats: Eidolon helvum, Eidolon dupraenum, Pteropus lylei, Pteropus hypomelanus-related bat, Lonchophylla thomasi, and one unassigned species (7, 8). Two kinds of betaherpesviruses have been isolated from 2 species (Miniopterus fuliginosus and Miniopterus schreibersii) (9, 10), whereas 3 gammaherpesviruses have been isolated from 3 species of bats (Myotis velifer incautus, Eptesicus fuscus, and Rhinolophus ferrumequinum) (11–13). Whole-genome sequencing was performed on 1 alphaherpesvirus isolated from P. hypomelanus-related bat (8), one betaherpesvirus isolated from M. schreibersii (10), and 3 gammaherpesviruses (11–13). Their biological properties, such as replication capacity in cell cultures and virulence in mice, were analyzed (8, 10–12). Bat alphaherpesvirus and gammaherpesviruses could replicate in bat and other mammalian cells, but the replication of the bat betaherpesvirus was restricted in specific types of cells (8, 10–12). Infection of mice with the bat alphaherpesvirus could be lethal (8). The other 4 alphaherpesviruses and 1 betaherpesvirus were characterized for their partial gene sequences and molecular phylogeny (7, 9). The herpesvirus reports of bat origin are summarized in Table 1.

TABLE 1.

Summary of the herpesviruses isolated from bats

Reference	Sampling information			Virus information
	Location	Bat species	Specimen	Subfamily	Genus	Genome sequence	Accession no.
Inagaki et al. (this study)	Vietnam	Pteropus lylei	Urine	Alphaherpesvirinae	Simplexvirus	Complete (144,008 bp)	LC492974.1
Razafindratsimandresy et al. (7)	Madagascar	Eidolon dupraenum	Throat swab	Alphaherpesvirinae	Simplexvirus	Partial (465 bp)	FJ040879.1
	Cameroon	Eidolon helvum	Organs	Alphaherpesvirinae	Simplexvirus	Partial (465 bp)	FJ040890.1
	Central African Republic	Unknown	Salivary gland	Alphaherpesvirinae	Simplexvirus	Partial (465 bp)	FJ040888.1
	Cambodia	Pteropus lylei	Throat swab	Alphaherpesvirinae	Simplexvirus	Partial (465 bp)	FJ040877.1
	Brazil	Lonchophylla thomasi	Blood	Alphaherpesvirinae	Simplexvirus	Partial (465 bp)	FJ040887.1
Sasaki et al. (8)	Indonesia	Pteropus hypomelanus-related bat	Spleen	Alphaherpesvirinae	Simplexvirus	Complete (149,459 bp)	NC_024306.1
Watanabe et al. (9)	Japan	Miniopterus fuliginosus	Spleen	Betaherpesvirinae	Unassigned	Partial (5,737 bp)	AB517983.1
Zhang et al. (10)	Australia	Miniopterus schreibersii	Lymph node	Betaherpesvirinae	Unassigned	Complete (222,870 bp)	JQ805139.1
Shabman et al. (11)	United States	Myotis velifer incautus	Skin tumor	Gammaherpesvirinae	Unassigned	Subcomplete (129,563 bp)	KU220026.1
Subudhi et al. (12)	Canada	Eptesicus fuscus	Lung	Gammaherpesvirinae	Rhadinovirus	Complete (166,748 bp)	MF385016.1
Noguchi et al. (13)	Japan	Rhinolophus ferrumequinum	Spleen	Gammaherpesvirinae	Percavirus	Complete (147,790 bp)	LC333428.1

In the present study, the properties of the sixth bat alphaherpesvirus newly isolated from P. lylei, which we propose to name “Pteropus lylei-associated alphaherpesvirus (PLAHV),” were explored. The characteristics of PLAHV were evaluated in comparison with those of another bat alphaherpesvirus, Pteropodid alphaherpesvirus 1 (PtAHV1), isolated from P. hypomelanus-related bats inhabiting Indonesia (8), and of herpes simplex virus 1 (HSV-1) of humans.

RESULTS

Isolation of PLAHV.

In total, 10 urine and 206 fecal samples were collected from fruit bats of P. lylei. Cytopathic effect (CPE) appeared in Vero cells inoculated with 3 of 10 urine samples but not with any fecal samples. Electron microscopy imaging revealed the capsid structure surrounded by a membrane (Fig. 1). Next-generation sequencing and a proteomics-based virus detection system detected nucleotide sequences related to herpesvirus UL30 and UL31 genes and peptide sequences related to herpesvirus UL19 and UL46 genes, respectively (Table 2 and data not shown). These CPE agents were confirmed to be a member of the herpesviruses by the whole-genome sequencing as described in next section. The single clone isolated from 1 of the 3 urine samples was established by a three-round plaque purification procedure in Vero cells.

FIG 1.

Electron microscopic appearance of PLAHV virion cultured in Vero cells. Exocytosis of virions with a diameter of approximate 160 nm (a, left arrowhead) and whole virions in a cytoplasmic vacuole (a, right arrow) are shown. The micropraph (b) shows virions possibly in the final stage of egress at the cell surface (b, arrow).

TABLE 2.

Genes predicted in the PLAHV genome

Gene	Product	Positions	Orientationa	% amino acid sequence identity to:
				PtAHV1	HSV-1
RL1	ICP34.5	1405–2292	+	80.4	34.6
RL2	Ubiquitin E3 ligase; ICP0		+	Not calculated	17.6
	Exon 1	3297–3686		Not calculated	Not calculated
	Exon 2	4936–5529		Not calculated	Not calculated
UL1	Envelope glycoprotein L	7226–7933	+	74.6	36.6
UL2	Uracil-DNA glycosylase	8328–8990	+	89.1	46.4
UL3	Nuclear protein	9074–9742	+	82.9	53.8
UL4	Nuclear protein	9789–10421	−	89.7	44.4
UL5	Helicase-primase helicase subunit	10455–13076	−	95.1	78.4
UL6	Capsid portal protein	13075–15120	+	94.0	64.1
UL7	Tegument protein	15071–15958	+	92.9	61.1
UL8	Helicase-primase subunit	16077–18083	−	77.8	42.8
UL9	DNA replication origin-binding helicase	18436–20985	−	96.7	78.0
UL10	Envelope glycoprotein M	20942–22297	+	94.9	58.3
UL11	Myristylated tegument protein	22917–23174	−	88.2	44.8
UL12	DNase	23099–25069	−	80.4	50.4
UL13	Tegument serine/threonine protein kinase	25072–26619	−	96.7	66.2
UL14	Tegument protein	26379–26891	−	90.6	49.5
UL15	DNA packaging terminase subunit 1		+	97.3	82.7
	Exon 1	27110–28135		Not calculated	Not calculated
	Exon 2	31526–32701		Not calculated	Not calculated
UL16	Tegument protein	28183–29274	−	92.9	57.9
UL17	DNA packaging tegument protein	29306–31465	−	87.9	57.7
UL18	Capsid triplex subunit 2; VP23	32805–33761	−	96.2	78.3
UL19	Major capsid protein; VP5	33904–38049	−	95.9	82.8
UL20	Envelope protein	38218–38880	−	92.7	62.6
UL21	Tegument protein	39328–40893	+	91.2	59.9
UL22	Envelope glycoprotein H	40993–43563	−	89.9	48.8
UL23	Thymidine kinase	43733–44722	−	84.2	41.9
UL24	Nuclear protein	44802–45593	+	83.7	58.8
UL25	DNA packaging tegument protein	45792–47540	+	95.0	76.3
UL26	Capsid maturation protease	47619–49502	+	84.7	55.5
UL27	Envelope glycoprotein B	49833–52052	−	94.7	59.0
UL28	DNA packaging terminase subunit 2	52477–54837	−	91.3	71.4
UL29	Single-stranded DNA-binding protein; ICP8	54976–58566	−	95.9	81.1
UL30	DNA polymerase catalytic subunit	59282–62989	+	92.2	73.5
UL31	Nuclear egress lamina protein	62916–63848	−	95.8	74.0
UL32	DNA packaging protein	63841–65592	−	92.1	70.2
UL33	DNA packaging protein	65591–65980	+	94.6	65.6
UL34	Nuclear egress membrane protein	66042–66872	+	87.7	57.6
UL35	Small capsid protein	66957–67286	+	92.7	56.6
UL36	Large tegument protein	67381–75249	−	76.1	46.9
UL37	Tegument protein	76461–79718	−	93.5	62.0
UL38	Capsid triplex subunit 1	80085–81473	+	83.3	59.6
UL39	Ribonucleotide reductase subunit 1	81831–85625	+	82.5	52.9
UL40	Ribonucleotide reductase subunit 2	85661–86668	+	94.9	73.8
UL41	Tegument host shutoff protein	86793–88286	−	94.6	64.5
UL42	DNA polymerase processivity subunit	88701–90164	+	74.7	46.1
UL43	Envelope protein	90326–91510	+	78.9	36.3
UL44	Envelope glycoprotein C	91687–93075	+	81.4	44.0
UL45	Membrane protein	93186–93698	+	86.5	44.8
UL46	Tegument protein VP11/12	93778–96186	−	86.9	49.9
UL47	Tegument protein VP13/14	96249–98378	−	87.7	55.8
UL48	Transactivating tegument protein VP16	98738–100186	−	91.9	65.5
UL49	Tegument protein VP22	100578–101447	−	77.2	39.4
UL49A	Envelope glycoprotein N	101767–102012	−	81.5	30.8
UL50	Deoxyuridine triphosphatase	102025–103089	+	89.8	41.3
UL51	Tegument protein	103210–103899	−	90.4	59.6
UL52	Helicase-primase primase subunit	103975–107115	+	87.0	63.1
UL53	Envelope glycoprotein K	107085–108092	+	93.7	64.8
UL54	Multifunctional expression regulator; ICP27	108732–110366	+	77.7	54.1
UL55	Nuclear protein	110705–111256	+	97.3	61.8
UL56	Membrane protein	111957–112835	−	67.5	26.8
RL2	Ubiquitin E3 ligase; ICP0		−	Not calculated	17.6
	Exon 1	116672–117061		Not calculated	Not calculated
	Exon 2	114829–115422		Not calculated	Not calculated
RL1	ICP34.5	118066–118953	−	80.4	34.6
RS1	ICP4	121128–124547	−	85.4	56.4
US1	Regulatory protein ICP22	126048–127286	+	68.3	37.0
US2	Virion protein	127413–128315	−	89.3	59.2
US3	Serine/threonine protein kinase	128549–129946	+	90.3	58.6
US4	Envelope glycoprotein G	130039–132231	+	54.2	11.1
US5	Envelope glycoprotein J	132460–133098	+	Not calculated	14.7
US6	Envelope glycoprotein D	133385–134557	+	88.0	57.6
US7	Envelope glycoprotein I	134720–136102	+	60.8	35.6
US8	Envelope glycoprotein E	136322–137989	+	71.7	39.9
US8A	PtAHV1 US8A-like protein	137931–138221	+	55.6	22.8
RS1	ICP4	139854–143273	+	85.4	56.4

^a+ and −, forward and reverse directions, respectively.

Whole-genome sequence of PLAHV.

The plaque-purified virus, PLAHV, was subjected to whole-genome sequencing, in which 130,302 reads with an average of 164 bases in length were generated with Ion PGM and were assembled and connected with the nucleotide sequences determined with the Sanger sequencing to generate 2 contig sequences (nucleotides 1007 to 22354 and 22871 to 143355 of the whole genome). Because the gap of 516 bp between the 2 contig sequences could not be determined, another high-throughput sequencer, MinION, was used. MinION generated 3,353 reads with an average of 6,852 bases in length, making it possible to fill the gap region. The complete genome sequence of PLAHV was thus determined to be 144,008 bp in length. The GC content was 62.087%. Compared with the genome structure of PtAHV1, the PLAHV genome had 2 unique nucleotide sequence regions (U_L and U_S), both of which were flanked by 2 inverted repeat sequence regions (TRL and IRL and IRS and TRS, respectively) (Fig. 2).

FIG 2.

Schematic diagram of the genome structure of PLAHV. The whole-genome sequence of PLAHV of 144,008 bp in length was determined, and 72 genes were predicted. The genomic sequence is depicted as black lines and dark and light gray boxes, which indicate unique regions (U_L and U_S), repeat regions flanking U_L (TRL and IRL), and those flanking U_S (IRS and TRS), respectively. The linear genome is depicted in a circular manner. Genes predicted are shown in red (forward direction) and in blue (reverse direction) boxes labeled with each name. The nucleotide position is shown outside the diagram.

The PLAHV genome was predicted to carry 72 genes, including duplication of RL1, RL2, and RS1 in the repeat regions (Fig. 2 and Table 2). The mRNA sequences of the RL2 and US15 genes, which each have an intron within their open reading frames (ORFs), were sequenced and their splicing sites were determined. The expression of US5 gene was further confirmed, because PtAHV1 has no such gene, by sequencing their mRNAs using 5′-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT-3′ as the primer for reverse transcription. The cDNA was synthesized by PCR using primers flanking the poly(A) sequence and sequenced. US9, which was carried in the PtAHV1 genome (NC_024306.1), could not be annotated, but US5, which was not carried in the PtAHV1 genome, was shown in the PLAHV genome.

The amino acid sequence identity of each PLAHV gene with the corresponding gene of PtAHV1 and HSV-1 strain 17 (NC_001806.2) was calculated and ranged from 54.2 to 97.3% and 11.1 to 82.8%, respectively. Thirty of 69 proteins were highly similar (>90% identity) to the corresponding PtAHV1 proteins, whereas 5 proteins had a similarity of <70% (Table 2).

The UL23, UL30, and US4 genes of isolates from the other 2 urine samples were determined by Sanger sequencing and were confirmed to be identical to these genes in the complete genome sequence (data not shown).

Phylogenetic analyses.

Phylogenetic trees were constructed based on the amino acid sequences corresponding to the UL27 and UL30 genes, which encoded glycoprotein B and the DNA polymerase catalytic subunit, respectively (Fig. 3 and Table 2). PLAHV was assigned to genus Simplexvirus within subfamily Alphaherpesvirinae in all of the trees constructed (Fig. 3). PLAHV was clustered with other simplexviruses isolated from pteropodid bats, which branched from the common ancestor of the present human and Old World primate simplexviruses (Fig. 3b and Table 1). The clade for pteropodid simplexviruses was subdivided into two branches, one of which was for the isolates from bats in Southeast Asia and the other of which was for those in Africa (Fig. 3b and Table 1). The amino acid sequence corresponding to the UL30 gene of PLAHV was identical to that of an isolate from P. lylei in Cambodia (GenBank accession number FJ040877.1) (Fig. 3b and Table 1) (7).

FIG 3.

Phylogenetic trees of herpesviruses. The phylogenetic trees were constructed based on the amino acid sequences of whole viral DNA polymerase (encoded in UL30) (a), partial viral DNA polymerase (b), and whole glycoprotein B (encoded in UL27) (c) using the neighbor-joining method. Scale bars indicate the branch length representing the number of amino acid substitutions per site. The bootstrap percentage of 1,000 replicate calculations is shown next to each branch. Viruses used to construct the trees are listed in Table 3.

In vitro replication of PLAHV in mammalian cells.

The replication capacities of PLAHV in several mammalian cells were compared to those of HSV-1. PLAHV replicated in all of the cells tested, although the replication capacities were different depending on the cell types (Fig. 4). PLAHV replicated less efficiently than HSV-1 F in Vero cells (Fig. 4a). PLAHV replicated as well in COS7 and HeLa cells as HSV-1 F did, although PLAHV generated more progeny at early time points (Fig. 4b and d). PLAHV replicated more efficiently in FBKT1 cells (established from kidney of Pteropus dasymallus yayeyamae [14]) than HSV-1 F but less efficiently in human embryonic lung (HEL) cells (Fig. 4c and e).

FIG 4.

Replication kinetics of PLAHV (orange) and HSV-1 (blue) in Vero (a), COS7 (b), HEL (c), HeLa (d), and FBKT1 (e) cells. These cells were inoculated with PLAHV or HSV-1 F at a multiplicity of infection of 5 and were cultured with maintenance medium. The cells with culture medium were harvested at 0, 2, 6, 12, 20, and 24 h postinfection, and the infectious dose of each sample was measured with the standard plaque assay. Means and SDs from 3 independent experiments are shown. *, statistical significance with a P value of <0.05 as calculated by Student’s t test.

Serological relationship between bat and human simplexviruses.

Anti-PLAHV sera inhibited the plaque formation of both PLAHV and PtAHV1, but the inhibitory efficacy against PLAHV was stronger than that against PtAHV1 (Fig. 5a). Anti-PLAHV sera did not inhibit plaque formation by HSV-1 F or HSV-2 186. Similar phenomena were seen in the neutralization activities of anti-PtAHV1 sera (Fig. 5b). In contrast, sera raised against each of HSV-1 F and HSV-2 186 did not inhibit the plaque formation of either PLAHV or PtAHV1, whereas they did inhibit the plaque formation of both HSV-1 F and HSV-2 186 (Fig. 5c and d).

FIG 5.

Serological analyses between bat and human alphaherpesviruses. The target virus was mixed with DMEM (control) or each serum sample serially diluted with DMEM and incubated at 37°C for 1 h. The mixtures were then inoculated in triplicate to Vero cells, and the inoculated cells were incubated for 2 days to form plaques. The neutralization activities of mouse sera raised against PLAHV (a), PtAHV1 (b), HSV-1 (c), and HSV-2 (d) to each virus are shown as the percentage of the plaque numbers to the control. Percentages are shown as means ± SDs calculated from 3 independent experiments.

Pathogenicity of PLAHV to mice.

The body weight of BALB/c mice decreased when the mice were intranasally inoculated with PLAHV in an infectious dose-dependent manner. Some mice showed ulcerative skin lesions around their eyes from 5 days postinfection. The 50% lethal dose (LD₅₀) values of PLAHV for mice were calculated to be 2.0 × 10⁴ PFU/mouse at 13 days postinfection (Fig. 6).

FIG 6.

Experimental infection of mice with PLAHV. Five and 4 mice were inoculated through the nasal cavity with virus solution containing 1 × 10⁴ or 1 × 10⁵ PFU of PLAHV, respectively, and the medium without virus as the control group (mock). Survival percentages of each group on each day are shown. The experiments were conducted twice independently.

Sensitivity of PLAHV to acyclovir.

Acyclovir (ACV) inhibited the replication of PLAHV in a dose-dependent manner (Fig. 7). The inhibitory effect of ACV on PLAHV was parallel to that on HSV-1 F. The 50% inhibitory concentration (IC₅₀) values of ACV for PLAHV, HSV-1 F, and HSV-2 186 were 0.64 μg/ml, 0.50 μg/ml, and 1.5 μg/ml, respectively. There was no significant difference in IC₅₀ values between PLAHV and HSV-1 F (P = 0.20, Student’s t test).

FIG 7.

Sensitivities of PLAHV, HSV-1, and HSV-2 to acyclovir (ACV) as determined with plaque reduction assay in Vero cells. Vero cells cultured in 24-well culture plates were inoculated with PLAHV, HSV-1, or HSV-2 in triplicate and were cultured for 2 days in DMEM-1FBS containing 1% (wt/vol) methylcellulose and the designated concentration of ACV. Data are shown as the means and SDs from 3 independent reactions.

DISCUSSION

PCR and high-throughput sequencing have made it possible to elucidate the genetic diversity of herpesviruses in bats in detail (15–25). In addition, infectious viruses were isolated from specimens of bats, and their characteristics, such as the genome sequence (7–13), replication capacity in several human and other mammalian cell lines (8, 10–12), and virulence in mice (8), were studied. Five bat alphaherpesviruses have been isolated (7, 8). However, their virologic properties have not been analyzed in detail except for PtAHV1 (8).

The complete genome sequence of PLAHV was determined, and 72 genes were predicted (Fig. 2 and Table 2). PLAHV included the US5 gene, whose respective gene was absent in PtAHV1, whereas PtAHV1 included the US9 gene, whose respective gene was absent in PLAHV. Furthermore, the identities in some viral proteins were relatively small (e.g., US4), suggesting that PLAHV and PtAHV1 can be considered independent species from each other. The partial genomic sequence (465 bp encoding 155 amino acid residues of the UL30 gene) of the isolate from P. lylei in Cambodia (GenBank accession number FJ040877.1) was reported previously (7). The corresponding gene sequence of PLAHV (nucleotides 61463 to 61927) was almost identical to that of the Cambodian isolate. The GC content in the PLAHV genome was 62.087%, while those in PtAHV1 (GenBank accession number NC_024306.1), macropodid herpesviruses 1 (MaHV-1; NC_029132.1), HSV-1 (NC_001806.2), HSV-2 (NC_001348.1), and varicella zoster virus were 60.856%, 52.920%, 68.301%, 70.379%, and 46.020%, respectively. MaHV-1 is an alphaherpesvirus of marsupial population origin. It was confirmed that PLAHV has a similar characteristic in terms of the GC content to that of bat-originated PtAHV1.

Several types of human cells were well competent for infection by a bat alphaherpesvirus (8). The present study showed, however, that the replication of PLAHV in HEL cells was severely impaired compared to that of HSV-1 (Fig. 4c). In contrast, PLAHV replicated more efficiently in bat kidney-derived FBKT1 cells than did HSV-1 (Fig. 4e). It is noteworthy that PtAHV1 efficiently replicated in CHO-K1 cells (8), in which HSV-1 cannot replicate because the cells lack HSV-1 entry receptors (26, 27). Future comparative studies are expected to reveal the replication mechanism of bat alphaherpesviruses and those of HSV-1.

Pteropodid bats are associated with the human community, in which they are recognized as a source of meat and medicines, and have been killed as agricultural pests and for sport hunting (28, 29). Thus, it is possible that humans are being exposed to bat alphaherpesviruses, including PLAHV. Herpesviruses rarely infect unnatural hosts, but some herpesviruses, such as Macacine alphaherpesvirus 1 (herpes B virus) and Suid alphaherpesvirus 1 (pseudorabies virus), cause severe disease when they do infect them (30, 31). Therefore, further studies about infection of humans by bat herpesvirus are needed. The neutralizing test using infectious PLAHV, HSV-1, and HSV-2 in parallel may differentiate PLAHV infections from infections caused by HSV-1 or HSV-2 (Fig. 5). PLAHV was sensitive to ACV (Fig. 7), suggesting that ACV might be a drug of choice if humans are infected with PLAHV.

The complete genome of PLAHV was determined. The genome-wide differences in sequences from another bat alphaherpesvirus, PtAHV1, the serological relationship between bat and human alphaherpesviruses, and the sensitivity of PLAHV to ACV were revealed for the first time. The virulence of PLAHV in BALB/c mice was elucidated. PLAHV lethally infected the mice as PtAHV1 did (8). There might be numbers of other alphaherpesviruses in geographically dispersed and diverse species of bats other than the 6 isolates documented in this and other studies (7, 8). The accumulation of knowledge on the genome and characteristics of bat alphaherpesviruses might unveil the evolutional history of simplexviruses in bats and other mammals and lead to a comprehensive understanding of herpesvirology.

MATERIALS AND METHODS

Samples (urine and feces) collected from fruit bats in Vietnam.

Urine and feces samples of fruit bats were collected at Ma Toc or Mahutup pagoda, in which the Mahutup pagoda temple known as Doi (Bat) pagoda is located, in Soc Trang province, Mekong Delta, Vietnam, between February 2012 and March 2013. Thousands of flying foxes (P. lylei) inhabit the forest in the precincts of the Mahutup pagoda temple. The bat species was identified through morphological observation and determination of the cytochrome b gene sequence in specimens from bats (data not shown). Clean plastic sheets were used for collection of samples as reported previously (32). Fresh samples were collected using cotton swabs. The swab samples were mixed with viral transport medium (KCl 5.37 mM, 0.44 mM KH₂PO₄, 4.17 mM NaHCO₃, 136.89 mM NaCl, 0.33 mM Na₂HPO₄, 5.55 mM dextrose, 0.05 mM phenol red, 10 g/liter of bovine albumin, 20,000 U/liter of penicillin G, and 10 mg/liter of streptomycin [pH 7.4]) and kept at –80°C until use.

Cells.

Vero and human rhabdomyosarcoma (RD) cells were used for virus isolation from urine samples. Vero, HeLa, COS7, and FBKT1 cells and human embryonic lung (HEL) fibroblasts were used for the characterization of PLAHV. Vero, RD, and HeLa cells were cultured in Dulbecco’s modified Eagle’s minimal essential medium (DMEM; Wako Pure Chemical Industries Ltd., Osaka, Japan) supplemented with 5% heat-inactivated fetal bovine serum (FBS; Biological Industries, Beit Haemek, Israel), 100 U/ml of penicillin, and 100 μg/ml of streptomycin (both antibiotics from Thermo Fisher Scientific, Waltham, MA). FBKT1 cells were cultured in RPMI 1640 medium (Wako) with 10% FBS and the antibiotics. COS7 and HEL cells were cultured in DMEM with 10% FBS and the antibiotics.

Virus isolation from samples of fruit bats and identification.

The urine samples were subjected to virus isolation. The samples soaked in the transport medium were centrifuged at 3,500 rpm for 10 min, and the supernatants were filtered through a 0.22-μm membrane. Vero and RD cells were inoculated with the supernatant fraction of each sample and cultured in DMEM supplemented with 1% FBS and the antibiotics (DMEM-1FBS) for 7 days. A blind passage procedure was introduced twice.

When CPE appeared in Vero cells and/or RD cells, the cells with CPE were analyzed for virion structure with transmission electron microscopy (JEM1010; JEOL Ltd., Tokyo, Japan). The culture fluid was also subjected to both next-generation sequencing analysis and a proteomics-based virus detection system using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI/MS/MS) (33) to detect nucleotide or peptide traces of viruses, respectively.

Transmission electron microscopy.

Twenty-four and 48 h after infection, the cells were detached from the culture flask with a cell scraper, and the cell suspension was centrifuged at 3,000 × g for 5 min. The cell pellet was resuspended and fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) and kept overnight at 4°C. The sample was then rinsed carefully in 0.1 M cacodylate buffer (pH 7.2) several times. The final pellet was fixed with 1% OsO₄ in the same buffer for 60 min, then rinsed carefully in 0.1 M cacodylate buffer (pH 7.2) several times, subsequently dehydrated in graded ethanol (50 to 100%), washed in propylene oxide, and infiltrated for 6 h in a 1:1 mixture of propylene oxide and epoxydic resin. The cells were then embedded in the resin. Thin sections were obtained with an ultramicrotome (Ultracut UC6; Leica Microsystems GmbH, Germany) and stained with uranyl acetate and lead citrate. The ultrathin section was then observed through a transmission electron microscope operating at 80 kV.

Viruses.

PLAHV used in this study was a novel alphaherpesvirus isolated from P. lylei as described in Results. PtAHV1, another bat-originated alphaherpesvirus, was also used (8), as were HSV-1 strain F and HSV-2 strain 186. All viruses were propagated and titrated in Vero cells cultured in DMEM-1FBS and stored at –80°C until use.

Whole-genome sequencing of PLAHV.

The viral DNA was obtained by lysing Vero cells infected with PLAHV using a QIAmp DNA minikit (Qiagen, Hilden, Germany) and sequenced with Ion PGM (Thermo Fisher Scientific) following the manufacturers’ instructions. The read was de novo assembled using SPAdes assembler to make the contig sequences (34). The gap sequences were amplified by PCR and sequenced with the designated primer sets using an ABI PRISM 3130 genetic analyzer (Thermo Fisher Scientific). The nucleotide sequence, which could not be determined with high-throughput next-generation sequencing and the Sanger sequencing method, was determined with another high-throughput sequencer, the MinION (Oxford Nanopore Technologies, Oxford Science Park, UK), which generated long read sequences. The culture supernatant of Vero cells infected with PLAHV was centrifuged at 20,000 rpm for 2 h at 4°C. The viral pellet obtained was gently suspended in phosphate-buffered saline solution and lysed with a mixture of phenol, chloroform, and isoamyl alcohol (25:24:1). The viral DNA was subjected to sequencing with MinION following the manufacturer’s instructions. The sequence data were de novo assembled with Canu software (35) and used for filling in the missing gaps. The reads from the PLAHV genome obtained with both sequencing methods were mapped onto the draft genome using Burrows-Wheeler Aligner software (36) and merged by extracting the consensus nucleotide sequence automatically using the Unipro Ugene platform (37). The consensus nucleotide sequence was validated with Sanger sequencing if needed to complete the genome sequence of PLAHV. Due to the structure of PLAHV genome (Fig. 2), nucleotide sequences from genome termini were obtained, but the terminal nucleotides were not determined. The genome termini were determined as follows. The genome termini of PLAHV were blunt ended with T4 DNA polymerase and ligated with a DNA fragment whose nucleotide sequence was known using T4 DNA ligase. The genome termini were amplified by PCR and sequenced with primers flanking the genome termini.

Genome analyses.

The PLAHV genome sequence was annotated referring to the PtAHV1 genome sequence (GenBank accession number NC_024306.1) using GATU software (38). Homologs of the RL2, US5, US10, US11, and US12 genes of HSV-1 strain 17 (NC_001806.2) were searched in the open reading frames of the PLAHV genome sequence detected by GATU software with the BLASTP program using those genes as query sequences. The UL15, RL2, and US5 genes of PLAHV that were predicted were further confirmed by Sanger sequencing of their mRNAs. Briefly, Vero cells were inoculated with PLAHV at a multiplicity of infection (MOI) of 5 per cell and harvested at 12 h after infection. The RNA in the infected cells was extracted using an RNeasy Plus minikit (Qiagen) according to the manufacturer’s instructions, and single-stranded cDNA was synthesized using SuperScript IV reverse transcriptase (Thermo Fisher Scientific) with the primers 5′-TTTTTTTTTTTTTTTTTTTT-3′ and 5′-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT-3′. The cDNAs of the UL15, RL2, and US5 genes were amplified with PCR and sequenced with the appropriate designed primers.

The amino acid sequence identities corresponding to each gene predicted in PLAHV to its homolog in PtAHV1 and HSV-1 strain 17 were calculated using the Needle program with default parameters (http://www.bioinformatics.nl/cgi-bin/emboss/needleall).

Phylogenetic analyses.

The phylogenetic trees were constructed using amino acid sequences of herpesviral proteins, including those of PLAHV predicted with the neighbor-joining method using the MEGA 7 program (39). The Poisson model was used as the amino acid substitution model and the alignment gaps were completely removed. The viral species and their GenBank accession numbers included in the analyses are listed in Table 3.

TABLE 3.

Viruses and their accession numbers included in the phylogenetic analysis

Virus	Abbreviation	Accession no.
Ateline alphaherpesvirus 1	AtHV-1	NC_034446.1
Bovine alphaherpesvirus 1	BoHV-1	NC_001847.1
Bovine alphaherpesvirus 2	BoHV-2
DNA polymerase		AF181249.1
Glycoprotein B		M21628.1
Cercopithecine alphaherpesvirus 2	CeHV-2	NC_006560.1
Chelonid alphaherpesvirus 5	ChHV-5	HQ878327.2
Equid alphaherpesvirus 1	EqHV-1	NC_001491.2
Felid alphaherpesvirus 1	FeHV-1	NC_013590.2
Pteropodid alphaherpesvirus 1	PtAHV1	NC_024306.1
Gallid alphaherpesvirus 1	GaHV-1	NC_006623.1
Gallid alphaherpesvirus 2	GaHV-2	NC_002229.3
Human alphaherpesvirus 1	HSV-1	NC_001806.2
Human alphaherpesvirus 2	HSV-2	NC_001798.2
Human alphaherpesvirus 3	HHV-3	NC_001348.1
Human betaherpesvirus 5	HHV-5	NC_006273.2
Human gammaherpesvirus 4	HHV-4	NC_007605.1
Leporid alphaherpesvirus 4	LeHV-4	NC_029311.1
Macacine alphaherpesvirus 1	McHV-1	NC_004812.1
Macropodid alphaherpesvirus 1	MaHV-1	NC_029132.1
Papiine alphaherpesvirus 2	PaHV-2	NC_007653.1
Saimiriine alphaherpesvirus 1	SaHV-1	NC_014567.1
Eidolon dupraenum simplexvirus 1		FJ040879.1
Eidolon helvum simplexvirus 1		FJ040890.1
Bat simplexvirus 1		FJ040888.1
Pteropus lylei simplexvirus 1		FJ040877.1
Lonchophylla thomasi simplexvirus 1		FJ040887.1

In vitro replication assay (one-step growth).

Vero, COS7, HEL, HeLa, and FBKT1 cells were inoculated with PLAHV or HSV-1 F at an MOI of 5 and incubated for 1 h at 37°C and 5% CO₂ for adsorption. The cells were then washed 3 times with phosphate-buffered saline solution and cultured for the desired times. Vero, COS7, HeLa, and HEL cells were cultured with DMEM-1FBS, whereas the FBKT1 cells were cultured with RPMI 1640 medium supplemented with 1% FBS. The cells with culture medium were harvested at the desired time points, and the infectious dose of each sample was measured as described previously (40), with some modifications.

Virulence of PLAHV in mice.

Four-week-old female BALB/c mice (Japan SLC, Inc., Shizuoka, Japan) were intranasally inoculated with 5 μl of DMEM containing the desired dose of PLAHV under anesthesia using isoflurane and monitored daily for their survival and body weight for 13 days. Mice were euthanized under excess CO₂ gas when they lost more than 20% of their initial body weight. Five mice were used for each infectious dose group, and the LD₅₀ was calculated by the Reed and Muench method (41).

Production of mouse anti-PLAHV, PtAHV1, HSV-1 F, or HSV-2 serum.

Four-week-old female BALB/c mice (Japan SLC, Inc.) were inoculated with either PLAHV, PtAHV1, HSV-1 F, or HSV-2 186 as described above at the respective dose of 1.0 × 10⁴, 1.0 × 10², 1.0 × 10², or 1.0 × 10² PFU/mouse. Two weeks later, mice of each group were again intraperitoneally inoculated with 200 μl of DMEM containing the respective virus at the respective dose of 1.0 × 10⁵, 1 × 10⁵, 1 × 10³, or 1 × 10³ PFU. The mice of the PtAHV1-inoculated group were euthanized under excess CO₂ gas, and their blood was collected and pooled 1 month after the second infection, whereas the mice in the other groups were intraperitoneally inoculated with the respective virus at the dose of 1 × 10⁵ PFU/mouse, and their blood was collected 2 weeks after the third inoculation. The condition of these mice was monitored until the day of blood collection. Each blood pool was stored at 4°C overnight and centrifuged at 5,000 rpm for 15 min at 4°C. The serum fractions were stored at –25°C until use.

Neutralization antibody assay.

Each serum sample obtained was incubated at 56°C for 30 min for heat inactivation and stored at –25°C until use. The serum samples were 2-fold serially diluted with DMEM. Then, 35 μl of each serum sample was mixed with 315 μl of virus solution containing 50 PFU of each virus/90 μl and incubated for 1 h at 37°C under a 5% CO₂ humidified condition. Vero cells in 24-well culture plates were then inoculated with 100 μl of each mixture in triplicate, and the number of plaques was counted as described above 2 days after inoculation.

Determination of PLAHV sensitivity to acyclovir.

Sensitivities of PLAHV, HSV-1 F, and HSV-2 186 to acyclovir (Tokyo Chemical Industry, Tokyo, Japan) in Vero cells were evaluated with the standard plaque reduction assay as described previously (40, 42).

Statistical analyses.

The differences in replication kinetics and antiviral sensitivity of PLAHV to those of HSV-1 were assessed using Student’s t test. All statistical calculations and data visualizations were performed with Microsoft Excel software.

Ethics statement.

All of the animal experiments in this study were approved by the Animal Care and Use Committee of the National Institute of Infectious Diseases (no. 116112) and conducted in compliance with animal husbandry and welfare regulations of Japan.

Data availability.

The genome sequence information for PLAHV was deposited in GenBank with accession number LC492974. All the materials and data on this study will be distributed if requested.