Partial molecular characterization of alphaherpesviruses isolated from tropical bats

Correspondence Jean-Marc Reynes jmreynes@pasteur.mg Unité de virologie, Institut Pasteur de Madagascar, route de l’Institut Pasteur, BP 1274, Antananarivo 101, Madagascar Unité de virologie, Institut Pasteur du Cambodge, 5 boulevard Monivong, BP 983, Phnom Penh, Cambodia Centro Colaborador da OMS para Referência e Pesquisa em Arbovı́rus, Instituto Evandro Chagas/ SVS/MS, Av. Almirante Barroso, 492, 66093-020 Belém, Pará, Brazil Centre Collaborateur OMS de Référence et de Recherche sur les arbovirus, Institut Pasteur de Dakar, 36 Avenue Pasteur, BP 220, Dakar, Senegal

The family Herpesviridae includes about 124 virus species (Bu ¨chen-Osmond, 2001).The diagnostic characters of this family are based on virus structure (large, enveloped virus, from 120 to 200 nm, icosahedral capsid, large doublestranded DNA of 120 000-220 000 bp). Viruses of this family exhibit considerable differences in their nucleotide composition and genome organization.However, they share several biological properties including their ability to be latent or persistent for life in their host.On the basis of differences in cellular tropism, genome organization and gene content, the family Herpesviridae has been divided into three subfamilies, Alphaherpesvirinae, Betaherpesvirinae and Gammaherpesvirinae.Each subfamily includes genera and species grouped together based on, for example, DNA polymerase sequences and similarities in genome sequence arrangement (Roizman, 1996;McGeoch et al., 2006).
Herpesviruses are highly disseminated in vertebrates, and most mammal orders have yielded at least one herpesvirus.No herpesvirus is listed in the universal virus database from the order Chiroptera or bats (Bu ¨chen-Osmond, 2001).However, bats are not an exception.Herpesviruses, among numerous viruses from different families, have been obtained from bats, including a cytomegalovirus isolated from an insectivorous bat (Myotis lucifugus, family Vespertilionidae) in the USA and Agua preta and Parixa viruses isolated from frugivorous bats (family Phyllostomidae) in Brazil (Calisher et al., 2006).Further isolates have been obtained from frugivorous bats (family Pteropodidae) in Cameroon (Dak An Y 6, Dak An Y 7 and Dak An Y 9) and from an unidentified bat species in the Central African Republic (Dak An B N27) (http:// www.pasteur.fr/recherche/banques/CRORA/virus/v1301010.htm).Allocation to the family Herpesviridae was based on serological tests or electron microscopy; however, these viruses were not studied in a molecular manner and have not been assigned to a genus in the family.Recently, herpesvirus sequences targeting the DNA polymerase gene were detected from European insectivorous bats (family Vespertilionidae) and related to members of the gammaherpesvirus genera Percavirus, Rhadinovirus and Macavirus and of the subfamily Betaherpesvirinae (Wibbelt et al., 2007).
In the present study, we report on the partial molecular characterization of Parixa (Be AN 422840), Dak An Y 6, Dak An Y 7, Dak An Y 9 and Dak An B N27 herpesviruses isolates and 10 additional herpesvirus isolates that we obtained recently from Malagasy and Cambodian frugivorous bats, in order to confirm their allocation to the family Herpesviridae and to assign a genus name to these isolates.
Fifteen isolates were analysed, including 10 undescribed isolates obtained on Vero E6 cells from samples collected in Cambodia and Madagascar (Table 1).The sample (throat swab) from Cambodia was collected from a Lyle's flying fox (Pteropus lylei; family Pteropodidae) captured and released in a tree roost located in the village/municipality of Svay Sach Phnum (Srey Santhor District, Kampong Cham Province; 11 u 54.9469 N 105 u 10.9079 E).Samples from Malagascar (throat swabs) were collected from endemic Malagasy fruit bats (Eidolon dupreanum; family Pteropodidae) captured and released in the Angavokely Caves (Nandihizana Carion Municipality, Manjakandriana District; 18 u 55.9479 S 47 u 45.4459 E) and Angavobe Caves (Sabotsy Anjiro Municipality, Moramanga District; 18 u 55.0839 S 47 u 56.6169 E).Isolates from these two countries were obtained during surveys for henipaviruses (Reynes et al., 2005;Iehle ´et al., 2007).They were suspected to belong to the family Herpesviridae because they induced cytopathic effects (CPE) on Vero E6 cells compatible with those produced by alphaherpesviruses (rapid mass destruction of the cells associated with polykaryocytosis and/or rounded cells).Furthermore, margination of the chromatin in the nucleus and vacuolization of the cytoplasm were observed in Giemsa-stained infected Vero E6 cells (results not presented).
Isolates were propagated in Vero E6 cells to obtain viruscontaining supernatants for molecular studies.DNA was extracted from these supernatants using the classical phenol/chloroform method or with the QIAamp DNA blood mini kit (Qiagen) following the manufacturer's instructions.DNA was then submitted to PCR using the degenerate consensus primers DFASA and GDTD1B targeting the UL30 catalytic subunit region of the DNA polymerase gene (Rose et al., 1997) using PCR cycling conditions described previously (Lacoste et al., 2001).PCR products of the expected size (~518 bp) were obtained from DNA of all isolates.The amplification products were sequenced on both strands by Cogenics (Meylan, France).Unverified sequences and chromatograms returned to us by the company were compared and corrected when needed.Sequences were aligned and fragments of 465 bp shared by all 13 samples and encoding amino acids 724 to 878 of the catalytic subunit of DNA polymerase (positions according to the DNA polymerase sequence from Human herpesvirus 1; GenBank accession no.CAA32323) were selected for the rest of the analysis.The partial nucleotide sequences from the Cameroonian isolates were identical, as were those from all nine Malagasy isolates.These results are not surprising, since the isolates were obtained in each country at the same locality, within a short period and from the same host.Interestingly, there was only one nucleotide difference (among 465) between the Cameroonian and Malagasy nucleotide sequences and and the available sequences of known herpesviruses from other host species, particularly those belonging to the subfamily Alphaherpesvirinae.Phylogenetic analysis of all the sequences was conducted with MEGA version 4 (Tamura et al., 2007), using the neighbour-joining method with the Poisson model correction.This analysis confirms the affiliation of these isolates with the subfamily Alphaherpesvirinae and placed all the isolates in the genus Simplexvirus (Fig. 1).
Bats have been described recently as natural hosts for betaherpesviruses and gammaherpesviruses in Europe (Wibbelt et al., 2007).The data presented in this paper expand the taxonomic range of herpesviruses within bat hosts to the subfamily Alphaherpesvirinae.Although not all of the virus genome was sequenced, four new members in the genus Simplexvirus have been uncovered.The nature of the CPE induced on Vero E6 cells by the Cambodian and Malagasy isolates supports their placement in this genus (Roizman, 1996).Furthermore, these data confirm and extend the geographical distribution of herpesvirus in bats to three more continents (Africa, South America and Asia) and indicate the presence of these viruses in frugivorous bats of the families Pteropodidae and Phyllostomidae.
Interestingly, the partial sequences of the Malagasy and Cameroonian isolates were identical, suggesting that they belong to the same member of the genus Simplexvirus.The Malagasy and Cameroonian isolates were obtained from the bats Eidolon dupreanum and Eidolon helvum, respectively, the only two recognized members of this genus.
During the henipavirus survey in Madagascar, we isolated the orbivirus Ife and detected neutralizing antibodies against the Lagos bat lyssavirus in E. dupreanum (J.-M.Reynes, unpublished data).These two viruses have also been isolated from bats belonging to E. helvum, which is strictly African in distribution (Calisher et al., 2006).The sharing of these viruses by the only two species in this genus is extraordinary, as the two populations are isolated from one another by the Mozambique Channel, a distance of 450 km between the closest points of Africa and Madagascar, and no exchange has been reported (S.M. Goodman, personal communication).
None of the herpesviruses described in European bats could be related consistently to a pulmonary lesion or any other distinct histopathology in the sampled animals (Wibbelt et al., 2007).The Cambodian isolate was obtained from an apparently healthy bat.There is no information on the health status of the sampled Cameroonian, Central African Republic and Brazilian bats.The Malagasy isolates were obtained from apparently healthy bats.However, in Madagascar, we noticed that the herpesviruses isolates were obtained from throat swabs collected in 2006 when keratitis (sometimes ulcerative) was observed in 15 of the 143 bats sampled (virus isolation tested negative using throat swabs from these 15 animals).No herpesvirus was isolated from throat swabs collected from 109 animals in 2007, when keratitis was observed in only one animal.This association is striking, since alphaherpesviruses such as Human herpesvirus 1 and Feline herpesvirus 1 have been responsible for keratitis in their host (Andrew, 2001).
Sampling in Madagascar continues, and virus isolation will be attempted from corneal swabs collected from bats exhibiting keratitis.

Table 1 .
Bat virus isolates analysedThe genera Eidolon and Pteropus belong to the family Pteropodidae, whereas Lonchophylla belongs to the family Phyllostomidae.