key: cord-0043738-thitcl8a
authors: James, Samantha; Donato, Damien; de Thoisy, Benoît; Lavergne, Anne; Lacoste, Vincent
title: Novel herpesviruses in neotropical bats and their relationship with other members of the Herpesviridae family
date: 2020-05-23
journal: Infect Genet Evol
DOI: 10.1016/j.meegid.2020.104367
sha: d18a2a7997c03370052cb93d66d5f6569b8ccbfa
doc_id: 43738
cord_uid: thitcl8a

In the past decade, a large number of studies have detected herpesvirus sequences from many bat species around the world. Nevertheless, the discovery of bat herpesviruses is geographically uneven. Of the various bat species tested to date, only a few were from the New World. Seeking to investigate the distribution and diversity of herpesviruses circulating in neotropical bats, we carried out molecular screening of 195 blood DNA samples from 11 species of three bat families (Phyllostomidae, Mormoopidae, and Molossidae). Using polymerase chain reaction amplification, with degenerate consensus primers targeting highly conserved amino acid motifs of the herpesvirus DNA polymerase and Glycoprotein B genes, we characterized novel viral sequences from all tested species. BLAST searches, pairwise nucleotide and amino acid sequence comparisons, as well as phylogenetic analyses confirmed that they all belonged to the Herpesviridae family, of the Beta- and Gammaherpesvirinae subfamilies. Fourteen partial DNA polymerase gene sequences, of which three beta- and 11 gamma-herpesviruses, were detected. A total of 12 partial Glycoprotein B gene sequences, all gamma-herpesviruses, were characterized. Every sequence was specific to a bat species and in some species (Desmodus rotundus, Carollia perspicillata, and Pteronotus rubiginosus) multiple viruses were found. Phylogenetic analyses of beta- and gammaherpesvirus sequences led to the identification of bat-specific clades. Those composed of sequences obtained from different bat species belonging to distinct subfamilies follow the taxonomy of bats. This study confirms the astonishing diversity of bat herpesviruses and broadens our knowledge of their host range. Nevertheless, it also emphasizes the fact that, to better appreciate the evolutionary history of these viruses, much remains to be done at various taxonomic levels.

With over 1,200 species, representing more than 20% of all registered mammal species, Chiroptera is the second-largest species-rich mammalian order (Fenton, 1997) . Bats are present on all continents except the poles and a few isolated oceanic islands. They are highly diverse in terms of their anatomy and lifestyles, and have different diets (insectivorous, frugivorous, nectarivoro us, carnivorous, piscivorous, or hematophagous) . Thanks to their biological traits, bats provide key ecosystem services (Kunz et al., 2011; López-Baucells et al., 2018) . At the same time, as a natural reservoir of many viruses, their global distribution, abundance, ability to fly and migrate over large distances, and the diversity of their diets and sociality are all factors that favor the acquisition and spread of viruses (Calisher et al., 2006; Drexler et al., 2012; Mackenzie, 2005; Wong et al., 2007) .

They are thus considered to play a major role in the emergence and transmission of zoonotic viruses such as lyssaviruses, coronaviruses, paramyxoviruses, or filoviruses (Baker et al., 2013b; Calisher et al., 2006; Davis et al., 2006; Freuling et al., 2011; Halpin et al., 2000; Kupferschmidt, 2013; Leroy et al., 2005; Li, 2005; Luby et al., 2009; Smith and Wang, 2013; Towner et al., 2009; Wong et al., 2007; Yob et al., 2001; Zaki et al., 2012) . Bats are also carriers of other viruses, includ ing herpesvirus species that are host specific. However, there are examples of cross-species transmission (Ehlers et al., 2008) . The first description of bat herpesvirus sequences dates back to 2007 (Wibbelt et al., 2007) . Over the past decade, dozens of herpesvirus sequences have been described from different bat species on every continent (Anthony et al., 2013; Baker et al., 2013a; Dacheux et al., 2014; Dietrich et al., 2018; Donaldson et al., 2010; Escalera-Zamudio et al., 2016; Ge et al., 2012; Geldenhuys et al., 2018; He et al., 2013; Holz et al., 2018; Hu et al., 2017; Jánoska et al., 2011; L. Li et al., 2010; Molnár et al., 2008; Mühldorfer et al., 2011; Noguchi et al., 2018; Paige Brock et al., 2013; Pozo et al., 2016; Razafindratsimandresy et al., 2009; Salmier et al., 2017; Sano et al., 2015; Sasaki et al., 2014; Shabman et al., 2016; Subudhi et al., 2018; Wada et al., 2018; Watanabe et al., 2010 Watanabe et al., , 2009 Wray et al., 2016; Wu et al., 2012; Yang et al., 2013; Zhang et al., 2012; Zheng et al., 2016 Zheng et al., , 2018 . Most of them were characterized from apparently healthy anima ls sampled during trapping campaigns in the frame of random surveillance programs. To date, two bat herpesviruses, fruit bat alphaherpesvirus 1 and bat gammaherpesvirus 8 (FBAHV1 and BGHV8, respectively) have been recognized as species by the International Committee for Taxonomy of Viruses (ICTV) according to the latest master species list (MSL# 34) released on March 8, 2019 (https://talk.ictvonline.org/files/master-species-lists/m/msl/8266). Their ICTV official names are Pteropodid alphaherpesvirus 1 (PtAHV1) and Vespertilionid gammaherpesvirus Nevertheless, the discovery of bat herpesviruses is geographically uneven. Most sequences are from Asian, African, and European bat species. Comparatively few data are available regarding herpesviruses of New World species. Indeed, of the 233 recorded sequences, only eight are from New World bat species, six from North and Central America and two from South America (Donaldson et al., 2010; Escalera-Zamudio et al., 2016; Razafindratsimandresy et al., 2009; Salmier et al., 2017; Shabman et al., 2016; Subudhi et al., 2018) . The ones from North and Central America were obtained from two insectivorous and two hematophagous bat species of the Vespertilionidae and Phyllostomidae families, respectively, and corresponded to beta-and gammaherpesvirus sequences (Donaldson et al., 2010; Escalera-Zamudio et al., 2016; Shabman et al., 2016; Subudhi et al., 2018) . Those from South America, one alpha-and one gammaherpesvir us, were derived from bats belonging to the Phyllostomidae and Molossidae families, respectively (Razafindratsimandresy et al., 2009; Salmier et al., 2017) . In addition, Wray et al. reported the characterization of two other herpesvirus sequences, DrHV-1 and DrHV-2, described as gammaand betaherpesvirus, respectively, from Desmodus rotundus individuals from Guatemala (Wray et al., 2016) . Unfortunately, these last sequences have not been released in the databases. Strikingly, with the exception of the two aforementioned sequences, there has been no prior organized effort to discover herpesviruses in neotropical bat species from South America and in particular from Amazonia, which hosts one of the highest diversity of bat species in the world (López-Baucells et al., 2016; Paglia et al., 2012) . To ascertain the distribution and diversity of herpesviruses circulating in neotropical bats, additional investigations were required. Taking advantage of a unique collection of bat samples collected during the past 10 years in French Guiana (South America) and Martinique (French West Indies), we addressed the presence of herpesvir uses in the different bat species collected by analyzing two distinct informative partial genes. The partial J o u r n a l P r e -p r o o f Journal Pre-proof characterization of DNA polymerase and Glycoprotein B sequences constitutes a powerful tool in the search for new herpesviruses, which in addition allows unambiguous classification of the newly detected sequences within the different Herpesvirinae subfamilies. Here, we report finding sequences of herpesviruses in all New World bat species tested and describe their phylogene tic relationships.

To look for the presence of herpesvirus sequences in our collection of bats, we attempted to amplify a fragment of the highly conserved herpesvirus DNA polymerase (DPol) gene from the PBMC DNA of each wild-caught bat using two different sets of degenerate primers and PCR conditions, as described previously (Ehlers et al., 1999; Rose et al., 1997; VanDevanter et al., 1996) . A total of 195 samples from 11 bat species were tested ( Table 1) . DNA samples (39/195 = 20%) from 10 species scored positive after the nested PCRs (nPCRs) ( Table 1) Table 1) .

All obtained sequences were species-specific and all but the two sequences from Molossus molossus viruses, MmolBHV1 and MmolGHV1, and the one from Pteronotus alitonus, PaliBHV1, were characterized from at least two individuals ( Table 1) . Thus, DrotGHV1, DrotGHV2, and DrotGHV3 were detected from 10, four, and three out of 22 D. rotundus individuals, respectively while DyouGHV1, AplaGHV1, and SangGHV1 were each identified from five individuals. We also characterized a few cases of co-infections: two D. rotundus individuals were co-infected with DrotGHV1 and DrotGHV2 and three with DrotGHV1 and DrotGHV3 as well as one C.

perspicillata individual infected with CperGHV1 and CperGHV2.

We then designed species-specific antisense primers to extend each of the newly identified sequences in upstream direction by about 300 bp with a one round of semi-nested semi-specific Table 1 ). This resulted in concatenated nucleotide sequences of 439 and 454 bp in size for betaherpesviruses and of 472 bp in size for gammaherpesviruses.

gene in bat samples.

belonged to the Herpesviridae family and revealed the presence of 12 distinct sequences, all from the Gammaherpesvirinae subfamily. Among them, three different sequences were found in D.

rotundus (DrotGHVA, DrotGHVB, and DrotGHVC) and two in P. rubiginosus (PrubGHVA and PrubGHVB). Each viral sequence detected was specific to a bat species and, even though some viruses were found in different individuals of the same species, we did not identify any case of coinfection ( Table 1 ).

Sequence identity was determined by comparing nucleotide and amino acid sequences of all herpesviruses detected in the different bat species with viruses of the same subfamily described in several mammalian orders (Supplementary Table 2 and Supplementary Table 3 Table 3 Table 3 ). The percentage of identity between the three viruses detected in D.

rotundus ranged from 49% to 55.1% at the nucleotide level and from 43.2% to 58% at the amino acid level. The two sequences detected in C. perspicillata showed 62.9% and 64.2% identity in nucleotides and amino acids, respectively. Comparison with the other bat gammaherpesvir uses yielded similar results, ranging from 44.1% to 86.9% nucleotide identity and from 40.7% to 91.4% amino acid identity. In addition, the level of nucleotide and amino acid identity with the other mammal gammaherpesviruses ranged from 42% to 69.4% and from 35.8% to 69.1%, respectively.

Sequence identity of Gb sequences was determined by comparing sequences of all bat gammaherpesviruses identified as well as with those of other mammal gammaherpesvir uses representative of the different gammaherpesvirus genera (Supplementary Table 4 Table 4 ). The percentage of identity between the J o u r n a l P r e -p r o o f Journal Pre-proof three viruses detected in D. rotundus ranged from 62.1% to 85.5% at the nucleotide level and from 59.1% to 93% at the amino acid level, while the two distinct sequences detected in P. rubiginosus showed 56.4% and 54.8-55.7% nucleotide and amino acid identities, respectively.

Overall, the percentages of nucleotide and amino acid identities between the newly obtained sequences with those already published from bats or other mammals were higher or on the same order of magnitude as for DPol sequences (Supplementary Table 3 Comparison with the other chiropteran gammaherpesviruses showed from 48.7% to 93.7% nucleotide identity and from 41.7% to 98.3% amino acid identity. Finally, the level of nucleotide and amino acid identity with the other mammal gammaherpesviruses ranged from 49.3% to 69.8% and from 45.2% to 74.8%, respectively.

Phylogenetic analyses performed on nucleotide sequences of the newly characterized betaherpesvirus partial DPol sequences with those of other betaherpesviruses available in the databases grouped the bat viral sequences in three well-supported monophyletic clades only lineages are close to the Cytomegalovirus genus. The first one is only composed of sequences derived from Vespertilionidae bats. In addition, this clade is divided into two different groups, (a) one containing sequences detected in bats belonging to the genus Myotis and the one identified 

The phylogenetic analysis based on DNA polymerase sequences of Gammaherpesvirinae shows that bat viral sequences are distributed all over the tree (Figure 2) . Based on this sequence dataset, all that could be obtained is a multifurcating tree with low support for the deepest nodes. Therefore, resolution of the branching pattern is incomplete and phylogenetic relationships between the different Gammaherpesvirinae genera are not supported. Nevertheless, in the terminal branchings, certain highly supported clades can be recognized. Two of them correspond to the 

The phylogenetic analysis between the newly characterized bat gammaherpesvirus Gb sequences and that of already published sequences from bats and of representative gammaherpesvirus sequences available in the databases is presented in Figure 3 . Almost all nodes to different families (clades a, b, d, f, and i). Some sequences do not belong to these clades: They correspond to previously published viral sequences from Scotophilus kuhlii and Rhinolophus blythi closely associated with bovine herpesvirus sequences (Bovine herpesvirus 6 and Bovine lymphotropic herpesvirus) that belong to the Macavirus genus and to the viral sequence derived from Hipposideros pomona that is associated with Sorex araneus gammaherpesvirus (SaraGHV1) and viral sequences from Carnivora (MusHV1, FcatGHV1 and LrufGHV1) but with a pp value of less than 0.7 (Watanabe et al., 2009; Zheng et al., 2016) . This last sequence and those of clades a and b from bat species of the Vespertilionidae, Rhinolophidae, and Miniopteridae families belong to a monophyletic well-supported (pp=0.96) lineage of Percaviruses.

All new sequences detected here fall within four distinct clades (d, e, g, and j). Clade d is 

This is the largest study conducted to date to assess the occurrence and diversity of herpesvir uses in New World bat species. A viral sequence was obtained for every tested species. Each obtained sequence was novel, species-specific and no case of cross-transmission between bat species was J o u r n a l P r e -p r o o f Journal Pre-proof identified. Except for P. rubiginosus, for which we did not generate any DPol sequence, and A. planirostris and P. alitonus, for which no Gb sequence was amplified, the two types of sequences were obtained for all other species. Nevertheless, for the species from which both sequences were obtained, PCR targeting Gb was less sensitive than the one targeting DPol with a lower number of positive samples (Table 1 ). In addition, for Gb, only gammaherpesvirus sequences were obtained.

These results prove that, to screen samples, the combinations of primers targeting DPol are a better tool both in terms of sensitivity and diversity of amplified sequences. This also suggests that for

Gb other combinations of primers should be used to allow for amplification of sequences of other subfamilies. Nevertheless, the fact that we preferentially amplified gammaherpesvirus sequences is not only a question of primer degeneracy, but also depends on the type of samples tested. Indeed, of all the bat herpesviruses currently described in the literature, most alpha-and betaherpesvir uses (globally underrepresented compared with gammaherpesviruses) were amplified from oral swabs (Pozo et al., 2016; Razafindratsimandresy et al., 2009 ). It will therefore be important for future studies to test not only other bat species but also different types of samples from the same species.

From a phylogenetic perspective, in view of the available data, it appears that differentia l clustering exists between bat betaherpesvirus sequences according to their host families ( Figure  betaherpesvirus sequences are available and that only one betaherpesvirus sequence is availab le for certain families while others are not represented.

Regarding Gammaherpesvirinae, the phylogeny based on Gb sequences is well supported with well-defined clades corresponding to the different known genera. Comparatively, the topology of the DPol tree is less reliable and exhibits poor overall support. The two phylogenies are based on alignments of similar size, and thus the lower support observed for DPol can be attributed to an overall higher level of sequence divergence. Nevertheless, the two phylogenies show that bat gammaherpesviruses are scattered over the entire tree (Figures 2 and 3) . They also demonstrate that viral sequences from Phyllostomidae are distributed in two distinct clades, while those of Molossidae fall together on a separate branch. Based on the Gb phylogeny, which is in agreement with the known phylogeny of Gammaherpesvirinae, we further observe that viruses of Phyllostomidae are close to the Rhadinovirus and Percavirus genera, while those of Molossidae possess a basal position relative to these genera. Finally, within the different clades (c, d, e, and h), composed of viral sequences obtained from bat species belonging to different subfamilies, the phylogenetic relationships of the different viral sequences demonstrate a good correlation with the taxonomy of the host species (Figure 3) (Agnarsson, 2011) . These results are in support of a coevolutionary scenario. Nevertheless, each bat-specific clade is only represented by a few sequences from different species (2 < n < 5). Owing to the paucity of the available data, considering the number of bat species, genera, subfamilies, and families that remain to be tested, this assumptio n will only be confirmed when more sequences become available.

This study greatly expands our knowledge on the distribution and genetic variation of bat herpesviruses. It adds new insights into the viral diversity hosted by bats from French Guiana and J o u r n a l P r e -p r o o f

Martinique and, as may be expected, confirms the astonishing diversity of bat herpesviruses.

However, we are still far from having deciphered the in-depth details of this diversity. Indeed, the number of New World bat species tested still accounts for only a tiny part. In French Guiana alone, 107 bat species are currently recognized, with the number evolving steadily, mostly due to the splitting of taxa on the basis of new genetic evidence (Catzeflis, 2017 (Catzeflis, , 2015 Simmons and Voss, 1998) . Therefore, the number of tested species accounts for just 10% of the local bats, suggesting a wide diversity of herpesviruses awaiting discovery. These results emphasize the crucial need for a better assessment of herpesvirus distribution in bats. Analysis of other species at a wider geographical scale, as well as of the same species but on other types of samples, should maximize our chances of detecting the whole diversity of bat herpesviruses and thereby expanding our understanding of the diversification processes and evolutionary history of these viruses. 

All bats examined in this study were collected as part of an investigation program on rabies virus circulation. In French Guiana, captures were implemented in bat communities during a 10-year period (de Thoisy et al., 2016) . In Martinique, bats were captured in January 2015. All captures were performed at night with Japanese mist nets erected near breeding sites, roosts, at forest edges, around livestock, or through putative foraging courses. Animals were kept in individual bags before being sampled for blood with a sterile needle and capillary at the brachial vein. Before release, external pressure was exerted on the vein with a sterilized absorbent hemostatic sponge to prevent bleeding and facilitate healing. Blood samples were preserved at 4°C until arrival at the laboratory and centrifuged at 6500 g for 10 min to separate sera. Sera and buffy coat samples were stored at -80°C for later use in the laboratory. The sex and age of all animals were recorded, and the anima ls were identified morphologically in the field. When possible errors of species identification were suspected, identification was molecularly confirmed by sequencing a fragment of the mitochondrial Cytochrome oxidase I or Cytochrome b genes (Borisenko et al., 2008) . Nucleic acids were extracted using the NucliSENS easyMAG® bio-robot (bioMérieux®, Marcy l'Etoile, France (Prepens et al., 2007; Rose et al., 1997; VanDevanter et al., 1996) . For the DNA polymerase amplification, two sets of primers targeting the same region of the gene but with different levels of Table 1 ) (Rose et al., 1997; VanDevanter et al., 1996) . For the Glycoprotein B amplification, one set of primers was used: 2759s/2762as for the first-round PCR and 2760s/2761as for the second-round PCR (66). All amplicons of approximately the expected size were purified, cloned via TA cloning, and sent for sequencing to Genewiz®, Takeley, UK (https://www.genewiz.com/). To help increase the possibility of identifying different herpesvir us sequences from each amplicon obtained, five to eight clones of the "screening amplicons" were sequenced on both strands.

To obtain the nucleotide sequence upstream of the VYGA or TGV motif, species-specific non-degenerate primers were derived from the complementary sequences of the small fragments and used in an nPCR amplification with the DFASA or DFA primer pools using the initial PCR products as templates (Supplementary Table   1 ). Overlapping amplicons were generated, cloned, and sequenced as described above. Each sequence corresponds to at least three independent clones sequenced on both strands. Contig sequences were then assembled using MEGA 5.05 software (Tamura et al., 2011 

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NC_024696), EEHV6 (HM060765) and EEHV7A (JQ300083). The Roseolovirus clade comprises sequences of MndHV (AF282942), PanHV6 (AY359407), HHV6A and HHV6B (NC_001664 and NC_000898, respectively), HHV7 (NC_001716), PtroHV7 (KJ843227 and KJ843228), PpanHV7 (KJ843230), GgorHV7 (KJ843231), and MneHV7 (NC_030200). The Muromegalovirus clade is composed of RatCMV/MuHV2/MuHV8 strains (AY728086, KP967684 and NC_019559

MCMV/MuHV1 (AM886412 and NC_004065), MarvCMV1 (EF125059), MglaCMV1 (EF125061), AflaCMV2 (EF125063), and RatCMV Maastricht (NC_002512). Finally, viruses of the Cytomegalovirus collapsed clade are: AoHV1 (FJ483970)

AsenCMV1 (KU963225), AmacCMV1 (KU963227), ApanCMV1 (KU963228)

BaCMV (NC_027016)

Phylogenetic tree of Gammaherpesvirus DNA polymerase sequences. The phylogene tic

tree was derived from the partial nucleotide sequences of the DNA polymerase gene (489 bp) of 80 representatives of gammaherpesviruses using the Bayesian method with the GTR + I + G model of nucleotide evolution. Herpes simplex virus type 1 sequence (HHV1 M10792) served as outgroup.The tree is shown as a majority rule consensus tree. Support for nodes was provided by the posterior probabilities of the corresponding clades. All resolved nodes have posterior probability greater than 0.75. A scale indicating divergence, as substitutions per site, is at the foot. Sequences generated in this study are in boldface. The virus names are associated with their accession numbers. For bat viruses, the host species from which the virus has been detected is indicated after the virus name.In addition, they are color-coded according to the bat families. Abbreviations of virus names use the first letter of the generic host name in uppercase and the first three letters of the specific host name followed by either GHV (Gammaherpesvirus), LCV (Lymphocryptovirus), RHV

The authors declare that they have no conflict of interest.J o u r n a l P r e -p r o o f