Here we report the isolation and characterization of a new betaherpesvirus from the bat M. schreibersii
. Similar to previously described betaherpesviruses, MsHV has a low rate of replication, reaching a peak 2 weeks after infection, which is similar to that of HCMV (61
). MsHV has a restricted species tropism in vitro
and is able to infect only one cell line of nonbat origin from a total of 14 cell lines representing 10 different animal species. The genome of MsHV was sequenced using 454 next generation sequencing and is 222,870 bp in length.
MsHV was initially identified in lymph node primary cells and then subjected to two rounds of purification and propagation with multiple passages in kidney primary cells. The number of passages was less than 10. Genetic stability of betaherpesviruses varies during in vitro
passaging. HCMV and GPCMV have been shown to undergo genetic alterations, including gene loss (24
), while MCMV was reported to be stable (15
). The genetic stability of MsHV is unknown. Given that our approach to identifying MsHV genes is based on finding homologues with the genes of other betaherpesviruses and the common criteria used for unique gene prediction, we acknowledge that data presented in this paper may not be a complete representation of MsHV genome content and that some of the predicted ORFs, especially those with splicing, may need revision in the future when we have transcription data to help better annotation.
Many herpesviruses have terminal repeat (TR) sequences, and according to the presence and location, herpesvirus genomes can be divided into six groups (61
). The pattern of TR sequences in betaherpesviruses varies significantly. HCMV and CCMV have large TR at both termini and the junction of UL
); RCMV (89
) and MCMV (64
) have 504-bp and 31-bp TR, respectively, and GPCMV has TR at the left terminus only (72
). A number of herpesviruses have no TR, including THV, RhCMV, CyCMV (48
), and the gammaherpesvirus RHVP (46
). MsHV also has no TR. We cannot exclude the possibility that only one copy of the TR unit was incorporated into the assembled genome, but taking the 428-bp average length of the sequencing reads into consideration, we think it is unlikely. MsHV, RCMV, MCMV, GPCMV, RhCMV, and CyCMV share a similar genome organization, which is colinear with the prototype isomer of the HCMV genome, while the THV genome is colinear with another isomer of the HCMV genome because its US22 family locus reversely translocates from one end to the other (). So far, of all the betaherpesvirus genomes sequenced, only HCMV and CCMV have a large TR and form four genomic isomers; none of the others, including RhCMV and CyCMV from primates in the Cercopithecidae, share these genomic features. The emergence of large TR and genomic isomers may have occurred very recently in betaherpesvirus evolutionary history, but this hypothesis needs to be validated with more genome sequences from different species.
One of the most significant findings in this study is that MsHV encodes multiple families of gene homologues implicated in immunity. One family encodes MHC class I homologues. Virus-encoded MHC class I homologues have previously been identified in some betaherpesviruses (21
) and a gammaherpesvirus RHVP (46
). HCMV evades T-cell recognition by downregulating expression of MHC class I molecules on the surface of infected cells, an effect mediated by the products of the virus-encoded genes US2, US3, US6, and US11 (85
). Although MsHV doesn't carry any homologues of US2, US3, US6, or US11, MsHV b40 is a homologue of HHV-7 U21, which downregulates classical and nonclassical MHC class I complexes from the cell surface (50
). The downregulation of endogenous MHC class I would expose infected cells to attack by natural kill (NK) cells.
MsHV also encodes two MHC class II homologues and one homologue of the HFE protein which is closely related to MHC class II. To our knowledge, virus-encoded MHC class II homologues have not previously been reported. However, HCMC US2 and US3 not only inhibit the MHC class I antigen presentation pathway, which allows infected cells to evade recognition by CD8+
T lymphocytes, but also inhibit presentation of exogenous protein antigens to CD4+
T lymphocytes, which is part of the MHC class II antigen presentation pathway (84
). Other viruses were also reported to target the MHC class II processing pathway for immune evasion (34
). Epstein-Barr virus, a gammaherpesvirus, downregulates MHC class II expression during its reactivation (42
). Future functional studies of MsHV MHC class II homologues are required to fully understand the significance of the roles they may play in potentially novel virus immune evasion pathways.
The term C-type lectin was originally introduced to distinguish between Ca2+
-dependent and Ca2+
-independent carbohydrate-binding lectins, but the C-type lectin superfamily now includes a large number of proteins that are homologous to the C type but don't bind carbohydrates (100
). Recent studies have identified C-type lectins as an important family of pattern recognition receptors (PRRs) that are involved in the induction of specific gene expression profiles in response to specific pathogens (29
). The human NK receptor complex includes a group of type II transmembrane proteins resembling C-type lectins, for which there is accumulating evidence to support crucial roles in the innate immune system (36
). MsHV b156, b161, b162, and b163 are related to C-type lectin or natural killer cell lectin-like receptors. The English strain of RCMV was reported to express a C-type lectin protein with high similarity with host C-type lectins (90
), but a genome-wide comparative analysis between this virus and MsHV was not possible because of the limited sequence available to RCMV. C-type lectin homologues have also been found in the genomes of other viruses, including gammaherpesviruses (46
), poxviruses (6
), and African swine fever virus (35
). While some of them are dispensable or play some role in virus infection, the EBV C-type lectin homologue gp42 was reported to bind to the MHC class II receptor HLA-DR1 (57
A gene family of 16 members, the b149 family was identified at the 3′ end of the MsHV genome and before the US22 family. The b149 family gene products have no significant homologues in GenBank, but three-dimensional (3D) structure predication analysis indicated they all contain immunoglobulin-like beta-sandwich folds. Interestingly, in the MCMV genome, the m145 family, a unique gene family of 12 ORFs, was predicted with MHC class I-like folds with a confidence of 70% to 90% (76
). Remarkably, in both the b149 and m145 families, the members located in the center of the gene cluster are more conserved with each other (66
). As reviewed by Revilleza et al. (66
), the m145 family plays an important role in disrupting NK-cell recognition. For example, m138, m145, m152, and m155 downregulate different ligands of NKG2D, and m157 binds both the inhibitory NK receptor Ly49I and the activating receptor Ly49H. And interestingly, the surface molecules of NK cells, NKG2D, Ly49I, and Ly49H, belong to the C-type lectin family. The prediction confidence of the b149 family is low, ranging from less than 10% to 35%, which could be due to the fact that there is little information available in the public database for bat immune molecules. If we can experimentally prove their functionality in immune evasion, these molecules may represent an important new class of immune modulators yet to be fully characterized in other mammalian viruses.