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Virus Genes
2009 Aug 01;391:90-3. doi: 10.1007/s11262-009-0368-8.
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Detection of a new bat gammaherpesvirus in the Philippines.
Watanabe S
,
Ueda N
,
Iha K
,
Masangkay JS
,
Fujii H
,
Alviola P
,
Mizutani T
,
Maeda K
,
Yamane D
,
Walid A
,
Kato K
,
Kyuwa S
,
Tohya Y
,
Yoshikawa Y
,
Akashi H
.
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A new bat herpesvirus was detected in the spleen of an insectivorous bat (Hipposideros diadema, family Hipposideridae) collected on Panay Island, the Philippines. PCR analyses were performed using COnsensus-DEgenerate Hybrid Oligonucleotide Primers (CODEHOPs) targeting the herpesvirus DNA polymerase (DPOL) gene. Although we obtained PCR products with CODEHOPs, direct sequencing using the primers was not possible because of high degree of degeneracy. Direct sequencing technology developed in our rapid determination system of viral RNA sequences (RDV) was applied in this study, and a partial DPOL nucleotide sequence was determined. In addition, a partial gB gene nucleotide sequence was also determined using the same strategy. We connected the partial gB and DPOL sequences with long-distance PCR, and a 3741-bp nucleotide fragment, including the 3'' part of the gB gene and the 5'' part of the DPOL gene, was finally determined. Phylogenetic analysis showed that the sequence was novel and most similar to those of the subfamily Gammaherpesvirinae.
Fig. 1. Overall scheme for direct sequencing with RDV primer sets
Fig. 2. A phylogenetic tree was constructed using a multiple alignment of 914 aa, consisting of concatenated gB and DPOL amino acid sequences. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test are shown next to the branches. Phylogenetic analyses were conducted in MEGA4 [11]. The tree was rooted to herpes simplex virus type 1 (HSV1) (X14112). The evolutionary distances were computed using the Poisson correction method and are given in units of the number of amino acid substitutions per site. All positions containing alignment gaps and missing data were eliminated from the dataset. The herpesviruses used for comparison and their accession numbers are as follows: alcelaphine herpesvirus 1 (AHV1), NC_002531; badger herpesvirus (BadgerHV), AF376034; bat gammaherpesvirus 1 (BatGHV1), DQ788623; BatGHV4, DQ788627; BatGHV5, DQ788629; caprine herpesvirus 2 (CPHV2), AF283477; Epstein-Barr virus 1 (EBV)(human herpesvirus 4), NC_007605; equine herpesvirus 2 (EHV2), NC_001650; human cytomegalovirus (HCMV), NC_006273; human herpesvirus 6 (HHV6), AF157706; HHV7, NC_001716; HHV8 (Kaposi’s sarcoma virus), NC_003409; retroperitoneal fibromatosis-associated herpesvirus (RFHV), AF005479; rhesus monkey rhadinovirus (RRV), AF083501; saimiriine herpesvirus 2 (HVS), NC_001350
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