The sequence of the unique short (U S ) region of monkey B virus (BV) was determined. The 13 genes identified are arranged in the same order and orientation as in herpes simplex virus (HSV). These results demonstrate that the BV U S region is entirely colinear with that of HSV type 1 (HSV-1), HSV-2, and simian agent 8 virus.Cercopithecine herpesvirus 1 (monkey B virus [BV]) is a member of the alphaherpesvirus subfamily indigenous in Asiatic macaque monkeys. The natural history of BV in macaques is similar to that of the human herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) in humans, of simian agent 8 virus (SA8) in green monkeys, and of Herpesvirus papio 2 (HVP-2) in baboons (11,12,38). When transmitted to nonmacaque primates, BV produces severe infections which usually involve the central nervous system and are frequently fatal (31,37,38). The molecular basis for the extreme neuropathology of BV in nonmacaque species is an intriguing question that remains unanswered.While sequences for a number of genes of BV have been reported (3,9,23,34,35,37), genomic characterization of BV has largely been limited to restriction analysis and gene mapping by hybridization with HSV gene probes (10,16,17). Such studies have suggested that, for the most part, the BV genome is colinear with that of HSV. However, it has been reported that the unique short (U S ) region of the BV genome may not be colinear with that of HSV (16). Here we present the sequence of the U S region of the BV genome and its genetic layout relative to that of HSV and SA8.The DNA sequence of the BV rhesus genotype (BVrh) strain E2490 (35) U S region was determined by a combination of cloning restriction fragments of the genome and PCR amplification of small gaps in the sequence with genomic BV DNA as a template. The BVrh U S region (GenBank accession no. AB 077432) is 14,447 nucleotides long, slightly longer than the 12,980 bp reported for HSV-1 strain 17 (26) but close to the 14,329-bp HSV-2 U S region (6). The BVrh U S sequence presented here does not include sequences aligning with the N-terminal 2 codons of the US12 open reading frame (ORF). The overall GϩC content of the BVrh U S region is 73.2%, close to the 75% GϩC estimated for the entire BV genome (19). The coding sequences in the U S region have a combined GϩC content of 74.4%, a value somewhat higher than that for the noncoding intergenic regions (68.9%). BV exhibits a strong bias toward use of codons with G or C in the third position (89.6%) and Arg and Leu codons with C in the first position (93.4%). While this GC bias is strong, it is not as extreme as in the SA8 U S region (76.8% GϩC overall, 92.9% GC in the third position, 97.2% C in the first position of the Arg and Leu codons [13]).Analysis of the BVrh U S sequence for ORFs, homology of predicted amino acid sequences with HSV polypeptides, promoter and transcriptional initiation sites, and mRNA termination sites [consensus poly(A) signals associated with mRNA termination motifs] indicates that the genetic layout of the BVrh U S regio...