Patient 1 worked in the macaque facility at a pharmaceutical research company. In 2019, the patient was hospitalized for headache, fever, and deterioration of consciousness. CSF samples were collected at the time of hospitalization and sent to the National Institute of Infectious Diseases (Tokyo, Japan), where we tested them for B virus infection. We extracted total DNA from the CSF samples and tested the samples by using real-time PCR with primers and a fluorescent probe targeting the B virus gB gene: forward primer, 5′- CGTGGCCAGGTAGTACTGCAC-3′; reverse primer, 5′- CTCGTTCCGCTTCTCCTCGTC-3′); AND fluorescent-labeled probe, 5′- FAM-TAGCGCCGGAGGAA-MGB-3′. The reaction mixture (total volume 25 µL) contained 12.5 µL TaqMan Universal PCR Master Mix (ThermoFisher, https://www.thermofisher.com), 2.0 µg/mL of sonicated salmon sperm DNA, 0.2 µmol/L of each primer and fluorescein amidite–labeled probe, and 4.0 µL of extracted DNA. We subjected the reaction mixture to real-time PCR by using an ABI-7500 Fast Real-Time PCR System (ThermoFisher). The reaction conditions were as follows: 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. We performed real-time PCR targeting the B virus gG gene as described previously (4). We performed conventional PCR by using primers targeting a gB gene region conserved among primate herpes viruses (herpes-PCR) as described previously (5). We detected the gB and gG genes in the CSF samples at a concentration of 5.1 × 10⁵ copies/mL for gB and 7.6 × 10⁵ copies/mL for gG. Sequencing of the 364-bp herpes-PCR product revealed 100% identity with a B virus sequence from GenBank (accession no. LC637778).

Figure 1

Figure 1. Brain biopsy from patient (patient 2) with Macacine alphaherpesvirus1 (herpes B virus) infection, Japan, 2019. A–C) Inflammatory cell infiltration and hemorrhage observed around blood vessels in the cerebellar...

Patient 2 had been diagnosed with chronic neurologic disease. The patient was tested for B virus infection because they worked at the same facility as patient 1 and had been working with macaques. In 2014, patient 2 was admitted to hospital with fever, headache, and neurologic symptoms. Brain biopsy was performed, but no pathogen was detected at that time. In 2019, after identification of patient 1, we collected a CSF sample and tested it for B virus infection in addition to testing a paraffin-embedded section of the brain biopsy collected in 2014. Real-time PCR of the CSF detected the gB (3.5 × 10⁵ copies/mL) and gG genes (2.0 × 10⁶ copies/mL) of B virus. Nucleotide sequencing of the 364-bp herpes-PCR product confirmed that it had 100% identity with the sequence detected in patient 1. Furthermore, real-time PCR of the DNA extracted from the paraffin-embedded section of brain tissue biopsied in 2014 was positive for the B virus gG gene. Real-time PCR did not detect herpes simplex virus 1, herpes simplex virus 2, or varicella zoster virus. Histologic analysis of the brain biopsy revealed inflammatory cell infiltration and hemorrhage in the white matter of the cerebellum (Figure 1). Careful observation revealed the presence of inclusion bodies in the nuclei. Immunohistochemical analysis using B virus rabbit polyclonal and gB mouse monoclonal antibodies generated positive signals in cells with inclusion bodies (6).

Figure 2

Figure 2. Phylogenetic tree of Macacine alphaherpesvirus1 (herpes B virus) gB gene in 2 patients with B virus infection, Japan, 2019. Nucleotide sequences of the herpes-specific PCR products (364 bp)...

From our results, we concluded that patients 1 and 2 had been infected with B virus. Although the 2 patients worked in the facility managing imported macaques, there was no direct evidence that they were infected from imported macaques. Given that no epidemiologic link between the 2 patients had been recorded, it seems that they were infected independently. Nucleotide sequence analysis identified B virus genotypes known to be carried by the macaque species (7,8). Recently, a case of human B virus infection was identified in China; however, the genotype was not identified (3). Phylogenetic analysis of the gB gene indicated that B virus from the 2 patients in Japan clustered with the genotype in rhesus macaques (Figure 2). The full-length nucleotide sequences of the gG, gB, and gJ genes also were obtained from the CSF of patient 2 and also were classified by phylogenetic analysis as the rhesus B virus genotype (data not shown). Although there is no direct epidemiologic evidence of zoonotic transmission of rhesus B virus in the monkey facility, the results indicated that workers in such facilities are at risk for infection with rhesus B virus infection, which is pathogenic in humans.