Prevalence of herpes B virus genome in the trigeminal ganglia of seropositive cynomolgus macaques.

Herpes B virus infection is almost asymptomatic in macaques (Macaca spp.), which are the natural hosts of this pathogen, but is the cause of high mortality in humans. Reactivation of the latent virus in the trigeminal ganglia (TG) results in the shedding of infectious particles into the oral mucosal membrane. Saliva contaminated with the reactivated virus from the ganglia of the natural host is considered to be important for viral transmission to humans and other monkeys. In the present study, we investigated the prevalence of the herpes B virus genome in the left and right TG of seropositive asymptomatic cynomolgus macaques. The latent virus genome was detected using a polymerase chain reaction and microplate hybridization assay. We found that the virus DNA was present in one or both TG of 12 of the 30 macaques (40%) tested, with the virus being detected from both TG in five of the 12 macaques and from a single TG in the remaining seven.

Herpes simplex lymphadenitis. Report of two cases with review of the literature.

We report two cases of herpes simplex lymphadenitis without widespread organ involvement in a 60-year-old man and a 67-year-old woman. Their complaints were high fever and generalized erythema followed after few days by generalized lymphadenopathy. This report describes the findings obtained by light and electron microscopy, immunohistochemistry, and in situ hybridization. In both instances, Cowdry's type A intranuclear inclusion bodies were found in T-immunoblasts in the background of T-zone hyperplasia with focal necrosis. Electron microscopic investigation revealed intranuclear and cytoplasmic virus particles with characteristics of the herpes group. Immunohistochemical staining utilizing anti-herpes simplex virus (HSV) antibody was positive and in situ hybridization with HSV-DNA probe revealed positive signals in the nucleus and in the cytoplasm of T-immunoblasts. Although rare, HSV lymphadenitis in the absence of generalized infection can occur.

Detection of viral antigens in formalin-fixed specimens by enzyme treatment.

Enzyme treatment (protease or trypsin) was applied to formalin-fixed paraffin-embedded materials and virus-infected cultured cells to detect viral antigens by immunofluorescence. The viral antigens were demonstrated in several organs of autopsy or biopsy cases of which diagnoses had been established by immunofluorescence or virus isolation using frozen materials, or suspected on the basis of serology and/or histopathological findings. These included herpes simplex, varicella-zoster, cytomegalo, subacute sclerosing panencephalitis, progressive multifocal leukoencephalopathy, Japanese B encephalitis, measles, acute hemorrhagic conjunctivitis, Lassa and Korean hemorrhagic fever. Antigen could be recovered also in virus-infected cells (herpes simplex, measles, Lassa, Ebola, Marburg, Rift Valley, Congo and Korean Hemorrhagic fever) by enzyme treatment after periods of formalin fixation of four weeks and storage of three months. In herpes simplex virus-infected mouse brain, antigen was detected after fixation for three months in formalin.

Detection of varicella-zoster virus DNA by polymerase chain reaction in cerebrospinal fluid of patients with herpes zoster meningitis.

In three of five patients with herpes zoster meningitis, varicella-zoster virus (VZV) DNA was detected by the polymerase chain reaction (PCR) in the initial samples of cerebrospinal fluid. DNA fragments of group A or B, following classification of VZV strains by the size of the variable region IV of VZV genome, were found at the 7th, 10th and 24th illness day in the three positive cases: one of these cases did not have skin lesions. These results suggest that the detection of VZV DNA by PCR is useful for the diagnosis of herpes zoster meningitis, as well as for its molecular epidemiology.

Differentiation of herpes simplex virus 1 and 2 in cerebrospinal fluid of patients with HSV encephalitis and meningitis by stringent hybridization of PCR-amplified DNAs.

Differentiation of herpes simplex virus (HSV) types 1 and 2 in cerebrospinal fluid of 17 patients with serologically diagnosed HSV encephalitis and meningitis or acute limbic encephalitis was determined by stringent hybridization of polymerase chain reaction--amplified DNAs. Ten of 17 patients were positive; six with HSV 1 isolates and four with HSV 2 isolates. We detected HSV type 1 in two cases of meningitis, although meningitis is generally thought to be caused by type 2. Additionally, HSV type 2 was found in one case of acute adult encephalitis, which is generally due to HSV type 1. HSV DNAs could be detected for over 1 month after onset, although our patients included several prolonged and recurrent cases. HSV DNA genomes were not detected in three cases of acute limbic encephalitis. Our study indicates that this method can be used for type differentiation in HSV CNS infections.

Polymerase chain reaction for the detection of the varicella-zoster genome in ocular samples from patients with acute retinal necrosis.

We used the polymerase chain reaction to detect the virus genome in ocular samples from patients with clinically diagnosed acute retinal necrosis. Four samples from four patients with acute retinal necrosis, and five samples from three patients with other ocular diseases (sarcoidosis, rhegmatogenous retinal detachment, and epiretinal membrane of unknown origin) were evaluated. The samples consisted of aqueous humor, vitreous, or subretinal fluid. Primers were specific for varicella-zoster virus, herpes simplex virus, or cytomegalovirus. The varicella-zoster virus genome was detected in three of the four samples from patients with acute retinal necrosis. Among these three positive samples, two had PstI-site-less point mutation, strains that have been described only in Japan and of low prevalence. Samples from patients with diagnoses other than acute retinal necrosis yielded negative results when varicella-zoster virus primer was used. No sample was positive for herpes simplex virus or cytomegalovirus primers.

Vanadium contamination and the relation between vanadium and other elements in wild birds.

There is little information on vanadium (V) contamination in wildlife. In the present study, the mean V contents in liver and kidney from 41 wild Japanese water birds were less than 3.69 and 8.11 microg/g dry wt, respectively. The V contents in the liver and kidney of the spotbill duck were more than two times higher than those of other species in Japan. Spotbill ducks obtained in a residential district had a strong correlation between the V contents in the kidneys and those in the livers (R=0.924), and also between V and Ti, Cd, and Li contents in the liver (R>0.8). These results suggest that V accumulation in wild birds reflects the degree of environmental contamination.

Male genital herpes complicated with urethral infection.

A 26-year-old man had a primary infection or reinfection of genital herpes due to herpes simplex virus (HSV) type 2 complicated with herpetic urethritis. By analysis of the restriction endonuclease digestion pattern of the viral DNA, two isolates, one from the penile lesion and one from the urethra, were identified as the same strain.

Classification of Listeria monocytogenes by PCR-restriction enzyme analysis in the two genes of hlyA and iap.

PCR-restriction enzyme analysis in the two virulence-associated genes was performed. The hlyA gene cording for listeriolysin O and the iap gene cording for an invasion associated factor were amplified with primers SH2 or SI3. The PCR products obtained were cleaved with 32 restriction enzymes, and restriction profiles from 12 strains, 6 each of serotypes 1/2a and 4b, were compared. We obtained two profiles for the hlyA using 4 restriction enzymes and eight profiles for the iap by using AluI, and the results of the profiles did not correlate with the serotypes. The polymorphism in the iap region was of a higher degree than that in the hlyA region, and the PCR-restriction enzyme analysis of the iap gene with primers SI3 and AluI was confirmed as one of the useful epidemiological analysis methods for listerosis outbreaks.

Characterization of iap gene in Listeria monocytogenes strains isolated in Japan.

Variation of the iap gene region (407bp) encoding an invasion-associated protein p60 was studied on 12 strains of Listeria monocytogenes of different origin in Japan. These 12 strains are known to have 2 types of serotype (1/2a and 4b) and have a diversity among the strains (Saito et al., 1998). The dye-primer cycle sequencing method was employed to determine the genomic structure, and the nucleotide sequences obtained were compared with those of reference strain SV 1/2a EGD. Differences found in the nucleotides were as follows; point mutations of 33 variations in 32 places; an insertion and 3 deletions of 3 bases; AAT position (po.) 1282-1283, and GCA po. 1307-1309, ACA po. 1412-1414, AAT po. 1439-1444, respectively. Different repeating numbers by 6 base unit, ACA AAT, were also found in the tandem repeat region (po. 1394-1423). Classification of 12 strains was attempted, then 8, 4 and 5 types were obtained from the point mutations, the insertions and deletions, and the repeating numbers, respectively. Consequently, 8 patterns were profiled regardless of each serotype. From these results, genomic structures were partially clarified in the iap gene 407bp of L. monocytogenes isolated in Japan. Then, the possibility of detailed epidemiology for L. monocytogenes infection using a combination of serotype and genome structure was suggested because of the previous polymorphism thought to be due to the nucleotide differences in the region.

Swift and definite serotyping for isolated Listeria monocytogenes strains.

The serotype is most important for molecular epidemiological analysis of Listeria monocytogenes (L.m.) contaminating marketed meats. An improvement on the traditional method was thus attempted in the present study because of the requirement of swift and definite serotyping. In the determination of O-antigen, definite judgement was allowed by an immediate cooling at 80 degrees C after autoclaving the bacteria. In the determination of H-antigen, use of a culture plate without Craigie's tube yielded the active bacteria only by single culture. The stable and clear agglutination in many samples was also obtained with a microplate using less antiserum. The availability was confirmed with 123 strains and the serovar 1/2b was dominant in the Japanese strains.

[Characteristics of the Shigella flexneri strains isolated from Japanese monkeys (Macaca fuscata)].

Shigella flexneri was isolated from 11 of 95 Japanese monkeys (Macaca fuscata) purchased by the Animal Research Center, Tokyo Medical and Dental University, during the period from 1988 through 1990. The serotypes of the isolates were 4a (5 isolates), 2a (4 isolates), 2b (1 isolate), and varY (1 isolate). Although some of the strains in the same serotypes were similar, others were different in their antibiotic susceptibility patterns or the plasmid profiles. Our results suggested that various types of Shigella flexneri had infected the Japanese monkeys. However, the source(s) and the routes of infection were unclear. Dysentery in the Japanese monkey has not yet been reported. So, to our knowledge, this may be the first report of dysentery among Japanese monkeys.

[Diagnosis of varicella-zoster virus (VZV) infection by using FITC-labeled monoclonal antibodies].

Anti-varicella zoster virus (VZV) mouse monoclonal antibodies conjugated with fluorescein isothiocyanate were evaluated for their usefulness as a practical diagnostic tool in the clinical field by examining cells infected with isolated herpes viruses and 431 clinical samples. The kit stained clearly the cells infected with 14 isolated VZV strains without cross reaction to 15 isolated herpes simplex virus type-1 strains (HSV-1) and 14 type-2 (HSV-2) strains. In clinical specimens, viral antigens of VZV were detected in 92/105 (87.6%) cases of varicella and in 176/190 (92.6%) cases of herpes zoster. Specific fluorescence of VZV was also observed in 5 out of 96 cases diagnosed as HSV infections, although these samples had no specific reaction to HSV when tested by the commercially available diagnostic kit. In 24 cases which could not be clinically diagnosed as herpes zoster or herpes simplex, the VZV antigen was demonstrated in 9 cases. All 109 VZV-positive cases in virus isolation by culture were also judged VZV-antigen positive by the kit, while all 69 HSV-positive cases in virus isolation were VZV-antigen negative. Furthermore, the VZV antigen was detected by the kit in 53/60 clinical diagnoses of varicella or herpes zoster without successful virus isolation. These results clearly indicate the usefulness of the kit as a practical VZV diagnostic reagent, especially in terms of specific sensitivity and easy technical manipulation.

[A case of herpes simplex encephalitis diagnosed by polymerase chain reaction].

A case of a 55-year-old male with herpes simplex encephalitis (HSE) was reported. He was admitted because of fever, headache and memory disturbance. T1 weighted MRI showed low signal intensity and T2 weighted imaging revealed high signal intensity in the medial portions of bilateral temporal lobes. Herpes simplex virus (HSV) antibody titer in cerebrospinal fluid (CSF) was not elevated. HSV DNA in CSF was amplified by polymerase chain reaction (PCR) and identified by the microplate hybridization method. The PCR technique would be useful for the diagnosis of HSE.

[Secretion and dynamics of herpes simplex virus in tear and saliva of patients with Bell's palsy].

Herpes simplex virus (HSV) involvement has been clarified as the cause of Bell's palsy by molecular biological techniques. To clarify the direct relationship between virus reactivation and paralysis development, both detection of the virus genome by DNA-diagnostic and quantitative analysis of its time-course change are needed. Using polimerase chain reaction (PCR) method and microplate-hybridization combined, we detected the virus genome in small amount of specimens from patients with Bell's palsy, quantified its number of copies, and examined time-course changes. We attempted to type of HSV in positive specimens. Subjects were 16 patients with Bell's palsy positive for serum anti-HSV IgG antibody (enzyme immunoassay) who visited the outpatient clinic of Department of Otorhinolaryngology, Itabashi Hospital, Nihon University School of Medicine, within 14 days after onset without having any treatment. Tears and saliva from the parotid gland and the submaxillary gland were separately collected from the affected side and healthy side twice or more. A total of 244 specimens was subjected to DNA extraction, PCR, and microplate-hybridization, and HSV DNA was detected in 38 specimens (11.8%) from 5 patients (31%). From sampling time, the highest detection (28.5%) was obtained within 2 weeks after onset. Detection at 3 weeks and later (2.8%) was significantly lower (p < 0.05). DNA was also found on the healthy side, but in comparison between sides, detection on the healthy side (18.9%) was significantly lower than that on the affected side (83.8%) (p < 0.01). In quantitative determination of the virus genome, the amount (number of copies) was large on the affected side and largest early after onset regardless of the clinical course. Positive specimens underwent HSV-typing, and HSV-1 was found in all of them, suggesting that HSV-1 reactivation is a cause of Bell's palsy.

[Detection of varicella-zoster virus DNA in tear fluid and saliva of patients with Ramsay Hunt syndrome].

Ramsay Hunt syndrome develops when the varicella-zoster virus (VZV) is reactivated. In the present study, we examined the secretion kinetics of VZV DNA in the tear fluid, submandibular gland saliva and parotid gland saliva of 15 patients with Ramsay Hunt syndrome. The presence of VZV DNA was detected using PCR and a microplate hybridization method. Hybridization signals were measured using the fluorescence density of an enzymatic reaction product using fluoroscan and a system involving streptavidin-conjugated beta-galactosidase. The results were converted into numerical values and used to estimate the number of virus DNA copies. VZV DNA was detected in the tear fluid, submandibular gland saliva and parotid gland saliva of the Ramsay Hunt syndrome patients. The rate of VZV DNA detection in the submandibular gland saliva was 72%, and the detection rate in the parotid gland saliva was 57%. The detection rate in the tear fluid was 27%, which is significantly lower than other two detection rates. Regarding the submandibular gland saliva and the parotid gland saliva, the VZV DNA was detected in samples collected at a comparatively early stage of onset. In the tear fluid, the detection rate increased significantly in samples collected 2 weeks after onset or later. Thus, differences in the detection rate were observed depending on the type of secretory gland and the timing of the sample collection. The VZV DNA in the tear fluid is thought to derive from the ganglion trigeminale. The increase and decrease in the number of VZV DNA copies detected in samples collected at different times is considered to substantiate VZV reactivation in Ramsay Hunt syndrome.

[DNA diagnosis of alpha-herpesvirinae infection].

Herpesviruses have a characteristic of the latency after the primary infection. Advanced study on the causal relation between the reactivation of latent virus and the appearance of the symptoms would need both of the detection of viral genome by the DNA diagnosis method and the analysis of the kinetics of the virus. We developed a new quantitative method for the detection and the titration of copy number in the viral genome using the combination of polymerase chain reaction and microplate hybridization. The availability of the method was confirmed by the several cases of alpha-herpesvirinae infections.