Clinicopathological characterization of monkey B virus (Cercopithecine herpesvirus 1) infection in mice

The purpose of this study was to establish a small animal model for monkey B virus (BV) infection. Mice were inoculated intramuscularly with several BV isolates. Comparisons were based upon the doses required to produce infection (ID50), non-central nervous system (CNS) clinical disease (CS50), CNS disease (CNSD50) and lethal effect (LD50). Strains differed in respect of the dose required to produce clinical disease in BALB/c mice. C57BL/6 mice were more resistant than BALB/c mice to CNS disease. Skin lesions at the inoculation site consisted of epidermal necrosis, ulceration, serocellular crusts and underlying dermatitis. CNS lesions included marked inflammation in the ipsilateral dorsal root ganglion and lumbar spinal cord (point of viral entry). The distribution of the lumbar spinal cord lesions suggested viral entry via sensory afferent neurons, ventral motor tracts, or both. The lesions in the more cranial spinal cord segments suggested ascension to the brain via bilateral spinothalamic and spinoreticular tracts. Brain lesions included encephalitis with neuronal necrosis and white matter destruction located consistently at the base of the brainstem, the reticular system, and rostrally to the thalamus and hypothalamus. Viral antigen was detected immunohistochemically in the lesions. The results indicated an ascending encephalomyelitis syndrome similar to that produced by BV in man.     

Rapid and sensitive detection of ostreid herpesvirus 1 in oyster samples by real-time PCR

Herpes and herpes-like virus infections have been reported in various marine mollusc species associated with high mortality rates. Following the characterisation and genome sequencing of ostreid herpesvirus 1 (OsHV-1), specific diagnostic tools have been developed based on conventional PCR techniques or in situ hybridisation. We have now developed a real-time PCR assay for rapid, sensitive and quantitative detection of OsHV-1, and compared it with a conventional PCR technique described previously. The new assay utilised SYBR((R)) Green chemistry with specific primers C(9)/C(10) targeting the C region. The melt curve analysis of OsHV-1 DNA or DNA extracted from infected material showed only one melting temperature peak (75.75+/-0.1 degrees C). The assay had a detection limit of 4 copies/microL of viral genomic DNA and a dynamic range of 5 logs. Using infected oyster samples as template, the assay was about 100-fold more sensitive than single PCR method using C(2)/C(6) primers. The assay was applied successfully for rapid diagnosis (100 min) and quantitation of OsHV-1 in different developmental stages of Crassostrea gigas. Although it already exists a competitive PCR method to quantify OsHV-1 DNA, quantitative data that will emerge in future using the new sensitive and reliable assay will illuminate aspects of pathogenesis, in particular the viral loads in asymptomatic oysters and the kinetics of infection in specific target tissues.     

Polymerase chain reaction for detection of herpesvirus simiae (B virus) in clinical specimens

Summary A polymerase chain reaction (PCR) was designed which is specific toMacaca fascicularis (cynomolgus monkey) isolates of B virus. The PCR primers produced the expected 188 basepair product from the Cyno 2 strain and seven other cynomolgus monkey isolates of B virus. Oligomer hybridization with a 31-mer oligonucleotide was used to confirm the origin of this product. The PCR failed to amplify DNA of Epstein-Barr virus, cytomegalovirus, varicella-zoster virus, and other alphaherpesviruses (herpes simplex virus types 1 and 2, four SA 8 isolates and three rhesus isolates of B virus). PCR testing of swabs obtained from four orally-infected cynomolgus monkeys confirmed the presence of B virus DNA in samples previously shown to be positive by culture. In addition, PCR detected B virus in several swabs from infected monkeys that were culture negative. Total DNA extracts from the trigeminal and sacral ganglia of these animals were tested by nested PCR and B virus DNA was detected in the trigeminal ganglia of 3 of the 4 orally-infected cynomolgus monkeys. Nested PCR did not detect B virus DNA in total DNA extracts obtained from the brains of the four monkeys.     

Evaluation of a real-time PCR assay for detection of Bordetella pertussis and B. parapertussis in clinical samples

A real-time PCR assay based on the TaqMan technology was developed for the detection of Bordetella pertussis and B. parapertussis in clinical samples. The assay was evaluated with 182 specimens from 153 patients with and without symptoms of pertussis. The analytical sensitivity ranged from 0.1 to 10 cfu for B. pertussis and B. parapertussis, respectively, and diagnostic sensitivity was 94.1% when culture was used as a reference. No sample from a patient without symptoms of pertussis was positive in PCR. Twenty-four of 28 patients who were negative by culture and positive by PCR assay met the CDC clinical case definition for pertussis; the remaining four patients had paroxysms of shorter duration. Intra- and inter-assay variation were <5% and results were available within 4 h.     

Quantitative real-time PCR on Lightcycler for the detection of human immunodeficiency virus type 2 (HIV-2)

Similar to HIV-1, the viral load in HIV-2 is a marker of the evolution of infection and the success of therapy. No approved or commercially distributed assay exists to determine the plasma viral load of HIV-2. We therefore developed a quantitative real-time PCR to determine the plasma RNA viral load of HIV-2, based on the reference strains ROD and EHO, which represent subtypes A and B of HIV-2, respectively. After testing several pairs of primers, a set was chosen that recognised the 5'-LTR region of a subtypes A and B consensus sequence. The quantification of the PCR reaction was done using SYBR-Green I as the fluorescent dye in a Lightcycler system and relying on an external standard curve. The method was then optimised with reference strains, an validated further using patient samples and an inter-laboratory comparison. Good specificity was obtained for both subtypes of HIV-2, and a linear range between 10 and 10(6) copies of viral RNA. The limit of quantitation was 250 copies per millilitre of plasma. The coefficient of variation ranged from 0.7 to 5.6%, depending on the concentration of the target sequences.     

Genome sequence of a pathogenic isolate of monkey B virus (species Macacine herpesvirus 1)

The only genome sequence for monkey B virus (BV; species Macacine herpesvirus 1) is that of an attenuated vaccine strain originally isolated from a rhesus monkey (BVrh). Here we report the genome sequence of a virulent BV strain isolated from a cynomolgus macaque (BVcy). The overall genome organization is the same, although sequence differences exist. The greatest sequence divergence is located in non-coding areas of the long and short repeat regions. Like BVrh, BVcy has duplicated Ori elements and lacks an ORF corresponding to the γ34.5 gene of herpes simplex virus. Nine of ten miRNAs and the majority of ORFs are conserved between BVrh and BVcy. The most divergent genes are several membrane-associated proteins and those encoding immediate early proteins.     

Rapid detection of human parechoviruses in clinical samples by real-time PCR.

BACKGROUND: Human parechoviruses (HPeVs) have been associated with severe conditions such as neonatal sepsis and meningitis in young children. Rapid identification of an infectious agent in such serious conditions in these patients is essential for adequate decision making regarding treatment and hospital stay. OBJECTIVES: We have developed an HPeV specific real-time PCR assay based on the conserved 5'untranslated region. STUDY DESIGN: To determine the detection limit of the assay, serial dilutions of HPeV in vitro RNA were tested in a background of HPeV and EV RNA-negative cerebrospinal fluid (CSF). The specificity was tested by analyzing culture isolates of HPeV 1-6, enterovirus (EV) types, human rhinoviruses (HRVs) and hepatitis A virus (HAV). To establish diagnostic relevance, 522 CSF samples from children <5 years were tested. RESULTS: The detection limit of the assay was 75 copies of HPeV cDNA per reaction. The assay was highly specific for HPeV, detecting all HPeV types. We identified HPeV infections in CSF of 20 children (3.8%), all with severe conditions such as sepsis and meningitis. CONCLUSIONS: These results suggest that HPeV screening of paediatric clinical samples should be included in viral diagnostics in suspected cases of neonatal sepsis and meningitis.     

New diagnostic real-time PCR for specific detection of Parachlamydia acanthamoebae DNA in clinical samples

Given the low sensitivity of amoebal coculture, we developed a specific real-time PCR for the detection of Parachlamydia. The analytical sensitivity was high, and the inter- and intrarun variabilities were low. When the PCR was applied to nasopharyngeal aspirates, it was positive for six patients with bronchiolitis. Future studies should assess the role of Parachlamydia in bronchiolitis.     

Dual-probe assay for detection of lamivudine-resistance hepatitis B virus by real-time PCR

A rapid and accurate minor groove binder (MGB) real-time PCR is described for detection lamivudine resistance mutations in hepatitis B virus. The real-time PCR was compared with direct Sanger sequencing in 53 clinical patients samples, the results of the real-time PCR correlate with the nucleotide sequence and the assay has the advantage of detecting a mixture of quasi-species with higher sensitivity than sequencing. As a simple, easy, rapid, accurate and high throughout method, MGB real-time PCR assay should be useful for detecting lamivudine resistance variants during lamivudine therapy in clinical settings.     

Quantitative real-time PCR assay for detection of Ehrlichia chaffeensis

A real-time PCR assay was developed for the detection of Ehrlichia chaffeensis. The assay is species specific and provides quantitative results in the range 10 to 10(10) gene copies. The assay is not inhibited by the presence of tick, human, or mouse DNA and is compatible with high sample throughput. The assay was compared with previously described assays for E. chaffeensis.     

Validation of a real-time PCR assay for the detection of bovine herpesvirus 1 in bovine semen

A real-time polymerase chain reaction (PCR) assay was developed for detection of the presence of bovine herpesvirus type 1 (BoHV-1) in extended bovine semen. The assay detects a region encoding a highly conserved glycoprotein B gene. The real-time PCR assay was validated for specificity, sensitivity and repeatability using spiked semen and semen from naturally infected animals. The real-time PCR was very rapid, highly repeatable and more sensitive (lower detection limits) than conventional virus isolation method for the detection of BoHV-1 in extended semen. The specificity of the assay is as expected. The assay had an analytical sensitivity of 0.38 TCID(50) virus spiked into negative semen. The second real-time PCR system for the detection of the bovine growth hormone (bGH) gene was applied as an internal control for the DNA extraction and PCR. The bGH PCR can be performed separately to BoHV-1 PCR, or in a duplex format. The real-time PCR assay is intended for use in international trade. The complete validation dossier based on this study and an international inter-laboratory ring trial has been accredited by the Office International des Epizooties (OIE) and has been recommended to be adopted as a prescribed test for international trade.     

Development of a real-time PCR for the detection and quantitation of caprine herpesvirus 1 in goats

Caprine herpesvirus 1 (CpHV-1) is an alphaherpesvirus interfering with goat reproductive performances. The virus is associated with neonatal mortality in kids and reproductive failure in adults. A real-time PCR assay based on TaqMan technology and targeting the gene encoding for glycoprotein C (gC) was developed for detection and quantitation of CpHV-1 in samples collected from infected goats. The detection limit of the assay was 1 x 10(2) standard DNA copies, with a sensitivity of 1-2 logs higher than the conventional gel-based PCR assay targeting the same gene. The real-time PCR was reproducible, as shown by satisfactory low intra-assay and interassay coefficients of variation. The quantitative assay was validated on clinical samples, including genital swabs and various tissue samples collected from goats either infected naturally or experimentally with CpHV-1. The high sensitivity, simplicity and reproducibility of the CpHV-1 fluorogenic PCR assay, combined with its wide dynamic range and high throughput, make this method especially suitable for studies on the pathogenesis and for trials with experimental vaccines and antiviral drugs.     

Quantitative, fluorogenic probe PCR assay for detection of human herpesvirus 8 DNA in clinical specimens

A quantitative, fluorescence-based PCR assay (TaqMan-based system) was developed for detection of human herpesvirus 8 (HHV-8) DNA in clinical specimens. Primers and probes chosen from each of five 10-kb segments from the unique region of the HHV-8 genome were evaluated for sensitivity with dilution series of DNA extracted from a cell line (BCBL-1) that harbors HHV-8 DNA. Although several of the primer-probe sets performed similarly with BCBL-1 DNA that had been diluted in water, their performance differed when target DNA was diluted in a constant background of uninfected cell DNA, an environment more relevant to their intended use. The two best primer-probe combinations were specific for HHV-8 relative to the other known human herpesviruses and herpesvirus saimiri, a closely related gammaherpesvirus of nonhuman primates. PCRs included an enzymatic digestion step to eliminate PCR carryover and an exogenous internal positive control that enabled discrimination of false-negative from true-negative reactions. The new assays were compared to conventional PCR assays for clinical specimens (saliva, rectal brushings, rectal swab specimens, peripheral blood lymphocytes, semen, and urine) from human immunodeficiency virus-positive patients with or without Kaposi's sarcoma. In all instances, the new assays agreed with each other and with the conventional PCR system. In addition, the quantitative results obtained with the new assays were in good agreement both for duplicate reactions in the same assay and between assays.     

Development of a SYBR Green I-based real-time PCR for detection of elephant endotheliotropic herpesvirus 1 infection in Asian elephants (Elephas maximus)

Elephant endotheliotropic herpesvirus 1 (EEHV1) can cause fatal hemorrhagic disease in Asian elephants (Elephas maximus). Several studies have described this virus as a major threat to young Asian elephants. A SYBR Green I-based real-time polymerase chain reaction (PCR) was developed to identify EEHV1 on trunk swabs and necropsied tissues. Two of 29 (6.9%) trunk swab samples from healthy Asian elephants were positive for EEHV1. The viruses were analyzed and classified as EEHV1A based on 231 nucleotides of the terminase gene. Necropsied spleen and heart tissue showed the highest level and second highest levels of DNA virus copy accumulation, respectively. The detection limit of the test was 276 copies/μl of DNA. There was no cross-reaction with other mammalian herpesviruses, such as herpes simplex virus 1 and equine herpesvirus 2. Inter- and intra-assay showed low coefficients of variation values indicating the reproducibility of the test. The results indicated that the test can be practically used for epidemiological study, clinical diagnosis, and management and control of EEHV1.     

Quantitative real-time PCR assay for detection of human polyomavirus infection

The human polyomaviruses BK (BKV) and JC (JCV) affect immunosuppressed patients and are associated with urogenital tract (BKV) and CNS disorders (JCV) and in humans, the pathogenic role of the rhesus monkey virus, Simian virus 40 (SV40), is uncertain. These three viruses have somewhat overlapping tissue pathogenicity and detection of all three polyomaviruses is desirable. A broadly targeted, simple, single tube real-time degenerated quantitative PCR (QPCR) technique for detection of JCV, BKV and SV40 DNA was developed. To avoid false positive results, due to contamination with commonly used SV40 T-antigen plasmids, a conserved region of the VP2 gene was targeted. Down to 1-10 copies of target DNA per PCR reaction were detected. The QPCR was compared with a nested PCR on 41 clinical samples (urine, serum and plasma): 24 (58.5%) tested positive by nested PCR, whereas 31 (75.6%) were positive with QPCR. One CSF sample, from a patient with progressive multifocal leukoencephalopathy, was negative with the nested PCR but determined as positive by QPCR. Sera from 24 blood donors were negative with QPCR. The QPCR described had a high sensitivity. Its specificity was confirmed sequencing. The QPCR is simple to perform and is valuable for diagnosis of polyomavirus infection.