G.M. Cleator

Molecular Typing of Enteroviruses: Current Status and Future Requirements

SUMMARY Human enteroviruses have traditionally been typed according to neutralization serotype. This procedure is limited by the difficulty in culturing some enteroviruses, the availability of antisera for serotyping, and the cost and technical complexity of serotyping procedures. Furthermore, the impact of information derived from enterovirus serotyping is generally perceived to be low. Enteroviruses are now increasingly being detected by PCR rather than by culture. Classical typing methods will therefore no longer be possible in most instances. An alternative means of enterovirus typing, employing PCR in conjunction with molecular genetic techniques such as nucleotide sequencing or nucleic acid hybridization, would complement molecular diagnosis, may overcome some of the problems associated with serotyping, and would provide additional information regarding the epidemiology and biological properties of enteroviruses. We argue the case for developing a molecular typing system, discuss the genetic basis of such a system, review the literature describing attempts to identify or classify enteroviruses by molecular methods, and suggest ways in which the goal of molecular typing may be realized.

New method for the extraction of viral RNA and DNA from cerebrospinal fluid for use in the polymerase chain reaction assay

A new, rapid, and simple method for the isolation of either RNA or DNA from cerebrospinal fluid samples for subsequent amplification by specific polymerase chain reaction (PCR) assays is described. The technique involves a single extraction with a guanidinium thiocyanate acid (GuSCN) buffer, and does not require the use of organic solvents. Applied to the recovery of enteroviral RNA, herpes simplex virus (HSV) and Varicella-zoster virus (VZV) DNAs the method has proved to be of equivalent or better efficiency than established methods of nucleic acid separation but is less laborious and time consuming. The simplicity of the procedure permits the processing of large numbers of samples and the use of a single preparative method for either RNA or DNA PCR makes it an attractive method for the routine laboratory.

The role of laboratory investigation in the diagnosis and management of patients with suspected herpes simplex encephalitis: a consensus report. The EU Concerted Action on Virus Meningitis and Encephalitis.

As effective therapies for the treatment of herpes simplex encephalitis (HSE) have become available, the virology laboratory has acquired a role of primary importance in the early diagnosis and clinical management of this condition. Several studies have shown that the polymerase chain reaction (PCR) of CSF for the detection of herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) DNA provides a reliable method for determining an aetiological diagnosis of HSE. The use of PCR in combination with the detection of a specific intrathecal antibody response to HSV currently represents the most reliable strategy for the diagnosis and monitoring of the treatment of adult patients with HSE. The use of these techniques has also led to the identification of atypical presentations of HSV infections of the nervous system and permits the investigation of patients who develop a relapse of encephalitic illness after an initial episode of HSE. A strategy for the optimal use of the investigative laboratory in the diagnosis of HSE and subsequent management decisions is described.

Association of human parvovirus B19 infection with acute meningoencephalitis

To find out the incidence and clinical presentation of parvovirus B19 meningoencephalitis, we tested samples of cerebrospinal fluid from 162 patients (one from each patient) with undiagnosed meningoencephalitis, who presented between March, 1997, and March, 1998 (an outbreak period) using nested PCR for B19 genes. Seven patients were positive; an incidence of 4.3%. Five additional cases of meningoencephalitis were detected from other years. Three patients with underlying disorders (haemophagocytic lymphohistiocytosis, Cockaynes syndrome, and Turners syndrome) died. Neurological sequelae were observed in three surviving patients, all of whom had had striking abnormalities detected on brain scans done during the acute phase.

Detection of enteroviral RNA and specific DNA of herpesviruses by multiplex genome amplification.

A reverse transcription (RT) multiplex polymerase chain reaction (PCR) assay was developed to allow rapid, sensitive and simultaneous detection of enteroviral RNA and herpesviral DNA specific sequences in a single tube. The method involves a reverse transcription step followed by a multiplex nested PCR in which the combination of primers amplifies cDNA from enteroviruses and specific herpesviruses DNA. Nested amplification utilises primers designed to anneal into the amplification product from the first reaction. Individual viruses were then detected and differentiated by the size of their PCR products determined using ethidium bromide stained agarose gels. To exclude false negatives due to sample inhibitors an internal amplification control, a cloned fragment of DNA from Pseudorabies virus (PRV DNA) was included in the reaction mixture. Detection levels between 0.01 and 0.001 TCID50 of prototype strains of Polio and Coxsackie type B viruses and between 1 and 100 molecules of cloned-DNA of herpesviruses prototype strains were achieved. The RT multiplex PCR method proved capable of detecting enteroviral RNA or herpesviral DNA in cerebro spinal fluid (CSF) samples from patients with aetiologically well characterized encephalitis or aseptic meningitis.

Mild forms of herpes encephalitis.

Three cases of herpes encephalitis are described. A definite diagnosis was established and all patients made a good recovery without specific antiviral chemotherapy. These reports are representative of those forms of herpes encephalitis with a good prognosis. It is suggested that the introduction of rapid non-invasive procedures will indicate a higher incidence of herpes encephalitis than is presently accepted. The relevance of repeated episodes of herpes encephalitis with respect to the aetiology of some psychiatric disorders and in the evaluation of antiviral agents is also discussed.

HSV‐1 and HSV‐2 in herpes simplex encephalitis: A study of sixty‐four cases in the United Kingdom

The incidence of herpes simplex virus type 1 (HSV‐1) and herpes simplex virus type 2 (HSV‐2) in herpes simplex encephalitis (HSE) was investigated using cerebrospinal fluid (CSF) samples from sixty‐four cases of HSE. A polymerase chain reaction (PCR) employing primers flanking a region of the HSV thymidine kinase gene common to both HSV‐1 and HSV‐2 was used to detect HSV in the CSF. HSV‐1 and HSV‐2 were differentiated by digestion with restriction enzymes. Two enzymes were employed; AvaI which cleaved only the HSV‐2 gene product and AvaII which cleaved only the HSV‐1 gene product. Sixty‐three cases of HSE were found to be due to HSV‐1; one case due to HSV‐2. These data confirm previous observations that HSV‐2 is a rare cause of post‐neonatal herpes encephalitis but indicates that a PCR procedure capable of detection of both viruses is essential for efficient diagnosis of HSE. J. Med. Virol. 53:1–3, 1997.

Diagnosis and clinical management of neurological disorders caused by cytomegalovirus in AIDS patients

Cytomegalovirus (CMV) infections are common and severe complications of HIV infection. The virus involves the nervous system, causing encephalitis, polyradiculomyelitis and peripheral neuropathies, Due to their limited sensitivity, traditional virological approaches, such as virus isolation or antigen detection in the CSF are useful only in limited instances, e.g. CMV polyradiculopathy. The aetiological diagnosis of these disorders relies on the analysis of cerebrospinal fluid by PCR and quantitative PCR may be important to establish the extent of CNS lesions and to monitor the efficacy of antiviral treatments, CMV is susceptible to various antivirals, including ganciclovir, foscarnet and cidofovir. CMV infections of the nervous system, in particular encephalitis, however, show only a poor response to standard treatments, Drug combination treatments i.e. ganciclovir plus foscarnet, are currently under evaluation in clinical trials.

Polymerase chain reaction in the investigation of “relapse” following herpes simplex encephalitis

Five cases of apparent relapse of herpes encephalitis were investigated. All patients recovered after antiviral and corticosteroid therapy. Samples of CSF taken from the patients at intervals through the initial and subsequent encephalitic episode were examined. PCR amplification of a 351 bp sequence from the Herpesvirus simplex (HSV) thymidine kinase gene demonstrated the presence of HSV DNA in CSF taken during the initial encephalitic illness but not during the second encephalitic episode. Intrathecal synthesis of HSV antibody (HSV antibody index > 1.9) was observed in all cases following the first episode, and there appeared to be no significant increase in intrathecal antibody synthesis in the second episode. High levels of CSF myelin basic protein were found during the acute phases of both the initial and the subsequent encephalitic illnesses. These data suggest that at least in our series of five patients, relapse following HSE may not be due to active viral replication.

Herpesvirus simplex in chronic human stromal keratitis

A method is described for the isolation of Herpesvirus simplex (HSV) from corneal discs of patients suffering from chronic stromal keratitis. The discs were removed during penetrating keratoplasty. Virus was successfully isolated from 2 out of 8 discs maintained in vitro.