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.

Molecular Typing of Enteroviruses: Current Status and Future Requirements

We examined the genetic and antigenic properties of Dobrava (DOB) virus, a hantavirus associated with severe haemorrhagic fever with renal syndrome in Europe. Cloning and sequence analyses revealed the DOB M segment to consist of 3644 nucleotides, with a coding capacity of 1134 amino acids in the virus complementary-sense RNA (cRNA). Seven potential asparagine-linked glycosylation sites were identified in the M segment gene product, one in the G2 and six in the G1 coding regions. The S segment is 1667 nucleotides long, and has a single ORF in the cRNA capable of encoding a protein of 428 amino acids. Phylogenetic comparisons of the M and S segments of DOB virus to those of other hantaviruses indicated that DOB virus is similar to, but clearly distinct from Hantaan (HTN) and Seoul (SEO) viruses. Certain G2-specific, but not G1-specific monoclonal antibodies to HTN virus reacted to the same titre with DOB and homologous viral antigen. Plaque-reduction neutralization tests indicated that, of the sera tested, only antisera to SEO virus were able to neutralize DOB virus to a titre greater than 1:10; however, this neutralization titre was eightfold lower than that observed with homologous SEO virus. The data reported here confirm that DOB virus is a unique species in the Hantavirus genus, family Bunyaviridae.

Genetic and antigenic properties of Dobrava virus: a unique member of the Hantavirus genus, family Bunyaviridae

Isolation, characterization and geographic distribution of Caño Delgadito virus, a newly discovered South American hantavirus (family Bunyaviridae)

The outbreak of deadly hantavirus pulmonary syndrome (HPS) in the United States in 1993 (CDC., 1993) changed the concept of hemorrhagic fever with renal syndrome (HFRS) (WHO, 1983) because the clinical manifestations of this newly described hantavirus infection (Duchin et al., 1994) are quite different from the known typical forms of HFRS caused by Hantaan, Seoul, and Puumala viruses and because it occurred in the United States, where hantavirus disease had not been known to exist previously. Recent data on hantavirus disease show that HFRS occurs in the Old World and HPS, caused by novel hantaviruses, occurs in the New World. Epidemiological studies of HFRS progressed rapidly after the discovery of Hantaan virus, the etiologic agent of Korean hemorrhagic fever (Lee and Lee, 1976; Lee et al., 1978) through seroepidemiologic surveys of hantavirus infections in humans and in animals. It was long believed that HFRS only occurs in certain limited rural areas of Eurasia, such as Korea, China, Far East of Russia, and Northern Europe (Lee, 1982). Recent findings (Lee and van der Groen, 1989; Lee et al., 1990b), however, have shown that HFRS patients can be found not only in rural areas but also in urban cities in many parts of the world.

Epidemiology and Pathogenesis of Hemorrhagic Fever with Renal Syndrome

Puumala virus (PUUV) is the endemic hantavirus in northern Sweden and causes nephropathia epidemica (NE), a milder form of hemorrhagic fever with renal syndrome. There is a need for fast and reliable diagnostics to differentiate the disease from other infections. By aligning virus RNA sequences isolated from 11 different bank voles and one human patient, we designed a real-time reverse transcriptase (RT) PCR method for detection of PUUV RNA. The real-time RT-PCR assay showed linearity from 20 to 2 x 10(6) virus copies with a correlation coefficient above 0.98 to 0.99 for all experiments. The detection threshold for PUUV cDNA was two copies per reaction. A two-step qualitative RT-PCR to detect PUUV RNA showed 100% concordance with the real-time RT-PCR assay. PUUV RNA viremia was detected in 33 of 34 PUUV immunoglobulin M (IgM)-positive patients with typical clinical NE disease from the region of endemicity. One PUUV IgM-negative sample had PUUV RNA, and 4 days later, the patient was IgM positive. Of samples with indeterminate IgM, 43% were PUUV RNA positive. The kinetics of antibody titers and PUUV viremia were studied, and five of six NE patients displayed a decrease in PUUV viremia a few days after disease outbreak coupled with an increase in PUUV IgM and IgG. In one patient with continuously high PUUV RNA levels but low IgM and no IgG response, the infection was lethal. These findings demonstrated that real-time RT-PCR is a useful method for diagnosis of PUUV viremia and for detecting PUUV RNA at early time points, before the appearance of IgM antibodies.

Wang Lee Ho

Papers

Typing of Hantaviruses from five continents by polymerase chain reaction