First isolation of a rabies-related virus from a Daubenton's bat in the United Kingdom.

On May 30, 1996, a sick Daubenton's bat (Myotis daubentonii) was recovered from the cellar of a public house in Newhaven, East Sussex. Its condition deteriorated rapidly, and it was euthanased and examined. Positive results, establishing the presence of a rabies or rabies-related virus in its brain, were obtained from the fluorescent antibody test, the rabies tissue culture isolation test, and a hemi-nested reverse-transcription PCR. The complete sequence of the nucleoprotein gene was determined and a phylogenetic analysis, based on the 470 nucleotide bases of the amino terminus of the nucleoprotein, established the genotype of the virus as European bat lyssavirus 2. Bat rabies had not previously been recorded in the UK but does occur in mainland Europe. A study of the back-trajectories of the wind on May 29 and 30, established that the infected bat possibly came from near the Franco-Swiss border.

Rabies virus in the decomposed brain of an Ethiopian wolf detected by nested reverse transcription-polymerase chain reaction.

Approximately 75 individuals from a population of 111 Ethiopian wolves (Canis simensis) died or disappeared from the Bale Mountains National Park (Ethiopia) between 1988 and 1992 during two significant population declines. Confirmation of rabies virus in two carcasses was based on the fluorescent antibody test (FAT) and the mouse inoculation test (MIT). In an Ethiopian wolf brain previously designated rabies negative by both FAT and MIT, rabies virus was identified by nested reverse transcription-polymerase chain reaction (RT-PCR) and confirmed by Southern blot hybridization. These methods were successfully used on a highly decomposed brain sample which had been stored in 20% dimethyl sulfoxide. This test system allows early and sensitive detection to be undertaken to more effectively prevent spread of disease and thus protect surviving animals.

Heminested PCR assay for detection of six genotypes of rabies and rabies-related viruses.

A heminested reverse transcriptase PCR (hnRT-PCR) protocol which is rapid and sensitive for the detection of rabies virus and rabies-related viruses is described. Sixty isolates from six of the seven genotypes of rabies and rabies-related viruses were screened successfully by hnRT-PCR and Southern blot hybridization. Of the 60 isolates, 93% (56 of 60) were positive by external PCR, while all isolates were detected by heminested PCR and Southern blot hybridization. We also report on a comparison of the sensitivity of the standard fluorescent-antibody test (FAT) for rabies antigen and that of hnRT-PCR for rabies viral RNA with degraded tissue infected with a genotype 1 virus. Results indicated that FAT failed to detect viral antigen in brain tissue that was incubated at 37 degrees C for greater than 72 h, while hnRT-PCR detected viral RNA in brain tissue that was incubated at 37 degrees C for 360 h.

Evolution of European bat lyssaviruses.

Forty-seven European bat lyssaviruses (EBL) and two African insectivorous bat lyssaviruses (Duvenhage viruses) were selected for a comparison to be made of their evolutionary relationships. Studies were based on direct sequencing of the PCR-amplified products of the 400 nucleotides coding for the amino terminus of the nucleoprotein. Phylogenetic relationships were analysed after bootstrap resampling using the maximum parsimony and the neighbour-joining methods. Analyses of both the nucleotide and amino acid sequences placed these viruses in three separate clusters, namely genotype 4 (Duvenhage), genotype 5 (EBL1) and genotype 6 (EBL2). Evolutionary analysis of the nucleoprotein gene of EBL1 and EBL2 indicated low intrinsic heterogeneity mainly due to synonymous substitutions. In addition, both EBL1 and EBL2 evolved into at least two genetically distinguishable lineages (a and b) following geographical drifting. We can speculate that subsequently the lineages EBL1a and EBL1b were introduced into parts of northern Europe from two different geographical directions; EBL1b was probably introduced most recently and was from North Africa. Eptesicus serotinus appears to be the principal reservoir for EBL1 and Myotis dasycneme and M. daubentonii the reservoirs for EBL2.

Rapid detection of rabies and rabies-related viruses by RT-PCR and enzyme-linked immunosorbent assay.

A rapid detection method for the six established genotypes of rabies and rabies-related viral RNA using RT-PCR-ELISA is described. The detection of digoxigenin-labelled amplified products is performed by solution hybridization to two specific, biotin-labelled, capture probes, which are complementary to the inner region of the amplification products. The capture probe and amplified product hybrid are then immobilised on a streptavidin-coated microtitre plate, bound products are detected by an anti-DIG Fab fragment conjugated to peroxidase, and colorimetric reaction automatically measured. This method was up to 100-fold more sensitive than Southern blot hybridization, detecting 0.00002 TCID50/ml of a genotype 1, classical rabies virus strain. The complete detection methodology from RT-PCR to PCR-ELISA detection could be completed within 10 h. Using this procedure, we were 100% successful in detecting 60 isolates from a representative selection of the six established genotypes from all over the world. This test is a useful additional tool for the detection of the rabies and rabies-related viruses, which is easy to perform, rapid and highly sensitive.

Ten-year survey of British bats for the existence of rabies.

In 1985, a notable increase in the number of recorded cases of rabies in European bats was observed, indicating a possible spread of the rabies virus in these bats. Because of concern that the disease could be introduced into the United Kingdom by bats crossing from mainland Europe, a programme of screening dead bats for the presence of rabies and rabies-related viruses was initiated at the Rabies Research and Diagnostic Unit at the Central Veterinary Laboratory. Over a period of 10 years (January 1986 to December 1995), 1882 bats belonging to 23 species from all parts of England, Scotland and Wales have been screened for rabies antigen. All of these bats were found to be negative. Forty-one serotine bats (Eptesicus serotinus), the species of bat most commonly infected in Europe, were included in the total. Subsequent to this survey, in June 1996, a European bat lyssavirus 2 was isolated from a Daubenton's bat (Myotis daubentonii) in Newhaven, East Sussex. It is possible that this bat originated from mainland Europe but this cannot be established with certainty.

Detection of the 5' region of the rubella virus genome in clinical samples by polymerase chain reaction.

BACKGROUND: Maternal rubella infection in early pregnancy has a high probability of causing congenital rubella infection. In some cases it may be difficult to establish the risk of congenital infection and polymerase chain reaction (PCR) based techniques are therefore being applied to prenatal diagnosis. OBJECTIVES: To investigate whether the non-structural region of the rubella virus (RV) genome can be detected in clinical samples using PCR, thereby providing a prenatal assay independent of those currently used to detect the structural protein coding region. STUDY DESIGN: Oligonucleotide primers coding for RV nucleotides 1-17 and 541-558 from the non-structural protein coding region of the RV genome were used in a reverse transcription polymerase chain reaction (NS RT-PCR) to amplify 558 nucleotides of RV cDNA. Amplification of RV specific sequences was confirmed by Southern hybridization. RESULTS: The specificity of the assay was confirmed by the detection of RV RNA from both wild-type and vaccine strains of RV, pharyngeal swabs from two adult males with acute rubella and products of conception from three women with serologically confirmed primary rubella in pregnancy. RV RNA was not detected in uninfected HEL and Vero cells or peripheral blood mononuclear cells. The results were concordant with those of an RT-PCR directed to the E1 protein coding region and with virus isolation. CONCLUSIONS: Detection of the non-structural coding region of the RV genome in clinical samples suggests that NS RT-PCR could be used as a confirmatory assay for prenatal diagnosis of congenital rubella, and that it will be of value for the identification of nucleotide changes in the 5' region of the RV genome.

Sequence variation in 5' termini of rubella virus genomes: changes affecting structure of the 5' proximal stem-loop.

Variation within a 523 nucleotide region proximal to the 5' terminus of seven rubella virus strains has been analysed. Compared to the Therien strain twenty sites of nucleotide variation have been identified, three of which are in the 5' untranslated region. Individual strains have between three and nine nucleotide differences, only three of which result in amino acid substitutions. TO-336 has a serine for threonine at amino acid (aa) 42 and CM arginine for histidine at aa 159. RA27/3 has arginine for lysine at aa 3 and serine for threonine at aa 42. Nucleotide differences which affect a stem-loop structure reported to be important for binding of host cell proteins have been identified.

Rapid detection of viruses of the tick-borne encephalitis virus complex by RT-PCR of viral RNA.

Studies were performed to identify a pair of primers, specific for the tick-borne encephalitis (TBE) virus complex of the Flaviviridae, with which to develop a rapid and specific identification system based on reverse transcription and the polymerase chain reaction (RT-PCR). The specificity of a putative primer pair was examined by RT-PCR of representative viruses from other antigenic complexes of the Flaviviridae and by computer sequence homology checks. All viruses of the TBE complex tested, with a single exception, were identified by RT-PCR using the identified primer pair. Accumulated data suggest that one of the putative primers identified in these studies may have flavivirus group specificity. The advantages of such a primer in the development of identification systems for all virus complexes of the Flaviviridae is discussed.

Nucleotide sequence of the envelope protein gene of the tick-borne flavivirus, Kumlinge A52.

The envelope protein gene of the tick-borne flavivirus, Kumlinge A52, the proto-type Finnish strain, has been amplified and sequenced.* The nucleotide and deduced amino acid sequence has been analyzed and compared with the closely related tick-borne encephalitis (TBE) virus, Western subtype, strain Neudoerfl, isolated in Austria. Although these two virus strains were isolated 12 years apart from different hosts and in different countries, the envelope proteins only differed by a single amino acid. It is likely, therefore, that strong selection pressures against antigenic variation exist. The possible reasons for this are discussed.

Nucleotide sequence of the envelope protein of a Turkish isolate of tick-borne encephalitis (TBE) virus is distinct from other viruses of the TBE virus complex.

Turkish tick-borne encephalitis (TTE) virus causes an acute form of meningoencephalomyelitis in sheep in the north-western region of Turkey. The clinical syndrome resembles louping ill (LI) and the viruses responsible for both LI and TTE are members of the tick-borne encephalitis (TBE) complex of the Flaviviridae. The envelope protein gene of TTE virus was reverse-transcribed, amplified, cloned and sequenced. Alignment of the resultant sequence with those from other viruses of the TBE complex reveals that TTE virus is more closely related, at both nucleotide and amino acid levels (84.6% and 96% respectively), to the Central European (CEE) subtype of the TBE virus, usually associated with human disease. The relationship with LI virus is more distant (83% and 93.5% respectively). These studies support the assertion that the ovine encephalomyelitis found in Turkey is caused by a virus that is genetically distinct from known strains of both LI and CEE viruses and from a number of other known viruses of the TBE complex.

Comparison of the nucleotide and deduced amino acid sequences of the envelope protein genes of the wild-type French viscerotropic strain of yellow fever virus and the live vaccine strain, French neurotropic vaccine, derived from it.

The envelope (E) protein genes of the wild-type yellow fever (YF) virus French viscerotropic virus and its' vaccine derivative, French neurotropic virus (FNV), were compared and found to differ by 13 nucleotides that coded for 8 amino acid substitutions. Comparison of the E proteins of FNV and 17D, the vaccine strain derived from wild-type strain Asibi, showed that there was no common nucleotide change or amino acid substitution between these two vaccine strains. However, changes are clustered around amino acid positions 52-56 and may represent the common vaccine epitope shared by 17D and FNV vaccine viruses. The molecular basis of any difference in neurotropism and viscerotropism of YF virus, attributable to the E protein, remains unclear.

Comparison of the nucleotide and deduced amino acid sequences of the structural protein genes of the yellow fever 17DD vaccine strain from Senegal with those of other yellow fever vaccine viruses.

The nucleic acid and deduced amino acid sequences of the structural proteins of the 17DD vaccine strain of yellow fever (YF) virus originating from Senegal are compared with those published for other vaccine strains of YF virus. Even though the 17D-204 and 17DD substrains of 17D diverged at passage 195 of 17D, they show a very high degree of nucleotide (99.5%) and amino acid (99.5%) homology over this region. The sequences are discussed with respect to monoclonal antibodies that can distinguish between 17DD and 17D-204 substrains of YF vaccines.

Examination of the envelope glycoprotein of yellow fever vaccine viruses with monoclonal antibodies.

Two panels of envelope glycoprotein reactive monoclonal antibodies (mAbs) were prepared against yellow fever (YF) 17D vaccine viruses. Five mAbs were prepared against the World Health Organization 17D-204 avian leukosis virus-free secondary seed virus and eight mAbs against 17DD vaccine manufactured in Brazil. The majority of these mAbs were type-specific and displayed differing reactions in neutralization tests. One, B14, would only neutralize YF vaccine virus grown in invertebrate cells. Others would differentiate 17D-204 and 17DD vaccines, from different manufacturers, in neutralization tests when the viruses were grown in vertebrate cells. The data indicate that heterogeneity exists between the epitopes that elicit neutralizing antibody on YF vaccine from different manufacturers.