Characterisation of an Australian bat lyssavirus variant isolated from an insectivorous bat

In 1996 a variant lyssavirus was isolated from an insectivorous bat (yellow bellied, sheath tail bat-Saccolaimus flaviventris) in Australia. The nucleocapsid protein (N), matrix protein (M), phosphoprotein (P), glycoprotein (G) and polymerase (L) genes of the Australian bat lyssavirus (ABL) insectivorous isolate were compared with that previously described from a frugivorous bat (Pteropus sp.), and showed sequence divergence at both the nucleotide and amino acid sequence level of 20% and 4-12%, respectively. Comparison of deduced protein sequences of ABL isolates from Pteropus and insectivorous bats, showed that viral isolates were homologous and varied by only a few percent. However, these viruses separated into two distinct clades; those isolated from Pteropus or those from Saccolaimus flaviventris bats, when comparisons were made at the nucleotide level. Nucleoprotein sequence comparisons also showed insectivorous isolates to be of the same putative genotype (genotype 7) as that isolated from frugivorous bats. Immediately after the isolation of ABL from an insectivorous bat, the first human case of ABL infection was identified. PCR and sequence analysis done on cerebrospinal fluid, brain and virus isolated from fresh brain tissue of this human case, was consistent with this infection originating from an insectivorous bat. Monoclonal antibody profiling studies of the virus isolated from the human brain tissues supported this conclusion. Sequence comparisons done on the nucleocapsid (N) gene of insectivorous or frugivorous bats showed no geographic associations between isolates but did delineate between the variants of ABL in Australia.     

Detection of Australian bat lyssavirus using a fluorogenic probe.

BACKGROUND: Australian bat lyssavirus (ABLV) has been transmitted to humans following a scratch or bite from an infected bat in two cases. Following a scratch or bite to a person, the bat is usually submitted for testing and diagnosis is made using a direct fluorescent antibody test on a brain smear. A nested RT-PCR assay has also been utilised to confirm diagnosis. If positive for lyssavirus, post-exposure prophylaxis is administered. OBJECTIVES: The TaqMan assay was developed to improve the diagnosis of ABLV infection, following problems encountered with the generation of spurious PCR products in the nested RT-PCR and also to reduce the high risk of contamination inherent with nested PCRs. STUDY DESIGN: RNA was extracted from 161 bat brains and the samples were compared using a conventional RT-PCR and the TaqMan based assay. Samples from a patient with an ABLV infection collected antemortem and postmortem were also tested. RESULTS: The sensitivity of the new TaqMan based PCR assay compared favourably with the nested PCR previously in use in our laboratory. This assay was able to detect RNA in samples collected antemortem and postmortem for the diagnosis of a human case of ABLV. CONCLUSIONS: The major advantage of the TaqMan based assay was the speed of diagnosis with a result within minutes of completing the PCR (a result within 4 h of receiving the specimen). This test greatly reduces the chance of false positives through the elimination of second-round PCR and the requirement for agarose gels. The assay is sensitive and specific and should be invaluable for future antemortem and postmortem diagnosis of ABLV infection in humans.     

Sequence analysis of an Australian isolate of sugarcane bacilliform badnavirus

Summary.  The genome of an Australian isolate of Sugarcane bacilliform virus (SCBV-IM) was cloned, sequenced and analysed. The genome consisted of 7687 nucleotides and contained three open reading frames which were similar in size and organisation to those of other badnaviruses. SCBV-IM was found to be most similar to the SCBV-Morocco isolate with amino acid sequence similarity of 91.4 %, 83.8 % and 85.3 % in the ORF I, II and III coding regions, respectively. Phylogenetic analysis of the SCBV-IM ORF III deduced amino acid sequence showed that SCBV isolates were more closely related to each other than to other badnaviruses. Amplification of SCBV sequences from three different sugarcane varieties revealed considerable variability in the viral populations, both within single infected plants as well as between infected plants, suggesting that the SCBV isolates sequenced to date may not be representative of the range of virus variability. Received February 19, 2002; accepted June 14, 2002     

Natural and experimental infection of sheep with European bat lyssavirus type-1 of Danish bat origin

In 1998 and 2002, European bat lyssavirus type-1 (EBLV-1) was demonstrated in brain tissue of five Danish sheep suffering from neurological disorders. Four of the five sheep also had encephalic listeriosis. The animals originated from four flocks on pastures within a limited area of western Jutland. In a serological investigation in two of the herds, from which three of the diseased animals originated, EBLV-1 neutralizing antibodies were detected in only one of 69 sheep. In follow-up surveys, 2110 sheep sera collected at Danish slaughterhouses during 2000 were all negative for EBLV-1-antibodies, and EBLV-1 was not demonstrated in 87 ruminants displaying neurological symptoms. To investigate the pathogenic effects of EBLV-1, four sheep were inoculated intralabially with either brain material from one of the naturally infected sheep or virus isolated from the same sheep. These animals developed EBLV-1 neutralizing antibodies at 5-9 weeks post-inoculation but did not exhibit neurological signs during a 33-week observation period. It was speculated that the immune response prevented viral dissemination to the brain, resulting in an abortive peripheral infection. It was concluded that EBLV-1 can infect sheep under natural conditions as an incidental event.     

A lyssavirus with an unusual antigenic structure isolated from a bat in southern Kyrgyzstan]

Examination of 191 specimens of Chiroptera in Osh Province of Kyrgyzstan yielded 1 strain of lyssavirus from Myotis blythi, the isolate not belonging to serotype 1. The virus was designated Aravan by the area of its isolation. Its antigenic structure was studied using antinucleocapsid monoclonal antibodies of the Wistar Institute (Philadelphia, ISA) and Central Veterinary Laboratory of Great Britain (Waybridge, Great Britain). The paper presents its antigenic profile, brief characteristics of similarity and differences of the Aravan strain and known lyssavirus serotypes.     

Molecular diagnostics for the detection of Bokeloh bat lyssavirus in a bat from Bavaria,Germany

A brain sample of a Natterer's bat tested positive for rabies with classical virological techniques. Molecular techniques confirmed the presence of Bokeloh bat lyssavirus (BBLV) in Germany for the second time. Sequence analysis revealed a close genetic relationship to the initial German BBLV case. Using a TaqMan RT-PCR specific for BBLV viral RNA was detected in various other organs albeit with differences in the relative viral load.     

Sequence analysis of segment a of a field virus isolate from an outbreak of Infectious bursal disease in India

Sequence analysis of genome segment A of an Indian Infectious bursal disease virus (IBDV) field isolate (KT1/99) revealed total 95 nucleotide substitutions, resulting in 17 amino acid changes. Of these, five amino acid changes, namely F60S, T137I, I374V, V519I and E682D were unique to the KT1/99 isolate. The amino acid change P222A and the proposed hot mutation spot 680Y, reported to be present in very virulent IBDV isolates were also found in KT1/99. This isolate had nucleotide divergence of 1.1% to 4.95% from the other reported serotype 1 IBDV isolates and 19.6% from serotype 2 strain OH in polyprotein gene sequence, while divergence at amino acid level was 0.6% to 2.9% and 11.4%, respectively. Based on both nucleotide and amino acid sequence analysis, KT1/99 was grouped phylogenetically with the reported Bangladesh isolate BD3/99 in one cluster along with other reported very virulent isolates in same lineage.     

T and B cell human responses to European bat lyssavirus after post-exposure rabies vaccination.

T and B cell human responses to European bat lyssavirus (EBL1) induced by post-exposure rabies vaccination (PM virus vaccine) were evaluated by measuring plasmatic titres of EBL1-specific neutralizing antibodies; specific EBL1-binding antibodies; and proliferation indices of peripheral blood lymphocytes stimulated in vitro with EBL1. These parameters for vaccination efficacy were compared with those obtained with vaccine-related viruses (CVS and ERA) and with a non-vaccine-related virus. Mokola virus, the last implicated in vaccination failures. Twenty-two patients exposed to rabies risk who received a reduced rabies post-exposure vaccination were involved in the study. On day 21, vaccine induced CVS-specific neutralizing antibodies in all patients; but EBL1-specific neutralizing antibodies were induced in only 73% of patients. No vaccine had Mokola-specific neutralizing antibodies. Patients having EBL1-specific neutralizing antibodies were usually those in whom vaccination induced high titres of CVS-specific neutralizing antibodies. On day 21, peripheral blood lymphocytes of 86% of patients could be restimulated in vitro with vaccine, 43% with EBL1 and 45% with Mokola. Patients exhibiting a high vaccine-specific proliferation response more likely developed an EBL1- or a Mokola-specific proliferative response. No correlation was found between T and B cell responses. Rabies vaccination induced neither T nor B cell EBL1-specific responses in 22% of patients.     

Phylogeny of Lagos bat virus: challenges for lyssavirus taxonomy

Lagos bat virus (LBV) belongs to genotype 2 of the Lyssavirus genus. The complete nucleoprotein (N), phosphoprotein (P), matrixprotein (M) and glycoprotein (G) genes of 13 LBV isolates were sequenced and phylogenetically compared with other lyssavirus representatives. The results identified three different lineages of LBV. One of these lineages demonstrated sufficient sequence diversity to be considered a new lyssavirus genotype (Dakar bat lyssavirus). The suggested quantitative separation of lyssavirus genotypes using the N, P, M and G genes was also investigated using P-distances matrixes. Results indicated that the current criteria should be revised since overlaps between intergenotypic and intragenotypic variation occur.     

Isolation of Bokeloh bat lyssavirus in Myotis nattereri in France

Bokeloh bat lyssavirus (BBLV) was found in Myotis nattereri for the first time in northeastern France in July 2012. The complete genome sequence of the virus from the infected Natterer’s bat was determined by whole-genome sequencing and compared to that of the first BBLV strain isolated in 2010 in Germany and with those of all currently identified lyssaviruses. The French isolate [KC169985] showed 98.7 % nucleotide sequence identity to the German BBLV strain [JF311903]. Several organs of the infected French bat were examined by classical rabies diagnostic methods: fluorescent antibody test, cell culture inoculation test and RT-qPCR. Antigen, infectious virus and high viral RNA levels were found in both the brain and salivary glands. Traces of genomic RNA were detected in the bladder, kidney and lung tissue. The results of an investigation of the distribution of lyssaviruses with the detection of infectious virus in the salivary glands suggest a possible mode of transmission of the virus.     

Case of fatal systemic infection with an Aureobacterium sp.: identification of isolate by 16S rRNA gene analysis.

The case of a 75-year-old man who succumbed to a disseminated infection most likely caused by a species of the genus Aureobacterium is reported. Identification of the isolate was achieved by comparative 16S rRNA gene analysis. Aureobacteria are commonly found in the environment. However, only recently have they been recognized as a cause of infections including septicemia and soft tissue infections. To our knowledge, this is the first documentation of a fatal infection caused by an Aureobacterium sp.     

A bat rabies isolate with an unusually short incubation period

Rabies viruses from two types of bats were inoculated intracerebrally into laboratory mice. The reactions of the mice differed markedly. Those inoculated with virus from a Mexican freetail bat, Tadarida brasiliensis mexicana, died a violent death after an incubation of 4 to 5 days. The pathology was marked, with much neuronal destruction noted. Those mice inoculated with virus from a vampire bat, Desmodus rotundus, died after incubation periods similar to those noted after inoculation of common “street” viruses, i.e., 7 days or more. The pathology corresponded to the limited amount usually seen after death from “street” virus.     

Generation of recombinant European bat lyssavirus type 1 and inter-genotypic compatibility of lyssavirus genotype 1 and 5 antigenome promoters

Bat lyssaviruses (Fam. Rhabdoviridae) represent a source for the infection of terrestial mammals and the development of rabies disease. Molecular differences in the replication of bat and non-bat lyssaviruses and their contribution to pathogenicity, however, are unknown. One reason for this is the lack of reverse genetics systems for bat-restricted lyssaviruses. To investigate bat lyssavirus replication and host adaptation, we developed a reverse genetics system for European bat lyssavirus type 1 (EBLV-1; genotype 5). This was achieved by co-transfection of HEK-293T cells with a full-length EBLV-1 genome cDNA and expression plasmids for EBLV-1 proteins, resulting in recombinant EBLV-1 (rEBLV-1). Replication of rEBLV-1 was comparable to that of parental virus, showing that rEBLV-1 is a valid tool to investigate EBLV-1 replication functions. In a first approach, we tested whether the terminal promoter sequences of EBLV-1 are genotype-specific. Although genotype 1 (rabies virus) minigenomes were successfully amplified by EBLV-1 helper virus, in the context of the complete virus, only the antigenome promoter (AGP) sequence of EBLV-1 was replaceable, as indicated by comparable replication of rEBLV-1 and the chimeric virus. These analyses demonstrate that the terminal AGPs of genotype 1 and genotype 5 lyssaviruses are compatible with those of the heterologous genotype.     

Sequence polymorphisms of the mtDNA control region in a human isolate: the Georgians from Swanetia

In this work, we analyzed the sequence diversity of the mtDNA control region (HVI and HVII) in a sample of 48 individuals from Swanetia (Georgia), using direct fluorescent-based sequencing methods. We identified 43 different mtDNA haplotypes resulting from 78 polymorphic sites (46 in HVI and 32 in HVII). Most of the variable positions identified in both HVI and HVII were transitions (82.6 and 71.9%, respectively). The frequency of length heteroplasmy in the homopolymeric C-stretch regions was the same for both segments (10.4%). The sequence diversity increased markedly when both hypervariable regions were analyzed jointly (HVI: 0.985, HVII: 0.975, HVI+HVII: 0.994). Accordingly, the probability of two randomly selected sequences matching (random match probability, RMP) decreased from 3.4% (HVI) to 2.6% (HVI+HVII), despite which the RMP values in Georgians remained higher than estimated in most Europeans. This suggests that the variability of maternal lineages tends to be lower in traditional human isolates and, therefore, the potential of discrimination of mtDNA in forensic analysis is more limited in this type of population. The incorporation of HVII data also contributed to the refinement of results regarding the genetic relationships among the samples included in the analyses, which stress the importance of considering HVII in both population and forensic genetics.M. A. Alfonso-Sánchez and C. Martínez-Bouzas contributed equally to this work and are listed alphabetically