European bat lyssavirus type 2 RNA in Myotis daubentonii.

Organ distribution of European bat lyssavirus type 2 viral RNA in its reservoir host, Myotis daubentonii (Daubenton's bat), was measured with a novel quantitative reverse transcription-polymerase chain reaction assay. High levels of genomic RNA were found in the brain and were also detectable in the tongue, bladder, and stomach.

**********

Bat-mediated rabies has been reported in Europe for more than 50 years. Two variants or genotypes are now recognized that are distinct from rabies viruses of terrestrial mammals and new world bats (1). These are known as European bat lyssaviruses (EBLVs). A third lyssavirus, West Caucasian bat lyssavirus, has been isolated in eastern Europe (2). EBLV type 1 (EBLV-1) is found throughout mainland Europe and principally associated with the Serotine bat (Eptesicus serotinus) (3). EBLV-2 is found in Myotis bats (Myotis daubentonii [Daubenton's bat] and Myotis dasycneme) and has been identified in 3 locations in Europe: the Netherlands, the United Kingdom, and Switzerland (Table 1).

Two reports detail isolation of EBLV-2 from humans who died of rabies encephalitis in Finland and the United Kingdom (4,5). In addition, 4 isolations from Daubenton's bat have been reported in the United Kingdom since 1996. Seroprevalence studies suggest that EBLV-2 is maintained at certain sites in the United Kingdom at low levels (6). However, the small number of bats infected with EBLV-2 and the nocturnal habits of insectivorous bats have hampered attempts to understand the distribution, prevalence, and transmission of the virus. Biting by Daubenton's bats was suspected in the 2 human cases from Finland and the United Kingdom because both persons had handled this species before symptoms developed. However, like rabies virus, EBLV-2 does not persist in the environment outside of an infected host, and alternative routes of infection should be considered. Investigation of the second bat detected viable EBLV-2 in the brain, and genomic RNA in the heart, stomach, tongue, intestine, liver, and kidney by using a sensitive nested reverse transcription-polymerase chain reaction (RT-PCR) (7). This approach was unable to quantify viral RNA within particular tissues. Since this study, 2 additional cases have occurred in the United Kingdom (Table 1). The investigation and quantification of viral load within the infected host could provide evidence for release of virus and methods of transmission.

The Study

In 2004, two EBLV-2 cases were identified in Daubenton's bats (Table 1). A diagnosis of EBLV-2 infection was confirmed on brain samples with a fluorescent-antibody test, the mouse inoculation test, and a rapid TaqMan assay (8). Attempts to culture EBLV-2 from organs in both cases failed because of cytotoxicity of the samples, which destroyed the cell monolayer. Sample dilution reduced the cytotoxic effects of the sample on the cell monolayer (used for virus isolation) and enabled the development of small foci of infection (bat 603/04). Heminested RT-PCR detected virus RNA in brain, tongue, thyroid gland, and bladder after the first round of amplification, and in salivary gland, heart, lung, intestine, and stomach after the second round of amplification. We suspect that inappropriate storage of bat 696/04 in a freezer with repeated freezing and thawing before submission resulted in inactivation of virus in this sample. Heminested RT-PCR detected virus RNA in samples of brain and stomach after the first round of PCR, and in samples of tongue, intestine, liver, and kidney after the second round of amplification.

An EBLV-2-specific real-time PCR was developed to measure virus genome to quantify the potential viral RNA load within organs. Analysis was only attempted on those organs with sufficient RNA within the sample (Figure). Primers EBLVNa (5'-CCTGGCAGATGATGGGAC-3') and EBLVNb (5'-GCCTTTTATCTTGGATCACT-3') are located within the nucleoprotein gene and amplify a 221-bp target. An amplified product from a previous case (5) was purified by using the RNeasy kit (Qiagen, Valencia, CA, USA) and quantified with a NanoDrop WD-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA). This procedure enabled the absolute number of copies of the amplicon to be calculated by its approximate molecular weight and Avogadro's number, as previously described (9).

[FIGURE OMITTED]

RNA was isolated from each organ with Trizol (Invitrogen, Carlsbad, CA, USA) and quantified. Dilutions were made to either 0.25 [micro]g/p[micro]L (bat 603/04) or 1 [micro]g/[micro]L (bat 696/04) to standardize the quantity of RNA used for reverse transcription. Primer EBLVNa was used for cDNA synthesis from the genomic (negative) sense strand as previously described (10). All PCRs were performed by using SYBR Green JumpStart Taq ReadyMix (Sigma, Saint Louis, MO, USA) and an MX3000P real-time thermal cycler (Stratagene, La Jolla, CA, USA). A dilution series of the control amplicon was amplified simultaneously with the organ samples to create a standard curve for comparison of the threshold value (Ct) with target copy number (Figure, panel A).

A representative plot of amplification curves from organ samples taken from bat 696/04 is shown in the Figure, panel B, with 10 [micro]L of product separated by electrophoresis on a 1% agarose gel included for comparison. The quantitative results for viral RNA load for both bats are shown in Table 2. In both cases, the brain had the highest viral genome load. Virus RNA was consistently detected in the tongue, intestine, and stomach. EBLV-2 was also found in the bladder of bat 603/04 but not in the kidney of bat 696/04 from which the bladder was not recovered because of carcass decomposition. Virus was not detected in the liver of either bat.

Conclusions

The detection and quantification of EBLV-2 RNA in bat organs by real-time PCR show the potential distribution of this virus. The choice of organ tested in both cases was severely limited by degradation of the carcass before investigation. Furthermore, live virus could not be recovered from many organs because of eytotoxicity of the samples and virus degradation caused by repeated freezing and thawing.

Viable virus was recovered from the brain of bat 603/04. Since the brain is the main site of EBLV-2 replication, this finding suggests that the virus displays a similar neurotropism to classical rabies virus. Rabies virus, especially in the late stages of disease, disseminates from the brain to other innervated sites within the host (11). For EBLV-2, the tongue was consistently found to contain detectable levels of viral RNA in this study and a previous study (7). Genomic RNA was also found in the stomach and intestines of 3 bats investigated (this study and [7]). All of these organs are highly innervated tissues, although virus RNA in the stomach could result from swallowing virus.

Dissemination of rabies virus to the salivary glands and subsequent virus shedding enables transmission through biting. Detection of EBLV-2 RNA in the tongue of infected bats leads us to conclude that transmission of EBLV-2 may occur through biting. However, since EBLV-2 genome was detected in a bladder sample, we cannot exclude the possibility of virus release from urine. In future cases, where possible, organs such as the salivary glands and lungs should be examined to provide further evidence for the route of virus transmission between bats.

Acknowledgments

We thank Denise Marston and Karen Mansfield for excellent technical assistance.

This work was supported by a Department for Environment, Food and Rural Affairs (United Kingdom) grant SE0524 and grant SV3500.

References

(1.) Bourhy H, Kissi B, Tordo N. Molecular diversity of the Lyssavirus genus. Virology. 1993; 194:70-81.

(2.) Botvinkin AD, Poleschuk EM, Kuzmin IV, Borisova TI, Gazaryan SV, Yager P, et al. Novel lyssaviruses isolated from bats in Russia. Emerg Infect Dis. 2003;9:1623-5.

(3.) Amengual B, Whitby JE, King A, Cobo JS, Bourhy H. Evolution of European bat lyssaviruses. J Gen Virol. 1997;78:2319-28.

(4.) Lumio J, Hillbom M, Roine R, Ketonen L, Haltia M, Valle M, et al. Human rabies of bat origin in Europe. Lancet. 1986; 1:378.

(5.) Fooks AR, McElhinney LM, Pounder DJ, Finnegan C J, Mansfield K, Johnson N, et al. Case report: isolation of a European bat lyssavirus type-2a from a fatal human case of rabies encephalitis. J Med Virol. 2003 ;71:281-9.

(6.) Brookes SM, Aegerter JN, Smith GC, Healy DM, Joliffe TA, Swift SM, et al. European bat lyssavirus in Scottish bats. Emerg Infect Dis. 2005;11:572-8.

(7.) Johnson N, Selden D, Parsons G, Healy D, Brookes SM, McElhinney LM, et al. Isolation of a European bat lyssavirus type 2 from a Daubenton's bat in the United Kingdom. Vet Rec. 2003;152:383-7.

(8.) Wakeley PR, Johnson N, McElhinney LM, Marston D, Sawyer J, Fooks AR. Development of a real-time, Taqman reverse transcriptase-PCR assay for detection and differentiation of lyssavirus genotypes 1, 5, and 6. J Clin Microbiol. 2005;43:2786-92.

(9.) Hammond JA, Pomeroy PP, Hall A J, Smith VJ. Identification and real-time PCR quantification of Phocine distemper virus from two colonies of Scottish grey seals in 2002. J Gen Virol. 2005;86:2563-7.

(10.) Johnson N, McElhinney LM, Smith J, Lowings P, Fooks AR. Phylogenetic comparison of the genus Lyssavirus using distal coding sequences of the glycoprotein and nucleoprotein genes. Arch Virol. 2002; 147:2111-23.

(11.) Jackson AC, Ye H, Phelan CC, Ridaura-Sanz C, Zheng Q, Li Z, et al. Extraneural organ involvement in human rabies. Lab Invest. 1999;79:945-51.

Nicholas Johnson, * Philip R. Wakeley, * Sharon M. Brookes, * and Anthony R. Fooks *

* Veterinary Laboratories Agency, Addlestone, Surrey, United Kingdom

Address for correspondence: Nicholas Johnson, Rabies and Wildlife Zoonoses Group, Veterinary Laboratories Agency, Weybridge, Woodham Lane, Addlestone, Surrey KT15 3NB, UK; email: n.johnson2@ vla.defra.gsi.gov.uk

Dr Johnson is a senior researcher in the Rabies and Wildlife Zoonoses Group at the Veterinary Laboratories Agency, Surrey, United Kingdom. His research interests include molecular epidemiology of rabies and host response to viral infection.

Table 1. Reports of European bat lyssavirus type 2 (EBLV-2)
in Europe, 1985-2004 *

Year                  County                   Description

1985 ([dagger])       Finland          30-year-old man admitted to
                                    department of neurology, Helsinki
                                    University Central Hospital, with
                                    ascending paralysis and radiating
                                       pain in right arm and neck;
                                           became agitated with
                                           hyperexcitability,
                                     hyperventilation, and spasms the
                                    following day; died 20 days after
                                                admission.
                                      Rabies diagnosis confirmed by
                                               FAT and MIT.
1986                  Denmark           Rabies in pond bat (Myotis
                                               dasycneme).
1986                  Denmark           Rabies in Daubenton's bat
                                            (M. daubentonii).
1986                  Germany           Rabies in Daubenton's bat.
1987                  Denmark           Rabies in Daubenton's bat.
1987 ([dagger])   The Netherlands    Virus isolated from pond bat in
                                                 Wommels.
1987 ([dagger])   The Netherlands    Virus isolated from pond bat in
                                                Tjerkwerd.
1987              The Netherlands     Virus isolated from pond bat.
1989 ([dagger])   The Netherlands    Virus isolated from pond bat in
                                                 Andijk.
1992 ([dagger])     Switzerland      Daubenton's bat found hanging on
                                    grill of ventilation shaft during
                                     daylight hours in Fribourg. Bat
                                    was weak, unable to fly, and died
                                       shortly afterwards. Rabies
                                      diagnosis confirmed by FAT and
                                                   MIT.
1993 ([dagger])   The Netherlands    Virus isolated from pond bat in
                                                  Roden.
1993 ([dagger])     Switzerland      Virus isolated from Daubenton's
                                             bat in Versoix.
1996 ([dagger])   United Kingdom      Sick Daubenton's bat found in
                                        cellar of public house in
                                      Newhaven bit a pregnant woman
                                     while it was being cared for in
                                      bat hospital. Bat deteriorated
                                     rapidly. Diagnosis confirmed by
                                       FAT, RTCIT, MIT, and RT-PCR.
2002 ([dagger])   United Kingdom     Juvenile female Daubenton's bat
                                     brought onto property adjoining
                                       Lancashire canal. Bat was in
                                      distress with wing damage, was
                                       treated for >7 weeks before
                                         signs of agitation and
                                      vocalization developed, became
                                       aggressive and tried to bite
                                      handler. Bat died 6 days after
                                      symptoms developed. Diagnosis
                                      confirmed by FAT, RTCIT, MIT,
                                               and RT-PCR.
2002 ([dagger])     Switzerland        Rabies in Daubenton's bat in
                                                 Geneva.

2002 ([dagger])   United Kingdom       55-year-old man admitted to
                                        Dundee hospital with acute
                                       hematemesis and upper limb
                                      parasthesia; became aggressive
                                     and required sedation on day 5;
                                     died on day 14. Man had history
                                      of exposure to bats in United
                                    Kingdom; postmortem PCR on saliva
                                     detected EBLV-2. Virus recovered
                                     from brain tissue after autopsy.
2003 ([dagger])   United Kingdom     Adult male Daubenton's bat found
(bat 696/04)                         Sep 2003 after flying into tree
                                         in daylight near Bury in
                                     Lancashire. Bat was cared for by
                                      volunteers and took water but
                                      attempted to bite carpet when
                                      placed there to feed. Bat died
                                      and was frozen until diagnosis
                                            was made Oct 2004.
2004 ([dagger])   United Kingdom        Grounded juvenile female
(bat 603/04)                          Daubenton's bat was found in
                                        Staines and cared for by
                                      volunteers. Its condition was
                                      poor and it displayed signs of
                                        aggression and lethargy.
                                       Diagnosis confirmed by FAT,
                                         RTCIT, MIT, and RT-PCR.

* FAT, fluorescent-antibody test; MIT, mouse inoculation test;
RTCIT; rabies tissue culture infection test, RT-PCR, reverse
transcription-polymerase chain reaction.

([dagger]) Virus identity confirmed by genomic sequence analysis.

Table 2. Quantification of European bat lyssavirus type 2 genome
copies in organs of 2 naturally infected Daubenton's bats *

                Genome copies/[micro]g total RNA

Organ            Bat 603/04             Bat 696/04

Brain           204,000,000            640,000,000
Tongue            292,800                136,533
Liver              61,760                 37,800
Bladder           839,680                   ND
Kidney               ND                   87,933
Intestine         277,067                680,667
Stomach           380,133               10,586,667

* ND, not done.