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Host Range and Genetic Diversity of Arenaviruses in Rodents, United Kingdom

Host Range and Genetic Diversity of Arenaviruses in Rodents, United Kingdom All animal research was conducted under license, accord- Host Range and ing to UK regulations. Serum samples were separated by centrifugation Genetic Diversity (10,000 rpm, 10 min) and tested for LCMV antibody by using the manufacturer’s protocol for commercial indirect of Arenaviruses in fl uorescent antibody assay slides (Charles River Laborato- ries, Wilmington, MA, USA). A 1:40 dilution of anti-rat Rodents, United or anti-mouse immunoglobulin G fl uorescein isothiocya- nate (Sigma-Aldrich, Gillingham, UK) or a combination of Kingdom both were used as secondary antibody. Ninety-three serum Kim R. Blasdell, Stuart D. Becker, Jane Hurst, samples (from the original serum samples tested for anti- Mike Begon, and Malcolm Bennett body) that were either antibody positive or from sites with high seroprevalence were tested for arenavirus RNA by During a study to extend our knowledge of the host PCR. Another 379 blood samples from the captive colony range and genetic diversity of arenaviruses in Great Brit- of house mice, which had not been previously tested for ain, 66 of 1,147 rodent blood samples tested for antibody, antibody, were also tested. The PCR targeted a fragment of and 127 of 482 tested by PCR, were found positive. All se- the glycoprotein precursor gene (GPC) (10). A selection of quences most closely resembled those of previously identi- samples found negative by the GPC PCR were subsequently fi ed lymphocytic choriomeningitis virus. retested by PCR targeted at a fragment of the nucleoprotein (N) gene (8), by using primers to sequences common to the iruses in the family Arenaviridae are separated into 2 Old World arenaviruses. Total RNA was extracted by us- Vdistinct serocomplexes, the New World serocomplex ing QIAamp viral RNA mini-kit (QIAGEN, Crawley, UK), and the Old World serocomplex (1). Several arenavirus converted to cDNA, and amplifi ed by using a single-step species are known to cause human disease, including lym- kit (Superscript III one-step RT-PCR with Platinum Taq phocytic choriomeningitis virus (LCMV), which causes polymerase system; Invitrogen, Paisley, UK) in conjunc- infl uenza-like clinical signs, occasionally with neurologic + – tion with oligonucleotides arena1 and LCMV322 (10) or complications. Infection may be asymptomatic in up to one 1010C and either OW1696R or NW1696R (8). Products third of patients (2), and serious complications often occur were separated and visualized by agarose gel electropho- in intrauterine infection (3). Less severe cases of adult hu- resis, and amplicons were purifi ed with the QIAquick PCR man infection are likely underreported and often misdiag- purifi cation kit (QIAGEN). Bidirectional sequencing was nosed (4). performed off-site (MWG Biotech AG, Ebersberg, Ger- LCMV is found worldwide, probably because of its as- many). The 97-nt sequences generated here were deposited sociation with its natural Old World host, the house mouse, with GenBank (accession nos. DQ275199–DQ275295). Mus musculus (5). Although antibodies have also been de- The software package MEGA version 4.0 (11) was tected in other rodent species (6,7), arenaviruses are known used to construct an alignment of a 283-nt fragment of the to be serologically cross-reactive. Few isolates of LCMV GPC gene nucleotide sequences and predicted amino acid have been obtained from wild rodents so little is known sequences, and for phylogenetic analysis with the neighbor- about its genetic diversity. Recent studies on American are- joining method (p distance model), with bootstrap support naviruses found that diverse arenaviruses co-evolved with based on 1,000 pseudoreplicates. Other GenBank sequenc- their rodent hosts (8), a fi nding that suggests that a more es included for comparison are listed in Table 2. Pairwise thorough study of European rodents might also identify genetic distances were calculated by using the p distance novel arenaviruses. The purpose of this study was therefore model; percentage sequence identities were calculated by to extend our knowledge of LCMV and LCMV-like arena- subtracting the genetic distances from 1.0 and multiplying viruses in rodents in Great Britain. by 100. Overall, 66 of 1,147 serum samples and 7 of 9 rodent The Study species had antibodies to arenaviruses. Sciurus vulgaris In total 1,147 blood samples were collected from ro- had the highest prevalence, 26%, although only 15 squir- dents: 1,060 were live-trapped, wild animals from <20 sites rels were tested. M. musculus had the second highest preva- (Table 1), and 87 blood samples were collected from a cap- lence, 17.5%. Antibodies were also detected in Apodemus tive colony of wild house mice (9) and tested serologically. sylvaticus, Microtus agrestis, Micromys minutis, captive- housed Cynomys ludovicianus, and Rattus norvegicus. Author affi liation: University of Liverpool, Liverpool, UK Seroprevalence varied between species (1.4%–26%) and between sites (0%–50%) (Table 1). DOI: 10.3201/eid1409.080209 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 9, September 2008 1455 DISPATCHES Table 1. Rodent species, numbers tested, seroprevalence, and viral RNA prevalence to LCMV at each UK and Republic of Ireland site* Site code and year Geographic location Species tested for antibody Species tested for viral RNA PHF 2004 Cheshire MMu (0/4), RN (0/2); SP = 0.0 PHF 2005 Cheshire MMu (8/26); SP = 30.8 BHF 2004 Cheshire MMu (4/10); SP = 40.0 MMu (2/9), RN (0/2) – 2 sequences BHF 2005 Cheshire MMu (2/2), RN (0/2); SP = 50.0 BGF Cheshire MMu (0/7), RN (0/2); SP = 0.0 CLF 2004 Cheshire MMu (0/6), RN (0/2); SP = 0.0 CLF 2005 Cheshire MMu (0/12), RN (0/4); SP = 0.0 MF 2002 Cheshire AS (0/10), MG (0/9), MA (0/2), MMu (0/30); SP = 0.0 AS (1/10), MG (0/1), MA (0/1), MMu (2/4) – 3 sequences MF 2004 Cheshire AS (4/10), MG (0/1), MA (0/1), MMu (2/4); SP = 37.5 CZ 2002 Cheshire AS (0/4), MG (0/4), CL (4/61), MMi (0/22), RN (0/2); AS (1/1), CL (0/4), MMi (0/1), SP = 4.3 MMu (0/1) – 0 sequences CZ spring 2003 Cheshire AS (0/9), MG (0/3), MA (0/4), MMi (1/3), SP = 5.3 CZ autumn 2003 Cheshire AS (1/18), MG (0/19), MA (0/4), MMu (0/1), SP = 2.9 CZ 2004 Cheshire MMu (1/19), RN (1/12), SP= 3.2 DF Cheshire MMu (1/69), SP = 1.4 MW Cheshire MG (0/105), AS (0/45), SP = 0.0 RH Cheshire MG (0/19), AS (0/49), SP = 0.0 LVFS Cheshire RN (0/2, SC (0/4), SP = 0.0 FA Merseyside MA (0/2), AS (0/24), SP = 0.0 KF Northumberland MA (2/104), SP = 1.9 LI North Devon RN (0/40), SP = 0.0 IOW Isle of Wight SV (1/18), SP = 5.6 TF Thetford SV (1/21), SP = 4.8 CF Cumbria SC (0/10), SV, (0/4), SP = 0.0 NI Northern Ireland AS (1/149), SP = 0.7 CA Republic of Ireland MG (0/15), SP = 0.0 CC Republic of Ireland AS (0/7), SP = 0.0 TW Republic of Ireland AS (0/10), SP = 0.0 Other Various locations RN (1/6), SC (0/1), SV (2/26), SP = NA AS (0/1), MA (0/2), MMu (0/31), RN (0/5), SV (1/4) – 0 sequences Captive colony Captive colony, MMu (30/87), SP = 34.5 MMu (122/403) – 92 Cheshire sequences *LCMV, lymphocytic choriomeningitis virus; SP, site prevalence (%). Species key: MMu, Mus musculus (house mouse); RN, Rattus norvegicus (brown or Norway rat); AS, Apodemus sylvaticus (wood mouse); MG, Myodes glareolus (bank vole); MA, Microtus agrestis (field vole); MMi, Micromys minutis (harvest mouse); CL, Cynomys ludovicianus (black-tailed prairie dog); SV, Sciurus vulgaris (red squirrel); SC, Sciurus carolensis (gray squirrel); NA, not available. Species containing positive animals are in boldface italics, with number positive and number tested in parentheses. GPC PCR amplicons were obtained from 127 of 472 in the GPC PCR, but seropositive or from high prevalence tested samples, and sequences were determined for 97 sam- sites, were tested by N gene PCR, and 2 were weakly posi- ples (Table 1). All positive samples were from Mus muscu- tive: 1 S. vulgaris and 1 A. sylvaticus. In neither case, how- lus except 1 from A. sylvaticus. Twenty samples negative ever, could a sequence be obtained from the amplicon. Table 2. Percentage nucleotide identities between the study sample sequences (all and from the captive colony only) and previously isolated LCMV and Lassa virus sequences* Sequence All study sequences, % Captive colony sequences only, % All study sequences 93.6–100 NS Captive colony sequences only NS 97.4–100 LCMV CIPV76001 Pasteur (AF095783; France) 78.7–80.5 78.7–80.5 LCMV CIP97001 (AF079517; France) 79.4– 83.1 80.9–83.1 LCMV Marseille (DQ286931; France) 82.8– 83.9 82.8–83.5 LCMV CH5871 (AF325215; Germany) 81.6–83.1 81.6–83.1 LCMV CH5692 (AF325214; Germany) 81.3–82.8 81.3–82.8 LCMV MX (EU195888; Slovakia) 78.7–80.5 78.7–80.5 LCMV Armstrong (M20869; USA) 82.8– 85.8 83.9–85.8 LCMV WE (M22138) 82.0–84.3 82.0–84.3 Lassa LP (AF181853) 58.1–59.9 58.8–59.9 *LCMV, lymphocytic choriomeningitis virus; NS, not shown. 1456 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 9, September 2008 Arenaviruses in Rodents, UK Nucleotide and amino acid GPC sequence identities BU22 DQ275216 sample for all the samples in this study ranged from 93.6%–100% BU66 DQ275259 sample BU37 DQ275231 sample (Table 2), and 94.5%–100%, respectively (data not shown). BU71 DQ275264 sample BU84 DQ275277 sample When compared with other arenaviruses, the nucleotide C(BU)6 DQ275285 sample BU67 DQ275260 sample sequences exhibited 78.7%–85.8% identity with LCMV BU50 DQ275243 sample C(BU)2 DQ275282 sample BU33 DQ275227 sample reference sequences and only 58.1%–59.9% identity with BU63 DQ275256 sample M(BU)20+ DQ275290 sample Lassa virus (online Appendix Figure, available from www. BU32 DQ275226 sample BU75 DQ275268 sample cdc.gov/EID/content/14/9/1455-appF.htm). BU14 DQ275211 sample Captive colony BU64 DQ275257 sample Although antibodies to arenaviruses have been re- BU06 DQ275205 sample BU48 DQ275241 sample ported in a range of European rodent species, our study BU51 DQ275244 sample BU47 DQ275240 sample BU77 DQ275270 sample provided evidence of arenaviruses infecting red squirrels 64 BU85 DQ275278 sample BU25 DQ275219 sample (S. vulgaris) and European harvest mice (M. minutis). An- BU65 DQ275258 sample BU49 DQ275242 sample tibodies to arenaviruses have been reported in introduced BU82 DQ275275 sample BU83 DQ275276 sample S. carolensis in Great Britain (12) but were not detected in BU1 DQ275199 sample BU86 DQ275279 sample MF this study. We also reported antibodies to arenaviruses in MM16 DQ275293 sample BU16 DQ275213 sample BU68 DQ275261 sample black-tailed prairie dogs (Cynomys ludovicianus): those Captive colony BU74 DQ275267 sample BU36 DQ275230 sample tested in this study were part of a colony in a zoo, how- BU76 DQ275269 sample MM10 DQ275294 sample MF ever, and had contact with wild mice, some of which were BU44 DQ275237 sample BU23 DQ275217 sample seropositive. As found in previous studies, Mus musculus BU24 DQ275218 sample BU34 DQ275228 sample BU45 DQ275238 sample was more likely to be infected with LCMV than other ro- BU39 DQ275232 sample BU81 DQ275274 sample dent species. BU21 DQ275215 sample BU35 DQ275229 sample The nucleotide sequences of most PCR amplicons BU79 DQ275272 sample BU31 DQ275225 sample clearly identifi ed LCMV as the most frequent cause of BU28 DQ275222 sample Captive colony C(BU)1 DQ275281 sample the antibody detected. However, the detection of arenavi- BU29 DQ275223 sample BU80 DQ275273 sample BU30 DQ275224 sample ral RNA in 2 animals by the N gene PCR, but not by the BU53 DQ275246 sample MC(BU)1 DQ275291 sample LCMV-specifi c GPC PCR, may suggest the presence of BU02 DQ275202 sample 64 BU87 DQ275280 sample another species of arenavirus. Further studies are needed to BU52 DQ275245 LCMV BU08 DQ275207 sample determine if other arenaviruses species are present in Euro- BU62 DQ275255 sample MF* MM9 DQ275295 sample pean rodent populations (8). BU56 DQ275249 sample BU70 DQ275263 sample BU72 DQ275265 sample Genetic heterogeneity was present within and between BU73 DQ275266 sample BU13 DQ275210 sample sites (Figure), as seen in previous studies of arenaviruses 86 C(BU)9 DQ275287 sample BU12 DQ275209 sample (13,14). Sequences from animals in the captive colony BU07 DQ275206 sample BU15 DQ275212 sample and a nearby farm (MF) clustered and were different from BU10 DQ275200 sample BU05 DQ275204 sample BU40 DQ275233 sample those from a more distant farm (BHF). Furthermore, all of BU04 DQ275203 sample C(BU)20 DQ275283 sample the British sequences clearly clustered separately from the BU78 DQ275271 sample BU41 DQ275234 sample reference strain sequences (from the United States, France, Captive colony BU58 DQ275251 sample BU46 DQ275239 sample Germany, or Slovakia). These fi ndings suggest spatial BU54 DQ275247 sample BU69 DQ275262 sample heterogeneity in sequence may be refl ected in host range BU57 DQ275250 sample BU11 DQ275208 sample BU55 DQ275248 sample and pathogenicity. Sequencing might be useful in tracing BU03 DQ275201 sample C(BU)8 DQ275286 sample sources of future human outbreaks. C(BU)21 DQ275284 sample BU26 DQ275220 sample BU27 DQ275221 sample BU60 DQ275253 sample Conclusions BU42 DQ275235 sample BU59 DQ275252 sample This study has increased the list of European (and MC(BU)24 DQ275292 sample BU43 DQ275236 sample BU20 DQ275214 sample North American) rodents that may be infected with LCMV BU61 DQ275254 sample HM13 DQ275288 sample BHF and that might therefore pose a risk to humans. The genetic 99 HM14 DQ275289 sample LCMV Armstrong M20869 variation observed and potential variations in pathogenicity 0.02 may indicate that some wildlife populations pose more of a public health risk than others. Further studies are needed to Figure. Unrooted neighbor-joining tree using the p-distance model (1,000 replicates) for a section of the glycoprotein precursor gene assess which mutations cause increased pathogenicity and gene, showing bootstrap values of >60 for all sequences identifi ed in to establish whether or not LCMV represents the only are- this study (283 bp) and indicating site of origin. Captive colony, MF navirus present in European rodent populations. 2004, and BHF 2005 as in Table 1. MF* is from Apodemus sylvaticus, and all other sequences are from Mus musculus. Scale bar indicates number of substitutions per site. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 9, September 2008 1457 DISPATCHES 6. Lledo L, Gegundez MI, Saz JV, Bahamontes N, Beltran M. Lym- Acknowledgments phocytic choriomeningitis virus infection in a province of Spain: We thank all those who contributed samples, allowed us ac- analysis of sera from the general population and wild rodents. J Med cess to their animals, and allowed us to trap on their land. Thanks Virol. 2003;70:273–5. DOI: 10.1002/jmv.10389 also to Alan Radford for his assistance with sequence analysis. 7. El Karamany RM, Imam IZ. Antibodies to lymphocytic choriomen- ingitis virus in wild rodent sera in Egypt. J Hyg Epidemiol Microbiol This research was funded by the European Union grant Immunol. 1991;35:97–103. QLK2-CT-2002-01358, a BBRSC grant to J.H. and studentship 8. Bowen MD, Peters CJ, Nichol ST. Phylogenetic analysis of the Are- naviridae: patterns of virus evolution and evidence for cospeciation to S.D.B. between arenaviruses and their rodent hosts. Mol Phylogenet Evol. 1997;8:301–16. DOI: 10.1006/mpev.1997.0436 Dr Blasdell is based at the Pasteur Institute, Phnom Penh, 9. Becker SD, Bennett M, Stewart JP, Hurst JL. Serological survey of Cambodia, where she is studying hantaviruses in Southeast Asia. virus infection among wild house mice (Mus domesticus) in the UK. Her research interests are in the ecology and evolution of natural Lab Anim. 2007;41:229–38. DOI: 10.1258/002367707780378203 host-virus systems. 10. Asper M, Hofmann P, Osmann C, Funk J, Metzger C, Bruns M, et al. First outbreak of callitrichid hepatitis in Germany: genetic character- ization of the causative lymphocytic choriomeningitis virus strains. References Virology. 2001;284:203–13. DOI: 10.1006/viro.2001.0909 11. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular evolu- 1. Charrel RN, Lemasson JJ, Garbutt M, Khelifa R, De Micco P, Feld- tionary genetics analysis (MEGA) software version 4.0. Mol Biol mann H, et al. New insights into the evolutionary relationships Evol. 2007;24:1596–9. DOI: 10.1093/molbev/msm092 between arenaviruses provided by comparative analysis of small 12. Greenwood AG, Sanchez S. Serological evidence of murine patho- and large segment sequences. Virology. 2003;317:191–6. DOI: gens in wild grey squirrels (Scuirus carolensis) in North Wales. Vet 10.1016/j.virol.2003.08.016 Rec. 2002;150:543–6. 2. Barton LL, Mets MB. Congenital lymphocytic choriomeningitis vi- 13. Garcia JB, Morzunov SP, Levis S, Rowe J, Calderon G, Enria D, rus infection: decade of rediscovery. Clin Infect Dis. 2001;33:370–4. et al. Genetic diversity of the Junin virus in Argentina: geographic DOI: 10.1086/321897 and temporal patterns. Virology. 2000;272:127–36. DOI: 10.1006/ 3. Bonthius DJ, Wright R, Tseng B, Barton L, Marco E, Karacay B, viro.2000.0345 et al. Congenital lymphocytic choriomeningitis virus infection: 14. Bowen MD, Rollin PE, Ksiazek TG, Hustad HL, Bausch DG, Dem- spectrum of disease. Ann Neurol. 2007;62:347–55. DOI: 10.1002/ by AH, et al. Genetic diversity among Lassa virus strains. J Virol. ana.21161 2000;74:6992–7004. DOI: 10.1128/JVI.74.15.6992-7004.2000 4. Davison KL, Crowcroft NS, Ramsay ME, Brown DW, Andrews NJ. Viral encephalitis in England, 1989–1998: what did we miss? Emerg Address for correspondence: Malcolm Bennett, National Centre for Infect Dis. 2003;9:234–40. Zoonosis Research, The University of Liverpool, Leahurst, Chester High 5. Salazar-Bravo J, Ruedas LA, Yates TL. Mammalian reservoirs of Rd, Neston, South Wirral CH64 7TE, UK; email: [email protected] arenaviruses. Curr Top Microbiol Immunol. 2002;262:25–63. Search past Issues 1458 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 9, September 2008 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Emerging Infectious Diseases Pubmed Central

Host Range and Genetic Diversity of Arenaviruses in Rodents, United Kingdom

Blasdell, Kim R.; Becker, Stuart D.; Hurst, Jane; Begon, Mike; Bennett, Malcolm
Emerging Infectious Diseases , Volume 14 (9) – Sep 1, 2008
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10.3201/eid1409.080209
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Abstract

All animal research was conducted under license, accord- Host Range and ing to UK regulations. Serum samples were separated by centrifugation Genetic Diversity (10,000 rpm, 10 min) and tested for LCMV antibody by using the manufacturer’s protocol for commercial indirect of Arenaviruses in fl uorescent antibody assay slides (Charles River Laborato- ries, Wilmington, MA, USA). A 1:40 dilution of anti-rat Rodents, United or anti-mouse immunoglobulin G fl uorescein isothiocya- nate (Sigma-Aldrich, Gillingham, UK) or a combination of Kingdom both were used as secondary antibody. Ninety-three serum Kim R. Blasdell, Stuart D. Becker, Jane Hurst, samples (from the original serum samples tested for anti- Mike Begon, and Malcolm Bennett body) that were either antibody positive or from sites with high seroprevalence were tested for arenavirus RNA by During a study to extend our knowledge of the host PCR. Another 379 blood samples from the captive colony range and genetic diversity of arenaviruses in Great Brit- of house mice, which had not been previously tested for ain, 66 of 1,147 rodent blood samples tested for antibody, antibody, were also tested. The PCR targeted a fragment of and 127 of 482 tested by PCR, were found positive. All se- the glycoprotein precursor gene (GPC) (10). A selection of quences most closely resembled those of previously identi- samples found negative by the GPC PCR were subsequently fi ed lymphocytic choriomeningitis virus. retested by PCR targeted at a fragment of the nucleoprotein (N) gene (8), by using primers to sequences common to the iruses in the family Arenaviridae are separated into 2 Old World arenaviruses. Total RNA was extracted by us- Vdistinct serocomplexes, the New World serocomplex ing QIAamp viral RNA mini-kit (QIAGEN, Crawley, UK), and the Old World serocomplex (1). Several arenavirus converted to cDNA, and amplifi ed by using a single-step species are known to cause human disease, including lym- kit (Superscript III one-step RT-PCR with Platinum Taq phocytic choriomeningitis virus (LCMV), which causes polymerase system; Invitrogen, Paisley, UK) in conjunc- infl uenza-like clinical signs, occasionally with neurologic + – tion with oligonucleotides arena1 and LCMV322 (10) or complications. Infection may be asymptomatic in up to one 1010C and either OW1696R or NW1696R (8). Products third of patients (2), and serious complications often occur were separated and visualized by agarose gel electropho- in intrauterine infection (3). Less severe cases of adult hu- resis, and amplicons were purifi ed with the QIAquick PCR man infection are likely underreported and often misdiag- purifi cation kit (QIAGEN). Bidirectional sequencing was nosed (4). performed off-site (MWG Biotech AG, Ebersberg, Ger- LCMV is found worldwide, probably because of its as- many). The 97-nt sequences generated here were deposited sociation with its natural Old World host, the house mouse, with GenBank (accession nos. DQ275199–DQ275295). Mus musculus (5). Although antibodies have also been de- The software package MEGA version 4.0 (11) was tected in other rodent species (6,7), arenaviruses are known used to construct an alignment of a 283-nt fragment of the to be serologically cross-reactive. Few isolates of LCMV GPC gene nucleotide sequences and predicted amino acid have been obtained from wild rodents so little is known sequences, and for phylogenetic analysis with the neighbor- about its genetic diversity. Recent studies on American are- joining method (p distance model), with bootstrap support naviruses found that diverse arenaviruses co-evolved with based on 1,000 pseudoreplicates. Other GenBank sequenc- their rodent hosts (8), a fi nding that suggests that a more es included for comparison are listed in Table 2. Pairwise thorough study of European rodents might also identify genetic distances were calculated by using the p distance novel arenaviruses. The purpose of this study was therefore model; percentage sequence identities were calculated by to extend our knowledge of LCMV and LCMV-like arena- subtracting the genetic distances from 1.0 and multiplying viruses in rodents in Great Britain. by 100. Overall, 66 of 1,147 serum samples and 7 of 9 rodent The Study species had antibodies to arenaviruses. Sciurus vulgaris In total 1,147 blood samples were collected from ro- had the highest prevalence, 26%, although only 15 squir- dents: 1,060 were live-trapped, wild animals from <20 sites rels were tested. M. musculus had the second highest preva- (Table 1), and 87 blood samples were collected from a cap- lence, 17.5%. Antibodies were also detected in Apodemus tive colony of wild house mice (9) and tested serologically. sylvaticus, Microtus agrestis, Micromys minutis, captive- housed Cynomys ludovicianus, and Rattus norvegicus. Author affi liation: University of Liverpool, Liverpool, UK Seroprevalence varied between species (1.4%–26%) and between sites (0%–50%) (Table 1). DOI: 10.3201/eid1409.080209 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 9, September 2008 1455 DISPATCHES Table 1. Rodent species, numbers tested, seroprevalence, and viral RNA prevalence to LCMV at each UK and Republic of Ireland site* Site code and year Geographic location Species tested for antibody Species tested for viral RNA PHF 2004 Cheshire MMu (0/4), RN (0/2); SP = 0.0 PHF 2005 Cheshire MMu (8/26); SP = 30.8 BHF 2004 Cheshire MMu (4/10); SP = 40.0 MMu (2/9), RN (0/2) – 2 sequences BHF 2005 Cheshire MMu (2/2), RN (0/2); SP = 50.0 BGF Cheshire MMu (0/7), RN (0/2); SP = 0.0 CLF 2004 Cheshire MMu (0/6), RN (0/2); SP = 0.0 CLF 2005 Cheshire MMu (0/12), RN (0/4); SP = 0.0 MF 2002 Cheshire AS (0/10), MG (0/9), MA (0/2), MMu (0/30); SP = 0.0 AS (1/10), MG (0/1), MA (0/1), MMu (2/4) – 3 sequences MF 2004 Cheshire AS (4/10), MG (0/1), MA (0/1), MMu (2/4); SP = 37.5 CZ 2002 Cheshire AS (0/4), MG (0/4), CL (4/61), MMi (0/22), RN (0/2); AS (1/1), CL (0/4), MMi (0/1), SP = 4.3 MMu (0/1) – 0 sequences CZ spring 2003 Cheshire AS (0/9), MG (0/3), MA (0/4), MMi (1/3), SP = 5.3 CZ autumn 2003 Cheshire AS (1/18), MG (0/19), MA (0/4), MMu (0/1), SP = 2.9 CZ 2004 Cheshire MMu (1/19), RN (1/12), SP= 3.2 DF Cheshire MMu (1/69), SP = 1.4 MW Cheshire MG (0/105), AS (0/45), SP = 0.0 RH Cheshire MG (0/19), AS (0/49), SP = 0.0 LVFS Cheshire RN (0/2, SC (0/4), SP = 0.0 FA Merseyside MA (0/2), AS (0/24), SP = 0.0 KF Northumberland MA (2/104), SP = 1.9 LI North Devon RN (0/40), SP = 0.0 IOW Isle of Wight SV (1/18), SP = 5.6 TF Thetford SV (1/21), SP = 4.8 CF Cumbria SC (0/10), SV, (0/4), SP = 0.0 NI Northern Ireland AS (1/149), SP = 0.7 CA Republic of Ireland MG (0/15), SP = 0.0 CC Republic of Ireland AS (0/7), SP = 0.0 TW Republic of Ireland AS (0/10), SP = 0.0 Other Various locations RN (1/6), SC (0/1), SV (2/26), SP = NA AS (0/1), MA (0/2), MMu (0/31), RN (0/5), SV (1/4) – 0 sequences Captive colony Captive colony, MMu (30/87), SP = 34.5 MMu (122/403) – 92 Cheshire sequences *LCMV, lymphocytic choriomeningitis virus; SP, site prevalence (%). Species key: MMu, Mus musculus (house mouse); RN, Rattus norvegicus (brown or Norway rat); AS, Apodemus sylvaticus (wood mouse); MG, Myodes glareolus (bank vole); MA, Microtus agrestis (field vole); MMi, Micromys minutis (harvest mouse); CL, Cynomys ludovicianus (black-tailed prairie dog); SV, Sciurus vulgaris (red squirrel); SC, Sciurus carolensis (gray squirrel); NA, not available. Species containing positive animals are in boldface italics, with number positive and number tested in parentheses. GPC PCR amplicons were obtained from 127 of 472 in the GPC PCR, but seropositive or from high prevalence tested samples, and sequences were determined for 97 sam- sites, were tested by N gene PCR, and 2 were weakly posi- ples (Table 1). All positive samples were from Mus muscu- tive: 1 S. vulgaris and 1 A. sylvaticus. In neither case, how- lus except 1 from A. sylvaticus. Twenty samples negative ever, could a sequence be obtained from the amplicon. Table 2. Percentage nucleotide identities between the study sample sequences (all and from the captive colony only) and previously isolated LCMV and Lassa virus sequences* Sequence All study sequences, % Captive colony sequences only, % All study sequences 93.6–100 NS Captive colony sequences only NS 97.4–100 LCMV CIPV76001 Pasteur (AF095783; France) 78.7–80.5 78.7–80.5 LCMV CIP97001 (AF079517; France) 79.4– 83.1 80.9–83.1 LCMV Marseille (DQ286931; France) 82.8– 83.9 82.8–83.5 LCMV CH5871 (AF325215; Germany) 81.6–83.1 81.6–83.1 LCMV CH5692 (AF325214; Germany) 81.3–82.8 81.3–82.8 LCMV MX (EU195888; Slovakia) 78.7–80.5 78.7–80.5 LCMV Armstrong (M20869; USA) 82.8– 85.8 83.9–85.8 LCMV WE (M22138) 82.0–84.3 82.0–84.3 Lassa LP (AF181853) 58.1–59.9 58.8–59.9 *LCMV, lymphocytic choriomeningitis virus; NS, not shown. 1456 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 9, September 2008 Arenaviruses in Rodents, UK Nucleotide and amino acid GPC sequence identities BU22 DQ275216 sample for all the samples in this study ranged from 93.6%–100% BU66 DQ275259 sample BU37 DQ275231 sample (Table 2), and 94.5%–100%, respectively (data not shown). BU71 DQ275264 sample BU84 DQ275277 sample When compared with other arenaviruses, the nucleotide C(BU)6 DQ275285 sample BU67 DQ275260 sample sequences exhibited 78.7%–85.8% identity with LCMV BU50 DQ275243 sample C(BU)2 DQ275282 sample BU33 DQ275227 sample reference sequences and only 58.1%–59.9% identity with BU63 DQ275256 sample M(BU)20+ DQ275290 sample Lassa virus (online Appendix Figure, available from www. BU32 DQ275226 sample BU75 DQ275268 sample cdc.gov/EID/content/14/9/1455-appF.htm). BU14 DQ275211 sample Captive colony BU64 DQ275257 sample Although antibodies to arenaviruses have been re- BU06 DQ275205 sample BU48 DQ275241 sample ported in a range of European rodent species, our study BU51 DQ275244 sample BU47 DQ275240 sample BU77 DQ275270 sample provided evidence of arenaviruses infecting red squirrels 64 BU85 DQ275278 sample BU25 DQ275219 sample (S. vulgaris) and European harvest mice (M. minutis). An- BU65 DQ275258 sample BU49 DQ275242 sample tibodies to arenaviruses have been reported in introduced BU82 DQ275275 sample BU83 DQ275276 sample S. carolensis in Great Britain (12) but were not detected in BU1 DQ275199 sample BU86 DQ275279 sample MF this study. We also reported antibodies to arenaviruses in MM16 DQ275293 sample BU16 DQ275213 sample BU68 DQ275261 sample black-tailed prairie dogs (Cynomys ludovicianus): those Captive colony BU74 DQ275267 sample BU36 DQ275230 sample tested in this study were part of a colony in a zoo, how- BU76 DQ275269 sample MM10 DQ275294 sample MF ever, and had contact with wild mice, some of which were BU44 DQ275237 sample BU23 DQ275217 sample seropositive. As found in previous studies, Mus musculus BU24 DQ275218 sample BU34 DQ275228 sample BU45 DQ275238 sample was more likely to be infected with LCMV than other ro- BU39 DQ275232 sample BU81 DQ275274 sample dent species. BU21 DQ275215 sample BU35 DQ275229 sample The nucleotide sequences of most PCR amplicons BU79 DQ275272 sample BU31 DQ275225 sample clearly identifi ed LCMV as the most frequent cause of BU28 DQ275222 sample Captive colony C(BU)1 DQ275281 sample the antibody detected. However, the detection of arenavi- BU29 DQ275223 sample BU80 DQ275273 sample BU30 DQ275224 sample ral RNA in 2 animals by the N gene PCR, but not by the BU53 DQ275246 sample MC(BU)1 DQ275291 sample LCMV-specifi c GPC PCR, may suggest the presence of BU02 DQ275202 sample 64 BU87 DQ275280 sample another species of arenavirus. Further studies are needed to BU52 DQ275245 LCMV BU08 DQ275207 sample determine if other arenaviruses species are present in Euro- BU62 DQ275255 sample MF* MM9 DQ275295 sample pean rodent populations (8). BU56 DQ275249 sample BU70 DQ275263 sample BU72 DQ275265 sample Genetic heterogeneity was present within and between BU73 DQ275266 sample BU13 DQ275210 sample sites (Figure), as seen in previous studies of arenaviruses 86 C(BU)9 DQ275287 sample BU12 DQ275209 sample (13,14). Sequences from animals in the captive colony BU07 DQ275206 sample BU15 DQ275212 sample and a nearby farm (MF) clustered and were different from BU10 DQ275200 sample BU05 DQ275204 sample BU40 DQ275233 sample those from a more distant farm (BHF). Furthermore, all of BU04 DQ275203 sample C(BU)20 DQ275283 sample the British sequences clearly clustered separately from the BU78 DQ275271 sample BU41 DQ275234 sample reference strain sequences (from the United States, France, Captive colony BU58 DQ275251 sample BU46 DQ275239 sample Germany, or Slovakia). These fi ndings suggest spatial BU54 DQ275247 sample BU69 DQ275262 sample heterogeneity in sequence may be refl ected in host range BU57 DQ275250 sample BU11 DQ275208 sample BU55 DQ275248 sample and pathogenicity. Sequencing might be useful in tracing BU03 DQ275201 sample C(BU)8 DQ275286 sample sources of future human outbreaks. C(BU)21 DQ275284 sample BU26 DQ275220 sample BU27 DQ275221 sample BU60 DQ275253 sample Conclusions BU42 DQ275235 sample BU59 DQ275252 sample This study has increased the list of European (and MC(BU)24 DQ275292 sample BU43 DQ275236 sample BU20 DQ275214 sample North American) rodents that may be infected with LCMV BU61 DQ275254 sample HM13 DQ275288 sample BHF and that might therefore pose a risk to humans. The genetic 99 HM14 DQ275289 sample LCMV Armstrong M20869 variation observed and potential variations in pathogenicity 0.02 may indicate that some wildlife populations pose more of a public health risk than others. Further studies are needed to Figure. Unrooted neighbor-joining tree using the p-distance model (1,000 replicates) for a section of the glycoprotein precursor gene assess which mutations cause increased pathogenicity and gene, showing bootstrap values of >60 for all sequences identifi ed in to establish whether or not LCMV represents the only are- this study (283 bp) and indicating site of origin. Captive colony, MF navirus present in European rodent populations. 2004, and BHF 2005 as in Table 1. MF* is from Apodemus sylvaticus, and all other sequences are from Mus musculus. Scale bar indicates number of substitutions per site. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 9, September 2008 1457 DISPATCHES 6. Lledo L, Gegundez MI, Saz JV, Bahamontes N, Beltran M. Lym- Acknowledgments phocytic choriomeningitis virus infection in a province of Spain: We thank all those who contributed samples, allowed us ac- analysis of sera from the general population and wild rodents. J Med cess to their animals, and allowed us to trap on their land. Thanks Virol. 2003;70:273–5. DOI: 10.1002/jmv.10389 also to Alan Radford for his assistance with sequence analysis. 7. El Karamany RM, Imam IZ. Antibodies to lymphocytic choriomen- ingitis virus in wild rodent sera in Egypt. J Hyg Epidemiol Microbiol This research was funded by the European Union grant Immunol. 1991;35:97–103. QLK2-CT-2002-01358, a BBRSC grant to J.H. and studentship 8. Bowen MD, Peters CJ, Nichol ST. Phylogenetic analysis of the Are- naviridae: patterns of virus evolution and evidence for cospeciation to S.D.B. between arenaviruses and their rodent hosts. Mol Phylogenet Evol. 1997;8:301–16. DOI: 10.1006/mpev.1997.0436 Dr Blasdell is based at the Pasteur Institute, Phnom Penh, 9. Becker SD, Bennett M, Stewart JP, Hurst JL. Serological survey of Cambodia, where she is studying hantaviruses in Southeast Asia. virus infection among wild house mice (Mus domesticus) in the UK. Her research interests are in the ecology and evolution of natural Lab Anim. 2007;41:229–38. DOI: 10.1258/002367707780378203 host-virus systems. 10. Asper M, Hofmann P, Osmann C, Funk J, Metzger C, Bruns M, et al. First outbreak of callitrichid hepatitis in Germany: genetic character- ization of the causative lymphocytic choriomeningitis virus strains. References Virology. 2001;284:203–13. DOI: 10.1006/viro.2001.0909 11. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular evolu- 1. Charrel RN, Lemasson JJ, Garbutt M, Khelifa R, De Micco P, Feld- tionary genetics analysis (MEGA) software version 4.0. Mol Biol mann H, et al. New insights into the evolutionary relationships Evol. 2007;24:1596–9. DOI: 10.1093/molbev/msm092 between arenaviruses provided by comparative analysis of small 12. Greenwood AG, Sanchez S. Serological evidence of murine patho- and large segment sequences. Virology. 2003;317:191–6. DOI: gens in wild grey squirrels (Scuirus carolensis) in North Wales. Vet 10.1016/j.virol.2003.08.016 Rec. 2002;150:543–6. 2. Barton LL, Mets MB. Congenital lymphocytic choriomeningitis vi- 13. Garcia JB, Morzunov SP, Levis S, Rowe J, Calderon G, Enria D, rus infection: decade of rediscovery. Clin Infect Dis. 2001;33:370–4. et al. Genetic diversity of the Junin virus in Argentina: geographic DOI: 10.1086/321897 and temporal patterns. Virology. 2000;272:127–36. DOI: 10.1006/ 3. Bonthius DJ, Wright R, Tseng B, Barton L, Marco E, Karacay B, viro.2000.0345 et al. Congenital lymphocytic choriomeningitis virus infection: 14. Bowen MD, Rollin PE, Ksiazek TG, Hustad HL, Bausch DG, Dem- spectrum of disease. Ann Neurol. 2007;62:347–55. DOI: 10.1002/ by AH, et al. Genetic diversity among Lassa virus strains. J Virol. ana.21161 2000;74:6992–7004. DOI: 10.1128/JVI.74.15.6992-7004.2000 4. Davison KL, Crowcroft NS, Ramsay ME, Brown DW, Andrews NJ. Viral encephalitis in England, 1989–1998: what did we miss? Emerg Address for correspondence: Malcolm Bennett, National Centre for Infect Dis. 2003;9:234–40. Zoonosis Research, The University of Liverpool, Leahurst, Chester High 5. Salazar-Bravo J, Ruedas LA, Yates TL. Mammalian reservoirs of Rd, Neston, South Wirral CH64 7TE, UK; email: [email protected] arenaviruses. Curr Top Microbiol Immunol. 2002;262:25–63. Search past Issues 1458 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 9, September 2008

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