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Mopeia virus-related arenavirus in Natal multimammate mice, Morogoro, Tanzania.

A renaviruses are segmented negative-strand RNA viruses. Their natural hosts are various rodent species. The virus family comprises several human pathogens causing hemorrhagic fever, namely Machupo, Guanarito, Junin, Sabia, and Chapare viruses in South America, and Lassa and Lujo viruses in Africa (1-3). In addition, Africa harbors arenaviruses that are not linked with human disease: Mobala, Ippy, Mopeia, and Kodoko viruses (4-7). We conducted a systematic search in wildlife in Tanzania to identify new African arenaviruses.

The Study

During 1985 through 1989, a total of 2,520 small mammals were live-trapped in different regions of Tanzania. After species determination, they were measured and bled by orbital puncture. Serum samples were tested by indirect immunofluorescent antibody (IFA) assay (8). Lassa virus was used as antigen due to its cross-reactivity with immune sera from animals infected with other arenaviruses (4,6). Clusters of seropositivity were found in Arvicanthis spp. rodents from the Iringa region (20%) and in Natal multimammate mice (Mastomys natalensis) from Arusha (18%) and Morogoro (17%) (Table 1), which suggests that these animals are reservoirs of arenaviruses. Titers ranged from 16 to 512 and 16 to 4,096 in Arvicanthis spp. rodents and M. natalensis mice, respectively. Peak prevalence in M. natalensis mice was found on the campus of the Sokoine University in Morogoro (23.7% of 746 animals collected over several seasons).

In 2004, M. natalensis mice were trapped in a mosaic of maize fields and fallow grassland at the university campus in the city of Morogoro (6[degrees]50'34.9794''S; 37[degrees]38'8.232"E) to identify the virus. The animal voucher specimens were deposited at the Royal Museum of Central Africa, Tervuren, Belgium. RNA was prepared from 10 [micro]L of rodent serum by using the QIAamp Viral RNA kit (QIAGEN, Valencia, CA, USA), and screening was performed by using a pan-Old World arenavirus reverse transcription-PCR (RT-PCR) specific for the large (L) gene (9). One of 96 serum samples was positive (no. 3017/2004) (Table 2), and sequencing of the PCR fragment showed a new arenavirus sequence. The virus was isolated in Vero cells and called Morogoro virus (strain 3017/2004).

For sequencing, the isolate was propagated in T75 flasks, virus particles in supernatant were pelleted by ultracentrifugation, and RNA was isolated by using the QIAamp Viral RNA kit (QIAGEN). The entire 3.5-kb small (S) RNA segment was amplified by RT-PCR as described previously (10). The 7-kb L RNA segment was amplified in 2 fragments by using a long-range RT-PCR protocol and primers targeting the conserved termini of L RNA and Morogoro virus--specific primers designed on the basis of the sequence of the fragment detected by RT-PCR screening. By using the PCR products as a template, short overlapping fragments were amplified and sequenced with a set of consensus primers for Old World arenaviruses, and S and L RNA sequences were assembled (GenBank accession nos. EU914103 and EU914104). (Sequences reported in this article have been submitted to GenBank and assigned the following accession numbers: full-length S and L RNA sequences of Morogoro virus, EU914103-04; partial L gene sequences of Morogoro virus, EU914107-22; cytochrome B gene of Morogoro viruspositive Mastomys natalensis, EU914105-06.)

Full-length amino acid sequences of glycoprotein precursor (GPC), nucleoprotein (NP), and L protein of Morogoro virus were aligned with published Old World arenavirus sequences and pairwise p distances were calculated. Morogoro virus showed genetic similarity to strains of Mopeia virus that were circulating in Mozambique (4) and Zimbabwe (5). A close relationship between both viruses was also demonstrated by phylogenetic analysis using GPC, NP, and L gene sequences (Figure 1, panel D, and data not shown). Both viruses are sister taxa, sharing a common ancestor with Mobala virus.

Although the distances between Morogoro and Mopeia virus in the amino acid sequence of GPC (12%), NP (12%-13%), and L gene (26%) were higher than intraspecies differences among known African arenaviruses (i.e., pairwise differences between strains of the same species; <11% in GPC and NP; <21% in L), they did not reach the level of interspecies distances (>20% in GPC and NP; >37% in L) (Figure 1, panels A-C). Therefore, we currently consider Morogoro virus a subspecies of Mopeia virus rather than a new arenavirus species. This classification is supported by the fact that both viruses share the same host. Sequencing of the mitochondrial cytochrome b gene of rodent liver samples positive for Morogoro virus confirmed that its natural host is M. natalensis mice (GenBank accession nos. EU914105 and EU914106).

An additional 303 ethanol-preserved liver samples and 63 serum samples were collected in 2004 and 2007, respectively. Liver tissue ([approximately equal to] 3 mg) was homogenized by using a bead mill. Cell debris was pelleted by centrifugation, and RNA was isolated from the homogenate with the RNeasy Mini kit (QIAGEN). Testing by L gene RT-PCR (9) showed 16 positive liver and serum samples, which indicated a virus prevalence in the M. natalensis population of [approximately equal to] 4% (Table 2). PCR fragments were sequenced (GenBank accession nos. EU914107-EU914122), and Morogoro virus was isolated in cell culture from all 4 PCR-positive serum samples obtained in 2007. Morogoro virus-specific antibodies in serum samples from 2004 and 2007 were measured by IFA assay using Vero cells infected with Morogoro virus. The antibody prevalence was [approximately equal to] 50%, which compares quite well with the 23% prevalence determined in this area 20 years before. In some animals, virus and antibodies were detected (Table 2).

The availability of Morogoro virus L gene sequences from 2004 and 2007, originating from the same host population (trapping sites <1 km apart), provided us with the opportunity to estimate the molecular clock rate for this virus. Phylogenetic reconstruction was performed with the BEAST version 1.4.8 package (http://beast.bio.ed.ac.uk) (11) under the assumption of a relaxed lognormal molecular clock and general time reversible (GTR) or HasegawaKishino-Yano (HKY) substitution model with gamma-distributed substitution rate variation among sites (Figure 2 and data not shown). Analysis was run for 2 million Markov chain Monte Carlo steps, which yielded a reliable set of data as verified with the TRACER program (http://tree.bio. ed.ac.uk/software/tracer). Based on GTR and HYK model, 3.2 x [10.sup.-3] and 3.4 x [10.sup.-3] substitutions per site and year (95% interval of highest posterior density 1.1-6.6 x [10.sup.-3]), respectively, were calculated.

Conclusions

A serologic survey in small mammals from Tanzania identified a hot spot of arenavirus circulation in Morogoro in the late 1980s. This work is being published now because early attempts to substantiate the existence of the virus failed. The identification of the virus was facilitated by a recently developed pan-Old World arenavirus PCR (9) that also led to the discovery of new arenaviruses in rodents from West Africa (7). Only a small percentage of M. natalensis mice carry Morogoro virus, and a large proportion shows specific antibodies, which indicates that most animals clear the virus during life. Viruses and antibodies, which are presumbably directed to nucleocapsid proteins, also co-circulate, as seen in hantavirus infection in rodents (12). Detection of Morogoro virus in the liver is consistent with the organ tropism of Lassa virus in M. natalensis mice (13). In agreement with studies on Lassa virus strains, the largest genetic distance between Morogoro and Mopeia virus was seen in L gene, which contains several highly variable regions (14).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

The clock rate estimate of 3 x 10-3 for Morogoro virus L gene is in agreement with that of other RNA viruses (15), although it must be interpreted with caution, given that the difference in date between the samples is not large. The tree topology did not correlate with geographic or ecologic sampling data.

The pathogenicity of Morogoro virus for humans is not known, though its phylogenetic clustering with African arenaviruses that are not linked with human disease (4-6) and the absence of hemorrhagic fever in the area suggest that it does not cause severe disease. Hospital-based investigations are required to estimate the public health relevance of this virus.

Acknowledgments

We thank the Tanzanian authorities and the academic authorities of the Sokoine University of Agriculture, Morogoro, who provided us with the necessary permits and working facilities; and the field staff as well as Mike Michiels, Jan Stuyck, and Bukheti S. Kilonzo for their help in collecting animals.

The early work was supervised and stimulated by the late Walter Verheyen, within the framework of the Tanzanian-Belgium Joint Rodent Research Project (supported by the Belgian General Administration for Developmental Cooperation); the recent work was undertaken under the SUA-VLIR Interuniversity Cooperation program of the Flemish Interuniversity Council and the VIZIER integrated project grant LSHG-CT-2004-511960 of the European Union 6th Framework. The Bernhard-Nocht-Institute is supported by the Bundesministerium fur Gesundheit and the Freie und Hansestadt Hamburg.

References

(1.) Gunther S, Lenz O. Lassa virus. Crit Rev Clin Lab Sci. 2004;41:339-90. DOI: 10.1080/10408360490497456

(2.) Delgado S, Erickson BR, Agudo R, Blair PJ, Vallejo E, Albarino CG, et al. Chapare virus, a newly discovered arenavirus isolated from a fatal hemorrhagic fever case in Bolivia. PLoS Pathog. 2008;4:e1000047. DOI: 10.1371/journal.ppat.1000047

(3.) Briese T, Paweska JT, McMullan LK, Hutchison SK, Street C, Palacios G, et al. Genetic detection and characterization of Lujo virus, a new hemorrhagic fever-associated arenavirus from southern Africa. PLoS Pathog. 2009;5:e1000455. DOI: 10.1371/journal. ppat.1000455

(4.) Wulff H, McIntosh BM, Hamner DB, Johnson KM. Isolation of an arenavirus closely related to Lassa virus from Mastomys natalensis in south-east Africa. Bull World Health Organ. 1977;55:441-4.

(5.) Johnson KM, Taylor P, Elliott LH, Tomori O. Recovery of a Lassa-related arenavirus in Zimbabwe. Am J Trop Med Hyg. 1981;30:1291-3.

(6.) Gonzalez JP, McCormick JB, Saluzzo JF, Herve JP, Georges AJ, Johnson KM. An arenavirus isolated from wild-caught rodents (Pramys species) in the Central African Republic. Intervirology. 1983;19:105-12. DOI: 10.1159/000149344

(7.) Lecompte E, ter Meulen J, Emonet S, Daffis S, Charrel RN. Genetic identification of Kodoko virus, a novel arenavirus of the African pigmy mouse (Mus Nannomys minutoides) in West Africa. Virology. 2007;364:178-83. DOI: 10.1016/j.virol.2007.02.008

(8.) van der Groen G, Kurata T, Mets C. Modifications to indirect immunofl uorescence tests on Lassa, Marburg, and Ebola material. Lancet. 1983;1(8325):654. DOI: 10.1016/S0140-6736(83)91831-7

(9.) Vieth S, Drosten C, Lenz O, Vincent M, Omilabu S, Hass M, et al. RT-PCR assay for detection of Lassa virus and related Old World arenaviruses targeting the L gene. Trans R Soc Trop Med Hyg. 2007;101:1253-64. DOI: 10.1016/j.trstmh.2005.03.018

(10.) Gunther S, Emmerich P, Laue T, Kuhle O, Asper M, Jung A, et al. Imported Lassa fever in Germany: molecular characterization of a new Lassa virus strain. Emerg Infect Dis. 2000;6:466-76. DOI: 10.3201/eid0605.000504

(11.) Drummond AJ, Ho SY, Phillips MJ, Rambaut A. Relaxed phylogenetics and dating with confidence. PLoS Biol. 2006;4:e88. DOI: 10.1371/journal.pbio.0040088

(12.) Essbauer SS, Schmidt-Chanasit J, Madeja EL, Wegener W, Friedrich R, Petraityte R, et al. Nephropathia epidemica in metropolitan area, Germany. Emerg Infect Dis. 2007;13:1271-3.

(13.) Walker DH, Wulff H, Lange JV, Murphy FA. Comparative pathology of Lassa virus infection in monkeys, guinea-pigs, and Mastomys natalensis. Bull World Health Organ. 1975;52:523-34.

(14.) Vieth S, Torda AE, Asper M, Schmitz H, Gunther S. Sequence analysis of L RNA of Lassa virus. Virology. 2004;318:153-68. DOI: 10.1016/j.virol.2003.09.009

(15.) Jenkins GM, Rambaut A, Pybus OG, Holmes EC. Rates of molecular evolution in RNA viruses: a quantitative phylogenetic analysis. J Mol Evol. 2002;54:156-65. DOI: 10.1007/s00239-001-0064-3

Author affiliations: Bernhard-Nocht-Institute for Tropical Medicine, Hamburg, Germany (S. Gunther, B. Becker-Ziaja); Institute of Tropical Medicine Leopold II, Antwerp, Belgium (G. Hoofd, G. van der Groen); Universite de la Mediterranee, Marseille, France (R. Charrel); Artus Company, Hamburg (C. Roser); Centre for Emergency Preparedness and Response, Salisbury, UK (G. Lloyd); Sokoine University of Agriculture, Morogoro, Tanzania (C. Sabuni, A. Katakweba, R. Machang'u, R. Makundi) University of Antwerp Department of Biology, Antwerp (R. Verhagen, J. Kennis, H. Leirs); and University of Aarhus Department of Integrated Pest Management, Kongens Lyngby, Denmark (H. Leirs)

DOI: 10.3201/eid1512.090864

Address for correspondence: Stephan Gunther, Bernhard-Nocht-Institute for Tropical Medicine Department of Virology, Bernhard-Nocht-Str 74, 20359 Hamburg, Germany; email: guenther@bni.uni-hamburg.de

Dr Gunther is head of the Virology Department at the BernhardNocht-Institute for Tropical Medicine, Hamburg, Germany. His research interests are molecular biology and epidemiology of arenaviruses, in particular, Lassa virus.
Table 1. Detection of African arenavirus-specific antibodies in small
mammals in Tanzania, 1985-1989 *

                       Antibody detection ([dagger]) by region
                            (no. positive/no. tested)

Genus               Arusha       Iringa       Lindi        Mbeya

Acomys                --          0/3          0/2          0/2
Aethomys              --          0/3          0/4           --
Arvicanthis          0/13        6/30#          --           --
Cricetomys            --           --           --           --
Lemniscomys          0/5          1/2#         1/2#          --
Lophuromys           0/3          0/1           --           --
Mastomys            7/39#         0/17        1/120#        0/12

Mus                   --          0/1           -           0/1
Praomys               --          0/3           -           0/1
Rattus                --           -           0/24         0/1
Tatera               0/1          0/1          0/32          --
Uranomys              --           --           --           --
Sciuridae             --           --          0/13          --
Crocidura             --           --           --           --
Petrodomus            --           --          0/9           --
13 other genera       --          0/1          0/2          0/7
Total               7/61#        7/62#        2/208#        0/24

                     Antibody detection ([dagger]) by region
                          (no. positive/no. tested)

Genus                  Morogoro          Mtwara       Ruvuma

Acomys                   0/57             0/2           --
Aethomys                 0/23             0/11         0/7
Arvicanthis               --               --           --
Cricetomys               0/35              --           --
Lemniscomys             1/30#             0/2          0/1
Lophuromys               0/3               --           --
Mastomys              181/1,054           0/81         0/8
                  ([double dagger])#
Mus                     1/47#              --           --
Praomys                  0/1               --           --
Rattus                   0/49             0/20         0/3
Tatera                  0/127             0/69         0/11
Uranomys                 0/11              --           --
Sciuridae                0/2               --          0/2
Crocidura               1/14#              --           --
Petrodomus                --              0/18          --
13 other genera          0/21             0/20          --
Total                 184/1,474#         0/223         0/32

                   Antibody detection ([dagger]) by
                   region (no. positive/no. tested)

Genus               Songea       Tanga        Total

Acomys                --           --          0/66
Aethomys             0/8           -           0/56
Arvicanthis           --          0/87        6/130#
Cricetomys            --           --          0/35
Lemniscomys           --           --         3/42#
Lophuromys            --          0/7          0/14
Mastomys             0/25         0/82      189/1,438#

Mus                   --           --         1/49#
Praomys               --          0/1          0/6
Rattus               0/15        0/196        0/308
Tatera               0/3           --         0/244
Uranomys              --           --          0/11
Sciuridae             --          0/10         0/27
Crocidura             --           --         1/14#
Petrodomus            --           --          0/27
13 other genera       --          0/2          0/53
Total                0/51        0/385      200/2,520#

* Positive samples as well as the respective sampling sites and
animals are indicated in boldface.

([dagger]) Immunofluorescent antibody (IFA) assay was performed with
Lassa virus-infected cells (cut-off titer 16).

([double dagger]) Fifty IFA assay-positive serum samples were randomly
selected and tested by immunoblotting. Presence of African
arenavirus-specific antibodies, as defined by reactivity with Lassa
virus nucleoprotein and glycoprotein 2, was confirmed in 47 serum
specimens.

Note: Positive samples as well as the respective sampling sites and
animals indicated with #.

Table 2. Prevalence of Morogoro virus and Morogoro virus-specific
antibodies in Mastomys natalensis mice from Morogoro University
campus, Tanzania

Specimen and
year of sampling   No. samples   No. (%) virus positive (PCR)

Serum 2004             96              1 (1) ([dagger])
Liver 2004             303             12 (4) ([dagger])
Serum 2007             63           4 (6) ([double dagger])

Specimen and       No. (%) antibody   No. (%) antibody plus
year of sampling      positive *         virus positive

Serum 2004             42 (44)                  0
Liver 2004                --                   --
Serum 2007             40 (63)          3 (5) ([section])

* By immunofluorescent antibody (IFA) assay, performed with Morogoro
virus-infected cells (cut-off 32).

([dagger]) Testing was performed with universal Old World arenavirus
large (L) gene reverse transcription-PCR (9).

([double dagger]) Testing was performed with Morogoro virus-specific L
gene RT-PCR using primers MoroL3359-forward
(5'-GGATTAGTGAGAGAGAGAGTAATTC-3) and MoroL3753-reverse
(5'-ACATCATTGGGCCCCACTTACTATGGTC-3).

([section]) Titers ranged from 64 to 512.
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Article Details
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Title Annotation:DISPATCHES
Author:Gunther, Stephan; Hoofd, Guy; Charrel, Remi; Roser, Christina; Becker-Ziaja, Beate; Lloyd, Graham; S
Publication:Emerging Infectious Diseases
Article Type:Report
Geographic Code:6TANZ
Date:Dec 1, 2009
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