Detection of the first incidence of Akodon paranaensis naturally infected with the Jabora virus strain (Hantavirus) in Brazil

SHORT COMMUNICATIONS

Detection of the first incidence of Akodon paranaensis naturally infected with the Jabora virus strain (Hantavirus) in Brazil

Renata Carvalho de OliveiraI, ⁺; Alexandro Guterres^I; Carlos Guerra Schrago^II; Jorlan Fernandes^I; Bernardo Rodrigues Teixeira^III; Suzana Zeccer^IV; Cibele R Bonvicino^{III, V}; Paulo Sérgio D'Andrea^III; Elba Regina Sampaio de Lemos^I

^ILaboratório de Hantaviroses e Rickettsioses, Instituto Oswaldo Cruz-Fiocruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brasil

^IIDepartamento de Genética, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil

^IIILaboratório de Biologia e Parasitologia de Mamíferos Silvestres Reservatórios, Instituto Oswaldo Cruz-Fiocruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brasil

^IVSecretaria Estadual de Saúde de Santa Catarina, Florianópolis, SC, Brasil ⁵Instituto Nacional do Câncer, Rio de Janeiro, RJ, Brasil

ABSTRACT

We characterised hantaviruses circulating in different Akodon rodent species collected in midwestern Santa Catarina (SC), southern Brazil, where the Jabora hantavirus (JABV) strain was first identified in Akodon montensis. Genetic and phylogenetic analyses based on a partial S segment indicated that, in SC, Akodon paranaensis and A. montensis carried the same type of hantavirus. Additionally, we conducted the first genomic characterisation of the complete S segment from the Brazilian JABV strain. This is the first report of A. paranaensis infected with the JABV.

Key words: phylogenetic analyses - rodents - hantavirus - Santa Catarina

Hantavirus pulmonary syndrome (HPS) is caused by the emerging rodent-borne viruses of the genus Hantavirus (Nichol et al. 1993). Since 1993, over 1,400 cases have been identified in 14 states in Brazil. Santa Catarina (SC) (southern Brazil, 27º10'S 51º44'W) is the second most affected Brazilian state, with a large number of cases (n = 226) (Brazilian Health Ministry Report on Hantavirus cases 1993-2011, unpublished data). Currently, there are five hantaviruses associated with HPS in Brazil: Juquitiba/Araucaria, Araraquara, Laguna Negra, Anajatuba and Castelo dos Sonhos; these are carried by Oligoryzomys nigripes, Necromys lasiurus, Calomys sp., Oligoryzomys fornesi and Oligoryzomys utiaritensis, respectively (Johnson et al. 1999, Suzuki et al. 2004, Raboni et al. 2005a, 2009a, Rosa et al. 2010, Travassos da Rosa et al. 2011). Two other hantaviruses, Rio Mearim and Jabora (JABV) have been identified in the rodent species Holochilus sciureus and Akodon montensis, respectively, but their roles in human disease have not been determined (Rosa et al. 2005, Oliveira et al. 2011).

Hantaviruses can co-circulate in the same locality and can be maintained side-by-side in different rodent species, as reported for Juquitiba and JABV related viruses and other Old World hantaviruses (Artois et al. 2007, Chu et al. 2009, Raboni et al. 2009b, Razzauti et al. 2009, Oliveira et al. 2011). In this study, we have identified JABV, which is associated with A. montensis and Akodon paranaensis, two related and sympatric rodent species in Midwestern SC (Jabora, 27º09'S 51º47'W). These two species of Akodon are widely distributed in the central Southern Cone of South America and may be locally abundant in their preferred habitat (Musser & Carleton 2005). Additionally, we conducted the first genomic characterisation of the complete S segment from the JABV strain to better characterise the hantaviruses circulating in these two related rodent species.

RNA was extracted from lung tissue samples of five antibody-positive rodents from A. paranaensis (Akp8032, Akp8048) and A. montensis (Akm6266, Akm6943 and Akm9635) species, according to the manufacturer's instructions from the Trizol^® Plus RNA Purification Kit. The rodent samples were analysed using the polymerase chain reaction with reverse transcription and nested reactions. For direct sequencing of overlapping amplimers, generic primer combinations (n = 8) were used for amplification and sequencing of the complete genomic S segment, including published (Raboni et al. 2005b, Oliveira et al. 2011) and unpublished (S Levis 2005, unpublished observations, A Guterres 2011, unpublished observations) oligonucleotide sequences. These sequences were designed based on the conserved regions of the S segment among South American hantaviruses (primers available on request). Amplicons of the expected size (approximately 1,800 bp) were recovered from two samples: one from A. montensis (Akm9635) and another from A. paranaensis (Akp8048). Sequence alignments were run in SeaView, using the MUSCLE algorithm. Obtained virus sequences (partial and complete) are accessible from GenBank (JN232078_Akm9635, JN232080_Akp8048 and JN232081_Akp8032). Phylogenetic relationships were estimated using (i) a maximum likelihood (ML) phylogenetic inference method with 1,000 bootstrap replicates, implemented in PhyML 3 (Guindon & Gascuel 2003) under the GTR+G model of sequence evolution, which was chosen after hierarchically testing alternative models by computing likelihood ratios and (ii) a Bayesian Markov chain Monte Carlo method implemented in MrBayes v3.1.2 (Ronquist & Huelsenbeck 2003), using the GTR+G model of nucleotide substitution. Two simultaneous runs of four chains each were run for 1 million generations and sample frequency = every 100th generation; a consensus tree (burn-in of 25%) was constructed from the remaining trees. Posterior probabilities above 0.95 and bootstrap values above 70% at the nodes were accepted as significant. We also assessed the phylogenetic relationships between JABV and other hantaviruses with a partial section of the S segment sequence (700 nucleotides due to the large number of partial sequences available).

Phylogenetic trees calculated by ML (not shown) and Bayesian methods, based on partial and complete sequences, indicated similar topology at the relevant nodes (Fig. 1). Phylogenetic analyses of these sequences (partial and complete) indicated that all hantaviruses carried by A. montensis and A. paranaensis form a distinct and monophyletic lineage. ML and Bayesian analyses based on partial S segment indicated that the sequences circulating in A. paranaensis are closely related to A. montensis viruses from Brazil, Argentine and Paraguay (JABV/AC210py/AAIV). Although A. montensis sequences are not monophyletic, A. paranaensis (Akp8032 and Akp8048) sequences are grouped with significant support and are tightly associated with the A. montensis virus strain (Fig. 1). The JABV-like virus clade could be divided into two well-supported subclades: one composed of Paraguayan and Argentinean viruses and the other composed of Brazilian viruses (Akp and Akm samples). The ML tree that was constructed using the amino acid sequences of the S segment indicated highly similar bootstrap values and a branching pattern obtained from the nucleotide sequence phylogenetic analysis (not shown).

In pairwise comparisons of a nucleotide sequence, calculated using MEGA 5 (Tamura et al. 2011), the genetic distance among JABV strains from Brazilian A. montensis and A. paranaensis ranged from 0.1-3.4% and amino acid derived differences ranged from 0-1.3%. This suggests that spillover infection of JABV-related viruses is actively occurring among Akondontini rodent species in southern Brazil. Furthermore, the nucleotide differences between JABV strains from Brazil and JABV/AAIV-related viruses from Paraguay and Argentina (Ac210py) ranged from 11.5-14.7% (Table).

All five Akodon specimens were karyotyped to confirm morphologic identification. Phylogenetic reconstructions of three rodent specimens (Akm6943, Akp8048, Akp8032), based on the mitochondrial DNA cytochrome b gene (Smith & Patton 1993), were also obtained. These reconstructions were used to confirm species identification and to estimate phylogenetic relationship of the hantavirus-positive specimens, using the same phylogeny model of evolution and parameters described above for hantaviruses. All rodent specimens collected were fixed in 10% formalin or prepared as skin and skull and placed as voucher specimens in the mammal collection of the National Museum of the Federal University of Rio de Janeiro.

Karyologic analyses confirmed that three Akodon specimens belong to A. montensis (2n = 24, FNa = 42) and the other two specimens to A. paranaensis (2n = 44, FNa = 44). The Bayesian and ML (not shown) trees indicated similar topologies for Akodontini (Fig. 2). These analyses grouped the haplotypes of Akodon sequenced specimens (Akm6943, Akp8048, Akp8032) with the GenBank sequence of A. montensis and A. paranaensis.

Genetic and phylogenetic analyses based on S partial and complete segments indicated that, in SC, A. paranaensis and A. montensis carried the same type of hantavirus. According to some ecological mathematics models, the presence of multiple hosts increases the possibility of disease emergence (McCormack & Allen 2007). In this study, JABV was identified for the first time in A. paranaensis. The phylogenies obtained from S segments indicate that the A. paranaensis strain is monophyletic and related to the virus circulating in the sympatric A. montensis. Spillover of JAB-like virus from its real host to other sympatric rodent species cannot be excluded and, therefore, further investigation of this issue is needed. Studies utilising phylogenetic methods to generate and compare evolutionary scenarios of hantaviruses and their rodent hosts are critical to better understanding the evolution of hantaviruses, especially in South America. Additionally, a longitudinal study and new rodent collection expeditions in different areas are needed to elucidate whether A. paranaensis rodents are true reservoirs or only sporadic hosts.

ACKNOWLEDGEMENTS

To Antônio Caldas, Epidemiologic Surveillance and Manager of the Zoonotic Control of the Santa Catarina State Health Department, for their dedication during the development of the studies in Jabora, and to Dalir Alberto Ruaro, secretary of Jabora Health Department, for their logistic and continued support.

Received 8 July 2011

Accepted 24 November 2011

Financial support: CNPq/FIOCRUZ (403050/2004-9)

• Artois M, Cochez C, Van Mele R, Heyman P 2007. Genetic evidence of Puumala and Tula hantaviruses in rodents in the Jura region, France-preliminary results. Euro Surveill 28: E070628.3
• Chu YK, Goodin D, Owen RD, Koch D, Jonsson CB 2009. Sympatry of 2 hantavirus strains, Paraguay, 2003-2007. Emerg Infect Dis 15: 1977-1980.
• Guindon S, Gascuel O 2003. A simple, fast and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52: 696-704.
• Johnson AM, Souza LTM, Ferreira IB, Pereira LE, Ksiazek TG, Rollin PE, Peters CJ, Nichol ST 1999. Genetic investigation of novel hantaviruses causing fatal HPS in Brazil. J Med Virol 59: 527-535.
• Kimura MA 1980. Simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16: 111-120.
• McCormack RK, Allen LJ 2007. Disease emergence in multi-host epidemic models. Math Med Biol 24: 17-34.
• Musser GG, Carleton MD 2005. Family Cricetidae. In DE Wilson, DM Reeder, Mammal species of the world. A taxonomic and geographic reference, The Johns Hopkins University Press, Baltimore, p. 955-1188.
• Nichol ST, Spiropoulou CF, Morzunov S, Rollin PE, Ksiazek TG, Feldmann H, Sanchez A, Childs J, Zaki S, Peters CJ 1993. Genetic identification of a hantavirus associated with an outbreak of acute respiratory illness. Science 262: 914-917.
• Oliveira RC, Padula PJ, Gomes R, Martinez VP, Bellomo C, Bonvicino CR, Freire e Lima DI, Bragagnolo C, Caldas AC, D'Andrea PS, de Lemos ER 2011. Genetic characterization of hantaviruses associated with sigmodontine rodents in an endemic area for hantavirus pulmonary syndrome in southern Brazil. Vector Borne Zoonotic Dis 11: 301-314.
• Raboni SM, Borba L, Hoffmann FG, Noronha L 2009a. Evidence of circulation of Laguna Negra-like hantavirus in the Central West of Brazil: case report. J Clin Virol 45: 153-156.
• Raboni SM, Hoffmann FG, Oliveira RC, Teixeira BR, Bonvicino CR, Stella V, Carstensen S, Bordignon J, D'Andrea PS, Lemos ER, Duarte dos Santos CN 2009b. Phylogenetic characterization of hantaviruses from wild rodents and hantavirus pulmonary syndrome cases in the state of Parana (southern Brazil). J Gen Virol 90: 2166-2171.
• Raboni SM, Probst CM, Bordignon J, Zeferino A, dos Santos CN 2005a. Hantaviruses in central South America: phylogenetic analysis of the S segment from HPS cases in Paraná, Brazil. J Med Virol 76: 553-562.
• Raboni SM, Rubio G, De Borba L, Zeferino A, Skraba I, Goldenberg S, Dos Santos CN 2005b. Clinical survey of hantavirus in southern Brazil and the development of specific molecular diagnosis tools. Am J Trop Med Hyg 72: 800-804.
• Razzauti M, Plyusnina A, Sironen T, Henttonen H, Plyusnin A 2009. Analysis of Puumala hantavirus in a bank vole population in northern Finland: evidence for co-circulation of two genetic lineages and frequent reassortment between strains. J Gen Virol 90: 1923-1931.
• Ronquist F, Huelsenbeck JP 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572-1574.
• Rosa ES, Lemos ER, Medeiros DB, Simith DB, Pereira A, Elkhoury MR, Mendes WS, Vidigal JR, Oliveira RC, D'Andrea PS, Bonvicino CR, Cruz AC, Nunes MR, Vasconcelos PF 2010. Hantaviruses and hantavirus pulmonary syndrome, Maranhão, Brazil. Emerg Infect Dis 16: 1952-1955.
• Rosa ES, Mills JN, Padula PJ, Elkhoury MR, Ksiazek TG, Mendes WS, Santos ED, Araújo GC, Martinez VP, Rosa JF, Edelstein A, Vasconcelos PF 2005. Newly recognized hantaviruses associated with hantavirus pulmonary syndrome in northern Brazil: partial genetic characterization of viruses and serologic implication of likely reservoirs. Vector Borne Zoonot Dis 5: 11-19.
• Smith MF, Patton JL 1993. The diversification of South American murid rodents: evidence from mitochondrial DNA sequence data for the Akodontini tribe. Biol J Linnean Soc 50: 149-177.
• Suzuki A, Bisordi I, Levis S, Garcia J, Pereira LE, Souza RP, Sugahara TK, Pini N, Enria D, Souza LT 2004. Identifying rodent hantavirus reservoirs, Brazil. Emerg Infect Dis 10: 2127-2134.
• Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S 2011. MEGA5: Molecular Evolutionary Genetics Analysis using maximum likelihood, evolutionary distance and maximum parsimony methods. Mol Biol Evol: doi: 10.1093/molbev/msr121.
• Travassos da Rosa ES, Medeiros DB, Nunes MR, Simith DB, de Souza Pereira A, Elkhoury MR, Lavocat M, Marques AA, Via AV, D'Andrea P, Bonvicino CR, Lemos ER, Vasconcelos PF 2011. Pygmy rice rat as potential host of Castelo dos Sonhos hantavirus. Emerg Infect Dis 17: 1527-1530.

Publication Dates

• Publication in this collection
  13 Apr 2012
• Date of issue
  May 2012

History

• Received
  08 July 2011
• Accepted
  24 Nov 2011