«Viruses 2014, 6, 1897-1910; doi:10.3390/v6051897 OPEN ACCESS viruses ISSN 1999-4915 Short Communication Molecular ...»
EU929078), Oxbow virus (OXBV Ng1453, S: FJ5339166; M: FJ539167; L: FJ593497) Viruses 2014, 6 1904 and Rockport virus (RKPV MSB57412, S: HM015223; M: HM015219; L: HM015221).
Also shown are the phylogenetic positions of representative rodent-borne hantaviruses, including Hantaan virus (HTNV 76-118, S: NC_005218; M: Y00386; L: NC_005222), Soochong virus (SOOV SOO-1, S: AY675349; M: AY675353; L: DQ056292), Dobrava virus (DOBV Greece, S: NC_005233; M: NC_005234L: NC_005235), Seoul virus (SEOV 80-39, S: NC_005236; M: NC_005237; L: NC_005238), Sangassou virus (SANG SA14, S:
JQ082300; M: JQ082301; L: JQ082302),Tula virus (TULV M5302v, S: NC_005227; M:
NC_005228; L: NC_005226), Puumala virus (PUUV Sotkamo, S: NC_005224; M:
NC_005223; L: NC_005225), Prospect Hill virus (PHV PH-1, S: Z49098; M: X55129; L:
EF646763), Sin Nombre virus (SNV NMH10, S: NC_005216; M: NC_005215; L:
NC_005217) and Andes virus (ANDV Chile9717869, S: NC_003466; M: NC_003467; L:
NC_003468). The numbers at each node are posterior node probabilities (left) based on 150,000 trees and bootstrap values (right) based 1000 replicates executed on the RAxML BlackBox web server, respectively. The scale bars indicate nucleotide substitutions per site.
2.4. Bats as Hosts of Hantaviruses
The phylogeny of bats is not fully resolved . The order Chiroptera was traditionally divided in two suborders, Megachiroptera and Microchiroptera. However, due to the paraphyly of the Microchiroptera, a new taxonomic nomenclature, comprising the suborder Yinpterochiroptera (megabats or fruit bats in the Pteropodidae family in Megachiroptera and a few Microchiroptera families) and Yangochiroptera (the remaining Microchiroptera families), has been proposed . In the former classification, bat species hosting hantaviruses belong only to the Microchiroptera suborder, Viruses 2014, 6 1905 but in the Yinpterochiroptera-Yangochiroptera classification, they belong to both suborders, suggesting that primordial hantaviruses may have emerged in an early common ancestor of bats.
Within the Microchiroptera, hantaviruses are found in bats belonging to four phylogenetically distant families, namely Hipposideridae (Old World leaf-nosed bats) and Rhinolophidae (horseshoe bats) in the suborder Yinpterochiroptera, and Nycteridae (hollow-faced bats) and Vespertilionidae (vesper bats) in the suborder Yangochiroptera. The families Hipposideridae and Vespertilionidae are among the most speciose insectivorous bats, with member species distributed across Africa, Europe, Asia, the Americas and Australia. Their vast geographic distribution provides unlimited opportunities to search for related bat-associated hantaviruses.
Compared to the multitude of hantaviruses reported from approximately 50% of soricomorph species tested [34,35], the cumulative number of newly recognized bat-borne hantaviruses is exceedingly low, if one considers the 533 bat samples tested in the present study, along with the nearly 1200 bat specimens analyzed in four other studies [27–30]. The modest proportion of hantavirus RNA detection in bat tissues may be attributed to the highly divergent nature of their genomes, as well as the very focal or localized nature of hantavirus infection, small sample sizes of bat species, primer mismatches, suboptimal PCR cycling conditions, and variable tissue preservation with degraded RNA [27,29].
Alternatively, bats may be less susceptible to hantavirus infection or may have developed immune mechanisms to curtail viral replication and/or persistence. For answers to such questions, and myriad others, reagents need to be developed and multidisciplinary collaborative studies must be designed to collect optimal specimens to isolate and characterize these newfound bat-borne hantaviruses. Only then will a better understanding be gained about their evolutionary origins and phylogeography, co-evolution history, transmission dynamics and pathogenic potential.
3. Experimental Section
3.1. Samples Archival frozen, ethanol-fixed and RNAlater®-preserved tissues from bats, captured during 1981–2012 in Brazil, China, Cote d’Ivoire, Guinea, Korea, Republic of Georgia, Vietnam and the United States (Figure 1 and Table 1), were tested for hantavirus RNA by RT-PCR, using newly designed and previously employed oligonucleotide primers [12,18,27,29]. Of the 533 samples tested, the majority consisted of lung (310) and kidney (51) tissues (Table 1). RNA extracted from rectal swabs and feces (79) were also tested. Bats were from seven families (Hipposideridae, Molossidae, Nycteridae, Pteropodidae, Phyllostomidae, Rhinolophidae and Vespertilionidae), 28 genera and 53 species (Figure 1). The University of Hawaii Institutional Animal Care and Use Committee approved the use of archival tissues as being exempt from protocol review.
3.2. Genome Detection and Sequencing
Total RNA extraction from tissues, using the PureLink Micro-to-Midi total RNA purification kit (Invitrogen, San Diego, CA, USA), and cDNA synthesis, using the SuperScript III First-Strand Synthesis Systems (Invitrogen) with random hexamers, were performed as described previously [9,12,18].
Oligonucleotide primers used to amplify S-, M- and L-genomic segments of bat-borne hantaviruses are Viruses 2014, 6 1906
3.3. Phylogenetic Analysis Maximum likelihood and Bayesian methods, implemented in RAxML Blackbox webserver  and MrBayes 3.1 , under the best-fit GTR+I+Γ model of evolution  and jModelTest version 0.1 , were used to generate phylogenetic trees. Two replicate Bayesian Metropolis–Hastings Markov Chain Monte Carlo runs, each consisting of six chains of 10 million generations sampled every 100 generations with a burn-in of 25,000 (25%), resulted in 150,000 trees overall. The S, M and L segments were treated separately in phylogenetic analyses. Topologies were evaluated by bootstrap analysis of 1000 iterations, and posterior node probabilities were based on 2 million generations and estimated sample sizes over 100 (implemented in MrBayes) .
Viruses 2014, 6 1907
Mammalian reservoirs of zoonotic viruses typically do not display host restrictions within a given taxonomic order. Also, infection is usually chronic, persistent and subclinical. For example, rodents of multiple genera and species, belonging to four subfamilies in the order Rodentia, serve as reservoirs of hantaviruses in Eurasia, Africa and the Americas and do not exhibit clinical disease or survival disadvantage. In addition, recently, hantaviruses exhibiting far greater genetic diversity have been detected in healthy-appearing shrews and moles representing many genera in six subfamilies within the order Soricomorpha in Eurasia, Africa and North America. Similarly, as mentioned earlier, bat species belonging to both suborders of Chiroptera host hantaviruses without evidence of apparent disease.
However, some might contend that the low prevalence of hantavirus RNA in a few bat species, and the absence of hantavirus infection in the majority of bat species analyzed to date, would argue against a long-standing hantavirus-reservoir host relationship, and instead support spillover or host switching.
That is, the gleaning feeding behavior of some bats, such as Nycteris, presents the possibility of acquired infection from excreta of well-established terrestrial reservoirs of hantaviruses. However, this seems highly improbable because bat-borne hantaviruses are among the most genetically diverse described to date.
With the discovery of divergent hantavirus lineages in three taxonomic orders of placental mammals, there is renewed interest in investigating their genetic diversity, geographic distributions, and evolutionary dynamics [34,35]. Newfound knowledge that insectivorous bats harbor a distinctly divergent lineage of hantaviruses emphasizes the truly complex evolutionary origins and phylogeography of a group of viruses once thought to be restricted to rodents. At this point, it would not be surprising if hantaviruses are found in small mammals belonging to other taxonomic orders, such as Erinaceomorpha (hedgehogs) and even Afrosoricida (tenrecs). Such anticipated discoveries may provide additional insights into the dynamics of hantavirus transmission, potential reassortment of genomes, and molecular determinants of hantavirus pathogenicity. As importantly, a sizable expansion of the hantavirus sequence database would provide valuable tools for refining diagnostic tests and enhancing preparedness for future outbreaks caused by emerging hantaviruses.
This work was supported by U.S. Public Health Service grants R01AI075057 and P20GM103516 from the National Institutes of Health, grant 24405045 from the Japan Society for the Promotion of Science, grant H25-Shinko-Ippan-008 for Research on Emerging and Re-emerging Infectious Diseases, and grant UE134020ID from the Agency for Defense Development of Korea. The services provided by the Genomics Core Facility, funded partially by the Centers of Biomedical Research Excellence program (P30GM103341), are gratefully acknowledged.
K.S., D.P., I.V.K. and M.Y.K. provided bat tissues. R.Y. conceived the project, and R.Y. and J.W.S.
provided overall scientific oversight. All authors contributed to the preparation of the final manuscript.
Conflicts of Interest The authors declare no conflict of interest.
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