isolation and genetic characterization of a hantavirus - Semantic Scholar

Am. J. Trop. Med. Hyg., 61(1), 1999, pp. 92–98 Copyright q 1999 by The American Society of Tropical Medicine and Hygiene

ISOLATION AND GENETIC CHARACTERIZATION OF A HANTAVIRUS (BUNYAVIRIDAE: HANTAVIRUS) FROM A RODENT, OLIGORYZOMYS MICROTIS (MURIDAE), COLLECTED IN NORTHEASTERN PERU ANN M. POWERS, DAVID R. MERCER, DOUGLAS M. WATTS, HILDA GUZMAN, CHARLES F. FULHORST, VSEVOLOD L. POPOV, AND ROBERT B. TESH Department of Pathology and Center for Tropical Diseases, University of Texas Medical Branch, Galveston, Texas; U.S. Naval Medical Research Institute Detachment, Lima, Peru

Abstract. This paper describes the isolation and partial genetic characterization of a hantavirus from a pygmy rice rat, Oligoryzomys microtis, collected within the urban area of Iquitos, Loreto Department, Peru. The virus, designated HTN-007, exhibited the highest degree of genetic similarity to Rio Mamore virus, which was originally described from the same rodent species in eastern Bolivia. Comparison of small and medium segment nucleotide sequence data from HTN-007 and Rio Mamore virus revealed 87% and 85% sequence identity, respectively. Based on these analyses, HTN-007 appears to be a variant of Rio Mamore virus. As such, it represents the first successful isolation of Rio Mamore virus and the first evidence for the existence of a hantavirus in Peru. Serologic studies done by immunofluorescence on blood samples of 56 O. microtis trapped at the collection site indicated that 21.4% had antibodies to hantavirus. In view of the proximity of this rodent species to humans and the close phylogenetic relationship of Rio Mamore virus to hantaviruses that have been associated with human disease, Rio Mamore virus may be a hantavirus of some public health importance in tropical South America. Viruses in the genus Hantavirus, family Bunyaviridae, are primarily rodent-borne. Some members of this genus are proven human pathogens, including the etiologic agents of hemorrhagic fever with renal syndrome in the Old World and hantavirus pulmonary syndrome (HPS) in the New World.1–3 In contrast to their pathogenicity for humans, these viruses cause no apparent pathology in their rodent reservoir hosts and are generally believed to establish chronic infections that allow continued shedding of virus into the environment.4 Evidence suggests that each specific hantavirus is maintained by a distinct rodent species, consistent with the concept of cospeciation.4,5 Since the first recognition of HPS in North America in 1993, there has been considerable interest in the distribution, host associations, and evolution of hantaviruses in the Americas.3 To date, at least 19 genetically or serologically distinct hantaviruses are known to exist in the New World. These include Bayou (BAY), Black Creek Canal (BCC), Blue River (BR), El Moro Canyon (ELMC), Isla Vista (ISLA), Muleshoe (MULE), New York (NY), Prospect Hill (PH), Seoul (SEO), and Sin Nombre (SN) viruses in North America; and Andes (AND), Bermejo (BER), Cano Delgadito (CDG), Laguna Negra (LN), Lechiguanas (LECH), Maciel (MAC), Oran (ORAN), Pergamino (PRG), and Rio Mamore (RM) viruses in South America (Table 1). For many of these viruses, little information is available regarding their prevalence, geographic distribution, natural host associations, or human disease potential. To further determine the relationships of South American hantaviruses, rodents and other small mammals have been collected in the northeastern Amazon Region of Peru during the past two years in search of new zoonotic viruses. This paper describes the isolation and characterization of a hantavirus from a pygmy rice rat, Oligoryzomys microtis, collected during this study.

1) in the Zona Marina district of the City of Iquitos, Loreto Department, Peru, during April 1996. Iquitos is a city of approximately 300,000 inhabitants located in the Amazon River Basin in northeastern Peru. The city is situated about 120 meters above sea level (73.28W, 3.78S) on the bank of the Amazon River. The climate of the region is tropical with an average temperature of 27.58C and a mean annual precipitation of 2.7 meters. Traps were placed randomly throughout the field. Much of the field study site was littered with old equipment and other discarded materials from the hospital. A few small trees were present, but most of the field was covered with thick grass about 1 meter in height. Rodent trapping. Rodents were collected and processed according to recommended safety procedures.6 The animals were captured live, using Sherman (Sherman Trap Company, Tallahassee, FL) or Tomahawk (Tomahawk Trap Company, Tomahawk, WI) traps baited with dried corn and banana, and processed at the trapping site, using methods described previously.7 Blood, spleen, and lung samples were collected aseptically, and specimens were placed in plastic vials for storage at 2708C. Carcasses of many of the rodents, including the ones yielding viruses, were subsequently submitted frozen to The Museum of Texas Tech University (Lubbock, TX) for definitive identification and archiving. The experiments reported herein were conducted according to the principles set forth in the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council, National Academy Press, 1996. Serologic studies. Rodent serum samples were tested at an approximate dilution of 1:20 by indirect fluorescent antibody test (IFAT), using Sin Nombre virus-infected Vero cells as antigen spotted onto 12-well, teflon-coated, glass microscope slides as previously described.7,8 The irradiated, Sin Nombre virus–infected Vero cells were provided by the Special Pathogens Branch, Centers for Disease Control and Prevention (Atlanta, GA). Virus isolation. Lung tissue from individual IFAT-anti-

MATERIALS AND METHODS

Study site. Rodents described in this paper were collected in a vacant field, located on the grounds of a hospital (Figure

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ISOLATION OF A HANTAVIRUS FROM PERU

TABLE 1 Summary of recognized and proposed South American hantaviruses Virus

Andes Bermejo Cano Delgadito Laguna Negra Lechiguanas Maciel Oran Pergamino Rio Mamore

Known distribution

Argentina Argentina Venezuela Paraguay Argentina Argentina Argentina Argentina Bolivia

Associated host

Oligoryzomys longicaudatus Oligoryzomys chacoensis Sigmodon alstoni Calomys laucha Oligoryzomys flavescens Bolomys obscurus Oligoryzomys longicaudatus Akodon azarae Oligoryzomys microtis

body positive rodents was homogenized in phosphate-buffered saline, pH 7.2, containing 30% (v/v) heat-inactivated (568C for 30 min.) fetal bovine serum, using sterile 7.0 ml Ten Broeck tissue grinders. About 0.5 ml of the crude homogenate was inoculated into a 12.5 cm2 flask of Vero cells and cultured for 14 days as previously described.8 On day 14 postinoculation, some of the cells were scraped from the flask and placed on 12-well glass microscope slides. After drying and acetone fixation, the cells were examined by IFAT for the presence of hantaviral antigen, using a Sin Nombre virus recombinant hyperimmune mouse ascitic fluid and a commercial, fluorescein isothiocyanate–conjugated goat anti-mouse IgG (Sigma, St. Louis, MO). Vero cell cultures that were negative by IFAT on the initial culture were blind passaged one additional time before being discarded. Genetic analysis of virus. The RNA was extracted from infected Vero cells, using methods described previously.7–10 For the small (S) genome segment, cDNA was synthesized using an oligonucleotide primer (59-GGTGGTGTGGTAGTAGTAGACTCC-39;11 designated HTS13) designed to anneal to the conserved genome termini. The RNA was combined with primer and incubated at 708C for 5 min. The reaction tube was chilled on ice while buffer, dithiothreitol, dNTPs, and RNase inhibitor were added to a final volume

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of 100 ml. The reaction was heated to 428C for 2 min. before 200 units of SuperscriptII reverse transcriptase (RT) (GibcoBRL, Gaithersburg, MD) were added and the reaction allowed to proceed for 1 hr. A subsequent polymerase chain reaction (PCR) amplification used HTS13 combined with HTS10 (59-CCTACAGACTTTGATGCCATDAT-39;12) to generate a product of 1,095 basepairs. The resulting PCR product was cloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA) and sequenced on an Applied Biosystems (Foster City, CA) Prism automated DNA sequencer using m13 and T7 plasmid specific primers. The medium (M) segment cDNA was generated using the conditions described earlier and primer HTM2 (59-TCAATATGTTTRCATACCTGYTC-39; genome position 2525). The PCR amplification of a 526-basepair (bp) fragment was performed using primer HTM1 (59-TGGGCTGCAAGTGC-39; genome position 1999) combined with HTM2. The M segment PCR products were sequenced directly using these same primers. Nucleotide sequences, minus the primer sequences, were aligned and compared with others previously determined8,11–19 using the GAP and PILEUP programs (Genetics Computer Group, Madison, WI). Phylogenetic analyses were conducted using the phylogenetic analysis using parsimony algorithm with both ordered (transition:transversion 5 1:4) and unordered characters.20 Puumala virus was used as the outgroup to root the trees. The neighbor joining program (Kimura 2-parameter distance formula) was used with the parsimony tree topology constraint to correct branch lengths for multiple substitutions. Bootstrap resampling analysis21 was performed using 100 replicates. The S and M segment sequences were deposited in Genbank with accession numbers AF133254 and AF133255, respectively. Transmission electron microscopy. Infected cell monolayers were fixed in situ in a mixture of 1.25% formaldehyde, 2.5% glutaraldehyde, and 0.03% trinitrophenol with 0.03% CaCl2 in 0.05 M cacodylate buffer, pH 7.3,22 for 1 hr at room temperature and then kept at 58C until further pro-

FIGURE 1. View of the vacant field where infected rodents were collected. The photograph was taken from behind hospital buildings, looking across the field.

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FIGURE 2. Immunofluorescence of Vero cells 12 days after infection with HTN-007 virus. The antibody used in this test was an ascitic fluid from mice hyperimmunized with a Sin Nombre virus recombinant antigen. Viral antigen is seen as discrete balls of fluorescence within infected cells (magnification 3 500).

cessing. Monolayers were washed in cacodylate buffer, scraped off the plastic, and further processed as a pellet. The pellet was post-fixed in 1% OsO4 in the same buffer for 1 hr at room temperature, stained en bloc in 1% uranyl acetate in 0.1 M maleate buffer, pH 5.2, dehydrated in ethanol, processed through propylene oxide, and embedded in Poly/Bed 812 (Polysciences, Warrington, PA). Ultrathin sections were cut on a Reichert-Ultracut S ultramicrotome (Leica, Deerfield, IL), stained with uranyl acetate and lead citrate, and examined in a Philips 201 electron microscope (Philips Electron Optics, Eindhoven, The Netherlands) at 60 kV. RESULTS

Rodent collections and serology. A total 60 rodents were live-trapped at the study site behind the hospital in Iquitos during April 1996. Fifty-six of the rodents were Oligoryzomys microtis; the remaining 4 were Rattus rattus. Sera from 12 of the 56 O. microtis (21.4%) gave a positive IFAT reaction against Sin Nombre antigen at the 1:20 screening dilution. All of the R. rattus sera were negative. Virus isolations. Lung tissue from 10 of the hantavirus antibody-positive rodents was triturated and cultured in Vero cells. One culture, designated HTN-007, of lung from an adult male O. microtis, demonstrated hantavirus antigen when examined by IFAT 14 days after inoculation (Figure 2). No viral cytopathic effect (CPE) was observed in the culture. The virus was subsequently passaged in Vero cells and genetically characterized as described below. The 9 other lung samples were hantavirus-negative by IFAT on primary culture and after a second blind culture. However, three of the O. microtis lung cultures (HTN005, HTN-014, and HTN-032) exhibited CPE between the fifth and seventh day after inoculation, on the second (blind) passage. The IFAT done on the infected cells, using a Na-

tional Institute of Allergy and Infectious Diseases (Bethesda, MD) polyvalent rabies, lymphocytic choriomeningitis, Newcastle disease, herpes, and vaccinia grouping ascitic fluid (G236-601-567),23 gave a strongly positive reaction. These samples were negative when tested with Sin Nombre virus recombinant ascitic fluid. Electron microscopic examination of Vero cells infected with isolate HTN-014 demonstrated the presence of herpes-like virus particles (Figure 3). Genetic characterization of HTN-007. The DNA fragments of 1,095 bp for the S segment and 526 bp for the M segment were amplified by RT-PCR. These PCR products were either sequenced directly or cloned into a plasmid vector before being sequenced. Pairwise comparison of S segment nucleotide sequence data with that of other New World hantaviruses indicated that the HTN-007 virus was most closely related to RM virus, sharing 87% nucleotide sequence identity. The viruses exhibiting the next highest degree of sequence identity were Laguna Negra and Andes with 82% and 79%, respectively (Table 2). Parsimony analysis of the S segment fragment confirmed that HTN-007 was most closely related to RM virus (Figure 4). Comparison of the M segment fragment of HTN-007 with other hantaviruses revealed that HTN-007 and RM share an 85% nucleotide sequence identity and a phylogram consistent with that of the S segment. The degree of divergence between virus isolate HTN-007 and RM presented here was not sufficiently high to consider HTN-007 as distinct from RM virus; therefore, HTN-007 should be tentatively considered a strain or variant of RM virus. Transmission electron microscopy. Approximately 96 hr after infection, both intranuclear and extracellular virions, resembling herpes simplex virus, were observed in thin sections. Different stages of virion formation were observed in the nucleus: empty capsids, capsids with internal protein, and capsids containing DNA (all about 80 nm in diameter),


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FIGURE 3. A, herpes-like virus in the nucleus of an infected Vero cell 96 hr postinfection. Scaffolding protein–containing capsid (small arrowhead), DNA-containing capsids (long arrowheads), and enveloped virions inside nuclear envelope pockets (arrows) are shown. Bar 5 100 nm. B, herpes-like virions at the surface of a Vero cell 96 hr after infection of the monolayer. The short arrowhead indicates an envelope, the long arrowhead shows a tegument, and the arrows point at a central DNA core. Bar 5 100 nm.

and virions inside enlargements (pockets) of nuclear envelope. These virions were about 110 nm in diameter (Figure 3A). Extracellular virions were larger (130–140 nm in diameter) and had clearly visible envelope and tegument (Figure 3B). DISCUSSION

A description of the genetic identification of Rio Mamore virus was first published in 199712 based on the complete sequence of the S genomic segment and a partial sequence of the M genome that were obtained by RT-PCR analysis of tissues from a hantavirus-seropositive museum specimen of O. microtus captured in the Beni Department of northeastern Bolivia in 1985. In the original description, Bharadwaj and others identified 5 RM virus–seropositve animals among 35 archived museum specimens of O. microtis examined from several different departments (Beni, La Paz, and Santa Cruz) in eastern Bolivia. The isolation of an RM-like virus from Iquitos, located about 1,500 km from the original Beni collection site in Bolivia, significantly extends the known geographic distribution of RM virus and establishes the presence of hantaviruses in a new region, the Amazon basin. Oligoryzomys microtis has a wide distribution in tropical South America and reportedly occurs in central Brazil and the con-

tiguous lowlands of Peru, Bolivia, Paraguay and Argentina;24 therefore, RM virus may be distributed over this entire range as well. Pygmy rice rats preferentially inhabit dense brushy or grassy habitats and are numerous in open areas such as river edges, gardens, secondary brush, rice fields, and plantations; they commonly occupy houses and barns in rural areas.25 The rodents examined in the current study were captured in a grassy vacant field behind a hospital within the city limits of Iquitos. The neighborhood immediately surrounding the collection site was mixed residential-industrial and consists of scattered houses interspersed with small factories and other commercial establishments. Although the public health importance of RM virus is unknown, clearly the putative rodent reservoir of the virus does occasionally come into contact with people, so RM virus has the potential to infect humans. Our initial genetic characterization of the S and M genome segments of HTN-007 suggested that it might represent a new hantavirus. The genetic difference between HTN-007 and its closest identified relative, RM virus, is approximately 15%. In some circumstances, this degree of genetic divergence is sufficient to consider a virus isolate as a unique virus species. Van Regenmortal and others recently published suggested guidelines to aid in the demarcation of viral species, listing 7 characteristics useful in discriminating be-

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TABLE 2 Percent nucleotide sequence identity of the small (S) genome segment among North and South American hantaviruses*

CDG AND LN SN RM PUU PH NY ISLA HTN-007 BCC BAY

CDG

AND

LN

SN

RM

PUU

PH

NY

ISLA

HTN-007

BCC

– 77.3 73.7 76.1 75.7 67.0 68.2 76.1 67.4 75.7 73.0 74.7

– – 79.4 76.5 79.3 67.8 69.6 77.0 67.8 79.2 76.0 76.3

– – – 76.4 83.0 68.3 69.6 76.1 70.1 81.9 77.3 77.3

– – – – 75.3 68.0 69.9 84.1 69.7 75.1 75.3 76.7

– – – – – 69.9 70.7 76.7 70.1 87.2 76.4 78.1

– – – – – – 72.2 68.4 73.2 70.4 69.1 69.2

– – – – – – – 70.2 77.8 71.2 69.6 72.4

– – – – – – – – 70.0 75.9 75.4 77.0

– – – – – – – – – 69.8 70.0 68.7

– – – – – – – – – – 76.6 78.0

– – – – – – – – – – – 81.3

* CDG 5 Cano Delgadito; AND 5 Andes; LN 5 Laguna Negra; SN 5 Sin Nombre; RM 5 Rio Mamore; PUU 5 Puumala; PH 5 Prospect Hill; NY 5 New York; ISLA 5 Isla Vista; BCC 5 Black Creek Canal; BAY 5 Bayou.

tween virus species allocated to a particular genus: genome sequence relatedness, natural host range, cell and tissue tropism, pathogenicity and cytopathology, mode of transmission, physiologic properties, and antigenic properties.26 The degree of genome sequence relatedness between HTN-007 and other recognized or proposed hantavirus species did not provide a clear indication of its relationship to other members of the genus; therefore, we attempted to use other criteria to determine whether HTN-007 represented a novel hantavirus species.

FIGURE 4. Phylogenetic tree of South American hantaviruses generated from the 1048-basepair small segment reverse transcriptase–polymerase chain reaction product (minus the primer sequences) using the phylogenetic analysis using parsimony branch and bound parsimony algorithm with unordered characters. Parsimony analysis with ordered characters produced a phylogram whose topology differed only in the placement of the Sin Nombre/New York and Bayou/Black Creek Canal groups. Numbers indicate bootstrap values for groups to the right. The bar represents nucleotide sequence divergence.

The concept of demarcating individual virus species within the genus Hantavirus is particularly difficult. Typically, viruses in this genus have been designated as distinct if they deviate from other recognized hantaviruses in any one of several species distinguishing characteristics. For example, BCC and MULE viruses are genetically related hantaviruses originating from southern Florida and western Texas, respectively. They were proposed as distinct species based on the degree of genetic diversity among their S segments (approximately 23%) and the geographic distance (about 2,200 km) between their original collection sites.27 However, both viruses have been associated with the cotton rat Sigmodon hispidis, a species complex with a wide geographic distribution in North America,28 so they could be considered as geographic variants of the same hantavirus. Similarly, the South American AND and ORAN hantaviruses are approximately 20% divergent in their M segment nucleotide sequences, but both have been associated with the pygmy rice rat O. longicaudatus in Argentina.29 In contrast, genetic analyses indicate a relatively high degree of M sequence identity (88.6%) between LECH and BER viruses; yet these two hantaviruses have been proposed as distinct, because they were identified in two different but related rodent species (O. flavescens and O. chacoensis) in Argentina.29 Interestingly, examples of conservancy in describing previously uncharacterized hantaviruses are more rare. Morzunov and others recently designated two strains of North American hantaviruses having 16% nucleotide divergence in the M genome segment as lineages of the same virus, BR, rather than propose their classification as distinct viruses.4 To date, the criteria for designating what constitutes a novel hantavirus (species) have been inconsistent and rather arbitrary. It has been proposed that a combination of three characters be evaluated to determine if a virus truly represents a novel species or is merely a divergent strain of a previously recognized hantavirus. Viruses in the genus Hantavirus present a unique taxonomic problem, since most of the aforementioned guidelines for species demarcation are not applicable at present because many recognized hantaviruses have never been isolated, so knowledge about them is limited mainly to genome sequences obtained by RT-PCR of viral RNA from tissues of infected humans or rodents.3,30 Consequently, little is known about their pathogenesis, host

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range, physiologic properties, or antigenic relationships, which eliminates the use of several species-demarcating characters to distinguish among these closely related hantaviruses. An additional problem is presented when attempting to use natural host range as one of the species demarcating characters. The use of this character assumes that there are sufficient isolations of the virus available to incriminate a particular animal as the probable host, and that the vertebrate host can be accurately identified to the level of species and does not represent a species complex. Identification is not a trivial issue in the case of hantaviruses, since proper rodent taxonomy is essential for examining concepts such as cospeciation and reservoir status. Considerable efforts have been made to elucidate the phylogenetic history of rodents in the genus Peromyscus, which serve as hosts of several North American hantaviruses; but the relationships of the various species and subspecies of mice in this genus still remain unclear at this time.4,31 Similar difficulties with establishing rodent evolutionary relationships are encountered when attempting to study cospeciation of arenaviruses with their rodent reservoirs.32 The taxonomy of most neotropical rodents is still poorly defined; for many rodents in this group, even less is known about their ecologic, taxonomic, genetic, and evolutionary relationships than is known about their associated viruses. The comparison of RM and HTN-007 viruses is a case in point. Both viruses have been associated with rodents identified by museum specialists as O. microtis. Musser and Carleton listed the known geographic distribution of O. microtis as central Brazil south of the Salimoes and Amazon Rivers and the contiguous lowlands of Peru, Bolivia, Paraguay and Argentina. They also listed species and synonym names for 57 additional Oligoryzomys rodents that have been described from South America, Central America, southern Mexico, and the Lesser Antilles.24 The geographic range of this genus represents a huge and poorly studied region of tropical America. Additionally, no molecular characterization has yet been performed on rodents of this genus to determine phylogenic relationships. Assuming that the hantaviruses share a long-term evolutionary relationship with their rodent hosts, it is then difficult to make meaningful decisions about neotropical hantavirus-host relationships without knowing more about the phylogeny of their respective rodent hosts. The recovery of three herpesvirus-like isolates from O. microtis lung tissue during blind-passage in Vero cell cultures may be significant. Similar results were obtained during hantavirus ecologic studies in eastern Texas; three additional herpesvirus isolates were obtained during blind passage of BAY virus–seropositive Oryzomys palustris lung tissue in Vero cell cultures (Tesh RB, Guzman H, Hice C, Poplov VL, unpublished data). In each case, viral CPE was observed during the second (blind) passage. We have not attempted to identify these viruses further, but our experience suggests that latent herpesvirus infection present in the rodent tissues was somehow activated during blind passage in monkey cell culture. The significance of this finding in regard to hantavirus infection is still undetermined. Acknowledgments: We thank Dr. Robert Bradley and Christine Hice (Texas Tech University, Lubbock, TX) for identifying the rodent

specimens, and Dr. Thomas Ksiazek (Centers for Disease Control and Prevention, Atlanta, GA) for providing the Sin Nombre virus antigen and antibody. We also thank Violet Han for assistance with the electron microscopy. Financial support: This work was supported by grants AI-10984 and AI-39800 from the National Institutes of Health. Ann M. Powers was supported by a post-doctoral fellowship from the McLaughlin Fellowship Fund, University of Texas Medical Branch. Disclaimer: The opinions and assertions contained herein are the private ones of the authors and do not reflect the official policy of the Department of the Navy, Department of Defense, or the U.S. Government. Authors’ addresses: Ann M. Powers, David R. Mercer, Hilda Guzman, Charles F. Fulhorst, Vsevolod L. Popov and Robert B. Tesh, Department of Pathology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0609. Douglas M. Watts, U.S. Naval Medical Research Institute Detachment, NAMRID/Unit 3800, Lima, Peru, APO AA 34031. Reprint requests: Robert B. Tesh, Department of Pathology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0609. REFERENCES

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