Genetic identification and characterization of limestone canyon virus, a unique Peromyscus-borne hantavirus

Hantaviruses, family Bunyaviridae, are rodent-borne RNA viruses that can cause hantavirus pulmonary syndrome (HPS) in various regions of the Americas. A coevolutionary relationship exists between hantaviruses and their specific rodent reservoir hosts; the phylogeny of the viruses generally matches that of the rodents. There are several Peromyscus-borne hantaviruses, including Sin Nombre virus, the most common cause of HPS in North America. This report describes the genetic detection and characterization of a newly discovered Peromyscus boylii-borne virus, Limestone Canyon (LSC) virus, the most divergent member of the Peromyscus-borne hantaviruses to date. Analysis of a 1209-nucleotide region of the S segment of LSC virus showed it to be more closely related to hantaviruses found in harvest mice (Reithrodontomys megalotis and R. mexicanus) than to other Peromyscus-associated hantaviruses (Sin Nombre, New York, and Monongahela). Phylogenetic analysis of virtually the entire M genome segment (3489 nucleotides) of LSC virus revealed a similar picture in which LSC virus was found to be very distinct from other Peromyscus-associated viruses, but its exact relationship to the other Peromyscus-borne and the Reithrodontomys-borne viruses was not resolved. These results indicate that hantavirus host species-jumping events can occur by which a hantavirus may switch to, and become established in, a rodent host belonging to a different genus. P. boylii are present throughout the southwestern United States and central Mexico. More extensive screening of HPS patients by using RT-PCR assays will be necessary to determine if LSC virus can cause human disease.     

Ecology of hantaviruses in Mexico: Genetic identification of rodent host species and spillover infection

In our recent epidemiological survey conducted in Mexico for hantavirus infection, we identified three distinct viruses circulating in Mexican wild rodents, namely Montano virus (MTNV), Huitzilac virus (HUIV), and Carrizal virus (CARV). To gain a detailed understanding of hantavirus epidemiology and its associated hosts, 410 rodents were captured at eight collecting points in Morelos and Guerrero, Mexico, and examined for hantavirus seroprevalence, the presence of viral RNA, and rodent host species identification using cytochrome b gene sequences. Of the 32 species captured, seven species were positive for hantavirus: Peromyscus beatae (31/127; 24.4%), Reithrodontomys sumichrasti (6/15; 40%), Reithrodontomys megalotis (2/25; 8%), Peromyscus aztecus evides (1/1; 100%), Peromyscus megalops (1/41; 2.4%), Megadontomys thomasi (1/9; 11.1%), and Neotoma picta (1/6; 16.7%), with an overall prevalence of 10.5%; virus genome persisted in the majority of seropositive rodents. Nucleotide sequence and phylogenetic analysis showed that the viruses belonged mainly to the three lineages previously identified. The data showed that MTNV and CARV were primarily carried by P. beatae and R. sumichrasti, respectively. In addition, the data revealed an apparent complex interaction between hantaviruses and their hosts, suggesting active transmission and/or spillover infections within sympatric rodent species.     

Development and evaluation of a broad reacting SYBR-green based quantitative real-time PCR for the detection of different hantaviruses

BackgroundHantaviruses are endemic in most parts of the world and cause hundreds of thousand human cases of hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) annually throughout Eurasia and the Americas. They are zoonotic viruses, most commonly transmitted to humans by aerosolized rodent excreta. New hantaviruses are frequently discovered in previously unknown reservoir species and geographic areas. Consequently, there is a need to improve hantavirus diagnostics.ObjectivesThis paper describes the design and evaluation of a rapid and robust quantitative real-time PCR (QRT-PCR) assay able to detect a wide range of hantaviruses.Study designPrimers with the potential to detect different hantaviruses were designed from conserved regions of different hantavirus L segments, as identified from multiple sequence alignments.ResultsBy using SYBR-green-based QRT-PCR 100–1000 target molecules of in vitro produced RNA and less than 100 copies of hantavirus RNA from different hantavirus clades and regions of the world were detected. When using the assay on clinical samples from patients with acute HFRS, Puumala hantavirus (PUUV) RNA was confirmed in all previously positive samples. Notably, the broad reacting L-segment QRT-PCR also detected viral RNA in HFRS patient samples, previously negative by a QRT-PCR targeting the S segment of PUUV.ConclusionsThis novel assay provides a powerful tool for diagnosis of hantaviruses from different clades and regions and may also be useful in surveys with the purpose of finding new hantaviruses in rodent or insectivore species.     

Sequence analysis of the complete S genomic segment of a newly identified hantavirus isolated from the white-footed mouse (Peromyscus leucopus): Phylogenetic relationship with other sigmodontine rodent-borne hantaviruses

Four Corner (FC) or Sin Nombre virus, a hantavirus harbored by the deer mouse (Peromyscus maniculatus), is the principal etiologic agent of hantavirus pulmonary syndrome (HPS). Recently, a hantavirus, designated New York (NY) virus, isolated from a white-footed mouse (Peromyscus leucopus) captured on Shelter Island, New York, was molecularly linked to a fatal case of HPS occurring in the northeastern United States. To clarify the genetic and phylogenetic relationship between NY and FC viruses and other sigmodontine rodent-borne hantaviruses, we amplified and sequenced the entire S genomic segment of NY virus. The S segment of NY virus was 2078 nucleotides long, with an open reading frame of 1284 nucleotides in the virus complementary strand, capable of encoding a protein of 428 amino acids, and with a 752-nucleotide long 3-noncoding region, comprised of numerous imperfect repeats. Pairwise analysis indicated that NY virus was more similar to FC virus than to other sigmodontine rodent-borne hantaviruses, differing from strains of FC virus by 16.6–17.8% and 7.0–8.2% at the nucleotide and amino acid levels, respectively. As determined by the maximum parsimony and neighbor-joining methods, NY virus formed a separate lineage from FC virus and was phylogenetically distinct from hantaviruses harbored by other sigmodontine rodents. Whether or not NY and FC viruses represent distinct viral species is unclear. Further analyses of hantaviruses harbored by white-footed mice are needed to clarify the genetic diversity and evolution of Peromyscus-borne hantaviruses.     

Reassortment events in the evolution of hantaviruses

Hantaviruses (order Bunyavirales, family Hantaviridae), known as important zoonotic human pathogens, possess the capacity to exchange genome segments via genetic reassortment due to their tri-segmented genome. Although not as frequent as in the arthropod-borne bunyaviruses, reports indicating reassortment events in the evolution of hantaviruses have been recently accumulating. The intra- and inter-lineage reassortment between closely related variants has been repeatedly reported for several hantaviruses including the rodent-borne human pathogens such as Sin Nombre virus, Puumala virus, Dobrava-Belgrade virus, or Hantaan virus as well as for the more recently recognized shrew-borne hantaviruses, Imjin and Seewis. Reassortment between more distantly related viruses was rarely found but seems to play a beneficial role in the process of crossing the host species barriers. Besides the findings based on phylogenetic studies of naturally occurring strains, hantavirus reassortants were generated also in in vitro studies. Interestingly, only reassortants with exchanged M segments could be generated suggesting that a high degree of genetic compatibility is required for the S and L segments while the exchange of M segment is better tolerated or is particularly beneficial. Altogether, the numerous reports on hantavirus reassortment, summarized in this review, clearly demonstrate that reassortment events play a significant role in hantavirus evolution and contributed to the currently recognized hantavirus diversity.     

Detection of hantaviruses in Brazilian rodents by SYBR-Green-based real-time RT-PCR

Current knowledge of the pathogenic hantavirus indicates that wild rodents are its primary natural reservoir. Specific primers to detect the presence of viral genomes were developed using an SYBR-Green-based real-time RT-PCR protocol. One hundred sixty-four rodents native to the Atlantic Forest biome were captured in S?o Paulo State, Brazil, and their tissues were tested. The presence of hantavirus RNA was detected in sixteen rodents: three specimens of Akodon montensis, three of Akodon cursor, two of Necromys lasiurus, one of Juliomys sp., one of Thaptomys nigrita, five of Oligoryzomys nigripes, and one of Oryzomys sp. This SYBR Green real-time RT-PCR method for detection of hantavirus may be useful for surveying hantaviruses in Brazil.     

The N-terminus of the Montano virus nucleocapsid protein possesses broadly cross-reactive conformation-dependent epitopes conserved in rodent-borne hantaviruses

The hantavirus nucleocapsid (N) protein is an important immunogen that stimulates a strong and cross-reactive immune response in humans and rodents. A large proportion of the response to N protein has been found to target its N-terminus. However, the exact nature of this bias towards the N-terminus is not yet fully understood. We characterized six monoclonal antibodies (mAbs) against the N protein of Montano virus (MTNV), a Mexican hantavirus. Five of these mAbs recognized eight American hantaviruses and six European and Asian hantaviruses, but not the Soricomorpha-borne Thottapalayam hantavirus. The N protein-reactive binding regions of the five mAbs were mapped to discontinuous epitopes within the N-terminal 13-51 amino acid residues, while a single serotype-specific mAb was mapped to residues 1-25 and 49-75. Our findings suggest that discontinuous epitopes at the N-terminus are conserved, at least in rodent-borne hantaviruses, and that they contribute considerably to N protein cross-reactivity.     

Rodent host specificity of European hantaviruses: evidence of Puumala virus interspecific spillover

In order to investigate rodent host specificity of European hantaviruses, experimental infection of colonized and wild-trapped rodents was performed. In addition to the natural rodent reservoir, Clethrionomys glareolus, Puumala hantavirus (PUUV) could infect colonized Microtus agrestis and Lemmus sibiricus, but not Syrian hamsters or Balb/C mice. Neither C. glareolus, nor M. agrestis, could be readily infected by Tula hantavirus (TULV). Wild-trapped Apodemus flavicollis and A. agrarius, the natural reservoirs of Dobrava (DOBV) and Saaremaa (SAAV) hantaviruses, respectively, could both be infected by SAAV. NMRI mice could also be infected by SAAV, but with lower efficiency as compared to Apodemus mice. Balb/C and NMRI laboratory mice, but not C. glareolus, could be infected by DOBV. To our knowledge, this is the first time DOBV and SAAV have been shown to infect adult laboratory mice. Moreover, potential hantavirus spillover infections were investigated in wild-trapped rodents. In addition to the natural host C. glareolus, we also found M. arvalis and A. sylvaticus with a history of PUUV infection. We did not find any C. glareolus or A. sylvaticus infected with TULV, a hantavirus which is known to circulate in the same geographical regions of Belgium.     

Analysis of the nucleocapsid gene brings new insights to the classification of Sigmodontinae-borne hantaviruses

Hantaviruses, members of the family Bunyaviridae, are the causative agents of hantavirus cardiopulmonary syndrome in South America. Hantaviruses are currently classified into species based on the guidelines provided by the International Committee on Taxonomy of Viruses. However, a new taxonomic system was proposed recently to classify Sigmodontinae-borne hantaviruses, which are divided currently into three phylogenetic clades corresponding to Andes, Laguna Negra, and Rio Mamore. Analyzing complete nucleocapsid gene sequences of all Sigmodontinae-borne hantaviruses, we propose the addition of a new clade and a fourth group to the already established Andes clade, allowing a better classification of the Sigmodontinae-borne hantaviruses.     

Detection of Dobrava-Belgrade hantavirus using recombinant-nucleocapsid-based enzyme-linked immunosorbent assay and SYBR Green-based real-time reverse transcriptase-polymerase chain reaction

Dobrava (DOBV) hantaviruses belong to the genus Hantavirus, family Bunyaviridae, and are carried by yellow-necked and striped field mice. The goal of this study was to detect DOBV using serological and genetic methods in Apodemus rodents in Hungary and in northern Croatia. During the study period, a total of 125 Apodemus sp. (67 A. agrarius, 58 A. flavicollis) were tested for the presence of hantaviruses, and 21 rodents (17%) were positive by rRT-PCR and/or ELISA. We conclude that the prevalence of DOBV is much higher than previously anticipated. The simultaneous use of molecular and serological techniques provides a highly reliable way to detect hantavirus infections.     

Susceptibility of laboratory and wild rodents to Rattus or Apodemus-type hantaviruses.

Adult Wistar rats (Rattus norvegicus), Apodemus agrarius, Meriones unguiculatus, Clethrionomys rufocanus, and Apodemus argenteus were inoculated with Rattus-type (SR-11, KI-262, and TB-314) or Apodemus-type (Hantaan 76-118) hantaviruses. Production of serum antibody to the inoculated virus (IAHA titres of 1:32 to 1:4 096) was obsersved in all rodent species 10 weeks after virus inoculation. Rattus-type virus was detected in some organs of all the rodent species employed except of Apodemus agrarius. Apodemus-type virus was found only in some organs of Apodemus agrarius. Newborn Wistar rats induced antibody in high titres to both Rattus- and Apodemus-type hantaviruses. Rattus-type virus was detected in all the organs examined for up to 6 weeks after inoculation, whereas Apodemus-type virus disappeared from all organs except of brain and lung tissues. The virulence of the three Rattus-type viruses to newborn rats was different. These findings indicate that the susceptibility of rodents may vary depending on the combination of rodent species and virus strains; they also suggest that the various species of rodents may be the reservoir animals of hantavirus infection in nature.     

Serotypic classification of hantaviruses by indirect immunofluorescent antibody and plaque reduction neutralization tests

Antisera prepared against 16 strains of hantaviruses isolated from patients with hemorrhagic fever with renal syndrome (HFRS) or from rodents captured in HFRS-endemic and nonendemic regions were titrated against Hantaan virus strain 76-118, Puumala virus strain Sotkamo, and Prospect Hill virus strain Prospect Hill-I by using the indirect immunofluorescent antibody and plaque reduction neutralization tests. Isolates fell into one of four distinct groups or serotypes. Serotype 1 included Apodemus-derived strains, serotype 2 included Rattus-derived strains, serotype 3 included Clethrionomys-derived strains, and serotype 4 included Microtus-derived strains. Serotypic classification of hantavirus infections was possible for humans and rodents in widely varied geographical areas, but in a few instances, sera from patients with HFRS did not conform to any of the four serotypes, suggesting the existence of as yet unidentified serotypes. A definitive serological classification of hantaviruses must await analysis of additional virus isolates.     

Genetics of hantaviruses: implications to taxonomy

Hantaviruses (genus Hantavirus, family Bunyaviridae) represent a prime example of emerging viruses. Since isolation of the prototype Hantaan virus in the late 70s more than 20 new species have been described and the number is increasing fast thus demanding for a more refined classification. Taking into account that hantaviruses are difficult to isolate in cell culture, one should not be surprised that most of the "newcomers" were first described as distinct hantavirus genotypes. Moreover, the only "solid" characteristics of many hantavirus species still exist in the form of nucleotide sequences of their genome. The relatively short history of hantavirology can thus be taken to illustrate how genetics can contribute to (and even, perhaps, dominate) discovery, characterization and classification of viruses. In this review the following aspects of hantavirus genetics are discussed: (i) genome structure; (ii) genetic diversity and evolution; and (iii) use of genetic criteria in current taxonomy of hantaviruses. In addition, several examples of classification of hantavirus species (New York virus, Saaremaa virus and Hokkaido virus) are given, and future prospects are analyzed.     

Co‐circulation of three pathogenic hantaviruses: Puumala,Dobrava, and Saaremaa in Hungary

Hantaviruses (Bunyaviridae) cause hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus (cardio)pulmonary syndrome (HCPS) in the Americas. HFRS is caused by Hantaan virus (HTNV), Seoul virus (SEOV), Dobrava virus (DOBV), Saaremaa virus (SAAV), and Puumala virus (PUUV). Of those, only HTNV is not present in Europe. In recent years, hantaviruses, described in other parts of Europe, were also detected at various locations in Hungary. To study the genetic properties of Hungarian hantaviruses in detail, sequences of the viral S and M segments were recovered from bank voles (Myodes glareolus), yellow‐necked mice (Apodemus flavicollis), and striped field mice (Apodemus agrarius) trapped in the Transdanubian region. As expected, the sequences recovered belonged, respectively, to PUUV (two strains), DOBV (one strain), and SAAV (one strain). On phylogenetic trees two new Hungarian PUUV strains located within the well‐ supported Alpe‐Adrian (ALAD) genetic lineage that included also Austrian, Slovenian, and Croatian strains. Analysis of the Hungarian SAAV and DOBV genetic variants showed host‐specific clustering and also geographical clustering within each of these hantavirus species. Hungarian SAAV and DOBV strains were related most closely to strains from Slovenia (Prekmurje region). This study confirms that multiple hantaviruses can co‐circulate in the same locality and can be maintained side‐by‐side in different rodent species. J. Med. Virol. 81:2045–2052, 2009. © 2009 Wiley‐Liss, Inc.     

Confirmation of Choclo virus as the cause of hantavirus cardiopulmonary syndrome and high serum antibody prevalence in Panama

Choclo virus (CHOV) was described in sigmodontine rodents, Oligoryzomys fulvescens, and humans during an outbreak of hantavirus cardiopulmonary syndrome (HCPS) in 1999–2000 in western Panama. Although HCPS is rare, hantavirus‐specific serum antibody prevalence among the general population is high suggesting that CHOV may cause many mild or asymptomatic infections. The goals of this study were to confirm the role of CHOV in HCPS and in the frequently detected serum antibody and to establish the phylogenetic relationship with other New World hantaviruses. CHOV was cultured to facilitate the sequencing of the small (S) and medium (M) segments and to perform CHOV‐specific serum neutralization antibody assays. Sequences of the S and M segments found a close relationship to other Oligoryzomys‐borne hantaviruses in the Americas, highly conserved terminal nucleotides, and no evidence for recombination events. The maximum likelihood and maximum parsimony analyses of complete M segment nucleotide sequences indicate a close relationship to Maporal and Laguna Negra viruses, found at the base of the South American clade. In a focus neutralization assay acute and convalescent sera from six Panamanian HCPS patients neutralized CHOV in dilutions from 1:200 to 1:6,400. In a sample of antibody‐positive adults without a history of HCPS, 9 of 10 sera neutralized CHOV in dilutions ranging from 1:100 to 1:6,400. Although cross‐neutralization with other sympatric hantaviruses not yet associated with human disease is possible, CHOV appears to be the causal agent for most of the mild or asymptomatic hantavirus infections, as well as HCPS, in Panama. J. Med. Virol. 82:1586–1593, 2010. © 2010 Wiley‐Liss, Inc.     

Host switch during evolution of a genetically distinct hantavirus in the American shrew mole (Neurotrichus gibbsii)

A genetically distinct hantavirus, designated Oxbow virus (OXBV), was detected in tissues of an American shrew mole (Neurotrichus gibbsii), captured in Gresham, Oregon, in September 2003. Pairwise analysis of full-length S- and M- and partial L-segment nucleotide and amino acid sequences of OXBV indicated low sequence similarity with rodent-borne hantaviruses. Phylogenetic analyses using maximum-likelihood and Bayesian methods, and host-parasite evolutionary comparisons, showed that OXBV and Asama virus, a hantavirus recently identified from the Japanese shrew mole (Urotrichus talpoides), were related to soricine shrew-borne hantaviruses from North America and Eurasia, respectively, suggesting parallel evolution associated with cross-species transmission.     

An efficient in vivo method for the isolation of Puumala virus in Syrian hamsters and the characterization of the isolates from Russia

Puumala virus (PUUV) and other Arvicolinae-borne hantaviruses are difficult to cultivate in cell culture. To isolate these hantaviruses efficiently, hantavirus nucleocapsid protein (NP)-positive but seronegative wild rodents were selected by NP-detection ELISA. Three of 68 Myodes glareolus captured in Samara, Russia, were NP-positive and seronegative. Syrian hamsters were inoculated with lung homogenates from NP-positive rodents for virus propagation. Virus isolation in vitro was carried out by inoculation of lung homogenates of NP-positive hamsters to Vero E6 cell monolayers. Two PUUV strains (Samara49/CG/2005 and Samara94/CG/2005) from M. glareolus were isolated in Vero E6 cells. Nucleotide and amino acid sequence identities of the S segment of these isolates to those of PUUV F-s808 from a fatal HFRS patient in Samara region were 96.7-99.3% and 99.3-100.0%, respectively. Morphologic features of Vero E6 cells infected with PUUV strain Samara49/CG/2005 were quite similar to those of Hantaan virus-infected cells. Isolation of Hokkaido virus from Myodes rufocanus captured in Hokkaido, Japan, was also performed. Hokkaido virus NP and RNA were recovered and maintained in hamsters. These results suggest that inoculation of Syrian hamsters with rodent samples is an efficient method for the isolation and maintenance of PUUV and other Arvicolinae-borne hantaviruses.     

Genetic and serotypic characterization of Sin Nombre-like viruses in Canadian Peromyscus maniculatus mice

In Canada, hantavirus infected deer mice (Peromyscus maniculatus) have been collected from British Columbia to Newfoundland. Partial sequencing of G1 and N protein encoding regions from Canadian Peromyscus maniculatus-borne hantaviruses demonstrated the existence of significant genotypic divergence among strains. Phylogenetic analysis showed that Sin Nombre (SN)-like viruses from eastern and western Canadian deer mice can be divided into at least two broad-based genogroups. Sequencing of mitochondrial DNA from infected deer mice originating from various eastern and western provinces showed that SN-like virus genogroups appeared to be associated with distinct haplotypes of mice. Sera from deer mice infected with eastern and western viral genotypes neutralized the Sin Nombre virus strain, Convict Creek 107, but not the New York 1 hantavirus. Despite the genetic heterogeneity of Canadian SN-like strains these hantaviruses do not appear to define unique hantavirus serotypes.     

New and Old World hantaviruses differentially utilize host cytoskeletal components during their life cycles

Recently we reported that the N protein of the Old World hantavirus, Hantaan (HTNV), traffics on microtubules to the ER-Golgi intermediate compartment (ERGIC) prior to its movement to the Golgi and requires an intact ERGIC for viral replication. We have extended these studies to the New World hantaviruses, Andes virus (ANDV) and Black Creek Canal virus (BCCV), and an additional Old World hantavirus, Seoul virus (SEOV). These studies support microtubule-dependent trafficking of the N protein to ERGIC within the perinuclear region for the New and Old World hantaviruses. However, we observed that early entry events were distinct for HTNV and ANDV with respect to the pathway for entry and the dependence on an intact actin (ANDV) versus microtubule (HTNV) cytoskeleton for viral replication. These studies show for the first time that while Old and New World hantaviruses share common features in their pathways, they have evolved differences in their interaction with host cell machinery.