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Background. Chronic kidney disease of unknown aetiology (CKDu) is a major public health problem in Sri Lanka, especially among agrarian communities. Although the cause of CKDu is still unknown, hantavirus infection has been proposed as a risk factor. Methods. This study was performed using serological samples collected from two CKDu-endemic areas, Anuradhapura (2010) and Badulla districts (2010 and 2016), and a non-endemic area, Matale (2016) district. The presence of anti-Thailand orthohantavirus IgG antibodies was investigated in serum samples. Hantavirus seroprevalence and demographic data were epidemiologically analysed. Results. Seroprevalence was higher in CKDu patients (40.6-60.0 %) and healthy individuals in CKDu-endemic areas (17.6-25.5 %) than in healthy individuals in non-endemic areas (3.0 %). Statistically significant odds ratios (ORs) for hantavirus infection in CKDu patients were detected in CKDu-endemic areas [ORs: 3.2 and 3.1; 95 % confidence interval (CI): 1.8-5.5 and 1.8-5.2 in Anuradhapura and Badulla districts in 2010; and OR: 4.4, 95 % CI: 2.3-8.5 in 2016 in Badulla district). Furthermore, the OR for hantavirus infection in Badulla district has increased in the last decade from 3.1 (95 % CI: 1.8-5.3) to 4.4 (95 % CI: 2.3-8.5). Conclusion. Hantavirus infection has been prevalent in two distant CKDu-endemic areas since 2010. The observed significant association of hantavirus seropositivity with CKDu indicates a possible role of hantavirus infection in CKDu pathogenesis.

We reported the genetic evidence of circulating hantaviruses from small mammals captured in a chronic kidney disease of unknown etiology (CKDu) hotspot area of Sri Lanka. The high seroprevalence of anti-hantavirus antibodies against Thailand orthohantavirus (THAIV) has been reported among CKDu patients and rodents in Sri Lankan CKDu hotspots. We captured 116 small mammals from CKDu endemic regions in the Polonnaruwa District of Sri Lanka. Seven animals (five out of 11 Mus booduga and two out of 99 Rattus rattus ) were PCR-positive for the hantavirus. A rat-borne sequence was grouped with a THAIV-like Anjozorobe virus. In contrast, Mus -borne sequences belonged to the THAIV lineage, suggesting a novel orthohantavirus species according to the phylogenetic analyses and whole-genome comparisons. Our genetic evidence indicates the presence of two THAIV-related viruses circulating in this CKDu endemic area, suggesting a basis for further investigations to identify the infectious virus in patients with CKDu and the CKDu induction mechanism of these viruses.

Rapid and simple serologic tests that require only a small amount of blood without the euthanization of animals are valuable for microbial control in colonies of laboratory animals. In this study, we developed a multiplex immunochromatographic assay (ICA) for detection of antibodies to Sendai virus (also known as hemagglutinating virus of Japan), hantavirus, and sialodacryoadenitis virus, which are causative agents of major infectious diseases in rats. For this assay, an ICA strip was placed into a microtube containing 150 µl PBS and either 0.75 µl of rat serum or 1.5 µl of whole blood. Binding antibodies were visualized by using anti-rat IgG antibody-conjugated colloidal gold. Under these conditions, the multiplex ICA simultaneously and specifically detected antibodies to multiple antigens. Positive serum samples for each infectious disease were used to evaluate the sensitivity and specificity of the multiplex ICA. The sensitivities of the multiplex ICA for Sendai virus, hantavirus, and sialodacryoadenitis virus were 100%, 100%, and 81%, respectively. No nonspecific reactions were observed in any of the 52 positive sera against heterologous antigens. In addition, 10 samples of uninfected sera did not show any bands except for the control line. These observations indicate high specificity of the multiplex ICA. Moreover, the multiplex ICA could be applied to diluted blood. These results indicate that the multiplex ICA is appropriate for rapid and simple serological testing of laboratory rats.

The infectivity of shrew-borne hantaviruses to humans is still unclear because of the lack of a serodiagnosis method for these viruses. In this study, we prepared recombinant nucleocapsid (rN) proteins of Seewis orthohantavirus, Altai orthohantavirus (ALTV), Thottapalayam thottimvirus (TPMV), and Asama orthohantavirus. Using monospecific rabbit sera, no antigenic cross-reactivity was observed. In a serosurvey of 104 samples from renal patients and 271 samples from heathy controls from Sri Lanka, one patient serum and two healthy control sera reacted with rN proteins of ALTV and TPMV, respectively. The novel assays should be applied to investigate potential infectivity of shrew-borne hantaviruses to humans.

We have reported high seroprevalence to Thailand orthohantavirus (THAIV) or THAIV-related orthohantavirus (TRHV) among patients with chronic kidney disease of unknown etiology in Girandurukotte, Sri Lanka. THAIV or TRHV infection is considered to be transmitted by rodent hosts in this area, but its reservoir rodents have not yet been identified. Hence, 116 rodents were captured, and seroprevalences were examined by indirect immunofluorescent antibody assay (immunofluorescence assay [IFA]) using antigens of THAIV strain Thai749-infected Vero E6 cells and recombinant nucleocapsid protein of THAIV expressed in Vero E6 cell. Molecular biological species identification of rodents was carried out by sequencing rag1 , irbp , and mitochondrial cytb genes. The majority (112/116) of the captured rodents were lineage Ib of black rats ( Rattus rattus ). Among them, 19.6% (22/112) of the rats possessed antibodies against THAIV. Also, a lesser bandicoot rat ( Bandicota bengalensis ), which belongs to the Sri Lankan endemic genetic lineage, was seropositive (1/1). Two Mus booduga and one Murinae sp. were seronegative. Rodent sera showed less cross-reactivities to antigens of Vero E6 cells infected with Hantaan orthohantavirus (HTNV), Seoul orthohantavirus (SEOV), and Puumala orthohantavirus (PUUV) in IFA. These results suggest that the hantavirus present in rodents in Sri Lanka is related to THAIV or TRHV rather than to SEOV, HTNV, or PUUV. However, it might be serologically distinct from the prototype THAIV strain, Thai749, used in this study. This study revealed that black rats and lesser bandicoot rats belonging to Sri Lankan endemic lineages are possible reservoirs for THAIV or TRHV in Girandurukotte. Further multiple geographical studies are needed to confirm the THAIV or TRHV reservoir status of black and lesser bandicoot rats in Sri Lanka.

Chronic kidney disease of unknown etiology (CKDu) imposes a substantial burden on public health in Sri Lankan agricultural communities. High seroprevalences of hantavirus have been reported in CKDu patients in several locations of Sri Lanka. We carried out a cross-sectional study followed by an unmatched case-control comparison in two geographically distinct areas of Sri Lanka, Girandurukotte (CKDu endemic) and Kandy (CKDu non-endemic) to determine whether exposure to hantaviruses is a potential risk factor in patients with kidney disease. An indirect immunofluorescent antibody assay using two antigens, Thailand orthohantavirus-infected and recombinant N protein-expressing Vero E6 cells, were used for serodiagnosis. Participants' demographic and other socio-economic data were collected through a structured questionnaire. Fifty kidney disease patients and 270 controls from Kandy and 104 kidney disease patients and 242 controls from Girandurukotte were examined. Seropositivities were 50% and 17.4% in kidney patients and controls, respectively, in Girandurukotte, and they were 18% and 7% in Kandy. The odds of exposure to hantaviruses were higher for kidney disease patients than for controls in both Girandurukotte (OR:3.66, 95% CI:2.01 to 6.64) and Kandy (OR:2.64, 95% CI:1.07 to 6.54) in binary logistic regression models. According to statistical analysis, individuals exposed to hantaviruses had a higher risk of developing renal impairment. Therefore, hantavirus infection might be an important risk factor for development of kidney disease in Sri Lanka., Competing Interests: The authors declare no conflict of interest.

Hantaviruses are causative agents of two rodent-borne zoonoses, hemorrhagic fever with renal syndrome (HFRS) and nephropathia epidemica (NE) in the Old World and hantavirus pulmonary syndrome (HPS) in the New World. Serological examinations to detect hantavirus antibodies have been most widely used for surveillance among humans and rodent reservoirs. Here, we will review antigenic structure of nucleocapsid (N) protein of hantaviruses and application of recombinant N protein as diagnostic antigen for screening and serotyping., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

Unlabelled: Hantavirus infections are characterized by vascular hyperpermeability and neutrophilia. However, the pathogenesis of this disease is poorly understood. Here, we demonstrate for the first time that pulmonary vascular permeability is increased by Hantaan virus infection and results in the development of pulmonary edema in C.B-17 severe combined immunodeficiency (SCID) mice lacking functional T cells and B cells. Increases in neutrophils in the lung and blood were observed when pulmonary edema began to be observed in the infected SCID mice. The occurrence of pulmonary edema was inhibited by neutrophil depletion. Moreover, the pulmonary vascular permeability was also significantly suppressed by neutrophil depletion in the infected mice. Taken together, the results suggest that neutrophils play an important role in pulmonary vascular hyperpermeability and the occurrence of pulmonary edema after hantavirus infection in SCID mice., Importance: Although hantavirus infections are characterized by the occurrence of pulmonary edema, the pathogenic mechanism remains largely unknown. In this study, we demonstrated for the first time in vivo that hantavirus infection increases pulmonary vascular permeability and results in the development of pulmonary edema in SCID mice. This novel mouse model for human hantavirus infection will be a valuable tool and will contribute to elucidation of the pathogenetic mechanisms. Although the involvement of neutrophils in the pathogenesis of hantavirus infection has largely been ignored, the results of this study using the mouse model suggest that neutrophils are involved in the vascular hyperpermeability and development of pulmonary edema in hantavirus infection. Further study of the mechanisms could lead to the development of specific treatment for hantavirus infection., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Background: Hantaviruses are causative agents of hemorrhagic fever with renal syndrome (HFRS) and nephropathia epidemica (NE) in the Old World and hantavirus pulmonary syndrome (HPS) in the New World. There is a need for time-saving diagnostic methods. In the present study, recombinant N antigens were used as antigens in an immunochromatography strip (ICG) test to detect specific IgG antibodies., Methods: The N-terminal 103 amino acids (aa) of Hantaan virus (HTNV), Puumala virus (PUUV) and Andes virus (ANDV) nucleocapsid (N) protein were expressed in E. coli as representative antigens of three groups (HFRS, NE and HPS-causing viruses) of hantavirus. Five different types of ICG test strips, one antigen line on one strip for each of the three selected hantaviruses (HTNV, PUUV and ANDV), three antigen lines on one strip and a mixed antigen line on one strip, were developed and sensitivities were compared., Results: A total of 87 convalescent-phase patient sera, including sera from 35 HFRS patients, 36 NE patients and 16 HPS patients, and 25 sera from healthy seronegative people as negative controls were used to evaluate the ICG test. Sensitivities of the three-line strip and mixed-line strip were similar to those of the single antigen strip (97.2 to 100%). On the other hand, all of the ICG test strips showed high specificities to healthy donors., Conclusion: These results indicated that the ICG test with the three representative antigens is an effective serodiagnostic tool for screening and typing of hantavirus infection in humans.

Hantavirus is a causative agent of rodent-borne viral zoonoses, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome. Seoul virus (SEOV) is a causative agent of urban and laboratory rat-associated HFRS worldwide. Surveillance of rodents has been done mainly by serological detection of hantavirus-specific antibodies by enzyme linked immunosorbent assay (ELISA) and immunofluorescent antibody assay (IFA). An immunochromatographic (ICG) test was developed with the N-terminal 103 amino acids of nucleocapsid protein of Hantaan virus expressed by Escherichia coli as an antigen to detect IgG antibody specific to hantavirus in sera from Rattus sp. animals. Antibody-detecting sensitivity of the ICG test was the same as that of ELISA and about 100-times higher than that of IFA. Overall sensitivities and specificities of the ICG test in comparison to ELISA and IFA for sera from 192 urban rats and 123 laboratory rats were 99.3% and 100%, respectively. Diluted whole blood samples without separation could be used for the ICG test. The ICG test enabled detection of antibodies to SEOV, Hantaan, Dobrava/Belgrade, and Thailand viruses, which are causative agents of HFRS throughout Eurasia. The ICG test is a rapid, simple and safe method for diagnosis of SEOV infection in rats., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Hemorrhagic fever with renal syndrome (HFRS) is a rodent-borne zoonotic disease caused by hantavirus infection. Many HFRS cases have been reported in East Asia and North Europe, while the situation in Southeast Asia remains unclear. In this study, the prevalence of hantavirus infection in rodents and humans in Thousand Islands regency, which is close to the port of Jakarta, one of the largest historic ports in Indonesia, was investigated. A total of 170 rodents were captured in 2005, and 27 (15.9%) of the rodents were antibody-positive against Hantaan virus antigen in an immunofluorescence assay (IFA) and Western blotting. Despite the high prevalence in rodents, human sera collected from 31 patients with fever of unknown origin and 20 healthy volunteers in the islands in 2009 did not show positive reaction to the antigen in IFA. To identify the virus in rodents genetically, a total of 59 rodents were captured in 2009. Sera from the rodents were screened for antibody by ELISA, and lung tissues were subjected to RT-PCR. 20 (33.9%) of the 59 rodents were antibody-positive, and 3 of those 20 rodents were positive for S and M genome segments of hantaviruses. Genetic analysis showed that the viruses belonged to Seoul virus and formed a cluster with those in Vietnam and Singapore. These results suggest that a unique group of Seoul viruses has spread widely in Southeast Asia.

We developed serological tools for the detection of hantavirus-specific antibodies and hantavirus antigens in shrews. The work was focussed to generate Thottapalayam virus (TPMV)-specific monoclonal antibodies (mAbs) and anti-shrew immunoglobulin G (IgG) antibodies. The mAbs against TPMV nucleocapsid (N) protein were produced after immunization of BALB/c mice with recombinant TPMV N proteins expressed in Escherichia coli, baculovirus and Saccharomyces cerevisiae-mediated expression systems. In total, six TPMV N-protein-specific mAbs were generated that showed a characteristic fluorescent pattern in indirect immunofluorescence assay (IFA) using TPMV-infected Vero cells. Out of the six mAbs tested, five showed no cross-reaction to rodent-associated hantaviruses (Hantaan, Seoul, Puumala, Tula, Dobrava-Belgrade and Sin Nombre viruses) in IFA and enzyme-linked immunosorbent assay (ELISA), although one mAb reacted to Sin Nombre virus in IFA. None of the mAbs cross-reacted with an amino-terminal segment of the shrew-borne Asama virus N protein. Anti-shrew-IgG sera were prepared after immunization of rabbits and BALB/c-mice with protein-G-purified shrew IgG. TPMV-N-protein-specific sera were raised by immunisation of Asian house shrews (Suncus murinus) with purified yeast-expressed TPMV N protein. Using these tools, an indirect ELISA was developed to detect TPMV-N-protein-specific antibodies in the sera of shrews. Using an established serological assay, high TPMV N protein specific antibody titres were measured in the sera of TPMV-N-protein-immunized and experimentally TPMV-infected shrews, whereas no cross-reactivity to other hantavirus N proteins was found. Therefore, the generated mAbs and the established ELISA system represent useful serological tools to detect TPMV, TPMV-related virus antigens or hantavirus-specific antibodies in hantavirus-infected shrews.

New World hantaviruses were divided into five groups based on the amino acid sequence variability of the internal variable region (around 230-302 amino acids) of hantavirus nucleocapsid protein (NP). Sin Nombre virus (SNV), Andes virus, Black Creek Canal virus (BCCV), Carrizal virus (CARV) and Cano Delgadito virus belong to groups 1, 2, 3, 4 and 5, respectively. Patient and rodent sera were serotyped successfully by an enzyme-linked immunosorbent assay (ELISA) with recombinant truncated NP lacking 99 N-terminal amino acids (trNP100) of SNV, CARV and BCCV. The trNP100 of BCCV showed lower reactivity to heterologous sera. In contrast, whole recombinant NP antigens detected both homologous and heterologous antibodies equally. The results together with results of a previous study suggest that trNP100 can distinguish infections among viruses in groups 1, 2, 3 and 4 of New World hantaviruses. The serotyping ELISA with trNP100 is useful for epidemiological surveillance in humans and rodents., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Antigenic diversity among different hantaviruses requires a variety of reagents for diagnosis of hantavirus infection. To develop a diagnostic method applicable to various hantavirus infections with a single set of reagents, we developed an enzyme-linked immunosorbent assay (ELISA) using recombinant nucleocapsid proteins of three hantaviruses, Amur, Hokkaido, and Sin Nombre viruses. This novel cocktail antigen-based ELISA enabled detection of antibodies against Hantaan, Seoul, Amur, Puumala, and Sin Nombre viruses in immunized laboratory animals. In wild rodent species, including Apodemus, Rattus, and Myodes, our ELISA detected antibodies against hantaviruses with high sensitivity and specificity. These data suggest that our novel diagnostic ELISA is a useful tool for screening hantavirus infections and could be effectively utilized for serological surveillance and quarantine purposes.

To investigate the distribution of hantaviruses among animals in Southern and Central Highland area of Vietnam, a total of 1311 serum samples were obtained from rats and Asian house shrews (Suncus murinus) captured at 11 locations between 2006 and 2009. A total of 1066 serum samples from rats were examined for IgG antibodies against Hantaan virus, and there were 30 antibody-positive serum samples from rats that had been captured mainly in a port area and urban area in Ho Chi Minh City (HCMC) (2.8%). All of the antibody-positive rats were Rattus norvegicus, and they had Seoul virus (SEOV) genome in their lungs. SEOV sequences detected from rats captured in Southern Vietnam belonged to the same lineage as those from rats captured at Haiphong Port and a market area in Hanoi City. SEOV strain CSG5 was isolated from a rat captured at Saigon Harbor. Strain CSG5 showed a cross-neutralization pattern almost the same as that of a representative strain of SEOV. A total of 245 Asian house shrews were captured in the Central Highland area and near HCMC. Sera were examined for IgG antibodies against Thottapalayam virus (TPMV), and 32 (13.1%) of the antibody-positive shrews were mainly from the Central Highland area and showed a neutralizing antibody against TPMV. These results indicated that SEOV is distributed among R. norvegicus inhabiting harbor and urban areas of Southern Vietnam and that TPMV or an antigenically related virus is distributed among Asian house shrews in Central Highland area.

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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Truncated recombinant nucleocapsid proteins (trNs) that lack N-terminally located cross-reactive epitopes of four Murinae rodent-associated hantaviruses, Seoul virus (SEOV), Thailand virus, Hantaan virus (HTNV) and Dobrava-Belgrade virus, were produced by using a baculovirus expression system. ELISA with the trNs as antigens enabled serotyping of immune sera from rats experimentally inoculated with the corresponding hantaviruses with cut-off OD values of 60% of those of whole N of HTNV. The trN-based ELISA could serotype 12 out of 13 sera obtained from wild rodents (Rattus norvegicus) naturally infected with SEOV using the 60% cut-off value. These results indicate that screening with whole N followed by serotyping with trNs using a cut-off OD value of 60% of that of whole N is a useful method for serological surveillance of Murinae-associated hantavirus infection among rodents.

A cross-sectional study was undertaken to determine the current prevalence of leptospirosis and hantaviral infections, and the socio-demographic characteristics and risk factors of infected patients, in Kandy, Sri Lanka. This report discusses the serological evidence of hantavirus infections among 105 suspected leptospirosis patients, 8 of whom had hantavirus antibodies. Serotyping ELISA showed that these 8 patients had high optical density values for Thailand virus. Most of the sera showed that the focus reduction neutralization test titer against Thailand virus was higher than that against Seoul virus, thereby suggesting that the hantaviral antibodies found in Sri Lanka are different from Seoul virus but closely related to Thailand virus. These findings imply that the hantaviral infection found in Kandy, Sri Lanka appears to be due to a virus similar to Thailand virus. Epidemiological analysis revealed that the association between hantavirus infection and socio-demographic characteristics was not statistically significant.

Sin Nombre virus (SNV), Andes virus (ANDV), and Laguna Negra virus (LANV) have been known as the dominant causative agents of hantavirus pulmonary syndrome (HPS). ANDV and LANV, with different patterns of pathogenicity, exist in a sympatric relationship. Moreover, there is documented evidence of person-to-person transmission of ANDV. Therefore, it is important in clinical medicine and epidemiology to know the serotype of a hantavirus causing infection. Truncated SNV, ANDV, and LANV recombinant nucleocapsid proteins (trNs) missing 99 N-terminal amino acids (trN100) were expressed using a baculovirus system, and their applicability for serotyping SNV, ANDV, and LANV infection by the use of enzyme-linked immunosorbent assays (ELISA) was examined. HPS patient sera and natural-reservoir rodent sera infected with SNV, ANDV, and LANV showed the highest optical density (OD) values for homologous trN100 antigens. Since even patient sera with lower IgM and IgG antibody titers were serotyped, the trN100s are therefore considered useful for serotyping with early-acute-phase sera. In contrast, assays testing whole recombinant nucleocapsid protein antigens of SNV, ANDV, and LANV expressed in Escherichia coli detected homologous and heterologous antibodies equally. These results indicated that a screening ELISA using an E. coli-expressed antigen followed by a serotyping ELISA using trN100s is useful for epidemiological surveillance in regions where two or more hantavirus species cocirculate.

The distribution of anti-hantavirus antibodies in humans and rodents in northern Vietnam was examined. In total, 837 serum samples from healthy humans (617) and patients with fever (220), living in six different areas were screened for IgG antibodies against Hantaan or Seoul virus (SEOV) by ELISA, IFA, and Western blot analysis. Antibody-positive sera were identified in 7/617 (1.1%) healthy donors, 5/150 port workers in the port of Hai Phong, and 2/185 residents of Ha Nam Province. In comparison, positive sera were detected in 5/220 (2.3%) fever patients in the provinces of Ha Nam (1/58) and Thanh Hoa (4/146). Antibody-positive Rattus norvegicus were found in the provinces of Ha Nam (7/52) and Thanh Hoa (1/67), in Haibatrung District (7/43) in Hanoi, and in Hai Phong Port (21/62), while antibody-positive R. rattus (2/17) were found in Hai Phong Port. Part of the Gc region from the viral genome was amplified by RT-PCR using lung tissue samples from R. norvegicus in Haibatrung (2/7) and Hai Phong Port (7/9), but not from R. rattus (0/2). Viral sequences were located in the SEOV clade and formed a single lineage with Indonesian SEOV, suggesting that Vietnamese SEOV is part of a distinct lineage among Asian SEOVs.

To distinguish Thailand virus infection from infections with other hantaviruses, we established an ELISA serotyping system using a truncated nucleocapsid protein of Thailand virus lacking 49 amino acids at the N-terminus. In evaluations using patient and rodent sera, Thailand virus infection was readily distinguished from Hantaan and Seoul virus infections. Therefore, this ELISA system is an effective alternative to neutralization tests.

Hantaviruses are etiological agents of hemorrhagic fever with renal syndrome in many parts of Asia and Europe. There has been no documented case of hantavirus disease from India, although serological evidence exists. We investigated the prevalence of hantavirus in the Indian population and tried to identify potential risk groups for hantavirus infections. The presence of hantavirus-specific IgG antibodies was prospectively evaluated in 661 subjects belonging to different groups, i.e. patients with chronic renal disease, warehouse workers and tribal members engaged in rodent trapping. Healthy volunteer blood donors were included as a control group. Thirty-eight seropositive samples were found using a combination of a commercial ELISA followed by an indirect immunofluorescence assay. Western blot using recombinant Hantaan virus nucleocapsid antigen confirmed the presence of anti-hantavirus IgG in 28 (74%) of the 38 sera tested. This study confirms the presence of hantaviruses in India and warrants increasing awareness of the problems of emerging pathogens and the threats they may pose to the public health system.

Hantaviruses are enveloped RNA viruses that belong to the Hantavirus genus of the family Bunyaviridae. These viruses persistently infect their rodent reservoirs without causing disease. The virus is transmitted to humans via the inhalation of infectious aerosols generated from contaminated animal secretions or through the contaminated saliva of animal bites. Hantaviruses cause haemorrhagic fever with renal syndrome in Euro-Asia, and hantavirus pulmonary syndrome (HPS) in North and South America. Here, we review the epidemiology and epizootiology of hantavirus infection in Asian countries.

Hantaviruses are causative agents of some severe human illnesses, including hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). The viruses are maintained by rodent hosts, and humans acquire infection by inhaling virus-contaminated excreta from infected animals. To examine the epidemiology of hantavirus infections in Japan and Far East Russia, we conducted epidemiological surveys in these regions. In Japan, anti-hantavirus antibodies were found in four rodent species, Clethrionomys rufocanus, Rattus norvegicus, R. rattus, and Apodemus speciosus. Although no new HFRS cases have been officially reported over the past 20 years in Japan, one member of the Japan Ground Self-Defense Force did test positive for hantavirus antibody. Repeated surveys in Far East Russia have revealed that two distinct hantavirus types cause severe HFRS in this region. Hantavirus sequences identified from A. peninsulae, fetal HFRS cases in Vladivostok, and Amur virus are highly similar to each other (> 92% identity), but they are less similar (approximately 84% identity) to the prototypical Hantaan virus, which is carried by A. agrarius. Phylogenetic analysis also indicates that Amur and A. peninsulae-associated viruses are distinct from Hantaan virus, suggesting that A. peninsulae is the reservoir animal for Amur virus, which causes severe HFRS. From HFRS patients in the Khabarovsk region, we identified viruses with nucleotide sequences that are more similar to Far East virus (> 96%identity) than to the Hantaan (88-89% identity) or Amur (81-83% identity) viruses. Phylogenetic analysis also indicates that the viruses from Khabarovsk HFRS patients are closely related to the Far East virus, and distinct from Amur virus.

Hokkaido virus (HOKV) is a member of the genus Hantavirus, in the family Bunyaviridae. To investigate HOKV infection in the host Myodes rufocanus, the grey red-backed vole, 199 animals were captured at Tobetsu (October 2004 and July 2005) and Nakagawa (October 2004) in Hokkaido, Japan, for detection of antibody, antigen, and viral RNA. In the surveys in Tobetsu (2004) and Nakagawa (2004), seropositive animals were detected at a frequency of 6.0% (5/84) and 10.4% (5/48), respectively. No seropositive animals were detected in Tobetsu in 2005. Seroprevalence in males in Tobetsu and Nakagawa in 2004 was 25% (1/4) and 45.5% (5/11), respectively, which was higher than in females, at 5.0% (4/80) and 0% (0/37), respectively (P<0.01). These results suggest that male animals play an important role in the maintenance of HOKV in M. rufocanus. Two females were seronegative but viral RNA-positive, indicating that these animals had acute infections before antibody was produced. Another five infected animals in Nakagawa were all male and had high levels of antibodies and viral RNA, suggesting that they had persistent infections. Viral RNA copies in organs of infected animals in Nakagawa were quantified by real-time polymerase chain reaction. Two acutely infected animals had > or = 10 times the number of RNA copies in their lungs compared to those of persistently infected animals. In most cases, lungs or spleen had the highest RNA copy number, regardless of infection status.

We examined whether a vesicular stomatitis virus (VSV) pseudotype bearing the hantavirus envelope glycoproteins (GPs) G1 and G2 (VSVdeltaG*HTN) could be used as a safe and effective alternative to native hantavirus. Mice were immunized with purified particles of VSVdeltaG*HTN. After the second immunization, all mice produced anti-GP antibody as detected in ELISA and a neutralization test. After the third immunization, the mice were challenged with Hantaan virus. Neither anti-NP antibody production nor Hantaan virus-specific CD8 T-cell reactions were detected in these mice. The present study demonstrated the potential of using a pseudotype VSV system as a tool for developing a hantavirus vaccine.

Hantaan (HTN) virus, the etiologic agent of clinically severe hemorrhagic fever with renal syndrome (HFRS), was first isolated in 1976 from lung tissue of a striped-field mouse (Apodemus agrarius) captured in Songnae-ri, Gyeonggi Province, Republic of Korea. Found primarily in mountainous areas, the Korean field mouse (A. peninsulae) is the second-most dominant field rodent species found throughout Korea. A new hantavirus, designated Soochong (SOO), was isolated in Vero E6 cells from four A. peninsulae captured in August 1997 at Mt. Gyebang in Hongcheon-gun, Mt. Gachil, Inje-gun, Gangwon Province, and in September 1998 at Mt. Deogyu, Muju-gun, Jeollabuk Province. The entire S, M, and L genomic segments of SOO virus, amplified by RT-PCR from lung tissues of seropositive A. peninsulae and from virus-infected Vero E6 cells, diverged from HTN virus (strain 76-118) by 15.6%, 22.8%, and 21.7% at the nucleotide level and 3.5%, 9.5%, and 4.6% at the amino acid level, respectively. Phylogenetic analyses of the nucleotide and deduced amino acid sequences, using the maximum parsimony and neighbor-joining methods, indicated that SOO virus was distinct from A. agrarius-borne HTN virus. SOO virus shared a common ancestry with Amur virus from Far East Russia, as well as with H5 and B78 hantaviruses, previously isolated from HFRS patients in China. Cross-focus-reduction neutralizating antibody tests showed that SOO virus, which is the first hantavirus isolated in cell culture from A. peninsulae, could be classified as a new hantavirus serotype., (Copyright 2005 Wiley-Liss, Inc.)

The virulence of hantaviruses that are antigenically related but have different genetic characteristics from the prototype of hantavirus, Hantaan (HTN) virus, was examined in newborn mice. The H5 and B78 strains of the Amur (AMR) genotype, the Bao14 strain of the Far East (FE) genotype, and the 76-118 strain of HTN virus were inoculated subcutaneously (1focus-forming unit; FFU) into newborn mice. All of the AMR and FE genotype viruses inoculated mice were died by 16 days post-infection (dpi) and 21 dpi, respectively, while 50% of the HTN virus inoculated mice survived until 30 dpi. The AMR and FE genotype viruses inoculated mice had high viral titers in the lung (1.3x10(6) to 1.3x10(8) FFU/gram [g] tissue) , brain (2.1x10(7) to 1.2x10(9) FFU/g tissue), and kidney(2.5x10(5) to 1.6x10(7) FFU/g tissue), and showed a detectable level of antibodies (titers 1:16-1:32) at 14 dpi. In contrast, the HTN virus infected mice had viruses only in the lungs at low titers (1.1-5.3x10(5) FFU/g tissue). Observations of body-weight changes revealed that the AMR and FE genotype viruses inoculated mice had lower growth rates than the HTN virus inoculated mice. These data suggest that the AMR and FE genotype viruses are more virulent than the HTN virus in newborn mice.

Epizootiological surveys on hantavirus infections in rodents were carried out in various areas of Japan, including the four major islands of Hokkaido, Honshu, Shikoku, and Kyushu from 2000 to 2003. A total of 1,221 rodents and insectivores were captured. Seropositive animals were found in Apodemus (A.) speciosus (5/482, 1.0%), Rattus (R.) norvegicus (4/364, 1.1%), R. rattus (3/45, 6.7%), and Clethrionomys (C.) rufocanus (7/197, 3.6%). The partial S segment was amplified from one seropositive R. rattus captured at Hakodate. The nucleotide sequence showed 96% identity with the Seoul virus (SEOV) prototype strain SR-11. In addition, we conducted an epidemiological survey on human hantavirus infection in a high-risk population, the personnel of the Japan Ground Self-defense Force on Hokkaido. One out of 207 human blood samples was positive for anti-hantavirus antibody by IFA, ELISA, and WB analysis. The result of the serotype specific ELISA indicates that this individual acquired SEOV infection. This study indicates that A. speciosus, R. norvegicus, R. rattus, and C. rufocanus carry hantaviruses as the reservoir animals in Japan. Infected R. rattus and R. norvegicus in port areas could be the sources of human SEOV infection and a threat to travelers and individuals working in seaports.

A recombinant nucleocapsid protein of Hantaan virus (HTN) 76-118 strain expressed in E. coli was applied as a serodiagnostic antigen in an enzyme-linked immunosorbent assay (rHTN-ELISA) for detection of hantavirus antibody in rat sera. The sensitivity and specificity of the rHTN-ELISA were compared with those of the indirect immunofluoresent assay (IFA) using virus-infected cells. The sensitivity of rHTN-ELISA was similar to that of the IFA both in experimentally SR-11 infected rat and naturally infected rat sera. Sera showing a low antibody titer in IFA and suspected to be negative by other methods were also found to be negative in rHTN-ELISA. These results indicate that rHTN-ELISA is effective as a screening method for serodiagnosis of hantaviruses, because of its high sensitivity, specificity, safety and suitability for processing large number of samples.

In an epizootiologic survey of 122 rodents captured in Vladivostok, Russia, antibodies positive for hantavirus were found in Apodemus peninsulae (4/70), A. agrarius (1/39), and Clethrionomys rufocanus (1/8). The hantavirus sequences identified in two seropositive A. peninsulae and two patients with hemorrhagic fever with renal syndrome (HFRS) from the Primorye region of Far East Russia were designated as Solovey and Primorye, respectively. The nucleotide sequences of the Solovey, Primorye, and Amur (obtained through GenBank) sequences were closely related (>92% identity). Solovey and Primorye sequences shared 84% nucleotide identity with the prototype Hantaan 76-118. Phylogenetic analysis also indicated a close relationship between Solovey, Primorye, Amur, and other viruses identified in Russia, China, and Korea. Our findings suggest that the Korean field mouse (A. peninsulae) is the reservoir for a hantavirus that causes HFRS over a vast area of east Asia, including Far East Russia.

The genetic and antigenic characteristics of the Amur (AMR) and Far East (FE) virus lineages, which are both within the genus Hantavirus, were studied. Representative viruses, H5 and B78 for AMR and Bao 14 for FE, were used. The entire small (S) and medium (M) segments, except for the 3′- and 5′-ends, were sequenced. The deduced amino acid sequences of AMR had 96.7 and 92.0–92.2% identities with the Hantaan (HTN) virus in the S and M segments, respectively. The amino acid sequences of FE had 99.1 and 97.9% identities in the S and M segments, respectively. The three viral strains and HTN virus had similar binding patterns to a panel of monoclonal antibodies (MAbs), except that one MAb did not bind AMR. However, sera from Apodemus peninsulae, naturally infected with AMR virus, neutralized homologous viruses at 1:160 to 1:320 dilutions and HTN at 1:20 to 1:40 dilutions. The anti-AMR serum neutralized homologous viruses at a 1:80 dilution and HTN at a 1:40 dilution. The anti-HTN serum did not neutralize AMR (<1:40 dilution), although it had a high neutralizing titer (1:320) against the homologous virus. Therefore, we suggest that AMR virus may constitute a distinct serotype within the genus Hantavirus. [Copyright &y& Elsevier]