Thailand orthohantavirus infection in patients with chronic kidney disease of unknown aetiology in Sri Lanka.

Chronic kidney disease of unknown aetiology (CKDu) reported in Sri Lanka and other countries is a mysterious and serious disease. Recently, we reported a high seroprevalence of antibodies to a hantavirus antigen among CKDu patients in Girandurukotte, Badulla district, Sri Lanka. However, the type of hantavirus with which the residents were infected was not determined. In this study, a total of 89 seropositive sera were examined to identify their serotypes using an indirect immunofluorescent antibody assay, a truncated-N-protein-based enzyme-linked immunosorbent assay, and a cross-neutralization test. These results indicated that the residents in this area were frequently infected with Thailand orthohantavirus or an antigenically related virus.

A survey of rodent-borne pathogens carried by wild Rattus spp. in Northern Vietnam.

To examine the prevalence of human pathogens carried by rats in urban areas in Hanoi and Hai Phong, Vietnam, we live-trapped 100 rats in January 2011 and screened them for a panel of bacteria and viruses. Antibodies against Leptospira interrogans (22·0%), Seoul virus (14·0%) and rat hepatitis E virus (23·0%) were detected in rats, but antibodies against Yersinia pestis were not detected. Antibodies against L. interrogans and Seoul virus were found only in adult rats. In contrast, antibodies to rat hepatitis E virus were also found in juvenile and sub-adult rats, indicating that the transmission mode of rat hepatitis E virus is different from that of L. interrogans and Seoul virus. Moreover, phylogenetic analyses of the S and M segments of Seoul viruses found in Rattus norvegicus showed that Seoul viruses from Hai Phong and Hanoi formed different clades. Human exposure to these pathogens has become a significant public health concern.

Hantavirus species in India: a retrospective study.

Hantaviruses cause hemorrhagic fever with renal syndrome in Europe and Asia. There are about 20 documented hantavirus species and newer species are being described worldwide, especially in non-rodent reservoirs, i.e shrews. Focus reduction neutralization test is the classical serotyping technique for hantavirus. However, this study employs a previously established serotyping ELISA, to retrospectively analyze known hantavirus IgG reactive samples for infecting serotypes. The result suggests presence of Thailand virus- like and Hantaan virus -like strains in India.

Prevalence of antibody to hepatitis E virus among wild sika deer, Cervus nippon, in Japan.

We examined 976 sika deer serum samples, 159 liver tissue samples and 88 stool samples collected from 16 prefectures in Japan, and performed ELISA and RT-PCR assays to detect antibodies to HEV and HEV RNA, respectively. Although 25 (2.6%) of 976 samples were positive for anti-HEV IgG, the antibody titers were very low. The OD values ranged between 0.018 and 0.486, forming a single distribution rather than a bimodal distribution, suggesting that the antibody detected in this study was not induced by HEV infection, or that deer have low sensitivity to HEV. HEV RNA was not detected in these samples, also suggesting that deer may not play a role as an HEV reservoir.

A pilot study for serological evidence of hantavirus infection in human population in south India.

BACKGROUND AND OBJECTIVES: Hantaviruses are rodent-borne viruses of the family Bunyaviridae that have been identified as aetiological agents of two human diseases, haemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). There are no reports of hantavirus infections in humans from India, hence this pilot study was undertaken to provide the serological evidence of hantavirus infections in humans in south India. METHODS: Serum samples were obtained from individuals with acute febrile illness and from voluntary blood donors, majority of whom were from south India. Serum samples were tested for anti-hantavirus IgM using a commercial enzyme immunoassay (EIA). Samples found positive by the EIA were tested by an indirect immunofluorescence assay (IFA) using slides coated with Seoul virus (SEOV) infected cells as substrate. RESULTS: Of the 152 serum samples from individuals with pyrexic illness, 23 (14.7%) were positive for anti-hantavirus IgM by EIA. In contrast, only 5.7 per cent of healthy blood donors were positive by this assay. Eighteen of the 22 (82%) EIA-positive samples from patients were positive by the IFA assay. In contrast, only 2 of the 5 (40%) blood donor EIA positive samples were positive in the IFA assay. INTERPRETATION AND CONCLUSION: The finding of this study indicated the possible presence of hantavirus infections in the human population of India presenting both as asymptomatic and symptomatic infections. Further studies need to be done to confirm the findings on a larger sample using molecular techniques.

Epitope analysis of monoclonal antibody E5/G6, which binds to a linear epitope in the nucleocapsid protein of hantaviruses.

Monoclonal antibody E5/G6 recognized a linear epitope common to hantavirus nucleocapsid proteins. Using synthetic peptides, we identified epitope E5/G6 as the 9 mer YEDVNGIRK (NP 165-173), in which D167, G170, I171, and R172 are indispensable. Furthermore, all the peptides synthesized using various hantavirus sequences bound MAb E5/G6 consistently, despite the existence of several amino acid variations in this region. These results indicate that MAb E5/G6 is a useful tool for detecting hantavirus antigen in rodent or patient tissues using Western blotting or other immunohistochemical assays.

Age-dependent hantavirus-specific CD8(+) T-cell responses in mice infected with Hantaan virus.

To investigate age-dependent differences in hantavirus-specific CD8(+) T-cell responses, mice were inoculated with 0.1 50% newborn mouse lethal dose of Hantaan virus (HTNV) at 0, 3, 7, 14, or 35 days after birth. HTNV-specific CD8(+) T cells producing gamma interferon (IFN-gamma) were measured on day 30 after HTNV inoculation. Although no IFN-gamma-producing HTNV-specific CD8(+) T cells were detected in most of the mice inoculated with HTNV on day 0 after birth, most mice inoculated at 3, 7, 14, or 35 days had HTNV-specific CD8(+) T cells. The production of tumor necrosis factor alpha (TNF-alpha) by IFN-gamma-producing CD8(+) T cells and the cytotoxic activity against HTNV-infected target cells were similar in immature and adult mice. However, the number of IFN-gamma-producing HTNV-specific CD8(+) T cells was significantly less in mice inoculated with HTNV at 3 days than in older mice. In addition, a strong correlation between HTNV persistence and a lack of HTNV-specific CD8(+) T cells was observed. These results suggest that mice over 7 days old have the ability to induce functional HTNV-specific CD8(+) T-cell responses that are indistinguishable from the responses of adult mice, and that HTNV-specific CD8(+) T cells are important for clearance of HTNV.

Detection of antibody for the serodiagnosis of hantavirus infection in different rodent species.

Peroxidase-labeled staphylococcal protein A, streptococcal protein G, and antibodies directed against Mus musculus (mouse), Rattus norvegicus (rat), Mesocretus auratus (hamster), and Peromyscus leucopus were examined for their reactivity with immunoglobulin G (IgG) from various rodent species. The purpose of this study was to identify the optimal secondary antibodies or reagents for specific serodiagnosis of hantavirus infection in various rodent species. Using ELISA, a total of 65 sera from 29 rodent species of the family Muridae and one serum sample from family Octodontidae were compared for IgG reactivity with the six different reagents. The results demonstrate that the reactivities of the secondary antibodies and reagents to the sera varied, even among sera from rodents of the same genus. Hantavirus-specific antibody ELISA revealed that hantavirus-infected rodent sera obtained from M. musculus, R. norvegicus, Apodemus agrarius, A. peninsulae, and Bandicota indica bound to the six different conjugates in a similar pattern as that detected in IgG ELISA. These results indicate that the applicability of secondary antibodies and protein A and G should be carefully evaluated before use for serodiagnosis in different rodent species.

Synthesis of Seoul virus RNA and structural proteins in cultured cells.

Seoul virus is a hantavirus that causes hemorrhagic fever with renal syndrome (HFRS). The virion has a tripartite (S, M, and L) negative-stranded RNA genome, which is characteristic of the family Bunyaviridae. However, the molecular basis of virus replication is not well known. We established a Northern blot hybridization (NB) procedure using digoxygenin-labeled RNA probes, to quantitate the hantaviral plus- and minus-strand RNAs separately. Virus RNA replication was analyzed in infected Vero E6 cells. When the Vero E6 cells were infected with Seoul virus strain KI-83-262 (KI) at m.o.i. = 0.25, the plus-strand RNA was detected within 1 h post-infection (hpi), and the minus-strand RNA was detected subsequently. Using laser confocal microscopy, the nucleocapsid protein (NP) was detected within 2 hpi, and accumulated as scattered granules in the cytoplasm until 24 hpi. In contrast, the G2 protein first appeared at 8 hpi, was immediately transported to the Golgi, and accumulated in the Golgi until 24 hpi. Infectious virus particles were released into the medium at 24 h hpi. These findings indicate that hantavirus RNA replication starts with the appearance of NP at 2 hpi, glycoproteins then accumulate gradually in the Golgi, and virion formation is initiated once the viral RNAs and proteins have accumulated.

Serological analysis of hemorrhagic fever with renal syndrome (HFRS) patients in Far Eastern Russia and identification of the causative hantavirus genotype.

Hemorrhagic fever with renal syndrome (HFRS) is endemic in East Asia and Europe. The disease is caused by several viruses belonging to the genus Hantavirus, including the Hantaan virus (HTNV), Seoul virus (SEOV), Dobrava Belgrade virus (DOBV), and Puumala virus (PUUV). Recently, HTNV-related viruses, Amur (AMR) and Far East (FE) genotypes were identified as causative agents of HFRS in Far Eastern Russia. To investigate the epidemiology of HFRS and virus transmission, we collected sera from 17 acute and 32 convalescent patients who were clinically diagnosed with HFRS in the Khabarovsk region of Far Eastern Russia, and detected anti-hantavirus antibodies using an ELISA that can differentiate the infected virus serotype using truncated hantavirus nucleocapsid protein antigen. Sixteen of the 17 acute phase patients had antibodies to hantavirus, and all the positive sera had higher optical densities for HTNV-specific antigen than for SEOV-, DOBV-, or PUUV-specific antigens. The partial M segment of the viral genome was amplified from blood clots from three acute patients by PCR. The nucleotide sequences had closer identities to the FE genotype (>96%) than to the prototype HTNV (88 to 89%) or AMR genotype (81 to 83%). A phylogenetic analysis found that the virus sequences from the patients clustered with the FE type, and were distinct from the AMR type. Thirty-one of 32 convalescent patient sera had antibodies to HTNV-specific antigen. These data suggest that our ELISA system can detect HTNV-specific antibodies to the FE type, which may be responsible for most of the HFRS in Khabarovsk.

No significant differences were observed in the amounts of DNA strand breaks produced by copper between male and female liver cells of long-evans cinnamon (LEC) strain rats.

The amounts of DNA single strand breaks that are oxidative damage produced by copper were examined by comet assay in the liver cells of an inbred strain of Long-Evans Cinnamon (LEC) rats that spontaneously develops fulminant hepatitis. At 4 weeks of age, copper contents in the liver of LEC rats were approximately 30-fold higher than those of WKAH rats that are control rats used in the present study. Copper accumulated in the liver of LEC rats in an age-dependent manner and no significant differences were observed between copper contents in the livers of males and females at each week of age from 4 to 15 weeks. No significant amounts of DNA strand breaks were found in the liver cells of both male and female WKAH rats from 4 to 15 weeks of age. DNA strand breaks were produced in the substantial population of LEC rat liver cells at 10 weeks of age and induced in an age-dependent manner from 10 to 15 weeks of age. The amounts of DNA strand breaks produced by copper accumulation in the liver cells of female LEC rats are not more abundant than those in the cells of male rats, although it has been reported that hepatitis in female rats is more serious than that in male rats.

Epizootiological survey of hantavirus among rodent species in Ningxia Hui Autonomous Province, China.

Hantaviral antibodies were detected in the sera from Apodemus (A.) agrarius and A. peninsulae captured in Ningxia province, China by several different serological diagnostic methods. A total of 409 sera from rodent and insectivore species were collected in 1999 and examined by indirect immunofluorescent antibody assay (IFA). Among them, 19 of 191 (9.9%) sera of A. agrarius and 1 of 13 (7.7%) sera of A. peninsulae were positive for hantaviral antibodies. The other species (Rattus norvegicus, Mus musculus, Cricetulus triton, and Sorex cylindricauda) were negative. The reaction pattern of positive serum was characterized as scattered and granular virus antigens in the cytoplasm of hantavirus infected Vero E6 cells. Some of the A. agrarius sera positive for hantavirus were further examined by Western blotting (WB), enzyme-linked immunosorbent assay (ELISA), and the focus reduction neutralization test (FRNT). By WB, positive sera showed the same specific reaction pattern of baculovirus-expressed recombinant hantaviral nucleocapsid protein, as shown in hantavirus-immune serum. By ELISA, IFA-positive sera showed significantly higher optical densities (around 1.0) than the negative A. agrarius sera. Hantaan type hantavirus was neutralized with the positive sera. These results suggest that A. agrarius have hantavirus infection and may play a role as a reservoir animal for hantavirus in Ningxia Hui Autonomous Province, China.

Epidemiology and epizootiology of hantavirus infection in Japan.

Hantaviruses cause two severe human diseases: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Various rodent species act as animal reservoirs for hantavirus. In Japan, urban rat- (Rattus norvegicus) and laboratory rat-derived human infections were reported during the 1960s and 1970s-1984, respectively. Although no human cases of infection have been reported since 1984, infected urban rats have been found throughout Japan, and infected grey red-backed voles (Clethrionomys rufocanus) have been identified in Hokkaido. These carriers can be considered to be potential sources of human infection. This review examines the epidemiology and epizootiology of this important zoonosis in Japan.

Characterization of in vitro and in vivo antiviral activity of lactoferrin and ribavirin upon hantavirus.

Mechanisms of anti-hantaviral activities of bovine lactoferrin (LF) and ribavirin (Rbv) were investigated. Hantavirus focus formation at 48 hr was 15% of the control in cells treated with 400 microg/ml LF for 1 hr at 37 degrees C prior to viral infection. Post infection treatment with 100 microg/ml Rbv also inhibited the focus formation to 2.5% of the control. Combined LF pre- and Rbv post-infection treatment completely inhibited focus formation. Viral glycoprotein (G2) and nucleocapsid protein (NP) syntheses were delayed in LF pretreated cells up to 24 hr post infection (hpi) but became comparable to the control by 48 hpi. Further, LF inhibited viral shedding at 24 hpi but did not inhibit shedding after 48 hpi. However, Rbv was able to inhibit synthesis of viral proteins, (+) and (-) strand RNAs also inhibited viral shedding after 24 hr. These results suggest that LF inhibits viral adsorption to cells, while Rbv inhibits viral RNA synthesis. For in vivo trials of LF and Rbv, LF pre- and Rbv post-treatment were evaluated in suckling mice infected with hantavirus, of which 7% survived. LF concentrations of 40 and 160 mg/kg administered prior to viral challenge improved survival rates to 15% and 70%, respectively for single administration and 85% and 94%, respectively, for double administration. Rbv concentrations of 25 and 50 mg/kg gave survival rates of 68% and 81%, respectively. This suggests that both LF and Rbv are efficacious in hantavirus infection in vivo.

Truncated hantavirus nucleocapsid proteins for serotyping Hantaan, Seoul, and Dobrava hantavirus infections.

Truncated recombinant nucleocapsid proteins (rNPs) of Hantaan virus (HTNV), Seoul virus (SEOV), and Dobrava virus (DOBV) were expressed by a baculovirus system. The truncated rNPs, which lacked 49 (rNP50) or 154 (rNP155) N-terminal amino acids of the NPs of HTNV, SEOV, and DOBV, were able to differentiate HTNV-, SEOV-, and DOBV-specific immune sera. Recombinant NP50s retained higher reactivities than rNP155s and were proven useful for enzyme-linked immunosorbent assay (ELISA). The ELISAs based on the rNP50s of HTNV, SEOV, and DOBV successfully differentiated three groups of patient sera, previously defined by neutralization tests: 17 with HTNV infection, 12 with SEOV infection, and 20 with DOBV infection. The entire rNP of Puumala virus (PUUV) distinguished PUUV infection from the other types of hantavirus infection. Serotyping with these rNP50s can be recommended as a rapid and efficient system for hantavirus diagnosis.

Mouse Mx2 protein inhibits hantavirus but not influenza virus replication.

The antiviral potential of Mx2 protein remains unknown, because the Mx2 gene in commonly used strains of laboratory mice is nonfunctional. Our previous study showed that functional Mx2 protein in some feral-origin strains was induced upon interferon treatment, was localized in the cytoplasm, and inhibited vesicular stomatitis virus replication. In the present study, we have demonstrated that the embryonic fibroblastic cells from a feral-origin strain (SPR) expressed 74 kDa Mx2 protein, which prevented the accumulation of viral transcripts and proteins of hantaviruses when the Mx2 gene was constitutively expressed in transfected Vero cells. Furthermore, the cells showed significantly lower titers of the virus than control cells. In contrast, influenza virus replication was not affected by the expression of Mx2 protein in the Vero cells.

Microbiological contamination in genetically modified animals and proposals for a microbiological test standard for national universities in Japan.

The Biosafety Committee of the Japanese Association of Laboratory Animal Facilities of National Universities (JALAN) investigated recent episodes of microbiological contamination in genetically modified mice (GMM), and the countermeasures taken when the contaminated GMM were introduced into animal facilities, by questionnaires addressed to 53 animal facilities belonging to JALAN and serological tests. Although almost all of the contaminated GMM were accepted with conditions such as rederivation after or before reception and housing in designated rooms, contamination with a spectrum of microorganisms was demonstrated in GMM transferred domestically and from abroad. In serological tests, Mycoplasma pulmonis, mouse parvovirus, and mouse encephalomylitis virus were detected in GMM transferred from domestic facilities and from abroad. The present results of the questionnaires and serological tests suggest that GMM are highly and widely contaminated with microorganisms compared with mice from commercial breeders. Thus, we propose a microbiological requirement, including microbiological status--excellent, common, and minimum--as a guide for the transfer and procurement of mice and rats in Japan.

Genetic diversity of hantaviruses isolated in china and characterization of novel hantaviruses isolated from Niviventer confucianus and Rattus rattus.

The antigenic and genetic properties of 46 hantaviruses from China, 13 from patients, 23 from rodents, and 10 from unknown hosts, were compared with those of other hantaviruses. The viruses were classified as either Hantaan (HTN) or Seoul (SEO) viruses. A phylogenetic analysis of the partial M (300 bp) and S (around 485 bp) genomes of HTN viruses identified nine distinct genetic subtypes, one consisting of isolates from Korea. The SEO viruses were divided into five genetic subtypes, although they had less variability than the HTN subtypes. There was a correlation between the subtype and province of origin for four subtypes of HTN viruses, confirming geographical clustering. Hantaan virus NC167 isolated from Niviventer confucianus and SEO virus Gou3 isolated from Rattus rattus were the basal clades in each virus. The phylogenetic trees constructed from the entire S and M segments suggested that NC167 was introduced to N. confucianus in a host-switching event. The reactivity of a panel of 35 monoclonal antibodies was almost exactly the same in NC167 and a representative HTN virus and in Gou3 and a representative SEO virus. However, there was a one-way cross-neutralization between them. These results confirm the varied nature of Murinae-associated hantaviruses in China.

Hepatic copper accumulation induces DNA strand breaks in the liver cells of Long-Evans Cinnamon strain rats.

Effects of accumulation of copper and iron on the production of DNA strand breaks were investigated in Long-Evans Cinnamon (LEC) strain rats that spontaneously develop fulminant hepatitis. Copper and iron accumulated in the liver of LEC rats in an age-dependent manner from 4 to 15 weeks. Low-copper food prevented the accumulation of copper in the liver, but did not prevent accumulation of iron. When the amounts of DNA single strand breaks were estimated by comet assay, the number of DNA strand breaks in the liver cells of rats fed standard food increased with age from 4 to 15 weeks. The number of DNA strand breaks in the liver cells from rats fed low-copper food were the same as those of rats at 4 weeks of age. Thus, the copper accumulation in the liver of LEC rats induced DNA strand breaks, but accumulation of iron did not.