Seroepidemiological Survey of Hantavirus Infection of Wild Rodents Trapped from 1994 to 1998 in Korea

Hantaviruses belong to the genus Hantavirus and Hantaan, Seoul, Puumala, Belgrade and Sin Nombre viruses are the etiolgic agents of two serious hantaviral diseases of humans. The rodent hosts and the specific etiologic agents of haemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) are known and many reported cases occurred in Eurasia and Americas. Wild rodents trapped in 13 different areas of Korea from 1994 to 1998 were investigated against hantavirus infection. A total of 718 wild rodents and 10 species were trapped and found 630 (87.7%) of them were Apodemus agrarius. Indirect immunofluorescent antibody technique (IFAT) was performed for hantaviruses infections using different hantavirus antigens. Hantavirus antibodies were found in 68 (10.8%) out of 630 A. agrarius, 8 (42.1%) of 19 Rattus norvegicus. Among 68 lungs and other tissues of antibody positive A. agrarius, 5 (7.4%) were antigen positive. IFA titers of 5 positive A. agrarius sera showed higher titers against Puumala or Sin Nombre viruses than Hantaan virus. These results suggest that there may be are possibilities of existence of a noble hantavirus in Korean wild rodents.

Molecular Characterization of the L Segment of Hantann Virus, Strain Howang

Hantaan virus (HTNV), the etiologic agent of hemorrhagic fever with renal syndrome (HFRS), belongs to the genus Hantavirus, and has three single negative straded RNA genome segments. HTNV strain Howang isolated from the blood of severe case of Korean HFRS is more virulent than HTNV 76/118 and the M and S genome segments' nucleotide sequence of Howang strain showed 93.5% and 94% homology to each segment of HTNV 76/118. We have obtained 6533 nucleotides long sequence of the L genome segment of Howang strain using reverse transcriptase in conjunction with PCR amplification and compared to other hantaviruses. The messenger sense of the L segment contains one long single long open reading frame of 2151 amino acids, which encodes a deduced RNA dependent RNA polymerase of 246.4 kDa caculated molecular weight protein. The nucleotide sequence of the Lsegment of Howang strain shows 93%, 74%, 66%,65% homology to HTNV 76/118, Seoul virus 80/39, Puumala virus Hallnas B1 and Sin Nombre virus, respectively. The amino acid sequence of the L segment of Howang strain shows 99%, 85%, 68%, 68% homology to HTNV 76/118, Seoul virus 80/39, Puumala virus Hallnas B1 and Sin Nombre virus, respectively.

Immune Reaction of the Vaccinated Hamsters with Combined Hantaan-Puumala Vaccine

A large number of viruses belonging to Genus Hantavirus in Family Bunyaviridae are etiologic agents for hemorrhagic fever with renal syndrome (HFRS), or hantavirus pulmonary syndrome (HPS). Hantaan (HTN), Seoul (SEO), Belgrade (BEL), Puumala (PUU) serotype viruses are well known causative agents for HFRS in Eurasian continent. Among those viruses Hantaan and Seoul serotypes are well known to cause HFRS in Korea, but there are some sporadic incidence by other than Hantaan or Seoul viruses. Recently we have developed the combined Hantaan-Puumala virus vaccine to prevent world-wide occurring HFRS. This combined vaccine is formalin inactivated, suckling mouse and suckling hamster brain extracts for Hantaan and Puumala viruses, respectively. Protein contents of this purified candidate vaccine is 27 microgram/ml, which contains 1,024 ELISA antigen units to each virus, but content of myelin basic protein which is causing experimental allergic encephalomyelitis is legs than 0.1 ng/ml. Thirty hamsters were given twice at one month interval intra-muscularly and bled on 30 days after each vaccination from retro-orbital sinus vein. Antibody titers were tested against 5 major serotype viruses, Hantaan, Seoul, Belgrade, Puumala and Sin Nombre viruses by IFA and PRNT. The mean IF antibody titers on 30 days after primary shot were 78.4, 68.8, 68.8, 37.9, and 15.6; mean neutralizing antibody titers were 65.4, 12, 6.1, 65.6 and 0.5 against Hantaan, Seoul, Belgrade, Puumala and Sin Nombre viruses, respectively. The mean IF antibody titers on 30 days after booster shot were 686.9, 567.5, 550.4, 516.3, and 430.9; and neutralizing antibody titers were 710.8, 41.9, 24.3, 409.9, and 1.6 against Hantaan, Seoul, Belgrade, Puumala and Sin Nombre viruses, respectively.

Discrimination of Hantaviruses from the Tissues of Infected Hamsters to 5 Different Serotype Hantaviruses by Nested RT-PCR using Hantavirus Serotype Specific Primers

We developed a sensitive, nested reverse transcription-polymerase chain reaction (RT-PCR) to detect Hantaan, Seoul, Belgrade, Puumala and Sin Nombre viruses in animal tissues. Total RNA was extracted from blood, lung or kidney samples of experimentally-infected hamsters by using the guanidine isothiocyanate buffer-acid phenol-chloroform method. Genus-reactive outer primers were derived from the consensus region of the G1 gene sequences of several hantaviruses. Serotype-specific primers were selected within the region amplified by the outer primers. To examine the sensitivity and specificity of the test, we diluted known quantities of Hantaan, Seoul, Belgrade, Puumala and Sin Nombre viruses in human or hamster immune sera before performing the nested RT-PCR. We could detect as little as 1 pfu of virus, even in the presence of high-titer neutralizing antibodies, and the serotype-specific primers amplified only homologous serotype viruses. RT-PCR with these primers demonstrated virus in the blood of experimentally-infected hamsters as early as four days to as late as 30 days after infection.4 comparison of a standard immunofluorescent antibody screening test (IFAT) to nested RT-PCR with RNA extracted from lung or kidney tissues of the hamsters, demonstrated that RT-PCR to be more sensitive for identifying viruses in these tissues.

A Case of Hantavirus Pulmonary Syndrome / 결핵

Hantavirus pulmonary syndrome(HPS) is a systemic disease that is caused by a newly discorved and characterized virus of the Hantavirus genus, which is most frequently referred to as the sin nombre virus. The clinical syndrome resembles other hantavirus syndromes worldwide, except that it is characterized by a brief prodromal illness followed by rapidly progressive, noncardiogenic edema, and that it is more deadly than any previously recognized hantavirus infection. The clinical manifestations of HPS are characterized by four clinical phases prodrome, pulmonary edema and shock, diuresis, and convalescence. Mortality is greatest in the first 24 hours of the pulmonary edema and shock phase of the illness. These phases are strikingly similar to the clinical phases of Hemorrhagic fever with renal syndrome(HFRS) induced by Hantaan virus, except that HPS has not been associated with renal failure and Disseminated intravascular coagulation(DIC). We here report a case of hantavirus pulmonary syndrome developed in a 58 year-old man. He had a flu-like illness followed by the rapid onset of respiratory failure due to noncardiogenic pulmonary edema. HPS was diagnosed by clinical manifestations, identification of high titer antibody to Hantaan virus antigen and histologic finding of transbronchial lung biopsy (TBLB) specimen. The patient was treated with mechanical ventilation and initial corticosteroid pulse therapy resulting in successful outcome.

The Role of alpha5beta1 Integrin on Infection of Hantaan Virus in Fibroblasts

Pathophysiological mechanism of hemorrhagic fever with renal syndrome(HFRS) is not fully understood. Major clinical findings of HFRS patients are widespread hemorrhage, acute renal failure and shock. Basic lesion is vascular injury with microvascular hemorrhage and relatively little inflammation. According to autopsy findings, renal medulla has focal hemorrhage, tubular necrosis and interstitial mononuclear infiltrates. Hantavirus pulmonary syndrome(HPS) caused by Sin Nombre virus which is known as a member of Hantavirus genus, characterized by rapidly progressive pulmonary edema, hypotension and interstitial pulmonary infiltrate with lymphocytes. The predominant cell type in the renal and pulmonary interstitium is a fibroblast and it participates in the healing process at injury site by secreting a large amount of extracellular matrix proteins. Cultured human lung fibroblasts and Mongolian gerbil fibroblasts were known as the good host cells to the hantaan virus. It is possible that not only endothelial cell but also fibroblast is a target of Hantaan virus and the fibroblast might be involved in the pathogenesis and the healing process in HFRS. Integrins are an adhesion molecule, and act as the receptors for many extracellular matrix proteins. Recently, there are many reports that cell surface integrins influence on some viral infections or reversely viruses influence on the expression of integrins. The alpha5beta1, integrin is a major receptor for the fibronectin which is an important extracellular matrix protein secreted by fibroblasts. In this study, the role of alpha5beta1 integrin on the infection of Hantaan virus was examined by using an anti-alpha5beta1 integrin antibody in chicken embryo fibroblasts(CEF) and Mongolian gerbil fibroblasts(MGF). CEF and MGF were treated with anti-alpha5beta1, integrin antibody during 30 minutes before viral infection and the culture supernatants were harvested 5 days after virus infection. The treatment of anti-alpha5beta1 integrin antibody on CEF reduced the viral titers about 30% when compared with the non-antibody treatment control CEF. In MGF, these viral titers were reduced by 36% and 68% according to the assay methods by FFU and ELISA, respectively. These results suggested that alpha5beta1 integrin might be act as a receptor for the Hantaan virus or blocking of alpha5beta1 integrin influence on the viral replication in CEF and MGF. To find out which suggestion is related with, further studies are going on.