First detection of group C rotavirus in children with acute gastroenteritis in South Korea.

Group C rotavirus (GpC RV) causes sporadic cases and outbreaks of acute diarrhoea in humans worldwide, but has not been detected among children in South Korea. The present study aimed to detect GpC RV among children hospitalized with gastroenteritis in South Korea and to perform a molecular characterization of GpC RV strains. From November 2003 to January 2006, 434 faecal samples were collected from children <10 years of age who were hospitalized for treatment of acute diarrhoea and screened for group C and A rotaviruses by enzyme immunoassay. GpC RV strains were characterized by sequence and phylogenetic analysis.Of the 434 samples screened, two were positive for GpC RV and one had a mixed GpC and GpA RV infection. One of the strains, Icheon, shared high sequence conservation in VP4, VP6 and VP7 genes with other published GpC RV. This is the first report describing the molecular characteristics of GpC RV among children in South Korea. Additional surveillance is needed to determine the burden of GpC RV gastroenteritis.

A dominant antigenic region of the hantaan virus nucleocapsid protein is located within a amino-terminal short stretch of hydrophilic residues.

The nucleocapsid (N) protein of the Hantaan virus (HTNV) is a major viral antigen that induces a strong antibody response during the acute phase of infection. By immunoblot analyses of the recombinant N proteins using human sera of the hemorrhagic fever with renal syndrome (HFRS), we have confirmed previous finding by other investigators of the presence of a highly antigenic region near the amino terminus of the HTNV N protein. We have further located the antigenic region within a short stretch of hydrophilic sequences between the 26 and the 46th amino acid residues. The recombinant glutathione S-transferase fusion proteins containing this region was expressed as a soluble form in a large quantity in Escherichia coli, and purified by a single-step affinity chromatography. The recombinant antigen also showed a similar, but a weaker reactivity with human antisera to Seoul virus (SEOV), the virus most closely related to HTNV.

Molecular genetic analysis of reovirus isolated in Korea.

Reovirus isolates from human, striped-field mouse (Apodemus agrarius) and Korean field mouse (A. peninsulae) in Korea showed extensive variability in the patterns of electrophoretic migration of the double-stranded RNA (dsRNA) genome segments. Hemagglutination inhibition (HI) test was performed for serotype determination of 12 reovirus isolates. To clarify genetic diversity and molecular phylogeny of Korean reoviruses, L1, S3 and S4 genomic segments of reoviruses were amplified by RT-PCR and directly sequenced. Among 12 reovirus strains, 9 strains were type 3 and 3 strains were type 2. The L1 was highly conserved showing 91.5-100%, 94.7-100% similarities among Korean isolates, and 77.5-97.9%, 92.6-96.8% similarities compared to other reference strains of each genotypes at nucleotide and amino acid levels, respectively. In S3 and S4 segments, 84.4-99.3%, 72.3-99% nucleotide sequence similarities and 92-99.3%, 89.1-98.4% amino acid sequence similarities among Korean isolates were observed, and 70.8-93.9%, 72.3-98.7% nucleotide sequence similarities and 81.8-100%, 88.3-97.7% amino acid sequence similarities compared with other reference strains of each genotype were observed, respectively. Phylogenetic analyses based on the S3 and S4 nucleotide sequences indicate that genotypes of reovirus are more related with geographic differences rather than host species or date of isolation.

Molecular epidemiology of hepatitis A virus in Korea.

BACKGROUND: The prevalence of antibodies for hepatitis A virus (anti-HAV) in adolescents and young adults has decreased remarkably following the economic growth in Korea. As a result, this age group has a high risk for HAV infection paradoxically, and over 1500 cases of clinically overt hepatitis A occurred in 1998. Human isolates of hepatitis A virus (HAV) are categorized within four genotypes (I, II, III, and VII). In some geographic regions, closely related isolates cluster, suggesting endemic spread of the virus, while in other regions multiple genotypes circulate. Virtually no data are available with regard to the genetic relatedness of Korean strains of HAV. METHODS AND RESULTS: A 168 base pair segment encompassing the putative VP1/2A junction of the HAV genome was amplified by RT-PCR and sequenced in sera of 18 Korean patients with a sporadic form of acute hepatitis A. Pairwise comparisons of the nucleic acid and amino acid sequences of 18 Korean isolates with one another revealed that the Korean isolates showed > 94.6% and > 96.4% identity, respectively. All of the 18 Korean isolates clustered within genotype IA, irrespective of the geographic locations and the time that hepatitis occurred. Unique amino acid sequence changes that had never been reported in genotype IA were found in nine of the 18 isolates. These changes were Gln-->Ser and Lys-->Arg in 2A-19 and 2A-10 amino acid positions. CONCLUSION: The presence of single genotype and unique mutations may be related with the circulation of endemic HAV over a long period of time in Korea.

Genetic diversity of Apodemus agrarius-borne hantaan virus in Korea.

Hantaan (HTN) virus, the etiologic agent of clinically severe hemorrhagic fever with renal syndrome (HFRS), was first isolated in 1976 from lung tissues of striped field mice (Apodemus agrarius) captured in Songnae-ri, Kyungki-do, Korea. To clarify the genetic diversity and phylogenetic relationship among Korean strains of HTN virus, viral sequences of the partial S and M segments were amplified from lung tissues of 24 seropositive striped field mice captured between 1989 and 1998 at 11 sites in South Korea. The 771-nucleotide (nt) S segment sequences (coordinates 432 to 1202) of HTN virus strains from Yangju-kun differed by 10 to 40 nt (1.3 to 5.2%) from virus strains from Pocheon-kun, Songnae-ri and Nonsan-kun. Similar degrees of genetic variation were found in the G1 and G2 glycoprotein-encoding M segment. Phylogenetic trees, based on the partial S and M segments and generated by the maximum parsimony and neighbor-joining methods, demonstrated that virus strains from various geographic regions in South Korea showed a tendency to form two phylogenetic subgroups and were evolutionarily distinct from HTN virus strains from the People's Republic of China.

Phylogenetic analysis of the small hydrophobic (SH) gene of mumps virus in Korea: identification of a new genotype.

Viral RNAs extracted from fifteen mumps virus isolated from throat swab, saliva, blood, urine or CSF during mumps epidemics between 1997-1998 in Korea were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) and compared by nucleotide sequencing of the small hydrophobic (SH) gene. The deduced amino acid sequences of the SH gene were aligned with the published sequences of mumps virus isolated in different geographic areas. A comparison of the SH gene of mumps viruses in Korea indicated 96.2-100% and 91.2-100% similarity at the nucleotide and amino acid levels, respectively. Phylogenetic analysis, using the neighbor-joining method, showed that Korean mumps virus strains formed a genetically distinct monophyletic group from previously reported genotypes based on the 315-bp length nucleotide and 57 deduced amino acid sequences of the SH gene, and possibly be designated as a new genotype (I).

Infection rate of Leptospira interrogans in the field rodent, Apodemus agrarius, in Korea.

Leptospirosis has significantly decreased in Korea since 1988, following the leptospiral vaccination programme initiated in 1988. Whether this wholly explains the decreased incidence is uncertain. As an initial step to answer this question, infection rates of Leptospira interrogans in field rodents, Apodemis agrarius, were examined and compared with previous data. Two hundred and twenty-two A. agrarius were captured during October-December 1996. Spirochaetes were isolated from 22 (9.9%) and leptospiral DNA was detected in an additional 6 rodents (12.6%). Subsequent microscopic agglutination tests (MAT) classified all these isolates as L. interrogans serogroup Icterohaemorrhagiae serovar lai. The above data did not significantly differ from previous surveys in 1984-7. There was no significant change of L. interrogans infection in field rodents following the introduction of the vaccination programme in Korea. Further studies are needed to determine the role of human vaccination in reducing incidence.

Genetic and phylogenetic analyses of hantaviral sequences amplified from archival tissues of deer mice (Peromyscus maniculatus nubiterrae) captured in the eastern United States.

The S and M segments of a hantavirus, enzymatically amplified from tissues of Cloudland deer mice (Peromyscus maniculatus nubiterrae) captured during 1985 in West Virginia, diverged from strains of Four Corners virus from the southwestern United States by more than 16% and 6% at the nucleotide and amino acid levels, respectively. Phylogenetic analysis suggested that this virus strain (designated Monongahela) forms a possible evolutionary link between the Four Corners and New York hantaviruses.

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 Corners (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.

Serological evidence of human Hantavirus infection in Argentina, Bolivia and Uruguay.

A serological survey was conducted in 1985-1987 to determine the presence of infection for Hantavirus in the general population in Argentina, Uruguay, Paraguay and Bolivia, as well as among rodent-exposed laboratory workers in Argentina. Out of 748 individuals tested by immunofluorescence 20 proved positive for Hantaan virus 76/118 strain of whom 16 also reacted against Seoul virus 80/39 strain and 2 against Puumala virus Sotkamo strain. Ten out of 72 Argentine laboratory workers were positive for the first 2 viruses by ELISA, immunofluorescence and/or plaque reduction neutralization test, in 4 of whom recent infection was demonstrated by IgM antibody presence. Inapparent Hantavirus infection was thus demonstrated for the first time in 2.7% of regional inhabitants, together with 13.9% among rodent-exposed laboratory workers. Our data established the existence of human Hantavirus infection nearly 10 years before the recognition of clinical cases of hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome in Argentina.

Comparison of nucleotide sequences among hantaviruses belonging to the same serotype: an analysis of amplified DNA by thermal cycle sequencing.

The hantavirus genus, belonging to the bunyaviridae family, is comprised of at least four serologically distinct types: Hantaan, Seoul, Puumala and Prospect Hill. Previously, we reported the use of the polymerase chain reaction (PCR) for grouping hantavirus isolates by using four sets of primers specific to each serotype. Our PCR typing results agreed with those of serological typing. The present study makes use of thermal cycle sequencing to sequence PCR-amplified DNA products in order to determine the level of similarity among members of the same serotype. We show that members of Hantaan and Seoul serotypes are over 92% homologous, irrespective of their host and geographical origin. Puumala sequences show a degree of homology ranging from 80 to 98%. Despite the variation in sequence at the nucleotide level, amino acids show an even higher level of conservation.

Identification of Hantaan virus-related structures in kidneys of cadavers with haemorrhagic fever with renal syndrome.

The etiologic agent of haemorrhagic fever with renal syndrome (HFRS), Hantaan virus, was first isolated in 1976. Since then numerous Hantaan-like viruses have been isolated and five serotypes of Hantavirus have been recognized. Serological studies indicate that these viruses are globally distributed, with each serotype occurring in specific areas. Hantaan virus has been intensively studied antigenically, biochemically, and genetically. However there is still a paucity of information on the pathogenesis of Hantaan virus in the human host. In this paper, we report the detection by thin section immune electron microscopy of the occurrence of numerous dense precipitates, typical inclusion bodies, a surface antigen layer, as well as Hantaan virion-like structures in the kidneys of patients that died during the acute phase of HFRS. These findings may shed some light on understanding the pathogenesis of HFRS in target organs most affected by the disease, such as the kidneys.

[Trombiculid mites (Acarina: Trombiculidae) from Apodemus agrarius coreae caught at Pochun-gun, Gyeonggi Province and Chinhae-City, Kyongnam Province].

During October-December 1987, chigger mites infested on the striped field mice (Apodemus agrarius coreae) collected at Pochun-gun, Gyeonggi Province and Chinhae city, Kyongnam Province were identified. 1. Of 171 back-striped mice collected, chigger mites were found from 58 mice, showing 33.9% of infestation rate. 2. Total 865 chigger mites collected were classified into seven species; Leptotrombidium pallidum (4.3%), L. palpale(23.0%), L. orientalis(20.2), L. zetum(19.0%). Neotrombicula tamiyai(32.9), N. japonica(0.2%) and Euschongastia koreaensis(0.3%). Though the most dominant species in number was N. tamiyai(32.9% of the total), this species was collected at very limited locality and for a short period. Therefore, most common species in Korea seems to be L. palpale which was found at all localities throughout the whole survey period. L. pallidum which is known as the vector species of tsutsugamushi disease in Korea was collected in a very low number(4.3% of the total chiggers collected). 3. The number of chigger mites infested on a host animal showed great variations from one chigger up to 207 chiggers. The present study has shown that there may be other Leptotrombidium species mite(s) for the vector and host of tsutsugamushi disease in Korea.

Leakey virus: a new hantavirus isolated from Mus musculus in the United States.

A hantavirus, designated Leakey virus, was isolated from a Mus musculus captured in Real County, Texas, U.S.A. in August 1986. Virus-specific fluorescence was first detected 13 days after inoculation of Vero-E6 cells with spleen tissue from the seropositive M. musculus. Ultrastructurally, the new isolate resembled other hantaviruses. Leakey virus induced a fatal meningoencephalitis in infant Fischer rats, with viral antigen detectable in brain, lung, liver, kidney and spleen. Serum dilution, plaque reduction neutralization tests indicated that Leakey virus was antigenically distinct from Hantaan, Seoul, Puumala and Prospect Hill viruses, and therefore constitutes a new serotype.

Serological survey of Prospect Hill virus infection in indigenous wild rodents in the USA.

We found serological evidence of infection with Prospect Hill virus, a Hantaan-like virus isolated from meadow voles (Microtus pennsylvanicus), in microtine and cricetid rodents trapped in Maryland, West Virginia, Minnesota and California, USA. Fluorescent antibodies were detected in sera from M. pennsylvanicus (74/277), M. californicus (39/185), Clethrionomys gapperi (5/51), Peromyscus maniculatus (4/22) and P. truei (1/11). Sera from seropositive P. maniculatus contained neutralizing antibodies against Prospect Hill virus, confirming that infection with Prospect Hill virus or antigenically related viruses is not restricted to microtine rodents in the USA. Despite the widespread distribution of Prospect Hill virus in indigenous rodents, the recent demonstration that American mammalogists are only rarely infected supports the view that the overall risk of Prospect Hill virus infection in man is low.

Positive serological evidence that Hantaan virus, the etiologic agent of hemorrhagic fever with renal syndrome, is endemic in Canada.

Hemorrhagic fever with renal syndrome is endemic in most parts of the world. The etiological agent in known as Hantaan virus. More than 2000 Canadian sera from all age groups and provinces were tested for the presence of antibody to this virus by the indirect immunofluorescent antibody technique. The frequency of seropositivity ranged from 0.3 to 4.0%, with the national average being 1.4%. Newfoundland and New Brunswick in the Atlantic region had the highest positivity, being higher than hyperendemic areas in Korea. The inland provinces of Ontario, Manitoba, and Alberta had only 0.5% positives to Hantaan virus. In contrast, approximately 1.5% of sera from Saskatchewan, Quebec, and British Columbia were positive. No correlation in positivity was found between geography or sex, but positivity did increase with age. The highest titres were found in the group aged 65 to 93 years.

Isolation of Hantaan virus, the etiologic agent of Korean hemorrhagic fever, from wild urban rats.

Urban rats captured in Seoul and four nearby Korean cities were found to have immunofluorescent antibodies reactive with Hantaan virus, the etiologic agent of Korean hemorrhagic fever (KHF). Serum antibodies were detected in 13% from 477 Rattus norvegicus and 11% of 47 Rattus rattus. Hantaan viral antigen was found in pulmonary tissues of 42 animals, and Hantaan virus was recovered from 23 rats, all but two of which were R. norvegicus. Wistar rats were qualitatively much more sensitive than Apodemus agrarius rodents for isolation of virus from tissues of wild rats. Wistar rats inoculated with one of these strains had virus in lung and spleen for at least 75 days. These results document the existence of an urban cycle for Hantaan virus, which had been suspected on the basis of the occurrence of sporadic urban cases in humans of KHF, and suggest that Rattus-borne Hantaan virus may be widely distributed in urban centers.

Intraspecific transmission of Hantaan virus, etiologic agent of Korean hemorrhagic fever, in the rodent Apodemus agrarius.

Experimental parameters of infection and intraspecific transmission of Hantaan virus, the etiologic agent of Korean hemorrhagic fever, in Apodemus agrarius rodents were determined. Mice inoculated by the intramuscular route experienced viremia for about 1 week beginning on day 7. After 3 weeks, immunofluorescent and neutralizing antibodies were present and no mouse ever developed signs of acute illness. Virus was recovered from lung, kidney, salivary gland, and liver, and virus excretion in urine, saliva, and feces occurred from about day 10 through day 360 (urine) post-inoculation. Antigen, but not infectious virus, was persistent in lung tissue for as long as 1 year. Horizontal contact infection occurred among cage-mates regardless of sexual pairing up to 360 days after infection and no evidence for participation of ectoparasitic arthropods in such transmission was obtained.

Observations on natural and laboratory infection of rodents with the etiologic agent of Korean hemorrhagic fever.

Studies were conducted to define the natural host range of the Korean hemorrhagic fever (KHF) agent in South Korea, and to identify colonized rodents susceptible to this infection. Eight species of field rodents were captured in areas of Korea endemic for KHF and their tissues were examined by immunofluorescence for the presence of KHF antigen. One hundred and fourteen of 817 Apodemus agrarius coreae captured between 1974 and 1978 had one or more positive organs. No positive organ was found in 239 rodents of the other seven species examined. Two hundred and thirty-eight specimens of Apodemus agrarius jejuensis captured on Jeju Island, an area thought to be free of disease, were also negative. Attempted laboratory infection of nine species of rodents captured in the field but maintained in the laboratory was successful only in the two subspecies of Apodemus. The 46 specimens of A. a. jejunesis tested in this manner were all uniformly susceptible to infection as determined by immunofluorescence. Serial sacrifice of experimentally infected A. a. jejuensis revealed viremia of short duration terminating on day 10 postinfection. In contrast, other tissues of this animal, including lung, kidney, liver and parotid gland were positive on day 10 and remained so through the 100-day observation period. When 12 species of colonized laboratory rodents were inoculated with KHF agent five were found to develop KHF antibody by indirect immunofluorescence and two, Calomys callosus and Apodemus agrarious ningpoensis, developed detectable KHF antigen in their tissues.