Detection of phylogenetically distinct Puumala-like viruses from red-grey vole Clethrionomys rufocanus in China

In order to investigate whether Puumala virus (PUUV) or PUUV-like virus is present in China, Clethrionomys rufocanus and C. rutilus were captured in the Jilin province during the spring and autumn of 2002-2003 for detection of PUUV viral RNA by RT-PCR and confirmation of PUUV-positive antigens by an immunofluorescence assay. PUUV-positive RNA was identified in six out of 121 C. rufocanus but not in any of the 41 C. rutilus. Complete S and partial M sequences (nt 1,316-1,598 and 2,687-3,089) were amplified by RT-PCR directly from some of the antigen positive lung tissues and subjected to nucleic acid sequencing. It was found that the Chinese PUUV-like viruses were related most closely with the PUUV strains with 77.7-81.7% identity at the nucleotide level and 91.7-97% identity at the amino acid level for S segment, and with 77-78.8% identity at the nucleotide level and 91.5-92.6% identity at the amino acid level for the partial M segment (nt 1,316-1,598). Genetic analysis indicated that the Chinese PUUV-like viruses shared the highest level of identity with the viruses which circulate in C. rufocanus in the Far East region of Russia with 85.1-87.4% identity at the nucleotide level and 95.9% identity at the amino acid level for the partial M segment (nt 2,687-3,089), respectively. Phylogenetic analysis revealed that the Chinese PUUV-like viruses are distinct from those identified from Japan, South Korea, Europe or Russia. These results indicate that PUUV-like virus is present in China in addition to Hantaan, Seoul and Dabieshan viruses.     

Soochong virus and Amur virus might be the same entities of hantavirus

Amur virus (AMRV) and Soochong virus (SOOV) were reported to be carried by Korean field mice (Apodemus peninsulae) in the Far East of Russia, China, and Korea. The distinction and demarcation between these two viruses have been a matter of debate. In order to clarify this issue and to confirm taxonomic position of AMRV and SOOV, the extensive phylogenetic analyses based on entire S segment and entire M segment sequences of AMRV, SOOV and other reference virus strains deposited in GenBank, were carried out using maximum likelihood and distant matrix methods. All inferred phylogenies revealed that all AMRV strains from China and Far East and SOOV (especially SOO-1/2 strains from Northeastern Korea) shared high identities of nucleotide sequences and were monophyletic distinct from Apodemus agrarius HTNV. Although two genetic sublineages of SOOV exist, these findings revealed that AMRV and SOOV might belong to the same entities of hantavirus.     

在中国发现普马拉型汉坦病毒

目的：确定我国是否存在普马拉（Puumala)病毒。方法：从我国东北地区棕背Bing肺标本中用RT－PCR扩增汉坦病毒S片段基因序列，对所扩增序列进行核苷酸序列测定和分析。结果：从我国东北地区棕背Bing肺标本中扩增出长度为926碱基对的cDNA片段，核苷酸序列测定证实为普马拉病毒S片段序列。与不同型别汉坦病毒代表株进行比较表明，此次发现为新的普马拉病毒株，系统发生分析结果表明，此次发现的病毒与普马拉病毒P360、K27、CG－820、CG－17株种系相近，同源性达到99％以上。结论：我国存在普马拉病毒，我国新发现的普马拉病毒核苷酸序列和俄罗斯远东地区普马拉病毒接近。     

Isolation and genetic characterization of hantaviruses carried by Microtus voles in China

To gain more insights into hantavirus distribution in China, Microtus fortis were caught in Jilin province and M. maximowiczii in the Inner Mongolia Autonomous Region. Hantavirus specific RNA was detected by RT-PCR in 3 out of 26 M. fortis and 5 out of 64 M. maximowiczii. Two hantaviruses (Fusong-Mf-682 and Yakeshi-Mm-59) were isolated successfully in cell culture and their S and M segment nucleotide sequences were determined. Phylogenetic analysis of the S and M segment sequences revealed that the Mf-originated strains from Fusong were closely related to Vladivostok hantavirus (VLAV) with 99% nucleotide identity, but differed from the Yakeshi-Mm strains, with an amino acid divergence of more than 8.8% for the N protein and 11.8% for the GnGc proteins. Yakeshi-Mm strains were closely related to the Khabarovsk hantavirus (KHAV) isolated earlier from M. fortis in Khabarovsk, with an amino acid sequence identity of more than 98.4% for the S segment and 95.6% for the M segment. On phylogenetic trees, Yakeshi-Mm strains clustered together with KHAV and Topografov virus (TOPV) carried by Lemmus sibiricus. The results suggest that the hantavirus carried by M. fortis in China belongs to VLAV type and should be considered as a distinct hantavirus species. They also suggest that M. fortis is the natural host of VLAV (including Fusong-Mf strains), whereas M. maximowiczii is the natural host of KHAV including Yakeshi-Mm strains. Thus, in addition to Hantaan, Seoul, Dabieshan and Puumala-like Hokkaido viruses, at least two other hantaviruses, namely KHAV and VLAV, are circulating in China.     

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

Summary. 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.Received August 29, 2002; accepted March 10, 2003 Published online June 2, 2003     

Rapid detection of genomic variations in different strains of hantaviruses by polymerase chain reaction techniques and nucleotide sequence analysis

The polymerase chain reaction (PCR) with subsequent nucleotide sequence analysis was employed to rapidly detect genomic variations among different Hantavirus strains. Using synthetic oligonucleotide primers derived from the M and S segment RNAs of nephropathia epidemica virus strain H?lln?s B1 (NEV) we succeeded in amplifying the corresponding sequences of Hantaan and Puumala viruses. The nucleotide sequences of the cDNAs derived from the Puumala M and S RNA segments were analyzed. It was found that the particular nucleotide sequences of Puumala M and S segments were 81% and 82% homologous to the corresponding genomic segments of NEV, respectively. The amino acid homology was 94% for both segments. In contrast, the degree of homology to the corresponding Hantaan M and S genomic RNA segments was 63% at the nucleotide level for both segments and 53 and 55% at the deduced amino acid level, respectively. This demonstrates that Puumala virus is very similar to NEV and significantly different from Hantaan virus at both the nucleotide and protein level.     

Soochong virus: an antigenically and genetically distinct hantavirus isolated from Apodemus peninsulae in Korea

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.     

Genetic and antigenic characterization of the Amur virus associated with hemorrhagic fever with renal syndrome

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.     

浙江东方田鼠分离汉坦病毒株S基因的克隆和序列分析

目的对国内首次从东方田鼠分离到的汉坦病毒（HV）ZT10S基因克隆进行序列分析。方法参考GenBank发表的汉坦病毒核蛋白基因序列，设计合成一对引物，提取分离株ZT10细胞培养物的总RNA，对ZT10S基因进行RT-PCR扩增，产物经琼脂糖电泳分析，呈现一条约1．7kb的条带，回收纯化后，将其克隆至pGEM．T载体中，然后进行核苷酸序列的测定及分析。结果与4株SEO型病毒（SR-11、R22、Gu03、8610）核苷酸和氨基酸同源性分别为87．9％～96．0％和96．9％～97．9％，与HTN型病毒株76-118的核苷酸和氨基酸同源性分别为71．3％和81．9％，与其他型汉坦病毒的同源性均很低，表明此分离株ZT10为SEO型汉坦病毒。结论为进一步研究其进化和变异提供了有利条件，也为生产SEO型汉坦病毒特异性核蛋白抗原，免疫血清学诊断试剂的制备和分子生物学研究奠定了基础。     

Genetic characterisation of a Hantavirus isolated from a laboratory-acquired infection

Seoul (SEO) viruses belong to the Hantavirus genus (family Bunyaviridae), cause the moderate form of haemorrhagic fever with renal syndrome, and have been associated with laboratory-acquired infections in many countries. To investigate the pedigree of an isolate, designated IR461, which was obtained from a laboratory-acquired infection in a UK research institute, we determined the nucleotide sequences of the small (S) and medium (M) genome segments. In addition, we determined the sequences of the S segments of two Chinese isolates (R22 and L99) and an American isolate (Tchoupitoulas [TCH]). The S segments range within 1769-1785 nucleotides in length and showed identities of >88% in nucleotide sequence and 97% in amino acid sequence to those of published S segment sequences. The M RNA segment of IR461 is 3651 nucleotides long and shows >84% identity at the nucleotide level and >98% at the amino acid level to the M segments of other SEO viruses. These data confirm that SEO viruses show the least diversity within the Hantavirus genus. Phylogenetic analyses of the sequences showed geographic clustering of the Chinese SEO viruses, and that IR461 was more closely related to SEO viruses isolated in the New World than to those from the Far East.     

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

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

Characterization of Tula Virus from Common Voles (Microtus Arvalis) in Poland: Evidence for Geographic-Specific Phylogenetic Clustering

Tula virus (TULV), a recently identified arvicolid rodent-borne hantavirus, is harbored by the European common vole (Microtus arvalis) in Central Russia and the Czech and Slovak Republics. We report the isolation and characterization of this hantavirus from M. arvalis captured in Poland, a country where human disease caused by hantaviruses has not been recognized. Of 34 arvicolid rodents (24 Clethrionomys glareolus, 9 M. arvalis, 1 Pitymys sp.) captured in Lodz and Tuszyn, Poland, during June to September 1995, sera from 3 M. arvalis and 3 C. glareolus contained IgG antibodies to Puumala virus (PUUV), as determined by an indirect immunofluorescent antibody assay. Alignment and comparison of the 1852-nucleotide S segment and a 1676-nucleotide region of the G2 glycoprotein-encoding M segment, amplified from lung tissues of two hantavirus-seropositive M. arvalis, revealed 83.9-85.2% and 82.3-83.5% sequence similarity, respectively, with TULV strains from Central Russia and the Czech and Slovak Republics. A > 98% sequence conservation was found at the amino acid level. Phylogenetic analysis indicated that the newly found TULV strains from Poland were closely related to, but distinct from, TULV from elsewhere in Europe.     

Coding strategy of the S and M genomic segments of a hantavirus representing a new subtype of the Puumala serotype

Summary The hantavirus strain Vranica was previously reported to have been isolated from a bank vole in Bosnia-Hercegovina and associated with the occurrence of hemorrhagic fever with renal syndrome (HRFS) in humans. The complete cDNA nucleotide sequences of the small (S) and medium (M) genomic RNA segments of this virus were determined. Major open reading frames were found in the S and M segment between nucleotide positions 43 and 1341 coding for a polypeptide of 433 amino acid residues and between nucleotide positions 41 and 3 484 coding for 1 148 amino acid residues, respectively. The analysis and the alignment of the nucleotide and the derived amino acid sequences with known sequences of other hantavirus strains demonstrate that Vranica resembles Swedish strains and represents a new virus subtype of the Puumala serotype distinct from the subtypes represented by virus strains CG18–20 and Sotkamo.The nucleotide sequence data reported in this paper have been deposited in Gen Bank under accession numbers U14137 and U14136.     

Molecular characterization of hantavirus Zhejiang isolate ZT10 strain from M. fartis]

OBJECTIVE: To learn about the complete genomic sequence of the Seoul virus strain ZT10 isolated from M. fartis. METHODS: The total RNA was extracted from the infected Vero E6 cells and amplified by RT-PCR. The purified PCR products were cloned into T-vector and sequenced. RESULTS: The results demonstrated that the complete genome of ZT10 was comprised of L(6530), M(3651) and S(1753) segments which encoded 2151-1133 and 429 amino acids respectively. CONCLUSION: Analysis of sequence revealed that the ZT10 belonged to Seoul virus. The nucleotide sequence identity of the M gene with Seoul virus was 84.0%-96.3%. The identity with Hantan vrisu (Prospect Hill virus, Tula virus) isolated from M. fartis was 57.5%-60.9%. The sequence identity of the S gene with Seoul virus was 87.9%-96.0% at nucleotide level and 96.9%-97.9% at amino acid level.     

Genetic Characterization of the M RNA Segment of Crimean-Congo Hemorrhagic Fever Virus Strains Isolated in Russia and Tajikistan

The data on the structure of the M genome segment of CCHF virus strains from Russia and Central Asia (Tajikistan) are presented. Data obtained have been compared with other available published sequences of the middle segment of strains from China, Nigeria, and Pakistan. It has been found that all the known strains can be divided into four genetic groups, based on the nucleotide sequence of the M genome segment and an amino acid sequence of the glycoprotein precursor it encodes, whereas VLG/TI29414 and STV/HU29223 strains from Russia form a separate group. The CCHF virus strain from Tajikistan, TADJ/HU8966, was genetically related to strains 7803 and 75024 from China, and together with these and the Nigerian IbAr 10200 strain, it forms another group.     

Dobrava hantavirus causes hemorrhagic fever with renal syndrome in central Europe and is carried by two different Apodemus mice species

In central Europe, hemorrhagic fevers with renal syndrome (HFRS) in humans are caused by the hantavirus species Puumala (transmitted by voles) and a second, Hantaan-related species (transmitted by mice). The second virus could be identified as Dobrava virus. To date, 19 clinical cases of Dobrava infection have been found in Germany and Slovakia. All patients exhibited a mild/moderate clinical course and no case fatality occurred. Screening for infected rodents revealed that the striped field mouse (Apodemus agrarius) represents the main reservoir for Dobrava virus in central Europe. Nucleotide sequence comparisons and phylogenetic analysis based on complete and partial genomic S segment nucleotide sequences placed the Slovakian A. agrarius-derived hantavirus strains within the Dobrava species, forming a cluster on the Dobrava phylogenetic tree. In east Slovakia, a single Dobrava virus-infected yellow-necked mouse (Apodemus flavicollis) was trapped in a locality that predominantly showed Dobrava-infected A. agrarius. Comparison of the S segment sequence (nucleotides 381-935) revealed that the Dobrava strain from A. flavicollis shows only 84.3% nucleotide homology to A. agrarius-derived strains from this location but 96.3% homology to A. flavicollis-derived Dobrava strains from the Balkans (southeast Europe). Phylogenetic analysis of the partial S segment placed the A. flavicollis-derived Dobrava strain from Slovakia on a distinct Dobrava lineage (DOB-Af) together with the south-east European A. flavicollis-derived strains. The results indicate that Dobrava strains from A. agrarius (DOB-Aa) vs. A. flavicollis (DOB-Af) could develop different degrees of virulence in humans.     

Complete Nucleotide Sequence of a Mumps Virus Genotype I Strain Isolated in Korea

The complete nucleotide sequence of mumps virus isolated in Korea, Dg1062/Korea/98 (Dg1062), was determined. As other mumps viruses, its genome was to be 15,384 nucleotides (nts) in length and encoded seven proteins. The both 5' and 3' ends were confirmed to be 55 and 24 nts by RACE method, respectively. The full-length nucleotide sequence of Dg1062 isolate differed from other strains by 2.9-6.8% in the nucleotide sequence level, resulting in 206 nucleotide and 54 amino acid substitutions which were observed in only Dg1062 isolate relative to the consensus sequences of other strains. Despite the variations of amino acids over the full genome including HN gene, it might be considered that this isolate have no significant variations in the antigenic sites. This result is the first report of full-length genome of genotype I strain and provides an overview on the diversity of genetic characteristics of circulating mumps virus.     

Molecular genetic analysis of a diversity of Getah virus strains isolated from mosquitoes in the North-Eastern Asia

A molecular genetic study was performed of the Getah virus strains isolated in Russia (Eastern Siberia and Far East) and Mongolia. A phylogenetic analysis was made, by examining the nucleotide sequences of genome fragments that had been obtained by RT-PCR and that included the portions of the E1 and E2 surface glycoprotein genes and the 6K gene. A genetic diversity of Getah virus strains in North-Eastern Asia is discussed.     

一株新型戊型肝炎病毒的部分克隆及分析

目的　对得自北京地区一例急性散发性戊型肝炎病人病毒 (ChinaT)作基因克隆分析。方法　用逆转录套式聚合酶链反应方法从患者粪便中克隆病毒并做核苷酸序列分析。结果　ChinaT株与 4株典型中国株HEV(ChinaA、ChinaB、ChinaC、ChinaD)、缅甸株、墨西哥株、美国株、非洲的乍得株核苷酸 /氨基酸同源性分别为 78% /92 %、78% /92 %、79% /90 %、78% /94%、76 % /92 %。结论　ChinaT株有较高的基因异质性 ,与其它HEV株有很大的不同 ,是一新型HEV。