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Objective: To analyze the viral genetic characteristics of hantaviruses carried by Microtus maximowixzii in Yakeshi of Inner Mongolia Autonomous Region and its relationship with Hantaan virus (HTNV) and Seoul virus (SEOV) viruses as well as to identify the natural host of Khabarovsk virus (KHAV)., Methods: HV specific RNAs were detected by RT-PCR. Complete S and M segment were amplified from the RNA-positive samples. Phylogenetic analysis were performed to estimate the genetic characterization and the relationship with other hantaviruses., Results: Fifty two Microtus maximowixzii voles were captured in Yakeshi areas. Of those voles, hantaviral RNA was tested positive in 5 samples (9.62%). Complete S and M segments sequences were obtained from 5 and 2 lung samples, respectively. The complete S segment was consisted of 1848 to 1861 bp, and the M segment consisted of 3662 bp. These viruses were closely related to each other with 92.5% - 96.4% for the S segment sequences and 88.9% - 95.4% for the M segment sequences. They shared a higher identity with KHAV found previously in Yakeshi and KHAV of Russia. However, they were obviously different from the other hantavirus species. The 5 strains had the consistent secondary structure of nucleocapsid protein (NP) and glycoprotein (GP). When further comparing their secondary structures with those of HTNV and SEOV, our results indicated that there were no obvious differences in NP between KHAV and both HNTV, SEOV but with obvious difference in GP. Based on the S and M segment sequences, phylogenetic analyses revealed that these 5 strains clustered together with KHAV and formed a distinct lineage. Furthermore, all known KHAV strains could be divided into two small branches with a nucleotide divergence more than 5.3%., Conclusion: Our research data revealed that KHAV was highly endemic among Microtus maximowixzii in Yakeshi area which supported the notion that Microtus maximowixzii had been the natural host of KHAV in the area.

Objective: To investigate the infection and genotype of hantaviruses in rodents from Wuhan area, Hubei province., Methods: Rodents were trapped in fields and residential areas of Xinzhou and Jiangxia districts of Wuhan in autumn and winter seasons, from 2000 to 2003 and from 2009 to 2011. Trapped rodents were identified, and hantavirus antigens were detected in the lung tissues with indirect immunofluorescence assay (IFA). Partial S segment sequences were amplified with RT-PCR in hantavirus antigen positive samples and then sequenced. Phylogenetic tree was constructed to analyze the genetic characteristics of hantaviruses., Results: From 2000 to 2003, 437 rodents were trapped, with 24 (5.49%) lung tissues showed hantavirus antigen positive. From 2009 to 2011, 173 rodents were trapped and 7 (4.05%) were hantavirus antigen positive. Rattus norvegicus were the dominant species of rodents. Partial S segment sequences were amplified from 22 samples with Hantaan and Seoul viruses specific primers and sequenced. Partial S segments of Seoul viruses (nucleotide 588 - 1147) were amplified from 17 rodents (13 R. norvegicus and 4 Apodemus agrarius). Seven of these sequences belonged to 3 genetic lineage, while two novel genetic lineages were formed by 9 and 1 sequences, respectively. Partial S segments of Hantaan viruses (nucleotide 615 - 1141) were amplified from 5 A. agrarius. One of these sequences belonged to 7 genetic lineages, and 4 sequences formed one novel genetic subtype., Conclusion: Hantaan and Seoul viruses co-circulated in Wuhan area, Hubei province. Novel genetic lineages were identified in this study and Seoul virus might have caused spillover infection in A. agrarius.

Objective: To investigate the situation of the natural infection of hantaviruses (HV) in small mammals and to provide evidence for the control and prevention of hemorrhagic fever with renal syndrome (HFRS) in Longquan area, Zhejiang province., Methods: Small mammals were captured by night trap, and lung tissue samples were collected and stored in liquid nitrogen. HV antigens were detected by indirect immuno-fluorescence assay (IFA). The partial S genome segment sequences were amplified by RT-PCR. DNAStar program was used for editing and comparing the sequences. Phylogeny was analyzed through PAUP*4.0 software., Results: 319 small animals were collected in Longquan, and 9 hantavirus antigen-positive samples were identified. The positive rate of hantavirus in Apodemus agrarius was 4.97%. Phylogenetic tree constructed by partial S segment (620 - 999 nt) showed that the 9 strains carried by A. agrarius from Longquan all belonged to HTNV, and had a closer evolutionary relationship with isolate Z251 from Zhejiang province., Conclusion: Our results indicated that the main host was A. agrarius and the infection rate of HTNV was high in Longquan area.

Objective: Genetic analysis was performed to infer the relationship between hantaviruses carried by Rattus norvegicus from Henan and Neimenggu provinces and the other known hantavirus and the vaccine strain., Methods: Total RNA was extracted from lung tissues with Trizol reagent. The complete M and S segment sequences of strains NM133 and Q12 were amplified by RT-PCR. The purified DNA fragments were directly subjected to sequencing, and then to sequence analysis and phylogenetic analysis., Results: The complete S segment sequences of strains NM133 and Q12 were found to be 1770 nt and 1772 nt in length respectively, with one open reading frame encoding 429 amino acids. The complete M segment sequences of both two strains are 3654 nucleotide in length encoding a protein of 1133 amino acids. The two strains shared a high degree of homology with most of known Seoul virus (SEOV) but quite different from Hantaan virus and other hantaviruses. Furthermore, the nucleoprotein and glycoprotein of the two strains had the congruent structure with the vaccine strain Z37. On the S- and M-phylogenetic trees, both strains (NM133 and Q12) were grouped into the first cluster of SEOV, and were more closely related to the strains, such as: Hb8610, R22, HB55, L99, and K24-e7., Conclusion: Both strains (NM133 and Q12) belonged to SEOV, and sharing a high degree of homology and similar secondary structure with strains including the vaccine strains Z37, our data suggested that the present vaccine used in China could effectively prevent HFRS caused by SEOV.

Objective: To determine the hosts of hantavirus (HV) and its molecular epidemiological characteristics, to provide evidence for prevention and control on hemorrhagic fever with renal syndrome (HFRS)., Methods: Rodents were captured by a special trap within the residential area. The antigens of HV in lung tissues were detected by direct immuno-fluorescence assay (DFA). Nucleotide sequences of HV were amplified by RT-PCR with HV genotype-specific primer. The amplified genes were then sequenced. Phylogenetic tree were built on nucleotide sequence with ClustalX 1.83 software., Results: 1421 rodents were captured and classified into 8 species of 4 Genera in the epidemic area within 10 counties of Chuxiong prefecture, Yunnan province, between 2005 and 2006. Out of the 1421 rodents, 1056 (74.31% ) of them were Rattus norvegicus and 280 (19.70%) belonged to Rattus flavipectus. The antigens of HV were detected by DFA in lung tissues and the total positive rate of HV was 5.15% (53/ 1029). After applying the sequencing nucleotide method to the 53 positive specimens, data showed that 21 specimens were positive and all of them belonged to Seoul type (15 samples were from Rattus norvegicus, 4 samples Rattus flavipectus, 2 samples Rattus nitidus). The partial S segments from 12 specimens were sequenced which appeared homologic with R22, L99 and HLD65 from GenBank in relatively high level (87.1% -99.7%). When compared to 76-118 strain of Hantaan type, their homologic degree was only 64.4%-69.1%. Results from Phylogenetic analysis showed that 12 specimens belonged to Seoul type. As for their homology, they were significantly similar to Seoul type and could be tentatively divided into two subtypes S1 and S3., Conclusion: It was confirmed that the Seoul type virus, as HFRS's pathogenetic agent mainly carried by rats, prevailed widely in Chuxiong prefecture. Owing to the local ecological environment, we also noticed the characteristics of different HV subtypes among Seoul type.

Objective: To isolate hantavirus from Lishui county--one of the epidemic regions for hemorrhagic fever with renal syndrome (HFRS), in Zhejiang province, and to identify the serotype and molecular/biological characteristics of a new HTN subtype hantavirus (HV) strains, hopefully to provide evidence for HFRS prevention and therapy., Methods: Data on the host animals was collected from Lishui, Zhejiang province in 2007. Direct immunofluorscece assay was adopted to determine HFRS antigens and the lung tissues from HV infected Vero-E6 cells for HV isolation, then total RNA was extracted from Hantavirus Lishui strains and amplified by RT-PCR M, S segments of strains genome were also cloned and sequenced and compared with those of other strains of HV., Results: 2 strains virus (ZLS6-11 and ZLS-12) were successfully isolated from 7 positive lung samples of mice and were identified as HTNV by anti-McAb and phylogenetic analysis. With sequence compation,we found that 2 strains with complete M and S segment had higher homology with HTN-type strains than with other types of HV, but 13.4%-20.7% and 10.3%-16.1% of the genes were found which were different from HTNV. The phylogenetic trees constructed by complete S and M segment showed that ZLS6-11 and ZLS-12 strains were located in HTNV group,and structured independent embranchment., Conclusion: ZLS6-11 and ZLS-12 Strains were believed to belong to HTN-type and phylogenetically different from the HTNV.

Objective: To study the epidemiological features of hantavirus in rodents in Wenzhou, Zhejiang province., Methods: Rodents were captured in Wenzhou, where hemorrhagic fever with renal syndrome (HFRS) had been endemic. Hantavirus antigens in the rat lungs were detected by immunofluorescence assay (IFA). Partial S segment (nt 620-999) and partial M segment (nt 2001-2301) sequences were amplified by RT-PCR, and then sequenced. Neighbor-joining method was used to construct for phylogenetic analysis., Results: A total of 96 rodents were trapped in the epidemic areas, and 6 hantavirus antigens were identified from these lung samples (6.3%). Partial S and partial M segment sequences were successfully recovered from 5 samples and determined. Phylogenetic analysis of these sequences indicated that all viruses belonged to Seoul virus (SEOV), regardless of the sources (Rattus norvegicus, Rattus tanezumi and Rattus rattoide) that they were derived. However, the clustering pattern in the partial S-tree was different from that in the partial M-tree, suggesting that the re-assortment between SEOVs had occurred., Conclusion: All Rattus rats carried SEOV in Wenzhou and the genetic reassortment with SEOV had occurred naturally.

Objective: To collect more data on the epidemiology of hantavirus in rodents in Cixi, Zhejiang province., Methods: Rodents were captured in Cixi, where hemorrhagic fever with renal syndrome (HFRS) appeared endemic. Hantavirus antigens in the rat lungs were detected by immunofluorescence assay. Partial S segment sequences were amplified by reverse transciption-polymerase chain reaction (RT-PCR) and then sequenced. The phyologenetic trees were constructed by maximum likelihood method to detect the genetic characteristics of hantavirus., Results: A total of 243 rodents were trapped in the epidemic areas, and hantavirus antigens were identified in 7 out of these lung samples (2.88%). Partial S segment sequences (620-999 nt) were recovered from 6 samples and sequenced. Data from phylogenetic analysis of these S segment sequences indicated that all viruses belonged to Seoul virus (SEOV), despite the origins of sources were either from Rattus norvegicus or from R. flabipectus. These viruses could further be divided into two distinct lineages but the viruses carried by R. norvegicus were different from those carried by R. flabipectus., Conclusion: Two distinct lineages of SEOV had been cocirculating in rodents in Cixi.

Objective: To understand the rate of viral carrying status among rodents as well as genotypes and distribution of Hantaviruses (HV) isolated in Hunan province., Methods: With DFA, the HV antigen in lung tissues of rodents was detected. The total viral RNA was extracted from the lung tissues of the HV infected rats and amplified with reverse transcrition-polymerase chain reaction (RT-PCR), using the HV genotype specific primers. The amplified genes were then sequenced and subjected to genotyping and homologic analysis., Results: The average density of rodents was 3.15% and the virus carrying rate among rodents was 1.31%. Data from genotype analysis showed that the HV isolated from seven lung specimens taken from Rattus norvgicus, Apodemus agraius, Mus musculus, Rattus flavipectus among indoor rodents in Shaodong and Liuyang belonged to HV type II (SEOV), and one isolated from Apodemus agraius in Shaungfen belonged to HV type I (HTNV) among outdoor rodents. Six strains were sequenced successfully and the homology between six srains was 88.3%-100%. The homology of HN1, HN2, HN4, HN6 came from Liuyang and the HN7 and HN8 from Shaodong were both 100% while the homology between L99 and the strains from Liuyang and Shaodong were 94.4% and 88.3% respectively., Conclusion: HV type II (SEOV) and the HV type I (HTNV) were all existed in Hunan province while SEOV was the main genotype.

Objective: To confirm the existence of Amur-like viruses in Apodemus peninsulae in China, and to understand the molecular characteristics of these viruses., Methods: Total RNA was extracted from lungs of A. peninsulae captured in Jilin of Northeast China with Trizol reagent. Complete S and partial M segments of Amur virus were amplified and sequenced. Phylogenetic analyses on multiple nucleotide sequences were performed with the Clustal method and DNASTAR software., Results: 383 bp cDNA of M segment and 1696 bp of S segment of Amur like virus were recovered from lung tissue of A. peninsulae, named JilinAP06. The full-length of its S gene comprised of 1696 nucleotides with ORF including 1287 nucleotides and encoding a protein which comprised 429 amino acids. The phylogenetic analysis of this sample with other hantaviruses revealed that the complete S and partial M segment sequence of JilinAP06 both were closely related to those Amur viruses such as AP63, AP61, AP1371 and AP1168 found in A. peninsulae from Far East region of Russia and B78 strain, Liu strain and H5 strain, which were all from Chinese patients. The complete S and partial M segment sequence of JilinAP06 had only 81.0% identities with the nucleotide sequences of HV prototype 76-118 strain., Conclusion: Amur-like viruses did exist in A. peninsulae from Northeasern China while A. peninsulae might be the natural reservoir of Amur-like viruses in China and was the important infectious source to HFRS patients which were caused by Amur-like viruses.

Objective: To explore the hantavirus infection and their genotype in rodents in Hunan., Methods: Hantavirus antigens in the rat lungs from Hunan province were detected by immunofluorescence assay. Partial S and M segment in antigen-positive samples were amplified by RT-PCR, and then sequenced. The phyologenetic trees were constructed for the analysis of genetic characters of hantavirus., Results: A total of 344 rats were trapped in the main epidemic area of Hunan province, and hantavirus antigens were found in 6 of the 344 rats( 1.74% ).The phylogenetic trees constructed by partial S segment( nt 620-990) or partial G2 segment (nt 2001- 2301) showed that the hantaviruses carried by Rattus norvegicus, R . flabipectus and R. rattoides from Xiangxiang district were genetic subtype SEOV4. The virus carried by R. norvegicus in Ningyuan district was phylogenetically different from the known SEOV. The hantavirus carried by Mus musculus from Shimen district was genetic subtype HTNV4., Conclusion: The hantaviruses in the main epidemic areas in Hunan province mainly belonged to SEOV, and R. flabipectus and R. rattoides carried the same genotype of SEOV as R. norvegicus.

Objective: For clarifying the situation of the natural infection of rodents having hemorrhagic fever with renal syndrome (HFRS) virus and to type Hantavirus (HV) using molecular technique in Shenzhen city in 2005, and offering guidance for prevention and control of HFRS., Methods: Data on the host animals was collected from the city of Shenzhen. ELISA and indirect immunofluorscent antibody(IFA) test were applied to the specific antibodies against HV in the sera of captured rats. Direct immunofluorscece assay was adopted to determine HFRS antigens and the lung tissues of the HV infected rats were inoculated into Meriones unguiculata to isolate HV. The whole viral RNA was extracted from the lung tissues of the HV infected rats and amplified the partial M fragments with RT-nested-PCR, using the HV genotype specific primers. The amplified genes were then sequenced, and subjected to genotyping and homology analysis., Results: 472 rodents were captured from Shenzhen in 2005. Surveillance on rats demonstrated 9.96% rats carrying HV (with a density of 8.25%) and the main host was Rattus norvegicus. In the blood samples of rats, anti-HV IgG antibodies were detectable in 56 cases by IFA, and proved to be positive in 76 cases by ELISA. We successfully isolated a HV strain designated as SZ2083 from Rattus norvegicus for the first time in Shenzhen and was identified to SEO type by RT-nested-PCR. Compared with the coding region of the M gene of HV L99 virus strain, the homologies of nucleotide among them were 97%, but the homology was 76% of the SZ2083 with HTN 76-118 virus strain., Conclusion: Results showed the existence of natural epidemic areas of HFRS in Shenzhen city. Based on the results of sequencing, it is possible that the Seoul strain of HV might be the predominant serotype of virus harbored.

Objective: To investigate the Hantavirus infection and their genotype in rodents in Huludao., Methods: Rodents were collected from the main epidemic areas to detect antigen of Hantavirus in rat lungs by indirect immunofluorescence assay. Antigen-positive samples were inoculated onto cultures of confluent Vero E6 cells for the isolation of virus. The genotypes of viruses in all antigen-positive samples were identified by reverse transcriptase-polymerase chain reaction (RT-PCR)., Results: 200 rats were collected in the main epidemic areas, and 11 Hantavirus-positive samples were tested. The positive rate of Hantavirus in rats was 5.5%. Three strains of Hantavirus were isolated in Vero E6 cell culture. Data from the phylogenetic trees constructed by partial S segment (620-999 nt) or partial G1 segment (180-580 nt) showed that the three isolates carried by rats from Huludao were all genetic subtype SEOV 3. Furthermore, the phylogenetic tree constructed by partial G2 segment (2003-2302 nt) divided SEOV strains into 7 genetic subtypes, and the three isolates were having a closer evolutionary relationship with isolates CP211, ch302 and dc501 from Beijing, and the isolates SD10 and SD227 form Shandong., Conclusion: Data indicated that the rate of carrying virus was high and the main genetic subtype of Hantavirus was S3 of Seoul virus in Huludao area.

Objective: In order to find out the factors related to hemorrhagic fever with renal syndrome (HFRS) infection, and to evaluate the probability of ecdemic hantaviruses (HV) infection in rodents in Beijing areas., Methods: Rodents were collected in a large-scale railway station and a produce market with 'trap nights' method from April to May, 2004. The IgG reacting sera to HV antigen were detected using ELISA. The partial M and S segment of HV from captured rodent lung samples were amplified with RT-PCR. The PCR products were purified and sequenced. BLAST program was then used to perform on nucleotide pairwise alignment with all available sequence in GenBank. The alignment of the multiply nucleotide and the deduced amino acid sequences, together with phylogenetic analysis were completed with DNASTAR software., Results: The average population density was 3.49% (24/690). The overall seroprevalence of HV infection was 8.3% (2/24). RT-PCR positive rates were 8.3% (2/24). The nucleotide sequences of 356 bp region (1958 - 2313) of M segment obtained from 2 samples were all identified to Seoul virus (SEOV), with 7.6% heterogeneity. The dc501 strain from railway station was closely related to SD227 and Hebei4 from Shandong and Hebei provinces respectively. BjFT01 strain from the farm product market had more special nucleotide transitional mutations than other known SEOV from Beijing in GenBank. This strain, together with known HN71 from Hainan province, K24-E7 from Zhejiang province, L99 from Jiangxi province and R22 from Henan province, represented a monophylogentic linkage., Conclusion: The higher HV prevalence of rodents in transportation center was the potential and important risk for HFRS epidemic in Beijing. The increasing prevalence of M. musculus should call for attention. It was possible that SEOV in Beijing was imported by infected rodents through vehicles from other provinces.

Objective: To investigate hantanvirus infection of captured rodents in Haidian district and Changping district of Beijing and to type hantavirus using molecular technique., Methods: The captured mice were classified and the density of distribution was calculated. Reverse transcription-polymerase chain reaction (RT-PCR) technique was used to amplify the partial M fragnments of hantaviruse. Several representative positive samples were sequenced and analysed by ClustalX (5.0) and DNAClub software., Results: A total of 414 animals were captured, among which Battus norvegicus was the dominant group. In Haidian district, the median infection rates with hantavirus were 13.14% in Battus norvegicus and 0 in Mus musculus Linnaeus. In Changping district, the average infection rates were 17.46% in Battus norvegicus and 3.57% in Mus musculus Linnaeus. Nucleotide sequences analysis showed that the virus detected all belonged to SEO-type. They clustered with Z37 virus and could be branched into 2 different subclades., Conclusion: The major hosts of hantavirus in Haidian and Changping district were Battus norvegicus and the epidemic strains in the two districts of Beijing were genotyped as SEO-type. Nucleotide sequence and deduced amino acid sequence from different rodents were highly homologous, while nucleotide mutation had also been observed. Further studies are required to explore the possible virus sequence mutation.

Objective: To understand the molecular and epidemic characteristics of Hantavirus S85-46 isolated from Sichuan province., Methods: RT-PCR products were amplified from S85-46 virus infected cells, cloned into T vector, sequenced and analyzed using DNASTAR software., Results: The homology of M segment of S85-46 strain and HTN type strain varied from 84.1%-99.7%, compared to 70.4%-70.9% with SEO strains. This result indicated that S85-46 strain belonged to hantavirus. The nucleotide sequence analyses indicated that S85-46 strain, LR1 strain and 76-118 strain were highly homologous. The two latter strains were isolated from Shaanxi province of China and South Korea respectively, which were far from Sichuan province of China. The nucleotide sequence analyses of S segment was similar to M segment., Conclusion: The epidemic strains isolated from different regions may have been highly homologous.

Serotyping with hemagglutination-inhibition test (HI) on 458 patients with EHF showed that 419 (91.48%) of them belonged to the house-rat type, 33 (7.21%) belonged to the wild-rat type, and 6 (1.31%) were undetermined. The majority (80-100%) of the patients in various regions belonged to the house-rat type. The type of the virus antigen and the type of the antibody of rats were in conformity with the species of rats. These indicate that the epidemic areas of EHF in Shandong Province are mixed-type epidemic areas, predominantly the house-rat type.

Between 1988 and 1990, the positive seroconversion rates of HFRS were tested with a random sample of 7,145 persons in Apodemus-type endemic area and Rattus-type endemic area(type A; type R) in Shandong Province. The results showed that: (1) the highest positive seroconversion rate was in the group of peasants of 20 to 49 years of age in the area of type A; (2) in the area of type R, there was no statistically significant difference in positive seroconversion rates of age, and housewives had the highest rate. The male/female ratio was 2.1-2.2:1 and 0.9:1, respectively in the two types of endemic area. The ratio of apparent infection to inapparent infection was 1:22.6 and 1:42, respectively in the two types of area. It is concluded that inapparent infection is dominant in HFRS infection and there are probably many potential inapparent infections among the population.

Using hemagglutination inhibition test (HI), we confirmed that Junan County is an endemic area of both EHF of R. norvegicus and A. agrarius, which indicated that the main host animals were R. norvegicus and A. agrarius in the area. Remarkable difference was shown between the infectious rates of pigs from the endemic area and that of pigs from the nonendemic area, which suggested that pigs could play certain roles in the transmission of EHF. We also proved that the main transmission mode of EHF virus in rats was horizontal transmission, and vertical transmission in rats also existed.

In this paper, we have used HI assay in an observation of the dynamic changes of serotype of EHF patients in a mixed on demic area in Shaoxing, Zhejiang province. The results showed that serotypes of EHF patients were strongly correlated with different endemic areas and had obvious seasonal fluctuation in the incidence of the disease. This further demonstrates that the difference between the two serotypes (Apodemus type and Rattus type) in different geographic areas.