Genetic Diversity and the Spatio-Temporal Analyses of Hantaviruses in Shandong Province, China.

Hemorrhagic fever with renal syndrome (HFRS) is a serious public health problem in Shandong Province, China. We conducted an epizootiologic investigation and phylogeographic and phylodynamic analyses to infer the phylogenetic relationships of hantaviruses in space and time, and gain further insights into their evolutionary dynamics in Shandong Province. Our data indicated that the Seoul virus (SEOV) is distributed throughout Shandong, whereas Hantaan virus (HTNV) co-circulates with SEOV in the eastern and southern areas of Shandong. Their distribution showed strong geographic clustering. In addition, our analyses indicated multiple evolutionary paths, long-distance transmission, and demographic expansion events for SEOV in some areas. Selection pressure analyses revealed that negative selection on hantaviruses acted as the principal evolutionary force, whereas a little evidence of positive selection exists. We found that several positively selected sites were located within major functional regions and indicated the importance of these residues for adaptive evolution of hantaviruses.

A new hantavirus from the stripe-backed shrew (Sorex cylindricauda) in the People's Republic of China.

Inspired by the recent discovery of genetically distinct hantaviruses from insectivore species worldwide, we performed a small-scale search for insectivore-borne hantaviruses. In this paper, we report the discovery of a new hantavirus, which was designated the Qian Hu Shan virus (QHSV). This virus was detected in the lung tissues of three stripe-backed shrews (Sorex cylindricauda), which were captured in the Yunnan Province, China. The full-length S genomic segment of the representative QHSV strain YN05-284 was 1661 nucleotides and is predicted to encode a nucleocapsid protein of 429 amino acids that starts at nucleotide position 48. It exhibited the highest similarity with other Sorex-related hantaviruses, with 68.1%-72.8% nucleotide and 71.9%-84.4% amino acid sequence identities. An analysis of a 1430-nucleotide region of the partial M segment exhibited approximately 54.4%-79.5% nucleotide and 43.2%-90.8% amino acid sequence identities to other hantaviruses. A comparison of a 432-nucleotide region of the L segment also showed similar degrees of identity, with 68.9%-78.4% nucleotide and 71.1%-93.8% amino acid sequence identities to other hantaviruses. Phylogenetic analyses using Bayesian methods indicated that QHSV shared the most recent common ancestor with other Sorex-related hantaviruses. The host was identified using a morphological assessment and verified using mitochondrial cytochrome b (mt-Cyt b) gene sequencing. A pair-wise comparison of the 1140-nucleotide mt-Cyt b gene sequence from the host demonstrated that the host was close to S. cylindricauda from Nepal with 94.3% identity. The virus-host association tanglegram, which was constructed using the Dendroscope software, indicated that the QHSV phylogeny and the host phylogeny were approximately matched, which suggests no evidence of host switching for QHSV. Our results contribute to a wider viewpoint regarding the heterogeneity of viruses that infect shrews.

Cytokine and chemokine responses to Japanese encephalitis live attenuated vaccine in a human population.

OBJECTIVES: The SA14-14-2 Japanese encephalitis (JE) live attenuated vaccine is licensed for use only in China, and has provided excellent efficacy in reducing the incidence of JE. The humoral immune response related to the JE vaccination has been well characterized, however cellular immune responses are less well known. METHODS: Thirty-four healthy males who had recently received inoculation with the SA14-14-2 live attenuated vaccine were recruited. Serum samples from these subjects were analyzed for cytokine and chemokine levels using the FlowCytomix method. RESULTS: Eighteen of 34 subjects were positive for JE virus-specific IgG antibodies. Levels of interleukin (IL)-8, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, and MIP-1ß were significantly higher in the vaccinees than in a control group (p<0.0001, p<0.0001, p=0.021, and p<0.0001, respectively). IL-6 was detectable in 64.7% of vaccinees, but was not detectable in any of the controls. IL-1ß, IL-2, IL-4, IL-5, IL-9, IL-10, IL-12p70, IL-13, IL-17A, IL-22, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ were detected in very few subjects or were undetectable in both groups. CONCLUSIONS: IL-6, IL-8, MCP-1, MIP-1α, and MIP-1ß may play important roles in the immune response to JE live attenuated vaccine.

Encephalitis temporally associated with live attenuated Japanese encephalitis vaccine: four case reports.

BACKGROUND: Japanese encephalitis (JE) vaccination is the most effective measure for preventing JE disease. The live attenuated JE vaccine, which has shown good efficacy and safety, has been widely used in China. CASE PRESENTATIONS: We report four laboratory-confirmed JE cases detected in JE-endemic areas during the JE virus (JEV) transmission season, who all received a first dose of live attenuated JE vaccine within 2 weeks prior to the onset of illness. All cases presented with acute encephalitis and rapidly reduced consciousness. All cerebrospinal fluid (CSF) samples from the patients were positive for JEV-specific immunoglobulin M (IgM) antibodies, but viral isolation and polymerase chain reaction (PCR) detection of JEV were both negative. CONCLUSIONS: It is difficult to identify a causal link between the disease and the vaccination, as the source of positive CSF JEV IgM antibodies might be natural JEV infection or possibly due to a traumatic lumbar puncture. Our observations highlight the need for public health officers and doctors to consider reasonable vaccination policies during the JE season. In addition, continued surveillance as well as thorough investigation of any events that occur after JE vaccination is necessary.

Isolation and genetic characteristics of human genotype 1 Japanese encephalitis virus, China, 2009.

BACKGROUND: Several studies have shown that the predominant genotype of Chinese Japanese encephalitis virus (JEV) is evolving from genotype 3 to genotype 1. However, in recent years, almost all genotype 1 isolates were from mosquitoes, and genotype 1 has been less associated with human disease than genotype 3. This study reports the isolation of human genotype 1 JEV and its genetic characteristics to provide additional insights into human JE pathogens that are currently circulating in China. METHODS AND RESULTS: In 2009, 31 cerebrospinal fluid samples were collected from patients living in Yunnan and Shanxi provinces and were used to inoculate Aedes albopictus C6/36 cells for virus isolation. The JEV strains were identified using immunofluorescent assays and the reverse transcription-polymerase chain reaction. Phylogenetic analyses based on the partial capsid/pre-membrane and full envelope (E) sequences were performed using Clustalx 1.8 software. Three JEV isolates were obtained from a 4-year-old girl and a 2-year-old boy living in Yunnan and an 82-year-old woman in Shanxi. The boy had been immunized with one dose of JE live attenuated vaccine. New isolates were grouped into genotype 1. Amino acid sequence for the viral E protein indicated 95% to 100% identity with each other and with other JEV strains. When compared with a consensus sequence of E protein, two amino acid substitutions were found: Ser(E-123)-Asn in the two Yunnan isolates and Lys(E-166)-Arg in the Shanxi isolate. CONCLUSIONS: Our findings indicate that the genotype 1 of JEV is causing human infections in China. Our observation of a previously vaccinated boy developing JE from genotype 1 virus infection also calls for more detailed studies, both in vitro and in vivo neutralization tests as well as active surveillance, to examine the possibility of a lack of complete protection conferred by the live attenuated JE vaccine against genotype 1 virus.

Genetic analysis of a hantavirus strain carried by Niviventer confucianus in Yunnan province, China.

Hantavirus genome sequences were recovered from lung tissues of Chinese white-bellied rats (Niviventer confucianus) captured in Yunnan province, China. Pairwise comparison of the nucleotide and deduced amino acid sequences of the entire S and partial M and L segments indicated that the newly discovered virus strain, which was designated as strain YN509, was very different from other rodent-borne hantaviruses. Phylogenetic analysis showed that the new strain fit into a clade containing Da Bie Shan virus (DBSV) (also carried by N. confucianus), which is mainly found in Anhui Province in mainland China. Strain YN509 appears to be in a sister taxa of the DBSV group described previously. These data suggest that strain YN509 is a new subtype of DBSV, which appears to be widely distributed in China with a higher genetic diversity than expected.

Tick-borne agents in rodents, China, 2004-2006.

A total of 705 rodents from 6 provinces and autonomous regions of mainland People's Republic of China were tested by PCRs for tick-borne agents (Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato, spotted fever group rickettsiae, and Francisella tularensis). Infection rates were 5.5%, 6.7%, 9.1% and 5.0%, respectively. Eighteen (2.6%) rodents of 10 species were positive for 2 or 3 agents. Sequence analysis of PCR products confirmed the presence and genotypes of detected agents. These findings demonstrate that these tick-borne agents cocirculate and that a variety of rodent species may be involved in their enzootic maintenance.

Genomic sequence of a Japanese encephalitis virus isolate from southern China.

We determined the complete nucleotide sequence of a Japanese encephalitis virus (JEV) isolate (designated SH17M-2007) from a pool of Culex tritaeniorhynchus collected in southern China in 2007. The genome consisted of 10,965 nucleotides and included a single open reading frame (10,296 nucleotides) that encodes a 3,432-amino-acid polyprotein. The SH17M-2007 had 97.3 to 98.4% nucleotide identity with two Korean strains (KV1899, K94P05) and two Japanese strains (Ishikawa, JEV/sw/Mie/40/2004), but only 88.8% identity with the Chinese vaccine strain SA14-14-2. Five unique amino acid substitutions including one in the envelope (E) protein (Glu(E-306)-Lys) were found in the SH17M-2007 strain. Phylogenetic relationships based on the full-length nucleotide sequences were similar to those based on the E gene.

Detection of Francisella tularensis in ticks and identification of their genotypes using multiple-locus variable-number tandem repeat analysis.

BACKGROUND: Tularemia was reported in China over 50 years ago, however, many epidemical characteristics remain unclear. In the present study, the prevalence of Francisella tularensis in ticks was investigated during an epidemiological surveillance in China and then we measured their genetic diversity by conducting multiple-locus variable- number tandem repeat analysis (MLVA). RESULTS: 1670 ticks from 2 endemic areas (Inner Mongolia Autonomous Region and Heilongjiang Province) and 2 non-endemic areas (Jilin and Fujian Provinces) were collected and tested for evidence of tularemia by nested PCR. The prevalence of Francisella tularensis in ticks averaged 1.98%. The positive rates were significantly different among tick species, with Dermacentor silvarum and Ixodes persulatus responsible for all positive numbers. All F. tularensis that were detected in ticks belonged to F. tularensis subsp. holarctica and MLVA disclosed genetic diversity. One subtype was identified in 17 of 33 positive tick samples in three different study areas. Another subtype belonging to F. tularensis subsp. holarctica genotype was described for the first time in the current study. CONCLUSION: The study showed two tick species, D. silvarum and I. persulatus harboring the pathogen of tularemia in natural environment, indicating these two tick species might have a role in tularemia existence in China. MLVA results disclosed the genetic diversity F. tularensis and identified one genotype as the most prevalent among the investigated ticks in China.

[Expression of nucleostemin mRNA and protein in the esophageal squamous cell carcinoma].

OBJECTIVE: To investigate the mRNA and protein expression of nucleostemin (NS) in human esophageal squamous cell carcinoma. METHODS: The mRNA and protein expression of NS were detected in 31 mucosal atypical hyperplasia specimens, 62 esophageal squamous cell carcinoma specimens and the matched normal esophageal mucosa samples by RT-PCR and immunohistochemistry method, respectively. RESULTS: The positive expression rate of NS protein in normal esophageal mucosa, atypical hyperplasia and esophageal squamous cell carcinoma was 17.7% (11/62), 41.9% (13/31) and 69.4% (43/62), respectively. There was a significant difference among the above three groups (chi2 = 33.676, P < 0.01). The expression levels of NS mRNA in esophageal squamous cell carcinoma (0.971 +/- 0.121) was significantly higher than that in the atypical hyperplasia (0.913 +/- 0.085) and also in the normal esophageal mucosa (0.866 +/- 0.103; F = 14.829, P < 0.01). The expression level of both NS protein and mRNA was positively correlated with histological grade, infiltration depth, and lymph node metastasis (P < 0.05), but not with age, gender or pathological type (P > 0.05). CONCLUSION: Our results indicate that nucleostemin mRNA and protein are over-expressed in human esophageal squamous cell carcinoma, and it may be related with its oncogenesis.

Novel genospecies of Borrelia burgdorferi sensu lato from rodents and ticks in southwestern China.

By using multilocus sequence analysis, five Borrelia valaisiana-related strains isolated from rodents and ticks in southwestern China were eventually classified as a new genospecies of B. burgdorferi sensu lato rather than B. valaisiana. The finding explained the differences in transmission cycle and phenotype between B. valaisiana strains from Europe and B. valaisiana-related strains from eastern Asia.

Presence of pathogenic Borrelia burgdorferi sensu lato in ticks and rodents in Zhejiang, south-east China.

A molecular epidemiological survey was conducted to investigate the presence of pathogenic Borrelia burgdorferi sensu lato (s.l.) species in the forest areas of Zhejiang province, south-east China. A total of 182 ticks of 6 species and 200 rodents of 8 species were collected and individually examined for the presence of B. burgdorferi s.l. DNA by nested PCR targeting the 5S-23S rRNA intergenic spacer. Forty-one ticks of four species, Haemaphysalis concinna, Haemaphysalis longicornis, Rhipicephalus microplus and Haemaphysalis warburconi, were infected with B. burgdorferi s.l., with an overall infection rate of 23 %. Sixteen rodents of four species, Nivivener confucianus, Nivivener coxingi, Apodemus sylvaticus and Rattus losea, were positive for B. burgdorferi s.l., with an overall prevalence of 8 %. MseI RFLP analysis and sequence analysis of the positive PCR products showed that Borrelia spirochaetes in specimens consisted of Borrelia garinii, Borrelia afzelii and Borrelia valaisiana-related group. Forty (98 %) of the B. burgdorferi s.l.-positive ticks were infected with B. garinii and one (2 %) was infected with B. afzelii. Twelve (75 %) of the positive rodents were infected with B. garinii and four (25 %) were infected with the Borrelia spirochaete belonging to B. valaisiana-related group.

Correlation of matrix metalloproteinase suppressor genes RECK, VEGF, and CD105 with angiogenesis and biological behavior in esophageal squamous cell carcinoma.

AIM: To explore the expression of reversion inducing cysteine-rich protein with Kazal motifs (RECK), vascular endothelial growth factor (VEGF) and endoglin (CD105) protein and its correlation with occurrence, development, invasion and metastasis in esophageal squamous cell carcinoma (ESCC). METHODS: Streptavidin-peroxidase (SP) immunohisto-chemistry was used to detect expression of RECK and VEGF in 62 cases of ESCC, 31 cases of adjacent atypical hyperplastic epithelium and 62 cases of normal esophageal epithelium. CD105 Mb was used to assess microvessel density (MVD). RESULTS: The expression of RECK was closely correlated with histological grade, infiltrative depth and lymphatic metastasis in ESCC (P < 0.05). The expression of RECK decreased during cancer development: normal esophageal epithelium (85.5%, 53/62), adjacent atypical hyperplastic epithelium (71.0%, 22/31), and carcinoma (59.7%, 37/62). There was a significant difference among the groups (P < 0.05). The expression of VEGF protein was closely correlated with infiltrative depth and lymphatic metastasis in ESCC (P < 0.05). The expression of VEGF protein increased during cancer development: normal esophageal epithelium (29.0%, 18/62), adjacent atypical hyperplastic epithelium (54.8%, 17/31), and carcinoma (67.7%, 42/62). There was a significant difference among the groups (P < 0.05). MVDCD105 increased in accordance with histological grade, but there was no significant difference (grade I, 36.92 +/- 10.85; grade II, 37.65 +/- 9.50; and grade III, 38.06 +/- 12.19). The MVDCD105 was closely correlated with infiltration and lymphatic metastasis in ESCC (P < 0.05). The expression of RECK was inversely correlated with the expression of VEGF and CD105. CONCLUSION: RECK, VEGF and CD105 play important roles in the infiltration, metastasis and carcinogenesis in esophageal carcinoma. Angiogenesis in ESCC may be promoted by over-expression of CD105.

[Identification of Amur like virus in Apodemus peninsulae and its molecular characteristics in China].

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.

[Investigation on Anaplasma phagocytophilum infection in rodents from forest areas in northeastern China].

OBJECTIVE: To investigate the prevalence of Anaplasma phagocytophilum in rodents from forest areas in northeastern China. METHODS: PCR amplification, followed by sequence analysis was carried out. The sequences of 16S rRNA and gltA gene fragment amplified from rodent specimens were compared with corresponding part of the sequences deposited in GenBank. RESULTS: A total number of 276 rodents were tested, including 102 in Jilin province, 61 in Helongjiang province and 113 in Inner Mongolia autonomous region. The positive rates were 8.82%, 1.64% and 0.00%, respectively. The infection rate in rodents infected by ticks was 11.30 times higher than that in rodents without ticks (P = 0.002). The S. A. phagocytophilum 16S rRNA sequences from rodents in Jilin and Heilongjiang were identical and differed in 3-5 bases compared with the corresponding parts of A. phagocytophilum from America, Sweden and Japan. Compared with the sequences registered in GenBank, the nucleotide sequence of gltA varied from 87%-97% and its deduced amino acid sequence changed from 84%-99%. CONCLUSION: A. phagocytophilum infection was presented in rodents from Jilin and Heilongjiang province.

[Investigation on Borrelia burgdorferi sensu lato in ticks and rodents collected in Da Xing-An Mountains Forest areas of China].

OBJECTIVE: To detect and study the types of Borrelia burgdorferi sensu lato in ticks and rodents from Da Xing-An Mountains Forest areas of China. METHODS: Nested PCR was performed to amplify 5S-23S rRNA intergenic spacer of B. burgdorferi. Positive products were analysed by restriction fragment length polymorphism (RFLP) and single strand conformation polymorphism (SSCP), specimens showing unique RFLP profile were sequenced and analysed. RESULTS: 1336 Ixodes persulcatus, 144 Dermacento silvarum, 144 Haemaphysalis concinna and 145 rodents of 9 species were collected from 16 sections of Da Xing-An Mountains Forest areas of China. Specific fragments were amplified from 293 I. persulcatus and 6 D. silvarum and 5 rodents of 4 species. B. burgdorferi was not detected in H. concinna. Among the positively tested I. persulcatus, 209 contained B. garinii genospecies and 45 contained B.afzelii genospecies based on RFLP. Moreover, B.garinii genospecies consisted of B. garinii 20047 and B. garinii NT29. 17 adult I. persulcatus were simultaneously infected with B. garinii 20047 and B. garinii NT29. Nine adult I. persulcatus were simultaneously infected with B. garinii 20047 and B. afzelii. Four adult I. persulcatus were simultaneously infected with B. garinii 20047 and B. garinii NT29 and B. afzelii. Two D. silvarum were infected with B. garinii 20047, 1 D. silvarum with B. garinii 20047, 2 D. silvarum with B. afzelii. 3 rodents were infected with B. garinii 20047 while 2 rodents were infected with B. garinii NT29. Mixed infection was not found in D. silvarum and rodents. In addition, nine I. persulcatus and one D. silvarum specimens showed unique RFLP pattern. Data from sequential analysis showed that they all belonged to B. garinii. PCR-SSCP profiles of 5S-23S rRNA intergenic spacer of B. burgdorferi in the positive specimens exceeded 36 types; B. garinii 20047 showed 16 types while B. garinii NT29 showing 11 types, B. afzelii showing 9 types. SSCP profiles of the specimens coinfected with multiple B. burgdorferi was relatively complex. CONCLUSION: The infection of B. burgdorferi was found in the ticks and rodents in Da Xing-An Mountains Forests areas. The infection rate of I. persulcatus was high. B. garinii was predominant genospecies, and the population of B. burgdorferi was heterogeneous in the area. Mixed infections of different B. burgdorferi genospecies in ticks were found. I. persulcatus and Clethrionomys rufocanus were possibly served as major vector and major host for B. burgdorferi, respectively, suggesting that further study is needed to confirm the coinfection in humans and animals in this region.

[Investigation on rodents' natural infection of Orientia tsutsugamushi in some areas of Inner Mongolia and Xinjiang, China].

OBJECTIVE: To investigate rodents' natural infection of Orientia tsutsugamushi (Ot) in some areas of Inner Mongolia and Xinjiang, China. METHODS: DNAs were extracted from spleens of the captured mice and nested-polymerase chain reaction (nPCR) technique was used to detect the Ot-Sta56 gene. Six positive samples were sequenced and analyzed by Clustal X (5.0) and DNA Club software. RESULTS: A total of 90 rodents were captured in Inner Mongolia, and the overall prevalence of Ot was 6.67%. There was no significant difference in infection rates among the positive rodents species. 20 rodents were captured in Xinjiang, and the prevalence of Ot was 5.00%. The geographical difference in infection rates was not statistically significant between Inner Mongolia and Xinjiang. 9 rodents were captured in farmlands of Inner Mongolia and Xinjiang but there was no positive samples found. 101 rodents were captured in grasslands, and the prevalence of Ot was 6.93%. The Sta56 gene nucleotide sequence homology to Karp strain of N59 (from Microtus maximowiczii), N69 (from Cricetulus barabensis) and X33(from Cricetus cricetus) was 99%. The sequence homology to Taitung-2 strain and TW461 strain of N65 (from C. barabensis) was 94%, and the sequence homology to Taitung-2 strain and TW461 strain of N88(from Apodemus agrarius) was also 94%. The sequence homology to Oishi strain of N90 (from A. agrarius) was 96.00%. CONCLUSION: Our findings indicated that infections of Ot did exist in rodents captured from Inner Mongolia and Xinjiang. The genotypes of Ot in Inner Mongolia and Xinjiang were quite complex, with some of them belonged to Karp type, and the others belonged to Taitung-2, TW461 and Oishi types which providing evidence for further investigation on the scrub typhus fuci in the two areas.

[Study on the coinfection status of Borrelia burgdorferi sensu lato and spotted fever group Rickettsia in ticks from Hunchun, Jilin province].

OBJECTIVE: To understand the coinfection status of Borrelia burgdorferi sensu lato (B.b.s.l) and spotted fever group Rickettsia (SFGR) in Hunchun of Jilin province, China. METHODS: Polymerase chain reaction (PCR) was used to detect the 5S-23S rRNA intergenic spacer of B. b. s. l and ompA of SFGR in ticks was collected in Hunchun,Jilin province. The amplification products of positive ticks were sequenced, and phylogenetic analysis was conducted by PHYLIP software package. RESULTS: The infection rate of B. b. s. l was 36.0% in Ixodes persulcatus ticks and the SFGR was discovered in I. persulcatus ticks,with an infection rate of 2.0%. The coinfection rate of both agents was 2.0%. In 327 Dermacentor siltarum ticks, the positive rates of B. b. s. l and SFGR were 30.9% and 29.1% respectively. 55 ticks (16.8%) were coinfected with the two pathogens. The sequence analysis of B. b. s. l showed that the B. b. s. l in Jilin area, which were highly homologous, all belonged to B. garinii genotypes. The sequence analysis of SFGR positive products showed that the DNA secquence of the newly detected agent (JL-95) was close to the two previously described rickettsiae which were detected in I. ricinus from Slovakia (called IRS3 and IRS4). Phylogenetic relationships inferred from the comparison of these sequences with those of other genus Rickettsiae indicated that JL-95, IRS3 and IRS4 constituted a new rickettsial genotype and formed a separate cluster among the spotted fever group Rickettsiae. CONCLUSION: Coinfection of B. b. s. l and SFGR existed in Hunchun, Jilin province. The sequencing of specific fragment confirmed a new SFGR which was different from other rickettsiae known in China.

Francisella tularensis in rodents, China.

A total of 420 rodents in China were examined for Francisella tularensis by polymerase chain reaction. The infection rates were 4.76% in total, and 11.65%, 10.00%, 6.56%, 1.77%, and 0% in Jilin, Xinjiang, Heilongjiang, Inner Mongolia, and Zhejiang, respectively. Sequence analysis showed that all the detected agents belonged to F. tularensis subsp. holarctica.

Molecular epidemiology of SARS-associated coronavirus, Beijing.

Single nucleotide variations (SNVs) at 5 loci (17564, 21721, 22222, 23823, and 27827) were used to define the molecular epidemiologic characteristics of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) from Beijing patients. Five fragments targeted at the SNV loci were amplified directly from clinical samples by using reverse transcription-polymerase chain reaction (RT-PCR), before sequencing the amplified products. Analyses of 45 sequences obtained from 29 patients showed that the GGCTC motif dominated among samples collected from March to early April 2003; the TGTTT motif predominanted afterwards. The switch from GGCTC to TGTTT was observed among patients belonging to the same cluster, which ruled out the possibility of the coincidental superposition of 2 epidemics running in parallel in Beijing. The Beijing isolates underwent the same change pattern reported from Guangdong Province. The same series of mutations occurring in separate geographic locations and at different times suggests a dominant process of viral adaptation to the host.