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.

Wide distribution and genetic diversity of "Candidatus Neoehrlichia mikurensis" in rodents from China.

"Candidatus Neoehrlichia mikurensis" was detected by PCR in 4.0% (34/841) of the rodents tested in this study. The 34 rodents represented nine species from seven regions of China. Phylogenetic analyses based on the partial groEL and nearly entire 16S rRNA gene sequences of the agent revealed genetic diversity, which was correlated with its geographic origins.

[Ecological-geographic landscapes of natural plague foci in China VI. Biological characteristics of natural vectors of Yesinia pestis].

OBJECTIVE: To characterize the biological characteristics of natural vectors of Yersinia (Y.) pestis in China. METHODS: Species and genera of natural vectors of Y. pestis in China and their faunal distribution were characterized with modern insect taxonomic techniques. The ecological roles of natural vectors of Y. pestis in natural plague foci were determined according to insect ecological experiments. RESULTS: There were 63 species of natural vectors of Y. pestis including 28 major reservoirs and 35 secondary ones. CONCLUSION: The biology characteristics of major vectors on Y. pestis and their roles in natural plague foci were defined.

[Ecological-geographic landscapes of natural plague foci in China VII. Typing of natural plague foci].

OBJECTIVE: To group and characterize natural plague foci in China. METHODS: A novel two-class typing method as well as a three-indication nomenclature method were established to group and characterize the natural plague foci, on the basis of eco-geographical landscapes of plague foci, genetics of Yersinia pestis, zoology of rodent reservoirs and the entomology of flea vectors. RESULTS: A total of 12 distinct natural plague foci (including 19 subtypes) as well as their biological features were characterized. CONCLUSION: Natural plague foci in China were grouped and characterized in this study.

Discovery of DNA viruses in wild-caught mosquitoes using small RNA high throughput sequencing.

BACKGROUND: Mosquito-borne infectious diseases pose a severe threat to public health in many areas of the world. Current methods for pathogen detection and surveillance are usually dependent on prior knowledge of the etiologic agents involved. Hence, efficient approaches are required for screening wild mosquito populations to detect known and unknown pathogens. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we explored the use of Next Generation Sequencing to identify viral agents in wild-caught mosquitoes. We extracted total RNA from different mosquito species from South China. Small 18-30 bp length RNA molecules were purified, reverse-transcribed into cDNA and sequenced using Illumina GAIIx instrumentation. Bioinformatic analyses to identify putative viral agents were conducted and the results confirmed by PCR. We identified a non-enveloped single-stranded DNA densovirus in the wild-caught Culex pipiens molestus mosquitoes. The majority of the viral transcripts (.>80% of the region) were covered by the small viral RNAs, with a few peaks of very high coverage obtained. The +/- strand sequence ratio of the small RNAs was approximately 7∶1, indicating that the molecules were mainly derived from the viral RNA transcripts. The small viral RNAs overlapped, enabling contig assembly of the viral genome sequence. We identified some small RNAs in the reverse repeat regions of the viral 5'- and 3' -untranslated regions where no transcripts were expected. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate for the first time that high throughput sequencing of small RNA is feasible for identifying viral agents in wild-caught mosquitoes. Our results show that it is possible to detect DNA viruses by sequencing the small RNAs obtained from insects, although the underlying mechanism of small viral RNA biogenesis is unclear. Our data and those of other researchers show that high throughput small RNA sequencing can be used for pathogen surveillance in wild mosquito vectors.

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.

Distribution of arboviruses and mosquitoes in northwestern Yunnan Province, China.

From July to September in 2005 and 2006, a survey was conducted to identify mosquito species and mosquito-borne arboviruses at elevations ranging from 900-3280 m between 24 degrees 00' N and 29 degrees 00' N latitude in the northwestern part of Yunnan Province, China. A total of 54,879 mosquitoes representing 15 species and 4 genera was collected using UV light traps at 59 sites. Culex tritaeniorhynchus and Anopheles sinensis were the most abundant species. The density of mosquitoes as well as the diversity of species decreased with increasing altitude. A total of 21,008 mosquitoes in 281 pools representing all of the 15 species was tested for the presence of viruses using cell culture. Viruses identified included Japanese encephalitis virus (13 isolates), Getah virus (five isolates), Banna virus (three isolates), Kadipiro virus (five isolates), and Densovirus (seven isolates). These isolates were obtained from Culex tritaeniorhynchus (20 isolates), Anopheles sinensis (three isolates), Armigeres subalbatus (six isolates), Culex pipiens quinquefasciatus (two isolates), and from unidentified, mixed mosquitoes (two isolates). Most of the isolates were from collections made at elevations below 2,500 m. Vector Borne Zoonotic Dis. 0, 000-000.

[Identification of Anaplasma phagocytophilum in small mammals from Hengduan Mountains of Southwest China].

OBJECTIVE: To investigate the prevalence of Anaplasma phagocytophilum in small mammals from the forest area of Hengduan Mountains in southwestern China. METHODS: Small mammals captured from Gaoligong and Xianggelila mountainous area of Yunnan province were detected by PCR amplification. The sequences of 16S rRNA and Msp4 gene fragments from positive samples were compared with corresponding sequences deposited in GenBank. RESULTS: A total number of 436 small animals, which belongs to 5 orders 18 genera 35 species were tested, 32 (7.34%) were positive in 6 genera 11 species. There were 8.64% (26/301) positive in 25 species at Goligong mountainous areas, and 4.44% (6/135) were positive in 19 species at the Xianggelila mountainous areas. Positive small mammals were most rodents. The nucleotide sequences of A.phagocytophilum 16S rRNA gene amplified from small mammals varied from 99% - 100% and were 99% - 100% similar with the corresponding segments of A. phagocytophilum from Jilin deposited in GeneBank. The sequences of A. phagocytophilum Msp4 gene showed that there was 95% - 97% similarity with the corresponding sequences registered in GenBank. CONCLUSION: A. phagocytophilum was firstly identified in 6 genera 11 species small mammals from a forest area of Hengduan Mountainous areas in southwestern China. Rodents might serve as the primary hosts indicating the potential risk to the domestic animals and human beings in this area.

[Fleas on small mammals in the surrounding area of Erhai Lake].

OBJECTIVE: To investigate the distribution pattern, species diversity and community structure of fleas on small mammals in the surrounding area of Erhai Lake, and the relationship between fleas and their hosts. METHODS: Different geographical areas surrounding the Erhai Lake in Yunnan were selected as investigated spots. Small mammals were captured with baited cages. The cage-traps were examined and re-baited each morning. All fleas on the hosts were collected and identified. The richness (S), evenness (J'), diversity index (H'), dominance index (C'), total ectoparasite infestation rate (Rpt), total ectoparasite infestation index (Ipt), and constituent ratio (Cr) were used to measure the community structure. RESULTS: Altogether, 3,303 small mammals and 3,243 fleas were collected. From the 21 species of small mammal hosts, 13 species of fleas were identified. In southern area of the Lake, the species richness (21 species of small mammals & 12 species of fleas) was highest among the three selected areas. Seventeen species of small mammals and 8 species of fleas were found in eastern area, and only 13 species of small mammals and 7 species of fleas found in the west. This implied the probable influences of ecological environments on the fleas and their corresponding hosts. The community structure of fleas on small mammals was complex. The species diversity, species composition, community structure and distribution pattern of fleas were simultaneously influenced by the hosts' body surface microenvironment and the macroenvironment (habitat). CONCLUSION: The fleas are commonly distributed in small mammals in the areas and their communities are related to host species and the habitats.

Isolation and characterization of the full coding sequence of a novel densovirus from the mosquito Culex pipiens pallens.

During an investigation of arboviruses in China, a novel densovirus (DNV) was isolated from the adult female Culex pipiens pallens. The virus, designated Culex pipiens pallens densovirus (CppDNV), caused cytopathic effect in C6/36 cells. The virus particles were icosahedral, non-enveloped and had a mean diameter of 24 nm. The complete coding region of CppDNV was found to be 3335 nt and it contained three open reading frames (ORFs). CppDNV shares 82-93 % identical nucleotides with isolates of the Aedes albopictus densovirus [isolates AalDNV-1, AalDNV-2 (C6/36 DNV) and AalDNV-3], Aedes aegypti densovirus (AaeDNV) and Haemagogus equines densovirus (HeDNV). The nucleotide sequence identity among CppDNV isolates exceeds 98 %. Phylogenetic trees based on non-structural (NS1 and NS2) and capsid (VP) genes show that CppDNV clustered with the species AaeDNV and represents a novel variant of this species within the genus Brevidensovirus.

[Study on the prevalence of Bartonella species in rodent hosts from different environmental areas in Yunnan].

OBJECTIVE: To investigate Bartonella infections in small mammalian reservoir hosts from different environments and types of climate in Yunnan. METHODS: Femoral blood samples were collected from the anesthetic captured animals from five counties including three types of climate. All isolates were grown on brain and heart infusion agar plates containing 5% defibrinated rabbit blood. The agar plates were incubated at 35 degrees C in a humidified with 5% CO2 environment for at least 4 weeks. Bartonella-like isolates were confirmed by the polymerase chain reaction and visualizing the target gene fragment by gel electrophoresis. RESULTS: Bartonella species were isolated from 69 of 176 small animals including 4 species of 3 genera from 4 counties and the total prevalence in rodents was 39.2%. The maximal prevalence was 42.0% of Rattus tanezumi flavipectus usually inhabiting indoors and courtyard and contacting closely to human. Moreover, Bartonella isolates were obtained from Rattus noruegicus, Eothenomys miletus and Mus pahari. Life environments of captured animals involved indoors, courtyard, brush and forest in mountain. CONCLUSION: The finding in this study suggested the characteristic of diversity of Bartonella infections in rodent hosts in southern China included Bartonella species parasiting in a wide range of animal hosts in different environments as well as climate types. Further investigations were needed in different areas in China to confirm more mammalian reservoir hosts with Bartonella infections.