Objective To evaluate a real-time detection technology for rodent-borne pathogens based on the MinION high-throughput nanopore sequencing platform with Bartonella spp. as an indicator. Methods The whole-genome DNA was extracted from the spleen and lung tissues of wild rodents with positive Bartonella culture results. Sanger sequencing following conventional PCR amplification (using universal primers of 16S ribosomal RNA [16S rRNA] gene) and nanopore sequencing was performed and compared, and then evaluate the feasibility and accuracy of MinION nanopore sequencing technology in the detection of rodent-borne pathogens. Results By using the first-generation Sanger sequencing after the conventional 16S rRNA gene amplification, Bartonella was not identified in 12 samples. In contrast by using the third-generation nanopore sequencing, Bartonella was identified in all samples. The number of reads per sample used for taxonomic analysis ranged from 4 to 609 424, and the reads length was mainly 1 500 bp, with the mean accuracies ranging from 79.2% to 92.0%. Bartonella was the main pathogen detected, and its number of reads ranged from 1 to 77 833. By using cloud real-time analysis software EPI2ME, the identification results were generated within 30 minutes after the sequencing was started. In addition, Brucella spp. was also identified in some samples. The concentration of the original samples had an impact on the amount of sequencing data, with evidence showing that low-concentration samples had significantly less total data volume and number of reads. The cleaning procedure significantly reduced the residual nucleic acid fragments in the sequencing chip, but could not completely remove them, with a 1.40% residual ratio. Conclusion Nanopore sequencing of 16S rRNA gene amplicons can be used for direct and real-time detection and identification of pathogens in organs and tissues from rodents. Nanopore MinION sequencing provides convenience for on-site detection and pathogen monitoring with its portable and simple operation, and real-time data transmission and analysis.

Objective To investigate the prevalence of Bartonella infection and gene polymorphisms of Bartonella in small mammals in Maixiu National Forest Park in the Qinghai-Tibet Plateau, China, and to provide a scientific basis for the control and prevention of local natural focal diseases. Methods The night trapping method was used to capture small mammals, whose liver and spleen tissues were collected and cultured for Bartonella isolation. The suspected positive colonies were confirmed using PCR amplification and sequencing of the citrate synthase (gltA) gene. BLAST and MEGA 7.0 softwares were used to perform the nucleotide sequence homology comparison and phylogenetic analysis, and DnaSP 5.10 software was used to analyze the genetic diversity. Results A total of 21 rodents were captured, including 10 Cricetulus longicaudatus rodents, 6 Apodemus speciosus rodents, 4 Mus musculus rodents, and 1 Microtus oeconomus rodent. In addition, one small mammal of Soricidea, which belonged to Insectivora, was also captured. Except M. oeconomus, Bartonella was detected in all the other four species of small mammals, with an overall positive rate of 59.09% (13/22). Specifically, nine cases showed positive results in both the liver and spleen tissues, one showed positive results in the liver tissue alone, and three showed positive results in the spleen tissue alone. There was no statistical difference in the positive rate between liver and spleen tissues (45.45% vs 54.55%, P=0.625). The phylogenetic analysis showed that the isolated Bartonella species were as follows: Bartonella grahamii (10 strains), B. taylorii (1 strain), B. khabarovsk (1 strain), and B. japonica (1 strain), among which B. grahamii was the dominant prevalent species with potential pathogenicity. In addition, the traceability analysis showed that the B. grahamii isolates from C. longicaudatus and M. musculus belonged to the same cluster as those from A. speciosus in Japan, and the B. grahamii isolates from A. speciosus belonged to the same cluster as those from Ochotona curzoniae in this area. The genetic diversity analysis showed that the nucleotide sequences in B. grahamii were quite different between various rodent species. There were 8 polymorphic loci in the 10 sequences, resulting in 3 haplotypes. The haplotype diversity (Hd) was 0.622±0.138; the average number of nucleotide differences (k) was 3.200, and the nucleotide diversity (π) was 0.010. The fragment diversity was highest between 152 bp and 251 bp. Conclusion The small mammals in Maixiu National Forest Park have a high infection rate with Bartonella, and B. grahamii is the dominant species, which has genetic diversity and may cause human infection and diseases.

Objective To study the characteristics of tick-borne pathogenic populations using 16S rDNA full-length sequencing and laboratory test data, and to provide a basis for pathogen control. Methods Fifty ticks were selected from the dominant tick species in Qi county, Shanxi province, China. Whole genome DNA was extracted after morphological and genetic identification. The species and taxonomic abundance of microorganisms in ticks were analyzed using 16S rDNA full-length high-throughput sequencing on the PacBio platform. According to the 16S rDNA sequence analysis, the specific genes of tick-borne pathogens were identified by PCR for a comprehensive analysis of tick-borne pathogens. Results The 16S rDNA full-length sequencing analysis showed that all the 50 ticks carried Coxiella burnetii and Pseudomonas, 8 ticks carried Anaplasma phagocytophilum, some ticks carried Rickettsia and Ehrlichia (only identified at the genus level), and Borrelia was not detected. The PCR results showed that C. endosymbiont, Borrelia burgdorferi, B. miyamotoi, and spotted fever group rickettsia were detected in 50 ticks. Conclusion High-throughput 16S rDNA full-length sequencing can simultaneously detect a variety of pathogens and identify the main tick-borne pathogens in the area, though some pathogens are identified at the genus level, which requires combination with pathogen-specific tests for comprehensive detection of tick-borne pathogens in the area.

Objective To establish a method for determining the virus titer of the Culex flavivirus (CxFV) strain (SDDM06-11) isolated in China. Methods C6/36 cells were cultured in a 6-well cell culture plate and were then inoculated with CxFV. Methylcellulose was added to the cells and cultured for 4 days. Crystal violet was used for staining, and the number of plaques was determined to calculate the plaque forming unit of the virus. Results Observation of plaques using methylcellulose and crystal violet showed that CxFV (SDDM06-11) strain formed clear round plaques with the size of a needle tip after infecting C6/36 cells, and the virus titer of SDDM06-11 strain was successfully determined as 10^6.85 pfu/ml. Conclusion The methylcellulose-crystal violet plaque forming assay can be used to determine the titer of CxFV.

Objective To investigate the species and prevalence of blood-sucking insects and their viruses in Qiongzhong county, Hainan province, China. Methods In August 2019, UV mosquito light-trapas (Gongfu Xiaoshuai) were used to collect specimens in the local environment (sheep pens and pig pens) where blood-sucking insects bred, and dry ice was used to transport the specimens to the laboratory. Virological and molecular biological methods were used to isolate viruses and identify the virus isolates. Results A total of 34 mosquitoes from 3 genera (including 26 from Culex, 1 from Anopheles, and 7 Armigeres subalbatus mosquitoes) and 1 450 midges (to be identified) were collected. A virus isolate (HNQZ1927) was isolated from the collected midge specimens, which could cause cytopathic effect in mosquito cells (C6/36), but did not cause cytopathic effect in BHK-21 cells. The gene amplification and nucleotide sequencing and analysis showed that HNQZ1927 was a 10-gene segment orbivirus. The nucleotides (amino acids) in the coding region of HNQZ1927 had 79.4%-98.7% (94.6%-100%) homology with those of Tibet orbivirus (TIBOV) from the genus Orbivirus, and had 1.2%-78.1% homology with those of other orbiviruses except TIBOV. The phylogenetic analysis of the viral genome showed that the HNQZ1927 virus was in the same evolutionary branch as TIBOV XZ0906, which was previously isolated from An. maculatus collected in Tibet, and the TIBOV isolated from Cx. tritaeniorhynchus, Cx. fatigans, and some species of midges in China. Conclusion The HNQZ1927 virus isolated from midges in Hainan province is TIBOV, which is the first time that TIBOV has been isolated from midge specimens collected from nature in Hainan province, China.

Objective To investigate the mosquito species and the distribution of mosquito-borne viruses in some regions of Guizhou province, China, and to provide a basis for the prevention and control of mosquito-borne diseases in this area. Methods Mosquitoes collected with trapping lamps were identified, mosquito-borne viruses were identified by molecular biological methods, and a phylogenetic tree of virus strains was constructed using bioinformatics software. Results A total of 27 169 mosquitoes (9 species, 3 genera) were collected in Zhongchao town of Liping county and Yangchang town of Wudang district (Guiyang city) of Guizhou province in June 2019. The dominant species was Anopheles sinensis (13 712/27 169,50.47%), followed by Culex tritaeniorhynchus (12 444/27 169,45.80%) and other species (70/27 169,0.26%). A virus strain isolated from a mosquito sample (LK6110) from Zhongchao town was identified as Japanese encephalitis virus genotype Ⅲ (JEV Ⅲ). Conclusion In this investigation, Yangchang town was dominated by An. sinensis and Zhongchao town was dominated by Cx. tritaeniorhynchus, and JEV Ⅲ was isolated from Cx. tritaeniorhynchus in Zhongchao town.

Objective To investigate the difference in the positive rate of Orientia tsutsugamushi between different organs of small mammals, and to provide a reference for the pathogen monitoring of scrub typhus. Methods Liver, spleen, lung, kidney, and ear samples were collected from 105 small mammals collected in Yongcheng city of Henan province, China in October 2017, and liver, spleen, and ear samples were collected from 324 small mammals collected in Pinggu district of Beijing, China from September to November 2018. DNA was extracted, and nested PCR amplification was used to detect the nucleic acid fragment of the 56 kDa protein gene of O. tsutsugamushi. The specimens with specific bands were genotyped by comparison of nucleotide sequence, and the positive rate was calculated. Results The positive rate of O. tsutsugamushi in small mammals was 10.48% in Yongcheng city and 12.96% in Pinggu district, with no significant difference (χ²=0.494, P=0.504). The positive rate of O. tsutsugamushi in different organs within the same site was 0.98%-14.81%; pairwise comparison showed that the positive rate of O. tsutsugamushi in the ear of the small mammals in Yongcheng city was higher than that in the other four organs, and the positive rate of O. tsutsugamushi in the liver of the small animals in Pinggu district was significantly higher than that in the spleen (χ²=5.818, P=0.022). The comparison of the two places showed that the positive rate of O. tsutsugamushi in the ear of the small mammals in Yongcheng city was significantly higher than that in Pinggu district (χ²=6.525, P=0.011). For the small mammals in Pinggu district, the positive rate of Shimokoshi-type O. tsutsugamushi in the spleen was lower than that in liver and the ear, while there was no significant difference in the positive rate of Kawasaki-type O. tsutsugamushi between the different organs (χ²=1.560, P=0.455). The pooled analysis of the detection results of the liver and the spleen in Yongcheng city and Pinggu district showed that the positive rate of O. tsutsugamushi in the liver was higher than that in the spleen. The results of consistency test showed no significant difference (Kappa=0.015, P=0.744), with poor consistency of positive infection in the liver and the spleen. Conclusion There are no significant differences in the positive rates of Kawasaki-type O. tsutsugamushi in the liver, spleen, lung, and kidney of small mammals between Yongcheng city and Pinggu district, while the positive rate of Shimokoshi-type O. tsutsugamushi in the liver and the ear is higher than that in the spleen. Detection of O. tsutsugamushi in the organ of various small mammals or in multiple organs can improve the detection rate of O. tsutsugamushi, and rodent ears can also be used as samples for the pathogen monitoring of scrub typhus.

Objective To investigate the species composition of edible rodents and rodent-borne pathogens in some ethnic minority areas in Guizhou province, and to provide basic data for the prevention and control of rodents and rodent-borne diseases. Methods Edible rodents were preliminarily investigated through interviews with local ethnic minority people. Rodents were captured by the night trapping method in the mountainous forest areas of three counties and four towns in Qiandongnan Miao and Dong autonomous prefecture. Rodents were identified and dissected and the viscera were stored at -20 ℃ and transported to the laboratory. PCR was used to detect rodent-borne Leptospira, Hantavirus, and Yersinia pestis. Bacteria were isolated and identified from two intestinal samples. Results A total of 141 (20.74%) rodents were captured, which belonged to 2 families, 5 genera, and 10 species, and the most frequently consumed rodents were Berylmys bowersi and Niviventer coninga. Leptospira (positive rate, 13/48, 27.08%), Hantavirus type I (positive rate, 1/141, 0.71%), and Hantavirus type Ⅱ (positive rate, 18/141, 12.77%) were detected, while Y. pestis was absent (0/125). Four non-diarrheagenic bacterial species were isolated from the duodenum of B. bowersi, while two of the four bacterial strains isolated from N. coninga were diarrheagenic. Conclusion The dominant species of edible rodents in mountain forests are B. bowersi and N. coninga. These species are major hosts of Leptospira and Hantavirus and present a high risk of disease transmission. We suggest to strengthen the surveillance of rodents and rodent-borne diseases in this area, and pay attention to the publicity and education of rodent-borne diseases to reduce the risk of infection.