Jing-Feng Yu

Introduction: Tacheng tick virus 1 (TcTV-1) is an emerging tick-borne nairovirus associated with human febrile illness. To date, no reliable detection method for TcTV-1 has been established. In this study, we developed and evaluated a rapid loop-mediated isothermal amplification (LAMP) assay for the detection of TcTV-1. Methods: The primers were designed based on the nucleocapsid protein (NP) gene of TcTV-1. Sensitivity was assessed using ten-fold serial dilutions of recombinant plasmids containing the target sequence. Specificity was evaluated using cDNA from Songling virus (SGLV), Yezo virus (YEZV), Tick-borne encephalitis virus (TBEV), Severe fever with thrombocytopenia syndrome virus (SFTSV), and Beiji nairovirus (BJNV). The assay was validated using field-collected tick samples. Results: The TcTV-1-specific LAMP assay detected as few as 1×10 ^-1 copies/μL within 60 minutes at 65 °C and specifically amplified TcTV-1, with no cross-reactivity to SGLV, YEZV, TBEV, SFTSV, or BJNV. Positive reactions exhibited a clear color change from purple to blue, indicating a robust colorimetric response. A total of eight tick specimens (16.0%; 95% CI: 7.2–29.1) tested positive for TcTV-1 using both the established LAMP assay and SYBR Green real time quantitative polymerase chain reaction (RT-qPCR), demonstrating 100% sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy for the LAMP assay. Discussion: We report a TcTV-1-specific LAMP assay with high sensitivity, specificity, and cost-effectiveness, making it a practical tool for use in field-based or resource-limited settings.

Background: Songling virus (SGLV) within the genus Orthonairovirus, family Nairoviridae, is an emerging tick-borne virus associated with human febrile illness. However, no rapid detection method for SGLV has been established. Methods: In this study, four primer sets targeting the nucleocapsid protein gene of SGLV were designed for use in the LAMP assay and evaluated to identify the optimal primer set. Recombinant plasmids were constructed and utilized for assessing the sensitivity of the assay. Tacheng tick virus 1 (TcTV-1)-, Beiji nairovirus (BJNV)-, Yezo virus (YEZV)-, severe fever with thrombocytopenia syndrome virus (SFTSV)-, and tick-borne encephalitis virus (TBEV)-positive tick samples were utilized to assess the specificity. Field-collected ticks were also evaluated as biological specimens to validate the assay. Results: A SGLV-specific LAMP assay was established with a detection limit of 1 × 10^–2 copies/μl and could be visually confirmed by a color change from purple to blue in SGLV-positive samples. No cross-reactivity was observed in the detection of TcTV-1, BJNV, YEZV, SFTSV, and TBEV using the LAMP assay. In addition to the detection of the same seven high-copy numbers of SGLV as the SYBR Green quantitative RT-PCR assay within a reduced timeframe, the developed LAMP method also effectively identified an additional sample with a low copy number in the field-collected tick samples. Conclusions: We successfully developed a sensitive, specific, and cost-effective visual method for the rapid detection of SGLV using the LAMP assay, which can be applied in pathogenesis and epidemiological surveillance studies of SGLV. Graphical

Spotted fever group (SFG) rickettsioses are important zoonoses, threatening human health seriously and gradually attracting more attention in the world. SFG rickettsiae are classified as neglected pathogens. If these pathogens are detected at all, they are usually recognized very late in the infection through indirect detection of specific antibodies. Previous studies have shown that Rickettsia raoultii (R. raoultii), a member of the SFG rickettsiae, occurs with increasing incidence in remote countries. Therefore, a rapid detection method for R. raoultii is in urgently need. In this study, a R. raoultii diagnosis method by closed dumbbell-mediated isothermal amplification (R-CDA) assay targeting a conserved sequence of the outer membrane protein A (OmpA) gene with high sensitivity and specificity was developed. This assay offered a rapid and simple method for on-site detection of R. raoultii. Firstly, four pairs of R-CDA primers were designed and the optimum primer set was selected to amplify target gene specifically and effectively. Then, a pair of outer primer was designed to accelerate the reaction based on the inner primers to establish the RO-CDA reaction. In addition, the results of real-time amplification curves, melting curves and end-point colorimetric judgements showed that the established visual RO-CDA reaction could accurately detect R. raoultii without cross-reaction with other closely related pathogens. Furthermore, the detection limit of visual RO-CDA assay was 10 copies/μL, which was feasible for on-site detection with merits of easy-operation, rapidity, high sensitivity, and specificity. In conclusion, the developed RO-CDA detection method could be helpful for pathogen screening and epidemic prevention at the point of care.

Background: The genus Rickettsia contains the lineages spotted fever group (SFG), typhus group (TG), and transitional group (TRG). The spotted fever group Rickettsia (SFGR) is transmitted by ticks. The tick species Dermacentor nuttalli is considered the main vector carrying SFGR in Inner Mongolia. Studying the genetic diversity and population structure of Rickettsia is essential for developing effective control strategies and predicting evolutionary trends of Rickettsia. Methods: In 2019 we collected 408 D. nuttalli in the Inner Mongolia Autonomous Region, detected the percentage of Rickettsia-positive specimens, and characterized the haplotypes. From the Rickettsia-positive ticks, the gltA and ompA genes were extracted, amplified, and sequenced. Results: Ten haplotypes of the gltA gene and 22 haplotypes of the ompA gene were obtained. The phylogenetic analysis showed that the haplotypes G1–G7 and G9 of the gltA gene cluster with Rickettsia raoultii, while G8 and G10 cluster with Rickettsia sibirica. Haplotypes O1–O15, O18 and O20–O22 of the ompA gene cluster with R. raoultii, while O16 and O19 cluster with R. sibirica. The average haplotype diversity was 0.3 for gltA and 0.7 for ompA. The average nucleotide diversity was greater than 0.05. Neutrality tests were nonsignificant for Tajima’s D results and Fu’s Fs results. The fixation index values (F_ST) showed that the degree of genetic differentiation between most sampled populations was small (F_ST < 0.05), whereas some populations showed a medium (F_ST > 0.05) or large (F_ST > 0.15) degree of differentiation. Analysis of molecular variance (AMOVA) revealed that the variation within populations was greater than that between populations. The mismatch analysis of Rickettsia showed double peaks. Conclusions: We found two Rickettsia spp. (R. raoultii and R. sibirica). The high genetic disparity of Rickettsia allows for easy adaption to different environments. Genetic differentiation between populations is small, and Rickettsia populations do not show a geographically differentiated structure. The high rates of retention and infection of Rickettsia in D. nuttalli together with the animal husbandry exchange in Inner Mongolia gradually led to the harmonization of genetic characteristics of Rickettsia across various regions. Overall, the significant genetic diversity and geographical structure of Rickettsia in D. nuttalli are critical for SFGR control. Graphical

Background: Ticks (Arthropoda, Ixodida), after mosquitoes, are the second most prevalent vector of infectious diseases. They are responsible for spreading a multitude of pathogens and threatening the health and welfare of animals and human beings. However, given the history of tick-borne pathogen infections in the Inner Mongolia Autonomous Region of China, surprisingly, neither the genetic diversity nor the spatial distribution of haplotypes within ticks has been studied.Methods: We characterized the haplotype distribution ofDermacentor nuttalliin four main pastoral areas of the Inner Mongolia Autonomous Region, by sampling 109 individuals (recovered from sheep) in April–August 2019. The 16S rRNA gene, cytochrome c oxidase subunit I (COI), and the internal transcribed spacer 2 region (ITS2) were amplified and sequenced from extracted DNA.Results: Twenty-six haplotypes were identified using 16S rRNA sequences, 57 haplotypes were identified with COI sequences, and 75 haplotypes were identified with ITS2 sequences. Among the three genes, total haplotype diversity was greater than 0.7, while total nucleotide diversity was greater than 0.06. Neutrality tests revealed a significantly negative Tajima’s D result, while Fu's Fs was not significantly positive. Fixation index values (F_ST) indicated that the degree of genetic differentiation among some sampled populations was small, while for others it was moderate. Analysis of molecular variance (AMOVA) revealed that the variation within populations was greater than that among populations. The mismatch analysis ofD. nuttalliexhibited double peaks.Conclusion: The genetic diversity ofD. nuttallipopulations in our region can likely adapt to different geographical environments, thereby leading to genetic diversity, and creating genetic differentiation among different populations. However, genetic differentiation is cryptic and does not form a pedigree geographical structure.Graphical