BackgroundThe 5’ region of cytochrome oxidase I (COI) is the standard marker for DNA barcoding. However, COI has proved to be of limited use in identifying some species, and for some taxa, the coding sequence is not efficiently amplified by PCR. These deficiencies lead to uncertainty as to whether COI is the most suitable barcoding fragment for species identification of ticks.MethodsIn this study, we directly compared the relative effectiveness of COI, 16S ribosomal DNA (rDNA), nuclear ribosomal internal transcribed spacer 2 (ITS2) and 12S rDNA for tick species identification. A total of 307 sequences from 84 specimens representing eight tick species were acquired by PCR. Besides the 1,834 published sequences of 189 tick species from GenBank and the Barcode of Life Database, 430 unpublished sequences representing 59 tick species were also successfully screened by Bayesian analyses. Thereafter, the performance of the four DNA markers to identify tick species was evaluated by identification success rates given by these markers using nearest neighbour (NN), BLASTn, liberal tree-based or liberal tree-based (+threshold) methods.ResultsGenetic divergence analyses showed that the intra-specific divergence of each marker was much lower than the inter-specific divergence. Our results indicated that the rates of correct sequence identification for all four markers (COI, 16S rDNA, ITS2, 12S rDNA) were very high (> 96%) when using the NN methodology. We also found that COI was not significantly better than the other markers in terms of its rate of correct sequence identification. Overall, BLASTn and NN methods produced higher rates of correct species identification than that produced by the liberal tree-based methods (+threshold or otherwise).ConclusionsAs the standard DNA barcode, COI should be the first choice for tick species identification, while 16S rDNA, ITS2, and 12S rDNA could be used when COI does not produce reliable results. Besides, NN and BLASTn are efficient methods for species identification of ticks.

A growing number of studies have demonstrated that autophagy has a diverse role in the infection process of different pathogens. However, to date, it is unknown whether autophagy is activated in encephalomyocarditis virus (EMCV)-infected host cells, and if so, what its role is in this process. In the present study, we first demonstrated that EMCV infection significantly increases the number of double- and single-membrane vesicles in the cytoplasm of host cells. It was then confirmed that these observed vesicles are indeed related to autophagy, and that EMCV replication is required for the induction of autophagy by examining LC3-I/-II conversion and p62/SQSTM1 degradation using immunoblotting. Next, we performed confocal immunofluorescence analysis and discovered that, during EMCV replication, both the nonstructural protein 3A and capsid protein VP1 colocalized with LC3. The colocalizations of both 3A and VP1 protein with autophagosome-like vesicles were further confirmed using immunoelectron microscopy, indicating that EMCV undergoes RNA replication on the membranes of these vesicles. Finally, we used pharmacological regulators and siRNAs to examine the role of autophagy in EMCV replication. Our results suggest that autophagy not only promotes the replication of EMCV in host cells, but it also provides a topological mechanism for releasing cytoplasmic viruses in a nonlytic manner. Noticeably, the autophagic pharmaceuticals we used had no significant effect on virus entry or cell viability, both of which may affect viral replication. To our knowledge, ours is the first strong evidence indicating that autophagy is involved in EMCV infection in host cells.

To control the spread of tick-borne diseases, there is an urgent need to develop a reliable technique that can distinguish different species of ticks. DNA barcoding has been proved to be a powerful tool to identify species of arthropods, but this technique has not yet been developed for identifying ticks. Here, we screened and analyzed 1082 sequences of ticks from BOLD system and GenBank, consisting of 647 16S, 325 COI, and 110 18S. These sequences are reported in previous studies and considered to be correctly identified at the species level. Through the analyses of genetic divergences and neighbor-joining (NJ) phylogenetic relationships between the species of ticks, our results show that COI and 16S are reliable in discriminating species of ticks and the 18S could discriminate ticks at the genera level. New universal primers for 16S, 18S, and COI of ticks were designed and a DNA barcoding system for the Ixodida was developed. To assess the performance of this system, 57 specimens of ticks were collected within China. Our results show that DNA barcoding system could correctly identify the species of specimens in adult and subadult stages. This system would assist non-taxonomists to conveniently identify the species of Ixodida based on DNA sequences rather than morphological traits. However, there are still serious deficiencies in the information of 16S and COI of some species of ticks, and additional research is needed to resolve this problem.

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Classical swine fever (CSF), formerly known as "hog cholera", is a highly contagious infectious disease that affects both domestic pigs and wild boars, with high morbidity and mortality. Due to its tremendous impact on the swine industry, CSF is therefore notifiable to the World Organization for Animal Health (OIE) (Zhou, 2019). Classical swine fever virus (CSFV), the causative agent of CSF, is classified in the genus Pestivirus within the family Flaviviridae. Currently, the genus Pestivirus includes eleven recognized species, such as CSFV, bovine viral diarrhoea viruses 1 and 2 (BVDV-1 and BVDV-2), and border disease virus. CSFV has a linear, non-segmented, single-stranded, positive-sense RNA genome of approximately 12.3 kb (Edwards et al., 2000). The genome consists

Peste des petits ruminants virus (PPRV) is an important pathogen that seriously influences the productivity of small ruminants worldwide. Although PPRV is known to induce apoptosis in infected cells, the interaction between PPRV and permissive cells requires further elucidation. Here, we provide the first evidence that PPRV infection triggered autophagy in Vero cells based on the appearance of abundant double- and single-membrane vesicles, the accumulation of LC3 fluorescent puncta, the enhancement of LC3-I/-II conversion, and autophagic flux. We further demonstrated that induction of autophagy with rapamycin significantly increased PPRV progeny yield and nucleocapsid (N) protein expression, while inhibition of autophagy with siRNA targeting ATG7 resulted in diametrically opposite results. Our data indicate that PPRV exploits the autophagy machinery to facilitate its own replication in host cells, thus the production efficiency of live attenuated PPRV vaccines may be improved by targeting the autophagic pathway.

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