Transient and tunable CRISPRa regulation of APOBEC/AID genes for targeting hepatitis B virus.

APOBEC/AID cytidine deaminases play an important role in innate immunity and antiviral defenses and were shown to suppress hepatitis B virus (HBV) replication by deaminating and destroying the major form of HBV genome, covalently closed circular DNA (cccDNA), without toxicity to the infected cells. However, developing anti-HBV therapeutics based on APOBEC/AID is complicated by the lack of tools for activating and controlling their expression. Here, we developed a CRISPR-activation-based approach (CRISPRa) to induce APOBEC/AID transient overexpression (>4-800,000-fold increase in mRNA levels). Using this new strategy, we were able to control APOBEC/AID expression and monitor their effects on HBV replication, mutation, and cellular toxicity. CRISPRa prominently reduced HBV replication (∼90%-99% decline of viral intermediates), deaminated and destroyed cccDNA, but induced mutagenesis in cancer-related genes. By coupling CRISPRa with attenuated sgRNA technology, we demonstrate that APOBEC/AID activation can be precisely controlled, eliminating off-site mutagenesis in virus-containing cells while preserving prominent antiviral activity. This study untangles the differences in the effects of physiologically expressed APOBEC/AID on HBV replication and cellular genome, provides insights into the molecular mechanisms of HBV cccDNA mutagenesis, repair, and degradation, and, finally, presents a strategy for a tunable control of APOBEC/AID expression and for suppressing HBV replication without toxicity.

CRISPR-Cas systems for diagnosing infectious diseases.

Infectious diseases are a global health problem affecting billions of people. Developing rapid and sensitive diagnostic tools is key for successful patient management and curbing disease spread. Currently available diagnostics are very specific and sensitive but time-consuming and require expensive laboratory settings and well-trained personnel; thus, they are not available in resource-limited areas, for the purposes of large-scale screenings and in case of outbreaks and epidemics. Developing new, rapid, and affordable point-of-care diagnostic assays is urgently needed. This review focuses on CRISPR-based technologies and their perspectives to become platforms for point-of-care nucleic acid detection methods and as deployable diagnostic platforms that could help to identify and curb outbreaks and emerging epidemics. We describe the mechanisms and function of different classes and types of CRISPR-Cas systems, including pros and cons for developing molecular diagnostic tests and applications of each type to detect a wide range of infectious agents. Many Cas proteins (Cas3, Cas9, Cas12, Cas13, Cas14 etc.) have been leveraged to create highly accurate and sensitive diagnostic tools combined with technologies of signal amplification and fluorescent, potentiometric, colorimetric, lateral flow assay detection and other. In particular, the most advanced platforms -- SHERLOCK/v2, DETECTR, CARMEN or CRISPR-Chip -- enable detection of attomolar amounts of pathogenic nucleic acids with specificity comparable to that of PCR but with minimal technical settings. Further developing CRISPR-based diagnostic tools promises to dramatically transform molecular diagnostics, making them easily affordable and accessible virtually anywhere in the world. The burden of socially significant diseases, frequent outbreaks, recent epidemics (MERS, SARS and the ongoing COVID-19) and outbreaks of zoonotic viruses (African Swine Fever Virus etc.) urgently need the developing and distribution of express-diagnostic tools. Recently devised CRISPR-technologies represent the unprecedented opportunity to reshape epidemiological surveillance and molecular diagnostics.

Analysis and insights into recombination signals in lumpy skin disease virus recovered in the field.

Wide spread incidences of vaccine-like strains of lumpy skin disease virus (LSDV) have recently been reported in a Russian region with a neighboring country that actively vaccinate with a live attenuated LSD vaccine. The use of live-attenuated viruses (LAVs) as vaccines during an active outbreak, creates potential ground for coinfection of hosts and emergence of a strain combining genetic fragments of both parental vaccine and field strains. In this study, we analyse the vaccine-like strain LSDV RUSSIA/Saratov/2017 detected in Saratovskaya oblast, a region sharing border with Kazakhstan. To gain insight into possible recombination signals, a full-genome next-generation sequencing of the viral genome was performed using the Illumina platform. The genome contains the backbone of a live-attenuated vaccine with a patchwork of wild-type field virus DNA fragments located throughout. A total of 27 recombination events were identified. The average distance between the recombination sites was 3400 base pairs (bp). The impact of the recombination events on the virulence and transmission capacity of the identified virus remains to be clarified. These findings provide evidence for the first time of genetic exchanges between closely related strains of capripoxviruses in the field and a vaccine strain, and prompt a revisiting of the vaccination issue for a safe and efficacious prevention and control strategy of LSD.

Seasonal population fluctuations of Culicoides midges in the Smolenskaya, Pskovskaya and Vladimirskaya provinces of Western Russia in 2013.

Light trapping for biting midges was performed on cattle farms in 3 provinces of Western Russia in 2013. A total of 9,272, 2,457, and 10,245 Culicoides midges were collected from farms in Smolenskaya, Pskovskaya, and Vladimirskaya provinces, respectively. More than 99% of collected midges belonged to either the Obsoletus or Pulicaris complexes. Species belonging to the Obsoletus complex were the most abundant in Pskovskaya, whereas species from the Pulicaris complex dominated the catches in Vladimirskaya and Smolenskaya provinces. The seasonal duration of adult activity in all 3 regions was approximately 4.5 months.