Abrogation of pRRSV infectivity by CRISPR-Cas13b-mediated viral RNA cleavage in mammalian cells Jin cui, navapon techakriengkrai, teerawut nedumpun & Sanipa Suradhat ✉ CRISPR/Cas9 enables dsDNA viral genome engineering. However, the lack of RNA targeting activities limits the ability of CRISPR/Cas9 to combat RNA viruses. The recently identified class II type VI CRISPR/ Cas effectors (Cas13) are RNA-targeting CRISPR enzymes that enable RNA cleavage in mammalian and plant cells. We sought to knockdown the viral RNA of porcine reproductive and respiratory syndrome virus (PRRSV) directly by exploiting the CRISPR/Cas13b system. Effective mRNA cleavage by CRISPR/ Cas13b-mediated CRISPR RNA (crRNA) targeting the ORF5 and ORF7 genes of PRRSV was observed. To address the need for uniform delivery of the Cas13b protein and crRNAs, an all-in-one system expressing Cas13b and duplexed crRNA cassettes was developed. Delivery of a single vector carrying double crRNAs enabled the simultaneous knockdown of two PRRSV genes. Transgenic MARC-145 cells stably expressing the Cas13b effector and crRNA mediated by lentiviral-based transduction showed a robust ability to splice the PRRSV genomic RNA and subgenomic RNAs; viral infection was almost completely abrogated by the combination of double crRNAs simultaneously targeting the ORF5 and ORF7 genes. Our study indicated that the CRISPR/Cas13b system can effectively knockdown the PRRSV genome in vitro and can potentially be used as a potent therapeutic antiviral strategy. RNA viruses remain a great threat to humans and animals around the world 1,2. Vaccination is the primary strategy to prevent viral infections in hosts 3. Unlike DNA viruses, the extremely high evolutionary rates of RNA viruses have led to the rapid emergence of variant virus strains 4,5. Heterologous infections often cause vaccine failure. In addition, vaccines against many RNA viruses have not been successfully developed 6. Following viral infection, antiviral drug treatments are the only clinical therapy to combat viruses. However, there are only a limited number of antivirals available targeting viruses that threaten public human health, such as HIV, HBV, HCV, herpesviruses and influenza viruses 7. Although certain compounds have the potential to be developed as broad-spectrum antiviral agents, the relatively high cost limits their widespread clinical use in veterinary medicine, RNA viral infection causes significant economic loss in the livestock industry. Porcine reproductive and respiratory syndrome (PRRS) has been one of the most important infectious viral diseases in swine worldwide for almost three decades 8,9. Porcine reproductive and respiratory syndrome virus (PRRSV), the aetiological agent of PRRS, is an enveloped single-stranded positive-sense RNA virus belonging to the family Arteriviridae 10. Presently, PRRSV is taxonomically classified into the genus Porarterivirus 11,12. The PRRSV genome is approximately 15 kb in length with at least 10 open reading frames (ORFs) 13. PRRSV utilizes two distinct transcr...

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The Chinese highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) has caused a severe threat to the pig population in Southeast Asian countries. The purpose of this study was to investigate the efficacy of a type 2 PRRSV modified live vaccine (PrimePac™ PRRS, lineage 7) against a Thai HP-PRRSV (10PL01, lineage 8). Three-week-old PRRSV-free pigs were randomly assigned into three groups. Vaccinated challenged group (group 1, n = 16) was immunized with PrimePac™ PRRS vaccine at 3 weeks old. The unvaccinated challenged group (group 2, n = 16) was injected with PBS at 3 weeks old, and unvaccinated unchallenged group (group 3, n = 10) was served as a negative control. At 9 weeks old, all groups, except the negative control group, were challenged with the Thai HP-PRRSV. All pigs were monitored daily during 10 days post-infection (dpi) and were necropsied at 10 and 17 dpi. The results revealed that vaccinated challenged pigs showed significantly lower (p < 0.05) mean rectal temperatures, clinical respiratory scores, lung lesion scores, and levels of virus load in serum and lung tissue compared with the unvaccinated challenged pigs. Moreover, vaccinated challenged pigs exhibited PRRSV-specific serum neutralizing antibodies at the end of the experiment. Our findings indicated that the studied type 2 PRRSV vaccine provided partial protection against the Thai HP-PRRSV infection based on the body temperature, levels of viremia, and the severity of lung lesions. These results demonstrated that partial protection of PrimePac™ PRRS vaccine might be useful for controlling HP-PRRSV infection in the endemic area.

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