AAV-CRISPR-Cas13 eliminates human enterovirus and prevents death of infected mice.

BACKGROUND: RNA viruses account for many human diseases and pandemic events but are often not targetable by traditional therapeutics modalities. Here, we demonstrate that adeno-associated virus (AAV) -delivered CRISPR-Cas13 directly targets and eliminates the positive-strand EV-A71 RNA virus in cells and infected mice. METHODS: We developed a Cas13gRNAtor bioinformatics pipeline to design CRISPR guide RNAs (gRNAs) that cleave conserved viral sequences across the virus phylogeny and developed an AAV-CRISPR-Cas13 therapeutics using in vitro viral plaque assay and in vivo EV-A71 lethally-infected mouse model. FINDINGS: We show that treatment with a pool of AAV-CRISPR-Cas13-gRNAs designed using the bioinformatics pipeline effectively blocks viral replication and reduces viral titers in cells by >99.99%. We further demonstrate that AAV-CRISPR-Cas13-gRNAs prophylactically and therapeutically inhibited viral replication in infected mouse tissues and prevented death in a lethally challenged EV-A71-infected mouse model. INTERPRETATION: Our results show that the bioinformatics pipeline designs efficient CRISPR-Cas13 gRNAs for direct viral RNA targeting to reduce viral loads. Additionally, this new antiviral AAV-CRISPR-Cas13 modality represents an effective direct-acting prophylactic and therapeutic agent against lethal RNA viral infections. FUNDING: Agency for Science, Technology and Research (A∗STAR) Assured Research Budget, A∗STAR Central Research Fund UIBR SC18/21-1089UI, A∗STAR Industrial Alignment Fund Pre-Positioning (IAF-PP) grant H17/01/a0/012, MOE Tier 2 2017 (MOE2017-T2-1-078; MOE-T2EP30221-0005), and NUHSRO/2020/050/RO5+5/NUHS-COVID/4.

Actin cytoskeleton remodeling primes RIG-I-like receptor activation.

The current dogma of RNA-mediated innate immunity is that sensing of immunostimulatory RNA ligands is sufficient for the activation of intracellular sensors and induction of interferon (IFN) responses. Here, we report that actin cytoskeleton disturbance primes RIG-I-like receptor (RLR) activation. Actin cytoskeleton rearrangement induced by virus infection or commonly used reagents to intracellularly deliver RNA triggers the relocalization of PPP1R12C, a regulatory subunit of the protein phosphatase-1 (PP1), from filamentous actin to cytoplasmic RLRs. This allows dephosphorylation-mediated RLR priming and, together with the RNA agonist, induces effective RLR downstream signaling. Genetic ablation of PPP1R12C impairs antiviral responses and enhances susceptibility to infection with several RNA viruses including SARS-CoV-2, influenza virus, picornavirus, and vesicular stomatitis virus. Our work identifies actin cytoskeleton disturbance as a priming signal for RLR-mediated innate immunity, which may open avenues for antiviral or adjuvant design.

Loss of the Nuclear Protein RTF2 Enhances Influenza Virus Replication.

While hundreds of genes are induced by type I interferons, their roles in restricting the influenza virus life cycle remain mostly unknown. Using a loss-of-function CRISPR screen in cells prestimulated with interferon beta (IFN-ß), we identified a small number of factors required for restricting influenza A virus replication. In addition to known components of the interferon signaling pathway, we found that replication termination factor 2 (RTF2) restricts influenza virus at the nuclear stage (and perhaps other stages) of the viral life cycle, based on several lines of evidence. First, a deficiency in RTF2 leads to higher levels of viral primary transcription, even in the presence of cycloheximide to block genome replication and secondary transcription. Second, cells that lack RTF2 have enhanced activity of a viral reporter that depends solely on four viral proteins that carry out replication and transcription in the nucleus. Third, when the RTF2 protein is mislocalized outside the nucleus, it is not able to restrict replication. Finally, the absence of RTF2 leads not only to enhanced viral transcription but also to reduced expression of antiviral factors in response to interferon. RTF2 thus inhibits primary influenza virus transcription, likely acts in the nucleus, and contributes to the upregulation of antiviral effectors in response to type I interferons.IMPORTANCE Viral infection triggers the secretion of type I interferons, which in turn induce the expression of hundreds of antiviral genes. However, the roles of these induced genes in controlling viral infections remain largely unknown, limiting our ability to develop host-based antiviral therapeutics against pathogenic viruses, such as influenza virus. Here, we performed a loss-of-function genetic CRISPR screen in cells prestimulated with type I interferon to identify antiviral genes that restrict influenza A virus replication. Besides finding key components of the interferon signaling pathway, we discovered a new restriction factor, RTF2, which acts in the nucleus, restricts influenza virus transcription, and contributes to the interferon-induced upregulation of known restriction factors. Our work contributes to the field of antiviral immunology by discovering and characterizing a novel restriction factor of influenza virus and may ultimately be useful for understanding how to control a virus that causes significant morbidity and mortality worldwide.

Genome-wide CRISPR screen identifies host dependency factors for influenza A virus infection.

Host dependency factors that are required for influenza A virus infection may serve as therapeutic targets as the virus is less likely to bypass them under drug-mediated selection pressure. Previous attempts to identify host factors have produced largely divergent results, with few overlapping hits across different studies. Here, we perform a genome-wide CRISPR/Cas9 screen and devise a new approach, meta-analysis by information content (MAIC) to systematically combine our results with prior evidence for influenza host factors. MAIC out-performs other meta-analysis methods when using our CRISPR screen as validation data. We validate the host factors, WDR7, CCDC115 and TMEM199, demonstrating that these genes are essential for viral entry and regulation of V-type ATPase assembly. We also find that CMTR1, a human mRNA cap methyltransferase, is required for efficient viral cap snatching and regulation of a cell autonomous immune response, and provides synergistic protection with the influenza endonuclease inhibitor Xofluza.

Antibody-mediated enhancement aggravates chikungunya virus infection and disease severity.

The arthropod-transmitted chikungunya virus (CHIKV) causes a flu-like disease that is characterized by incapacitating arthralgia. The re-emergence of CHIKV and the continual risk of new epidemics have reignited research in CHIKV pathogenesis. Virus-specific antibodies have been shown to control virus clearance, but antibodies present at sub-neutralizing concentrations can also augment virus infection that exacerbates disease severity. To explore this occurrence, CHIKV infection was investigated in the presence of CHIKV-specific antibodies in both primary human cells and a murine macrophage cell line, RAW264.7. Enhanced attachment of CHIKV to the primary human monocytes and B cells was observed while increased viral replication was detected in RAW264.7 cells. Blocking of specific Fc receptors (FcγRs) led to the abrogation of these observations. Furthermore, experimental infection in adult mice showed that animals had higher viral RNA loads and endured more severe joint inflammation in the presence of sub-neutralizing concentrations of CHIKV-specific antibodies. In addition, CHIKV infection in 11 days old mice under enhancing condition resulted in higher muscles viral RNA load detected and death. These observations provide the first evidence of antibody-mediated enhancement in CHIKV infection and pathogenesis and could also be relevant for other important arboviruses such as Zika virus.

Use of multiplex allele-specific polymerase chain reaction (MAS-PCR) to detect multidrug-resistant tuberculosis in Panama.

The frequency of individual genetic mutations conferring drug resistance (DR) to Mycobacterium tuberculosis has not been studied previously in Central America, the place of origin of many immigrants to the United States. The current gold standard for detecting multidrug-resistant tuberculosis (MDR-TB) is phenotypic drug susceptibility testing (DST), which is resource-intensive and slow, leading to increased MDR-TB transmission in the community. We evaluated multiplex allele-specific polymerase chain reaction (MAS-PCR) as a rapid molecular tool to detect MDR-TB in Panama. Based on DST, 67 MDR-TB and 31 drug-sensitive clinical isolates were identified and cultured from an archived collection. Primers were designed to target five mutation hotspots that confer resistance to the first-line drugs isoniazid and rifampin, and MAS-PCR was performed. Whole-genome sequencing confirmed DR mutations identified by MAS-PCR, and provided frequencies of genetic mutations. DNA sequencing revealed 70.1% of MDR strains to have point mutations at codon 315 of the katG gene, 19.4% within mabA-inhA promoter, and 98.5% at three hotspots within rpoB. MAS-PCR detected each of these mutations, yielding 82.8% sensitivity and 100% specificity for isoniazid resistance, and 98.4% sensitivity and 100% specificity for rifampin resistance relative to DST. The frequency of individual DR mutations among MDR strains in Panama parallels that of other TB-endemic countries. The performance of MAS-PCR suggests that it may be a relatively inexpensive and technically feasible method for rapid detection of MDR-TB in developing countries.

Prevalence of antibodies against seasonal influenza A and B viruses during the 2009-2010 and 2010-2011 influenza seasons in residents of Pittsburgh, PA, USA.

Seroprevalence of antibodies against influenza viruses from 1000 people between the ages of 0 to 90 years of age (100 samples for each decade of life) in the Pittsburgh, PA, USA was measured. One year removed from the outbreak of novel H1N1 influenza into the human population in the Northern Hemisphere and following the emergence of a new H3N2 influenza isolate, sera was collected to determine the hemagglutination-inhibition antibodies against influenza A/H1N1, A/H3N2, and B viruses representative of viruses in the vaccine used for the 2010-2011 influenza season. The seroprevalence of antibodies to influenza virus, A/California/7/2009 (H1N1), increased from the previously reported November 2009 samples and the samples collected at the end of the 2010 influenza season (June 2010) during the 2010-2011 season in all age groups, but people the under the age of 20 had the highest rise in the number of positive samples. The number of individuals positive for H1N1 stayed the same through the entire influenza season. In contrast, there were little to no positive serum samples against the H3N2 virus, A/Perth/16/2009, from samples collected during the 2009-2010 influenza season, however, titers against these viruses rose significantly during the early months of the 2010-2011 season with the highest number of positive samples detected in the very young and very old populations. However, these titers waned by May, 2011 in those over the age of 40. There was a rise in adults to the B/Brisbane/60/2008 influenza virus in adults in samples collected in October, 2010, but these titers quickly declined. The highest titers to B influenza were detected in people between the ages of 10-30 years of age. These findings may have implications for the development of vaccination strategies aiming at the protection against seasonal and/or pandemic influenza virus infection and pre-pandemic preparedness activities.