Cas9-expressing chickens and pigs as resources for genome editing in livestock.

Genetically modified animals continue to provide important insights into the molecular basis of health and disease. Research has focused mostly on genetically modified mice, although other species like pigs resemble the human physiology more closely. In addition, cross-species comparisons with phylogenetically distant species such as chickens provide powerful insights into fundamental biological and biomedical processes. One of the most versatile genetic methods applicable across species is CRISPR-Cas9. Here, we report the generation of transgenic chickens and pigs that constitutively express Cas9 in all organs. These animals are healthy and fertile. Functionality of Cas9 was confirmed in both species for a number of different target genes, for a variety of cell types and in vivo by targeted gene disruption in lymphocytes and the developing brain, and by precise excision of a 12.7-kb DNA fragment in the heart. The Cas9 transgenic animals will provide a powerful resource for in vivo genome editing for both agricultural and translational biomedical research, and will facilitate reverse genetics as well as cross-species comparisons.

Molecular surveillance revealed no SARS-CoV-2 spillovers to raccoons (Procyon lotor) in four German federal states.

Raccoons (Procyon lotor), which are closely related to the family Mustelidae, might be susceptible to natural infection by SARS-CoV-2. This assumption is based on experimental evidence that confirmed the vulnerability of farmed fur-carnivore species, including Procyon lotor to SARS-CoV-2. To date, there are no reports of natural SARS-CoV-2 infections of raccoons in Germany. Here, we use RT-PCR to analyze 820 samples from raccoons hunted in Germany with a focus on 4 German federal states (Saxony-Anhalt, Thuringia, Hesse, North Rhine-Westphalia). Lung tissues were homogenized and processed for RNA extraction and RT-qPCR for detecting SARS-CoV-2 was performed. No viral RNA was detected in any samples (0/820). Next, we compared raccoons and human ACE-2 residues that are known to serve for binding with SARS-CoV-2 receptor binding domain (RBD). Interestingly, we found only 60% identity on amino acid level, which may have contributed to the absence of SARS-CoV-2 infections in raccoons. In conclusion, the chance of raccoons being intermediate reservoir hosts for SARS-CoV-2 seems to be very low.

Impact of animal saliva on the performance of rapid antigen tests for detection of SARS-CoV-2 (wildtype and variants B.1.1.7 and B.1.351).

SARS-CoV-2 infects several animal species and SARS-CoV-2 variants of concern (VOCs) may even show (as in humans) enhanced inter- and intra-species transmission rates. We correlated sensitivity data of SARS-CoV-2 rapid antigen tests (RATs) to viral RNA genome equivalents analyzed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Further, we checked their suitability for testing animals by assessing saliva and VOC effects. Viral loads up to 2 logs (RNA copy number) under the hypothetical SARS-CoV-2 infectivity threshold were detected by most analyzed RATs. However, while saliva from various animal species showed generally no adverse effects on the RATs' analytical sensitivities, the detection of VOCs B.1.1.7 and B.1.351 was in some RATs inferior to non-VOC viruses.

Characterization of a Novel Viral Interleukin 8 (vIL-8) Splice Variant Encoded by Marek's Disease Virus.

Marek's disease virus (MDV) is a highly cell-associated oncogenic alphaherpesvirus that causes lymphomas in various organs in chickens. Like other herpesviruses, MDV has a large and complex double-stranded DNA genome. A number of viral transcripts are generated by alternative splicing, a process that drastically extends the coding capacity of the MDV genome. One of the spliced genes encoded by MDV is the viral interleukin 8 (vIL-8), a CXC chemokine that facilitates the recruitment of MDV target cells and thereby plays an important role in MDV pathogenesis and tumorigenesis. We recently identified a novel vIL-8 exon (vIL-8-E3') by RNA-seq; however, it remained elusive whether the protein containing the vIL-8-E3' is expressed and what role it may play in MDV replication and/or pathogenesis. To address these questions, we first generated recombinant MDV harboring a tag that allows identification of the spliced vIL-8-E3' protein, revealing that it is indeed expressed. We subsequently generated knockout viruses and could demonstrate that the vIL-8-E3' protein is dispensable for MDV replication as well as secretion of the functional vIL-8 chemokine. Finally, infection of chickens with this vIL-8-E3' knockout virus revealed that the protein is not important for MDV replication and pathogenesis in vivo. Taken together, our study provides novel insights into the splice forms of the CXC chemokine of this highly oncogenic alphaherpesvirus.

Detection of SARS-CoV-2 variant B.1.1.7 in a cat in Germany.

Several non-variant of concern SARS-CoV-2 infections in pets have been reported as documented in the OIE and GISAID databases and there is only one fully documented case of an alpha variant of concern (VOC)(B.1.1.7) in the United States so far. Here, we describe the first case in a cat infected with the alpha SARS-CoV-2 variant in Germany. A cat suffering from pneumonia was presented to a veterinary practice. The pneumonia was treated symptomatically, but 16 days later the cat was presented again. Since the owner had been tested positive for a SARS-CoV-2 infection in the meantime, swab samples were taken from the cat and analyzed for SARS-CoV-2 specific nucleic acids. The various RT-qPCR analyses and whole-genome sequencing revealed the presence of the SARS-CoV-2 B.1.1.7 variant in this cat. This study shows that pets living in close contact with SARS-CoV-2 B.1.1.7 infected owners can contract this virus and also suffer from a respiratory disease. It is not clear yet whether onward transmissions to other cats and humans can occur. To minimize transmission risks, pet owners and veterinarians should comply to the hygienic rules published by OIE and others. It must be stated, that infections of cats with SARS-CoV-2 is still a rare event. Cats with clinical signs of a respiratory disease should be presented to a veterinarian, who will decide on further steps.

Abrogation of Marek's disease virus replication using CRISPR/Cas9.

Marek's disease virus (MDV) is a highly cell-associated alphaherpesvirus that causes deadly lymphomas in chickens. While vaccination protects against clinical symptoms, MDV field strains can still circulate in vaccinated flocks and continuously evolve towards greater virulence. MDV vaccines do not provide sterilizing immunity, allowing the virus to overcome vaccine protection, and has increased the need for more potent vaccines or alternative interventions. In this study, we addressed if the CRISPR/Cas9 system can protect cells from MDV replication. We first screened a number of guide RNAs (gRNAs) targeting essential MDV genes for their ability to prevent virus replication. Single gRNAs significantly inhibited virus replication, but could result in the emergence of escape mutants. Strikingly, combining two or more gRNAs completely abrogated virus replication and no escape mutants were observed upon serial passaging. Our study provides the first proof-of-concept, demonstrating that the CRISPR/Cas9 system can be efficiently used to block MDV replication. The presented findings lay the foundation for future research to completely protect chickens from this deadly pathogen.