Generation and Characterization of an Influenza D Reporter Virus.

Influenza D virus (IDV) can infect various livestock animals, such as cattle, swine, and small ruminants, and was shown to have zoonotic potential. Therefore, it is important to identify viral factors involved in the broad host tropism and identify potential antiviral compounds that can inhibit IDV infection. Recombinant reporter viruses provide powerful tools for studying viral infections and antiviral drug discovery. Here we present the generation of a fluorescent reporter IDV using our previously established reverse genetic system for IDV. The mNeonGreen (mNG) fluorescent reporter gene was incorporated into the IDV non-structural gene segment as a fusion protein with the viral NS1 or NS2 proteins, or as a separate protein flanked by two autoproteolytic cleavage sites. We demonstrate that only recombinant reporter viruses expressing mNG as an additional separate protein or as an N-terminal fusion protein with NS1 could be rescued, albeit attenuated, compared to the parental reverse genetic clone. Serial passaging experiments demonstrated that the mNG gene is stably integrated for up to three passages, after which internal deletions accumulate. We conducted a proof-of-principle antiviral screening with the established fluorescent reporter viruses and identified two compounds influencing IDV infection. These results demonstrate that the newly established recombinant IDV reporter virus can be applied for antiviral drug discovery and monitoring viral replication, adding a new molecular tool for investigating IDV.

Time-resolved characterization of the innate immune response in the respiratory epithelium of human, porcine, and bovine during influenza virus infection.

Viral cross-species transmission is recognized to be a major threat to both human and animal health, however detailed information on determinants underlying virus host tropism and susceptibility is missing. Influenza C and D viruses (ICV, IDV) are two respiratory viruses that share up to 50% genetic similarity, and both employ 9-O-acetylated sialic acids to enter a host cell. While ICV infections are mainly restricted to humans, IDV possesses a much broader host tropism and has shown to have a zoonotic potential. This suggests that additional virus-host interactions play an important role in the distinct host spectrum of ICV and IDV. In this study, we aimed to characterize the innate immune response of the respiratory epithelium of biologically relevant host species during influenza virus infection to identify possible determinants involved in viral cross-species transmission. To this end, we performed a detailed characterization of ICV and IDV infection in primary airway epithelial cell (AEC) cultures from human, porcine, and bovine origin. We monitored virus replication kinetics, cellular and host tropism, as well as the host transcriptional response over time at distinct ambient temperatures. We observed that both ICV and IDV predominantly infect ciliated cells, independently from host and temperature. Interestingly, temperature had a profound influence on ICV replication in both porcine and bovine AEC cultures, while IDV replicated efficiently irrespective of temperature and host. Detailed time-resolved transcriptome analysis revealed both species-specific and species uniform host responses and highlighted 34 innate immune-related genes with clear virus-specific and temperature-dependent profiles. These data provide the first comprehensive insights into important common and species-specific virus-host dynamics underlying the distinct host tropism of ICV and IDV, as well as possible determinants involved in viral cross-species transmission.

Susceptibility of Well-Differentiated Airway Epithelial Cell Cultures from Domestic and Wild Animals to Severe Acute Respiratory Syndrome Coronavirus 2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally, and the number of worldwide cases continues to rise. The zoonotic origins of SARS-CoV-2 and its intermediate and potential spillback host reservoirs, besides humans, remain largely unknown. Because of ethical and experimental constraints and more important, to reduce and refine animal experimentation, we used our repository of well-differentiated airway epithelial cell (AEC) cultures from various domesticated and wildlife animal species to assess their susceptibility to SARS-CoV-2. We observed that SARS-CoV-2 replicated efficiently only in monkey and cat AEC culture models. Whole-genome sequencing of progeny viruses revealed no obvious signs of nucleotide transitions required for SARS-CoV-2 to productively infect monkey and cat AEC cultures. Our findings, together with previous reports of human-to-animal spillover events, warrant close surveillance to determine the potential role of cats, monkeys, and closely related species as spillback reservoirs for SARS-CoV-2.

Consensus PCR protocols for the detection of amphibian herpesviruses (Batrachovirus).

Amphibians have been disappearing at an unprecedented rate worldwide. Among the proposed contributing factors are infectious diseases. Investigations have focused mainly on ranavirus and chytrids; however, additional agents may be relevant stressors. Two novel batrachoviruses have been discovered (ranid herpesvirus 3 [RaHV-3] and bufonid herpesvirus 1 [BfHV-1]). Their clinical role is still to be clarified; however, both have been associated with obvious skin lesions in their respective hosts. Herein we present 2 consensus PCR protocols that can be used to detect all of the known and, possibly, yet to be discovered batrachoviruses. We targeted a 200 nt long, highly conserved region of the DNA terminase gene. We established a sensitive protocol, which can detect both European batrachoviruses (European batrachovirus PCR protocol; RaHV-3 and BfHV-1) and a panbatrachovirus PCR protocol detecting all known batrachoviruses, including ranid herpesvirus 1 and 2 (RaHV-1, -2). The limit of detection (LOD) for the European batrachovirus protocol was 101 copies of RaHV-3 and 102 copies of BfHV-1 per reaction. The panbatrachovirus protocol could detect all known batrachoviruses with LODs of 103 (RaHV-3, BfHV-1, RaHV-1) to 104 copies (RaHV-2) per reaction. These novel detection tools can be used as a first line of detection when herpesviral infection in amphibians is suspected, followed by additional PCRs with herpesvirus-specific primers in the case of known viral species, or sequencing as in the case of novel batrachoviruses.