Studying the entire virus replication cycle of SARS-CoV-2 is essential to identify the host factors involved and treatments to combat infection. Quantification of released virions often requires lengthy procedures, whereas quantification of viral RNA in supernatant is faster and applicable to clinical isolates. Viral RNA purification is expensive in terms of time and resources, and is often unsuitable for high-throughput screening. Direct lysis protocols were explored for patient swab samples, but the lack of virus inactivation, cost, sensitivity, and accuracy is hampering their application and usefulness for in vitro studies. Here, we show a highly sensitive, accurate, fast, and cheap direct lysis RT-qPCR method for quantification of SARS-CoV-2 in culture supernatant. This method inactivates the virus and permits detection limits of 0.043 TCID₅₀ virus and <1.89 copy RNA template per reaction. Comparing direct lysis with RNA extraction, a mean difference of +0.69 ± 0.56 cycles was observed. Application of the method to established qPCR methods for RSV (-ve RNA), IAV (segmented -ve RNA), and BHV (dsDNA) showed wider applicability to other enveloped viruses, whereby IAV showed poorer sensitivity. This shows that accurate quantification of SARS-CoV-2 and other enveloped viruses can be achieved using direct lysis protocols, facilitating a wide range of high- and low-throughput applications.

SUMMARY: Continual low-level exposure of sheep to the helminth Teladorsagia circumcincta elicits a temporary protective immunity, where factors in the immune abomasal mucosa prevent penetration of infective larvae, but which is essentially lost within 6 weeks of cessation of parasite challenge. Here, a proteomic approach was used to identify proteins that are differentially regulated in immune compared to naïve sheep, as potential key mediators of immunity. Six naïve sheep and 12 sheep trickle-infected with T. circumcincta were treated with anthelmintic, and the naïve (control) and 6 immune sheep were killed 7 days later. The remaining 6 sheep (immune waning) were killed 42 days after anthelmintic treatment. Abomasal tissue samples were subjected to 2D-gel electrophoresis and densitometric analysis. Selected spots (n=73) were identified by peptide mass fingerprinting and confirmatory Western blotting was carried out for 10 proteins. Spots selectively up-regulated in immune versus control, but not immune waning versus control sheep, included galectin-15 and thioredoxin, which were confirmed by Western blotting. In immune sheep, serum albumin was significantly down-regulated and albumin proteolytic cleavage fragments were increased compared to controls. Unexpectedly, albumin mRNA was relatively highly expressed in control mucosa, down-regulated in immune, and was immunolocalized to mucus-producing epithelial cells. Thus we have identified differential expression of a number of proteins following T. circumcincta trickle infection that may play a role in host protection and inhibition of parasite establishment.

African Swine Fever virus (ASFV) is the causative agent of a deadly, panzootic disease, infecting wild and domesticated suid populations. Contained for a long time to the African continent, an outbreak of a particularly infectious variant in Georgia in 2007 initiated the spread of the virus around the globe, severely impacting pork production and local economies. The virus is highly contagious and has a mortality of up to 100% in domestic pigs. It is critical to track the spread of the virus, detect variants associated with pathology, and implement biosecurity measures in the most effective way to limit its spread. Due to its size and other limitations, the 170-190kbp large DNA virus has not been well sequenced with fewer than 200 genome sequences available in public repositories. Here we present an efficient, low-cost method of sequencing ASFV at scale. The method uses tiled PCR amplification of the virus to achieve greater coverage, multiplexability and accuracy on a portable sequencer than achievable using shotgun sequencing. We also present Lilo, a pipeline for assembling tiled amplicon data from viral or microbial genomes without relying on polishing against a reference, allowing for structural variation and hypervariable region assembly other methods fail on. The resulting ASFV genomes are near complete, lacking only parts of the highly repetitive 3’- and 5’telomeric regions, and have a high level of accuracy. Our results will allow sequencing of ASFV at optimal efficiency and high throughput to monitor and act on the spread of the virus.

In vitro investigations of host-virus interactions are reliant on suitable cell and tissue culture models. Results are only as good as the model they are generated in. However, choosing cell models for in vitro work often depends on availability and previous use alone. Despite the vast increase in coronavirus research over the past few years, scientists are still heavily reliant on: non-human, highly heterogeneous or not fully differentiated, or naturally unsusceptible cells requiring overexpression of receptors and other accessory factors. Complex primary or stem cell models are highly representative of human tissues but are expensive and time-consuming to develop and maintain with limited suitability for high-throughput experiments.Using tissue-specific expression patterns, we identified human kidney cells as an ideal target for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and broader coronavirus infection. We show the use of the well-characterized human kidney cell line Caki-1 for infection with three human coronaviruses (hCoVs): Betacoronaviruses SARS-CoV-2 and Middle Eastern respiratory syndrome coronavirus and Alphacoronavirus hCoV 229E. Caki-1 cells show equal or superior susceptibility to all three coronaviruses when compared to other commonly used cell lines for the cultivation of the respective virus. Antibody staining against SARS-CoV-2 N protein shows comparable replication rates. A panel of 26 custom antibodies shows the location of SARS-CoV-2 proteins during replication using immunocytochemistry. In addition, Caki-1 cells were found to be susceptible to two other human respiratory viruses, influenza A virus and respiratory syncytial virus, making them an ideal model for cross-comparison for a broad range of respiratory viruses. IMPORTANCE: Cell lines remain the backbone of virus research, but results are only as good as their originating model. Despite increased research into human coronaviruses following the COVID-19 pandemic, researchers continue to rely on suboptimal cell line models of: non-human origin, incomplete differentiation, or lacking active interferon responses. We identified the human kidney Caki-1 cell line as a potential target for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). This cell line could be shown to be infectable with a wide range of coronaviruses including common cold virus hCoV-229E, epidemic virus MERS-CoV, and SARS-CoV-2 as well as other important respiratory viruses influenza A virus and respiratory syncytial virus. We could show the localization of 26 SARS-CoV-2 proteins in Caki-1 cells during natural replication and the cells are competent of forming a cellular immune response. Together, this makes Caki-1 cells a unique tool for cross-virus comparison in one cell line.