Inosine Triphosphate Pyrophosphatase Dephosphorylates Ribavirin Triphosphate and Reduced Enzymatic Activity Potentiates Mutagenesis in Hepatitis C Virus.

A third of humans carry genetic variants of the ITP pyrophosphatase (ITPase) gene (ITPA) that lead to reduced enzyme activity. Reduced ITPase activity was earlier reported to protect against ribavirin-induced hemolytic anemia and to diminish relapse following ribavirin and interferon therapy for hepatitis C virus (HCV) genotype 2 or 3 infections. While several hypotheses have been put forward to explain the antiviral actions of ribavirin, details regarding the mechanisms of interaction between reduced ITPase activity and ribavirin remain unclear. The in vitro effect of reduced ITPase activity was assessed by means of transfection of hepatocytes (Huh7.5 cells) with a small interfering RNA (siRNA) directed against ITPA or a negative-control siRNA in the presence or absence of ribavirin in an HCV culture system. Low ribavirin concentrations strikingly depleted intracellular GTP levels in HCV-infected hepatocytes whereas higher ribavirin concentrations induced G-to-A and C-to-U single nucleotide substitutions in the HCV genome, with an ensuing reduction of HCV RNA expression and HCV core antigen production. Ribavirin triphosphate (RTP) was dephosphorylated in vitro by recombinant ITPase to a similar extent as ITP, a naturally occurring substrate of ITPase, and reducing ITPA expression in Huh 7.5 cells by siRNA increased intracellular levels of RTP in addition to increasing HCV mutagenesis and reducing progeny virus production. Our results extend the understanding of the biological impact of reduced ITPase activity, demonstrate that RTP is a substrate of ITPase, and may point to personalized ribavirin dosage according to ITPA genotype in addition to novel antiviral strategies.IMPORTANCE This study highlights the multiple modes of action of ribavirin, including depletion of intracellular GTP and increased hepatitis C virus mutagenesis. In cell culture, reduced ITP pyrophosphatase (ITPase) enzyme activity affected the intracellular concentrations of ribavirin triphosphate (RTP) and augmented the impact of ribavirin on the mutation rate and virus production. Additionally, our results imply that RTP, similar to ITP, a naturally occurring substrate of ITPase, is dephosphorylated in vitro by ITPase.

SARS-CoV-2 replicates and displays oncolytic properties in clear cell and papillary renal cell carcinoma.

The SARS-CoV-2 virus is currently causing a global pandemic. Infection may result in a systemic disease called COVID-19, affecting primarily the respiratory tract. Often the gastrointestinal tract and kidneys also become involved. Angiotensin converting enzyme 2 (ACE2) serves as the receptor for SARS-CoV-2. The membrane proteins, Transmembrane serine protease 2 (TMPRSS2) and Neuropilin 1 (NRP1) are accessory proteins facilitating the virus entry. In this study we show that the human proximal kidney tubules, express these factors. We hypothesized that cancers derived from proximal tubules as clear cell (CCRCC) and papillary renal cell carcinoma (PRCC), retain the expression of the SARS-CoV-2 entry factors making these cancers susceptible to SARS-CoV-2 infection. We used bioinformatics, western blotting, and assessment of tissue micro arrays (TMA) including 263 cases of CCRCC, 139 cases of PRCC and 18 cases of chromophobe RCC to demonstrate that the majority of CCRCC and PRCC cases retained the RNA and protein expression of the entry factors for SARS-CoV-2. We furthermore show that SARS-CoV-2 virus propagated robustly in primary cultures of CCRCC and PRCC cells with a visible virus cytopathogenic effect correlating with viral RNA expression levels. We also noted that the delta-variant of SARS-CoV-2 causes cancer cells to form syncytia in-vitro. This phenomenon was also identified histologically in CCRCC tissue from a patient that had been hospitalized for COVID-19, twelve months prior to nephrectomy. Our data provide insights into SARS-CoV-2 infectivity in renal cell carcinoma and that the virus causes a distinct cytopathogenic effect.

Optimization of cerebrospinal fluid microbial DNA metagenomic sequencing diagnostics.

Infection in the central nervous system is a severe condition associated with high morbidity and mortality. Despite ample testing, the majority of encephalitis and meningitis cases remain undiagnosed. Metagenomic sequencing of cerebrospinal fluid has emerged as an unbiased approach to identify rare microbes and novel pathogens. However, several major hurdles remain, including establishment of individual limits of detection, removal of false positives and implementation of universal controls. Twenty-one cerebrospinal fluid samples, in which a known pathogen had been positively identified by available clinical techniques, were subjected to metagenomic DNA sequencing. Fourteen samples contained minute levels of Epstein-Barr virus. The detection threshold for each sample was calculated by using the total leukocyte content in the sample and environmental contaminants found in the bioinformatic classifiers. Virus sequences were detected in all ten samples, in which more than one read was expected according to the calculations. Conversely, no viral reads were detected in seven out of eight samples, in which less than one read was expected according to the calculations. False positive pathogens of computational or environmental origin were readily identified, by using a commonly available cell control. For bacteria, additional filters including a comparison between classifiers removed the remaining false positives and alleviated pathogen identification. Here we show a generalizable method for identification of pathogen species using DNA metagenomic sequencing. The choice of bioinformatic method mainly affected the efficiency of pathogen identification, but not the sensitivity of detection. Identification of pathogens requires multiple filtering steps including read distribution, sequence diversity and complementary verification of pathogen reads.