Studies Progression on the Function of Autophagy in Viral Infection

Autophagy is a conservative lysosomal catabolic pathway commonly seen in eukaryotic cells. It breaks down proteins and organelles by forming a two-layer membrane structure of autophagosomes and circulating substances and maintaining homeostasis. Autophagy can play a dual role in viral infection and serve either as a pro-viral factor or an antiviral defense element dependent on the virus replication cycle. Recent studies have suggested the complicated and multidirectional role of autophagy in the process of virus infection. On the one hand, autophagy can orchestrate immunity to curtail infection. On the other hand, some viruses have evolved strategies to evade autophagy degradation, facilitating their replication. In this review, we summarize recent progress of the interaction between autophagy and viral infection. Furthermore, we highlight the link between autophagy and SARS-CoV-2, which is expected to guide the development of effective antiviral treatments against infectious diseases.

Direct lysis RT-qPCR of SARS-CoV-2 in cell culture supernatant allows for fast and accurate quantification of virus, opening a vast array of applications

An enormous global effort is being made to study SARS-CoV-2 and develop safe and effective treatments. Studying the entire virus replication cycle of SARS-CoV-2 is essential to identify host factors and treatments to combat the infection. However, quantification of released virus often requires lengthy procedures, such as endpoint dilution assays or reinfection with engineered reporter viruses. Quantification of viral RNA in cell supernatant is faster and can be performed on clinical isolates. However, viral RNA purification is expensive in time and resources and often unsuitable for high-throughput screening. Here, we show a direct lysis RT-qPCR method allowing sensitive, accurate, fast, and cheap quantification of SARS-CoV-2 in culture supernatant. During lysis, the virus is completely inactivated, allowing further processing in low containment areas. This protocol facilitates a wide array of high- and low-throughput applications from basic quantification to studying the biology of SARS-CoV-2 and to identify novel antiviral treatments in vitro.

In-depth characterization of the chikungunya virus replication cycle

The chikungunya virus has spread globally with a remarkably high attack rate. Infection causes arthralgic sequelae that can last for years. Nevertheless, there are no specific drugs or vaccines to contain the virus. Understanding the biology of the virus, such as its replication cycle, is a powerful tool to identify new drugs and comprehend virus-host interactions. Even though the chikungunya virus has been known for a long time (first described in 1952), many aspects of the replication cycle remain unclear. Furthermore, part of the cycle is based on observations of other alphaviruses. In this study, we used electron and scanning microscopy, as well as biological assays, to analyze and investigate the stages of the chikungunya virus replication cycle. Based on our data, we found other infection cellular activities than those usually described for the chikungunya virus replication cycle, i.e. we show particles enveloping intracellularly without budding in a membrane-delimited morphogenesis area; and we also observed virion release by membrane protrusions. Our work provides novel details regarding the biology of chikungunya virus and fills gaps in our knowledge of its replication cycle. These findings may contribute to a better understanding of virus-host interactions and support the development of antivirals. IMPORTANCE The understanding of virus biology is essential to containing virus dissemination, and exploring the virus replication cycle is a powerful tool to do this. There are many points in the biology of the chikungunya virus that need to be clarified, especially regarding its replication cycle. Our incomplete understanding of chikungunya virus infection stages is based on studies with other alphaviruses. We systematized the chikungunya virus replication cycle using microscopic imaging in the order of infection stages: entry, replication, protein synthesis, assembly/morphogenesis, and release. The imaging evidence shows novel points in the replication cycle of enveloping without budding, as well as particle release by cell membrane protrusion.

Cool temperatures limit Zika virus genome replication

Zika virus is a mosquito-borne flavivirus known to cause severe birth defects and neuroimmunological disorders. We have previously demonstrated that mosquito transmission of Zika virus decreases with temperature. While transmission was optimized at 29°C, it was limited at cool temperatures (< 22°C) due to poor virus establishment in the mosquitoes. Temperature is one of the strongest drivers of vector-borne disease transmission due to its profound effect on ectothermic mosquito vectors, viruses, and their interaction. Although there is substantial evidence of temperature effects on arbovirus replication and dissemination inside mosquitoes, little is known about whether temperature affects virus replication directly or indirectly through mosquito physiology. In order to determine the mechanisms behind temperature-induced changes in Zika virus transmission potential, we investigated different steps of the virus replication cycle in mosquito cells (C6/36) at optimal (28°C) and cool (20°C) temperatures. We found that cool temperature did not alter Zika virus entry or translation but reduced the amount of double-stranded RNA replication intermediates. If replication complexes were first formed at 28°C and the cells were subsequently shifted to 20°C, the late steps in the virus replication cycle were efficiently completed. These data suggest that cool temperature decreases the efficiency of Zika virus genome replication in mosquito cells. This phenotype was observed in the Asian-lineage of Zika virus, while the African-lineage Zika virus was less restrictive at 20°C.

Multidose evaluation of 6,710 drug repurposing library identifies potent SARS-CoV-2 infection inhibitors In Vitro and In Vivo.

The SARS-CoV-2 pandemic has caused widespread illness, loss of life, and socioeconomic disruption that is unlikely to resolve until vaccines are widely adopted, and effective therapeutic treatments become established. Here, a well curated and annotated library of 6710 clinical and preclinical molecules, covering diverse chemical scaffolds and known host targets was evaluated for inhibition of SARS-CoV-2 infection in multiple infection models. Multi-concentration, high-content immunocytofluorescence-based screening identified 172 strongly active small molecules, including 52 with submicromolar potencies. The active molecules were extensively triaged by in vitro mechanistic assays, including human primary cell models of infection and the most promising, obatoclax, was tested for in vivo efficacy. Structural and mechanistic classification of compounds revealed known and novel chemotypes and potential host targets involved in each step of the virus replication cycle including BET proteins, microtubule function, mTOR, ER kinases, protein synthesis and ion channel function. In the mouse disease model obatoclax effectively reduced lung virus load by 10-fold. Overall, this work provides an important, publicly accessible, foundation for development of novel treatments for COVID-19, establishes human primary cell-based pharmacological models for evaluation of therapeutics and identifies new insights into SARS-CoV-2 infection mechanisms.

Structure and Activities of the NS1 Influenza Protein and Progress in the Development of Small-Molecule Drugs

The influenza virus causes human disease on a global scale and significant morbidity and mortality. The existing vaccination regime remains vulnerable to antigenic drift, and more seriously, a small number of viral mutations could lead to drug resistance. Therefore, the development of a new additional therapeutic small molecule-based anti-influenza virus is urgently required. The NS1 influenza gene plays a pivotal role in the suppression of host antiviral responses, especially by inhibiting interferon (IFN) production and the activities of antiviral proteins, such as dsRNA-dependent serine/threonine-protein kinase R (PKR) and 2′-5′-oligoadenylate synthetase (OAS)/RNase L. NS1 also modulates important aspects of viral RNA replication, viral protein synthesis, and virus replication cycle. Taken together, small molecules that target NS1 are believed to offer a means of developing new anti-influenza drugs.

The Role of Apolipoproteins in Dengue Infection: A Review

The re-emergence of the dengue virus in recent decades has significantly increased with almost 40%-50% of the world’s population being at risk. Meanwhile, cholesterol and its components, apolipoproteins, were found to play a vital role in dengue infectivity and the development of severe dengue. This review attempts to address the functional importance of cholesterol and related apolipoproteins in dengue virus pathogenesis and to identify the potential utilisation of this relationship in future diagnosis and management of dengue. The literature search was conducted using a computer-based electronic search on dengue infection with cholesterol and human lipoproteins from September 2017 to June 2019 through three main search engines: MEDLINE (OVID), PubMed, and Science Direct using the keywords including Flaviviruses, characteristics of dengue virus, the pathogenesis of dengue, enhancement of dengue, metabolism of cholesterol, cholesterol pathway and human lipoproteins in association with dengue. Dengue virus manipulates lipid raft integrity and utilizes cholesterol components and apolipoproteins for virus internalisation through LDLr and SR-BI receptors. Infectivity of the dengue virus correlated with a decrease in the cholesterol content of the virions. High cholesterol levels in the endoplasmic reticulum promote replication complexes formation of dengue virus. Cholesterol is needed for NS1 secretion which is essential in viral replication, dengue pathogenesis, and host immune evasion. Levels of cholesterol and its related components contributed to the development of severe dengue. The interplay between cholesterol and cellular proteins lead to significant effect in all aspects of the dengue virus replication cycle from viral entry to release.

The Antiviral Activities of Poly-ADP-Ribose Polymerases

The poly-adenosine diphosphate (ADP)-ribose polymerases (PARPs) are responsible for ADP-ribosylation, a reversible post-translational modification involved in many cellular processes including DNA damage repair, chromatin remodeling, regulation of translation and cell death. In addition to these physiological functions, recent studies have highlighted the role of PARPs in host defenses against viruses, either by direct antiviral activity, targeting certain steps of virus replication cycle, or indirect antiviral activity, via modulation of the innate immune response. This review focuses on the antiviral activity of PARPs, as well as strategies developed by viruses to escape their action.

Cell-based culture of SARS-CoV-2 informs infectivity and safe de-isolation assessments during COVID-19

Background The detection of SARS-CoV-2 RNA by real-time polymerase chain reaction (PCR) in respiratory samples collected from persons recovered from COVID-19 does not necessarily indicate shedding of infective virions. By contrast, the isolation of SARS-CoV-2 using cell-based culture likely indicates infectivity, but there are limited data on the correlation between SARS-CoV-2 culture and PCR. Methods One hundred and ninety-five patients with varying severity of COVID-19 were tested (outpatients [n=178]), inpatients [n=12] and critically unwell patients admitted to the intensive care unit [ICU; n=5]). SARS-CoV-2 PCR positive samples were cultured in Vero C1008 cells and inspected daily for cytopathic effect (CPE). SARS-CoV-2-induced CPE was confirmed by PCR of culture supernatant. Where no CPE was observed, PCR was performed on day four to confirm absence of virus replication. Cycle threshold (Ct) of the day four PCR (Ctculture) and the PCR of the original clinical sample (Ctsample) were compared, and positive cultures were defined where Ctsample - Ctculture was ≥3. Findings Of 234 samples collected, 228 (97%) were from the upper respiratory tract. SARS-CoV-2 was only successfully isolated from samples with Ctsample ≤32, including in 28/181 (15%), 19/42 (45%) and 9/11 samples (82%) collected from outpatients, inpatients, and ICU patients, respectively. The mean duration from symptom onset to culture positivity was 4.5 days (range 0-18). SARS-CoV-2 was significantly more likely to be isolated from samples collected from inpatients (p&lt;0∙001) and ICU patients (p&lt;0∙0001) compared with outpatients respectively, and in samples with lower Ctsample. Conclusion SARS-CoV-2 culture may be used as a surrogate marker for infectivity and inform de-isolation protocols.