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Vincent Racaniello: Fecal shedding in COVID-19 patients and function of SARS-CoV-2 nsp4 and ORF8 proteins (covid-19 symposium)

4/1/2020

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Summary by: Arooba Ahmed (CC '23)

     Dr. Racaniello works in the field of virology, and is currently working on two projects. One is clinical, which looks at COVID-19 patients, and the other looks at the molecular level at viral proteins.
     Sars-CoV-2 is a respiratory pathogen that originated from a Bat virus. Bats consist of about 20% of all species and harbor more viruses than any other organism, yet they do not get sick from them. What is interesting is that in its passage to the human species, the virus has also retained some aspect of its original diversity in attack. The virus can also infect the GI tract, and so it has not lost its intestinal tropism when transitioning to humans.
Therefore, Dr. Racaniello decided to look at fecal shedding. Two papers previously found that pediatric patients with the virus also experienced fecal shedding in the form of diarrhea and loose stool. Using RT PCR, viral presence was determined in stool. However, the exact role of fecal shedding in transmission is not known. 
     In Hong Kong in 2003, there was a small SARS outbreak due to faulty plumbing and sewage which aerosolized feces. They don’t know the pathogenesis for this, therefore it is possible that the fecal shedding is a secondary infection, or it could just be another symptom passed along with infection. To specify exactly its role, important questions to ask while researching include the following: Does shedding occur in asymptomatic patients? Does it correlate temporally with shedding from the respiratory tract or completely independent? To answer these, Dr. Racaniello’s group will assay samples for SARS-COV2 genome using RT PCR. In select cases for positive results, they will also look for virus isolation in cell culture. 
     Their second project addresses the function of viral proteins NSP4 and ORF8. A paper previously showed that deletion of NSP4 gene reduces replication of SARS-1 (classic sars). In addition, halfway through the 2001 SARS outbreak, they found the ORF8 gene deleted in circulating strain. This disruption in the circulation of SARS-1 had severe effects. The gene loss caused reduced replication and reduced pathogenesis of the virus, and possibly lengthened survival time so it was more easily transmitted. 
     Now, Dr. Racaniello and his group want to understand what the proteins do and their role in pathogenesis. When the viral genome enters cytosol, the translated proteins 
are involved in replication. They also form for double membrane vesicles, which are sites of viral genome synthesis. 
     NSP4 participates in formation of double vesicle membranes derived from ER, golgi, and other membraned organelles. These double vesicle membranes are where RNA synthesis occurs. ORF8 is located in the ER, and so the two proteins interact with similar sets of resident ER proteins from the ERAD pathway (ER- Associated Degradation pathway). This is linked to viral replication, because the virus exploits ER derived vesicles and ERAD regulators. Therefore, they want to confirm that these proteins interact with ERAD proteins in infected cells and also identify pathways that regulate lipid metabolism and vesicle trafficking.
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