Antiviral Response Detected Via RNA-Seq From Nasal Swabs Predicts Respiratory Viral Infection

Viral infections are a common cause of acute respiratory illnesses. However, it is always a diagnosis of exclusion since current tests to rule in viral infection are generally prohibitive in cost and time. The simple pan-viral test can be conducted in viral cause to see the respiratory symptoms. This could have a great positive impact if facilitated by rapid diagnosis, enhanced patient care, and a better use of medical resources for a large number of patients with respiratory illness.

The current diagnostic measures to rule in viral infection need testing for a lot of different viruses that cause same symptoms. This is because tests identify features specific to every virus. The polymerase chain reaction (PCR) based identification of viral genomes or viral antigen detection is most commonly used. PCR tubes of good quality play a big role here. To test a panel of potential viruses, it can take a lot of time and incur great costs. Also, it can be falsely negative if the patient has a virus that is not in the panel. Even if the virus is identified that is causing respiratory illness, it does not mean that the treatment will be impacted because virus specific therapies are only available for influenza. However, a better approach is to develop a pan-viral test that emphasises on biomarkers showing that the body is responding to the viral infection. 

A plethora of recent studies have shown that gene expression patterns in blood cells or plasma can show the presence of viral infection. Another report showed that levels of a particular host mRNAs identified on respiratory swabs had a relationship with symptomatic viral infection. All these findings prove that it’s promising to utilise the host response to develop a pan-viral diagnostic test. 2 studies were done to examine if the biomarkers of the antiviral response were able to identify the virus. Positive nasopharyngeal swabs having swabs were sent to the medical facility for respiratory virus testing. Host proteins were identified along with mRNAs that could show viral infection in the sample type because immunoassay-based tests are commonly used in labs. 

Using the RNA sequencing experiments on nasal epithelial cells in the first study, scientists evaluated the performance of a signature of 3 host mRNAs to help in predicting viral infection. Moreover, these samples were also used to retrospectively show the potential protein biomarkers that can be detected in the swab-associated viral transport medium. The second study, CXCL10 and CXCL11 proteins were examined. It was found out that there was a high correlation between levels of both the proteins and the viral infection was present. The findings indicate great potential for making simple pan-viral diagnostic tests to identify respiratory virus infections in patients. 


Methods Used

The primary nasal epithelial cells were taken commercially and developed in BEGM medium, also called Lonza. Epinephrine and Hydrocortisone supplements were taken out before for better stimulation. Cells were then tranfected with 14-hp. This was done using the Lipofectamine 2000 and then it was incubated for 7 hours at 370C. 


The raw reading of RNAseq experiments were removed from the sequencing adaptors and low-quality regions through the help of btrim. These trimmed readings were put on the human genome (GRCh37) by tophat2. The counts of reads for every gene depended mostly on Ensembl annotation and differential expression analysis was carried out by Deseq2 that calculated the adjusted p values. 


Sample Selection Process

The sample collection windows were chosen during the high test volume winter months, when several viruses were present. This was based on availability of people to help in processing samples for research. Samples were only included in both studies if direct fluorescent antigen (DFA) testing was ordered, and the respiratory PCR panel for 9-viruses was ordered. Also, the samples should be of good quality in order to perform the DFA testing as required by microscopy. (Landy and Foxman 2018)

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