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Identification of Required Host Factor for SARS-CoV-2 infection in human cell research article

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Background

Current times of the 21st century have seen the world turn into a small village. As people live in a very interconnected and globalized environment, people and diseases they carry and suffer from can be easily transmitted from one corner of the planet to the other in a matter of hours. Similarly, SARS-CoV-2 begun normally as any other virus does at the ordinary moment when a small cluster of patients in Wuhan China were admitted to different hospitals and diagnosed with pneumonia, which later turned to a have a similar strain of coronavirus. Suddenly, the virus with a high transmission rate turned into an epidemic in China and very fast, turning to a global pandemic after different countries in the world started reporting new infection cases and deaths1.

However, with the challenge of the speed at which viruses spread in the population, the current global advancement of science in health care has greatly helped in detection, taking measures, and controlling the spread of the virus. The early measures and response adopted by the Chinese, such as large-scale surveillance, situation monitoring, supply, and preparation of medical facilities, successfully aid in reducing the epidemic in Wuhan, which was the epicenter of the coronavirus1. However, a very interesting situation at the same time was that the coronavirus didn't hit China super hard despite beginning the epicenter as it was in other countries like United States, Spain, Italy and German, and Brazil. Africa was the last continent to be hit by the coronavirus's effects, with countries like South Africa and Egypt experiencing the adverse effect.

Currently, the coronavirus global pandemic has affected almost every country with millions of infections, deaths, job loss, and economic rapid down pull. Nevertheless, many health experts in different countries are working either separately or jointly to develop new ways of addressing the challenges of transmission. This is while at the same time working hard to develop vaccines which prevent the harsh effects of the virus2. Although health experts have been successfully able to alter the course of the outbreak, there are still concerns about possible pandemic recurrence which stress the need for continuous intervention as the pandemic is still ongoing, and most of the current reported cases are only mild symptoms.

Introduction

To begin with, coronaviruses are one of the families from the very contagious viruses which scientists have proven to have a range of mild, severe respiratory illnesses. The virus can rapidly mutate from new types of coronaviruses like acute respiratory syndrome coronavirus 2 identified in Wuhan China, which is responsible for the current global pandemic (COVID-19 disease). The Chinese have a history of fighting against coronavirus, as well as seen from both MERS and SARS outbreaks. In 2002, China was suffering from SARS outbreak, a very highly contagious coronaviruses which resulted in 8,098 infections and 774 deaths in 32 countries. Although the SARS was contained, the preparation done in both China and the affected countries were insufficient to deal with the virus ranging from; inadequate isolation and quarantine centers, late reporting and response, the vulnerability of frontline health workers to poor hygiene precautions.

Lately, Wuhan, China, the largest and capital city of Hubei province, also referred to as "Chicago of China," was the epicenter of the new coronavirus; COVID -19. In December of 2019, few cases were reported as emerging coronaviruses that presented with pneumonia signs and were first referred to as 2019-nCoV2. Later in mid-January, 2020, other provinces in China started to report more cases of the virus, and later due to the rapid mass international travels, other countries such as Japan, the USA, Thailand, and South Korea were now affected. The speedy escalation of new cases of infection and consequent deaths made the global situation abnormal and new measures like; lockdowns, restriction of both local and international flights, isolation, and quarantine of suspected cases were adopted.

According to the John Hopkins research center (SARS-CoV-2), a virus that causes C0VID-19 had affected 40 million people in the world and killed more than a million people as of October 2020. The SARS-CoV-2 virus, which is a member of the coronaviridae family shows low fatality rate, higher recovery rate but an increased infection rate3. As COVID-19 has taken a major toll on humanity, governmental institutions, pharmaceutical companies, and research institutions have been on the forefront to develop a vaccine which reduce the effect caused by the virus, and as of October 2020, 30 different vaccines against COVID-19 were under clinical trials. Hereafter is a discussion of a recent study carried on by (Daniloski et al., 2020) that identified small molecules that greatly antagonized the infection and replication of the SARS-CoV-2 virus by testing nearly 12,000 FDA approved and clinical stage inhibitor.

Currently, there are no genome studies in the world that have shown the required viral infection, which could be of great utility and interest for the scientific community. This study performed a broad genome-scale CRISPR loss of function on the patient's alveolar basal epithelial carcinoma cells to establish the specific genes whose loss confers huge resistance to SARS-CoV-2 viral infection. The validation of the genes to have resistance on SARS-CoV-2 infection was done on orthogonal cells perturbation like; RNA interference knockdown, CRISPR knockout, and small molecule inhibitors3. Furthermore, for every top gene hits the study, explore reasonable mechanisms of their different activities using; flow cytometry, immunofluorescence, and single-cell transcriptomics. Through the use of single-cell transcription, several groups of genes were identified, among them being (NPC1, CCD22, RAB7A, NPC1, ATP6V1A, AND ATP6AP1); all these gene type had a knockout induced transcription changes in every biosynthesis pathway.

The research findings indicated that perturbation of the cholesterol biosynthesis pathway possessing single small molecule amlodipine had an ability to reduce viral infection. Additionally, RAB7A reduced viral entry by sequestering ACE2 receptors in the cell by altering the endosomal trafficking.

It is important to note that before this scientific study, our knowledge of important host genes for SARS-CoV-2 was limited and based on a handful of genes, including cathepsin and ACE2. However, the study provided complete quantitative resources of the influence of each gene's loss due to viral infection on every protein-coding gene in the patient genome3.

Another critical stage of the study was to infect the GeCKOv2 pool cells with that of the SARS-CoV-2 virus; this was done at either low (0.01) to a high (0.3) MOI. The study experiment verified that the SARS-CoV-2 infects A549ACE2 cells through carefully staining for the nucleocapsid protein 24 hours before infection, and 6 days post the infection, cell survival was measured for both low MOI conditions3. As expected, the experiment indicated that with high MOI infection lead to a fewer cell to survive. Genomic DNA extraction was the next stage of the experiment, where through amplicon sequencing, a quantified guided abundance in every biological condition was achieved. Here the experiment found a contrasting factor where after contracting the SARS-CoV-2 virus, there was a high degree of guide dropout as expected given that A549ACE2 was quickly killed by SARS-CoV-2 cells without CRISPR perturbations.

The experiments in this study used robust-rank aggregation (RRA) to guide relative enrichment and compute gene-level scores that identify genes loss-of-function mutations which led to enrichment with the pool. The experiment also used two more previously approved methods to compute gene enrichment. The experiment found a higher degree of overlap among the enriched gene in all three methods. The experiment findings indicated a higher degree of shared genes in both high and low SARS-Cov-2 MOI conditions.

The findings of this study suggested that many gene hosts involved a viral pathogenesis that is independent of viral dosage. Also, a single genome-scale CRISPR screen for coronavirus infection resulted in A549, which overexpress ACE2; however, with similar ranked genes for the robustness of the experimental findings3.

The study finding lastly carried on cross-examination of the different but most enriched genes. The study found that most genes were involved in significant aspects of replication and viral entry. For instance, the established entry receptor-like angiotensin-converting enzyme (ACE) ranks the number 8th most-enriched gene in low MOI while 12th in the highest MOI screen. Additionally, among the 50 enriched genes, the experiment identified some sets of related genes which had a similar function in complexes, thus giving more confidence and reliability in the genome-scale screen4.

Before reading the study article, here is the perception regarding COVID-19 reported in the original news article, which was based on minimal scientific study. The COVID-19 symptoms are non-specific, yet the disease presented a wide range of asymptomatic (no symptoms), resulting in severe pneumonia or death. As of February 2020, based on nearly 55,924 confirmed laboratory tests, typical symptoms included, among others, shortness of breath, dry cough, fatigue, arthralgia or myalgia, nasal congestion, vomiting, or nausea conjunctival hemoptysis, fever, and headache. Subsequently, people living with COVID-19 developed symptoms including fever and mild respiratory symptoms on an average of 4-6 days after contracting the virus and a mean incubation period range of 14 days.

The majority of the people who contracted the virus had mild disease and recovered. 80% of the laboratory-confirmed patients had a mild to moderate disease, which composes of both pneumonia and Non-pneumonia cases; a small percentage had a mild to severe disease that is; blood oxygen saturation, dyspnea, respiratory frequency, and lung infiltration. On the other hand, the asymptomatic infection was reported in huge numbers, and these patients showed no sign of pneumonia or any corona-virus relate symptoms from the date of identification to on to develop the disease5. Although truly asymptomatic infections are unclear, it appears to be rare and lacks a high rate of transmission.

The majority of the patients at the highest risk for severe death and disease included people aged 6 years and above, in addition to those patients with underlying conditions like diabetes, cardiovascular disease, cancer, chronic respiratory disease, and hypertension. Although children were seen to have a higher risk for severe disease and deaths, the virus appears to be relatively low and mild 2.4% of globally reported cases among children below 19 years.

The mortality rate was high with age, where the highest mortality was reported among patients over 80 years of age. Also, the mortality rate was seen to be higher in males compared to females. In terms of occupation, people at the retiree stage were mostly affected. Lastly, for patients with no comorbid conditions showed much higher mortality than those with no comorbid conditions.

In a global emergency like a pandemic, it's normal for decisions to change as new and approved information emerges rapidly. Although this is normal, it's like whiplash to keep up with the research. Thus, it's harder to change people's first impressions once they have learned about a topic and change the way of life; this phenomenon is called anchoring bias. For instance, the majority of researchers thought that the virus was spread entirely through drops of spit or mucus among people who are in close proximity. It's now known that the virus can spread through the air. Also, initially, researchers thought that the virus could only be transmitted between people with visible symptoms; however, research has indicated that even the asymptomatic patient can also spread the disease5.

With the COVID-19 global pandemic, a better understanding of the different and complex relations between the virus genetic and host dependencies advance. The Daniloski lead research article reported a genome-wide loss of function in patient lung cells, which shows the host gene required for SARS-CoV-2 viral infection. The main finding of the research was to support the screen's ability to show key dependencies, and the well know host gene. This includes CoV-2 protein entry and biddings like cathepsin and ACE2 receptors, which are the top-ranked scoring genes.

It's important to highlight that the overlap between these studies and the different screens performed in Africa may be due to its limitation to CTSL and ACE2 genes, which are responsible for identifying the viral entry. Also, due to the technical difference, biological difference, and laboratory variations there might be overlaps between these two studies. The variation is an exciting opportunity for near future studies to investigate if SARS-CoV-2 also uses the many cell-type in genetic circuits5. However, the critical element of this study was to utilize genome-scale-loss-of-function to create a more therapeutic hypothesis.

The Daniloski study identified various limitations that should be expanded on in the future, among them being the requirement to identify how SARS-Cov-2 infection in patient (human) cell overexpress ACE2. Also, the study didn't uncover the main human variant related to COVID-19. This was due to the fact that such variants are completely non-coding regions.

Conclusively, the majority of approaches for the therapeutic association have largely focused on large-scale screens of various libraries. Even if the promising and potential therapeutic candidates are well identified, it's also challenging to comprehend the different mechanism to reducing viral pathogenesis. However, this study has showed progress in explaining critical aspects of the corona-virus disease which will be of great help in the process of examining and developing a safe vaccine which ultimately reduce the effect of the virus and save humanity!

References

1. Dashraath, P., Jeslyn, W., Karen, L., Min, L., Sarah, L., Biswas, A. and Lin, S., 2020. Coronavirus Disease 2019 (COVID-19) Pandemic and Pregnancy. [online] www.ajog.org. Available at: <https://www.ajog.org/article/S0002-9378(20)30343-4/fulltext> [Accessed 12 November 2020].

2. Kowalski, L. P., Sanabria, A., Ridge, J. A., Ng, W. T., de Bree, R., Rinaldo, A., ... & Paleri, V. (2020). COVID‐19 pandemic: effects and evidence‐based recommendations for otolaryngology and head and neck surgery practice. Head & neck, 42(6), 1259-1267.

3. Daniloski, Z., Guo, X. and Sanjana, N., 2020. Identification Of Required Host Factors For SARS_Cov2 Infection In Human... [online] Cell.com. Available at: <https://www.cell.com/action/showPdf?pii=S0092-8674%2820%2931394-5> [Accessed 12 November 2020].

4. Myers, K.R., Tham, W.Y., Yin, Y. et al. Unequal effects of the COVID-19 pandemic on scientists. Nat Hum Behav 4, 880–883 (2020). https://doi.org/10.1038/s41562-020-0921-y.

5. Dashraath, P., Jeslyn, W., Karen, L., Min, L., Sarah, L., Biswas, A. and Lin, S., 2020. Coronavirus Disease 2019 (COVID-19) Pandemic and Pregnancy. [online] www.ajog.org. Available at: <https://www.ajog.org/article/S0002-9378(20)30343-4/fulltext> [Accessed 12 November 2020].