# Elif Hilal Akcıl (SARS-COV VIRUS)
# SARS-COV2 VIRUS

With the outbreak of unknown pneumonia in Wuhan, China in December 2019, a new coronavirus, SARS-CoV-2 has been attracting tremendous attention. Rapid and accurate laboratory testing of SARS-CoV-2 is essential for early discovery, early reporting, early quarantine, early treatment, and cutting off epidemic transmission. The genome structure, transmission, and pathogenesis of SARS-CoV-2 are basically similar to SARS-CoV and MERS-CoV, the other two beta-CoVs of medical importance.


**Figure1:** Sars-CoV2 (Source:https://commons.wikimedia.org/wiki/File:Fmicb-11-01818-g001.jpg)

CoVs are enveloped single-stranded positive-sense RNA (+ssRNA) viruses that can infect respiratory, gastrointestinal, hepatic, central nervous systems of humans, other mammals and birds.
CoV comes from the subfamily Coronavirinae in the family Coronaviridae. This family consists of the four alpha, beta, gamma and delta-CoV species. CoV causes respiratory diseases in humans.
The genome of a typical CoV contains a 5′ untranslated region (UTR), a conserved replicase domain (ORF 1ab), four genes S, E, M, and N to encode structural proteins spike, envelope, membrane, and nucleocapsid proteins, a 3′ UTR, and several unidentified nonstructural ORFs.

**Figure2** : Genome structures of SARS-CoV, MERS-CoV, and SARS-CoV-2. All three CoVs contain a conserved replicase domain (ORF 1ab) (blue). The structural genes (green) S, E, M and N are common features to all CoVs, they encode the structural proteins spike, envelope, membrane, and nucleocapsid, respectively. The accessory genes (orange) are unique to different CoVs.

:::danger
At the very beginning of the symptoms, fever is the most common symptom occurring in 86% to 97% of SARS-CoV-2 infected patients, followed by dry cough (59-76%), fatigue (34-68%), and dyspnea (20-40%). is doing. Time to first symptoms dyspnea was only 5 days. Apart from acute respiratory disease, SARS also has some organ dysfunctions such as gastrointestinal tract, hepatic dysfunction, spleen atrophy, seizures and lymphadenopathy. Since SARS-CoV-2 succeeds the same transforming receptor universityensin enzyme 2 (ACE2) as SARS-Co, it may be in SARS-CoV-2 that it can be found in this organ function.
:::

**SARS-CoV-2 genome sequencing and phylogenetic analysis**
To investigate the genomic architecture of the virus, the genomes of 5,085 SARS-CoV-2 strains dated to COVID-19 cases were sequenced in two stages. Analysis of the disease-causing SARS-CoV-2 strains in the first phase (March 5 to May 11) revealed the presence of many different virus genomes, in total representing the major branches identified globally to date. Clades G, GH, GR, and S were the four most abundantly represented phylogenetic groups. Strains with the Gly614 amino acid variant in the spike protein represented 82% of SARS-CoV-2 strains at stage 1 and 99.9% at stage 2. This spike protein variant is characteristic of the G, GH and GR strands. More importantly, strains with the Gly614 variant represented only 71% of the samples sequenced in March, the first part of phase 1. The decrease in the number of strains with this variant observed in the first 2 weeks of March is thought to be due to the fluctuation caused by the relatively few COVID-19 cases that occurred at that time.

---

## Sample

---

Proper specimen collection and processing is the first and crucial step in laboratory diagnosis. The sample types of patients with respiratory virus infection are diverse. Viral RNA can be detected in the upper respiratory tract (URT), lower respiratory tract (LRT), stool, blood, and urine of people infected with SARS-CoV, MERS-CoV, and SARS-CoV-2. Among these, URT samples, as well as existing LRT samples from severely affected patients, should be collected for diagnosis and retested if the initial test is negative in patients with an epidemiological history and suspected symptoms of SARS-CoV-2 infection. It has been reported that SARS-CoV-2 RNA can be detected in stool, blood or urine even though it is not detectable in URT samples, so taking stool, blood or urine samples if the situation permits helps increase the positive rate when LRT samples are not available.

**Figure 3 :** Smpling for corona virus
*(Source: https://hsgm.saglik.gov.tr/depo/kurumsal/plan-ve-faaliyetler/numune-alma-el-kitabi.pdf)*

:::info
The quality of samples can be affected by several steps such as sample collection, transport and storage. For URT and sputum samples, sample quality is highly dependent on the work of the collectors.
:::

:::danger

Second, the swabs must be inserted deep enough to obtain enough virus-infected cells. In detail, the nasopharyngeal (NP) swab should be inserted through the nostrils parallel to the palate and the oropharyngeal (OP) swab placed in the posterior pharynx and tonsillar areas.
:::

## Nucleic Acid Tests

Specific primers and standard operating procedures for nucleic acid tests (NAT) can be established as soon as the full genome of the virus has been sequenced, making NAT the most suitable method for diagnosis. Current NAT tests for RNA viruses mainly include RT-PCR, alternative isothermal amplification methods, and the CRISPR-Cas13a-based Specific High Sensitivity Enzymatic Reporter Unlocking (SHERLOCK) system.
Because of its simplicity, easy methodology, and extensively validated standard operating procedure, RT-PCR is now the preferred and most widely used method for NAT. RT-PCR assays targeting ORF 1a, ORF 1b, S gene, N gene of SARS-CoV can detect <10 genome equivalents.Various commercial and in-house tests have been developed to detect SARS-CoV-2 RNA. While reporting two one-step quantitative rRT-PCR assays targeting the ORF1b and N gene, which can detect SARS-CoV-2 < 10 copies/reaction, the N gene assay was approximately 10 times more sensitive than the ORF1b gene assay. detection of positive clinical specimens. It is possible that clinical samples contain infected cells that express subgenomic mRNA, resulting in more N gene copies.

---

| Internal control type | Nucleic acid type | Preparation |
| --------------------- | ----------------- | ----------- | --- |
| RNase P | RNA | Easy |
|Housekeeping gene of airway epithelial cells| mRNA| Easy
|Plasmid DNA| DNA |Complex|
|Virus-like particles| RNA| More complex
Tablo1 : Internal control of nucleic acid test

---
## Serological Tests

In rare cases where NAT is not possible, serological testing may be performed for diagnosis, including the investigation of an ongoing outbreak or the retrospective assessment of the extent of an outbreak. Although antibody seroconversion provides reliable evidence of infection, it is not suitable for early diagnosis as serological testing lags behind detection of the viral genome by molecular testing. A higher neutralizing antibody response in SARS was associated with a longer illness.
A variety of serological assays were established for detecting SARS-CoV and MERS-CoV, mainly including enzyme-linked immunosorbent assay (ELISA), chemiluminescence assay (CLIA), immunofluorescence assay (IFA), western blot (WB), protein microarray, and neutralization. Of these methods, ELISA and CLIA are suitable for first line screening because of the large throughput, short processing time, and simple operating procedure, while neutralization assay is used as the gold standard for confirmation in many laboratories.

In SARS, IgG seroconversion was documented in 93% patients at mean of 20 days.43 Positive antibody, neutralizing antibody titer of most patients was shown to peak between Weeks 5 and 8 after onset and to decline with a half-life of 6.4 weeks. In most MERS patients, robust antibody responses developed by the third week of illness, and were delayed further in severely ill patients requiring mechanical ventilation. An IgG and IgM ELISA test which used bat SARS-related-CoV (SARSr-CoV) Rp3 nucleocapsid protein (NP) as antigen was developed previously. This SARSr-CoV NP is 92% identical to SARS-CoV-2 NP, thus this assay was used for serological testing of SARS-CoV-2. Both IgM and IgG titres were relatively low or undetectable in first sampling (not first day of illness). On Day 5, an increase of viral antibodies can be seen in nearly all patients. IgM positive rate increased from 50% to 81%, whereas IgG positive rate increase from 81% to 100%. This is in contrast to a relatively low detection positive rate from molecular test. Based on this result, serological testing might improve the detection positive rate. However, the clinical records of patients involved in this study were not available. Although the target patients were those who received around 10 days of medical treatments upon admission, it is not sure on which days of illness the high positive rate of IgG and IgM occurred. Diverse assays and more evidence are needed to confirm the IgG and IgM kinetics of SARS-CoV-2.

---
**Figure 4:** How a Covid-19 Serology Test Works And Obstacles to its Use
(Source:https://www.google.com/search?q=sars+cov+2+serological+tests&safe=strict&sxsrf=ALeKk01yGZ_4AzR15vC2N13ZxFw3lNHTcA:1622758827839&source=lnms&tbm=isch&sa=X&ved=2ahUKEwi85PTbv_zwAhUI_rsIHTT7CaAQ_AUoAXoECAIQAw&biw=1536&bih=754#imgrc=ULa6Y-8n7-DrEM )

---

## Conclusion
---
:::info
With the progress of global integration, emerging and reemerging infectious diseases are becoming easier to transmit all over the world. The very important and first key to respond to outbreaks is early discovery. Laboratory testing plays the major role in early detection of infected persons, enabling recognition of the infection source and cutting off the transmission route. The results of laboratory testing are affected by various factors. To improve NAT capacity of SARS-CoV-2 RNA, we summarized the literature and guidelines and suggested that: (a) If LRT specimens are unavailable, collect stool and blood samples at later period of illness to improve the positive rate of NAT. (b) Increase template volume to raise the sensitivity of detection. (c) Put samples in reagents containing guanidine salt such as TRIZOL, TRIZOL LS, or AVL buffer to inactivate virus as well as protect RNA. (d) Set proper positive, negative and inhibition controls for extraction and amplification to ensure quality results. (e) Simultaneously amplify human RNase P gene as internal control to avoid false-negative results. At the time of writing this review, serological evidence of SARS-CoV-2 is still poor. Diverse assays targeting different antigens are needed. Also, collecting paired samples would help to monitor the kinetics and positive rates of serological testing in different periods of diseases.
:::
---

Figure 5: Coronavirus Transmission in Human