| Abstract|| |
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) responsible for the current pandemic has resulted in over 5 million deaths globally. More than a year has passed, still SARS-CoV-2 panic the public life. Virus isolation is of paramount importance for development of vaccines, in-vitro screening of antiviral compounds, pathogenesis studies, etc., Many cell lines were studied for amplification and replication of SARS-CoV-2 and Vero cells were found to be ideal cell lines for isolation. In May 2020, ICMR-Regional Medical Research Centre, NE region, India, successfully established the SARS-CoV-2 culture system in Vero CCL-81 cell lines. Phylogenetic analyses of the whole genome sequences of the SARS-CoV-2 isolate (EPI_ISL_2501532 | 2020-05-19) showed monophyletic clade G and lineage B.1.1.
Keywords: COVID-19, SARS-CoV-2, virus isolation
|How to cite this URL:|
Jakharia A, Borkakoty B, Pait S, Baruah G, Hazarika R, Biswas D. First Isolation and Characterization of SARS-CoV-2 From COVID-19 Patient of North East India. Indian J Pathol Microbiol [Epub ahead of print] [cited 2023 Mar 24]. Available from: https://www.ijpmonline.org/preprintarticle.asp?id=367704
| Introduction|| |
Coronavirus belongs to respiratory virus family that causes diverse symptom such as fever, pneumonia, lung infection, acute respiratory distress, etc. The World Health Organization (WHO) announced officially the virus as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and was declared a pandemic on March 11, 2020. SAR-CoV-2 is a non-segmented, enveloped, positive sense RNA virus of 29.9 kb that is included in the sarbecovirus, ortho corona virinae subfamily which is largely distributed in humans and other mammals.,
The unprecedented spread of this pandemic warrants an urgent need for reference reagents/viral isolates. The medical science researchers require viral isolates as references for diagnostic, to screen anti-viral compounds, develop new vaccines, vaccine efficacy study for newly immerging strains, etc., Moreover, virus isolates are also essential for doing basic research, such as pathogenesis research, virus stability research, etc.
SARS-CoV-2 was first isolated in Wuhan, China, cultured in human airway epithelial cells from clinical specimens as part of early attempts to study the aetiologic agent of infection. ICMR-NIV, Pune, was the first research center to isolate the SARS-CoV-2 virus in India. In this study, we report successful establishment of the SARS-CoV-2 virus culture system in Vero-CCL-81 at a suboptimal temperature (35°C), from the first case of COVID-19 detected in Dibrugarh, Assam, on May 19, 2020. This is the first known report of the SARS-CoV-2 virus isolation from clinical sample in North-East India and third Research center from India.
| Materials and Methods|| |
Nasopharyngeal swab (NPS) was collected in an in-house prepared viral transport media (VTM) from suspected COVID-19 cases for detection of SARS-CoV-2 infection by Quantitative reverse transcription PCR (RT-qPCR). Vero CCL-81 cells were cultured in T25 flasks at 37°C in a 5% CO2 environment, in 1 × Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum (FBS) and penicillin-streptomycin. The Vero CCL-81 was grown to a ~ 80% confluent monolayer (2 to 3 days) in a 25-cm2 (T25) flask for further inoculation with clinical sample. The collected SARS-CoV-2 positive NPS sample was stored at 4°C, for 1-h prior to inoculation. The supernatant from Vero CCL-81 cells cultured T25 flask was decanted, followed by three times wash with virus growth medium (DMEM supplemented with TPCK trypsin and antibiotics). The cell line was than inoculated with the clinical samples and incubated at 35°C, 5% CO2 for 2 h. Similarly, 1 mL plain VTM was used as a control/mock inoculum in another Vero CCL-81 cell line in a T25 flask [Figure 1]a. After the incubation, the inoculums specimens along with the control were decanted and the inoculated cell lines were washed with virus growth medium. Fresh DMEM supplemented with 5% FBS and antibiotics was added into the T25 flask and incubated in CO2 incubator at 35°C, with 5% CO2. The cells were observed daily for cytopathic effects (CPEs) under an inverted microscope (Evos, Life Technologies) and recorded daily morphological changes of the Vero CCL-81 cell line. On post infection day (PID)-7, CPE was observed and on PID-8, the supernatants were harvested and aliquots were stored at –80°C. RNA was extracted from the harvested supernatant and subjected to qRT-PCR for SARS-CoV-2 E-gene, Orf gene, and RdRp genes., The cycle threshold (CT-value) of the clinical sample was compared with the harvested isolate for recording increase in virus titre. SARS-CoV-2 virus culture was conducted in the Bio-safety Level-3 facility available at the investigators institute according to standard laboratory bio-safety guidelines.
|Figure 1: Cytopathic effect (CPE) of SARS-CoV-2 on VERO-CCL81 cells. (a) Mock/control inoculated cells (b) SARS-CoV-2 inoculated cells with CPE. (c) Logarithmic increase in viral load detected based on CT-value in SARS-CoV-2 specific qRT-PCR.|
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The virus isolate along with nine clinical samples were subjected to whole genome sequencing of SARS-CoV-2 virus using Illumina platform. The sequencing data were used for the whole genome sequence assembly and variant calling analysis. All the 10 strains were categorized by the Pangolin analysis.,
The evolutionary history was inferred by using the Maximum Likelihood method and General Time Reversible model after model testing.
| Results|| |
Virus replication and isolation were confirmed through CPE, logarithmic increase in viral load detected based on CT-value in qRT-PCR [Figure 1]c, partial Sanger sequencing followed by next generation sequencing. This confirmed SARS-CoV-2 amplification and replication in Vero CCL-81 cell line. An approximately 5 log increase (CT-Value of isolate 9.2 vs 26.6 in clinical sample) in titre was recorded in isolate compared with the clinical sample by qRT-PCR.
Genome size of 29,903 nucleotides was obtained in each strain (both isolate and clinical sample) through the reference-based assembly. However, on average, 17 SNPs were identified per strain compared with reference strain (NC_045512.2). Overall, 119 total SNPs were detected from all the strains (one isolate and nine clinical samples). The virus isolate strain and the clinical sample of the same [RMRC-DIB-4/2020] were categorized into one lineage (B.1.1), under monophyletic clade G. The clinical samples collected during May–June 2020 were categorized into one lineage (B.1.1) and samples collected during August–November 2020 were categorized into another lineage (B.1.36).
Phylogenetic analysis involved 34 nucleotide sequences including reference, vaccine strain, and nearest sequences as shown in [Figure 2]. The average mean distance of SARS-CoV-2 whole genome between the Dibrugarh isolated strain (EPI_ISL_2501532 l 2020-05-19) and reference strain (NC 045512.2) was 0.033% and Covaxin vaccine strain (MT577010.1/NIV-770/2020) was 0.023%.
|Figure 2: Evolutionary tree involved 34 whole genome nucleotide sequences of SARS-CoV-2 (~29.9 kbp). Nine clinical samples (bulleted in blue circle), one cell culture isolate [EPI ISL 2501532|2020-05-19] (bulleted in blue diamond), Covaxin/BBV152 vaccine strain (bulleted in green diamond), SARS-CoV-2 reference strain (bulleted in red square), and other nearest SARS-CoV-2 whole genome sequences. The tree was rooted to the ancestral clade S. The evolutionary history was inferred by using the Maximum Likelihood method and General Time Reversible (GTR +G+i) model conducted based on best fit model in MEGA X|
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| Discussion|| |
China first isolated SARS-CoV-2 virus from human sample followed by Australia, Korea, Germany, and USA.,,, India became the 6th country to isolate the virus, and ICMR-NIV, Pune, was the first Indian Institute to isolate the virus successfully, followed by CSIR-CCMB, Hyderabad. ICMR-RMRCNE, Dibrugarh, was the third government Institute from India and first Institute from Northeast India to successfully isolate the SARS-CoV-2 virus from confirmed COVID-19 clinical sample.
Virus isolates are very much essential for further study on virus host interactions, in vitro screening of anti-viral compounds, development of vaccines, diagnostic kits, neutralization studies, etc. It is very much crucial to recognize different target cells and culture system for SARS-CoV-2. Earlier investigations established various cell lines where both SARS-CoV-2 and SARS-CoV-1 reproduce well; however, CPE were only observed in the Vero cells and Fetal Rhesus Kidney-4., Elsewhere, similar inclination findings for SARS-CoV-1 was observed; however, other familiar culture systems do not support SARS-CoV-2 replication like MRC-5 cells, HEP-2, HeLa, MDCK, and embryonated eggs. Moreover, bat EFK3B cells are also not susceptible to SARS-CoV-2, whereas susceptible to MERS-CoV. Both Vero CCL-81 and Vero E6 as well as human upper airway epithelial cell lines were found to be the best cell lines which support amplification of SARS-CoV-2 and Vero E6 is more suitable for amplification and quantification.,
This study determined that SARS-CoV-2 can be grown in Vero CCL-81 cell line at 35°C, that is, 2°C lower than normal human body temperature (37°C). Lower temperature culture system could be useful for virus attenuation; this may be supportive for developing live attenuated vaccine. The phylogenetic analysis showed that SARS-CoV-2 isolate (DIB-4) closely clustered with the Covaxin vaccine strain (MT577010.1/NIV-770/2020) and monophyletic clade G.
The study was approved by the Institutional Human Ethics Committee (IEC)/Institutional Review Board (IRB) of ICMR-Regional Medical Research Centre, NE region, Dibrugarh, India, under the scheme “Establishment of a network of laboratories for managing epidemics and natural calamities (Regional-VRDL)” vide letter no. RMRC/Dib/IEC (Human)/2019-20/135.
Financial support and sponsorship
Present investigation is supported by Department of Health Research (DHR), Ministry of Health and Family Welfare, Govt. of India. Under the scheme entitled “Establishment of a Network of Laboratories for Managing Epidemics and Natural calamities.”
Conflicts of interest
There are no conflicts of interest.
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Indian Council of Medical Research- Regional Medical Research Centre, North East Region (ICMR-RMRCNE), Lahowal, Dibrugarh - 786010, Assam
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]