Sir, The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has, as on March 31, 2020, spread to over 207 countries around the world12, with a total of 896,475 confirmed cases and 45,525 deaths2. The number of reported SARS-CoV-2 cases in India is also on an increase with 1,636 cases and 38 deaths2. In the current pandemic situation, the isolation of SARS-CoV-2 is important for developing and evaluating diagnostic reagents, for antiviral studies and for screening of vaccine candidates. Earlier studies showed that SARS-CoV-2 could not replicate in several cell lines, which are routinely used for isolation of respiratory viruses3. Human and animal cell lines that were found to support SARS-CoV-1 replication during the first outbreak of SARS in China, 20024, are currently being studied. The virus was first isolated in the human airway epithelial cells from clinical specimens as part of early attempts to identify the aetiologic agent of infection5. We describe here the successful isolation and characterization of SARS-CoV-2 from clinical samples in India using Vero CCL-81 cells by observing cytopathic effects (CPEs) and cycle threshold (Ct) values in real-time reverse transcription-polymerase chain reaction (RT-PCR), electron microscopy and next-generation sequencing (NGS). The first three SARS-CoV-2 cases were reported from Kerala during January 27-31, 2020. Later during March 2020, cases were also reported from a group of Italian tourists (n=15) and their contacts in New Delhi, India. Simultaneously, cases were reported in Agra, Uttar Pradesh, which was the outcome of close contact of an infected Delhi-based individual who returned from Italy. The designated COVID-19 testing laboratories of Virus Research Diagnostic Laboratory network (All India Institute of Medical Sciences, New Delhi; Sawai Man Singh Medical College, Jaipur; and King George's Medical University, Lucknow) referred the specimens (throat swab/nasal swab, oropharyngeal swab/sputum) to the Indian Council of Medical Research-National Institute of Virology (ICMR-NIV), Pune, after screening for envelope (E) gene by real-time RT-PCR was done6. A total of 12 SARS-CoV-2 positive specimens having a Ct <30 for the E gene were included in the study. Of these, eight samples were from positive cases of Italian tourists and their contacts in New Delhi. The rest of the specimens were from four positive cases at Agra, Uttar Pradesh, and the close contact cases of an infected Delhi-based individual who returned from Italy. The clinical specimens of the 12 cases were used for infecting Vero CCL-81 which was maintained in Eagle's minimum essential medium (MEM; Gibco, UK) supplemented with 10 per cent foetal bovine serum (FBS) (HiMedia, Mumbai), penicillin (100 U/ml) and streptomycin (100 mg/ml). Likewise, 100 μl was inoculated onto 24-well cell culture monolayers of Vero CCL-81, before growth medium was decanted. The cells were incubated for one hour at 37°C to allow virus adsorption, with rocking every 10 min for uniform virus distribution. After the incubation, the inoculum specimen was removed and the cells were washed with 1X phosphate-buffered saline (PBS). The MEM supplemented with two per cent FBS was added to each well. The cultures were incubated further in five per cent CO2 incubator at 37°C and observed daily for CPEs under an inverted microscope (Nikon, Eclipse Ti, Japan). Cellular morphological changes were recorded using a camera (Nikon, Japan). From each well of cell culture plate, on the third post-infection day (PID-3) of passage-1 (P-1), 50 μl of supernatant was taken and tested for SARS-CoV-2 using real-time RT-PCR for E and RNA-dependent RNA polymerase (RdRp) (2) genes as described earlier78. Similar testing was repeated on the cell supernatant of passage-2(P-2) at PID-4 for observing viral copy number. Cultures that showed CPE on PID-4 were centrifuged at 4815 × g for 10 min at 4°C; the supernatants were processed immediately or stored at −86°C. Further, those that showed CPE were grown in T-25 cm2 flasks at P-2 and titration was done after serial dilution. Tissue culture infective dose 50 per cent (TCID50) values were calculated by the Reed and Muench method9. CPEs were observed in 9 of 12 cultures in the P-1. The TCID50 values ranged from 105.5 to 106.4/ml for the different clinical specimens passaged in Vero CCL-81 at P-2. The cells were examined microscopically for cellular morphological changes following inoculation. Vero CCL-81 cells infected with SARS-CoV-2 strain NIV-2020-770 and uninfected cells (CC) were transferred onto microcavity slides and fixed with acetone. Serum samples (1:25 dilution) from the confirmed COVID-19 cases (POD nCOV-S11, nCOV-S13 and nCOV-S7) and negative serum samples were added followed by incubation at 37°C for 1.5 h10. Antibody reactivity was visualized using anti-human immunoglobulin fluorescein-isothiocynate. In immunofluorescence assay of COVID-19 positive patients, three serum samples exhibited specific reactivity against SARS-CoV-2 virus isolate (Fig. 1).Fig. 1: Immunofluorescence images (red panel) showing uninfected Vero CCL-81 cells probed by positive patient serum samples after post infection day of 13th (left), 11th (middle) and seventh (right) and with SARS-CoV-2 strain NIV-2020-770 infected Vero CCL-81 cells probed by positive patients serum (green panel) showing the reactivity of virus and antibody.Vero CCL-81 cells that were inoculated with the samples showed evidence of cell rounding and detachment from 9 of 12 clinical samples in P-1 at PID-4. Syncytial cells formed large cell masses that increased in size and number as the infection progressed. Enhanced CPE was noted in P-2 at PID-2. The cells were detached from the tissue culture plate surfaces by PID-3. Similar cellular morphological changes were observed after passaging of the above nine samples up to P-2. No cellular changes were observed in the cell control during both passages. Figure 2 depicts the day-wise changes during the passage of a representative clinical isolate (NIV-2020-770). Virus replication was confirmed using real-time RT-PCR with RNA extracted from the cell culture medium on PID-3. The Ct values ranged from 9.79 to 15.41 (in Vero CCL-81 cells) for the isolates at P-2, which were lower than the Ct values of 16-25.1 in the clinical samples (Table I). The number of virus copies in the isolates at P-1 in Vero CCL-81 cells ranged from 5.18×107 to 8.12×108 copy/ml and increased 1-26 fold to a range of 1.69×108 to 6.77×109 in the cell culture supernatants at P-2 (Table I).Fig. 2: Cytopathic effect of the SARS-CoV-2 isolate (NIV-2020-770) demonstrated in Vero CCL-81 cells on different post-infection days (PID).Table I: Cycle threshold (Ct) of SARS-CoV-2 positive clinical specimens and respective viral copy number in isolates in different passages for two different cell culture types using real-time reverse transcription-polymerase chain reaction (RT-PCR). E gene was targeted in allOn PID-4, enhanced CPE was observed. The P-1 material was reinoculated in a new batch of cells, and it showed progressive enhancement of CPE as observed day-wise. Further, an aliquot of cell culture supernatant was harvested from infected Vero CCL-81 showing CPE and the supernatant used for negative staining as described elsewhere1112. Distinct CoV particles with an average size of 95±10 nm having a distinct envelope fringe could be detected in the fields scanned (Fig. 3), as observed earlier13.Fig. 3: Transmission electron microscopy imaging of SARS-CoV-2. A negative-stained SARS-CoV-2 viral particle, demonstrating spike morphology of glycoprotein along with peplomeric projections, a feature typical to the family Coronaviridae, is seen.Next-generation sequencing was performed on SARS-CoV-2 positive clinical samples (100 μl) included in the study and the tissue culture fluid (50 μl) of virus isolates at PID-3 as described earlier1415. Reference-based mapping as implemented in the CLC genomics workbench 11.0 (CLC, Qiagen) was used to retrieve the sequence of the SARS-CoV-2. BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) identification of the viral genome sequences retrieved from the clinical samples and their isolates had 99.98 per cent identity with the SARS-CoV-2 isolate Wuhan-Hu-1 (Accession No. NC_045512). Details of the sequences obtained including the per cent of the reads mapped, total reads and the per cent of genome coverage recovered for the clinical samples and the isolates are provided in Table II. Partial sequences were retrieved from the clinical samples (nCoV-C 132 and nCoV-C 31) and were not included in the analysis.Table II: Per cent of the reads mapped, total reads and the per cent of genome coverage recovered for the clinical samples and the isolatesMEGA software version 7.0.1116 was used for the multiple alignments of the sequences retrieved in this study and the sequences from the Global Initiative on Sharing All Influenza Data (GISAID) database (https://www.gisaid.org/) (Supplementary Table (available from http://www.ijmr.org.in/articles/2020/151/2/images/IndianJMedRes_2020_151_2_244_282559_sm7.pdf)). A neighbour-joining tree was generated using the best substitution model (Kimura 2-parameter model) with a bootstrap of 1000 replicates. As per Tang et al17, the circulating SARS-CoV-2 can be grouped into two types (S and L type) based on the two different single-nucleotide polymorphisms (SNPs) at positions 8782 and 28144 in the genome. The S type possesses TC SNPs while the L type possesses CT SNPs at positions 8782 and 28144, respectively. In the present study, it was observed that two sequences from clinical samples (nCoV-763 and nCoV-770) had TT SNPs, while the other sequences had CT as the SNP (L type) (Table II). The TT SNPs have been observed in few of the GISAID sequences, including one of the Kerala genome sequences (nCoV-19/India/31 January 2020) submitted by us earlier. All the isolates of the clinical samples were of L type. Specific amino acid mutations in the nsp3 region, spike protein and ORF8, in general, lead to the formation of V, G and S genetic variants/clades, respectively, as per the recent classification followed by GISAID. It was observed that the clinical samples, as well as the isolates, had the mutation D614G in the spike protein, classifying the study samples and isolates into the G clade (Table II and Fig. 4). No specific substitutions were observed in any of the isolate sequences with respect to the corresponding clinical sample sequences, as these were sequences from a low passage. The sequences of the clinical samples and the isolate from the contact of the infected Delhi-based individual, who returned from Italy, further showed two mutations, R203K and G204R in the nucleocapsid protein (N). Although all strains demonstrated 99.6 per cent identity with the original Wuhan Hu-1 sequence, the role of unique SNPs and mutations in identifying the source of infection needs to be explored.Fig. 4: Neighbour-joining tree of SARS-CoV-2. The phylogenetic tree is generated using the best substitution model. A bootstrap of 1000 replicates was used to assess the statistical robustness of the tree. Same colours are used for sequences derived from a clinical sample and the respective isolate. Clinical samples are labelled with initials as nCoV while the isolates are labelled with initials as NIV. The clades are represented by different colours in the core region (S - yellow, V - pink, G - blue and unclassified - not coloured).After the first isolation of the virus in the human airway epithelial cells reported by China5, countries such as Australia18, Korea19, Germany20 and the USA21 have also isolated the SARS-CoV-2 strain. In India, initial attempts to isolate the virus from the first three cases did not succeed due to low titres in the clinical specimens. This is the first successful virus isolation of SARS-CoV-2 in the Vero CCL-81 cells in India from nasal and throat swabs of persons with a travel history from Italy and their contacts. Isolation of SARS-CoV-2 from clinical samples will be helpful to address key questions of correlating the differential cell line susceptibility and viral replication efficiency, especially important for clinical samples with low viral titres. Isolation of the virus in such a pandemic situation would help to develop indigenously designed reagents such as positive controls, virus antigen and antibodies, which could lead to the indigenous development of sero-diagnostic assays. These assays would be critical for conducting population-based serosurveys. Propagation in culture will also facilitate antiviral susceptibility studies and vaccine efforts in India.
Highlights With the emergence of the Variant of Concern, omicron (B.1.1.529), India has enhanced genomic surveillance in international travellers. The omicron variant was detected in 59 cases from different States; 40 from Maharashtra, 17 from Rajasthan and one each from Gujrat and Tamil Nadu. The positive cases and their contacts were asymptomatic and genomic surveillance could identify two clusters, one from Maharashtra and another from Rajasthan.
A cluster of SARS-CoV-2 infection occurred among Italian tourists visiting India. We report here the epidemiological, clinical, radiological and laboratory findings of the first cluster of SARS-CoV-2 infection among the tourists.Information was collected on demographic details, travel and exposure history, comorbidities, timelines of events, date of symptom onset and duration of hospitalization from the 16 Italian tourists and an Indian with laboratory-confirmed SARS-CoV-2 infection. The clinical, laboratory, radiologic and treatment data was abstracted from their medical records and all tourists were followed up till their recovery or discharge or death. Throat and deep nasal swab specimens were collected on days 3, 8, 15, 18, 23 and 25 to evaluate viral clearance.A group of 23 Italian tourists reached New Delhi, India, on February 21, 2020 and along with three Indians visited several tourist places in Rajasthan. By March 3, 2020, 17 of the 26 (attack rate: 65.4%) had become positive for SARS-CoV-2 infection. Of these 17 patients, nine were symptomatic, while eight did not show any symptoms. Of the nine who developed symptoms, six were mild, one was severe and two were critically ill. The median duration between the day of confirmation for COVID-19 and RT-PCR negativity was 18 days (range: 12-23 days). Two patients died with a case fatality of 11.8 per cent.This study reconfirms higher rates of transmission among close contacts and therefore, public health measures such as physical distancing, personal hygiene and infection control measures are necessary to prevent transmission.
Sir, The single-stranded RNA genome of the 2019 novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) about 29.9 kb in length and encoding about 9860 amino acids, was annotated to possess 14 open reading frames (ORFs) and 27 proteins12. The orf1ab and orf1a genes at the 5´-terminus of the genome encode the pp1ab and pp1a proteins, respectively, together form 15 non-structural proteins (nsps), nsp1-nsp10 and nsp12-nsp16. The 3´-terminus of the genome encodes four structural proteins, the spike surface glycoprotein (S), the small envelope protein (E), membrane protein (M) and nucleocapsid protein (N). There are eight accessory proteins denoted as 3a, 3b, p6, 7a, 7b, 8b, 9b and ORF142. The epidemiology of the SARS-CoV-2 since its emergence in December 2019 has been ever expanding, with increase in the number of cases and its spread globally34. The number of SARS-CoV-2 cases in India as on March 31, 2020 was 1,071, with mortality crossing 294. In this context, it is vital to understand the genetic nature of circulating SARS-CoV-2. In India, as per the guideline of the Ministry of Health and Family Welfare, suspected samples of SARS-CoV-2 were collected and tested at the designated Viral Research and Diagnostic Laboratories (VRDL)5. As a part of this activity, a total of 15 SARS-CoV-2 positive specimens were obtained during the first week of March 2020, from Italian tourists and travellers from Italy and their contact cases in India. Further, in an effort to screen Indian nationals in Iran to enable their evacuation, during March 5 to 17, 2020, throat swabs were collected from 1,920 individuals; of whom 281 were positive. In addition, a team of Indian doctors visited Italy and collected a total of 380 swabs of Indian citizens; of whom four positive specimens were identified. In an earlier study, the authors identified the first three cases of SARS-CoV-2 in Kerala, India, as imported cases from Wuhan, China, and presented the first two full-genome sequences along with the potential B-cell and T-cell epitopes on the spike protein6. Further, in another study, the SARS-CoV-2 viruses were isolated in Vero CCL-81 cells7. The present study was undertaken to understand and compare the genetic makeup of representative samples of the imported cases of SARS-CoV-2 to India from Wuhan, China, those of Italian tourists in India and the Indians evacuated from Iran and Italy. Throat swab/nasal swab specimens collected from the 1,920 individuals in Iran were tested at the Indian Council of Medical Research-National Institute of Virology (ICMR-NIV) Pune, using real-time reverse transcription-polymerase chain reaction (RT-PCR) protocols to detect RdRp (1), RdRp (2), E and N genes as described elsewhere8. Next-generation sequencing (NGS) was performed on a total of 41 SARS-CoV-2 positive clinical samples from Italy and Iran. Table I presents the details of the full genomes obtained (n=19) as a part of this study as well as the two earlier genomes retrieved from the Kerala samples (n=2) from those who had the travel history from China67.Table I: Cycle threshold (Ct) values of real-time reverse transcription-polymerase chain reaction (RT-PCR) for the E gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) along with the per cent of the reads mapped and the genome size recovered for the clinical samplesMultiple sequence alignment of 21 full genomes obtained and 1563 full-genome sequences (Supplementary Table (available from http://www.ijmr.org.in/articles/2020/151/2/images/IndianJMedRes_2020_151_2_255_283159_sm5.pdf)) available at the Global Initiative on Sharing All Influenza Data (GISAID) database (as of March 26, 2020) was carried out in MAFFT v.7.4509. The phylogenetic tree was constructed using MEGA v.610, employing the neighbour-joining method with the composite likelihood method and 1000 bootstrap replications. An initial tree was constructed based on a total of 1586 sequences. This tree was used to reduce the dataset to 121 sequences, on the basis of country and the genetic variant identified based on the GISAID classification. Comparison of the sequences of this study with respect to the Wuhan Hu-1 reference strain was done to identify unique mutations, if any. Phylogenetic trees based on full-genome sequences deposited and available at GISAID revealed the diversification and the clustering of sequences into groups, based on the genetic variants. Specific amino acid substitutions in the nsp3 region, spike protein and ORF8, in general, lead to the formation of V, G and S genetic variants/clades, respectively. The S clade corresponds to the C28144T nucleotide polymorphism that results in a non-synonymous substitution Leu84Ser in ORF8. Clades V, G and a group of unclassified strains possess mainly C28144 and are referred to as the L type11. The phylogenetic analyses of the study strains and the other global sequences revealed that the SARS-CoV-2 sequences derived from Italy (n=8) in this study, clustered in clade G, while the SARS-CoV-2 sequences (n=11) of Indians evacuated from Iran belonged to the unclassified group which also included one of the SARS-CoV-2 sequences imported from Wuhan (hCoV-19/India/1-27/2020) (Figure). The other sequences imported from Wuhan (hCoV-19/India/1-31/2020) possessed Leu84Ser in ORF8b, classifying it in clade S.Figure: Phylogenetic tree of selected representative full-genome sequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-derived from coronavirus disease 2019 positive patients' clinical samples who had travel history of Wuhan, China, Italy and Iran by neighbour-joining method. Strains sequenced at ICMR-NIV are shown in magenta colour. The clades as per Global Initiative on Sharing All Influenza Data (GISAID) nomenclature are indicated in blue (clade G), red (clade V), green (clade S) and black (unclassified).The sequences of Italy origin were noted to segregate into at least two subgroups. The percentage nucleotide divergence (PND) within these sequences was found to be 0.01 per cent. The SARS-CoV-2 sequences from the Italian tourists (n=6) showed relatedness to other European SARS-CoV-2 sequences from Scotland, Finland, England, Spain, Ireland and the Czech Republic along with a Shanghai, China, strain as the outgroup (Figure). Two other sequences (hCoV-19/India/3118/2020 and hCoV-19/India/3239/2020) clustered more closely with sequences from Belgium and Switzerland. The two sequences (hCoV-19/India/31/2020 and hCoV-19/India/32/2020) from the Agra contacts of the Italy-returned Delhi based individuals were more distinct and showed clustering in a strongly supported subgroup consisting of strains from Brazil and the European countries including Switzerland, Germany, France, Hungary and The Netherlands. The variable amino acid sites based on the alignment of the 21 sequences of this study with respect to Wuhan Hu-1 strain are shown in Table II. All the Italy-origin sequences possessed the substitution D7711G/D614G in the S protein, characteristic of the G clade, along with another mutation P4715L (nsp12-323) that is also shared with many other countries. Mutation S1515F (nsp3-697) was specific to the Italian cohort strain; D8726G (M-3) was specific to hCoV-19/India/3118/2020 and hCoV-19/India/3239/2020 (Indian contacts of an Indian citizen having travel history to Italy), similar to sequences from Scotland, Belgium, Finland, Switzerland and England. The mutations, R9455K and G9456R (N-203 and 204), were found to be specific to the two strains, hCoV-19/India/31/2020 and hCoV-19/India/32/2020 but shared with a few more countries. A recent study has identified the earliest Italian importation of SARS-CoV-2 to a case from Shanghai, China, and has also identified at least two circulating variants in Italy12. Thus, it is likely that the former strain (Italian cohort) has its origin from China, whereas the latter strain (contacts in Agra, n=2) appears to have been from a European cluster involving an entry into Germany that preceded the first cases in Italy by almost a month1213.Table II: Variable amino acid positions in the Indian full-genome sequencesAnalysis of the strains from the SARS-CoV-2 positives in Iran (Figure) showed that these sequences (n=11) clustered with other strains having a global spread inclusive of Canada, USA, several European countries, New Zealand, Australia and Southeast Asian countries noted in this group (moderate support of 64%). The PND among these study sequences was found to be 0.24 per cent. Common mutations shared among SARS-CoV-2 sequences in the group included R207C (nsp2-27), V378I (nsp2-198), M2796I (nsp4-33) and L3606F (nsp6-37). A mutation V9082F (ORF7a-74) was unique to four of the study sequences (hCoV-19/India/1073/2020, hCoV-19/India/1093/2020, hCoV-19/India/1115/2020 and hCoV-19/India/1100/2020) that clustered with a strain from Kuwait, KU12. The KU12 strain was also noted to possess this mutation. To date, there are no other sequences from Iran in the GISAID database. However, a phylogenetic study14 of full-genome sequences has identified distinct SARS-CoV-2 link to travellers returning from Iran to Australia and New Zealand. Some of these representative sequences were included in this study as well. In terms of the overall divergence of SARS-CoV-2, the strains in this study were 99.97 per cent identical to the earliest strain Wuhan Hu-1. However, it is vital to track the evolutionary dynamics of the strains vis-à-vis the strains circulating globally and monitor any specific changes in the functional sites of the major viral proteins. Delineation of circulating strains into three major evolving clades has been reflected in GISAID, with clade G apparently being one of the dominant ones. From the start of the pandemic, severity or transmission patterns have not been associated with any clade in particular. A limitation of this study was the non-availability of full genomes from other parts of India. This would enable a pan-India comparison of the circulating strains in the country. Overall, the present study revealed genetic variants in India that were similar to strains circulating in the specific regions of their origin. Continued surveillance of SARS-CoV-2 strains in India is warranted to get the complete picture of all circulating strains and identify changes that could be associated with increased virulence. Acknowledgment Authors thank Prof. (Dr) Balram Bhargava, Director-General, Indian Council of Medical Research (ICMR) & Secretary, Department of Health Research (DHR), Ministry of Health & Family Welfare (MoHFW), New Delhi for the support. Authors acknowledge the support from Dr P. Ravindran, Director, Emergency Medical Response (EMR), MoHFW, Dr R. Lakshminarayan, ICMR and the team from the DHR, MoHFW, for the logistic support. The National Centre for Disease Control (NCDC) team is acknowledged for sample collection from Italy. Shri Santosh Jadhav, Bioinformatics Group, ICMR-National Institute of Virology, Pune, is thanked for his inputs.
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