PMC:7366549 / 14145-22384 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/7366549","sourcedb":"PMC","sourceid":"7366549","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7366549","text":"Sequencing of the positive coronavirus specimens\nSanger sequencing of bat coronavirus: Using the Sanger sequencing protocol, partial RdRp sequences of BtCoV were retrieved from two (out of 4 amplicons) specimens of Rousettus spp. One of the sequences (MCL-19-bat-588/2) showed close identity to BtCoV HKU9-5-2 (AN): HM211099.1; sequence identity (SI): 99.2 per cent, whereas the second RdRp sequence (MCL-20-bat-76/10) had an SI of 98.8 per cent with BtCoV HKU9-1 (AN: EF065513.1), both from China.\nSanger's sequencing protocol led to retrieval of eight partial RdRp sequences which belonged to Pteropus spp. These bats were collected from Kerala (n=5) and Tamil Nadu (n=3) States. One of the three partial RdRp sequences from Tamil Nadu had 97.93 per cent SI with BtCoV/B55951/Pte_lyl/CB2-THA (AN: MG256459.1, Thailand). The other two sequences had a minimum of 99.48 per cent SI with the CoV PREDICT_CoV-17/PB072 (AN: KX284942.1, Nepal). One of the five partial RdRp sequences from Kerala had 98.88 per cent SI with BtCoV/B55951/Pte_lyl/CB2-THA (AN: MG256459.1, Thailand). The remaining four partial RdRp sequences had \u003e97 per cent SI with CoV PREDICT_CoV-17/PB072 (AN: KX284942.1, Nepal).\nNext-generation sequencing of bat coronavirus: NGS was performed on 10 specimens [4 RS, 2 kidney and 4 intestinal tissue) of the five Rousettus bats to retrieve the complete genome of the BtCoV. Kidney and intestine tissues of the bats from Karnataka State (MCL-20-Bat-76) and RS along with intestine tissue of bats from Kerala State (MCL-19-Bat-606) were used for sequencing and analysis.\nTwo different viruses were retrieved based on the BLAST analysis of the sequences from the kidney and intestine tissues of the bats from Karnataka. Kidney specimen of MCL-20-Bat-76 had an SI of 94 per cent and query coverage (QC) of 94 per cent with CoV BtRt-BetaCoV/GX2018 (AN: MK211379.1), whereas the intestine tissue of the MCL-20-Bat-76 had an SI of 96.8 and 95 per cent QC with the BtCoV HKU9-1 (AN: EF065513.1). The sequences from RS and intestine tissue of the MCL-19-Bat-606 from Kerala, had 93.69 and 93.99 per cent SI to CoV BtRt-BetaCoV/GX2018 (AN: MK211379.1), respectively, with 100 per cent QC. Further, 99.8 per cent of the CoV BtRt-BetaCoV/GX2018 sequences were retrieved from the intestine specimen of the MCL-19-Bat-606. The details of the genome recovered reads mapped and the per cent of reads mapped are summarized in Table II.\nTable II Details of the genome recovered reads mapped and the per cent of reads mapped from the Rousettus bat samples\nSample details Sample type Virus retrieved Relevant reads Per cent of reads Per cent of genome recovered\nMCL-20-Bat-76 Kidney Coronavirus BtRt-BetaCoV/GX2018 1632 0.015 94.39\nIntestine BtCoV HKU9-1 4499 0.056 95.75\nMCL-19-Bat-606 Rectal swab Coronavirus BtRt-BetaCoV/GX2018 13,973 0.114 99.53\nIntestine Coronavirus BtRt-BetaCoV/GX2018 10,214,492 93.476 99.87 Phylogenetic analysis of partial and complete genome sequences of bat coronavirus: A neighbour-joining tree was generated using the partial RdRp region sequences derived from Pteropus and Rousettus spp. bat specimens. It was observed that all the BtCoV sequences were clustered within the L_D sequences of beta CoVs. A distinct subclustering of the sequences retrieved from Pteropus and Rousettus spp. bats is shown in Figure 1. The sequences in the light pink colour are retrieved from the Pteropus spp., whereas those in the dark pink region belong to Rousettus spp. The sequence divergence of 0.35 was observed between Pteropus spp. and Rousettus spp., which was obtained by averaging over all the sequence pairs between the two species, determining those to be distinct sequences to each species.\nFig. 1 Neighbour-joining tree for the RNA-dependent RNA polymerase (RdRp) partial sequence (genomic location: 14,701-15,120) generated from Sanger sequencing. The tree was constructed using the RdRp sequence available in the GenBank sequences. Kimura 2-parameter model was used as the substitution model to generate the tree. A bootstrap replication of 1000 cycles was performed to generate the tree to assess the statistical robustness. The complete genome sequences of four BtCoV obtained from Rousettus spp. specimens were used for generating a neighbour-joining tree (Fig. 2). These sequences were also clustered within L_D of β-CoVs as observed for partial RdRp sequence tree. These complete genome sequences were grouped into gene pairs to identify the gene with higher and lower divergence. The complete genomes of the Indian BtCoV sequences were grouped under L_D. The evolutionary divergence of ORF 1b was \u003c0.54 between the different β-CoV lineages with a maximum score of 0.7 between different BtCoV sequences used in this study (Table III). E gene sequences had larger divergence within the β-CoV genus ranging from 2.18 to 0.94. Lineages L_A and L_C had the maximum divergence of 2.18, whereas the L_B and L_C were the least (0.94). N gene has an overall higher divergence among different lineages (ranging: 2.08-0.75). Overall, evolutionary divergence for the sequences of each gene pair demonstrated that S, N, E and M genes from the α- and δ-CoV highly diverged across the different genus. In contrast, the ORF 1b was less divergent across the genera (Table III).\nFig. 2 Phylogenetic tree for the complete genome tree: A neighbour-joining tree was generated using the representative complete genome sequence available in the GenBank sequences. Kimura 2-parameter model was used as the substitution model to generate the tree. A bootstrap replication of 1000 cycles was performed to generate the tree to assess the statistical robustness.\nTable III Evolutionary divergence for ORF 1b, S, N and M genes for the retrieved sequences with other reference sequences. The lower right-check hand matrix of the table depicts the divergence and the upper left-check matrix of the matrix (blue colour) depicts the variation observed in the bootstrap replication\nN gene Alpha Delta Gamma L_A L_B L_C L_D M gene Alpha Delta Gamma L_A L_B L_C L_D\nAlpha 0.15 0.09 0.10 0.08 0.08 0.09 Alpha 0.11 0.12 0.05 0.06 0.05 0.06\nDelta 2.08 0.11 0.16 0.09 0.11 0.10 Delta 1.50 0.26 0.08 0.16 0.10 0.11\nGamma 1.57 1.49 0.08 0.08 0.09 0.08 Gamma 1.53 1.84 0.10 0.12 0.11 0.09\nL_A 1.84 1.73 1.37 0.05 0.05 0.06 L_A 0.92 1.24 1.30 0.06 0.05 0.05\nL_B 1.48 1.37 1.32 1.09 0.03 0.04 L_B 1.05 1.51 1.37 0.92 0.05 0.05\nL_C 1.57 1.52 1.42 1.07 0.75 0.04 L_C 0.99 1.35 1.27 0.80 0.82 0.05\nL_D 1.64 1.46 1.36 1.27 0.90 0.97 L_D 0.99 1.42 1.23 0.84 0.79 0.82\nORF 1b Alpha Delta Gamma L_A L_B L_C L_D ORF 1a Alpha Delta Gamma L_A L_B L_C L_D\nAlpha 0.01 0.01 0.01 0.01 0.01 0.01 Alpha 0.02 0.02 0.01 0.02 0.02 0.03\nDelta 0.70 0.01 0.02 0.01 0.01 0.01 Delta 1.32 0.03 0.02 0.03 0.03 0.04\nGamma 0.62 0.67 0.01 0.01 0.01 0.01 Gamma 1.14 1.33 0.02 0.03 0.02 0.04\nL_A 0.61 0.69 0.60 0.01 0.01 0.01 L_A 1.22 1.01 1.30 0.02 0.02 0.04\nL_B 0.60 0.70 0.65 0.54 0.01 0.01 L_B 1.26 1.42 1.41 1.19 0.01 0.02\nL_C 0.58 0.69 0.62 0.53 0.50 0.01 L_C 1.35 1.41 1.44 1.19 0.97 0.03\nL_D 0.60 0.67 0.61 0.53 0.50 0.52 L_D 1.26 1.27 1.39 1.09 0.90 1.03\nS gene Alpha Delta Gamma L_A L_B L_C L_D E gene Alpha Delta Gamma L_A L_B L_C L_D\nAlpha 0.02 0.02 0.03 0.03 0.03 0.02 Alpha 0.12 0.18 0.09 0.15 0.15 0.12\nDelta 0.86 0.03 0.04 0.04 0.06 0.03 Delta 1.14 0.47 0.22 0.41 0.28 0.17\nGamma 1.14 0.96 0.04 0.05 0.06 0.04 Gamma 1.59 1.64 0.22 0.24 0.32 0.19\nL_A 1.36 1.28 1.43 0.03 0.03 0.02 L_A 1.03 1.58 1.57 0.23 0.21 0.25\nL_B 1.33 1.23 1.34 1.19 0.04 0.02 L_B 1.24 1.75 1.40 1.83 0.11 0.14\nL_C 1.42 1.32 1.46 1.17 1.03 0.03 L_C 1.37 1.64 1.83 2.18 0.94 0.17\nL_D 1.34 1.24 1.41 1.16 1.00 1.11 L_D 1.25 1.42 1.52 1.95 1.16 1.37\nORF 1a, open reading frame 1a polyprotein; ORF 1b, ORF 1b polyprotein; S, spike glycoprotein; N, nuclocapsid phospoptotein; M, membrane glycoprotein; E, envelope 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