Metagenomics identification of genetically distinct tick virome in India unveils signs of purifying selection, and APOBEC and ADAR editing

Abstract

Recently, several tick-borne zoonotic viruses have been identified through the application of virus metagenomics. However, the tick virome in South Asia and the factors driving the evolution of these viruses remain largely unknown. In this study, we report the complete genomes of the genetically distinct Nairobi sheep disease virus (NSDV), Jingmen tick virus (JMTV), Lihan tick virus (LTV), and Mivirus, along with nearly complete genomes of turnip mosaic virus (TMV) and turnip yellows virus (TYV). We also present partial genomes of Tamdy orthonairovirus, Nayun tick nairoviruses (NTNV), PTV-like viruses, Xinjiang tick-associated virus-1 (XTAV1), Totivirus, Kismayo viruses, Quaranjavirus, and Brown dog tick phlebovirus-2 (BDTPV-2), identified from Indian ticks through virus metagenomics. The diversity was categorized into distinct groups specific to particular host organisms and/or geographical regions. Our findings also indicated that selection pressure for codon usage in these viruses was influenced by purifying selection, which induces transition mutations potentially through apolipoprotein B mRNA editing enzyme, catalytic polypeptide (APOBEC) and adenosine deaminases acting on RNA (ADAR) editing.

Keywords: Microbial genomics; Virology.

Graphical abstract

Figure 1

Genetic diversity in the Nairoviridae viruses (A) NSDV complete segment L nucleotide sequences based on phylogenetic analysis separated them into China, Kenya, and India-1954 genotypes, and India-2020 formed an out-group. The details are illustrated in Figure S1A and Data S2. (B) NBGMD analysis based on whole segment L nucleotide sequences exhibited more than 10% nucleotide diversity among genotypes of the NSDV. The standard error calculated in the NBGMD analysis was displayed above the diagonal. Details of sequences are provided in Data S3. (C) NBGMD analysis based on complete segment L amino acid sequences exhibited around 2.5%–3.4% amino acid diversity among genotypes of the NSDV. The standard error calculated in the NBGMD analysis was displayed above the diagonal. Details of sequences are provided in Data S4. (D) NSDV segment L coding region nucleotide mismatches were displayed between the genotypes compared to the Kenya genotype. The EU697951.1/NSDV/708/Kenya virus was used as a reference. Details are illustrated in Figure S1E; Data S4. (E) NSDV segment L coding region silent and non-silent mutations displayed between the genotypes compared to the Kenya genotype. The EU697951.1/NSDV/708/Kenya viruses were used as a reference. Details are illustrated in Figure S1F; Data S4. (F) NSDV segment L coding region amino acid mismatches were displayed between the genotypes compared to the Kenya genotype. The EU697951.1/NSDV/708/Kenya viruses were used as a reference. Details are illustrated in Figure S1G; Data S4. (G) NSDV complete segment S nucleotide sequences, based on phylogenetic analysis, separated them into China, Kenya, and India-1954 genotypes, and the India-2020 formed an out-group. Details are illustrated in Figure S2A; Data S5. (H) NBGMD analysis based on whole segment S nucleotide sequences exhibited more than 10% nucleotide diversity among genotypes of the NSDV. The standard error calculated in the NBGMD analysis was displayed above the diagonal. Details of sequences are provided in Data S6. (I) NBGMD analysis based on complete segment S amino acid sequences exhibited around 1.9%–3.4% amino acid diversity among genotypes of the NSDV. The standard error calculated in the NBGMD analysis was displayed above the diagonal. Details of sequences are provided in Data S7. (J) NSDV segment S coding region nucleotide mismatches were displayed between the genotypes compared to the Kenya genotype. The AF504293.1/NSDV/708/Kenya virus was used as a reference. Details of sequences are provided in Figure S2C; Data S7. (K) NSDV segment S coding region silent and non-silent mutations displayed between the genotypes compared to the Kenya genotype. The AF504293.1/NSDV/708/Kenya virus was used as a reference. Details of sequences are provided in Figure S2D; Data S7. (L) NSDV segment S coding region amino acid mismatches were displayed between the genotypes compared to the Kenya genotype. The AF504293.1/NSDV/708/Kenya virus was used as a reference. Details of sequences are provided in Figure S3C; Data S7. (M) Based on phylogenetic analysis, NSDV complete segment M nucleotide sequences separated them into China, Kenya, and India-1954 genotypes, and the India-2020 formed an outgroup. Details of sequences are provided in Data S8. (N) NBGMD analysis based on whole-segment M nucleotide sequences exhibited more than 10% nucleotide diversity among genotypes of the NSDV. The standard error calculated in the NBGMD analysis was displayed above the diagonal. Details of sequences are provided in Data S9. (O) NBGMD analysis based on complete segment Mamino acid sequences exhibited around 5.6%–18.7% amino acid diversity among genotypes of the NSDV. The standard error calculated in the NBGMD analysis was displayed above the diagonal. Details are illustrated in Data S10. (P) NSDV segment M coding region nucleotide mismatches were displayed between the genotypes compared to the Kenya genotype. The EU697952.1/NSDV/708/Kenya viruses were used as a reference. Details are illustrated in Figure S4D; Data S10. (Q) NSDV segment M coding region silent and non-silent mutations displayed between the genotypes compared to the Kenya genotype. The EU697952.1/NSDV/708/Kenya virus was used as a reference. Details are illustrated in Figure S4E; Data S10. (R) NSDV segment M coding region amino acid mismatches were displayed between the genotypes compared to the Kenya genotype. The EU697952.1/NSDV/708/Kenya viruses were used as a reference. Details are illustrated in Figure S4F; Data S10. (S) Tamdy orthonairovirus partial segment S nucleotide sequences based on phylogenetic analysis out-grouped India-2020-Tamdy orthonairovirus. Details of sequences are provided in Data S11. (T) NTNV partial nucleotide sequences based on phylogenetic analysis grouped NTNV/Dog/R.sanguineus/India-2020 with the Romanian virus. Details of sequences are provided in Data S11. (U) NTNV partial nucleotide sequences based on phylogenetic analysis grouped NTNV/Goat/H. bispinosa/India-2020 with China virus. Details of sequences are provided in Data S11.

Figure 2

Genetic diversity in the JMTV (A) The complete segment 1/NS5 nucleotide sequences of the JMTV group of viruses, based on phylogenetic analysis, separated them into different genotypes, and JMTV/India/2020 formed an outgroup. Details of sequences are provided in Data S12. (B) NBGMD analysis based on JMTV complete segment 1/NS5 nucleotide sequences reveals nucleotide diversity among genotypes and sub-genotypes. The standard error calculated in the NBGMD analysis was displayed above the diagonal. Details of sequences are provided in Data S12. (C) NBGMD analysis based on JMTV group of viruses’ complete segment 1/NS5 amino acid sequences diversity among different viruses. The standard error calculated in the NBGMD analysis was displayed above the diagonal. Details of sequences are provided in Data S13. (D) JMTV segment 1/NS5 coding region nucleotide mismatches displayed between the genotypes and sub-genotypes compared to JMTV/Segment-1/India/2020. Details are illustrated in Figure S7A; Data S14. (E) JMTV segment 1/NS5 coding region silent and non-silent mutations displayed between the genotypes and sub-genotypes compared to JMTV/Segment-1/India/2020. Figure S7B; Data S14. (F) JMTV segment 1/NS5 coding region amino acid mismatches displayed between the genotypes and sub-genotypes compared to JMTV/Segment-1/India/2020. Figure S12A; Data S14. (G) JMTV segment 3 (NS3) coding region silent and non-silent mutations displayed between the genotypes and sub-genotypes compared to JMTV/Segment-3/India/2020. The JMTV/Segment-1/India/2020 was used as a reference Figure S9B; Data S14. (H) JMTV segment 3 (NS3) coding region amino acid mismatches displayed between the genotypes and sub-genotypes compared to JMTV/Segment-3/India/2020. Figure S12C; Data S14. (I) JMTV segment two coding region silent and non-silent mutations displayed between the genotypes and sub-genotypes compared to JMTV/Segment-2/India/2020. Figure S8B; Data S14. (J) JMTV segment two coding region amino acid mismatches displayed between the genotypes and sub-genotypes compared to JMTV/Segment-2/India/2020. Figure S12B; Data S14. (K) JMTV segment 4C coding region silent and non-silent mutations displayed between the genotypes and sub-genotypes compared to JMTV/Segment-4C/India/2020. The JMTV/Segment-1/India/2020 was used as a reference. Figure S10B; Data S14. (L) JMTV segment 4C coding region amino acid mismatches displayed between the genotypes and sub-genotypes compared to JMTV/Segment-4C/India/2020. The JMTV/Segment-1/India/2020 was used as a reference. Figure S13A; Data S14. (M) JMTV segment 4M coding region silent and non-silent mutations displayed between the genotypes and sub-genotypes compared to JMTV/Segment-4M/India/2020. Figure S11B; Data S14. (N) JMTV segment 4M coding region amino acid mismatches displayed between the genotypes and sub-genotypes compared to JMTV/Segment-4M/India/2020. Figure S13B; Data S14. (O) The (O) JMTV group of viruses’ partial segment L nucleotide sequences, based on phylogenetic analysis, separated them into different genotypes, including JMTV/Goat/H. bispinosa/India/2020 formed out-group. Detail of sequences provided in Data S15.

Figure 3

Evolutionary origin of segmented Flaviviridae viruses (A) The un-rooted phylogenetic tree exhibiting the genetic relationship between different genera in the family Flaviviridae at complete NS5 amino acid sequence levels. Detail of sequences provided in Data S16. (B) The rooted phylogenetic tree exhibits a genetic relationship between different viruses in the JMTV group of viruses at complete NS5 amino acid sequence levels. Detail of sequences provided in Data S16. (C–L) CLANS (pairwise similarity network) genetic relationship between different genera in the family Flaviviridae at complete NS5 amino acid sequence levels.Different p value threshold of CLANS is utilized to display the lines connecting the sequences to indicate levels of genetic relationships, (C) the lines connecting the sequences at p value threshold of 1e-140; (D) the lines connecting the sequences at p value threshold of 1e-120; (E) the lines connecting the sequences at p value threshold of 1e-90; (F) the lines connecting the sequences at p value threshold of 1e-80; (G) the lines connecting the sequences at p value threshold of 1e-75; (H) the lines connecting the sequences at p value threshold of 1e-70; (I) the lines connecting the sequences at p value threshold of 1e-50; (J) the lines connecting the sequences at p value threshold of 1e-22; (K) the lines connecting the sequences at p value threshold of 1e-20; and (L) the lines connecting the sequences at p value threshold of 1e-17. Detail of sequences provided in Data S16.

Figure 4

Genetic diversity in the Phenuiviridae viruses (A) LTV complete segment L nucleotide sequences base phylogenetic analysis separated them into LTV-1, LTV-2, and LTV-3 genotypes, and LTV/India/2020 formed out-group. Detail of sequences provided in Data S17. (B) NBGMD analysis based on LTV whole segment L nucleotide sequences exhibited more than 8% nucleotide diversity among genotypes. The standard error calculated in the NBGMD analysis was displayed above the diagonal. Detail of sequences provided in Data S17. (C) Uukuviruses complete segment L amino acid sequences based on the genetic relationship displayed in the phylogenetic tree. Detail of sequences provided in Figure S5A; Data S18. (D) NBGMD analysis based on Uukuviruses complete segment L amino acid sequences diversity among different Uukuviruses. The standard error calculated in the NBGMD analysis was displayed above the diagonal. Detail of sequences provided in Data S18. (E) LTV segment L coding region nucleotide mismatches displayed between the genotypes compared to the LTV-1 genotype. The MN095537.1/LTV/Thailand/phlebovirus was used as a reference. Details are illustrated in Figure S15B; Data S19. (F) In the LTV segment L coding region, silent and non-silent mutations were displayed between the genotypes compared to the LTV-1 genotype. The MN095537.1/LTV/Thailand/phlebovirus was used as a reference. Details are illustrated in Figure S15C; Data S19. (G) LTV segment L coding region amino acid mismatches displayed between the genotypes compared to the LTV-1 genotype. The MN095537.1/LTV/Thailand/phlebovirus was used as a reference. Details are illustrated in Figure S15D; Data S19. (H) LTV complete segment S nucleotide sequences based on phylogenetic analysis separated them into LTV-1 and LTV-2 genotypes, and LTV/India/2020 formed out-group. Detail of sequences provided in Data S20. (I) NBGMD analysis based on LTV complete segment S nucleotide sequences exhibited more than 10% nucleotide diversity among genotypes. The standard error calculated in the NBGMD analysis was displayed above the diagonal. Detail of sequences provided in Data S20. (J) LTV segment S coding region nucleotide mismatches displayed between the genotypes compared to the LTV-1 genotype. The MN095538.1/LTV/Thailand/phlebovirus was used as a reference. Details are illustrated in Figure S16C; Data S21. (K) LTV segment S coding region silent and non-silent mutations displayed between the genotypes compared to the LTV-1 genotype. The MN095538.1/LTV/Thailand/phlebovirus was used as a reference. Details are illustrated in Figure S16D; Data S21. (L) LTV segment S coding region amino acid mismatches were displayed between the genotypes compared to the LTV-1 genotype. The MN095538.1/LTV/Thailand/phlebovirus was used as a reference. Details are illustrated in Figure S6E; Data S21. (M) BDTPV-2 partial segment L nucleotide sequences based on phylogenetic analysis grouped BDTPV-2/India/2020-Tamdy with China virus. Detail of sequences provided in Data S22. (N) Kismayo virus partial nucleotide sequences based on phylogenetic analysis grouped Kismayo virus/Goat/H. bispinosa/India-2020 between Kismayo virus and Palma viruses. Detail of sequences provided in Data S23. (O) Kismayo virus coding region amino acid mismatches displayed between the Kismayo virus and Palma viruses compared to Kismayo virus/Goat/H. bispinosa/India-2020. The Kismayo virus/Goat/H. bispinosa/India-2020 was used as a reference. Detail of sequences provided in Data S23.

Figure 5

Genetic diversity in the Miviruses viruses, Turnip mosaic virus, Turnip yellows virus, Xinjiang tick-associated virus-1, Totivirus, and Quaranjavirus (A) WMV complete genome nucleotide sequences, based on phylogenetic analysis, separated them into different genotypes and sub-genotypes, with WMV/India/2020 forming an out-group. Detail of sequences provided in Data S24. (B) NBGMD analysis based on WMV complete genome nucleotide sequences displaying nucleotide diversity among genotype and sub-genotypes. The standard error calculated in the NBGMD analysis was displayed above the diagonal. Detail of sequences provided in Data S24. (C) HMV’s complete genome nucleotide sequences, based on phylogenetic analysis, separated them into different genotypes, and HMV/India/2020 formed the out-group. Detail of sequences provided in Data S25. (D) NBGMD analysis based on HMV complete genome nucleotide sequences displaying nucleotide diversity among genotypes. The standard error calculated in the NBGMD analysis was displayed above the diagonal. Detail of sequences provided in Data S25. (E) Complete RdRp protein amino acid sequences of different viruses in the genus Mivirus, with a genetic relationship displayed in the phylogenetic tree. Detail of sequences provided in Data S26. (F) NBGMD analysis based on complete RdRp protein amino acid sequences of various viruses in genus Mivirus. The standard error calculated in the NBGMD analysis was displayed above the diagonal. Detail of sequences provided in Data S26. (G) The partial genome nucleotide sequences of TMV viruses, based on phylogenetic analysis, grouped TMV/India/2020 with Iranian viruses. Detail of sequences provided in Data S27. (H) The partial genome nucleotide sequences of TYV viruses, based on phylogenetic analysis, grouped TYV/India/2020 with Australian viruses. Detail of sequences provided in Data S27. (I) The phylogenetic tree illustrates the genetic relationship of PTV-like RNA/India/2020 with related viruses at the level of partial nucleotide sequences. Detail of sequences provided in Data S27. (J) The phylogenetic tree displays the genetic relationship of XTAV1 in cattle and R. micoplus/India/2020 with related viruses at partial nucleotide sequence levels. Detail of sequences provided in Data S27. (K) The phylogenetic tree displaying the genetic relationship of XTAV1/sheep/R. Haemaphysaloides/India/2020 with related viruses at the partial nucleotide sequence level. Detail of sequences provided in Data S27. (L) The phylogenetic tree displays the genetic relationship of Totivirus/India/2020 with related viruses at the partial nucleotide sequence level. Detail of sequences provided in Data S27. (M) The phylogenetic tree displays the genetic relationship of Quaranjavirus/India/2020 with related viruses at the nucleotide sequence level of partial segment 3. Detail of sequences provided in Data S27. (N) The phylogenetic tree displays the genetic relationship of Quaranjavirus/India/2020 with related viruses at the partial five-nucleotide sequence level. Detail of sequences provided in Data S27.

Figure 6

Host codon usage bias, host adaptation indexes, selection pressure, and purifying selection in the coding regions (A) The graph depicts the percentages of A, T, G, and C nucleotides in the coding regions of the viruses. Data are represented as mean ± SD. Detail of sequences provided in Data S28 and S29. (B) The graph illustrates the ENc values in the coding regions of the viruses. Data are represented as mean ± SD. Detail of sequences provided in Data S28 and S29. (C–F) The graphs represent the codon usage fraction and RSCU values of different coding regions of viruses: (C) NSDV L, (D) LTV L, (E) JMTV segment 1, and (F) Mivirus L. Details of the sequences are provided in Data S28 and S29. (G–J) The graphs representing the neutrality plot analysis of the GC12 and GC3 values for different coding regions of viruses are shown in (G) NSDV L, (H) LTV L, (I) JMTV segment 1, and (J) Mivirus L. Details of the sequences are provided in Data S28 and S29. (K) The graphs representing the ENC plotted against GC3s values of different coding regions of viruses. Detail of sequences provided in Data S28 and S29. (L) The graphs represent the parity rule 2 (PR2)-bias plot values of different coding regions of viruses. Detail of sequences provided in Data S28 and S29. (M) The graphs represent the CAI values of different coding regions of viruses. Data are represented as mean ± SD. Detail of sequences provided in Data S28 and S29. (N) The graphs representing the eCAI values of different coding regions of viruses. Detail of sequences provided in Data S28 and S29. (O) The graphs represent the RCDI values of different coding regions of viruses. Data are represented as mean ± SD. Detail of sequences provided in Data S28 and S29. (P) The graphs represent the eRCDI values of different coding regions of viruses. Detail of sequences provided in Data S28 and S29. (Q–T) The graphs representing the cumulative dN/dS ratio in the different coding regions of viruses, (Q) NSDV L; (R) LTV L; (S) JMTV segment 1; and (T) Mivirus L. (U) The graphs representing the dN/dS ratio in the different coding regions of viruses. Data are represented as mean ± SD. Details of sequences are provided in Data S33 and S30.

Figure 7

APOBEC and ADAR editing in the virus evolution (A) The graphs represent the transition and transversion mutations in the different coding regions of NSDV. Data are represented as mean ± SD. Details of sequences are provided in Data S28 and S31. (B) The graphs represent the transition and transversion mutations in the various coding regions of LTV. Data are represented as mean ± SD. Details of sequences are provided in Data S28 and S31. (C) The graphs represent the transition and transversion mutations in the different coding regions of JMTV. Data are represented as mean ± SD. Details of sequences are provided in Data S28 and S31. (D) The graphs represent the transition and transversion mutations in the various coding regions of WMV. Data are represented as mean ± SD. Details of sequences are provided in Data S28 and S31. (E) The graphs represent the transition and transversion mutations in the different coding regions of HMV. Data are represented as mean ± SD. Details of sequences are provided in Data S28 and S31. (F) The graphs represent the APOBEC motif mutations in the various coding regions of NSDV. Data are represented as mean ± SD. Details of sequences are provided in Data S33 and S32. (G) The graphs represent the APOBEC motif mutations in the various coding regions of LTV. Data are represented as mean ± SD. Details of sequences are provided in Data S28 and S32. (H) The graphs represent the APOBEC motif mutations in the various coding regions of JMTV. Data are represented as mean ± SD. Details of sequences are provided in Data S28 and S32. (I) The graphs represent the APOBEC motif mutations in the different coding regions of WMV. Data are represented as mean ± SD. Details of sequences are provided in Data S33 and S38. (J) The graphs represent the APOBEC motif mutations in the various coding regions of HMV. Data are represented as mean ± SD. Details of sequences are provided in Data S33 and S32. Figures 7F–7J, indicates GG-to-AG, GA-to-AA, GC-to-AC, GT-to-AT, CC-to-CT, TC-to-TT, GC-to-GT, and AC-to-AT.