Main Figures

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posted on 2025-03-12, 18:23 authored by Dhanasekaran ShanmugamDhanasekaran Shanmugam, Manali BajpaiManali Bajpai, Ajinkya Khilari

Figure 1: LSDV sampling and detection. A, photographs showing the presence of skin scab and lesions on limbs and other parts of dairy cattle. Swab samples were collected from these scabs for isolation of DNA. B, Details of the number of samples collected as skin scab swabs and nasal swabs sample types from different districts of Maharashtra and Odisha. C, amplification of ~500 bp fragment obtained from the diagnostic multiplexed nested PCR reaction. Lane M - 100 bp DNA size ladder; lane 1 and lane 2 - representative samples showing positivity for LSDV. D, PCR products obtained using LSDV_WGSPP3.5kb primer panel for the amplification of the entire LSDV genome as ~3.5 kb fragments. Lane M - 1 kb DNA size ladder; lane 1 and lane 3 - Pool A PCR products of two different samples; lane 2 and lane 4 - Pool B PCR products of two different samples.
Figure 2: Number of samples collected and analysis outcome: The plots illustrate the sample numbers for which nested PCR based virus detection was successful and genome sequencing of LSDV was achieved. A, B and C, samples from Maharashtra state; D and E, samples from Odisha state. In each plot the outer, middle and inner circles indicate the total number of samples, number of samples showing positive detection of virus by multiplexed nested PCR, and number of samples for which LSDV genome sequence was obtained after multiplexed PCR amplification of genomic fragments using the LSDV_WGSPP3.5kb primer panel. The red line highlight in Ahmednagar plot indicates the four pairs of skin scab and nasal samples collected from four animals shown in order.
Figure 3: Read Depth plots for nanopore sequence data of LSDV genomic fragments obtained by multiplexed amplification: The LSDV genome was amplified by multiplexed PCR using two different primer panels, LSDV_WGSPP3.5kb (A, B, E & F) and LSDV_WGSPP7.5kb (C & D). Samples PP488409 (A, C & E) and PP488410 (B, D & F) were evaluated, using two different Taq DNA polymerases, Quantabio replica hifi tough mix (A, B, C & D) or Takara primestar GXL master mix (E & F), for PCR amplification. The resolution of the plot is at nucleotide level and red marks indicate the positions at which the read depth was <20X. The quality of the nanopore sequence data obtained with the LSDV_WGSPP3.5kb primer panel multiplexed PCR was suitable for variant calling (see Supplementary Figure 2 data). G, sequence coverage (blue) and percent identity (orange) plots for the field isolate genomes with the reference sequence NC003027.1.
Figure 4: Mapping of novel mutations identified from the genome sequence of field samples: Schematic representation of the LSDV reference genome showing the location of all 156 genes. The genomic bp position is shown within the red box at the end of each line. Each gene is shown as a coloured box (scaled relative to gene size in bp) and the numbers shown inside the gene boxes indicates the order in which the genes occur on the chromosome. Each gene is coloured by function and the position of the genes above or below the horizontal line indicates the forward and reverse direction of coding. Inverted terminal repeats (ITRs) are shown as green bars at the two ends of the linear genome. Novel mutations are shown as vertical lines and are mapped to genomic position; green - synonymous mutation; red - non-synonymous mutation; blue - indels. Gene function colour code: pink - RNA transcription and modification (26 genes); yellow - viral virulence and host range (37 genes); green - structure and assembly (33 genes); blue - DNA replication and nucleotide metabolism (10 genes); white - unknown function (50 genes).
Figure 5: Phylogenetic relationship of LSDV field strains with all previously reported isolates. Maximum likelihood analysis was performed using the LSDV genome sequence of 41 field samples generated in this study along with 133 other LSDV genomes which are accessible from NCBI. The resulting tree file was used to render rooted (A) and unrooted (B) tree structures. In the rooted radial tree (A), the NCBI accession ID of each LSDV genome is given. The coloured dots next to the sample name indicate the year of isolation/sampling of the virus from the field. The sequences reported from India which are grouped in clade A are indicated with a red asterisk. The isolate developed as live-attenuated LSD vaccine (Lumpi-ProVacInd) in India is highlighted in clade A with orange background. In the unrooted tree (B), the geographic region from which the viruses were reported is shown. In both trees, the clade branches are colour coded and the major clades are labelled A - E. Clade A includes the reference strain NC003027.1 and clade B contains the African vaccine strains (sample name in red font). Majority of the LSDV strains reported from India during the years 2022 and 2023 (including those from this study) are grouped within clade E. A few Indian isolates, including those from 2019 and 2020, are grouping within clade A. Clade E sequences are most closely related to Clade D sequences.