Genomic diversity of vaccinia virus strain Cantagalo isolated in southeastern Brazil during the early years of the outbreak, 1999-2006

Outbreaks of a vesiculopustular disease in dairy cattle and milkers have been frequently reported in Brazil since 1999 when the vaccinia virus strain Cantagalo was first isolated in the State of Rio de Janeiro. However, the genomic diversity of the viral isolates associated with these outbreaks is not well known, particularly in the southeastern states that represent the focal point of virus spread to other regions. Here, we report the genomic sequences and an analysis of the polymorphic site profiles and genotypic diversity of four clinical isolates of vaccinia virus strain Cantagalo collected from 1999 to 2006 in southeastern Brazil.

Key words:
virus diversity; poxvirus; zoonosis; Cantagalo virus

Authorship

Aline RV Souza

Universidade Federal do Rio de Janeiro, Instituto de Biofísica Carlos Chagas Filho, Rio de Janeiro, RJ, BrasilUniversidade Federal do Rio de JaneiroBrazilRio de Janeiro, RJ, BrazilUniversidade Federal do Rio de Janeiro, Instituto de Biofísica Carlos Chagas Filho, Rio de Janeiro, RJ, Brasil

Matheus Nobrega Luques

Clarissa R Damaso ⁺+ Corresponding author: damasoc@biof.ufrj.br

http://orcid.org/0000-0001-6299-2816

+ Corresponding author: damasoc@biof.ufrj.br

AUTHORS’ CONTRIBUTION

ARVS conducted all biological assays and bioinformatics analyses and prepared the manuscript draft; MNL contributed to the bioinformatics analyses; CRD conceived the study and wrote the manuscript with input and edits from all authors. The authors declare that there are no conflicts of interest.

SCIMAGO INSTITUTIONS RANKINGS

Figures | Tables

Figures (4)
Tables (1)

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Fig. 1:
schematic map of the sites and dates of sample collection in the southeast region. In 1999, two outbreaks of vaccinia virus (VACV) infection in dairy cattle were reported in the State of Rio de Janeiro (Cantagalo virus isolate CM01) and in the State of São Paulo (Araçatuba virus). Other outbreaks were reported in subsequent years, but only the location of outbreaks caused by Cantagalo virus (CTGV) isolates MI233, VI-04, CG04, and ALEH2 are shown. The locations where Serro2 virus and CTGV-CM01 were collected, as well as Araçatuba virus, are also indicated together with the date of collection.

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Fig. 2:
phylogenetic inference of Cantagalo virus (CTGV) isolates. A multialignment of the conserved region of 40 orthopoxvirus genomes was generated by Mafft vs 7.0 and used to construct a maximum likelihood tree using MEGA 6, opting for the kimura-2p model of substitution and a uniform rate. Numbers indicate the percentage of 1,000 replicates of bootstrap support. The scale bar indicates the number of substitutions per site. Coloured boxes indicate the three main clusters in the vaccinia lineage. The isolates sequenced in this work are indicated with red circles. Genbank accession numbers are indicated following the virus name.

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Fig. 3:
median-joining network of the sequences of Cantagalo virus (CTGV) isolates. The whole genomes of CTGV isolates CM01, MI233, VI04, CG04, ALEH2, and Serro2 virus were aligned using Mafft vs. 7.0, and the alignment was edited using Jalview version 2.11.1.1 to select sites with sequence variations. The resulting alignment was used as the input for Network version 5.0.1.1 software (https://www.fluxus-engineering.com/) to generate a median-joining network using default parameters. Branch length is proportional to the number of accumulated single-nucleotide polymorphisms (SNPs). The small red nodes (median vectors) indicate unsampled or extinct common ancestors.

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Fig. 4:
visualisation of the nucleotide substitutions, deletions, and insertions distributed along the genomes of Cantagalo virus (CTGV) isolates. The whole genomes of CTGV isolates CM01, MI233, VI04, CG04, ALEH2, and Serro2 virus were aligned using Mafft vs. 7.0, and the alignment was visualised using Base-by-base software (https://4virology.net/). Sequence variations per site single-nucleotide polymorphisms (SNPs) and insertions and deletions (INDELs) were highlighted in reference to the genome of CTGV-CM01 (A) or Serro2 virus (B). Asterisks indicate insertions in the A51R gene detected in some genomes. Black arrowheads indicate a 3.7 kb insertion shared by the genomes of CTGV-ALEH2 and Serro2 virus (A) or deletions shared by the genomes of CTGV-CM01, CTGV-MI233, CTGV-VI04, and CTGV-CG04 (B). White arrowheads indicate the 1.5 kb deletion unique to Serro2 genome (A), which is reflected as insertions in the other genomes when Serro2 virus is used as the reference (B).

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TABLE
Sequencing statistics and genomic features of the Cantagalo virus (CTGV) isolates MI233, VI04, CG04, and ALEH2

Fig. 1: schematic map of the sites and dates of sample collection in the southeast region. In 1999, two outbreaks of vaccinia virus (VACV) infection in dairy cattle were reported in the State of Rio de Janeiro (Cantagalo virus isolate CM01) and in the State of São Paulo (Araçatuba virus). Other outbreaks were reported in subsequent years, but only the location of outbreaks caused by Cantagalo virus (CTGV) isolates MI233, VI-04, CG04, and ALEH2 are shown. The locations where Serro2 virus and CTGV-CM01 were collected, as well as Araçatuba virus, are also indicated together with the date of collection.

Fig. 2: phylogenetic inference of Cantagalo virus (CTGV) isolates. A multialignment of the conserved region of 40 orthopoxvirus genomes was generated by Mafft vs 7.0 and used to construct a maximum likelihood tree using MEGA 6, opting for the kimura-2p model of substitution and a uniform rate. Numbers indicate the percentage of 1,000 replicates of bootstrap support. The scale bar indicates the number of substitutions per site. Coloured boxes indicate the three main clusters in the vaccinia lineage. The isolates sequenced in this work are indicated with red circles. Genbank accession numbers are indicated following the virus name.

Fig. 3: median-joining network of the sequences of Cantagalo virus (CTGV) isolates. The whole genomes of CTGV isolates CM01, MI233, VI04, CG04, ALEH2, and Serro2 virus were aligned using Mafft vs. 7.0, and the alignment was edited using Jalview version 2.11.1.1 to select sites with sequence variations. The resulting alignment was used as the input for Network version 5.0.1.1 software (https://www.fluxus-engineering.com/) to generate a median-joining network using default parameters. Branch length is proportional to the number of accumulated single-nucleotide polymorphisms (SNPs). The small red nodes (median vectors) indicate unsampled or extinct common ancestors.

Fig. 4: visualisation of the nucleotide substitutions, deletions, and insertions distributed along the genomes of Cantagalo virus (CTGV) isolates. The whole genomes of CTGV isolates CM01, MI233, VI04, CG04, ALEH2, and Serro2 virus were aligned using Mafft vs. 7.0, and the alignment was visualised using Base-by-base software (https://4virology.net/). Sequence variations per site single-nucleotide polymorphisms (SNPs) and insertions and deletions (INDELs) were highlighted in reference to the genome of CTGV-CM01 (A) or Serro2 virus (B). Asterisks indicate insertions in the A51R gene detected in some genomes. Black arrowheads indicate a 3.7 kb insertion shared by the genomes of CTGV-ALEH2 and Serro2 virus (A) or deletions shared by the genomes of CTGV-CM01, CTGV-MI233, CTGV-VI04, and CTGV-CG04 (B). White arrowheads indicate the 1.5 kb deletion unique to Serro2 genome (A), which is reflected as insertions in the other genomes when Serro2 virus is used as the reference (B).

TABLE Sequencing statistics and genomic features of the Cantagalo virus (CTGV) isolates MI233, VI04, CG04, and ALEH2

Sample	City/State/year	Genome size (bp)	ITR size	No. ORFs	Genome coverage^a	No. SNPs^b	% identity^c	Genbank accession
CTGV-MI233	Miracema (RJ), 1999	181,064	7,025	218	26,098 x	2 (41)	99.94 (97.10)	MW018153
CTGV-VI04	Vieiras (MG), 2001	181,894	7,393	219	27,637 x	13 (52)	99.95 (97.10)	MW018154
CTGV-CG04	Campos dos Goytacazes (RJ), 2003	180,989	7,024	223	26,600 x	14 (53)	99.98 (97.13)	MW018155
CTGV-ALEH2	Alegre (ES), 2006	184,841	7,023	225	23,493 x	62 (81)	97.86 (99.09)	MW018156
CTGV-CM01	Cantagalo (RJ), 1999	181,774	7,418	216	-	- (41)	100	KT013210
Serro2	Serro (MG), 2005	184,572	6,794	209	-	41 (-)	97.14	KF179385

a: genome coverage was calculated using Tablet 1.17.08.17. Total number of reads was 33,710,700 (MI233), 35,756,458 (VI04), 34,215,020 (CG04), and 30,971,724 (ALEH2), with an average percentage of mapping > 90%; b: the number of single-nucleotide polymorphisms (SNPs) was determined using Base-by-base and is expressed in relation to CTGV-CM01 or Serro2 virus (in parentheses); c: identity matrix was calculated using CLC version 7.9.1 and is expressed in relation to CTGV-CM01 or Serro2 virus (in parentheses). ITR: inverted terminal repeat; ORFs: open reading frames.

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Instituto Oswaldo Cruz, Ministério da Saúde Av. Brasil, 4365 - Pavilhão Mourisco, Manguinhos, 21040-900 Rio de Janeiro RJ Brazil, Tel.: (55 21) 2562-1222, Fax: (55 21) 2562 1220 - Rio de Janeiro - RJ - Brazil
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Genomic diversity of vaccinia virus strain Cantagalo isolated in southeastern Brazil during the early years of the outbreak, 1999-2006

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[1] + Corresponding author: damasoc@biof.ufrj.br