Constant testing for <i>Pestivirus</i> in cell lines reveals different routes of contamination

Pestivirus can contaminate cell cultures and sera and cause serious problems that evolve the integrity of studies, confidence in diagnostic results, and safety of human and animal vaccines. Contaminations by Pestivirus and other viruses may occur at any time and regular assays of monitoring in cell cultures and your supplies are necessary. This study aimed to analyze the phylogeny of Pestivirus detected from cell cultures, calf serum, and standard strains of three laboratories in Brazil that carry out frequent tests for the monitoring of cellular contaminations. These samples were submitted to phylogenetic analysis to understand the genetic relationship between contaminants occurring in these facilities. As result, the Pestivirus found in samples were Bovine viral diarrhea virus (BVDV-1 and BVDV-2), Hobi-like viruses (often named BVDV-3), and Classical swine fever virus (CSFV), and the phylogenetic analysis help us to infer at three possible routes of contamination in this work.

Key words
Pestivirus; phylogeny; cell line; contamination

Authorship

TATIANA F.P. DE OLIVEIRA

RNA extraction, RT-PCR, sequecing

Laboratório Nacional Agropecuário de Minas Gerais, Laboratório de Biologia Molecular/Laboratório de Diagnóstico de Doenças Virais, Avenida Rômulo Joviano, s/n, 33600-000 Pedro Leopoldo, MG, BrazilLaboratório Nacional Agropecuário de Minas GeraisBrazilPedro Leopoldo, MG, BrazilLaboratório Nacional Agropecuário de Minas Gerais, Laboratório de Biologia Molecular/Laboratório de Diagnóstico de Doenças Virais, Avenida Rômulo Joviano, s/n, 33600-000 Pedro Leopoldo, MG, Brazil

https://orcid.org/0000-0001-8805-9039

ANTÔNIO A.F. JÚNIOR

experiment design, sequecing, phylogenetic analysis, manuscript preparation

Centro Universitário de Sete Lagoas, Av. Marechal Castelo Branco, 2765, Santo Antonio, 35701-242 Sete Lagoas, MG, BrazilCentro Universitário de Sete LagoasBrazilSete Lagoas, MG, BrazilCentro Universitário de Sete Lagoas, Av. Marechal Castelo Branco, 2765, Santo Antonio, 35701-242 Sete Lagoas, MG, Brazil

https://orcid.org/0000-0002-2962-5616

ANAPOLINO M. DE OLIVEIRA

Tcell culture preparation

https://orcid.org/0000-0001-3256-2258

MARCELO F. CAMARGOS

cell culture preparation, manuscript preparation

https://orcid.org/0000-0001-8457-7875

MARCOS B. HEINEMANN

experiment design, manuscript preparation.

Universidade de São Paulo, Faculdade de Medicina Veterinária e Zootecnia, Departamento de Medicina Veterinária e Saúde Animal, Av. Prof. Dr. Orlando Marques de Paiva, 87, Butantã, 05508-270 São Paulo, SP, BrazilUniversidade de São PauloBrazilSão Paulo, SP, BrazilUniversidade de São Paulo, Faculdade de Medicina Veterinária e Zootecnia, Departamento de Medicina Veterinária e Saúde Animal, Av. Prof. Dr. Orlando Marques de Paiva, 87, Butantã, 05508-270 São Paulo, SP, Brazil

https://orcid.org/0000-0001-6444-9788

Correspondence to: Antônio Augusto Fonseca Júnior E-mail: antonio.biomol@gmail.com

SCIMAGO INSTITUTIONS RANKINGS

Figures

Figures (2)

Thumbnail

Figure 1
The evolutionary history by the Neighbor-Joining method. The optimal tree with the sum of branch length = 1,24082590 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Kimura 2-parameter method and are in the units of the number of base substitutions per site. The rate variation among sites was modeled with a gamma distribution (shape parameter = 5). The analysis involved 62 nucleotide sequences. Sequences from Pestivirus detected in □ cell lines from GO2 ▲ cell lines from RT1 ● cell lines and CSF from GO1○ reference strains form GO1.

Thumbnail

Figure 2
The evolutionary history by using the Maximum Likelihood method based on the Kimura 2-parameter model. The tree with the highest log likelihood (-887,5220) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0,8912)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 62 nucleotide sequences. Sequences from Pestivirus detected in □ cell lines from GO2 ▲ cell lines from RT1 ● cell lines and CSF from GO1○ reference strains from GO1.

Figure 1 The evolutionary history by the Neighbor-Joining method. The optimal tree with the sum of branch length = 1,24082590 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Kimura 2-parameter method and are in the units of the number of base substitutions per site. The rate variation among sites was modeled with a gamma distribution (shape parameter = 5). The analysis involved 62 nucleotide sequences. Sequences from Pestivirus detected in □ cell lines from GO2 ▲ cell lines from RT1 ● cell lines and CSF from GO1○ reference strains form GO1.

Figure 2 The evolutionary history by using the Maximum Likelihood method based on the Kimura 2-parameter model. The tree with the highest log likelihood (-887,5220) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0,8912)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 62 nucleotide sequences. Sequences from Pestivirus detected in □ cell lines from GO2 ▲ cell lines from RT1 ● cell lines and CSF from GO1○ reference strains from GO1.

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Constant testing for <i>Pestivirus</i> in cell lines reveals different routes of contamination

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[1] Correspondence to: Antônio Augusto Fonseca Júnior E-mail: antonio.biomol@gmail.com