Lourenço et al. Virol J (2021) 18:124 https://doi.org/10.1186/s12985-021-01595-z RESEARCH Open Access Zoonotic vaccinia virus strains belonging to diferent genetic clades exhibit immunomodulation abilities that are proportional to their virulence Karine Lima Lourenço1, Leandro Andrade Chinália1, Lethícia Ribeiro Henriques2, Rodrigo Araújo Lima Rodrigues3,4 and Flávio Guimarães da Fonseca1* Abstract Background: The vaccinia virus (VACV) isolates, Guarani P1 virus (GP1V) and Passatempo virus (PSTV), were isolated during zoonotic outbreaks in Brazil. Each one of them belongs to two diferent VACV clades, defned by biological aspects that include virulence in mice and phylogenetic analysis. Considering that information about how vaccinia viruses from diferent groups elicit immune responses in animals is scarce, we investigated such responses in mice infected either by GP1V (group 2) or PSTV (group 1), using VACV Western Reserve strain (VACV-WR) as control. Methods: The severity of the infections was evaluated in BALB/c mice considering diverse clinical signs and defned scores, and the immune responses triggered by GP1V and PSTV infections were analysed by immune cell phenotyp- ing and intra-cytoplasmic cytokines detection. Results: We detected a reduction in total lymphocytes (CD3 ), macrophages (CD14 ), and NK cells (CD3-CD49 ) in animals infected with VACV-WR or GP1V. The VACV-WR and +GP1V viruses, belonging +to the most virulent group in+ a murine model, were able to down-modulate the cell immune responses upon mice infection. In contrast, PSTV, a virus considered less virulent in a murine model, showed little ability to down-modulate the mice immune responses. Mice infected with VACV-WR and GP1V viruses presented signifcant weight loss and developed lesions in their spleens, as well as damage to liver and lungs whereas mice infected with PSTV developed only moderate clinical signs. Conclusions: Our results suggest that VACV immunomodulation in vivo is clade-related and is proportional to the strain’s virulence upon infection. Our data corroborate the classifcation of the diferent Brazilian VACV isolates into clades 1 and 2, taking into account not only phylogenetic criteria, but also clinical and immunological data. Keywords: Poxvirus, Vaccinia virus, Immune response, Immune downmodulation, Guarani P1 virus, Passatempo virus Background Members of the Poxviridae family present a double- stranded DNA genome that varies from 140 to 300 kbp in size, depending on the virus species. Teir large genomes *Correspondence: [email protected] encode more than 150 genes, including some involved in 1 Laboratory of Basic and Applied Virology, Department of Microbiology, immunomodulatory mechanisms [1–3]. In Brazil, vac- Institute of Biological Sciences, Federal University of Minas Gerais, Belo cinia virus (VACV) has been circulating in rural and wild Horizonte, MG, Brazil Full list of author information is available at the end of the article environments for decades[4]. Brazilian VACV isolates are © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Lourenço et al. Virol J (2021) 18:124 Page 2 of 12 currently classifed into two groups: group 1 (less viru- elements of their host’s antiviral responses. It has been lent in a murine model of infection), and group 2 (more proposed that the poxviruses’ ability to down-modulate virulent in a murine model of infection) (Fig. 1). In addi- the host’s immune response is directly proportional to tion to virulence in mice, this division refects biological their virulence in vivo [9]. Previous studies have already properties of the isolates such as plaque phenotype in shown a diference in the modulation of the immune BSC-40 cells and genetic diferences, including the pres- response in mice infected with non-replicating (Modifed ence or absence of an 18 nucleotide deletion in the viral vaccinia virus Ankara), attenuated (vaccinia virus Lister), hemagglutinin protein (HA A56R) coding gene (see inset and replicative (vaccinia virus Western Reserve) virus on Fig. 1) [5]. strains [9, 10]. Te Guarani P1 virus (GP1V) and Passatempo virus Te evaluation of infections caused by naturally circu- (PSTV), used in this study, were isolated in Minas Gerais, lating VACV strains GP1V (group 2) and PSTV (group 1) Brazil, in 2001 and 2003 respectively, from outbreaks in represents an opportunity to analyse immune responses rural properties involving cattle and humans [6, 7]. Te triggered in the host as a result of infections with viruses infections by these viruses caused lesions on the udder, presenting diferent virulence patterns. Such studies can, teats, snout and oral mucosa of cows and calves. Milk- then, be compared to those including either laboratory ers usually develop hand lesions after unprotected con- strains of VACVs or vaccine strains. Here we investigated tact with cows infected with these viruses and clinical the immune responses triggered in BALB/c mice infected signs such as high fever, severe headache, back pain and with the GP1V or PSTV VACV strains, originated from lymphadenopathy are frequently reported by individuals zoonotic outbreaks in Brazil, compared to infections handling infected cows [7, 8]. by a laboratory reference strain, vaccinia virus Western Much of the replicative success of poxviruses is related Reserve (VACV-WR). Our results are important not only to their capacity to obstruct, escape or subvert essential to better defne patterns of immunomodulation in vivo, Fig. 1 Phylogenetic analysis of VACV-BR based on nucleotide sequences of the A56R gene (hemagglutinin). VACV-BR group 1 is highlighted with light yellow and group 2 with light blue. White arrows indicate the Brazilian viruses used in this study. The alignment was built using Muscle with standard parameters [45]. The maximum likelihood tree was built with IQ-TREE [46]. The best-ft replacement model selected by ModelFinder was HKY F I. The branch supports were estimated using 1000 replicates of standard bootstrap and the tree was visualized with iTOL [47]. Bootstrap + + values lower than 50 were suppressed. The clade of variola viruses has been collapsed. The scale bar indicates the number of substitutions per site. A schematic alignment was included (inset) to indicate the genomic signature that divides VACV-BR into two distinct groups, an 18-nt deletion between positions 740 and 757 of the A56R gene. The representation was based on the VACV-WR (AY243312) gene Lourenço et al. Virol J (2021) 18:124 Page 3 of 12 caused by zoonotic vaccinia viruses, but they also cor- characterization of these slide preparations was per- roborate the genetic classifcation of feral VACVs into formed, through the attribution of clinical scores, con- two separate clades, which has been subject of criticism. sidering the presence and distribution of infammation, edema, pulmonary hemorrhage and infammation along Methods with hepatic and splenic reactive degeneration [12–14]. Viruses Samples of GP1V and PSTV strains were kindly provided Splenocyte preparation, immunophenotyping by Dr. Erna Kroon (Universidade Federal de Minas Ger- and detection of intracellular cytokines ais, Belo Horizonte, Brazil) and VACV-WR was gently In order to evaluate the production of interferon-γ (IFN- provided by Dr. Bernard Moss (NIAID/NIH, Bethesda, γ) and tumor necrosis factor α (TNF-α) by CD4 and + 6 EUA). Te three viral samples were grown and titrated CD8 + T lymphocytes, mice were inoculated with 10 (PFU/ml) in BSC-40 cells through plaque assay and puri- PFUs of PSTV, GP1V or VACV-WR through the intra- fed in sucrose cushions, as described before [11]. nasal route. Teir spleens were harvested 7 days post infection and the splenocytes were obtained after tis- Infection of mice and clinical signs sue maceration. For erythrocyte lysis, cell extracts were BALB/c mice used in this study were obtained from resuspended in deionized water and incubated on ice. UFMG’s central animal facility (Belo Horizonte, Bra- PBS 10X was used to stop the lysis process. Cell prolifera- zil) and maintained in our experimental animal facil- tion assays were performed through splenocyte labelling ity (Departamento de Microbiologia, Belo Horizonte, with Bromodeoxyuridine (BrdU). 96-well plates contain- 5 4 Brazil). Animals were kept into ventilated cages with ing 2 × ­10 spleen cells per well received stimulus of 10 food and water ad libitum. All in vivo procedures were PFU of purifed, UV-inactivated VACV-WR; concanava- approved by the Committee of Ethics for Animal