microorganisms Communication New Variants of Squash Mosaic Viruses Detected in Human Fecal Samples Fabiola Villanova 1,† , Roberta Marcatti 2,†, Mayara Bertanhe 2,3,† , Vanessa dos Santos Morais 2 , Flavio Augusto de Padua Milagres 4,5, Rafael Brustulin 4,5, Emerson Luiz Lima Araújo 6 , Roozbeh Tahmasebi 2 , Steven S. Witkin 2,7, Xutao Deng 8,9, Eric Delwart 8,9 , Ester Cerdeira Sabino 2, Cassio Hamilton Abreu-Junior 10 , Élcio Leal 1,*,‡ and Antonio Charlys da Costa 2,‡ 1 Laboratório de Diversidade Viral, Instituto de Ciências Biológicas, Universidade Federal do Pará, Belem 66075-000, PA, Brazil; [email protected] 2 Departamento de Moléstias Infecciosas e Parasitárias and Instituto de Medicina Tropical da Faculdade de Medicina da Universidade de São Paulo, São Paulo 05403-000, SP, Brazil; [email protected] (R.M.); [email protected] (M.B.); [email protected] (V.d.S.M.); [email protected] (R.T.); [email protected] (S.S.W.); [email protected] (E.C.S.); [email protected] (A.C.d.C.) 3 School of Veterinary Medicine and Animal Science, University of Sao Paulo, São Paulo 05508-270, SP, Brazil 4 Instituto de Ciências Biológicas, Universidade Federal do Tocantins, Palmas 77001-090, TO, Brazil; fl[email protected] (F.A.d.P.M.); [email protected] (R.B.) 5 Public Health Laboratory of Tocantins State (LACEN/TO), Palmas 77016-330, TO, Brazil 6 General Coordination of Public Health, Laboratories of the Strategic Articulation, Department of the Health, Surveillance Secretariat, Ministry of Health (CGLAB/DAEVS/SVS-MS), Brasília 70719-040, DF, Brazil; [email protected] 7 Department of Obstetrics and Gynecology, Weill Cornell Medicine, 1300 York Avenue, New York, NY 10065, USA 8 Citation: Villanova, F.; Marcatti, R.; Vitalant Research Institute, 270 Masonic Avenue, San Francisco, CA 94143, USA; [email protected] (X.D.); Bertanhe, M.; Morais, V.d.S.; Milagres, [email protected] (E.D.) 9 Department Laboratory Medicine, University of California San Francisco, San Francisco, CA 94143, USA F.A.d.P.; Brustulin, R.; Araújo, E.L.L.; 10 Center of Nuclear Energy in Agriculture, Universidade de São Paulo, Piracicaba 3400-970, SP, Brazil; Tahmasebi, R.; Witkin, S.S.; Deng, X.; [email protected] et al. New Variants of Squash Mosaic * Correspondence: [email protected] Viruses Detected in Human Fecal † These authors contributed equally to this work. Samples. Microorganisms 2021, 9, 1349. ‡ These authors jointly supervised this work. https://doi.org/10.3390/ microorganisms9071349 Abstract: Squash mosaic virus (SqMV) is a phytovirus that infects great diversity of plants worldwide. In Brazil, the SqMV has been identified in the states of Ceará, Maranhão, Piauí, Rio Grande do Norte, Academic Editor: Maria Cristina Mele and Tocantins. The presence of non-pathogenic viruses in animals, such as phytoviruses, may not be completely risk-free. Similarities in gene repertories between these viruses and viruses that affect Received: 1 May 2021 Accepted: 6 June 2021 animal species have been reported. The present study describes the fully sequenced genomes of Published: 22 June 2021 SqMV found in human feces, collected in Tocantins, and analyzes the viral profile by metagenomics in the context of diarrhea symptomatology. The complete SqMV genome was obtained in 39 of Publisher’s Note: MDPI stays neutral 253 analyzed samples (15.5%); 97.4% of them belonged to children under 5 years old. There was with regard to jurisdictional claims in no evidence that the observed symptoms were related to the presence of SqMV. Of the different published maps and institutional affil- virus species detected in these fecal samples, at least 4 (rotavirus, sapovirus, norovirus, parechovirus) iations. are widely known to cause gastrointestinal symptoms. The presence of SqMV nucleic acid in fecal samples is likely due to recent dietary consumption and it is not evidence of viral replication in the human intestinal cells. Identifying the presence of SqMV in human feces and characterization of its genome is a relevant precursor to determining whether and how plant viruses interact with host cells Copyright: © 2021 by the authors. or microorganisms in the human gastrointestinal tract. Licensee MDPI, Basel, Switzerland. This article is an open access article Keywords: Squash mosaic virus; plant viruses; next generation sequencing; virome; public health distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Microorganisms 2021, 9, 1349. https://doi.org/10.3390/microorganisms9071349 https://www.mdpi.com/journal/microorganisms Microorganisms 2021, 9, 1349 2 of 8 1. Introduction One of the greatest challenges in agricultural production is the control of viral diseases. Plant viruses are responsible for decreasing the quantity and quality of crop yield, leading to significant economic losses. Squash mosaic virus (SqMV) is an example of a phytovirus (family Secoviridae genus Comovirus) that infects melons and a diversity of plants of the family Cucurbitaceae [1,2]. SqMV is non-enveloped, 28–30 nm in diameter, and the capsid is icosahedral com- posed of 60 small proteins (18–26 kDa) and 60 large proteins (37–49 kDa). Genomic RNAs are encapsidated separately into two different types of particles of similar size [3]. The SqMV genome is segmented with bipartite linear ssRNA (positive sense) and is composed of RNA-1 (6–8 kb) and RNA-2 (4–7 kb). Each genomic segment has a VPg linked to its 50 end and a 30 poly(A) tract. The 50- and 30-UTRs of RNA-1 and RNA-2 are similar in sequence composition. The virion RNA is infectious and serves as both the genome and mRNA. Viral proteins are usually expressed as large polyproteins, which are cleaved by virus-encoded 3C-like proteinases. Both RNA-1 and RNA-2 segments are translated into two polyproteins, which are further processed to form functional proteins [3]. RNA-1 is translated into a single polyprotein that is processed into five functional proteins: the N-terminal 32K protein (also known as protease co-factor), the 58K protein with sequence motifs characteristic of an NTP-binding helicase, the Vpg, the protease, and the polimerase. The 32K Co-Pro and 58K NTB proteins induce cytopathic structures by the proliferation of ER-derived membranes. The 32K protein also curbs the processing of the RNA-1-encoded polyprotein and affects the processing of the RNA-2-encoded polyprotein [4–6]. RNA-2 is translated into three functional proteins: the movement protein (MP), the large capsid protein (LCP), and the small capsid protein (SCP). The MP and the CPs are required for cell-to-cell movement of the virus. The MP is a structural component of tubular structures containing virus-like particles that traverse the cell wall [7,8]. There are two different groups of SqMV, serotypes I and II [9], both of which cause plant diseases worldwide. SqMV is spread mainly by infected seeds, and can be transmitted by chrysomelid beetles, including Acalymma trivittata and Diabrotica spp. [1,2]. Symptoms and severity of the plant disease vary, depending on many factors, including the strain of the virus, the species and/or variety of plant, and the population of vectors [10]. In Brazil, SqMV has been reported in melons, watermelons, and squash, in the states of Ceará, Maranhão, Piauí, Rio Grande do Norte, and Tocantins [10,11]. Melons and watermelons are commonly consumed by human and animal populations in their raw form, without prior chemical or thermal treatment that inactivates potential pathogenic microorganisms. Among microorganisms that infect these fruits, plant viruses are not considered pathogenic to humans and animals [12]. Some similarities exist between phytoviruses and viruses that affect animal species, including gene repertories [12,13]. While the genes responsible for viral replication and expression may be conserved among plant and animal viruses, genes that determine interactions with their hosts are unique [13]. Indeed, some plant and animal viruses relevant to public health belong to the same viral families, for example, the Rhabdoviridae family, which includes Lyssavirus (infecting vertebrates) and Tenuivirus (infecting plants and insects) [12]. Phytoviruses have been identified in mammals, including humans. Their presence in the feces of non-human mammals has been reported [12]. These viruses are stable in the gastrointestinal tract, and, therefore, animal carriers can assume the role of disseminators, contaminating the environment with the virus when defecating [12]. Sequence similarities between viroids and human microDNA were recently identified and pose the need for further investigation on the potential of viroids and their derived small RNAs to cross kingdoms and interact with nucleic acids in humans [14]. There is a case study involving disruption of type III effector-mediated phagocytosis in a human cell line following a plant bacterial infection (Pseudomonas syringae)[15]. Studies that identify plant viruses in humans are necessary to better understand their potential to interact with human cells, which can Microorganisms 2021, 9, 1349 3 of 8 involve modulation of gene expression through interference of their RNA [14,16]. The use of metagenomics analysis is a powerful tool that can contribute to the identification of new viruses relevant to public health. There is a lack of studies involving the possible presence of phytoviruses in humans and potential health consequences [12]. The purpose of the present study is to describe the completely sequenced genomes of SqMV found in human feces, using samples collected in the state of Tocantins, northern Brazil, through a metagenomics approach. In addition, the profile of all viruses found in samples from individuals with acute gastroenteritis symptoms is delineated. 2. Materials and Methods 2.1. Population and Specimen Collection The current cross-sectional surveillance study was performed from 2010 to 2016 in Tocantins (Central region of Brazil). Fecal samples were collected in 38 different localities. A total of 238 stool specimens were obtained from children aged 1–5 years, 3 from children aged 8–15 years, and 7 from adults aged 20–78 years, all with gastroenteritis symptoms.