The third wave of Seneca Valley virus outbreaks in pig herds in southern Brazil

Abstract

Seneca Valley virus (SVV) is the only representative member of the Senecavirus genus of the Picornaviridae family. Since 2014, SVV has been identified as a causative agent of vesicular disease outbreaks in pigs of different ages from Brazil, the USA, Canada, China, Thailand, Colombia, Vietnam, and India. From May 2020, several pig herds, from the Brazilian states Parana and Santa Catarina reported vesicular disease in different pig categories. This study aimed to report the third wave of SVV outbreaks in pig herds in southern Brazil. A total of 263 biological samples from 150 pigs in 18 pig herds were evaluated. The samples were obtained from pigs with clinical signs of vesicular disease (n = 242) and asymptomatic animals (n = 21). Seneca Valley virus RNA was detected in 96 (36.5%) of the biological samples evaluated, with 89 samples from symptomatic and 7 from asymptomatic pigs. The data show that asymptomatic pigs, but in viremia, are possible sources of infection and can act as carriers and possibly spreaders of SVV to the herd. In this study, we report the third wave of vesicular disease outbreaks caused by SVV in different categories of pigs from herds located in southern Brazil.

Introduction

Seneca Valley virus (SVV) is the only representative member of the Senecavirus genus of the Picornaviridae family [1]. SVV is a non-enveloped virus with a single-stranded, positive-sense RNA genome. The first isolation of SVV occurred accidentally in 2002, in a PER.C6 (transformed fetal retinoblast) cell culture and was possibly introduced through the widespread use of contaminated fetal bovine serum or porcine trypsin [2]. Initially, SVV was not associated with any pathology [3]. However, in Canada in 2008 and the USA in 2012, the agent was identified in pigs with vesicular disease [4, 5].

Since 2014, SVV has been identified as a causative agent of vesicular disease outbreaks in pigs of different ages in Brazil [6, 7] and other countries, including the USA [8], Canada [9], China [10], Thailand [11], Colombia [12], Vietnam [13], and India [14]. Clinical signs presented by finished pigs and breeding animals include vesicles and/or ulcerated lesions in the coronary bands, interdigital space, and snouts. Anorexia, cutaneous hyperemia, fever, and neurological signs have been observed in newborn piglets (1–5 days old) [15–17]. Additionally, an increase in diarrhea and neonatal mortality rates, characterized as a neonatal multisystemic syndrome, have been described [15].

In Brazil, the first wave of SVV outbreaks occurred in 2014–2015, affecting pigs of different age groups [6, 7]. Since then, it has been circulating in Brazilian pig farms and clinical manifestations of the vesicular disease have been reported associated with SVV. In the second half of 2018, an increase in the number of SVV outbreaks was reported in piglets and finishing pigs, characterizing the second wave of disease [18]. Again in 2020, more specifically in May, several pig herds, mainly from the southern states (Paraná and Santa Catarina) of Brazil, started to report vesicular disease in different pig categories. Fluid-filled and ulcerative vesicular lesions in the snout and coronary bands have been reported in weaned and finishing pigs, breeders and neonatal multisystemic syndrome. Therefore, this study reports the third wave of SVV outbreaks in pig herds in southern Brazil.

Materials and methods

Animals and biological samples

A total of 150 pigs, of which 132 were symptomatic and 18 asymptomatic, were evaluated. The sampling included animals from the following categories: suckling piglets (n = 10), weaned (n = 33), finishing (n = 16), and breeders (n = 91) from 18 pig herds located in the Parana (n = 3) and Santa Catarina (n = 15) states in southern Brazil. The number of pigs evaluated ranged from 3 to 19 animals per herd. All pig herds sampled showed clinical signs of vesicular disease and animals sampled with intact or newly ruptured vesicles were prioritized. A total of 263 biological samples were collected, comprising 37 tissue fragments (heart, lungs, spleen, and tonsils), 89 vesicular lesions (scrapping, vesicular fluids, and/or swabs), and 137 serum samples. The samples were stored at − 80 °C until processing.

Molecular detection of SVV

Nucleic acids were extracted from biological samples using a combination of phenol/chloroform/isoamyl alcohol and silica/guanidinium isothiocyanate methods described previously [19, 20]. The extracted nucleic acids were eluted in 50 μL of ultra-pure RNase-free, diethylpyrocarbonate (DEPC)-treated sterile water (Invitrogen™ Life Technologies, Carlsbad, CA, USA), and stored at − 80 °C until used for molecular analysis. Sterile water was included as a negative control during nucleic acid extraction and subsequent procedures. All the samples were tested for the presence of SVV-RNA by qRT-PCR assay designed to amplify a 118 bp fragment of the VP1 protein of the SVV [21].

Sequencing and phylogenetic analysis

Positive samples from ulcerative lesions were randomly selected for sequencing analysis after performing conventional RT-PCR [6] for the partial amplification of the viral genome VP1 (542 bp). The PCR products were purified using a PureLink® Quick Gel Extraction and PCR Purification Combo Kit (Invitrogen® Life Technologies, Carlsbad, CA, USA), quantified using a Qubit® Fluorometer (Invitrogen® Life Technologies, Eugene, OR, USA), and sequenced in both directions with the forward and reverse primers used in the PCR assay on an ABI3500 Genetic Analyzer sequencer using a Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems®, Foster City, CA, USA). Sequence quality analyses were performed using PHRED and contig assemblies using CAP3 software (http://asparagin.cenargen.embrapa.br/phph/). Similarity searches were performed with nucleotide (nt) sequences deposited in GenBank using the Nucleotide Basic Local Alignment Search Tool (BLASTn) software (http://blast.ncbi.nlm.nih.gov/). The nt sequence identity matrix was constructed using BioEdit software, version 7.2.5 [22]. The phylogenetic tree was constructed from the nt sequence, using the Maximum Likelihood method and the Kimura 2-parameter model [23] in MEGA v.7 software [24], providing statistical support with 1000 bootstrap replicates.

Results

SVV RNA was detected in all the analyzed pig herds (18/18). Of the 263 biological samples analyzed, 96 (36.5%) were positive for SVV RNA. Regarding the positive samples, 89/242 (36.8%) were symptomatic, and 7/21 (33.3%) were asymptomatic. The results are presented in Table 1 according to the type of sample and pig category.

Table 1.

Detection of RNA of Seneca Valley virus according to the animal category and clinical manifestation

Animal category	Biological samples evaluated
	Tissue fragments	Ulcerative or vesicular lesions	Serum	Total
Symptomatic
Suckling piglets (n = 7)	10/13	nc	nc	10/13
Weaned (n = 33)	8/20	nc	6/28	14/48
Finishing pigs (n = 16)	nc	15/25	0/16	15/41
Breeders (n = 76)	nc	39/64	11/76	50/140
Asymptomatic
Suckling piglets (n = 3)	0/4	nc	1/2	1/6
Breeders (n = 15)	nc	nc	6/15	6/15
Total (n = 150)	18/37	54/89	24/137	96/263

nc, not collected

Sequencing analysis confirmed the specificity of the SVV amplicons obtained from ulcerative lesions. The four nt sequences from the partial VP1 of SVV were named BRA/UEL-SVV-PR1775/20, BRA/UEL-SVV-PR1778/20, BRA/UEL-SVV-SC1950/20, and BRA/UEL-SVV-SC2103/20 (GenBank accession numbers MZ032150-MZ032153). The SVV strains identified in this study were compared with 36 other SVV nt sequences available in GenBank. Comparisons with other Brazilian SVV strains showed nt similarities of 96.1 to 98.6% with SVV strains identified in 2015, 2016, and 2018 [6, 18, 25]. When compared with sequences of other countries, the nt similarity varied from 90.6 to 95.9%, including historical (90.6 to 91.8%) and contemporary (93 to 95.9%) SVV strains (Table 2). As expected, the phylogenetic analysis showed clusters according to geographical origin, with Brazilian strains grouped in a separate branch from contemporary strains from other countries. However, temporal groupings were also observed. Brazilian strains originating in 2018 and 2020 formed a new cluster of SVV strains detected in the country during 2015 and 2016 (Fig. 1).

Table 2.

Similarity to the partial nucleotide sequences VP1 (546 nt) of Seneca Valley virus identified in this study and compared to other sequences identified in Brazil, China, USA, Colombia, Thailand, Vietnam, and Canada

Year	Country	SVV strain	BRA/UEL-SVV-PR1775/20	BRA/UEL-SVV-PR1778/20	BRA/UEL-SVV-SC1950/20	BRA/UEL-SVV-SC2103/20
2018	Brazil	MK487483	98.6	98.6	97.6	97.4
2016	Brazil	MF615502	97.2	97.2	96.3	96.1
2016	Brazil	MF615503	97.8	97.8	96.8	96.7
2016	Brazil	MF615504	97.8	97.8	96.8	96.7
2015	Brazil	MF615501	97.6	97.6	96.7	96.5
2015	Brazil	MF615506	98.2	98.2	97.2	97.0
2015	Brazil	MF615507	97.8	97.8	96.8	96.7
2015	Brazil	MF615508	97.8	97.8	96.8	96.7
2015	Brazil	MF615509	97.8	97.8	96.8	96.7
2015	Brazil	MF615510	97.6	97.6	96.7	96.5
2015	Brazil	KR063107	97.8	97.8	97.2	97.0
2015	Brazil	KR063108	97.8	97.8	97.2	97.0
2015	Brazil	KR063109	97.8	97.8	96.8	96.7
2015	Brazil	KR075677	97.8	97.8	97.2	97
2015	Brazil	KR075678	98.2	98.2	97.2	97
2015	Brazil	KT445973	97.8	97.8	97.2	97.0
2015	Brazil	KT445974	97.8	97.8	97.2	97.0
2015	Brazil	KT445975	97.6	97.6	96.7	96.5
2015	Brazil	KT445976	97.6	97.6	96.7	96.5
2015	Brazil	KT445977	98.2	98.2	97.2	97.0
2018	China	MK357115	94.9	94.9	93.9	93.7
2016	China	KX377924	95.9	95.9	94.9	94.7
2016	China	MF460448	94.1	94.1	93.2	93
2015	China	KT321458	95.9	95.9	94.9	94.7
2017	USA	MH634509	94.3	94.3	93.6	93.7
2017	USA	MH634522	95.5	95.5	94.5	94.3
2016	USA	MK333636	94.9	94.9	93.9	94.1
2015	USA	MH634527	94.9	94.9	93.9	93.7
2015	USA	MK333629	95.3	95.3	94.3	94.1
2002	USA	DQ641257	91.8	91.8	90.8	90.6
2002	USA	NC011349	91.8	91.8	90.8	90.6
2016	Colombia	KX857728	94.7	94.7	93.7	93.6
2016	Thailand	MF416218	95.1	95.1	94.1	93.9
2016	Thailand	MF416219	95.1	95.1	94.1	93.9
2018	Vietnam	MH704432	95.9	95.9	94.5	94.3
2015	Canada	KY486156	96.5	96.5	95.5	95.3
2015	Canada	KY486166	96.5	96.5	95.5	95.3

Fig. 1.

Molecular phylogenetic analysis of partial (546 nt) VP1 gene Seneca Valley virus strains by Maximum Likelihood method. The evolutionary history was inferred using the Maximum Likelihood method based on the Kimura 2-parameter model [23]. 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 approach, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 37 nucleotide sequences, including old (NC_011349, DQ641257) and contemporary Seneca Valley virus strains from Brazil (KR063107-KR063109, KR075677, KR075678, KT445973-KT445977, MK487483, MF615501-MF615504, MF615506- MF615510), China (MK357115, KX377924, KT321458, MF460448), the USA (MK333629, MK333636, MH634509, MH634522, MH634527), Colombia (KX857728), Thailand (MF416218 and MF416219), Vietnam (MH704432), and Canada (KY486156 and KY486166). A foot-and-mouth disease virus sequence was used as an out-group (AY593829). Evolutionary analyses were conducted in MEGA7 [24]

Discussion

Brazil is the fourth-largest producer and exporter of pork. The South, Southeast and Center-West regions concentrate 99.91% of Brazilian pork-production. Among the regions, the three states in the southern region are responsible for 70.91% of the pork produced in the country, followed by the central-west region (16.06%), and southeast (12.94%) [26]. From 2014 to 2015, SVV was first described relating to outbreaks of vesicular diseases in Brazilian pig herds, affecting animals of different categories [6, 7]. Since then, SVV has been circulating in Brazilian pig farms and associated with vesicular outbreaks. Another major outbreak of SVV in Brazil occurred in the second half of 2018, affecting animals from the states of the southern, southeast, and midwest regions, mainly finishing pigs [18]. In this study, pigs of different ages were affected in herds from states located in southern Brazil, which is considered the main pork-producing region.

In Brazil, waves of SVV outbreaks are occurring at increasingly shorter intervals, 3 years between 2015 [6, 7] to 2018 [27], and 1.5 years between the outbreaks in late 2018 and the described by the present study in 2020. Three possible explanations for these recurring waves of SVV outbreaks can be identified: the emergence of new variants of the virus, duration of immunity against the virus, and/or breeding stock replacement rate. First, the partial VP1 analysis showed 1.4 to 3.9% nt difference among SVV strains identified in the present study and Brazilian strains detected in the 2015 to 2018 SVV outbreaks. Recently, a study observed that the historical and contemporary SVV strains (detected between 1988 and 2017) are in molecular evolution [25], and thus may have an influence in increasing the pathogenicity of contemporary strains [28]. Based on this information, the mutations found in the VP1 region suggest the emergence of SVV variants that possibly evolved to escape the immune response or with differentiated pathogenic potential. However, studies on the interference of these genomic changes and the relationship between the occurrence of outbreaks are necessary.

Regarding the immune response, there are no longitudinal studies that indicate the duration of humoral immunity induced by SVV infection. However, naturally infected animals produce high titers of neutralizing antibodies for a previously undetermined period [29]. The drop in antibody titers probably favors the occurrence of new infections. Finally, as the recommended annual breeding stock replacement rate in pig herds is approximately 40%, after 2 to 2.5 years, all the sows in the herd are replaced. Pig genetic multiplication farms have high levels of biosecurity, preventing the entry and circulation of many pathogens, as well as SVV; therefore, replacement animals susceptible to the virus may have been introduced into commercial farms. All these factors can justify the new waves of outbreaks and, due to the decrease in the time between intervals the virus may have become endemic in pig herds in the evaluated region.

Natural and experimental SVV infections in weaned pigs have been previously reported [30]. In our study, 40% (8/20) of the tissue from weaned pigs (4–9 weeks of age) were SVV-positive. It is possible that the decrease in maternal antibodies and stress generated by weaning contribute to the increased susceptibility of that animal category.

In this study, 33.3% (7/21) of serum samples from asymptomatic pigs were positive for SVV RNA, indicating that one-third of the animals were viremic. In an experimental SVV infection in pigs, RNA was detectable in the serum 3 to 10 days post-infection (dpi), with a viremia peak at the third dpi and viral shedding via oronasal and fecal occurred one day before the appearance of clinical signs [28, 31]. Collectively, these data show that asymptomatic pigs, albeit in viremia, are sources of infection and can act as carriers and possibly spreaders of the virus within the herd. In contrast, in a small number of serum samples from symptomatic animals, viremia was detected, reinforcing the idea that after viral clearance from the bloodstream, clinical signs such as vesicles and ulcerative lesions are manifested.

As vesicular diseases associated with SVV and foot-and-mouth disease virus are visually indistinguishable, notification and laboratory diagnosis are mandatory. Economic and production losses affect pig farmers and the industry. Direct losses are related to decreased production and increased animal mortality rate, while indirect losses are related to the implementation of biosafety measures to prevent the virus from entering farms, labor costs, and laboratory diagnostics [32].

Vaccination is a promising tool for the prevention of SVV infection in pigs. To date, there are no commercial vaccines against SVV available; however, studies have shown positive results with the development of inactivated vaccines [33] and virus-like particle vaccines [34]. However, further viral challenge studies are needed to demonstrate a robust immune response to ensure an effective vaccine.

Conclusions

In this study, we report the third wave of outbreaks caused by SVV in different categories of pigs from herds located in southern Brazil. Owing to its recent association with disease in pigs and the lack of little knowledge about the dynamics of infection, additional longitudinal studies directed mainly at understanding the immunity induced by SVV and its duration are of significant importance.

Author contribution

Conceptualization and design: AMD and AAA; Methodology, MVV, CYY and AMD; Writing—original draft preparation, MVV and AMD; Writing—review & editing: RAL, AFA and AAA. All authors have read and agreed to the published version of the manuscript.

Funding

The authors thank the following Brazilian Institutes for their financial support: the National Council of Technological and Scientific Development (CNPq); the Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES); the Financing of Studies and Projects (FINEP); and the Araucaria Foundation (FAP/PR). AAA and AFA are recipients of CNPq Fellowships. AMD is a recipient of the INCT-Leite/CAPES fellowship (grant number 88887.495081/2020–00). CYY is a recipient of the Araucaria Foundation (FAP/PR) fellowship.

Data availability

Sequences determined in this study were submitted to GenBank database under the accession numbers MZ032150-MZ032153.

Declarations

Ethics approval and consent to participate

This study was approved by the Ethics Committee on the Use of Animals in Teaching and Research of the Universidade Estadual de Londrina (UEL), Londrina, Brazil, under number 11363.2015.16. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Conflict of interest

The authors declare no competing interests.