The aim of our investigation was to detect WSSV in Litopenaues vannamei from farms in Rio Grande do Norte (RN) state, as well as perform genotyping of the isolates. A total of five farms were surveyed, from which 10 samples were collected in each one. We selected two ponds in each of the farms; these were chosen because of clinical suspicion of WSSV. Genomic DNA was extracted to detect the virus by polymerase chain reaction (PCR). Subsequently, we performed, through PCR, genotyping of the positive DNA samples analyzing a variable number of tandem repeats - VNTR in three molecular markers (ORFs 75, 94 and 125). The results revealed the presence of the virus in only one of the 5 analyzed farms. The loci ORF 75 and ORF 94 were identified in isolates from the positive samples and no amplification failure for ORF 125. The number of repeating units (RUs) was calculated, and found to 9 repetitions ORF 75 and 10 RUs to ORF 94, a pattern that had not been described, stressing the need for more strain typing for screening and surveillance of the disease

Araújo, Bruno Vinicios Silva de

Câmara, Rebeca Jéssica Falcão

Peixoto, Emanuelly Cristina Rodrigues

Rebouças, Germana Guimarães

Sakamoto, Sidnei Miyoshi

English

Brazilian Journals

Brazilian Journal of Development     Braz. J. of  Develop., Curitiba, v. 6, n. 7, p.45379-45385 jul. 2020.                            ISSN 2525-8761  45379  Identification and genotyping of white spot syndrome virus (WSSV) isolated from Litopenaeus vannamei in the state Rio Grande do Norte  Identificação e genotipagem do vírus da síndrome da mancha branca (WSSV) isolado de Litopenaeus vannamei no estado do Rio Grande Do Norte  DOI:10.34117/bjdv6n7-236 Recebimento dos originais: 03/06/2020 Aceitação para publicação: 10/07/2020  Emanuelly Cristina Rodrigues Peixoto Bacharela em Aquicultura, Mestre pelo Programa de Pós-Graduação em Ciência Animal da Universidade Federal Rural do Semi-Árido Instituição: Universidade Federal Rural do Semi-Árido (UFERSA) Endereço: Rua Francisco Mota - 572, Bairro Pres. Costa e Silva, Mossoró - RN, 59625-900 E-mail: emanuelly.peixoto@gmail.com  Rebeca Jéssica Falcão Câmara Médica Veterinária, Doutoranda no Programa de Pós-Graduação em Ciência Animal pela Universidade Federal de Minas Gerais Instituição: Universidade Federal de Minas Gerais (UFMG) Endereço: Av. Antonio Carlos – 6627, Bairro Pampulha, Belo Horizonte - MG, 31270901 E-mail: rebeca.jfc@gmail.com  Germana Guimarães Rebouças Biotecnologista, Doutoranda no Programa de Pós-Graduação em Ciência Animal pela Universidade Federal Rural do Semi-Árido Instituição: Universidade Federal Rural do Semi-Árido (UFERSA) Endereço: Rua Francisco Mota - 572, Bairro Pres. Costa e Silva, Mossoró - RN, 59625-900 E-mail: ggreboucas@gmail.com  Bruno Vinicios Silva de Araújo Graduando em Medicina Veterinária pela Universidade Federal Rural do Semi-Árido Instituição: Universidade Federal Rural do Semi-Árido (UFERSA) Endereço: Rua Francisco Mota - 572, Bairro Pres. Costa e Silva, Mossoró - RN, 59625-900 E-mail: brunovinicios.araujo@hotmail.com  Sidnei Miyoshi Sakamoto Médico Veterinário, Prof. Dr. do Departamento de Ciências da Saúde (DCS) da Universidade Federal Rural do Semi-Árido Instituição: Universidade Federal Rural do Semi-Árido (UFERSA) Endereço: Rua Francisco Mota - 572, Bairro Pres. Costa e Silva, Mossoró - RN, 59625-900 E-mail: sakamoto@ufersa.edu.br  ABSTRACT The aim of our investigation was to detect WSSV in Litopenaues vannamei from farms in Rio Grande do Norte (RN) state, as well as perform genotyping of the isolates. A total of five farms were surveyed, from which 10 samples were collected in each one. We selected two ponds in each of the Brazilian Journal of Development     Braz. J. of  Develop., Curitiba, v. 6, n. 7, p.45379-45385 jul. 2020.                            ISSN 2525-8761  45380  farms; these were chosen because of clinical suspicion of WSSV. Genomic DNA was extracted to detect the virus by polymerase chain reaction (PCR). Subsequently, we performed, through PCR, genotyping of the positive DNA samples analyzing a variable number of tandem repeats - VNTR in three molecular markers (ORFs 75, 94 and 125). The results revealed the presence of the virus in only one of the 5 analyzed farms. The loci ORF 75 and ORF 94 were identified in isolates from the positive samples and no amplification failure for ORF 125. The number of repeating units (RUs) was calculated, and found to 9 repetitions ORF 75 and 10 RUs to ORF 94, a pattern that had not been described, stressing the need for more strain typing for screening and surveillance of the disease.  Keywords: ORF 75, ORF 94, ORF 125, VNTR, WSSV.  RESUMO O objetivo de nossa investigação foi detectar o WSSV em Litopenaues vannamei de fazendas no estado do Rio Grande do Norte (RN), bem como realizar a genotipagem dos isolados. Um total de cinco fazendas foram submetidas ao estudo, das quais foram coletadas 10 amostras de cada. Foram selecionados dois viveiros de cada uma das fazendas, escolhidos mediante suspeita clínica de WSSV. O DNA genômico foi extraído para investigação da presença ou ausência do vírus através da reação em cadeia da polimerase (PCR). Posteriormente, foi realizada por PCR a genotipagem dos isolados utilizando as repetições em tandem de número variável – VNTR (ORFs 75, 94 e 125). Os resultados revelaram a presença do vírus em apenas uma das 5 fazendas analisadas. As regiões (ORF 75 e ORF 94) foram identificadas nos isolados da fazenda positiva e não houve amplificação para o ORF 125. O número de unidades repetidas (RUs) foi calculado, sendo verificado 9 repetições para o ORF 75 e 10 para o ORF 94, um padrão que ainda não havia sido descrito, alertando para a necessidade de mais isolados caracterizados geneticamente para o rastreamento e vigilância epidemiológica da doença.  Palavras-chave: ORF75, ORF94, ORF125, VNTR, WSSV.    1 INTRODUCTION The white spot syndrome is, characteristically, an acute contagious infection, responsible for great economic losses in the global shrimp farming industry (LIGHTNER, 1996). The causative agent is the White Spot Syndrome Virus (White Spot Syndrome Virus - WSSV), which belongs to the genus Whispovirus, and is the sole representative of the family Ninaviridae (ICTV, 2015). Brazil is one of the 25 largest producers of shrimp in the world (FAO, 2016), and we have to highlitght Rio Grande do Norte (RN), who is responsible for approximately 33% of the national production (IBGE, 2014). The WSSV combined with other factors is responsible for a reduction in Rio Grande do Norte’s shrimp production by 45%, from 37,400 tons in 2003 to 16,900 tons in 2013 (ARAÚJO, 2015). Given the importance of controlling this disease for the shrimp industry, we aimed to investigate the presence of WSSV in Litopenaues vannamei shrimp coming from farms in RN, as well as perform the genotyping of isolates using variable number tandem repeats - VNTR (ORFs 75, 94 and 125) as markers. Intending to collaborate with further epidemiological studies.  Brazilian Journal of Development     Braz. J. of  Develop., Curitiba, v. 6, n. 7, p.45379-45385 jul. 2020.                            ISSN 2525-8761  45381   2 METHODS In total, we collected 50 samples of shrimp Litopenaeus vannamei from five farms in Rio Grande do Norte (Table 1). The farms were chosen because their shrimp presented clinical symptoms for WSSV in the ponds. Shrimp sampled were desensitized to collect gills and pleopods with the aid of properly sterilized tweezers; next, the material was placed in 2mL micro tubes with alcohol and kept under refrigeration. Samples were then transported to the Molecular Biology Laboratory of the Federal Rural University of Semi-Arid - UFERSA, where we preceded with the extraction using a kit from Quiagen - QIAmp DNA Mini Kit - according to the manufacturer’s instructions.  Table 1 – Description of the Litopenaeus vannamei shrimp samples collected in the state of Rio Grande do Norte Farms Cities/ Towns Collection period Amount of shirmp A São Gonçalo do Amarante September/2015 10 B São Gonçalo do Amarante September/2015 10 C Macaíba May/2016 10 D Extremoz May/2016 10 E Arêz May/2016 10  We extracted DNA and performed Polymerase Chain Reaction (PCR) to detect the virus using a pair of primers and following reaction conditions described by Dieu et al. (2004). The positive samples were genotyped using the markers ORF 75, ORF 94 and ORF 125, according to Pradeep et al. (2008). From these, we calculated repeating units (RUs) to compare with other geographical isolates of WSSV.  3 RESULTS AND DISCUSSION From the farms investigated, only farm B was positive for WSSV. The presence of white spot syndrome virus (WSSV) was confirmed in 90% of the sampled animals from farm B. This property has a history of high mortalities in shrimp since the year 2013, associated to the characteristic clinic signs of this disease, such as pink color in shrimp and calcium deposits on the carapace. In the other farms, we could not detect the WSSV. The nine samples positive for WSSV underwent VNTR essay (ORFs 75, 94 and 125). We observed amplification in 7 (77.78%) of the samples for the ORF75 marker; in 5 (55.56%) samples for ORF94; and, no amplification from any of the samples for the marker ORF 125. The number of RUs calculated is shown in Table 2.    Brazilian Journal of Development     Braz. J. of  Develop., Curitiba, v. 6, n. 7, p.45379-45385 jul. 2020.                            ISSN 2525-8761  45382  Table 2 – Variation of the number of repeating units in ORF75, RF94 and ORF125 of the WSSV in different geographical regions Geographical origin Variety of RUs in VNTR loci References ORF75 RUs (45, 57 bp) ORF94 RUs (54 bp) ORF125 RUs (69 bp) China NA 6-14 NA (TAN et al., 2009) Thailand NA 6-20 NA (KIATPATHOMECHAI et al., 2005) Vietnam 5 (4.1) – 14(10.4)* 4-17 4-10 (DIEU et al., 2010) USA 6 (5.1) – 14 (11.3)* 5-8 10-11 (MULLER et al., 2010) India 6 (5.1) – 13(10.3) 2-16 2-14 (PRADEEP et al., 2008) Mexico 14 19 7 (MULLER et al., 2010) Brazil     Santa Catarina-2005, 2007 e 2008 10(7.3)* 16 8 (MULLER et al., 2010) Bahia 11(8.3)* 4 9 (MULLER et al., 2010) São Gonçalo do Amarante -RN 9** 10 failed This experiment *RUs calculated based in 45 and 102 bp. NA – non analyzed. **based on the size of the amplified fragment (≈700 bp).  The number of repeating units (RUs) was calculated, and we found 9 repetitions for ORF 75 and 10 for ORF 94, a pattern that had not been previously described. This highlights the need for more isolates to be genetically profiled, in order to better track and surveil this disease. Although unlikely, it can not be ruled out, that the failure in the PCR of ORF 125 in all the samples was caused by polymorphism in the hybridizing region of the primers for this marker. It does not seem likely that these issues were due to the presence of PCR inhibitors or to low concentrations of DNA – under the detection threshold -, seeing as there was amplification for VP26 and ORF’s 75 and 94. In Santa Catarina, the viral isolates from outbreaks in 2005, 2007 and 2008 (MULLER et al., 2010) maintained the same VNTR pattern, demonstrating the stability of the proposed markers (ORF’s 75, 94 and 125). Such stability has been experimentally demonstrated by Sindhupriya et al. (2014), who performed multiple essays on two strains of WSSV in three different species of shrimp, and no difference in the VNTR profile for ORF’s 75, 94 and 125 was detected. Thus, it is possible to perform studies that associate the phylogeny and geography of the virus and observe identical or very similar VNTR profiles from different locations, which suggests clonal connection and, therefore, require an epidemiological essay, for example, isolates from Madagascar, Mozambique and Saudi Arabia identified by Tang et al. (2013). However, Waikhom et al. (2006) found that the passing through different host species causes selective pressure for the variation of the virus. Brazilian Journal of Development     Braz. J. of  Develop., Curitiba, v. 6, n. 7, p.45379-45385 jul. 2020.                            ISSN 2525-8761  45383  The pattern found in the outbreak of São Gonçalo do Amarante indicates a geographical signature of the virus. Nevertheless, it is necessary to seek more isolates in fieldwork and add new markers such as ORF 23/24 (DIEU et al., 2010) and a greater power of resolution. For example, through DNA sequencing (SIMROUNI et al., 2014), to be able to place the origin of outbreaks in time and space by observing the dynamics of virus evolution (DIEU et al, 2010). The lack of this kind of information leaves doubt if the origin of this outbreak is the introduction of a new genotype or the reintroduction of the same virus type believed to be under control. The first case would imply several routes of infection, including the possibility of mixed infections with more than one genotype (WALKER et al., 2011). On the other hand, the recurrence of a certain viral genotype in the farm suggests the existence of reservoirs in the environment – other host species (COSTA et al., 2012) – or latent forms of infection (SANCHEZ-PAZ, 2010). The role of the environment in the transmission of WSSV through indirect route proved to be more efficient than through direct contact between L. vannamei shrimp (TUYEN et al., 2014). In addition, temperatures between 22 and 30°C increased the replication efficiency of WSSV (SUN et al., 2014) in the early stages of acute infection, which coincides with the optimum temperature range for juvenile shrimp when uninfected (RAHMAN et al., 2006). Molecular interactions between the virus and the activity of constituent genes, important for the development of Penaeus monodon, such as hsp70 and ATP synthetase, were identified; thereby, it is suggested that the WSSV benefits from the phase in which there is more development of the host (BIRADAR et al., 2013). The study of these factors becomes important to understand the cyclic episodes of infection, whether by the recurrence of the same genotype or by the introduction of new viral variants.  4 CONCLUSIONS Finding the white spot syndrome virus in samples of Litopenaeus vannamei shrimp in farms of Rio Grande do Norte state is not representative of the presence of an epidemic in the state, but alerts to the possibility of transmission. We must know the prevalent viral diversity in the region to generate hypotheses about what are the shortcomings of the measures we adopt to manage this disease, either during breeding, harvesting or transportation; and what actions should be taken to prevent further contamination by WSSV in the state.      Brazilian Journal of Development     Braz. J. of  Develop., Curitiba, v. 6, n. 7, p.45379-45385 jul. 2020.                            ISSN 2525-8761  45384  REFERENCES ARAÚJO, R. Produção de camarão reduz 54%. Tribuna do norte. 2015. Available in: <http://www.tribunadonorte.com.br/noticia/produa-a-o-de-camara-o-reduz-54/318300>.  BIRADAR, V; NARWADE, S; PAINGANKAR, M E DEOBAGKAR, D. White spot Syndrome Virus infection in Penaeus monodon is facilitated by housekeeping molecules.  Journal Biosci, 38(5): 917-924, 2013. COSTA, S. W.; FRAGA, A. S.; ZAMPARETTI, A. S.; MARQUES, M. R. F. E ANDREATTA, E. R. Presença do vírus da síndrome da mancha branca em crustáceos decápodes silvestres em lagoas costeiras no Sul do Brasil. Arquivo Brasileiro de Medicina Veterinária e Zootecnia 64(1): 209-216, 2012. DIEU, B. T. M.; MARKS, H.; SIEBENGA, J. J.; GOLDBACH, R. W.; ZUIDEMA, D.; DUONG, T. P.; VLAK, J. M. Molecular epidemiology of white spot syndrome virus within Vietnam.  Journal of General Virology 85: 3607-3618, 2004. DIEU, B. T. M.; MARKS, H.; ZWART, M. P.; VLAK, J. M.  Evaluation of White spot syndrome vírus variable DNA loci as molecular markers of vírus spread at intermediate spatiotemporal scales. 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Identification and genotyping of white spot syndrome virus (WSSV) isolated from Litopenaeus vannamei in the state Rio Grande do Norte / Identificação e genotipagem do vírus da síndrome da mancha branca (WSSV) isolado de Litopenaeus vannamei no estado do Rio Grande Do Norte

https://ojs.brazilianjournals.com.br/ojs/index.php/BRJD/article/download/12978/10980

Identification and genotyping of white spot syndrome virus (WSSV) isolated from Litopenaeus vannamei in the state Rio Grande do Norte / Identificação e genotipagem do vírus da síndrome da mancha branca (WSSV) isolado de Litopenaeus vannamei no estado do Rio Grande Do Norte

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