As enfermidades virais sÃo as que causam maiores prejuÃzos registrados na carcinicultura. O surgimento do IMNV no Nordeste do Brasil em 2002 colocou os produtores de camarÃo frente a uma enfermidade desconhecida que causou perdas significativas para o setor, tornando imperativo buscar um maior conhecimento sobre a doenÃa e de como podem se comportar outras espÃcies de camarÃo ao vÃrus do IMN. O objetivo deste trabalho foi avaliar a susceptibilidade do camarÃo rosa Farfantepenaeus subtilis, nativo da costa brasileira, atravÃs de teste de desafio frente ao IMNV. Para realizaÃÃo do bioensaio foram utilizados 200 camarÃes jovens saudÃveis mantidos em tanques individuais de 4L durante 30 dias. No experimento os camarÃes foram separados em dois grupos: controle e desafiado; o grupo desafiado foi alimentado com mÃsculo de L. vannamei contaminado com IMNV e o controle alimentado com mÃsculo de L. vannamei livre do vÃrus em estudo, durante 3 dias via âper osâ. ApÃs o desafio foram realizadas coletas aleatÃrias de 40 animais, 20 camarÃes controle e 20 camarÃes desafiados, a cada cinco dias (5Â; 10Â; 15Â; 20 e 30Â) onde durante cada coleta, foram extraÃdas hemolinfa para contagem total de hemÃcitos (CTH), brÃnquias para anÃlise de RT/PCR e em seguida cada camarÃo foi fixado com soluÃÃo de Davidson para anÃlise histolÃgica. As anÃlises moleculares, utilizando a metodologia do Kit IQ2000, mostraram que dos 100 animais desafiados apenas dez (10 %) se mostraram positivos à infecÃÃo. Nas anÃlises histolÃgicas foi observado uma baixa infiltraÃÃo hemocÃtica nos mÃsculos; uma leve coagulaÃÃo e presenÃa de hemolinfa entre as fibras musculares caracterizando uma infecÃÃo leve. De acordo com o teste t de Student para dados independentes, houve diferenÃa estatisticamente significativa (p≥ 0,05) entre a contagem total de hemÃcitos do grupo desafiado e grupo controle na amostragem do 30 dia. O estudo demonstrou que a espÃcie nativa Farfantepenaeus subtilis à susceptÃvel ao vÃrus IMN. / Viral diseases are causing great damage in shrimp farming. With the IMN virus emergence in Northeastern Brazil in 2002, the shrimp producers faced an unknown disease that caused significant losses for the industry. Thus, a better knowledge about this disease and the investigation of how other species of shrimp are affected by the IMN virus will be an important contribution for the aquaculture industry. The objective of this study is to evaluate the susceptibility of Farfantepenaeus subtilis from the Brazilian coast, to the IMN virus. To conduct the bioassay it was used 200 healthy young shrimps kept in individual tanks of 4L for 30 days. The experimental design was composed by two separated groups: a control group and a challenged one which was exposed to the IMN virus. The challenged group was fed on L. vannamei muscle contaminated with IMNV and the control group was fed on muscle of L. vannamei free of the virus, for 3 days per os. After the challenge procedures, random samples of 40 animals, 20 control and 20 challenged were taken, every five days (5Â, 10Â, 15Â, 20 and 30Â). On each sampling hemolymph was drawn for the total count of hemocytes (THC), gills were taken for analysis by RT / PCR and then each animal was fixed with Davidson solution for histological analysis. The molecular analysis, using the methodology of Kit IQ2000, showed that among 100 animals challenged only ten (10%) were positive for infection. In histological analysis a low hemocyte infiltration was observed in muscles and a slight presence of hemolymph coagulation was detected between muscle fibers characterizing a mild infection. According to the Student t test for independent data, there was a statistically significant difference (p ≥ 0.05) between the THC of challenged group and the control group sample of 30 days. The study showed wild specimens of F. subtilis to be susceptible to infection with IMNV.

Since 2002, a new pathogen called Infectious myonecrosis virus (IMNV) is causing significant economic losses in shrimp farms in the Northeast of Brazil. Unlike vertebrates, the shrimp do not have an immune system with adaptive memory, however have an innate immune system, giving the defense less complex answers. This study aimed to determine the effectiveness of a diet with high doses of L-ascorbic acid-2-monophosphate (VITC) in combination with a Ã-1, 3 / 1 ,6-glucan (BetG) on survival, growth and immune responses of shrimp Litopenaeus vannamei challenged with IMNV. The study was conducted on 30 tanks of 500 l, operated with clear water and subjected to aeration and renewal of water constant. Cameroon with 2.58 Â 0.39 g were populated with 100 animais/m2 and cultured for a period of 10 weeks. The experimental design was composed of three treatments and three controls. For every five tanks were designated treatment, so called: Ref, IMNV negative and feed business, Neg and Pos, IMNV negative and positive respectively, fed an experimental diet containing normal levels of L-ascorbic acid-2-monophosphate (VITC, 250 mg / kg) and no Ã-1, 3 / 1 ,6-glucan; VITC (IMNV positive, fed an experimental diet containing 1160 mg / kg of L-ascorbic acid-2-monophosphate), VitCBetG (IMNV positive , fed an experimental diet containing 1160 mg / kg of VITC and 600 mg / kg BetG), and BetG (IMNV positive, fed an experimental diet containing 600 mg / kg of Ã-1.3 / 1.6 -glucan and normal levels of VITC). The experimental diets were manufactured in the laboratory and the infection of shrimp made by per os administration of tissue infected by IMNV (1.82 x 103 copies of IMNV / Â l RNA) for three consecutive days, twice daily. The challenge began in the per 27 days of exposure of shrimp to feed when the animals reached between 4.93 g and 6.92 g. A total count of hemocytes (CTH), the concentration of serum total protein and specific activity of the enzyme phenol (OP) were evaluated at L. vannamei two days before oral challenge (27 th day of cultivation), 17 days after challenge per os (48 th day of cultivation) and despesca (70 th day of cultivation). The cultured shrimp were actually contaminated with the IMNV. Although mortality of 100% have not been observed, the animals were highly susceptible to the virus 29 days after the first day of the challenge. In despesca the shrimp reached a weight of 9.07 Â Sabry, H.-N. 1.48 g (BetG) and 11.11 Â 1.86 g (Pos). Survival ranged from a minimum of 22.8 Â 4.9% (VITC) to a maximum of 69.5 Â 5.7% (Ref). The weight gain of shrimp was progressive weekly growth ranged from 0.56 g on the 14th day of culture to 0.77 g in the last week, with no statistically significant difference between treatments. The survival decreased over the culture, regardless of treatment used. The CTH showed an increase in all treatments after the challenge with the per IMNV (ie, 17 days), however not all were significant. On this day of sampling, a larger number of cells/mm3 was observed in treatment voluntarily infected, which reached a lower final survival of shrimp (Pos and VITC). Also, the treatment is not infected, Ref, also exhibited a significant increase in hematopoietic stem cells from post-viral infection. In general, the concentration of serum protein of shrimp and activity on the enzyme phenol did not change during the cultivation or between treatments evaluated. In this study we can conclude that the inclusion of 600 mg / kg of Ã-1, 3 / 1, 6 -- glucan in a diet for shrimp L. vannamei provided a significant increase in survival of the species when exposed to IMNV. In contrast, the inclusion of 1160 mg / kg of L-ascorbic acid-2-monophosphate in diets for species infected with IMNV not resulted in increased growth or survival of the species. In this study, were not detected signs of fatigue or immunological decline in growth of the species when exposed in a continuous manner to a diet containing 600 mg / kg of Ã-1, 3 / 1 ,6-glucan. / Desde 2002 que um novo agente patogÃnico denominado de VÃrus da Mionecrose Infecciosa (IMNV) vem causando significativas perdas econÃmicas em fazendas de camarÃo na RegiÃo Nordeste do Brasil. Ao contrÃrio dos vertebrados, os camarÃes nÃo possuem um sistema imunolÃgico com memÃria adaptativa, no entanto possuem um sistema imune inato, apresentando respostas de defesa menos complexas. O presente estudo teve como objetivo determinar a eficÃcia de uma dieta com dosagens elevadas de Ãcido L-ascÃrbico-2-monofosfato (VitC) em combinaÃÃo com um Ã-1,3/1,6-glucano (BetG) sobre a sobrevivÃncia, o crescimento e as respostas imunolÃgicas do camarÃo Litopenaeus vannamei desafiado com o IMNV. O estudo foi realizado em 30 tanques de 500 l, operados com Ãgua clara e submetidos a aeraÃÃo e renovaÃÃo de Ãgua constante. CamarÃes com 2,58 Â 0,39 g foram povoados com 100 animais/m2 e cultivados por um perÃodo de 10 semanas. O desenho experimental foi composto por trÃs tratamentos e trÃs controles. Para cada tratamento foram designados cinco tanques, assim denominados: Ref, IMNV negativo e raÃÃo comercial; Neg e Pos, IMNV negativo e positivo respectivamente, alimentados com uma raÃÃo experimental contendo nÃveis normais de Ãcido L-ascÃrbico-2-monofosfato (VitC, 250 mg/kg) e sem Ã-1,3/1,6-glucano; VitC, (IMNV positivos, alimentados com uma raÃÃo experimental contendo 1.160 mg/kg de Ãcido L-ascÃrbico-2-monofosfato), VitCBetG, (IMNV positivos, alimentados com uma raÃÃo experimental contendo 1.160 mg/kg de VitC e 600 mg/kg de BetG); e, BetG, (IMNV positivos, alimentados com uma raÃÃo experimental contendo 600 mg/kg de Ã- 1,3/1,6-glucano e nÃveis normais de VitC). As raÃÃes experimentais foram fabricadas em laboratÃrio e a infecÃÃo dos camarÃes se deu atravÃs da administraÃÃo per os de tecido infectado por IMNV (1,82 x 103 copias de IMNV/μl RNA) durante trÃs dias consecutivos, duas vezes ao dia. O desafio per os teve inÃcio no 27Â dia de exposiÃÃo dos camarÃes Ãs raÃÃes, quando os animais alcanÃaram entre 4,93 g e 6,92 g. A contagem total de hemÃcitos (CTH), a concentraÃÃo de proteÃnas totais do soro e a atividade especÃfica da enzima fenoloxidase (PO) foram avaliadas no L. vannamei dois dias antes do desafio oral (27Â dia de cultivo), 17 dias apÃs o desafio per os (48Â dia de cultivo) e na despesca (70Â dia de cultivo). Os camarÃes cultivados foram efetivamente contaminados com o IMNV. Embora mortalidades de 100% nÃo tenham sido observadas, os animais mostraram-se altamente susceptÃveis ao vÃrus 29 dias apÃs o primeiro dia do desafio. Na despesca, os camarÃes alcanÃaram um peso entre 9,07 Â Sabry, H.-N. 1,48 g (BetG) e 11,11 Â 1,86 g (Pos). A sobrevivÃncia variou de um mÃnimo de 22,8 Â 4,9% (VitC) a um mÃximo de 69,5 Â 5,7% (Ref). O ganho de peso dos camarÃes foi progressivo e o crescimento semanal variou de 0,56 g no 14Â dia de cultivo para 0,77 g na Ãltima semana, nÃo havendo diferenÃa estatÃstica significativa entre tratamentos. A sobrevivÃncia decresceu ao longo do cultivo, independente do tratamento adotado. A CTH apresentou um aumento em todos os tratamentos apÃs o desafio per os com IMNV (i.e., 17 dias apÃs), entretanto nem todos foram significativos. Neste dia de amostragem, um maior nÃmero de cÃlulas/mm3 foi observado nos tratamentos voluntariamente infectados, os quais alcanÃaram uma menor sobrevivÃncia final de camarÃes (Pos e VitC). Igualmente, o tratamento nÃo infectado, Ref, tambÃm exibiu um aumento significativo na CTH no perÃodo de pÃs-infecÃÃo viral. De um modo geral, a concentraÃÃo protÃica do soro dos camarÃes e a atividade relativa da enzima fenoloxidase nÃo se alteraram ao longo do cultivo ou entre os tratamentos avaliados. AtravÃs do presente estudo pode-se concluir que a inclusÃo de 600 mg/kg de Ã-1,3/1,6- glucano em uma dieta para o camarÃo L. vannamei proporcionou um aumento significativo na sobrevivÃncia da espÃcie quando exposto ao IMNV. Ao contrÃrio, a inclusÃo de 1.160 mg/kg de Ãcido L-ascÃrbico-2-monofosfato em dietas para espÃcie infectada com IMNV nÃo resultou em um maior crescimento ou sobrevivÃncia da espÃcie. No presente estudo, nÃo foram detectados indÃcios de fadiga imunolÃgica ou diminuiÃÃo no crescimento da espÃcie quando exposta de forma contÃnua a uma dieta contendo 600 mg/kg de Ã-1,3/1,6-glucano.

Infectious myonecrosis, caused by infectious myonecrosis virus (IMNV), is an important disease of shrimp that has adversely affected the production of cultured Litopenaeus vannamei. The studies reported here were centered on development and/or validation of alternative diagnostic methods for detection of IMNV. Hence, two manuscripts were published in the Journal of Aquaculture and one manuscript was published in the Journal of Fish Diseases. Chapter 2 describes the development of a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) method using TaqMan probe. The results showed that this real-time RT-PCR assay can detect as little as 10 IMNV copies/μl RNA, while the nested RT-PCR can detect 1000 copies/μl RNA. These findings suggest that the TaqMan real-time RT-PCR is “the gold standard” for screening shrimp to protect aquaculture production systems from losses caused by IMNV, because it provides quantification, higher sensitivity and specificity, and because it is less time consuming and less prone to contamination compared to conventional gel-based RT-PCR. In Chapter 3 I evaluated if prolonged storage of infectious myonecrosis virus (IMNV) infected shrimp in Davidson’s AFA fixative can degrade its double-stranded RNA genome resulting in false negative ISH reactions. Shrimp were collected at Day 12 post-injection and fixed in Davidson’s AFA for five different preservation times (1, 2, 4, 7 and 10 days). Hence, in the present report it was found that the length of time (up to 10 days) in Davidson’s AFA did not have a deleterious effect on the ISH reaction for IMNV. The Chapter 4 describes the development of a reverse transcription loop-mediated isothermal amplification and nucleic acid lateral flow (RT-LAMP-NALF) for detection of IMNV. The RT-LAMP-NALF method combines simplified nucleic acid extraction, a reverse-transcription loop-mediated isothermal amplification platform, and one-step visual colorimetric confirmation of the IMNV amplified sequences using a generic NALF qualitative detection test strip. The RT-LAMP-NALF was found to be 100 and 10 times more sensitive than one-step RT-PCR and RT-LAMP (two primer pairs), respectively. These results clearly demonstrate that the RT-LAMP-NALF method is specific, sensitive, can shorten the time for analysis, and has potential application for IMNV diagnosis in resource-poor diagnostic settings.

Made available in DSpace on 2014-12-17T14:03:44Z (GMT). No. of bitstreams: 1 MarciaDAD_DISSERT.pdf: 4264187 bytes, checksum: 68a1d188a3ddcbd9b3e88211ae1a47e7 (MD5) Previous issue date: 2014-08-01 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Shrimp farming is one of the activities that contribute most to the growth of global aquaculture. However, this business has undergone significant economic losses due to the onset of viral diseases such as Infectious Myonecrosis (IMN). The IMN is already widespread throughout Northeastern Brazil and affects other countries such as Indonesia, Thailand and China. The main symptom of disease is myonecrosis, which consists of necrosis of striated muscles of the abdomen and cephalothorax of shrimp. The IMN is caused by infectious myonecrosis virus (IMNV), a non-enveloped virus which has protrusions along its capsid. The viral genome consists of a single molecule of double-stranded RNA and has two Open Reading Frames (ORFs). The ORF1 encodes the major capsid protein (MCP) and a potential RNA binding protein (RBP). ORF2 encodes a probable RNA-dependent RNA polymerase (RdRp) and classifies IMNV in Totiviridae family. Thus, the objective of this research was study the IMNV complete genome and encoded proteins in order to develop a system differentiate virus isolates based on polymorphisms presence. The phylogenetic relationship among some totivirus was investigated and showed a new group to IMNV within Totiviridae family. Two new genomes were sequenced, analyzed and compared to two other genomes already deposited in GenBank. The new genomes were more similar to each other than those already described. Conserved and variable regions of the genome were identified through similarity graphs and alignments using the four IMNV sequences. This analyze allowed mapping of polymorphic sites and revealed that the most variable region of the genome is in the first half of ORF1, which coincides with the regions that possibly encode the viral protrusion, while the most stable regions of the genome were found in conserved domains of proteins that interact with RNA. Moreover, secondary structures were predicted for all proteins using various softwares and protein structural models were calculated using threading and ab initio modeling approaches. From these analyses was possible to observe that the IMNV proteins have motifs and shapes similar to proteins of other totiviruses and new possible protein functions have been proposed. The genome and proteins study was essential for development of a PCR-based detection system able to discriminate the four IMNV isolates based on the presence of polymorphic sites / A carcinicultura ? uma das atividades que mais contribui para o crescimento da aquicultura mundial. Entretanto, esta atividade vem sofrendo perdas econ?micas significativas devido ao surgimento de doen?as virais como a Mionecrose Infecciosa (IMN). A IMN j? est? disseminada em toda regi?o Nordeste do Brasil e atingiu outros pa?ses como Indon?sia, Tail?ndia e China. O principal sintoma da doen?a ? a mionecrose, que consiste na necrose dos m?sculos estriados do abd?men e do cefalot?rax do camar?o. A IMN ? causada pelo v?rus da mionecrose infecciosa (IMNV), um v?rus n?o envelopado que apresenta protrus?es ao longo de seu caps?deo. O genoma viral ? formado por uma ?nica mol?cula de RNA dupla fita e possui duas Open Reading Frames (ORFs). A ORF1 codifica a prote?na principal do caps?deo (MCP) e uma poss?vel prote?na de liga??o a RNA (RBP). A ORF2 codifica uma prov?vel RNA polimerase dependente de RNA (RdRp) e classifica o IMNV dentro da fam?lia Totiviridae. Assim, o objetivo desse estudo foi estudar o genoma completo do IMNV e as prote?nas codificadas no intuito de desenvolver um sistema que identificasse diferentes isolados do v?rus com base na presen?a de polimorfismos. A rela??o filogen?tica entre alguns totiv?rus foi investigada e mostrou um novo grupo para o IMNV dentro da fam?lia Totiviridae. Dois novos genomas foram sequenciados, analisados e comparados a outros dois genomas j? depositados no GenBank. Os novos genomas foram mais semelhantes entre si do que com aqueles j? descritos. Regi?es vari?veis e conservadas do genoma foram identificadas atrav?s de gr?ficos de similaridade e alinhamentos utilizando as quatro sequ?ncias do IMNV. Esta an?lise possibilitou o mapeamento de s?tios polim?rficos e revelou que a regi?o mais vari?vel do genoma se encontra na primeira metade da ORF1 e coincide com as regi?es que possivelmente codificam a protrus?o viral, enquanto que as regi?es mais est?veis se encontraram em dom?nios conservados de prote?nas que interagem com o RNA. Al?m disso, estruturas secund?rias foram preditas para todas as prote?nas empregando diversos softwares e modelos estruturais proteicos foram calculados usando modelagens por threading e simula??es ab initio. A partir dessas an?lises foi poss?vel observar que as prote?nas do IMNV possuem motivos e formas similares ?s prote?nas de outros totiv?rus, e novas poss?veis fun??es proteicas foram propostas. O estudo do genoma e das prote?nas foi essencial para o desenvolvimento de um sistema de detec??o baseado em PCR capaz de discriminar os quatro isolados do IMNV com base na presen?a de s?tios polim?rficos