optimized rtpcr assay for the detection of infectious myonecrosis virus (imnv)

CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES Department of Aquatic Pathology OPTIMIZED RT-PCR ASSAY FOR THE DETECTION OF INFECTIOUS MYONECROSIS VIRUS IMNV By NGUYEN KHANH

CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES

Department of Aquatic Pathology

OPTIMIZED RT-PCR ASSAY FOR THE DETECTION OF

INFECTIOUS MYONECROSIS VIRUS (IMNV)

By

NGUYEN KHANH LINH

A thesis submitted in partial fulfillment of the requirements for

the degree of Bachelor of Aquaculture

Can Tho, December 2013

CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES

OPTIMIZED RT-PCR ASSAY FOR THE DETECTION OF

INFECTIOUS MYONECROSIS VIRUS (IMNV)

By

NGUYEN KHANH LINH

A thesis submitted in partial fulfillment of the requirements for

the degree of Bachelor of Aquaculture Science

Supervisor

Dr TRAN THI TUYET HOA

Can Tho, December 2013

I would love to express my sincere appreciation to many of my friends, especially Duong Thanh Long, Hong Mong Huyen, Tran Thanh Can and Le Tan Hieu for their unconditionally kind help throughout the experimental period

Last but not least, I really want to thank my academic adviser, Dr Duong Thuy Yen, who was guiding and encouraging me over the last four years, and my family for their great lifetime support which makes everything possible for me

The author,

Nguyen Khanh Linh

APPROVE BY SUPPERVISOR

The thesis “Optimized RT-PCR assay for detection of Infectious myonecrosis virus” which edited and passed by the committee, was defended by Nguyen Khanh Linh in 27/12/2013

Student sign Supervisor sign

Nguyen Khanh Linh Tran Thi Tuyet Hoa

ABSTRACT

The purpose of this study is aim to apply RT-PCR procedure (OIE, 2009) for

detection of Infectious myonecrosis virus on Penaeus vannamei The procedure was

amplified positive band at 328 bp in the first step and 139bp in the nested-step The research was focused to optimize the nested-step with components reaction as 1X PCR buffer; 1.0mM MgCl2; 200µM dNTP mix; 0.465µM primer NF and 0.465µM primer NR; Taq polymerase 2.0U; 0.5µl template (cDNA) Thermal cycling PCR condition consisted of initial denaturation at 95ºC for 2 minutes followed by 35 cycles

of 95ºC for 30 seconds, 65ºC for 20 seconds, 72ºC for 30 seconds, 72ºC for 7 minutes Total amplification time is 2 hours 11 minutes Protocol also showed that the optimal RNA concentration using in study has given at 200ng/µl and 100ng/µl by sensitivity

test The specific test was also conducted with common virus in Penaeid shrimp as

Monodon baculovirus, White spot syndrome virus, Infectious hypodermal and hematopoietic necrosis virus, Gill-associated virus Besides, shrimp samples showed white necrotic muscles in the distal abdominal segments were also employed for the application test

Table of Contents ACKNOWLEDGEMENT I

CHAPTER I 1

INTRODUCTION 1

1.1 General introduction 1

1.2 Research objective 1

1.3 Research activities 1

CHAPTER 2 2

LITERATURE REVIEW 2

2.1 Overview global white leg shrimp industry 2

2.2 Overview of white leg shrimp farming in Vietnam 4

2.3 Common diseases in P.vannamei 4

2.3.1 Taura syndrome 4

2.3.2 White spot disease 5

2.3.3 Yellow Head Disease 6

2.3.4 Infectious hypodermal and haematopoietic necrosis disease 7

2.3.5 Infectious myonecrosis disease 8

2.3.6 Diagnostic methods of IMNV 9

2.3.7 Research of IMNV detection by RT-PCR method 10

2.4 Reverse transcriptase-polymerase chain reaction (RT-PCR) 10

2.5 Factors affecting RT-PCR protocol 13

2.5.1 RT primer 13

2.5.2 One-step and Two-step RT-PCR 13

2.5.3 Quantitating the RNA 13

2.5.4 Check the RNA Integrity 13

2.5.5 Sequence of primer 14

2.5.6 Primer annealing 14

2.5.7 Magnesium Concentration 14

2.5.8 Deoxynucleotide triphosphate (dNTPs) 14

2.5.9 Enzyme Taq pol 14

2.5.10 Thermal cycle 15

2.5.11 Primer concentration and DNA concentration 15

CHAPTER 3 16

RESEARCH METHODOLOGY 16

3.1 Research place 16

3.2 Materials 16

3.3 IMNV infected shrimp: 16

3.4 RNA extraction procedure: 16

3.5 Determining yield of RNA 17

3.6 cDNA synthesis with Randome Hexamers primer 17

3.7 Amplified components (IMNV detection followed by OIE, 2009) 17

3.8 Electrophoresis 19

3.9 Test result 19

3.10 Determination of detection limit of the RT-PCR 19

3.11 Determination of specificity of the RT-PCR 19

3.12 Amplification of IMNV from clinical samples by the RT-PCR (OIE, 2009) 20 CHAPTER 4 21

RESULTS AND DISCUSSION 21

4.1 IMNV detection using OIE procedure (OIE, 2009) 21

4.2 Optimization of the chemical components and thermal cycles for the RT-PCR (IMNV - OIE, 2009) 21

4.2.1 Optimization of the chemical components and thermal cycles of first step (IMNV - OIE, 2009) 21

4.2.2 Optimization of the chemical components and thermal cycles of Nested step for the RT-PCR (IMNV - OIE, 2009) 22

4.2.2.1 Optimization of the chemical components and thermal cycles of Nested step for the RT-PCR (IMNV - OIE, 2009) 22

4.2.1.2 Optimization of Taq polymerase concentration 23

4.2.1.3 Optimization of the magnesium concentration and annealing time 24

4.2.1.4 Optimization of the MgCl2 concentration 26

4.2.1.5 Optimization of the thermal cycles of nested step 28

4.3 Determination of detection limit of the RT-PCR 30

4.4 Determination of specificity of the RT-PCR 31

4.5 Amplification of IMNV from clinical samples by the RT-PCR (OIE, 2009) 32 CHAPTER 5 33

CONCLUSIONS AND RECOMMENDATION 33

5.1 CONCLUSIONS 33

5.2 RECOMMENDATION 33

REFERENCES 34

List of tables

Table 3.1: Mixture A component 17

Table 3.2: Mixture B component 17

Table 3.3: First step RT-PCR component 18

Table 3.4: Nested RT-PCR component 18

Table 4.1: PCR reagents of nested PCR for the detection of IMNV 22

Table 4.2: PCR reagents of nested PCR for the detection of IMNV (change in Taq polymerase concentration) 23

Table 4.3: Optimization of RT-PCR assay for the detection of IMNV (magnesium concentration and annealing time) 24

Table 4.4: Optimization of RT-PCR assay for the detection of IMNV (magnesium concentration) 26

Table 4.5: Optimization of RT-PCR assay for the detection of IMNV (magnesium concentration) 26

Table 4.6: Optimization of RT-PCR assay for the detection of IMNV (number of cycles and final extension time) 28

List of figures

Figure 2.1 World shrimp aquaculture by species from 1991 to 2013 2

Figure 2.2 Shrimp aquaculture in Asia by species: 1991 - 2013 3

Figure 2.3: The template RNA prior to initiating reverse transcription 10

Figure 2.4: Priming for reverse transcription 11

Figure 2.5: First strand synthesis 11

Figure 2.6: Removal of RNA 12

Figure 2.7: PCR reaction 12

Figure 4.1: Result of 1st step of RT-PCR assay for the detection of IMNV 21

Figure 4.2: Result of 2st step of RT-PCR assay for the detection of IMNV 22

Figure 4.3: Result of 2st step of RT-PCR assay for the detection of IMNV

(change in Taq polymerase concentration) 23

Figure 4.4: Optimization of RT-PCR assay for the detection of IMNV (magnesium concentration and annealing time) 25

Figure 4.5: Optimization of RT-PCR assay for the detection of IMNV (magnesium concentration) 27

Figure 4.5a: Magnesium concentration at 1.5mM 27

Figure 4.5b: Magnesium concentration at 1.0mM 27

Figure 4.6: Optimization of RT-PCR assay for the detection of IMNV (number of cycles and final extension time) 29

Figure 4.7: Detection limit of the RT-PCR with dilution series of extracted RNA in agarose gel 30

Figure 4.8: Result of RT-PCR specific test with GAV, TSV, IHHNV, MBV infected samples 31

Figure 4.9: PCR amplification of IMNV extracted from Penaeus vannamei 32

CHAPTER I INTRODUCTION 1.1 General introduction

Penaeus vannamei is native to the Pacific coast of Mexico and Central and

South America as far south as Peru (Wyban and Sweeny, 1991; Rosenberry, 2002) This species was commonly cultured because it reaches commercial size in a short cultured period, and has high economic value and high resistance against diseases

Vietnam has allowed culturing P.vannamei after removing official ban concerning

possible negative impacts of this species However, white leg shrimp farming only

actually increases when P.monodon production drops down due to disease and market

demand

The change from wild life to controlled culture systems can cause many problems, such as culturing at high density, and environmental degradation, which facilitate outbreaks of serious disease, especially viral diseases such as Taura syndrome virus (TSV), Infectious hypodermal and haematopoietic necrotic virus (IHHNV), and Infectious myonecrosis virus (IMNV)

Infectious myonecrosis virus (IMNV) was found fist in 2004 in Brazil and second in 2006 in Indonesia There are anecdotal reports and informations about this virus as well as treatment for Infectious myonecrosis caused by IMNV Although IMNV is a new virus, mortalities from Infectious myonecrosis go up to 70% and change feed conversion ratios (FCR) from normal value to very high value (Andrade

et al., 2007)

A rapid and sensitive method for definitive diagnosis of the disease was developed using reserve-transcriptase polymerase chain reaction (RT-PCR) (Poulos, Lightner, 2006) This method was developed and tested to provide a rapid, sensitive

and specific test to detect IMNV in penaeid shrimp (Poulos et al., 2006)

1.2 Research objective

The research aim to apply a specific and sensitive protocol for the detection of

IMNV from Penaeus vannamei

1.3 Research activities

This research was conducted two following contents:

 Optimization of chemical ingredients of RT-PCR for the detection of IMNV

 Optimization of cycling conditions of RT-PCR for the detection of IMNV

CHAPTER 2 LITERATURE REVIEW 2.1 Overview global white leg shrimp industry

In the early 1970s, Penaeus vannamei were introduced to Pacific Islands for

breeding and aquaculture During the late 1970s and early 1980s they were introduced

to Hawaii and the Eastern Atlantic Coast of the Americas from South Carolina and Texas in the North to Central They have become the primary culture species in Ecuador, Mexico, Venezuela, Brazil, and Central America over the past 20-25 years Their production in Latin America areas is approximately 576 thousand metric tons, accounting for 17% of world total of marine shrimp (David, 2010)

Figure 2.1 World shrimp aquaculture by species from 1991 to 2013

Figure 2.2 Shrimp aquaculture in Asia by species: 1991 - 2013

Penaeus vannamei was introduced to Asia in the late 1980s However,

commercial shrimp industry really developed in the early 2000s Production increases sharply from 7% in 2001 to 37% in 2003 compared to total aquaculture shrimp production in Asia (Diego and James, 2011) (Figure 2.2) Production of this species is approximately 2.7 million metric tons, accounting for 82% of world total of marine shrimp (David, 2010) China, Thailand, Vietnam, Indonesia, and India are the countries with the largest shrimp production in Asia areas Especially, Thailand

converts nearly 100% of its total surface areas of P.monodon to culture white leg

shrimp, becoming the country with top production in South Asia Mainland China produces more than 270,000 metric tons in 2002 and estimated 300,000 metric tons in

2003, which is higher than the current production of the whole of the America areas

(Matthew et al., 2004), reaching 1,000,000 metric tons in 2009 (David, 2010) Other

South Asia countries also develop this species industry including Thailand with 120,000 metric tons in 2003, Vietnam and Indonesia with 30,000 metric tons for each country in 2003 (David, 2010) In 2009, Thailand reached 300,000 metric tons while production of Indonesia was approximately 250,000 metric tons and higher 150,000

metric tons in Vietnam (Matthew et al., 2004)

Although Asia contributed 37% of total shrimp production in 2003 to 67% in

2011 and world contributed 45% P P.vannamei production in 2003 to 71% in

2011(Diego and James, 2011) However, both production of Asia and world changed

in this period because of environment degradation as water quality, temperature, high quality seed, and habitat changed from natural to aquaculture, outbreak diseases Especially, viral disease threatens sustainable shrimp industry of worldwide

2.2 Overview of white leg shrimp farming in Vietnam

Penaeus vannamei have been cultured in Thailand, and Indonesia and achieved

high efficiency in late 1990s and early 2000s However, since June 2002, the Ministry

of Fisheries of Vietnam adopted to culture this shrimp in small scale tests because of

the concern that P.vannamei are capable of transmitting disease to the native shrimp

Since 2008, the agriculture rural development has allowed the culture of

P.vannamei in farming and immediately white leg shrimp farming quickly spread out

in coastal central areas such as Quang Nam, Quang Ngai, Ninh Thuan, Binh Thuan

and the Mekong Delta In 2012, areas for P.vannamei farming increased 28,169 ha

(15.5% total area), reaching 177,817 tons (3.2% production) compared to 2011, accounting for 5.9% cultural areas and 27.3% production Especially, the Mekong Delta contributed 77,380 tons, covering 15,727 ha of total area (VASEP, 2012)

Although P.monodon are dominant species of shrimp for the exported industry of Vietnam, farming areas and production of P.vannamei have increased in recent years

Both the total farming area and production increased from 1,170 ha and 10,000 tons in

2002 to 4,000 ha and 30,000 tons in 2007 The total farming area reached 8,000 ha in

2008, then to 14,500 ha in 2009 before occupying 25,300 ha in 2010 The North and the Center areas have 17,960 ha, accounting for 72% of the total farming area, while the Mekong Delta has smaller areas Recent year later, farming white leg shrimp area increase because of high economic value However, virus outbreak disease damages shrimp industry cause by culturing at high density and environmental degradation in short time

2.3 Common diseases in P.vannamei

In 1989, the first major crash in the production of farmed-raised shrimp was found in Asia Further crashes in production threaten shrimp industry, largely due to viral diseases (FAO, 2004)

Since 1980, at least four viruses were discovered that caused pandemics have adversely affected the global penaeid shrimp farming industry, those are Infectious hypodermal and hematopoietic necrosis virus (IHHNV), Yellow Head Virus (YHV), Taura Syndrome Virus (TSV), White Spot Syndrome Virus (WSSV), and recently is Infectious Myonecrosis (IMN) These viruses are major causes that seriously impact the socioeconomic in Asian and American countries Some reasons are international trade or movement of infected crustaceans that pose a significant disease threat to cultured and wild crustaceans

2.3.1 Taura syndrome

Taura syndrome was a major new disease in P.vannamei farming and was first

described in 1992 in Ecuador The virus and its viral etiology of TS were confirmed in

1994 and was named Taura syndrome virus (TSV) (Hasson et al., 1995) TSV is

similar infectious cuticular epithelial necrosis virus (ICENV) on the epizootiology of

the disease in Ecuador (Jimenez et al., 2000)

Taura syndrome is simple structural virus, virion was described is a 32 nm diameter, nonenveloped icosahedron with a buoyant density of 1.338 g/ml Its

genome includes a linear, positive-sense single-stranded RNA of 10,205 nucleotides, excluding the 3' poly-A tail, and it contains two large open reading frames (ORFs) ORF 1 contains the sequence motifs for nonstructural proteins, such as helicase, protease and RNA-dependent RNA polymerase ORF 2 contains the sequences for TSV structural proteins, including the three major capsid proteins VP1, VP2 and VP3

(55, 40, and 24 kDa, respectively) (Bonami et al., 1997; Mari et al., 1998; Mari et al., 2002; Robles-Sikisaka et al., 2001) Taura syndrome virus replicates in the cytoplasm

of host cells, so the International Committee on Taxonomy of Viruses (ICTV) assigned this virus in newly genus Cripavirus in new family Dicistroviridae (in the superfamily of picoranviruses) (Mayo 2002a, 2000b) TSV transmits horizontal by sea gulls when the latter eat infected shrimp and release feces with infected shrimp carcasses or pathogen in feces that can lead to rapidly spread out disease from

infectious farming to other areas (Garza et al., 1997; Lightner, 1999) There is still no

more documents that reported Taura Syndrome in wild shrimp since infected wild

shrimps were found near shrimp farm (Lightner et al., 1995) According to Brock

(1997), this disease may not impact wild shrimp population

2.3.2 White spot disease

White spot disease was first described in East Asia in 1992-1993 and has become a major economic and ecological threat for global shrimp industry WSSV is potentially lethal to most of the commercially cultivated penaeid shrimp species (OIE, 2003) This disease affect seed and broodstock, and quickly dispersed across the Asian continent to South East Asia and India where it became a major pandemic, and continues to cause significant losses in some regions The first report about WSD

outbreaks was from Metanephrops japonicus in Japan in 1993 Although WSD

impacted seriously shrimp farms, mostly in Asia regions, definition and name of disease has been a complex issue since it was found The causative agent was named

penaeid rod-shaped DNA virus (PRDV) or rod-shaped nuclear virus of M japonicus

(RV-PJ)

WSSV has a wide host range among decapod crustaceans (Lo et al., 1996;

Flegel, 1997; Flegel and Alday-Sanz, 1998), and is potentially lethal to most of the commercially cultivated penaeid shrimp species (OIE, 2003) Negatively stained

virions purified from shrimp hemolymph show unique, tail-like appendages (Wang et al., 1995) The virions are generated in hypertrophied nuclei of infected cells without

the production of occlusion bodies

In initial reports, WSSV was described as a non-occluded baculovirus, but WSSV DNA sequence analysis has shown that it is not related to the baculoviruses

(van Hulten et al., 2001; Yang et al., 2001) There are different isolates resulted in

size of genomes from other studies The Chinese isolate and Taiwan isolate were sequenced 305,107 bp (GenBank Accession No AF332093), 292,967 bp (GenBank Accession No AF369029) and 307,287 bp (GenBank Accession No AF440570) while a genome size of ~300 kb, a total of 531 putative open reading frames (ORFs) were identified by sequence analysis, among which 181 ORFs are likely to encode functional proteins Thirty-six of these 181 ORFs have been identified by screening and sequencing a WSSV cDNA library or else have already been reported to encode

functional proteins many of which show little homology to proteins from other viruses (OIE, 2003)

Despite the lack of study and evidence of live shrimp introductions from Asia

to the Americas, WSD had a severe impact on the shrimp industries of both Central

and South America during 1999 (Durand et al., 2000; Vidal et al., 2001; Lightner,

2003) WSSV was diagnosed at several sites in 1995-1997 in captive wild shrimp or crayfish and in cultured domesticated shrimp stocks in the eastern and southeastern

U.S (Nunan et al., 1998; Durand et al., 2000; Lightner et al., 2001) Early in 1999,

WSSV was diagnosed as the cause of serious epizootics in Central American shrimp farms By mid to late 1999, WSSV was causing major losses in Ecuador and, export

of shrimp in 2000-2001 from Ecuador was down nearly 70% before WSD (Rosenberry, 2001; Lightner, 2003)

During 1999, WSD also had a severe impact on the shrimp industries of both

Central and South America (Durand et al., 2000; Vidal et al., 2001; Lightner, 2003)

Despite the absence of evidence of live shrimp introductions from Asia to the Americas, WSSV was diagnosed at several sites in 1995-1997 in captive wild shrimp

or crayfish and in cultured domesticated shrimp stocks in the eastern and southeastern

U.S (Nunan et al., 1998; Durand et al., 2000; Lightner et al., 2001) Early in 1999,

WSSV was diagnosed as the cause of serious epizootics in Central American shrimp farms By mid to late 1999, WSSV was causing major losses in Ecuador (then among the world’s top producers of farmed shrimp), and by 2000-01, export of shrimp from Ecuador was down nearly 70% from pre-WSSV levels (Rosenberry, 2001; Lightner

2003) WSSV has been found in wild shrimp stocks in the Americas (Nunan et al.,

2001) In the US, the virus was successfully eradicated from shrimp farms and it has not been reported from farmed shrimp stocks since 1997

2.3.3 Yellow Head Disease

Yellow Head Virus is closely related Gill-Associated virus (GAV), and has been reported from Australian shrimp farms In laboratory condition, YHV can cause high mortality in representative cultured and wild penaeid species from the Americas American penaeids challenged with YHV did not develop yellow heads or signs of marked discoloration in laboratory studies (Lightner and Redman, 1998) YHV can cause high mortality in representative cultured and wild penaeid species from the

Americas (Lu et al., 1994, 1997; Lightner, 1999; Pantoja & Lightner, 2003) in P.monodon rearing farm

YHV has enveloped bacilliform virions They range from approximately 150

nm to 200 nm in length and from 40 nm to 50 nm in diameter and are located within vesicles in the cytoplasm of infected cells and in intercellular spaces The virions arise from longer, filamentous nucleocapsids (approximately 15 nm x 130-800 nm), which accumulate in the cytoplasm and obtain an envelope by budding at the endoplasmic reticulum into intracellular vesicles Negatively stained YHV virions show regular

arrays of short spikes on the viral envelope (Boonyaratpalin et al., 1993; Chantanachookin et al., 1993; Lightner, 1996a) YHV was originally described mistakenly as a granulosis-like virus (Boonyaratpalin et al., 1993; Chantanachookin et al., 1993), but it was later found to be a single-stranded, positive sense RNA (ssRNA)

virus (Tang and Lightner, 1999) related to nidoviruses in the Coronaviridae and

Arteriviridae (Sittidilokratna et al., 2002) GAV, the Australian strain of YHV has

been recognized as the type species for the new virus genus Okavirus in the new family Roniviridae (Mayo, 2002a, 2002b; OIE, 2003) YHD was first described as an epizootic from Thai shrimp farms (Limsuwan, 1991), and spread out in cultivated shrimp in many locations in Asia (OIE, 2003) Severe necrosis of the lymphoid organ,

a lesion once thought to be pathognomonic for YHD show that is diagnosis of YHV

(Lightner, 1996a; Lightner et al., 1998; Lightner and Redman, 1998) Frozen imported

commodity shrimp having the same diagnosis was reported in the United States

(Nunan et al., 1998; Durand et al., 2000) However, the diagnosis of YHV infection in

these cases was not confirmed with a second diagnostic method until after the errant reports were published A more recent study has shown that the presumptive histological diagnoses were due to severe infections with white spot virus, which can cause histopathology in the lymphoid organ which mimics that occurring in severe acute YHD (Pantoja and Lightner, 2003) YHV is not only harmful frozen commodity

shrimp still remains (Nunan et al., 1998, Durand et al., 2000), but also concurrent

WSSV/YHV infections may occur so RT-PCR and ISH method were using as detail analyzed one to confirm the presence of YHV

2.3.4 Infectious hypodermal and haematopoietic necrosis disease

IHHNV is the smallest of the known penaeid shrimp viruses The virion of this virus is a 22 nm diameter, having nonenveloped icosahedron with a density of 1.40 g/ml in CsCl IHHNV has been classified as a member of the Parvoviridae and a

probable member genus Brevidensovirus (Bonami et al., 1990; Bonami and Lightner, 1991; Mari et al., 1993; Nunan et al., 2000) because its genome is linear single-

stranded DNA of 4.1 kb in length, and its capsid consists of four polypeptides with molecular weights of 74, 47, 39, and 37.5 kD

The first case of IHHN disease was described as the cause of acute epizootics and mass mortalities (> 90%) in juvenile and subadult L stylirostris farmed in super-

intensive raceway systems in Hawaii (Brock et al., 1983; Lightner, 1983, 1988; Lightner et al., 1983a, 1983b; Brock and Lightner, 1990) in and widely distributed in

cultured shrimp in the Americas and in wild shrimp along the Pacific coast After that

this disease was found in P.vannamei being cultured at the same facility in Hawaii and these P.vannamei were shown to be asymptomatic carriers of the virus (Lightner et al., 1983b; Bell and Lightner, 1984) Some individuals of populations of L stylirostris and P.vannamei that survive IHHNV infections and/or epizootics may carry the virus

for life and pass the virus on to their progeny and other populations by vertical and horizontal transmission (Bell and Lightner, 1984; Lightner, 1996a; Morales-

Covarrubias et al., 1999, Morales-Covarrubias and Chavez-Sanchez, 1999; Motte et al., 2003) A few years after motality of P.vannamei was reported that not significant (Lightner et al., 1983b; Bell and Lightner, 1984) However, present ‘runt deformity

syndrome’ (RDS) caused by IHHNV, this syndrome affect growth and economic value due to small size and not same size Choosing SPF (specific pathogen-free) stock is recommended for preventing this disease

2.3.5 Infectious myonecrosis disease

In 2004, Infectious myonecrosis disease (IMN) was reported first in

P.vannamei in Nothern Brazil This disease is caused by Infectious myonecrosis virus

(IMNV) (Lightner and Pantoja, 2004), with gross signs being necrosis in skeletal muscle tissues in the distal abdominal segments that are visible as opaque, whitish,

discolorations (Lightner et al., 2004a,b) In 2006, similar signs were reported from

Indonesian shrimp farmers of high mortality Moribund shrimp exhibited similar signs

to white shrimp infected previously and reported from Brazil (Polous et al., 2006)

Penaeus vannamei is recognized as the definitive host of IMNV by potentially

high mortality in this species Commonly mortality rates range from 40 to 70% of populations However, mortality rates of the Infectious myonecrosis can be up to 100% or increase suddenly after stressful activities Mortality rates from 35% to 55%

and economic value was estimated to be US$20 million in Brazil (Nunes et al., 2004) Mortalities range from 40% to 70% in P.vannamei (Andrade et al., 2007)

In the acute phase, infected shrimp often have pathological signs, for example, part of the skeletal tail muscle necrosis has white opaque discoloration, so the phenomenon can lead to necrosis and reddened in this muscle In some cases, lymphoid organs increase and are 2-4 times bigger than their normal size IMNV pond severe infection may die suddenly and lasts for several days Necrosis of the high rate

of sudden death, usually occurring at or after the time of the operation may be shocking for example shrimp fishing, salinity or temperature changes suddenly Some dead shrimp was found after feeding time with full gut

Although IMN was found in 2004, the damage caused by this disease crash production of white shrimp as well as revenue threaten the sustaintability of white shrimp farming worldwide including South American countries, and Asian countries such as Thailand, Indonesia, and Vietnam There is anecdotal information about this disease but there is no effective vaccination as well as no effective chemotherapy Mortality rates from 35% to 55% and economic value was estimated to be US$20

million in Brazil (Nunes et al., 2004) Feed conversion ratios (FCR) can increase

from 1.5 up to 4.0 or higher in populations and mortalities range from 40% to 70% in

P.vannamei (Andrade et al., 2007)

IMNV particles are icosahedral in shape and 40 nm in diameter, has enveloped and double-stranded RNA The genome comprised 7560 nucleotides containing two open frames include ORF1 and ORF2 In the first half of ORF1locates RNA-binding proteins and contains s dsRNA-bingding motif in the first 60 amino acids A capsid protein was encoded in the second half of ORF1 and determined by amino acid sequencing The ORF2 codes putavate RNA-dependent RNA polumerase (RdRp)

non-For the above reasons, we need to test larvae prior to stocking in order to limit the risk of crop and test infectious shrimp which have symptoms of INM disease Besides that, keeping temperature, pH, and salinity in farm system; reducing the amount of food or stop feeding and aeration can prevent disease spread out seriously

In case a high mortality by disease occurs, water can be treated with 30ppm Chlorine for some days

2.3.6 Diagnostic methods of IMNV

Only acute-phase Infectious myonecrosis can be presumptively diagnosed from clinical signs and present behavioural changes The diseased shrimp which are infected IMNV become lethargic during or soon after stressful events such as capture

by cast-netting, feeding, sudden changes in water temperature, sudden reductions in water salinity, etc.) Severely affected shrimp may have been feeding just before the onset of stress and often have a full gut (OIE, 2012)

Shrimp in the acute phase of Infectious myonecrosis present focal to extensive white necrotic areas in striated (skeletal) muscles, especially in the distal abdominal segments and tail fan, which can become necrotic and reddened in some individual shrimp These signs may have a sudden onset following stresses: These signs may have a sudden onset following stresses (e.g capture by cast-netting, feeding, and sudden changes in temperature or salinity) Such severely affected shrimp may have been feeding just before the onset of stress and may have a full gut Severely affected shrimp become moribund and mortalities can be instantaneously high and continue for several days

When infected shrimp was dissection, the paired lymphoid organs (LO) show

that they are hypertrophied (Lightner et al., 2004; Poulos et al., 2006)

Infectious myonecrosis can can be presumptively diagnosed using histology in

the acute and chronic phases (Bell & Lightner, 1988; Lightner, 2011; Lightner et al., 2004; Poulos et al., 2006) However, requiring more diagnostic information from

other sources (e.g history, gross signs, morbidity, mortality, or RT-PCR findings) to confirm a diagnosis of IMN because some disease has same gross sign of Infectious myonecrosis such as white tail disease of penaeid shrimp caused by the nodavirus

PvNV (Tang et al., 2007)

By histology using routine haematoxylin–eosin (H&E) stained paraffin sections (Bell & Lightner, 1988), tissue sections from shrimp with acute-phase IMN present myonecrosis with characteristic coagulative necrosis of striated (skeletal) muscle fibers, often with marked oedema among affected muscle fibers Some shrimp may present a mix of acute and older lesions

Significant hypertrophy of the LO caused by accumulations of LO spheroids (LOS) is a highly consistent lesion in shrimp with acute or chronic-phase Infectious myonecrosis lesions Often, many ectopic LOS are found in other tissues not near the main body of the LO Common locations for ectopic LOS include the haemocoelom in the gills, heart, near the antennal gland tubules, and ventral nerve

cord (Lightner et al., 2004; Poulos et al., 2006)

By using wet mount stained or unstained tissue squashes of affected skeletal muscle or of the LO may show abnormalities Tissue squashes of skeletal muscle when examined with phase or reduced light microscopy may show loss of the normal striations Fragmentation of muscle fibres may also be apparent Squashes of the LO may show the presence of significant accumulations of spherical masses of cells (LOS) amongst normal LO tubules

The sensitivity was approximately tenfold lower than that of a one-step PCR assay using the same sample Other clinical methods such as clinical chemistry, smear, electron microscopy were not recommended because of not acceptable or not suitable for research purpose

RT-2.3.7 Research of IMNV detection by RT-PCR method

All PCR tests have proven to be specific to IMNV As the sensitivity of the nested and real-time RT-PCR is greater than any other diagnostic method available currently, these tests, which approach a detection limit of 10 viral genome copies, are

the gold standard for IMNV (Andrade et al., 2007; Poulos et al., 2006) The molecular

detection of IMNV by in-situhybridisation (ISH), nested RT-PCR and quantitative

real-time qRT-PCR are available published methods (Andrade et al., 2007; Poulos et al., 2006; Tang et al., 2005) The disease status of all shrimp was also determined by a

combination of PCR tests, histology and in situ hybridization (OIE, 2003) transcriptase polymerase chain reaction (RT-PCR) was noticed as a rapid and sensitive method for detecting IMNV The first-step PCR can detect as little as 100 IMNV RNA copies and the two-step PCR can detect in the order of 10 IMNV RNA copies (Poulos & Lightner, 2006) Sensitivity of the nestedRT-PCR for IMNV was determined using 10-fold serial dilutions of RNA extracted from purified virions

Reverse-(Poulos et al., 2006)

2.4 Reverse transcriptase-polymerase chain reaction (RT-PCR)

The polymerase chain reaction (PCR) is a technique for copying a piece of DNA a billion-fold As the name suggests, the protocol creates a chain of many pieces In this case the pieces are nucleotides, and the chain is a strand of DNA

The purpose of this method is similar to the PCR except it allows amplification

of small amounts of ribonucleic acid (RNA) RT-PCR is used for detecting viruses

with an RNA genome and RNA transcription (Lon V Kendall et al., 2000) The

principle RT-PCR protocol is summarized in the figures below

The template RNA must be isolated from the sample to be tested prior to initiating reverse transcription (Figure 2.3)

Figure 2.3: The template RNA prior to initiating reverse transcription

Priming for reverse transcription to generate cDNA using the enzyme reverse transcriptase (RT), a primer is annealed to the template RNA The primer can be gene specific primers, random primers or oligo-dT primers for mRNA In this figure, oligo-

dT primers are used to initiate cDNA synthesis from mRNA (Figure 2.4)

Figure 2.4: Priming for reverse transcription The first strand synthesis of cDNA is synthesized using RT Beginning at the primer annealing site (A), RT adds complementary nucleotide bases to the mRNA strand creating a strand of cDNA (B) (Figure 2.5)

Figure 2.5: First strand synthesis

The template strand of RNA is removed by treatment with RNAse H The cDNA can now be used for amplification by PCR (Figure 2.6)

Figure 2.6: Removal of RNA The oligonucleotide primer is allowed to anneal to the template cDNA (A) Taq polymerase adds complementary nucleotides beginning at the primer annealing site (B) The resultant product is a double stranded cDNA (C) The three step protocol of denaturation, primer annealing and extension are prepared to yield a detectable PCR product (D) The product can be visualized on an ethidium bromide stained agarose gel following electrophoresis (Figure 2.7)

Figure 2.7: PCR reaction RT-PCR not only has high sensitivity due to exponential amplification of the template RNA, very specific when using gene specific primers in the synthesis of cDNA, but also can be completed in one to two working days, which provides rapid results

2.5 Factors affecting RT-PCR protocol

2.5.1 RT primer

There are three RT primers: oligo dT, random primers, or a gene specific primer The most popular primer is OligodT primer so they can get full length copies

of the mRNA Random primer may use if the message is long (>4kb) or does not have

a poly A tail (prokaryotic mRNA), however, will enable you to transcribe 5′ ends of long genes, but your cDNAs may not be full length copies of the entire gene Two companies that provide an optimized primer including a mix of long random primers and oligo-dT such as Qiagen QuantiTect Rev Transcription Kit and the BioRad iScript Kit Gene A primer used for one-step RT-PCR is specific primers enhance sensitivity by directing all of the RT activity to a specific message instead of transcribing everything in the mix (Suzanne, 2009)

2.5.2 One-step and Two-step RT-PCR

With a one-step RT-PCR, a sample transfer step is eliminated, eliminating a potential source for contamination of controls And with a one-step RT-PCR, because the primer is gene-specific, the sensitivity may be higher With two-step RT-PCR, a larger batch of RNA can be converted to cDNA and then aliquoted into many reactions Two-step RT-PCR allows for greater choice in primers and also allows you

to use the same batch of RT for analysis of multiple genes This will help eliminate a potential contributor of variation when comparing different genes from the same source (Suzanne, 2009)

2.5.3 Quantitating the RNA

The yield of RNA can be affected by measuring instrument, contamination with DNA, contamination with salts, and level of degradation, UV spectrophotometers UV is use as popular instrument to meansure the yield Advantages of this instrument is not required dilution of the sample and has a very wide range for measuring RNA, however, UV quantification is that genomic DNA will also be measured in the sample and if salts or phenol left over from the prep are contaminating the RNA, then there can also be added absorbance giving a false higher reading of the RNA Fluorescent dyes is also used as a good way to prevent contamination (Suzanne, 2009)

2.5.4 Check the RNA Integrity

RNA quality will have a big impact on the results of cDNA synthesis And batch to batch variation in RNA quality will lead to inconsistent results Running an agarose gel and check it intact eukaryotic RNA should show a 28s and 18s rRNA with the larger band looking close to double in intensity compared to the smaller It is good

if have the same intensity However, if sample is limitted, the Agilent Bio Analzyer is also use to determine the quality of RNA (Suzanne, 2009)