Khóa luận tốt nghiệp Công nghệ sinh học: Evaluating specificity and determining the limit of detection of TopSPEC® FMDV RT-qPCR KIT and TopSPEC® EMCV RT-qPCR KIT

MINISTRY OF EDUCATION AND TRAININGNONG LAM UNIVERSITY - HO CHI MINH CITYFACULTY OF BIOLOGICAL SCIENCESEVALUATING SPECIFICITY AND DETERMINING THE LIMIT OF DETECTION OF TopSPEC® FMDV RT-qP

MINISTRY OF EDUCATION AND TRAININGNONG LAM UNIVERSITY - HO CHI MINH CITYFACULTY OF BIOLOGICAL SCIENCES

EVALUATING SPECIFICITY AND DETERMINING THE LIMIT OF DETECTION OF TopSPEC® FMDV RT-qPCR KIT

and TopSPEC® EMCV RT-qPCR KIT

MINISTRY OF EDUCATION AND TRAININGNONG LAM UNIVERSITY - HO CHI MINH CITYFACULTY OF BIOLOGICAL SCIENCES

Dr DINH XUAN PHAT TRAN NGUYEN MINH THANH

Thu Duc City, 08/2023

First of all, I would like to thank the Board of Nong Lam University Ho Chi Minh

City and the Board of Faculty of Biological Sciences along with all the teachers who

taught and imparted knowledge to me during my time studying at the school

In addition, I also want to thank Biomin Veterinary Diagnostics Laboratory,Branch of Biomin Vietnam in Binh Duong (belonging to DSM Group)

I really appreciate my advisor, PhD Dinh Xuan Phat, whose expertise was

invaluable in formulating the research questions and methodology Your insightful

feedback pushed me to sharpen my thinking and brought my work to a higher level

I am so grateful to Ms Nguyen Thi Mi Mi, Ms Cao Tran Quynh Nhu, Mr LeThanh Binh, Mr Hoang Gia Lam and other members of Gene Technology LaboratoryBIO313 for supporting me during the completion of my graduation thesis

And last but not least, I want to send thanks of my family for always listening,trusting and accompanying over the past time You are always there for me, at my worst,

at my best I promise that I will try harder to become successful and make you proud

CONEIRMATION COMMITMENT

My name is Tran Nguyen Minh Thanh, Student ID: 19126163, Class: DH19SHA,

Faculty of Biological Sciences, Nong Lam University Ho Chi Minh City I guarantee

that this research was conducted by myself and that the results presented are utterly

truthful and objective

Thu Duc City, August 2023

Student’s signature

il

The purpose of this study was performed to evaluate the specificity and determine

the limit of detection for two trial kits: TopSPEC® FMDV RT-qPCR KIT and

TopSPEC® EMCV RT-qPCR KIT The qPCR of the trial kit was performed

determining the limit of detection and checking specificity with some bacteria and

viruses commonly appearance in samples The results showed that the limit of detection

of two kits was 5 copies/reaction and its specificity with target pathogens The standard

curve parameters of trial kits were a slope of -3.3969, a linear correlation (R2) of 0.9969,

efficiency (E%) of 97% for FMDV’s kit, and a slope of -3.4606, a linear correlation (R?)

of 0.9997, and efficiency (E%) of 95% for EMCV’s kit After that the two trial kitswere applied to investigate the presence of FMDV and EMCV in clinical samplescollected from pig farms in Southern Vietnam Eighty field samples were examined andall samples were confirmed to be negative with each of pathogens Additionally, it’sindicated that TopSPEC® FMDV RT-qPCR KIT and TopSPEC® EMCV RT-qPCR

KIT can be utilized in routine diagnostics of these pathogens in pig farms

Keywords: EMCV, FMDV, limit of detection, real-time PCR (qPCR), specificity, trialKit

il

TÓM TẮT

Đánh giá độ đặc hiệu và giới hạn phát hiện của hai bộ kit thử nghiệm TopSPEC®

FMDV RT-qPCR KIT và TopSPEC® EMCV RT-qPCR KITNghiên cứu này được thực hiện nhằm đánh giá độ đặc hiệu và giới hạn phát hiện

của hai bộ kit thử nghiệm 7opŠSPEC® FMDV RT-qPCR KIT và TopSPEC® EMCV

RT-qPCR KIT Realtime PCR (qPCR) của các bộ kit được xác định giới han phát hiện

và kiểm tra độ đặc hiệu với một số vi khuân và virus Kết quả cho thấy rằng, giới hạn

phát hiện của hai bộ kit thử nghiệm đều là 5 copies/phản ứng và đặc hiệu với các tác

nhân muc tiêu Các thông số đường chuẩn của TopSPEC® FMDV RT-qPCR KIT: hệ

số dốc (Slope) -3,3969, hệ số tương quan R? = 0,9969 và hiệu quả khuếch đại (E%) 97% TopSPEC® EMCV RT-qPCR KIT với hệ số dốc (Slope) là -3,4606, hệ số tương quan R7 = 0,9997 và hiệu quả khuếch đại (E%) = 95% Sau đó, hai bộ kit này đã được

ứng dụng dé kiểm tra sự hiện điện của FMDV và EMCV với 80 mẫu thực địa được thu

thập từ các tỉnh phía Nam và tất cả các mẫu đều cho kết quả âm tính Tóm lại, kết quả nghiên cứu cũng cho thay hai bộ kit thử nghiệm TopSPEC® FMDV RT-qPCR KIT và TopSPEC® EMCV RT-qPCR KIT có thé ứng dụng trong chan đoán thường quy hai bệnh Lở mồm long móng và Viêm não-viêm cơ tim gây ra ở trên heo.

Từ khóa: EMCV, FMD, giới hạn phát hiện, real-time PCR (qPCR), độ đặc hiệu, kit

thử nghiệm

IV

TABLE OF CONTENTS

PageACKNOW LEDGEMEN TS sssssesssnssssenes venssuasenanssnannnennennavncensaenasas tneaasnenteoceunreseensnmiennenaane 1CONFIRMATION COMMITMENT c0sceccsssssesssssosssnsossenssseseenesssssnessessonessesessesnens i

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2,2 1, Structure Of EMG nugngbpogi61105303155544E04355EG5151XESEXSESSESISSGE4SEAEEASEASLESS4B.XEESSSS1464E 11

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3 2le CHEMICALS 87id:6dPIHGIĂneeeeddeni gh ghi thai GIAHfitĐSh4HRiUNSB4GTEEUEBIGEGIRU2GIBhEBCI0.S1000.86 21E000 J0 1177 223.3.15 RÌNA GTfAaGHỌibsessooiiHistolCGGIEGSESEEERRSEEAHBSSINHIREESESLSHDAGSSIIRSSSRHBSEISSEISSESRUSSERHDAine 323.3.2 Reverse transcription Real-time PCT - - - 5- 322222 E*2+sEEssessrrrrrsrrrrresee 233.3.3 TopSPEC® EMCV RT-qPCR KÍT -7 c5 cSs+sererrrrrrrrrrrrrrrrrrrrrrrrree 23

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3.3.4.2 Determine for the limit of detection (LOID) - -55 5-5 5+22<<++sc+sseeeseeezes 243.3.5 Investigate the presence of FMDV and EMCV in field samples 243.4 Data nh 25Chapter 4 RESULTS AND DISCUSSION so bi D B2 E2 EV HD d5 20850812801838e8E 26

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PCR : Polymerase Chain Reaction

PCV2 : Porcine Circovirus type 2

PRRSV : Porcine reproductive and respiratory syndrome virus

RNA : Ribonucleic acid

RT-qPCR : Reverse transcription Real-time Polymerase Chain Reaction

WOAH : World Organisation for Animal Health

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LIST OF TABLES

PageTable 3.1 Evaluating of results qPCR to detect FMDV and EMCV 23Table 4.1 Repetitions of the qPCR for determining the limit of detection Table 4.2 Repetitions of the qPCR for determining the limit of detection 31

IX

LIST OF FIGURES

PageFigure 2.1 Genome and structure of FMDV 0.00 ececccecceeseeseeseeeseesteseeseeeseeseeeseeseeeseees 4Figure 2.2 The geographical distribution of FMDV c7 5-5 S+cS+ccseeeseerrrexres 6Fisure:2.3 Theroute of spreading the diséa$ © scccsseccsserensaernscmmeenma naene ỶFigure 2.4 Vesicular lesions on feet of swine experimentally mnfected - 9Figure 2.5 Pigs with FMDV infection displaying clinical sign and lesions 10

Figure 2.6 Genome compositions of recombinant EMCV -+-c+-c+-cseces 12

Figure 2.7 Heart of pig with EMCV Imf€cfion - 2 5-2222 s+sssrrsrrrsrrsres 14

Figure 2.8 Sagittal section of the heart of pig with EMCV Infection 15

Figure 2.9 The action of SYBR Green I ÏDye - - 5-52 +s+c+scsererrrrrrrrrrrrrrrree 16Figure 2.10 The action of TaqMan probe - + 55222222 +23 +22 +2E£2eseesrrerkrrrrrrres 17Figure 2.11 Amplification pÏO£ - - - 5522211131221 1531231 131211 1111 KH 1kg ke 18Figure 4.1 The specificity evaluation of EMCV RT-qPCR KIt eee 26Figure 4.2 The specificity evaluation of EMCV RT-qPCR KIt - 5 - 27Figure 4.3 Determine LOD of the EMCV RT-qPCR KIt 55555 +55<<+<=+<<s2 27Figure 4.4 Determine LOD of the EMCV RT-gPCR KIt - 555555 55s<++=+ss>+ 28Figure 4.5 Detection limit and standard curves of EMCV RT-qPCR kit 29Figure 4.6 The specificity evaluation of FMDV RT-qPCR kit eee 30Figure 4.7 The specificity evaluation of FMDV RT-qPCR kit eee 30

Figure 4.8 Determine LOD of the FMDV RT-qPCR KIC - eee dl

Figure 4.9 Determine LOD of the FMDV RT-qPCR KIt 5-5 5-++<<+s<+ec+s 31Figure 4.10 Detection limit and standard curves of FMDV RT-qPCR kit 32

Chapter 1 INTRODUCTION

1.1 Introduction

In recent years, pork consumption has been decreasing in some countries due to

many factors, but it is still quite high because pork 1s the main ingredient of many dishes

in East and Southeast Asia, and the Western world, particularly in Central Europe Theswine industry is not only an essential food source for the people but also played animportant role in economic development Nonetheless, the livestock industry is still

facing many difficulties due to the impact of a number of reasons such as input costs,

biosecurity, especially diseases as FMDV and EMCV

Foot-and-mouth disease (FMD) is a severe highly contagious disease caused bypicornavirus FMD 1s the first disease for which World Organisation for Animal Health(WOAH) established an official list of disease-free countries FMD in swine caused aserious impact on the economy of the global livestock and trade industries, it had to lead

to the slaughter of swine or the destruction of infected herds to stop the spread of thedisease The economic impacts of FMD may be estimated revenue losses of between

$49.3 billion and $51.8 billion (in present value terms) were expected over 10 years.Control costs were estimated at between $60 million and $373 million, with $6.3 million

to $60.2 million representing compensation to farmers for animals destroyed during

control procedures (Australian Government Department of Agriculture, Fisheries andForestry: www.agriculture.gov.au) Preventing FMD disease in time is crucial to preventdamage caused by epidemics

Encephalomyocarditis virus (EMCV) was also a widely distributed picornavirusthat caused substantial pig mortalities due to heart disease In piglet, virus could beexpressed as acute myocarditis and sudden death, whereas causing abortion, fetal

mummification, stillbirth, and neonatal death in transplacental infection of sows

Because of similarities between EMC and FMD myocarditis in swine, they could bespecifically diagnosed via virus isolation or molecular methods (Yuan ef al., 2016)

The most common molecular method to diagnose these two viruses 1s RT-qPCR.technique Currently, there are many qPCR kits that had been developed and practicallyused in the diagnosis of FMDV and EMCV, including the JTopSPEC® FMDV RT-qPCR

KIT and 7opSPEC® EMCV RT-qPCR KIT which were in testing the presence of thesepathogens in pig farm Therefore, to determine the usability of these two kits, the thesiscalled: “Evaluating the limit of detection and specificity of TopSPEC® FMDV RT-qPCR KIT and TopSPEC® EMCV RT-qPCR KIT” was performed.

Chapter 2 LITERATURE REVIEW

2.1 Introduction to FMDV

2.1.1 Classification

In 1546, Foot-and-Mouth Disease (FMD) was first recognized by HieronymusFracastorius, who described an outbreak in cattle in Italy that caused clinical signssimilar to FMD It was also the first animal disease demonstrated to be caused by afilterable agent in 1897 by Loeffler and Frosch (Segundo ef a/., 2014) FMD 1s a highly

contagious viral disease of cloven-hoofed domestic and wild animals (Bachrach, 1968)

According to the World Organisation for Animal Health (WOAH), FMD ranks firstamong list A, including 15 dangerous infectious animal diseases for buffaloes, goats,cows, sheep, and swine

The causative agent of the disease is the Foot-and-Mouth Disease virus (FMDV)which is a species within the genus Aphthovirus of the family Picornaviridae The virus

is antigenically variable and identified as having seven serotypes: A, C, O, Asia-1, and

the South African Territories (SAT) serotypes (including SAT1, SAT2, and SAT3) with

multitudinous subtypes and strains within each serotype (Grubman and Baxt, 2004).There is considerable diversity within serotypes and no cross-immunity betweenserotypes (Robson ef a/, 1977) The FMDV serotype A was one of the mostantigenically different among the seven serotypes (Kitching, 2005) The FMDVserotype A had been classified into 10 major genotypes (designated as I to X) grounded

on the VP1 phylogenetic trees (Tosh et zí., 2002) FMDV serotype O was classified into

11 topotypes, designated as Europe-South America (Euro-SA), Cathay (CHY), WestAfrica (WA), East Africa 1 (EA-1), East Africa 2 (EA-2), East Africa 3 (EA-3), EastAfrica 4 (EA-4), Middle East-South Asia (ME-SA), Southeast Asia (SEA), Indonesia-1

(ISA-1), and Indonesia-2 (ISA-2) (Knowles ef a/., 2005); and FMDV serotype Asia 1

was Classified into seven genotypes (designated as I to VID) (Jamal et a/., 2011)

2.1.2 Structure of FMDV

The FMDV is a non-enveloped virus, with an icosahedral capsid about 25 - 30

nm in diameter The virion consists of a single-stranded, positive sense RNA genomeabout 8400 nucleotides long surrounded by a protein shell or capsid As for other,

picornaviruses the complete viral capsid of FMDV is composed of 60 copies of thecapsomers Each capsomer contains four structural polypeptides (VP1 - 4) The VPI,VP2, and VP3 are exposed on the surface of the virus while the VP4 is disposed on theinner surface of the particle (Jamal and Belsham, 2013) However, FMDV’s structure isunique, the external surface is smoother and the protein shell is generally thinner than

that of in other picornaviruses (Rowlands, 2008)

The nucleotide sequences of the VP1 region had been used to determine thegenetic characterization of FMDV strains because of their significance for virus

attachment and entry, serotype specificity, and protective immunity (Jamal and

Figure 2.1 Genome and structure of FMDV (Jamal and Belsham, 2013)

The FMDV genome included a single large open reading frame (ORF), flanked

by highly conserved and untranslated regions (5’ and 3’ UTRs) (Grubman and Baxt,

2004) A small protein, VPg, is linked with the 5’ end of the genome by a phosphodiester

bond The VPg peptide is made in 3 distinct forms and each acts as the primer for RNAsynthesis (Arias eft al., 2010).

The 5’ UTR has several distinct structural elements: a poly (C) tract (Cn), 3 or 4

pseudoknots (PK), and the internal ribosome entry site (IRES) The function of theinternal ribosome entry site is cap-independent translation initiation of viral proteinsynthesis (Mason ef a/., 2003)

The major portion of the viral genome is a long coding region After translation,four primary products are the L?®, P1-2A, P2, and P3 (Jamal and Belsham, 2013).Leader protease (L?"°) have a proteolytic function leading to the inhibition of host cellprotein synthesis (Devaney ef a/., 1988) The P1-2A region consist of the structuralproteins 1A, 1B, 1C, and 1D, also call VP4, VP2, VP3, and VPI, respectively(Rowlands, 2008) The P2 and P3 of the long coding region are converted to non-structural proteins (NSPs) (Belsham, 2005)

The 3’ UTR is about 90 nucleotides in length and folds to form a stem-loop

structure, followed by a poly(A) (Pilipenko ef aí., 1992) The role of the 3’ UTR in viralgenome replication is very important (Rohll ef a/., 1995)

2.1.3 Epidemiology

2.1.3.1 Geographic distribution

According to the World Organisation for Animal Health (WOAH), FMDV was

a transboundary animal disease (TAD) that obstructed regional and international trade

in animals and animal products thus important economic losses The disease wasendemic in several parts of Asia, most of Africa, and the Middle East Presently,Australia, continental Western Europe or Central, and North America are free of FMDV.However, FMD could occur sporadically in any typically free area because it had anextraordinary ability to cross international boundaries and caused epidemics inpreviously free areas As illustrated by the epidemic in the United Kingdom andcontinental Europe in 2001 (Davies, 2002), as well as the outbreaks in the year 2000 inSouth Africa, Japan, and South Korea (Sugiura ef al., 2001; Briickner ef al., 2002; Joo

et al., 2002)

Among seven serotypes of FMDV, serotypes O and A have been distributedwidely and caused outbreaks in Africa and Asia (Kitching ef a/., 2007) The serotypeAsia | has never been discovered outside of Asia, as well as three SAT serotypes have

5

been generally restricted in their distribution to Africa (Jamal and Belsham, 2013).

Besides, Serotype C seems to have disappeared (Paton ef a/., 2021)

MeEndemic

Sporadic

M@Free with vaccination

@Free without vaccination

» ~ 17008 0 1,700 3400 $.100 6290

Figure 2.2 The geographical distribution of FMDV (Freimanis ef a/., 2016)

In Vietnam, detecting concomitant circulation of FMDV serotypes O, A, and

Asia 1, which serotype O remained the most widespread and was responsible for the

highest number of outbreaks (Phan ef a/., 2010; Lee ef al., 2011) The FMDV serotype

O broke out in Vietnam and was first described in academic research between 1996 and

2001 (Gleeson, 2002) Later, The FMDV serotype Asia 1 and A were identified in 2005and 2009, respectively (Phan ef al., 2010)

2.1.3.2 Susceptible species

FMD affected members of the order Artiodactyla (cloven - hoofed animals), bothdomestic and wild (Coetzer ef al., 1994) Cattle, swine, small ruminants (goats andsheep), and water buffalo, particularly in Asia and South America were the species ofgreatest significance in the field (Thomson ef a/., 2003) Besides, more than 70 species

of wild mammals belonging to more than 20 families may also be involved in the naturalepidemiology of FMDV (Alexandersen and Mowat, 2005) Clinical signs are most

severe in cattle and swine, while sheep and goats usually experience subclinicalinfections (MacLachlan and Dubovi, 2017) On occasion, FMDV had also infectedanimals that are not members of the Artiodactyla, such as dogs, hedgehogs, bears,elephants, armadillos, kangaroos, nutrias, and capybaras (Spickler and Roth, 2015).Therefore, all animals could serve as mechanical vectors if they become contaminated

with the virus and subsequently came in close contact with susceptible livestock (Straw

et al., 2013)

2.1.3.3 Transmission

Cattle and swine usually become infected with FMDV by direct or indirectcontact with infected animals through contaminated fomites, and occasionally, viaconsumption of FMD - contaminated products (Siembieda ef a/., 2011) The main route

of spreading the disease was by inhalation of virus particles from the breath of acutelyinfected animals (Paton ef a/., 2018)

Because FMDV could persist in the environment for a prolonged period if thereare favorable conditions (Colenutt ef a/., 2020) Ideal conditions for virus survival wereneutral pH, temperatures below 50°C, and relative humidity above 55% (Colenutt ef aÍ.,2018)

2.1.4 Pathogenesis

FMDV had variable serotypes (7 serotypes) and a broad host range Therefore,the clinical symptoms, pathogenesis, and immune response varied with the hosts and

serotypes (Li ef a/., 2021) The pharyngeal area was the site for early localization, andwas also the primary site of infection, except when the virus has entered directly throughabrasions on the mucous membranes or skin (Stenfeldt et a/., 2014; Helke et a/., 2015).

FMDV was internalized into host cells by interaction between FMDV capsidproteins and host integrins, for example, alphaV - beta6 was found on the surface of the

target cell Integrin alphaV - beta6 was the major site for virus attachment on the transfected cells, and binding to alphaV - beta6 serves to increase the rate of virus entryinto these cells (Jackson ef al., 2000)

beta6-After initial replication in the mucosa of the pharynx, or in the skin through a

damaged integument, the virus spread to the regional lymph glands and into thecirculation (Boden, 2004) Viral seeding of secondary sites was followed by multiplecycles of viral replication and spread, with the cornified epithelia of the skin, tongue,

and mouth as are main sites of viral amplification (Alexandersen ef a/., 2001)

2.1.5 Clinical signs

The incubation period for FMDV and the severity of clinical signs depended onthe virus strain, the animal species, the exposure dose, and the route of spreading Swinewas readily infected via direct contact and had an incubation period is usually two days

or more The incubation period in swine could be as short as 18 - 24 hours and up to 9days (Kitching and Alexandersen, 2002) Typical signs of FMD were fever and theappearance of vesicles (blisters) in and around the mouth and on the feet (Zimmerman

et al., 2019) Vesicles often rupture easily and produce erosions A variety of symptomsinclude depression, anorexia, excessive salivation, lameness, and reluctance to move orrise, by painful and uncomfortable lesions (World Organisation for Animal Health)

Clinical disease is usually severe in swine The early clinical symptoms may be

lameness and blanching of the skin around the coronary bands The pain from footlesions by the development of vesicles may reluctant for the infected swine to stand orwalk, and adoption of a dog - sitting posture (Heinritzi and Bollwahn, 2001) Mouth

lesions are usually small and less apparent but severely affected swine may inappetence

become lethargic, and huddle together Fever may be present, with between 39 - 40°C(102 - 104°F) Although it may be as high as 42°C (107.6 °F), the fever is often short

and in the low degree range, making it hard to distinguish the infected from the herd

(Kitching and Alexandersen, 2002) Consequently, body temperature in swine should

not be used to exclude the possibility of FMDV infection (Zimmerman ef a/., 2019)

In adult animals, mortality was generally low (1 - 5%), but higher in young

animals (20 - 25%), especially piglets, due to acute myocarditis (World Organisation forAnimal Health) Despite negligible mortality in animals with vesicular lesions,

secondary bacterial infections in vesicular lesions may lead to chronic lameness,wasting, or mortality (Zimmerman ef al., 2019).

2.1.6 Lesion

The initial lesions often appear as blanched patches After these patches spread,they will develop into vesicles They might be found in the snout, teats, mammary gland,

prepuce, vulva, and other areas of the skin, but were most commonly found around the

mouth and on the feet (Kitching and Alexandersen, 2002) When the vesicle rupture,bacteria easily enter the wound causing secondary bacterial infection, which may

Figure 2.5 Pigs with FMDV infection displaying clinical sign and lesions (A)Grower

pigs died from the FMDV infection (B) Vesicles on the udders of sows (D) Sloughed hooves

on the front feet of a pig (E) Lesion on the tongue (F) A myocarditis lesion characterized bywhite stripes and spots (arrows) in a grower pig (Diep et al., 2020)

Vesicles on the feet of swine often appear on the heel, coronary band, andinterdigital cleft of the feet Coronitis may be found on the hooves, and the shedding of

claws may present 1f the lesion is severe (World Organisation for Animal Health) Orallesions of swine are most commonly seen on the tongue, either far back or on the

dorsum Vesicles are sometimes found on the snout or udder, as well as on the hock or

elbows if the swine are housed on rough concrete floors (Zimmerman et al., 2019)

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The criteria can be used to determine the age of lesions: development of vesicles

on days 0 - 2, rupture of vesicles on days 1 - 3, sharply marginated erosion (days 2 - 3),with the sharpness lost about day 3, sero-fibrinous exudation on days 4 - 6, andbeginning of repair with a marked fibrous tissue margin on day 7 or more (Zimmerman

et al., 2019)

According to WOAH, young animals (swine less than 8 weeks of age) usuallydie due to acute myocarditis Visual examination often reveals the heart to be soft andflaccid, with white or grayish stripes (the so-called tiger heart) or spots, seen mainly inthe left ventricle and interventricular septum

2.2 Introduction to EMCV

2.2.1 Structure of EMCV

In 1945, the Encephalomyocarditis virus (EMCV) was first isolated by Helwigand Schmidt (1945) The EMCV is belonging to the genus Cardiovirus of the familyPicornaviridae This genus contained two species: the Theilovirus andEncephalomyocarditis virus (EMCV) There are two known serotypes of the virus:

Encephalomyocarditis virus 1 1) and Encephalomyocarditis virus 2

(EMCV-2), with the second serotype lately discovered in a wood mouse in Germany(MacLachlan and Dubovi, 2017) In swine, EMCV caused acute myocarditis which

resulted in piglet’s mortality and reproductive failure in sows

As for other picornaviruses, the EMCV 1s a small non-enveloped virus, with anicosahedral shape about 30 nm diameter (Luo ef a/., 1987) The viral genome consisted

of a positive single-stranded RNA of roughly 7800 base that allows RNA to translatedirect into a polyprotein (Carocci and Bakkali-Kassimi, 2012) Because viral RNA wasinfectious in and of itself, viral proteins were not required to 1nitiate gene expression.Therefore, viral RNA was introduced into cells by transfection that is sufficient toproduce infectious viral particles During translation, viral RNA could act as an mRNAand during genome replication as a template

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Genes encoding Genes encoding structural proteins non-structural proteins

„ò2 EE ET — cr

| |

| ORF |sura—C—{ PI ] P2 l P3 F—:ut

CC ves Hf vr2}{ ves {ver Hf 2a 2p ff 2c fsa 3p fac Rap f=

Figure 2.6 Genome compositions of recombinant EMCV

(Rabouw ef a/., 2020)

The genome of EMCV also has a unique coding region flanked by two

untranslated regions (5’ and 3’ UTRs) The 5’ UTR is between 800 to 1,200 nucleotideslong, while the 3’ UTR is shorter with about 120 nucleotides long (Palmenberg ef al.,

1984) and composed of short stem-loop structures followed by a poly(A) tail of variablelength So, the viral RNA is polyadenylated at the 3’ end, but cellular mRNA is cappedwith a 7-methyl-guanosine There is also VPg, linked with the 5’ extremity of the viralRNA by a phosphodiester bond (Hruby and Roberts, 1978) Downstream, a poly(C) hasbeen found and 1s specific to picornaviruses as well as EMCV Regarding coding region,there is research showing that the EMCV genome does not have just one, but at leasttwo ORF, encoding for 13 mature proteins (Loughran ef al., 2011)

The proteins and precursors of EMCV as well as of the other picornaviruses

include the leader protein, the precursor P1, consisting of capsid proteins 1A, 1B, 1C,and 1D, which are corresponding to VP4, VP2, VP3, and VP1, respectively The P2 and

P3 are converted into non-structural proteins (Palmenberg ef a/., 1984)

2.2.2 Epidemiology

Encephalomyocarditis used to be considered a disease of subtropical or tropicalregions, but now it has a large number of susceptible hosts Currently, EMCV infectioncan be identified by virus isolation or the presence of antibodies test (Maurice ef al.,

2007) EMCV has been found in over 30 species of mammals and birds by virus

isolation The host range was mammals which includes chimpanzees, elephants, lions,mongooses, monkeys, raccoons, squirrels, and swine (both domestic and wild boar)(Zimmerman et al., 2019)

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Because infected swine can excrete the virus, the routes of virus transmissionhidden on the farm were largely due to direct contact and also contact with dead swine

by infection (Kitching and Alexandersen, 2002) Transplacental transmission alsopossible (Christianson ef a/., 1992) Despite the scarcity of rigorous risk studies, a factorassociated with the introduction or spread of EMCV into pig farms includes the presence

of rodents, which are thought to be the reservoir species and contribute to the spreadeither in their feces or as infected carcasses (Spyrou ef al., 2004) Therefore, the presence

of mice was an important risk factor for clinical EMCV infection

2.2.3 Pathogenesis

The EMCV virions entery 1s course, depends on virus strain, exposure dose, route

of transmission, and host susceptibility For instance, some strains cause bothreproductive failure and myocardial death, while others can give rise to only each of thetwo types of lesions (Koenen and Vanderhallen, 1997)

In nature, swine are most likely to be infected through the oral route So, in theexperiment, piglets are also oral infection by moculation in the intestinal tract after thatthe virus was demonstrated as early as 6 hours During the first 30 hours post-inoculation, only in the cytoplasm of isolated macrophages and myocardial cells werepositive reactions After 30 hours, some animals died with typical postmortem lesions

Necropsy detected predominantly myocardial lesions (Gelmetti ef a/., 2006)

The heart is the target organ because this is the place for virus replication, whichresulted in myocarditis (Zimmerman ef al., 2019) If myocarditis is severe, the infectedswine will die suddenly Proinflammatory cytokines such as IL-18, tumor necrosis

factor-a (TNF-a), and IL-6 have been implicated in the pathogenesis of myocarditis

caused by EMCV infections (Robinson ef al., 2009)

2.2.4 Clinical signs

Many species are infected with EMCV that are not lethal and subclinical Pigletsare usually more susceptible to clinical disease in the first weeks of life Infected pigletsare often characterized by acute disease and sudden death from heart failure Besides,other clinical signs include listlessness, loss of appetite, tremors, paralysis, grogginess,and shortness of breath Experimental infection of swine resulted in transient fever, with

a temperature of 41°C After 3 - 5 days, the infected swine might die or if they recover,they might develop chronic myocarditis Swine before weaning age are found to have a

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high fatality rate, close to 100% Infection in swine from post-weaning age to adulthood

commonly developed into subclinical disease, but mortality can still occur In

experimental infections, extremely high mortality was found at in swine 20 - 40 days

old, however none of the swine at 105 days old died

In pregnant animals, clinical signs may vary from inapparent infection to various

forms of reproductive failure, including abortion and increased numbers of stillbornfetuses and mummified (Koenen and Vanderhallen, 1997)

2.2.5 Lesion

Swine dying during of the acute phase of cardiac failure may show only

epicardial hemorrhage and no gross lesions Necropsy frequently observed are

hydropericardium, hydrothorax, and pulmonary edema The heart is usually enlarged,pale, and soft The most marked lesions are seen in the myocardium where multiple foci

of various sizes are found, especially in the right ventricle, which may extend to varying

depths within the myocardium They are often ill-defined, circular, linear, and grayish/

white in color Fattening pigs appear more frequently with these lesions than sucklingpiglets (Littlejohns and Acland, 1975)

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2.3 Real-time PCR technique

The polymerase chain reaction (PCR) was used to amplify target DNAsequences If the genetic material is RNA, single-stranded RNA is transcribed into

complementary DNA (cDNA) by a reverse transcriptase enzyme then employs this in

the process of PCR amplification This a method is called reverse transcription PCR(RT-PCR) At the end of the PCR reaction, this product is most commonly analyzed byagarose gel electrophoresis Nowadays, real-time polymerase chain reaction (Real-

time PCR) allows monitoring of the PCR progress (Artika ef al., 2020)

Real-time PCR application in research and diagnosis is usually used thequalitative (testing whether or not the target gene is present in the sample) and

quantitative purposes (determining amount of genes present in the sample)

Quantification is the outstanding advantage of the Real-time PCR method, there are two

quantification methods: relative quantification and absolute quantification (Arya ef

al., 2005)

Real-time PCR also known as quantitative PCR (qPCR) is a combination of theprinciple of traditional PCR and the detection of the fluorescence produced by a reporter

molecule which increases, as the reaction proceeds This occurs due to the PCR product's

accumulation with each amplification These fluorescent reporter molecules includedyes that bind to the double-stranded DNA (such as SYBR Green I and EvaGreen) or

sequence specific probes (such as Molecular Beacons or TaqMan Probes) (Navarro efal., 2015)

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2.3.1 DNA binding dye

SYBR Green I is the most commonly used double-stranded DNA intercalatingagent This dye attached to the minor groove of double-stranded DNA, irrespective ofits sequence It fluoresced only if inserted into double-stranded DNA Therefore, thequantity of double-stranded DNA present in the reaction is the determining factor in the

strength of the fluorescence signal The SYBR Green I have cost, convenience, and

sensitivity superiorities Besides that, the drawbacks of this dye are that they are notspecific because the probe binds to any amplified product as long as there are double-stranded DNA, targeted or non-targeted (for example primer dimers) Other DNA-binding dyes available commercially include ethidium bromide, YO-PRO-I, SYBR®Gold, SYTO, BEBO, BOXTO, and EvaGreen (Navarro et a/., 2015)

se * > k

Xu Ssð SOC PCO

>> ?e#eesipsd.

Figure 2.9 The action of SYBR Green I Dye (Artika e al., 2022)

2.3.2 Target Specific Probes

TaqMan probes consist of a 24 to 30 bases oligonucleotide with sequences

specific to the target DNA sequence that is labeled with a fluorescent dye and quencher

The 5’ end is attached to fluorescein called “reporter” and the 3’ end is attached to a

fluorescent adsorbent called a “quencher” The mechanism action of this probe depends

on the 5’ - 3’ exonuclease activity of the enzyme Taq polymerase, which hydrolyzes the

attached probe during PCR amplification If the amplification product is not present,little or no fluorescence will emit because the Taqman probe is still intact, therefore, the

quencher can absorb all the fluorescence signal emitted from the reporter However, inthe presence of PCR amplification product, the TaqMan probe will be specifically pairedwith the target DNA sequence, behind the primer binding site, and will be cleaved by

the 5’ - 3’ exonuclease activity of the enzyme Taq polymerase to synthesize

complete stand Because the cleavage separates the 5’ reporter and the 3’

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quencher, the quencher will be unable to absorb the fluorescence signal of the emitted

report Therefore, when receiving an excitation light source, the reporter will emit a

fluorescence signal proportional to the amplified amount of the targeted product (Arya

et al., 2005; Navarro et al., 2015)

a R = Reporter Polymerization Ca Onanhat

2.3.3 Amplification plot

2.3.3.1 Phases

The progress and results of the real-tme PCR reaction are monitored through areal-time PCR amplification curve plot In this plot, the vertical axis represents thefluorescence intensity emitted, and the horizontal axis represents the thermal cycles.This plot shows three main phases: linear (at the start), exponential (logarithmic-linear),and plateau phase In the first phase, the sequence of the target DNA has been amplifiedinto many copies, but the number is small, not enough for the machine to record the

emitted fluorescence signal In the exponential phase, the amplification product of the

reaction is large enough for the machine to recognize, the product after each cycleproducts increase exponentially causing the amplification curve to be pushed high Inthe final phase, the free nucleotides are exhausted, and the activity of Taq polymerase is

no longer effective, thus fluorescence intensity slowly increases and reaches a plateau(Tichopad et a/., 2003)

threshold cycle is the cycle at which the fluorescence signal of the sample is observed

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