Variable RNA expression from recently acquired, endogenous viral elements (EVE) of white spot syndrome virus (WSSV) in shrimp

Accepted Manuscript Variable RNA expression from recently acquired, endogenous viral elements (EVE) of white spot syndrome virus (WSSV) in shrimp Heny Budi Utari, Chumporn Soowannayan, Timothy W. Flegel, Boonsirm Whityachumnarnkul, Maleeya Kruatrachue PII:

S0145-305X(17)30239-2

DOI:

10.1016/j.dci.2017.07.011

Reference:

DCI 2939

To appear in:

Developmental and Comparative Immunology

Received Date: 27 April 2017 Revised Date:

9 July 2017

Accepted Date: 10 July 2017

Please cite this article as: Utari, H.B., Soowannayan, C., Flegel, T.W., Whityachumnarnkul, B., Kruatrachue, M., Variable RNA expression from recently acquired, endogenous viral elements (EVE) of white spot syndrome virus (WSSV) in shrimp, Developmental and Comparative Immunology (2017), doi: 10.1016/j.dci.2017.07.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Variable RNA expression from recently acquired,

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endogenous viral elements (EVE) of white spot

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syndrome virus (WSSV) in shrimp

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Heny Budi Utari1, 2, Chumporn Soowannayan1,3*, Timothy W. Flegel1,3, 4,

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Boonsirm Whityachumnarnkul1,5,6 and Maleeya Kruatrachue2

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CENTEX SHRIMP, Faculty of Science, Mahidol University, Rama VI Rd, Bangkok,

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10400, Thailand.

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Department of Biology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok, Thailand.

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National Center for Genetic Engineering and Biotechnology (BIOTEC), National

Science and Technology Development Agency, Thailand Science Park, Klong Luang,

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Patumthani, 12120, Thailand.

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Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd.,

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Department of Anatomy, Faculty of Science, Mahidol University, Rama VI Rd.,

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Bangkok, 10400, Thailand.

Bangkok, 10400, Thailand. 6

Shrimp Genetic Improvement Center (SGIC), Surathani, Thailand.

*Author for correspondence: [email protected]; [email protected] 1

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ABSTRACT

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The viral accommodation hypothesis proposes that endogenous viral elements (EVE)

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from both RNA and DNA viruses are being continually integrated into the shrimp

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genome by natural host processes and that they can result in tolerance to viral infection by

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fortuitous production of antisense, immunospecific RNA (imRNA). Thus, we

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hypothesized that previously reported microarray results for the presence of white spot

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syndrome virus (WSSV) open reading frames (ORFs) formerly called 151, 366 and 427

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in a domesticated giant tiger shrimp (Penaeus monodon) breeding stock might have

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represented expression from EVE, since the stock had shown uninterrupted freedom from

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white spot disease (WSD) for many generations. To test this hypothesis, 128 specimens

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from a current stock generation were confirmed for freedom from WSSV infection using

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two nested PCR detection methods. Subsequent nested-PCR testing revealed 33/128

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specimens (26%) positive for at least one of the ORF at very high sequence identity (95-

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99%) to extant WSSV. Positive results for ORF 366 (now known to be a fragment of the

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WSSV capsid protein gene) dominated (28/33 = 84.8%), so 9 arbitrarily selected 366-

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positive specimens were tested by strand-specific, nested RT-PCR using DNase-treated

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RNA templates. This revealed variable RNA expression in individual shrimp including no

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RNA transcripts (n = 1), sense transcripts only (n = 1), antisense transcripts only (n = 2)

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or transcripts of both sense (n = 5). The latter 7 expression products indicated specimens

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producing putative imRNA. The variable types and numbers of the EVE and the variable

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RNA expression (including potential imRNA) support predictions of the viral

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accommodation hypothesis that EVE are randomly produced and expressed. Positive

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nested PCR test results for EVE of ORF 366 using DNA templates derived from shrimp

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sperm (germ cells), indicated that they were heritable.

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KEYWORDS: white spot syndrome virus, WSSV, Penaeus monodon, endogenous viral

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elements (EVE), viariable RNA transcripts, variable EVE

47 INTRODUCTION

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It is well-known that viral infections in crustaceans may result in disease and mortality or

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in persistent, tolerated infections without gross signs of disease, often for a lifetime

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(Chayaburakul et al., 2005; Flegel, 1997; Flegel et al., 2004; Flegel and Pasharawipas,

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1998; Withyachumnarnkul et al., 2006). The process that results in tolerance to viral

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infection has been called viral accommodation (Flegel, 2007; Flegel and Pasharawipas,

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1998), but the mechanisms governing it are not well understood and constitute a

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challenging field of research. An updated hypothesis for the mechanism of viral

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accommodation was proposed by Flegel (2009) (Fig. 1). According to this hypothesis,

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mRNA from either DNA or RNA viruses is recognized as foreign mRNA (either directly

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or indirectly) by host reverse transcriptases that generate variable complementary DNA

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(cDNA) fragments from it. The resulting cDNA fragments are then integrated into the

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host genome, also in a variable manner, by the action of host integrase enzymes. The

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hypothesis proposes that some of these inserted fragments generate anti-sense RNA

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called immunospecific RNA (imRNA) that can bind to mRNA of the virus and induce its

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degradation by the host RNA interference (RNAi) mechanism, suppressing viral

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replication but not eliminating it. It was proposed that this would lead to low levels of

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infection, where the virus is still active but where the host exhibits no signs of disease.

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This would constitute an adaptive and heritable type of immune response, if the viral

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inserts (now called endogenous viral elements or EVE) could be transmitted via germ

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cells. Those interested in a detailed explanation of the viral accommodation hypothesis

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and in the rationale behind its formulation are encouraged to refer to the publication by

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Flegel (2009).

71 EVE for retroviruses and retroviral-like elements are well known, but recent studies have

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shown that many non-retrovirus sequences have also been naturally inserted into host

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genomes (Bézier et al., 2009a; Bézier et al., 2009b; Crochu et al., 2004; Federici and

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Bigot, 2003; Feschotte and Gilbert, 2012; Katzourakis and Gifford, 2010; Liu et al., 2011;

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Tanne and Sela, 2005; Thézé et al., 2011; Thézé et al., 2014). Examples of EVE in

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invertebrates are sequences of a discistrovirus found in the genome of bees (Apis

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mellifera) (Maori et al., 2007) and polydnaviruses (PDVs) found in the genome of

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parasitic wasps (Bézier et al., 2009b). Some hosts with EVE have been shown to be

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resistant to infection by the originating virus (Maori et al., 2007). Detailed mechanisms as

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to how these EVE might arise or how they lead to resistance, especially for non-

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retroviruses, have not yet been demonstrated except for a study described below on a

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positive sense, single-stranded RNA virus in insect cells (Goic et al., 2013).

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In a recent study on a positive sense, single-stranded RNA virus in Drosophila cells (Goic

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et al., 2013), it has been shown that complimentary strands of the viral RNA (cDNA) are

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formed by host reverse transcriptase (RT) early after infection and that these cDNA are

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integrated with a transposon element situated either in the host chromosome or extra-

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chromosomal DNA. These recombinant transposon-virus sequences were found to be the

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source of virus derived RNA interference molecules that induce viral RNA degradation

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by the RNA interference (RNAi) pathway of the host, leading to innocuous, persistent

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cell infections instead of cell death. Inhibition of RT activity led to mortality rather than

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persistent infections (Goic et al., 2013). The results of the study supported predictions of

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the updated viral accommodation hypothesis (Flegel, 2009).

95 Several EVE have been found in genomes of shrimp species. For example, sequences

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belonging to infectious hypodermal and hematopoietic necrosis virus (IHHNV) (also

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called Penaeus stylirostris densovirus or PstDNV) have been found in black tiger

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shrimp (P. monodon) (Jaroenram and Owens, 2014; Saksmerprome et al., 2011; Tang and

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Lightner, 2006) and whiteleg shrimp (P. vannamei) (Saksmerprome et al., 2011). White

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spot syndrome virus-like sequences have also been found by genome sequencing in the

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giant tiger shrimp and in kuruma shrimp Marsupenaeus japonicas (de la Vega, 2006;

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Huang et al., 2011; Koyama et al., 2010). For WSSV, the relatively low sequence

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identities of the EVE to genes of extant WSSV led the authors to suggest that they may be

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current remnants of ancient EVE of ancient WSSV or that they may be evolved forms of

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current host genes that were transferred in ancestral form to WSSV in the distant past.

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In contrast to the large WSSV-like gene fragments discovered by genome sequencing,

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WSSV cDNA sequences discovered in P. monodon in 2003 and 2004 using microarray

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technology (Hossain et al., 2004; Khadijah et al., 2003) had nucleic acid sequences

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almost identical to matching regions in the WSSV genome (i.e., regions called ORFs 89,

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151, 366 and 427). The P. monodon used in these two studies were obtained from the

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Shrimp Genetic Improvement Center (SGIC) in Thailand where they were regarded as

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specific pathogen free (SPF) for WSSV because of continual negative PCR testing for

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WSSV and lack of white spot disease for several generations. The authors (Hossain et al.,

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2004; Khadijah et al., 2003) disagreed and concluded that the shrimp they obtained from

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SGIC were not truly free of WSSV as claimed, but were instead infected with WSSV at a

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low level. They further proposed that the sequences identified arose from WSSV latency-

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related genes that were responsible for the low level of WSSV infection and that stress

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might activate the latent WSSV to cause disease. For us, these conclusions were not

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consistent with the stock history of very long freedom from WSD for several generations

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before and after the work reported in 2003 and 2004. However, there seemed to be no

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alternative explanation at the time for the presence of the expressed WSSV genome

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fragments that had very high sequence identity to matching regions of the extant WSSV

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genome.

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In succeeding years, publications about non-retroviral, shrimp EVE with high sequence

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identity to extant viruses (see above) led us to hypothesize that the WSSV cDNA from

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the microarray studies in 2003 and 2004 (Hossain et al., 2004; Khadijah et al., 2003) may

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have arisen from EVE in the shrimp genome rather than a latent virus infection. To test

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this hypothesis, we screened the current descendants of black tiger shrimp (i.e., that

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originated from the same source as those shrimp used in the previous microarray studies)

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for the presence, prevalence and expression of EVE for the WSSV ORFs called 151, 366

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and 427 in the previous studies (Hossain et al., 2004; Khadijah et al., 2003). It is

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important to note that different names have been used for these three ORFs by different

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research groups. The list of names that have been used and their references are given in

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Supplementary Table 1. ORF 366 was later found to be a small portion of the gene

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(ORF 419) for a mega protein of approximately 664 kDa which was first believed to be a

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krr1 family protein with an inferred function in 40S ribosome genesis (Tsai et al., 2004)

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but was later found to be the major protein of the stacked-ring, nucleocapsid of WSSV

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(Leu et al., 2005). For convenience in this report and for comparison to the earlier

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publications in 2003 and 2004 (Hossain et al., 2004; Khadijah et al., 2003), the original

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ORF names of 151, 366 and 427 will be used.

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144 MATERIALS AND METHODS

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Specific pathogen free (SPF) giant tiger shrimp (Penaeus monodon)

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SPF P. monodon (128 shrimp) were obtained from the Shrimp Genetic Improvement

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Center (SGIC), Surathani Province, Thailand. These shrimp were provided arbitrarily at

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the convenience of SGIC without a selection protocol, and with no distinction made as to

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whether the shrimp were male or female. These shrimp came from a stock that has been

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free of WSSV since 2003 by absence of white spot disease and by continual negative

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PCR test results for WSSV by screening using a commercial nested PCR detection

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method (IQ2000 detection system, GeneReach Corp, Taiwan) certified for WSSV

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detection by the World Organization for Animal Health (OIE).

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The origin of the SGIC stock was wild broodstock that were sourced predominantly from

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the Andaman Sea and the Gulf of Thailand prior to 2003 and subjected to stringent

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screening in a strict quarantine facility following the principles of establishing SPF stocks

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(Lotz, 1992; Lotz et al., 1995). The objective was to provide founder stocks free of a list

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of diseases including white spot syndrome virus (WSSV) before transfer to SGIC. Since

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white spot disease outbreaks were first reported in Thailand in 1995 (Wongteerasupaya et

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al., 1995), it is probable that the captured shrimp population sourced for the broodstock

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program had previously been exposed to WSSV.

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The 128 shrimp were not obtained in one lot but spread out in 9 lots over a period of 2

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years at the convenience of SGIC. Thus, they were processed in successive lots over time.

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Spermatophores from a separate group of 23 additional, arbitrarily-selected, male shrimp

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were provided by SGIC in 2 lots (preserved in 95% ethanol) late in the study to screen by

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PCR for the presence of endogenous viral elements. The relationship between these

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shrimp and those in the first group of 128 shrimp was not recorded.

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171 Nucleic acid extractions

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DNA samples were extracted from approximately 100 µl of shrimp hemolymph or 50 mg

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of shrimp tissues including shrimp pleopods or spermatophores using the phenol

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chloroform extraction method (Sambrook et al., 1989) with modifications at the cell lysis

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step in which approximately 50 mg of shrimp tissue was homogenized in 100 µl TF lysis

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buffer (50 mM Tris-HCl pH 9.0, 50 mM NaCl, 100 mM KCl, 100 mM EDTA, 2 % SDS,

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1 µg/ml Proteinase K) before extraction with phenol/chloroform/isoamyl alcohol as

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described previously (Soowannayan and Phanthura, 2011). DNA pellets obtained were re-

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suspended in 20 µl TE buffer (10 mM Tris-Cl, pH 7.5. 1 mM EDTA) buffer and treated

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with RNase A by adding 1 µl RNase A (10 mg/mL) to the re-suspended DNA before

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incubation at 37ºC for 30 min to digest any RNA that might contaminate the DNA

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samples. The total DNA concentrations were measured by a UV/Vis spectrophotometer at

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260/280 nm and stored at -20ºC. To test by PCR for EVE of WSSV or for the possible

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presence of WSSV itself, pleopod samples were usually employed

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RNA samples were extracted from hemolymph of the same shrimp used to prepare DNA

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templates for WSSV gene insert detection and also from WSSV infected shrimp samples

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which provided positive control RNA. To extract RNA from hemocytes, hemolymph

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(500 µl) was centrifuged for 10 min at 1000xg to collect the cells. The cell pellet obtained

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was homogenized in 500 µl TRIzol® LS. The extraction was done by using the protocol

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suggested by the TRIzol® LS reagent manufacturer. The RNA pellets obtained were re-

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suspended in 20 µl DEPC water and stored at -80oC.

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Before use in RT-PCR reactions, any possible traces of genomic DNA were removed by

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incubation of total RNA (1 µg) with a solution containing 1 µl DNase I solution (1000

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U/ml, New England Biolab, USA), 1 µl 10xDNase I buffer and 6 µl H2O at 37ºC for 30

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min. Then 1 µl 50 mM EDTA was added to the mixture followed by termination at 60°C

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for 10 min. The total RNA concentration was then measured by a UV/Vis

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spectrophotometer at 260/280 nm.

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PCR tests to insure absence of WSSV infection

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It was critical in this study to establish that the shrimp used to screen for EVE were not

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infected with WSSV, even at very low levels that might be characteristic of latent

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carriers. To do this, we calibrated the PCR methods used for detection of WSSV and for

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detection of EVE to ensure that detection sensitivity was higher for viral infection than

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for putative EVE. Our principle was that any detection method that gave a positive result

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for a WSSV DNA target with one detection method of known sensitivity must give

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positive results for all other WSSV DNA targets when using methods of the same

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sensitivity. The main standard used for detection of WSSV infection was the commercial,

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nested PCR detection method (IQ2000 detection system, GeneReach Corp, Taiwan)

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certified for WSSV detection by the World Organization for Animal Health (OIE) with a

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sensitivity of detection of approximately 20 viral targets per PCR reaction vial. A second,

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nested PCR method used to confirm absence of WSSV was developed based on the

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WSSV gene for envelope protein Vp28. All shrimp specimens used to screen for EVE

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were confirmed to give negative test results with both the IQ2000 and the Vp28 nested

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PCR methods (i.e., 2 distinct methods). They were subsequently tested using less

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sensitive nested PCR methods for ORFs 151, 366 and 427, such that any positive test

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results could not possibly have arisen from WSSV infections. Details for the process of

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optimization and sensitivity comparison for these 4 PCR methods are given in

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Supplementary Information 1. The following paragraphs summarize only the final

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optimized protocols for the 4 PCR detection methods used.

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To ensure that nucleic acids extracted were of good quality, the host housekeeping gene

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beta actin was also amplified from both DNAs and cDNAs using β-actin primers: β-actin

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F: 5’-CCC CAT TGA GCA CGG TAT CA-3’) and β-actin R: 5’- ACG CTC AGG AGG

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AGC AAT GA-3’). The PCR reaction conditions were as follows, 94°C for 2 min

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followed by 30 cycles of 94°C 30 sec, 60°C 30 sec, 72°C 30 sec, 72°C 1 min and final

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extension at 72°C for 7 min. Examples of results are shown in Supplementary Fig. 2 and

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3a. These tests were additional to tests done with the same samples using the IQ2000 kit

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that also included a β-actin housekeeping gene control. The kit gives a clear PCR

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amplicon band with host DNA containing no WSSV DNA and progressively weaker

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amplicon bands when there are increasingly intense WSSV amplicon bands with

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increasing concentrations WSSV target DNA. Thus, the β-actin control amplicon is not

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visible with DNA samples derived from severe infections (see below).

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RNAse-treated DNA samples were screened for WSSV infection by nested PCR using

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the IQ-2000TM detection kit (GeneReach Corp, Taiwan) that has been certified by the

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World Organization for Animal Health (OIE) as a standard PCR kit for WSSV detection.

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The detection procedure was performed according to the kit instructions, except that a

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fixed quantity of 200 ng of shrimp template DNA was used. The PCR products were

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analyzed on 2% agarose gel with ethidium bromide staining. Positive samples showed 2

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bands at 295 and 550 bp, while negative samples had only one band at 848 bp for the

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house-keeping ß-actin gene.

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To screen for WSSV using the Vp28 method, the first-step PCR was performed in 25 µl

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of final reaction mix containing 200 ng of shrimp DNA template, 2.5 µl of 10xPaq5000

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reaction buffer (Stratagene, USA), 0.3 µl of 10 mM dNTPs, 0.5 µl of 10 mM of each

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outer primers, forward VP28F-O, 5’-CTC GTG GTT TCA CGA GGT TG-3’) and reverse

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(VP28R-O, 5’-TCC GCA TCT TCT TCC TTC AT-3’), 0.25 µl of 500 U/µl of

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Paq5000TM DNA polymerase (Stratagene) and 19.95 µl of distilled water. The nested

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PCR reaction was prepared as with the first PCR, but with inner forward primer (VP28F-

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I, 5’-TGT GAC CAA GAC CAT CGA AA-3’) and reverse (VP28R-I, 5’-ATT GCG

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GAT CTT GAT TTT GC-3’) together with 1 µl of the first PCR product used as template.

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All PCR reactions were carried out using a T Professional Basic Gradient thermal cycler

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(Biometra, Germany) with the following PCR conditions for both PCR reactions: one

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cycle of 94°C for 2 min, followed by 25 cycles of 94°C for 30s for the first PCR (35

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cycles for the nested PCR), 58 oC for 30s and 72°C for 30s followed by a final extension

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step at 72°C for 7 min. The first PCR reaction generated a 400 bp amplicon and the

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second a 157 bp amplicon.

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261 Similar PCR methods to the Vp28 method were applied to the three targeted WSSV

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ORFs 151, 366 and 427 except that the shrimp DNA template was increased to 400 ng.

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The primer sequences and the expected target sizes for each pair are listed in Table 1. For

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the nested PCR step, the template consisted of 1µl of the final reaction solution from the

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first PCR step. PCR products were analyzed by 2% agarose gel electrophoresis with

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ethidium bromide staining followed by digital photography on a UV transilluminator.

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PCR tests for presence of EVE of ORFs 151, 366 and 427

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To test our hypothesis that results from the microarray studies in 2003 and 2004 (Hossain

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et al., 2004; Khadijah et al., 2003) may have arisen from EVE in the shrimp genome

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rather than a latent virus infection, we used the primer sequences described for ORFs 151,

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366 and 427 in the Khadijah and colleagues (2003) publication. These protocols were

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found to be less sensitive than those of the WSSV test protocols described in the previous

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section. This would insure that any positive PCR amplicon obtained could not have arisen

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from the presence of WSSV itself, since the pre-screening method using two other, more

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sensitive WSSV detection methods (IQ2000 and Vp28) had already been carried out and

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given negative results.

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For ORF 151, the first-step PCR protocol was 94°C for 2 min followed by 25 cycles of

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94°C for 30s, 60°C for 30s and 72°C for 4 min followed by final extension at 72°C for 7

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min yielding an amplicon of 4307 bp. The nested-step protocol was 94°C for 2 min

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followed by 35 cycles at 94°C for 30s, 60°C for 30s and 72°C for 30s followed by a final

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extension step at 72°C for 7 min yielding an amplicon of 510 bp.

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285 For ORF 366, the first-step protocol was 94°C for 2 min followed by 25 cycles 94°C for

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30s, 58°C for 30 sec and 72°C for 30s followed by a final extension step at 72°C for 7

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min yielding an amplicon of 252 bp. The nested-step protocol was 94°C for 2 min

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followed by 35 cycles of 94°C for 30s, 67°C for 30s and 72°C for 30s followed by a final

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extension step at 72°C for 7 min yielding an amplicon of 160 bp.

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For ORF 427, the first-step protocol was 94°C for 2 min followed by 25 cycles of 94°C

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for 30s, 60°C for 30s and 72°C for 1.50 min followed by a final extension step at 72°C

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for 7 min yielding an amplicon of 1866 bp. The nested-step protocol was 94°C for 2 min

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followed by 35 cycles of 94°C for 30s, 62°C for 30s and 72°C for 40s, followed by a final

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extension step at 72°C for 7 min yielding an amplicon of 900 bp. PCR products were

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analyzed by 2% agarose gel electrophoresis with ethidium bromide staining followed by

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digital photography on a UV transilluminator.

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RNA expression from the putative EVE of WSSV ORF 366

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Since putative EVE for ORF 366 were clearly the most prevalent in the shrimp screened

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in this study, sense-specific RT-PCR tests were carried out for it only. To prepare cDNA,

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a SuperScript® III reverse transcriptase cDNA synthesis kit (Invitrogen, USA) was

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employed using the protocol recommended by the manufacturer and using strand specific

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RT primers for ORF 366 (Table 1). This protocol has been shown to be effective in

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detecting sense and anti-sense RNA transcripts in a highly specific manner, with no

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interference from primer-independent cDNA synthesis (Haddad et al., 2007; Mehra and

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Wells, 2015). Each RNA sample extracted from hemolymph was subjected to RT using a

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sense primer in one reaction and an antisense primer in another reaction followed by

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separate PCR reactions using 1 µl of the cDNA reaction solution as the template in PCR

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tests for ORF 366. Antisense RNA expression was indicated by a positive PCR result

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obtained using cDNA resulting from a sense-primer while sense RNA expression was

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indicated by a positive PCR result obtained using cDNA resulting from an antisense

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primer. No positive result with either primer type indicated no detectable RNA transcript

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from the target EVE.

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316 Heritability of EVE of ORF 366

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DNA was extracted from spermatophores of an independent set of 23 arbitrarily selected

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male broodstock that were obtained from SGIC and pre-screened for absence of WSSV

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infection as described above. The samples were then subjected to PCR testing for the

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EVE of ORF 366 target. Spermatophores were used as the target tissue for DNA

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extraction because they have a thin chitin shell that contains only shrimp sperm that are

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non-motile (they have a spike only) and very rarely, if ever, contain mitochondria. Thus,

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DNA extracted from shrimp spermatophores is overwhelmingly dominated by

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chromosomal DNA so that any positive PCR test result for ORF 366 would strongly

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indicate that the target sequence arose from chromosomal DNA (i.e., excluding

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mitochondrial DNA).

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Cloning and sequencing of PCR products

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PCR products obtained from gels yielding positive bands for WSSV target sequences

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were excised, purified, and cloned into a plasmid vector (pCR®2.1 vector) and the

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plasmid DNA with inserts were sequenced by Macrogen, Korea. Three clones were

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sequenced from each specimen in both directions and the 6 sequences obtained were

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aligned to produce one consensus sequence for each specimen, so that any differences

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reported should be real. Sequences obtained were manually curated and subjected to

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homology searches using the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST).

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Multiple sequence alignments of nucleotide sequences were performed using Clustal W

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(http://www.genome.jp/tools-bin/clustalw).

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339 RESULTS AND DISCUSSION

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EVE for WSSV ORFs 151, 366 and 427 were detected

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Of the 128 DNA samples from broodstock specimens, all tested negative for WSSV using

343

both the IQ2000 nested PCR kit and the Vp28 detection method. Examples of gels

344

analyzing these PCR products are shown in Figs. 2a and b, respectively. The relatively

345

uniform amplicon bands obtained for the host β-actin gene internal control using the

346

IQ2000 kit with template DNA extracts from all 128 specimens verified the quality of

347

their DNA extracts for use as templates in all subsequent PCR reactions. Of these

348

samples, 33/128 (25.8%) gave positive test results for at least one of the 3 target ORF

349

(Table 2). Of these 33 samples, 30 (90.9%) were positive for sequences of only one ORF,

350

2 (6.0%) were positive for two ORFs (366 and 151) and 1 (3.0%) was positive for all 3

351

ORFs. Examples of gel photographs for the nested PCR tests are shown in Figs. 3a-c.

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When the nested PCR products obtained were excised from agarose electrophoresis gels,

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extracted, purified, cloned and sequenced, it was found (after removal of vector and

355

primer sequences) that nested PCR products amplified using primers targeting WSSV

356

ORF 151 (3 shrimp samples) had high identity (one 97% and two 98%) to ORF 151 of

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WSSV (GenBank accession number AF332093.2). For ORF 366, bands from 3 arbitrarily

358

selected shrimp samples also had high identity (two 96% and one 100%), while 3 from

359

ORF 427 all showed 95% identity to matching regions from the same accession number

360

(Supplementary Table 2). Overall the results revealed that the 128 individual shrimp

361

gave either negative results (95/128 samples = 74.2%) for the target sequences or gave

362

variable positive results (33/128 samples 25.8%) for 1 putative EVE (30/33 samples), for

363

2 putative EVE (2/33 samples) or for all 3 putative EVE (1/33). Since 3 shrimp had more

364

than 1 putative EVE, the total number of 37 putative EVE detected came from 33 shrimp

365

tested, and the target for ORF 366 was present in 28 of these 33 shrimp (85%) either

366

alone (25/33 shrimp), together with ORF 427 (2/33) or together with both ORFs 427 and

367

151 (1/33).

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357

368

We have done no work to determine the reason why ORF366 was the most prevalent of

370

the three ORF studied. However, if the viral accommodation hypothesis is correct, a

371

variety of EVE would arise from a single viral mRNA in millions of individuals in a

372

population and their succeeding generations. The population would be subjected to

373

selective pressure favoring individuals with EVE that confer host ability to become

374

infected but not develop disease (Flegel, 2009). Following this reasoning, the higher

375

prevalence of ORF 366 would suggest that it was more prevalent because of some

376

selective advantage. That is why our ongoing work is now focused on ORF366. At the

377

same time, we believe that maintenance of a domesticated shrimp breeding stock for

378

many generations in a high quarantine environment without any viral selective pressure

379

and without knowledge of protective EVE (if they existed in that population) might lead

380

to their random loss over time as other desirable traits were selected for.

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381 In summary, we have shown that DNA extracts from approximately 25% of individuals in

383

a WSSV-free breeding population of giant tiger shrimp P. monodon contained at least one

384

of three previously reported target ORF (151, 366 and 427) of WSSV (Hossain et al.,

385

2004; Khadijah et al., 2003) in variable assortments (single to multiple occurrence). The

386

single occurrences outnumbered the double and the double outnumbered the triple as

387

would be expected for independent random events, and as was found with multiple EVE

388

of IHHNV in P. monodon (Brock et al., 2013; Saksmerprome et al., 2011).

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By careful calibration of PCR testing sensitivity (See supplementary information 1 and

391

Supplementary Fig. 1), we showed that our positive test results for these 3 ORFs could

392

not have resulted from sub-clinical infections of WSSV. All of the shrimp we employed

393

tested negative for WSSV using an OIE-approved, commercial, nested PCR detection

394

method plus an additional in-house nested PCR method targeting the WSSV Vp28

395

envelope protein gene. It is highly improbable that these negative test results arose

396

because of simultaneous mutations of primer target regions for both methods in so many

397

individual shrimp specimens. In addition, both of these methods could detect WSSV at

398

lower concentrations than our methods for detecting the three target ORFs. Also, the test

399

results in Table 3 re-confirm these facts since positive test results for only one of the 3

400

target ORF (30/33 specimens) would have been impossible to obtain with WSSV-infected

401

shrimp that would necessarily had to give positive results for all 3 ORF.

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402 403

Put in another way, all 33 specimens were each subjected to 5 PCR tests for widely

404

separated genes in the WSSV genome and 30/33 gave negative test results for 4/5 of these

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tests, 2 gave negative results for 3/5 tests and 1 gave negative results for 2/5 tests.

406

Further, our PCR testing was done from 2009 to 2013 with shrimp stocks that have

407

continually tested free of WSSV using the IQ2000 method and have produced no WSSV

408

disease outbreaks from 2005 up to the time of submission of this manuscript (April 2017).

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Altogether, the results revealed that some of the individual shrimp in the stocks under

411

study contained target DNA of WSSV ORFs 151, 366 and 427, with ORF 366 being by

412

far the most prevalent EVE of the three. Similar results for variable numbers and types of

413

EVE in shrimp DNA have been reported for infectious hypodermal and hematopoietic

414

necrosis virus (IHHNV) in P. monodon and P. vannamei (Brock et al., 2013;

415

Saksmerprome et al., 2011). In addition, two identified fragments from one of these

416

studies were proven to be EVE by genome walking to determine their insertion points in

417

the genome of P. monodon (Saksmerprome et al., 2011). Thus, the simplest explanation

418

for our results is that the 3 identified fragments of WSSV ORF constitute EVE in the

419

shrimp DNA extracts examined. This supports our hypothesis that earlier reports on the

420

presence of WSSV gene sequences for ORFs 151, 366 and 427 in Thai P. monodon

421

stocks from SGIC arose from WSSV EVE and not from latent WSSV infections (Hossain

422

et al., 2004; Khadijah et al., 2003).

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We believe that these EVE are most likely located in genomic DNA of the tested shrimp

425

based on previous publications regarding EVE of WSSV in shrimp chromosomal DNA

426

(Huang et al., 2011; Koyama et al., 2010) and regarding EVE of IHHNV in shrimp DNA

427

(Saksmerprome et al., 2011), combined with evidence for Mendelian inheritance of the

428

latter (Brock et al., 2013). Although we carried out additional tests (see below) showing

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evidence for ORF 366 PCR targets in shrimp sperm (germ cells), absolute proof of the

430

chromosomal location of the targets for detection of ORFs 151, 366 and 427 by PCR

431

would depend on confirmation by host genome sequencing or by tests for Mendelian

432

inheritance. In any case, the fact that the EVE occur in an apparent random manner in

433

terms of numbers and types in individual shrimp fulfills one of the predictions of the viral

434

accommodation hypothesis (Flegel, 2009).

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RNA expression from EVE of ORF 366 is individually variable

437

Using 8 specimens arbitrarily selected from the group of 25 shrimp specimens positive

438

for ORF 366 only, RNA expression analysis by strand specific RT-nested PCR

439

amplification revealed only 1 shrimp sample with RNA of sense polarity, 2 with anti-

440

sense polarity, 5 with both polarities and 1 with neither polarity (i.e., no detectable RNA

441

expression). Examples of the sense-specific RT-PCR results are shown in Fig. 4. To re-

442

confirm absence of WSSV infection in the original shrimp samples, these RNA samples

443

were also tested for Vp28 mRNA expression by nested RT-PCR and all gave negative

444

results.

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In summary, expression patterns for the EVE of WSSV ORF 366 were variable, ranging

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from no expression to single sense or antisense expression and to expression of both

448

senses. There appeared to be no particular pattern, except for the unexpected fact that,

449

simultaneous expression of both sense and antisense targets dominated (5/8 = 63%)

450

(Table 3).

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This variation in expression suggested that the EVE had been inserted into the host

453

genome in a random manner with respect to orientation and presence or absence of host-

454

recognized promoters. These results are consistent with predictions of the viral

455

accommodation hypothesis that cDNA produced from viral mRNA via RT of host origin

456

would be inserted into the host genome by host integrases in a random manner and lead to

457

variable RNA expression (Flegel, 2009). However, the high proportion of shrimp (5/8)

458

that gave both positive sense and negative sense expression in our study was unexpected.

459

This could have resulted from simultaneous expression from both strands of a single EVE

460

or from expression of two independent EVE of opposite orientation. Dual to multiple

461

EVE from IHHNV in P. monodon and P. vannamei have been reported, but no work was

462

done on their RNA expression patterns (Saksmerprome et al., 2011). Although WSSV has

463

a double-stranded DNA genome and has genes expressed from both strands, there have

464

been no reports of transcript expression from both strands in the same region. Neither has

465

this been reported for genes of shrimp. Thus, our working hypothesis is that the results

466

obtained indicate the presence of at least 2 different EVE of opposite orientation, either as

467

different alleles of a chromosome pair, as different EVE in tandem on a single

468

chromosome or as EVE on different chromosomes. It is hoped that this will be

469

determined in further work on breeding tests. Whatever the case, the presence of both

470

suggests that they might bind with one another to produce dsRNA that might, in turn, be

471

capable of directly activating the host RNAi mechanism against mRNA for ORF 366 of

472

WSSV.

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473 474 475

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EVE of WSSV ORF 366 are present in shrimp sperm DNA

477

PCR testing for EVE of ORF 366 in spermatophores from 23 arbitrarily selected

478

broodstock shrimp that had been pre-screened for absence of detectable WSSV using both

479

the IQ-2000 and the Vp28 detection methods, revealed that 14 out of 23 samples (60.9%)

480

were positive (Fig. 5a). Cloning, sequencing and comparison with the matching sequence

481

of WSSV ORF at GenBank (accession number AF332093.2) revealed that amplicons

482

from 9/12 shared 100% identity, 1/12 shared 99.3% identity, 1/12 shared 98.1% identity

483

and 1/12 shared 97.5% identity (Fig. 5b). Since shrimp sperm do not carry mitochondria,

484

the results strongly suggested that the PCR target sequences were present in chromosomal

485

rather than mitochondrial DNA.

486

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These results support the proposal that the EVE for ORF 366 can be transmitted to shrimp

488

offspring, probably in a Mendelian fashion. Mendelian inheritance in shrimp has been

489

demonstrated for an EVE of infectious hypodermal and hematopoietic necrosis virus

490

(IHHNV) in P. monodon (Brock et al., 2013). Similar tests would need to be carried out

491

to prove chromosomal location of the EVE for ORF 366 in the breeding stock used.

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With respect to the higher prevalence of ORF 366 in the arbitrarily selected

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spermatophore group of shrimp (60.9%) when compared to the prevalence (21.9%) in the

495

earlier, arbitrarily-selected group of 128 shrimp, we can only speculate. We did do a Chi

496

square test using the “expected” prevalence as the proportion of overall ORF 366 positive

497

samples (28 + 14 = 42) in the total number of both groups (128 + 23 = 151) as 42/151 =

498

27.8%. The Chi square test revealed that the proportion of ORF 366 positive samples in

499

the spermatophore group (60.9%) was significantly higher than expected (p = 0.037)

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while that in the original group of 128 shrimp was not significantly different (p = 0.312).

501

We suspect that the significant difference in prevalence arose because the group of 23

502

spermatophore shrimp was obtained in 2 lots and came from a proportionally smaller

503

number of parental pairs than did the group of 128 shrimp obtained in 9 lots. This

504

variable could affect the prevalence greatly. For example, a single parental pair with one

505

heterozygous parent would yield offspring 50% positive for 366 while a pair with both

506

parents heterozygous would yield offspring 75% positive and a pair with a single

507

homozygous parent would yield offspring 100% positive. Variation in the number of

508

parental pairs with various combinations of homozygosity, heterozygosity or absence of

509

ORF 366 in the parents could lead to unlimited variation in pooled prevalence. In any

510

case, these issues are not relevant to the objective of our experiment, which was simply to

511

determine whether EVE 366 could be found in spermatophore DNA and indicate

512

probable Mendelian heritability.

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500

Old and new EVE of WSSV

515

An interesting feature of the revealed EVEs was their high sequence identity (95-100%)

516

to matching sequences of currently existing WSSV genomes. This contrasted with the

517

low sequence identity of WSSV EVE previously reported from genome sequencing work

518

using P. japonicus (Koyama et al., 2010) and P. monodon (de la Vega, 2006; Huang et

519

al., 2011). We agree with those publications that high EVE sequence identity would

520

indicate relatively recent acquisition from extant WSSV while low identity would

521

indicate ancient acquisition from ancient WSSV. There are also reports for EVE of

522

IHHNV in P. monodon that have very high sequence identity (~96% up) (Saksmerprome

523

et al., 2011) to sequences of extant, infectious IHHNV in addition to reports of EVE that

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have much lower sequence identity (~80%) (Tang and Lightner, 2006) that may be

525

interpreted in the same manner. It was found that dsRNA silencing of ancient EVE of

526

WSSV in P. japonicus could either decrease or improve survival after WSSV challenge,

527

indicating that individual EVE may or may not be protective (Dang et al., 2010).

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528

The polymerase enzyme used in this study was Paq5000 (former Stratagene now Agilent)

530

which according to the manufacturer, is a robust enzyme derived from a Pyrococcus

531

(hypothermophilic archeabacterium) species that provides equivalent and/or better

532

performance than Taq DNA polymerase. According to Lundberg and colleagues (1991)

533

the error rates of Pyrococcus furiosus (Pfu) DNA polymerase is 1.6 x 10-6 per nucleotide

534

which is about 10 times less than that of Taq polymerase (2x10-5 per nucleotide) after

535

approx. 105 folds amplification. According to McInerney and colleagues (2014) the error

536

rate of the Pfu polymerase is 2.8x10-6/nucleotide while that of Taq polymerase is 3.0 x 10-

537

5

538

test sample (2 reads each for 3 clones) could have arisen from either our PCR reactions or

539

PCR sequencing errors, but we believe that the consensus sequences derived from the 6

540

reads for each sample represent actual sequences from the specimens used and that

541

differences between these consensus sequences and the GenBank reference sequences we

542

used are real. At the same time, the sequence differences between the EVE

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/nucleotide. We believe that the small differences in our 6 raw sequence reads for each

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EVE reported from honey bees (Maori et al., 2007) and wasps (Bézier et al., 2009b), have

545

high sequence identity to extant viruses and are associated with host tolerance to those

546

viruses. However, there is as yet no evidence that EVE with high sequence identity to

547

extant IHHNV and WSSV reported for P. monodon and P. vannamei are associated with

23

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protection against disease from these viruses. The proven existence in breeding stocks of

549

EVE with high sequence identity to extant WSSV (herein) and IHHNV of P. monodon

550

(Brock et al., 2013) and P. vannamei (Saksmerprome et al., 2011) opens the way to test

551

whether such EVE are protective. For example, stocks could be screened to identify one

552

parent with a high identity EVE that gives rise to a negative sense RNA transcript and it

553

could be mated with a parent lacking that EVE. Analysis of the offspring from this cross

554

would reveal whether or not the EVE was inherited in a Mendelian fashion. Ideally, the

555

EVE-positive parent would be heterozygous for the EVE so that 50% of the offspring

556

would be positive for it and 50% not. Separating the offspring into two groups based on

557

presence or absence of the EVE followed by challenge with the appropriate virus (in

558

sufficient replicates and accompanied by appropriate controls) would reveal a protective

559

EVE if survival in the EVE positive group was significantly higher than in the negative

560

group.

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CONCLUSIONS

563

Our overall results reveal that EVE of WSSV occur in individual host shrimp DNA in

564

random types and numbers for fragments of the 3 WSSV ORF and that the EVE of ORF

565

366 was most prevalent in the shrimp breeding stock examined. In addition, expression of

566

RNA from EVE of ORF 366 was variable in 9 individual shrimp, ranging from no

567

expression (1), positive-sense expression (1), negative sense expression (2) and both

568

positive-sense and negative-sense expression (5). The 2 negative sense RNA transcripts

569

for ORF 366 would constitute candidates for potential binding to WSSV mRNA to form

570

dsRNA and activate the RNAi defense mechanism while the 5 dual-sense transcripts may

571

be able to activate the mechanism directly. These results constitute support for predictions

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572

of the viral accommodation hypothesis and lay a foundation for further work focused on

573

EVE 366 and their potential to protect shrimp against WSD.

574 Acknowledgements

576

This project was supported by the Office of the Higher Education Commission, Thailand

577

and Mahidol University under the National Research Universities Initiative. HB was the

578

recipient of a PhD scholarship from Central Proteinaprima Company of Indonesia and

579

from the CENTEX shrimp, Mahidol University of Thailand. The authors wish to thank

580

Dr. Pattira Pongtippatee of Prince of Songklanakharin University and Miss Somjai

581

Wongtreepob of the shrimp genetic improvement center, Chaiya, Surathani and the Thai,

582

National Center for Genetic Engineering and Biotechnology for providing spermatophore

583

samples and SPF shrimp samples respectively.

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584 References

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Liu, H., Fu, Y., Xie, J., Cheng, J., Ghabrial, S.A., Li, G., Peng, Y., Yi, X., Jiang, D., 2011. Widespread endogenization of densoviruses and parvoviruses in animal and human genomes. J Virol 85, 9863-9876 Lotz, J.M., 1992. Developing specific-pathogen-free (SPF) animal populations for use in aquaculture: A case study for IHHN virus of penaeid shrimp, in: Fulks, W., Main, K.L. (Eds.), Diseases of Cultured Penaeid Shrimp in Asia and the United States The Oceanic Institute, , Honolulu. HI. USA. Lotz, J.M., Browdy, C.L., Carr, W.H., Frelier, P.F., Lightner, D.V., 1995. USMSFP suggested procedures and guidelines for assuring the specific pathogen status of shrimp broodstock and seed, in: Browdy, C.L., Hopkins, J.S. (Eds.), Swimming Trough Troubled Water, Proceedings of the Special Session on Shrimp Farming. . World Aquaculture Society, Boca Raton, USA, pp. 66-75. Lundberg, K.S., Shoemaker, D.D., Adams, M.W., Short, J.M., Sorge, J.A., Mathur, E.J., 1991. High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. 108, 1-6. Maori, E., Tanne, E., Sela, I., 2007. Reciprocal sequence exchange between non-retro viruses and hosts leading to the appearance of new host phenotypes. Virology 362, 342349. McInerney, P., Adams, P., Hadi, M.Z., 2014. Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase. 2014, 8. Mehra, P., Wells, A.D., 2015. Long-Range Transcriptional Control of the Il2 Gene by an Intergenic Enhancer. Mol Cell Biol 35, 3880-3891. Saksmerprome, V., Jitrakorn, S., Chayaburakul, K., Laiphrom, S., Boonsua, K., Flegel, T.W., 2011. Additional random, single to multiple genome fragments of Penaeus stylirostris densovirus in the giant tiger shrimp genome have implications for viral disease diagnosis. Virus Res 160, 180-190. Sambrook, J., Fritschi, E.F., Maniatis, T., 1989. Molecular cloning: a laboratory manual, first ed. Cold spring harbor laboratory press, New York. Soowannayan, C., Phanthura, M., 2011. Horizontal transmission of white spot syndrome virus (WSSV) between red claw crayfish (Cherax quadricarinatus) and the giant tiger shrimp (Penaeus monodon). Aquaculture 319, 5-10. Tang, K.F.J., Lightner, D.V., 2006. Infectious hypodermal and hematopoietic necrosis virus (IHHNV)-related sequences in the genome of the black tiger prawn Penaeus monodon from Africa and Australia. Virus Res 118, 185 - 191. Tanne, E., Sela, I., 2005. Occurrence of a DNA sequence of a non-retro RNA virus in a host plant genome and its expression: evidence for recombination between viral and host RNAs. Virology 332, 614-622. Thézé, J., Bézier, A., Periquet, G., Drezen, J.-M., Herniou, E.A., 2011. Paleozoic origin of insect large dsDNA viruses. P Natl Acad Sci USA 108, 15931-15935. Thézé, J., Leclercq, S., Moumen, B., Cordaux, R., Gilbert, C., 2014. Remarkable diversity of endogenous viruses in a crustacean genome. Genome Biol Evol 6, 2129-2140. Tsai, J.M., Wang, H.C., Leu, J.H., Hsiao, H.H., Wang, A.H.J., Kou, G.H., Lo, C.F., 2004. Genomic and proteomic analysis of thirty-nine structural proteins of shrimp white spot syndrome virus. J Virol 78, 11360-11370. Withyachumnarnkul, B., Chayaburakul, K., Supak, L.A., Plodpai, P., Sritunyalucksana, K., Nash, G., 2006. Low impact of infectious hypodermal and hematopoietic necrosis virus (IHHNV) on growth and reproductive performance of Penaeus monodon. Dis Aquat Organ 69, 129-136.

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652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699

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700 701 702 703 704 705

Wongteerasupaya, C., Vickers, J.E., Sriurairatana, S., Nash, G.L., Akarajamorn, A., Boonsaeng, V., Panyim, S., Tassanakajon, A., Withyachumnarnkul, B., Flegel, T.W., 1995. A non-occluded, systemic baculovirus that occurs in cells of ectodermal and mesodermal origin and causes high mortality in the black tiger prawn Penaeus monodon. Dis Aquat Organ 21, 67-76.

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706 707

SC

708 709

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710 711 712

716 717 718

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715

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714

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713

719 720 721

28

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Figures

723

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722

Figure 1. Diagram of the viral accommodation hypothesis showing steps in the dedicated

725

host-cell mechanism for development of endogenous viral elements upon infection by

726

DNA or RNA viruses 1) The process begins with entry of foreign viral messenger RNA

727

into the host cell cytoplasm. 2) The foreign mRNA is recognized either directly or

728

indirectly by host reverse transcriptase that then produces random cDNA fragments from

729

it in terms of length and portion of genome sequence. 3) The resulting cDNAs are

730

inserted into the host genomic DNA as EVE in a random manner and orientation in

731

compatible, non-coding regions. 4) Antisense RNA fragments (imRNA) are generated

732

from some of the EVE. 5) The imRNA fragments bind with viral mRNA, producing

733

dsRNA and inducing the host RNA interference (RNAi) mechanism.

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734 29

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ACCEPTED MANUSCRIPT

736

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735

Figure 2.

Examples of electrophoresis gels of PCR products obtained from

738

broodstock screening for WSSV. (a) Example of a gel showing negative results

739

obtained using the IQ2000 detection kit (i.e., one band for the beta actin gene control at

740

848 bp to confirm DNA template integrity). M = molecular weight markers showing three

741

bands of 848 bp, 630 bp and 333 bp. Neg = negative control with uninfected shrimp DNA

742

as template, Pos = DNA from WSSV infected shrimp was used as template. (b) Example

743

of gel showing negative results for detection of the Vp28 gene in all broodstock

744

hemolymph samples (157 bp product) using DNA templates from the same extracts

745

verified for integrity by the IQ2000 tests. All samples are negative. The negative control

746

(Neg) was ddH2O and the positive control (Pos) was template DNA from WSSV-infected

747

shrimp. M = 100 bp marker (Invitrogen, USA).

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737

30

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748

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749

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750

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751

Figure 3. Agarose gels showing amplicons for WSSV gene inserts. (a) Example

754

electrophoresis gel showing amplicons for WSSV ORF 151 obtained by nested PCR.

755

using template DNA from specimens 2, 7, 8 and 11 (amplicon 510 bp). Other samples

756

showed a faint non-specific band at approximately 650 bp. Sequencing confirmed no

757

homology to genes of WSSV. (b) Example gel showing amplicons of ORF 366 (160 bp)

758

for specimens 1, 4, 5, 7, 9, 11, 12 and 14. M = 100 bp marker (Invitrogen, USA). (c)

759

Example of gel showing amplicons of ORF 427 (900 bp) for samples 46, 52 and 55. For

760

all tests the negative control was ddH2O and the positive control was DNA template from

761

a WSSV-infected shrimp sample.

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752 753

31

ACCEPTED MANUSCRIPT

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762

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763 764

Figure 4. RNA expression of EVE 366 by 9 arbitrarily selected specimens. Example

766

agarose gel showing amplicons from sense specific RT-PCR. Amplicons obtained with

767

the antisense primer indicate sense RNA expression while those obtained with the sense

768

primer indicate antisense RNA expression. Shrimp samples 1 and 2 gave amplicons

769

indicating and RNA template of antisense polarity, sample number 4 gave amplicons

770

indicating RNA templates of both sense and antisense polarity and sample 3 gave no

771

amplicons indicating no complementary RNA template. All RNA samples used were

772

extracted from hemolymph. M = 100 bp marker (Invitrogen, USA).

774 775 776

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773

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765

777 778 779 780 32

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781

ClustalW multiple sequence alignment of putative WSSV ORF 366 insert amplificons

783

from shrimp spermatophore DNA.

784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835

M24 M22 M13 M10 M9 M7 M4 M1 M2 59_9 80_14 08_6 WSV_ORF366

----------------------------------------GAGACGTCGCTCATCAAAGA ----------------------------------------GAGACGTCGCTCATCAAAGA ----------------------------------------GAGACGTCGCTCATCAAAGA ----------------------------------------GAGACGTCGCTCATCAAAGA ----------------------------------------GAGACGTCGCTCATCAAAGA ----------------------------------------GAGACGTCGCTCATCAAAGA ----------------------------------------GAGACGTCGCTCATCAAAGA ----------------------------------------GAGACGTCGCTCATCAAAGA ----------------------------------------GAGACGTCGCTCATCAAAGA ----------------------------------------GAGACGTCGCTCATCAAAGA ----------------------------------------GAGACGTCGCTCATCAAAGA ----------------------------------------GAGACGTCGCTCATCAAAGA ATGAGGAAAATGACCTCTATGAAGAAGAACAAGAAAGGAGGAGACGTCGCTCATCAAAGA ********************

20 20 20 20 20 20 20 20 20 20 20 20 60

M24 M22 M13 M10 M9 M7 M4 M1 M2 59_9 80_14 08_6 WSV_ORF366

TGGGGAAGATCCTTAGAGATCTTCATGAGAGTGATGATGACGACGATGACTACTTTGATG TGGGGAAGATCCTTAGAGATCTTCATGAGAGTGATGATGACGACGATGACTACTTTGATG TGGGGAAGATCCTTAGAGATCTTCATGAGAGTGATGATGACGACGATGACTACTTTGATG TGGGGAAGATCCTTAGAGATCTTCATGAGAGTGATGATGACGACGATGACTACTTTGATG TGGGGAAGATCCTTAGAGATCTTCATGAGAGTGATGATGACGACGATGACTACTTTGATG TGGGGAAGATCCTTAGAGATCTTCATGAGAGTGATGATGACGACGATGACTACTTTGATG TGGGGAAGATCCTTAGAGATCTTCATGAGAGTGATGATGACGACGATGACTACTTTGATG TGGGGAAGATCCTTAGAGATCTTCATGAGAGTGATGATGACGACGATGACTACTTTGATG TGGGGAAGATCCTTAGAGATCTTCAGGAGAGTGATGATGACGACGATGACTACTTTGATG TGGGGAAGATCCTTAGAGATCTTCATGAGAGTGATGATGACGACGATGACTACTTTGATG TGGGGAAGATCCTTAGAGATCTTCATGAGAGTGATGAGGACGACGATGACTACTTTGATG TGGGGAAGATCCTTACAGATCTTTATGAGAGTGATGATGACGACGATGACTACTTTGATG TGGGGAAGATCCTTAGAGATCTTCATGAGAGTGATGATGACGACGATGACTACTTTGATG *************** ******* * *********** **********************

80 80 80 80 80 80 80 80 80 80 80 80 120

M24 M22 M13 M10 M9 M7 M4 M1 M2 59_9 80_14 08_6 WSV_ORF366

ACGAATTTGATGGCGAACGTTCAATGTCAGAAACTATTGCAACCAGAAGAGCTGGCCGTA ACGAATTTGATGGCGAACGTTCAATGTCAGAAACTATTGCAACCAGAAGAGCTGGCCGTA ACGAATTTGATGGCGAACGTTCAATGTCAGAAACTATTGCAACCAGAAGAGCTGGCCGTA ACGAATTTGATGGCGAACGTTCAATGTCAGAAACTATTGCAACCAGAAGAGCTGGCCGTA ACGAATTTGATGGCGAACGTTCAATGTCAGAAACTATTGCAACCAGAAGAGCTGGCCGTA ACGAATTTGATGGCGAACGTTCAATGTCAGAAACTATTGCAACCAGAAGAGCTGGCCGTA ACGAATTTGATGGCGAACGTTCAATGTCAGAAACTATTGCAACCAGAAGAGCTGGCCGTA ACGAATTTGATGGCGAACGTTCAATGTCAGAAACTATTGCAACCAGAAGAGCTGGCCGTA ACGAATTTGATGGCGAACGTTCAATGTCAGAAACTATTGCAACCAGAAGAGCTGGCCGTA ACGAATTTGATGGCGAACGTTCAATGTCAGAAACTATTGCAACCAGAAGAGCTGGCCGTA ACGAATTTGATGGCAAACGTTCAATGTCAGTAACTAATGCAACCAGAAGAGCTGGCCGTA ACGAATTTGACGGCGAACGTTCAATGTCAGAAACTATTGTAACCAGAAGAGCTGGCCGTA ACGAATTTGATGGCGAACGTTCAATGTCAGAAACTATTGCAACCAGAAGAGCTGGCCGTA ********** ***.***************:*****:** ********************

140 140 140 140 140 140 140 140 140 140 140 140 180

M24 M22 M13 M10 M9 M7

TTCAATATGGTCCAGGTTTC TTCAATATGGTCCAGGTTTC TTCAATATGGTCCAGGTTTC TTCAATATGGTCCAGGTTTC TTCAATATGGTCCAGGTTTC TTCAATATGGTCCAGGTTTC

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160 160 160 160 160 160

33

ACCEPTED MANUSCRIPT

M4 M1 M2 59_9 80_14 08_6 WSV_ORF366

TTCAATATGGTCCAGGTTTC TTCAATATGGTCCAGGTTTC TTCAATATGGTCCAGGTTTC TTCAATATGGTCCAGGTTTC TTCAATATGGTCCAGGTTTC TTCAATATGGTCCAGGTTTC TTCAATATGGTCCAGGTTTC ********************

160 160 160 160 160 160 200

RI PT

836 837 838 839 840 841 842 843 844

b

846

Figure 5. Detection of EVE 366 in shrimp spermatophores. (a) Example of agarose

847

gel showing PCR amplicons (167 bp) for EVE 366 for DNA extracted from

848

spermatophores of 5 different shrimp. M = marker; Neg = negative control; Pos =

849

Positive control. (b) ClustalW multiple sequence alignment of PCR amplicons of EVE

850

366 from 12 spermatophore samples compared with WSV 366 (GenBank AF332093.2)

851

and showing 98-100% sequence identity. Bases Mis-matched bases with the reference

852

sequence are highlighted in grey background.

856 857 858 859 860

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861 862 863

34

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864

Tables

865

Table 1.

866

(Khadijah et al., 2003) and primers for the housekeeping gene beta-actin. O = outer primer;

867

I = inner primer; F = forward primer; R = reverse primer.

868 Primer names

Sequences (5’ to 3’)

β-actin F

ORF151

ORF366

VP28-OF VP28-OR VP28-IF VP28-IR 151-OF 151-OR 151-IF 151-IR 366-OF 366-OR 366-IF 366-IR

ORF427

427-OF 427-OR 427-IF

M AN U

Vp28

β-actin R

871 872 873

Tm (°C) 55.0

55.0 59.7 59.8 58.4 56.3 58.0 61.8 58.0 61.8 57.8 57.6 66.7 62.0 56.0 49.4 52.6 57.6

Product size (bp) 799 400 157 4,305 510 252 160 1,866 900

AC C

870

EP

427-IF

869

CCCCATTGAGCACGGTATCA ACGCTCAGGAGGAGCAATGA CTCGTGGTTTCACGAGGTTG TCCGCATCTTCTTCCTTCAT TGTGACCAAGACCATCGAAA ATTGCGGATCTTGATTTTGC ATGGATTTTGAAGGAAGAACTACCA CTTCTTTTGTTTTCTTTG GTGGTCACATCTGACATGGA GCATAATGCAGTAGCGTCAACGGC ATGAGGAAAATGACCTCTATGA TCAAGAAAGCGCGTGCTTTAG GAGACGTCGCTCATCAAAGATGGGGAAG GAAACCTGGACCATATTGAATACGGCCAG ATGGCATGGACCGTAATGGC TTCCTTGATCTAGAGCT GAGCTGGCAAAGGAAACC ACAGACAACAGAACCTCCTTC

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Beta-actin

SC

Genes

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Primers used to amplify WSSV ORFs 151, 366 and 427 obtained from

874 875 876

35

ACCEPTED MANUSCRIPT

877

Table 2. Summary of 128 shrimp specimens screened for WSSV ORFs 151, 366, and

878

427.

879 ORF 427

+ + + 28 21.9 75.7

+ + 5 3.9 13.5

+ + + 4 3.1 10.8

880

Percentage

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ORF 151

(%)

74.2 19.5 3.1 0.8 1.6 0.8 100.0 28.9 100.0

SC

ORF 366

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Number of samples (=128) 95 25 4 1 2 1 Number of inserts % of 128 shrimp % of 37 inserts

881

Table 3

882

Summary of the expression pattern of ORF 366 gene insert in selected samples. Polarity of expression

Sense

Antisense

+ + + + + + 11

+ + + + + + + 22

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WSSV ORF 366 Sample number

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EP

2 3 5 10 12 13 18 20 23 Percent (%) Both sense (%)

No expression

+ 11

56

36

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Supplementary Information 1 Sensitivity tests with the primers of Vp28 and WSSV ORFs 151, 366 and 427 Before use, the primers for targets Vp28 and WSSV ORFs 151, 366 and 427 were tested for their sensitivities and were optimized using DNA templateS extracted from

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WSSV-infected shrimp. These tests were carried out using various annealing temperatures and varied numbers of PCR cycles. The first PCR reaction mixture contained 1xPaq5000 reaction buffer, 0.2 mM dNTPs, 0.2 mM of both forward and reverse primers and 0.02 U/µl of Paq5000TM DNA polymerase (Stratagene) in a 25-µl

SC

reaction volume. RNase A-treated WSSV infected DNA (100ng /µl) was used as the template. The sensitivity of primers for Vp28 and for ORFs 151, 366 and 427 were

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determined. This was done to ensure that these primers were specific and sensitive for amplification of WSSV DNA only. To test all primers, WSSV infected DNA was prepared in various concentrations as a template in PCR reactions. Serial dilutions were set from 2µg/ µl to 10-4 ng/µl of template DNA.

The following PCR conditions were used to amplify the target Vp28 gene fragment.

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The first PCR was performed in 25 µl of final reaction containing 1 µl of WSSV infected DNA template (approx. 100 ng), 2.5 µl of 10xPaq5000 reaction buffer (Stratagene, USA), 0.3 µl of 10 mM dNTPs, 0.5 µl of 10 mM of both forward outer (VP28F-O, 5’-CTCGTGGTTTCACGAGGTTG-3’) and reverse outer primers of Vp28

EP

gene (VP28R-O, 5’-TCCGCATCTTCTTCCTTCAT-3’), 0.25 µl of 500 U/µl of Paq5000TM DNA polymerase (Stratagene, USA) and 19.95 µl of distilled water. The

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nested PCR reaction was prepared as first PCR, but the primers used were the forward inner (VP28F-I, 5’-TGTGACCAAGACCATCGAAA-3’) and the reverse inner primers (VP28R-I, 5’-ATTGCGGATCTTGATTTTGC-3’). The First and nested PCR optimization was employed in thermal cycler TProfessional Basic Gradient (Biometra, Germany) with the following PCR condition for both PCRs: one cycle of 94°C for 2 min, number of cycles were varied at 25 to 34 cycles of 94°C for 30s, at various annealing temperatures ranging from 56 to 62°C for 30s, then 72°C for 30s followed by final extension at 72°C for 7 min. The first PCR amplification was generated 400 bp product size whereas the nested was 157 bp.

37

ACCEPTED MANUSCRIPT Primers for WSSV ORFs 151, 366 and 427 were tested for both first and nested PCR amplification steps. For the first PCR amplification step, the reactions were carried out in 25 µl reaction solution containing 1 µl of WSSV infected DNA as a positive control (200ng), 0.3 µl of 10 µM dNTPs, 0.5 µl of 10 µM of each primer (the primer

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sequences were listed in Table 1), 0.25 µl of 500 U/µl of Paq5000 DNA polymerase (Stratagene, USA), 2.5 µl of 10X Paq5000 PCR buffer, and 19.95 µl of ultrapure distilled water. The product from the first PCR (1 µl) was used as the template for the

SC

nested PCR with the same reaction components. Primer test was performed in a thermal cycler TProfessional Basic Gradient (Biometra, Germany). Each primers of

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WSSV inserted genes was optimized in various of annealing temperatures ranging from 52°C to 67°C, at different of PCR cycles (25 to 34 cycles) and various extension times (30s to 4 min) both for the first and nested PCRs.

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Supplementary Fig. 1

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Sensitivities of primers targeting WSSV Vp28, ORF151, ORF366 and ORF427

38

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Supplementary Fig. 1 Detection limits of PCR using primers targeting the WSSV Vp28 gene and potential WSSV insert genes ORF 151, 366 and 427. Agarose gel electrophoresis analysis of PCR products amplified using primers targeting WSSV Vp28 gene and potential WSSV inserted genes ORFs 151, 366 and 427 with varying quantities of DNA template from WSSV-infected crayfish. The lowest quantities of template DNA that gave positive results by nested PCR from WSSV-infected shrimp

39

ACCEPTED MANUSCRIPT using primers targeting VP28 (a), ORF 151 (b), ORF 366 (c) and ORF 427 (d) were 2 pg, 2 ng, 20 ng and 20 ng, respectively. M = 100 bp marker (Invitrogen, USA).

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Supplementary Fig. 2

Supplementary Fig. 2 Example of a gel showing PCR products amplified from DNA

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extracted from broodstock hemolymph samples using primers targeting the beta actin gene (799 bp). Similar band intensities were observed across the nine DNA samples tested suggesting comparable quantities and qualities of all DNA tested. Neg = negative control with water (no DNA template), Pos = shrimp DNA from previous

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Supplementary Fig. 3

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study was used as template and M = 100 bp marker (Invitrogen, USA).

40

ACCEPTED MANUSCRIPT Supplementary Fig. 3 Example of electrophoresis gels of PCR products obtained from spermatophore screening for WSSV. (a) Example of a gel showing negative results obtained using the IQ2000 detection kit. A single band for the beta actin gene control at 848 bp of comparable thickness was observed in each sample indicating the

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acceptable/comparable quality and quantities of the extracted DNA samples. M = molecular weight markers showing three bands of 848 bp, 630 bp and 333 bp. Pos = DNA from WSSV infected shrimp was used as template showing two bands at 550 bp

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and 295 bp. (b) A gel showing negative results for detection of the Vp28 gene in the same spermatophore samples (157 bp product). All samples are negative. The negative

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control (Neg) was ddH2O and the positive control (Pos) was template DNA from

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WSSV-infected shrimp. M = 100 bp marker (Invitrogen, USA).

41

ACCEPTED MANUSCRIPT Supplementary Tables Supplementary Table 1. Name

genes

number

Isolate

according to

Location

Length

the acc.

(nt)

(bp)

number NC_003225* AF332093.2 AF369029.2 AF440570.1 JX515788.1

China China Thailand Taiwan Korea

WSV 151 WSV 151 ORF 89 WSSV 207 WSV 151

ORF 366

NC_003225* AF332093.2 AF369029.2 AF440570.1 JX515788.1 AF440570

China China Thailand Taiwan Korea Taiwan

WSV 366 WSV 366 ORF 167 WSSV 425 WSV 366 WSSV 419

ORF 427

NC_003225* AF332093.2 AF369029.2 AF440570.1 JX515788.1

China China Thailand Taiwan Korea

79065 - 83375 79065 - 83375 128334 - 132644 112978 - 117288 77731 - 82041

WSV 427 WSV 427 ORF 3 WSSV 486 WSV 427

4,310 4,310 4,310 4,310 4,310

216623 – 216874 216623 – 216874 265672 – 265923 251568 – 251819 213926 – 214177 244562 – 262795

252 252 252 252 252 18,234

247360 – 249231 247360 - 249231 3118 - 4989 281982 - 283853 244019 - 245890

1,872 1,872 1,872 1,872 1,872

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ORF 151

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Accession

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Name of

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Note: *The original acc. no. NC_003225 (Gao et al. 2001) have replaced by the accession number of AF332093.2 (Yang et al. 2001); AF369029.2 (Marcks et al, 2005; van Hulten et al. (2001); AF440570.1 (Tsai et al., 2000); JX515788.1 (Choi, 2012).

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Choi, T. J. Direct Submission, Submitted (22-AUG-2012) Department of Microbiology, Pukyong National University, 45, Yongso-Ro, Nam-Gu, Busan 608-737, South Korea Gao, M., Yang, F., Xu, L. and Li, F. Direct Submission, Submitted (07-JAN-2016) Key Laboratory of Marine Genetic Resources,Third Institute of Oceanography, State Oceanic Administration (SOA), Daxue Road 184, Xiamen 361005, P. R. China Marks, H., van Duijse, J.J.A., Zuidema, D., van Hulten, M.C.W., Vlak, J.M., 2005. Fitness and virulence of an ancestral white spot syndrome virus isolate from shrimp. Virus Res 110, 9-20. Tsai, M.F., Lo, C.F., van Hulten, M.C.W., Tzeng, H.F., Chou, C.M., Huang, C.J., Wang, C.H., Lin, J.Y., Vlak, J.M., Kou, G.H., 2000. Transcriptional analysis of the ribonucleotide reductase genes of shrimp white spot syndrome virus. Virology 277, 92-99. van Hulten, M.C., Witteveldt, J., Peters, S., Kloosterboer, N., Tarchini, R., Fiers, M., Sandbrink, H., Lankhorst, R.K., Vlak, J.M., 2001. The white spot syndrome virus DNA genome sequence. Virology 286, 7-22. Yang, F., He, J., Lin, X., Li, Q., Pan, D., Zhang, X., Xu, X., 2001. Complete genome sequence of the shrimp white spot bacilliform virus. J Virol 75, 11811-11820. 42

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Supplementary Table 2. BlastN results from selected clones representing amplicons of WSSV ORFs 151, 366 and 427 with significant similarity to the

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sequence in GenBank accession number AF332093.2.

Clone

Sequence length (bp)

Identity (%)

E-value

ORF 151

P1_2 P1_6 P1_10

481 507 507

98 97 98

3 x 10-66 9 x 10_65 8 x 10-66

ORF 427

P3_6 P3_7 P3_11

860 864 908

ORF 366

P2_6 P2_9 P2_14

SC

Genes

6 x 10-139 6 x 10-139 6 x 10-139

96 100 96

3 x 10-21 1 x 10-22 6 x 10-21

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95 95 95

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160 160 158

43

ACCEPTED MANUSCRIPT Highlights * WSSV endogenous viral elements (EVE) were found in genomic DNA of 33/128 SPF shrimp *Distribution of 3 EVE types found was random in the shrimp, but EVE 366 was dominant * EVE 366 expression was variable: none (1), + sense (1), - sense (2), +/- sense (5)

*Detection of EVE 366 in shrimp sperm DNA indicated heritability

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*Negative and dual sense RNAs could potentially induce RNAi against WSSV

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*These results support predictions of the viral accommodation hypothesis