Biology, genome organization, and evolution of parvoviruses in marine shrimp.

CHAPTER THREE

Biology, Genome Organization, and Evolution of Parvoviruses in Marine Shrimp Arun K. Dhar*,1, Refugio Robles-Sikisaka†, Vanvimon Saksmerprome{,}, Dilip K. Lakshman}

*BrioBiotech, Glenelg, Maryland, USA † University of California, San Diego, California, USA { Centex Shrimp, Faculty of Science, Mahidol University, Bangkok, Thailand } National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Thailand Science Park, Pathum Thani, Thailand } USDA-ARS, Floral & Nursery Plants Research Unit, Beltsville, Maryland, USA 1 Corresponding author: e-mail address: [email protected]; [email protected]

Contents 1. Introduction 2. Clinical Signs, Histopathology, Transmission, and Detection 2.1 Infectious hypodermal and hematopoietic necrosis virus 2.2 Hepatopancreatic parvovirus 2.3 Spawner-isolated mortality virus 2.4 Lymphoidal LPV 3. Biophysical Properties, Genome Organization, and Gene Expression 3.1 Virus morphology 3.2 Genome organization 3.3 Virus gene expression 3.4 Integration of IHHNV DNA in the host genome and implication in virus detection and disease resistance 4. Evolution of Shrimp Parvoviruses 4.1 Genetic diversity of IHHNV 4.2 Evolutionary mechanisms of IHHNV 4.3 Genetic diversity and phylogeny of HPV 5. Management of Parvovirus Infection 5.1 Virus prevention 5.2 Therapeutic approach: Viral inhibition by RNAi 6. Conclusion Acknowledgments References

Advances in Virus Research, Volume 89 ISSN 0065-3527 http://dx.doi.org/10.1016/B978-0-12-800172-1.00003-3

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2014 Elsevier Inc. All rights reserved.

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Abstract As shrimp aquaculture has evolved from a subsistent farming activity to an economically important global industry, viral diseases have also become a serious threat to the sustainable growth and productivity of this industry. Parvoviruses represent an economically important group of viruses that has greatly affected shrimp aquaculture. In the early 1980s, an outbreak of a shrimp parvovirus, infectious hypodermal and hematopoietic necrosis virus (IHHNV), led to the collapse of penaeid shrimp farming in theAmericas. Since then, considerable progress has been made in characterizing the parvoviruses of shrimp and developing diagnostic methods aimed to preventing the spread of diseases caused by these viruses. To date, four parvoviruses are known that infect shrimp; these include IHHNV, hepatopancreatic parvovirus (HPV), spawnerisolated mortality virus (SMV), and lymphoid organ parvo-like virus. Due to the economic repercussions that IHHNV and HPV outbreaks have caused to shrimp farming over the years, studies have been focused mostly on these two pathogens, while information on SMV and LPV remains limited. IHHNV was the first shrimp virus to be sequenced and the first for which highly sensitive diagnostic methods were developed. IHHNV-resistant lines of shrimp were also developed to mitigate the losses caused by this virus. While the losses due to IHHNV have been largely contained in recent years, reports of HPVinduced mortalities in larval stages in hatchery and losses due to reduced growth have increased. This review presents a comprehensive account of the history and current knowledge on the biology, diagnostics methods, genomic features, mechanisms of evolution, and management strategies of shrimp parvoviruses. We also highlighted areas where research efforts should be focused in order to gain further insight on the mechanisms of parvoviral pathogenicity in shrimp that will help to prevent future losses caused by these viruses.

1. INTRODUCTION Aquaculture is a major global industry with a total annual production of farm-raised food reaching almost 63 million tonnes with an estimated value of US$130 billion in 2011 (www.fao.org/fishery/topic/16140/en). Over the past four decades, aquaculture has grown at an average of approximately 6% annually and represents the fastest growing animal foodproducing sector (www.fao.org/fishery/topic/16140/en). China and other developing nations in the Asia-Pacific region contribute the most to aquaculture activities globally. Shrimp is one of the top aquaculture species that are farmed at a commercial scale worldwide. Among different aquaculture species, shrimp aquaculture is a major source of export revenue in the countries with large coastal lines in South-East Asia and South America. With an

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ever increasing demand for seafood, shrimp aquaculture is now a major global industry with total annual production exceeding 3.4 million metric tonnes with an estimated value of US $15.2 billion in 2010 (www.fao.org/ fishery/topic/16140/en). The unprecedented growth of shrimp aquaculture has not been realized without addressing major challenges. Diseases caused by viruses have become one of the major hurdles that negatively impact the growth of this industry. Periodic outbreaks of viral diseases in shrimp aquaculture have resulted in large economic losses—it has been estimated that over the past 15 years global losses due to diseases is over US $15 billion (Flegel, 2012; Flegel et al., 2008). The challenges in developing sustainable shrimp aquaculture have been exacerbated by the periodic emergence of hitherto unknown viral diseases or more lethal versions of existing viruses. The first report of a viral disease in marine shrimp was published in the early 1970s when a baculo-like virus was isolated from wild Penaeus duorarum from the Florida Gulf Coast (Couch, 1974a, 1974b). Since then, over 20 viral diseases have been reported and the list is still growing (Table 3.1). Although the first viral disease in shrimp was reported in 1974, the negative impact of viral diseases was not realized until the 1980s when mass mortalities (>90%) occurred in juveniles and subadults of Penaeus stylirostris farmed in superintensive raceway systems in Hawaii (Brock, Lightner, & Bell, 1983; Lightner, Redman, & Bell, 1983). Since then, a number of other viral epizootics have occurred in shrimp aquaculture globally, including outbreaks of white spot disease in Asia (reviewed by Flegel, 2006; Sanchez-Paz, 2010), Taura syndrome disease (reviewed by Dhar, Cowley, Hasson, & Walker, 2004; Lightner, 2011), yellow head disease (Flegel, 2006; Walker & Winton, 2010), and more recently infectious myonecrosis disease in Brazil and Indonesia (Lightner, 2011; Lightner et al., 2004; Senapin, Phewsaiya, Briggs, & Flegel, 2007; Wilkinson, 2006). Besides IHHN, three other parvoviruses infect marine shrimp. These include hepatopancreatic parvovirus (HPV), spawner-isolated mortality virus (SMV), and lymphoid organ parvo-like virus (LPV) (Table 3.1). These viruses alone or in combination with other viruses have caused major losses in shrimp aquaculture. This review provides a comprehensive, overview of the biology, genome organization, gene expression, and evolution of parvoviruses in shrimp. Additionally, diagnostic tools that are now available for detecting diseases caused by shrimp parvoviruses are summarized. Finally, different management strategies are discussed for prevention and treatment to reduce losses caused by parvovirus infections in marine shrimp.

Table 3.1 A list of known viral pathogens of marine and freshwater shrimp Virus morphology Genomic Disease Virus and size properties

Classification

Year of Geographic emergence distribution

OIE listed

I. Diseases caused by double-stranded DNA viruses

Family: Nimaviridae, Genus: Whispovirus

1992

Asia, America, Yes Middle-East, Mediterranean

Rod-shaped, enveloped, dsDNA Baculovirus penaeid (BP) (also 55–75 300 nm, occluded called Penaeus vannamei singly enveloped nuclear polyhydrosis virus, PvSNPV)

Baculovirus, unclassified

1974

America

No

Rod-shaped, enveloped, dsDNA Monodon 68–77 265–282 nm, baculovirus (MBV) (also called occluded Penaeus monodon singly enveloped nuclear polyhydrosis virus, PmSNPV)


1977

Asia-Pacific, America, Africa

No

White spot syndrome

White spot syndrome viruses (WSSV)

Baculovirus penaei infection

Monodon baculovirus infection

Rod-shaped to elliptical with a tail-like projection, enveloped, 80–120 250–380 nm

dsDNA, circular, 300 kb

Hemocytic rodshaped virus infection

Rod-shaped, enveloped, dsDNA Hemocyte rod90 640 nm, shaped virus nonoccluded (HRV) (also known as Penaeid hemocytic rodshaped virus, PHRV)


1993

Australia

Baculoviral midgut gland necrosis

Rod-shaped, Baculoviral 75 300 nm, midgut gland nonoccluded necrosis virus (BMNV), Penaeus japonicus nonoccluded baculovirus (PjNOBV)

dsDNA


1981

Asia, Australia No

NDa

Iridovirus, unclassified

1993

Ecuador

No

America, Asia-Pacific, Africa, Madagascar, Middle-East

Yes

Shrimp iridovirus Iridovirus infection (IRIDO)

Icosahedral, 80 nm nucleoid region surrounded by 20 nm amorphous layer

No

II. Diseases caused by single-stranded DNA viruses

Infectious hematopoietic necrosis

Infectious hematopoietic necrosis virus (IHHNV) or Penaeus stylirostris

Icosahedral, nonenveloped, 20 nm

1981 Single, linear, Family: Parvoviridae, ssDNA, Subfamily: 4.1 kb Densovirinae, Genus: Brevidensovirus

Continued

Table 3.1 A list of known viral pathogens of marine and freshwater shrimp—cont'd Virus morphology Genomic Disease Virus and size properties Classification


OIE listed

densonucleosis virus (PstDNV) Hepatopancreatic Hepatopancreatic Icosahedral, parvovirus (HPV) nonenveloped, parvovirus 22–24 nm or Penaeus infection monodon densovirus (PmDNV)

1983 Single, linear, Family: Parvoviridae, ssDNA, Subfamily: 6.3 kb Densovirinae, Genus: Brevidensovirus

America, Asia-Pacific, Africa, Madagascar, Middle-East

No

Spawner-isolated mortality

Spawner-isolated mortality virus (SMV)


ssDNA

Parvovirus, unclassified

1993

Australia

No

Lymphoidal parvo-like virus infection

Lymphoidal parvo-like virus (LPV)

Icosahedral, nonenveloped, 25–30 nm

ssDNA

Parvovirus, unclassified

1991

Australia

No

Mid-crop mortality syndrome

Mid-crop mortality syndrome (MCMV)associated virus

Icosahedral, nonenveloped, 20–25 nm

ND

Parvo-like virus, unclassified

1994

Australia

No

III. Diseases caused by double-stranded RNA viruses

Infectious myonecrosis

Icosahedral, Infectious myonecrosis virus nonenveloped, 40 nm (IMNV)

Single, linear, Totivirus, unclassified dsRNA, 7.6 kb

2002

Brazil, Indonesia

Shrimp reovirus infection

Reo-like viruses Icosahedral, (REO III and IV) nonenveloped, 50–70 nm

ND

REO III1984, REO IV-1996

No REO III: Hawaii, Japan, Malaysia, France, Ecuador, US; REO IV: Yellow sea region of Asia

Reovirus, unclassified

Yes

IV. Disease caused by single-stranded positive-sense RNA virus


1992 Single, linear, Family: (+) ssRNA, Dicistroviridae, Genus: 10.2 kb Cripavirus

America, East Yes and SouthEast Asia

Taura syndrome

Taura syndrome virus (TSV)

Yellow head disease

Yellow head virus Rod-shaped, enveloped Single, linear, Family: (YHV) Type I with surface projections, (+) ssRNA, Roniviridae, Genus: 26.7 kb 70 180 nm Okavirus

1990

East and South-East Asia, Mexico

Yes

Family: Roniviridae, Genus: Okavirus

1996

Australia

No

Yellow head virus Rod-shaped, enveloped Single, linear, with surface projections, (+) ssRNA, (YHV) Type II 26.2 kb 70 180 nm Gill-associated virus/lymphoid organ virus (GAV/LOV)

Continued

Table 3.1 A list of known viral pathogens of marine and freshwater shrimp—cont'd Virus morphology Genomic Disease Virus and size properties Classification


OIE listed

White tail disease Macrobrachium rosenbergii nodavirus (MrNV); Extra small virus (XSV)

Icosahedral, nonenveloped, 26 nm; icosahedral, nonenveloped, 15 nm

Two, linear, Family: (+) ssRNA, Nodaviridae, unclassified RNA1 is 2.9 kb and RNA2 is 1.3 kb; single, linear, (+) ssRNA

1995

Yes Thailand, China, Taiwan, India, Australia, Caribbean

Lymphoid organ vacuolization

Lymphoid organ vacuolization viruses (LOVV)

Icosahedral, 55 nm

Not yet Toga-like characterized virus, unclassified

1995

Australia

No

Penaeid rhabdovirus infection

Rhabdovirus of penaeid shrimp (RPS)

Bullet-shaped, Not yet Rhabdovirus, 1991 65–77 nm 115–138 nm characterized unclassified

Hawaii and Ecuador

No

V. Disease caused by single-stranded negative-sense RNA virus

Mourilyan virus Mourilyan virus infection (gut and (MoV) nerve syndrome?)

Spherical to ovoid, 85 100 nm

Four, () ssRNA

Bunyaviruslike, unclassified

1996

Asia, Australia No

ssRNA

Luteo-like virus, unclassified

2003

South and South-East Asia

VI. Presumptive viral disease

Monodon slow Laem-Singh virus ND growth syndrome (LSNV) a

ND, not determined.

No


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2. CLINICAL SIGNS, HISTOPATHOLOGY, TRANSMISSION, AND DETECTION 2.1. Infectious hypodermal and hematopoietic necrosis virus 2.1.1 Clinical signs, host range, and prevalence of the disease Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is also called P. stylirostris densovirus (Tattersall et al., 2005). The disease it causes was first reported from Hawaii in 1981 as an acute epizootic with mass mortality (up to 90%) in juvenile blue shrimp (P. stylirostris) in super-intensive raceways (Lightner, Redman, & Bell, 1983; Lightner, Redman, Bell, & Brock, 1983). Subsequently, the disease was found in Penaeus vannamei where it did not cause mortalities but instead deformities referred to as runt deformity syndrome (RDS) (Bell & Lightner, 1984). In 1987, IHHNV was introduced into Mexico through a shipment of infected postlarvae of P. vannamei from Hawaii (Lightner, 1996a; Lightner et al., 1992). By 1990, the virus caused unprecedented losses in shrimp farms rearing P. stylirostris in Sinaloa and Sonora states in Mexico (Bell & Lightner, 1984; Lightner et al., 1992). Subsequently, the virus was introduced to wild populations of shrimp in the Gulf of California, Mexico, which eventually led to the collapse of wild fishery of P. stylirostris in the Northern Gulf of California (Lightner, 1996a). A decade later, the wild P. stylirostris fishery recovered, and subsequent surveys of wild populations of P. stylirostris from the Gulf of California revealed that the virus is well established in the natural population with prevalence reaching as high as 100% (Morales-Covarrubias et al., 1999; Robles-Sikisaka, Bohonak, McClenaghan, & Dhar, 2010). The increase in the capture fisheries in spite of a high prevalence of the virus suggests that an IHHNV resistance has evolved in the surviving population (Lightner, 1996a). At present, IHHNV is widely distributed in the wild populations of the Pacific Americas (i.e., western coast of Mexico, Guatemala, Honduras, Panama, and Ecuador) (Lightner, 1996a). After the initial outbreak of IHHNV and the identification of the etiologic agent, no IHHNV epizootics were reported until 2010 when a viral epizootic was reported from South Korea (Kim, Choresca, et al., 2011). IHHNV is now widely prevalent in wild penaeid shrimp in the Americas (P. vannamei, P. stylirostris, and Penaeus californiensis) and in South-East Asia (Penaeus monodon) (Flegel, 2006; Nunan, Arce, Staha, & Lightner, 2001). In Thailand, until recently IHHNV appeared to cause no obvious problems in

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P. monodon culture. However, after the introduction of P. vannamei, IHHNV became an issue of concern (Chayaburakul, Nash, Pratanpipat, Sriurairatana, & Withyachumnarnkul, 2004). Although IHHNV may be lethal to juvenile P. stylirostris (Lightner, Redman, & Bell, 1983), shrimp that survive the infection can become carriers and pass virus on by vertical and horizontal transmission (Lightner, 1996a). In P. vannamei, the virus does not cause lethal infections; instead it causes reduction in growth and a variety of cuticular deformities of the rostrum (Fig. 3.1), antennae, and other thoracic and abdominal areas. These clinical signs together are known as RDS (Kalagayan et al., 1991). RDS can cause substantial economic losses and the extent of this loss varies from 10% to 50% depending on the level of infection (Bell & Lightner, 1987; Wyban, Swingle, Sweeney, & Pruder, 1992). There is a wide distribution of sizes in

Figure 3.1 Clinical sign of IHHNV infection in Penaeus vannamei shrimp. Deformed rostra, one curved down and the other curved up, are marked by arrows.


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IHHNV-infected shrimp populations and often the proportion of smaller shrimp (“runts”) is higher than expected. The coefficient of variation (CV) in size for shrimp populations with RDS is typically greater than 30% and may approach 90%, while IHHNV-free (and thus RDS-free) populations of juvenile P. vannamei and P. stylirostris usually show CVs of 10–30% (Bray, Lawrence, & Leung-Trujillo, 1994; Browdy et al., 1993; Primavera & Quinitio, 2000). IHHNV has been demonstrated to infect all life stages (i.e., eggs, larvae, postlarvae, juveniles, and adults) of P. vannamei and the virus can be transmitted vertically (Motte et al., 2003). It has been demonstrated that the eggs produced by IHHNV-infected females with high virus loads generally fail to develop and hatch and that nauplii produced from heavily infected broodstock have a high prevalence of IHHNV infection (Motte et al., 2003). In Asia, black tiger shrimp (P. monodon), which used to be the most economically important shrimp species, IHHNV infections are endemic and usually asymptomatic. Genetic studies suggest that IHHNV was introduced to the Americas from the Philippines in the early 1970s most likely through the import of IHHNV-contaminated broodstock of P. monodon imported for experimental aquaculture. It was then transmitted to P. vannamei and P. stylirostris (Lightner, 2011; Tang et al., 2003). IHHNV is transmitted via horizontal or vertical routes (Lightner, Redman, & Bell, 1983; Lightner, Redman, Bell, & Brock, 1983; Lightner et al., 1985). Horizontal transmission occurs when healthy shrimp cannibalize moribund or dead shrimp or via contaminated water, whereas vertical transmission occurs via infected eggs, as described above (Motte et al., 2003). 2.1.2 Histopathology and virus detection IHHNV infects tissues of ectodermal and mesodermal origin. Histological diagnosis of IHHNV infection is routinely confirmed by the presence of intranuclear, Cowdry type A inclusions in tissues of ectodermal origin (epidermis, hypodermal epithelium of fore- and hindgut, nerve cord, and nerve ganglia) and mesodermal origin (hematopoietic organs, antennal gland, gonads, lymphoid organ, and connective tissue). The inclusions occur in hypertrophied nuclei of cells as eosinophilic, often haloed, inclusions surrounded by marginated chromatin (Alday de Graindorge & Flegel, 1999; Lightner, 1996b). Although Cowdry type A inclusions are quite characteristic of IHHNV infection, they are also produced at the early stages of infection by the double-stranded DNA-containing virus white spot syndrome

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virus (WSSV) which may cause confusion with IHHNV. In that event, in situ hybridization using an IHHNV-specific probe provides a definitive diagnosis (Lightner & Redman, 1998). In fact, in P. monodon Cowdry type A inclusions are rarely seen (Fig. 3.2) and in situ hybridization using an IHHNV-specific probe may be needed for definitive diagnosis. Routine IHHNV detection is done by histopathology, in situ hybridization using an IHHNV-specific gene probe and by polymerase chain reaction (PCR) (Lightner & Redman, 1998). The protocols for IHHNV detection are described in the Aquatic Animal Health Manual of the World Organization for Animal Health, OIE (Anonymous, 2003, 2006). In situ hybridization and dot blot assay using digoxigenin-labeled probes for the detection of IHHNV were the first to be developed for the diagnosis of shrimp disease (Lightner & Redman, 1998; Mari, Bonami, & Lightner, 1993). These methods are more sensitive than IHHNV detection by histology and are therefore used widely for virus detection. In addition, these methods are routinely used to detect the virus in a noninvasive manner using hemolymph and pleopod samples (Bell, Lightner, & Brock, 1990) that are particularly useful in screening broodstock (Carr et al., 1996). Monoclonal antibodies (mAbs) have been developed for the detection of IHHNV (Poulos, Lightner, Trumper, & Bonami, 1994) but have not been used as much as in situ hybridization and dot blot assay for disease diagnosis. Several methods for single, nested, and multiplex PCR are now available for the detection of IHHNV (Anonymous, 2006; Flegel, 2006; Khawsak,

Figure 3.2 Histopathology of IHHNV-infected Penaeus monodon antennal gland. Hematoxylin- and eosin-stained section showing Cowdry type A inclusion body.


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Deesukon, Chaivisuthangkura, & Sukhumsirichart, 2008; Lightner & Redman, 1998; Mrotzek et al., 2010; Nunan, Poulos, & Lightner, 2000; Tang et al., 2000; Tang & Lightner, 2006; Yang et al., 2006). These methods are applicable for surveillance and virus screening of broodstock and populations in the hatchery, documenting the specific-pathogen-free (SPF) status of animals, and monitoring the prevalence of the virus in commercial ponds and in the wild. A number of commercial PCR kits are now available for the detection of IHHNV. The existence of many genotypes of IHHNV and the integration of IHHNV genomic DNA into the host genome may require careful consideration in using and qualifying appropriate primer sets for IHHNV detection. Different primers (primers 389F/389R to amplify a 389 bp amplicon, 77012F/77353R to amplify a 356 bp amplicon, and 392F/392R to amplify a 392 bp amplicon) were successfully used to amplify all genetic variants of IHHNV (Lightner, 2011; Tang et al., 2000; Tang, Navarro, & Lightner, 2007). However, these primer sets do not distinguish between infectious and genome-integrated forms of IHHNV. As a result, additional primers (309F/R and 831F/R, see Table 3 in Lightner, 2011) have been developed that can distinguish shrimp genome-integrated forms of IHHNV from the free infectious virus form. Recently, a combination of PCR and reverse-transcription PCR was reported for distinguishing infectious from noninfectious forms of IHHNV in P. vannamei (Teixeira et al., 2010). Since PCR-based assays need sophisticated laboratory conditions and expensive equipment, efforts have been made to develop virus detection methods that are rapid, highly sensitive, and very specific but do not need expensive equipment, to enable their use in shrimp farms. Loop-mediated isothermal amplification (LAMP) is one such method that has been developed for the detection of IHHNV (Arunut, Prombun, Saksmerprome, Flegel, & Kiatpathomchai, 2011). These authors combined LAMP with a chromatographic lateral flow dipstick (LFD) that enables amplicon visualization. The sensitivity was comparable to other methods of IHHNV detection, such as nested PCR and the whole process can be completed in less than an hour without the need of a sophisticated laboratory set up. Therefore, LAMP-LFD is very attractive for field application (Fig. 3.3). Multiple virus infections are common in wild and cultured shrimp. PCR methods have now been developed to detect multiple viral pathogens of shrimp, including IHHNV, simultaneously (Khawsak et al., 2008; Tan et al., 2009; Xie et al., 2007; Yang et al., 2006). However, ensuring the sensitivity and specificity of amplifications needs to be further evaluated before

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A

M

N

1

2

3

4

5

6

7

8

M

500 bp 200 bp

B

N

1

2

3

4

5

6

7

8

Control line

Test line

Figure 3.3 Detection of IHHNV in Penaeus vannamei by combining loop-mediated isothermal amplification (LAMP) with a lateral flow dipstick (LFD) method. Panel (A) shows the amplicons upon LAMP followed by gel electrophoresis and the panel (B) shows the detection of the same samples of IHHNV by LFD. Lanes 1–8: templates DNA dilutions of 101 to 108 from IHHNV-infected P. vannamei, lane M: 2-log DNA ladder, and lane N: no template control.

such methods can be widely used in screening for IHHNV and other shrimp viral pathogens in hatcheries, commercial shrimp farms, and in field conditions. Since conventional PCR-based virus detection methods do not allow for quantification of viral loads, highly sensitive real-time quantitative PCR was developed for the detection and quantification of IHHNV (Dhar, Roux, & Klimpel, 2001; Tang & Lightner, 2001). Real-time PCR detection is rapid and high throughput, and can be used for large-scale screening and virus surveillance. Real-time PCR methods, using a generic DNA-binding dye (e.g., SYBR Green) and Taqman probe (Tang & Lightner, 2001), have been described for the detection and quantification of IHHNV. Recently, a multiplex real-time PCR method using Taqman probes for the detection and quantification of IHHNV, WSSV, and Taura syndrome virus (TSV) has been published (Xie et al., 2010). The limits of detection varied depending on the virus template used giving


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20 copies for IHHNV and 2000 copies for both WSSV and TSV (Xie et al., 2010). This method, however, needs further optimization and validation using field samples of shrimp with multiple virus infections. Since real-time PCR detection requires expensive equipment and high technical expertise for interpretation of the results, the possibility for routine application at the farm level may be limited but could be supported by a service provider format. However, real-time PCR detection is valuable for broodstock screening, for the development of disease-free stocks, studying IHHNV gene expression and pathogenesis, and developing antiparvoviral therapies in shrimp.

2.2. Hepatopancreatic parvovirus 2.2.1 Clinical signs and host range HPV was first reported by Chong and Loh (1984) from farmed marine prawns in Singapore. Subsequently, Lightner and Redman (1985) described a disease in postlarvae of Penaeus chinensis that was similar to the disease reported from Singapore. HPV is now known to infect several wild and cultured penaeid species and is widely distributed in Asia, Africa, Australia, and North and South America (Flegel, 1997, 2006; Flegel, Nielsen, Thamavit, Kongtim, & Pasharawipas, 2004; Flegel & Sriurairatana, 1993; Gangnonngiw et al., 2009; Lightner & Redman, 1992; Manivannan, Otta, Karunasagar, & Karunasagar, 2002; Safeena, Rai, & Karunasagar, 2012; Spann et al., 1997). Shrimp affected by HPV usually show nonspecific gross signs, including atrophy of the hepatopancreas, anorexia, poor growth rate, reduced preening activities and (as a consequence) increased tendency for surface, and gill fouling by epicommensal organisms (Lightner et al., 1992; Lightner & Redman, 1985; Sukumsirichart et al., 1999). Mortalities associated with HPV infection during the larval stages in P. chinensis have been reported from Australia (Spann et al., 1997). A strong correlation between HPV infection and growth stunting in P. monodon was reported from Thailand (Flegel et al., 2004). Shrimp positive for HPV were much smaller (mean weight 6–6.5 g) than those from the same pond that were negative for HPV (mean weight 8.3–9.3 g) and the difference increased with cultivation time (Fig. 3.4). These very small shrimp had no market value and reduced crop profits (Flegel et al., 2004). Although HPV can cause considerable losses to farmers due to stunted growth, even heavy infections cause no visible inflammatory response (Flegel, 2001). Transmission of HPV is believed to occur by both horizontal and vertical routes. Until recently, studies involving HPV were hampered due to the lack of laboratory challenge methods. However, this has now been

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Figure 3.4 Clinical sign of HPV infection showing reduction in size in Penaeus monodon.

overcome with the development of a laboratory bioassay in which the virus was shown to be transmitted by the oral route in P. monodon postlarvae (Catap, Lavilla-Pitogo, Maeno, & Travina, 2003). The availability of a reproducible bioassay will enable virus transmission studies under natural conditions and help in determining the reservoir of the virus in nature. In addition, an insect model using the house cricket (Acheta domesticus) has been developed to study Penaeus merguiensis densovirus (PmergDNV) pathogenesis (La Fauce & Owens, 2008). Considering the lack of availability of an immortal cell line in crustaceans, the accessibility of an insect model will be helpful in studying parvoviruses of shrimp. Vertical transmission of HPV from broodstock to F1 progeny was first reported in P. chinensis (Lightner et al., 1996). This observation is now supported by the detection of HPV infection in hatchery larvae in P. monodon (Manivannan et al., 2002; Umesha, Uma, Otta, Karunasagar, & Karunasagar, 2003). 2.2.2 Histopathology and virus detection Since HPV-infected shrimp do not display distinctive gross signs of disease, HPV infection is routinely confirmed by histopathological methods and PCR analysis. While IHHNV infects tissues of ectodermal origin (epidermis, hypodermal epithelium of fore- and hindgut, nerve cord, and nerve ganglia) and mesodermal origin (hematopoietic organs, antennal gland, gonads, lymphoid organ, and connective tissue), HPV infects the tubule epithelial cells of the hepatopancreas only, and these are of endodermal origin.


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Generally, actively dividing cells, the E-cells, at the distal end of the hepatopancreatic tubules show the most HPV inclusions (Fig. 3.5). Similar to IHHNV, the molecular mechanisms governing the differences in tissue tropism in HPV remain an open area of research. In a recently published paper involving bioinformatic analysis IHHNV and HPV encoded proteins, it was shown that both non-structural proteins of IHHNV contain nuclear localization sequences (NLS) but capsid protein does not contain any functional NLS. However, in HPV the NS proteins as well as the capsid protein contain functional NLS (Owens, 2013). The difference in the presence or absence of NLS in the viral encoded proteins between IHHNV and HPV probably contribute to the difference in the nature of inclusion bodies and the site of virion assembly in these two parvoviruses. IHHNV produces eosinophilic Cowdry A type inclusion body and the nuclear hypertrophy it causes is smaller than those caused by HPV which is probably due to the lack of capsid protein in the inclusion body. In HPV, on the other hand, a large intranuclear basophilic inclusion bodies are produced which might be due to the importation of all three viral encode protein into the nucleus. A field test using Giemsa-stained smears of hepatopancreatic tissue has been reported as a rapid means of identifying HPV infection in shrimp,

Figure 3.5 Histopathology of HPV infection in the epithelial cells of hepatopancreatic tubules in Penaeus monodon. Left-hand panel shows an H&E section from a healthy animal and the right-hand panel represents an H&E section from an HPV-infected shrimp.

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although the sensitivity and accuracy of the method is low (Lightner, 1996b). Traditionally, HPV infection is detected by the presence of intranulear inclusions by hematoxylin and eosin (H&E) staining of histological sections of the hepatopancreas of presumed HPV-infected samples (Flegel & Sriurairatana, 1993; Lightner, 1996b). The sensitivity of H&E histology was further improved with the development of in situ hybridization using an HPV-specific gene probe (Bonami, Mari, Polous, & Lightner, 1995; Mari, Lightner, Poulos, & Bonami, 1995). Since histology-based diagnostic methods involve destruction of the sample (broodstock, postlarvae, etc.), development of noninvasive virus detection method(s) became necessary. In recent years, more sensitive and nondestructive molecular techniques, such as PCR and nested PCR, were developed (Pantoja & Lightner, 2000; Phromjai, Boonsaeng, Withyachumnarnkul, & Flegel, 2002; Phromjai, Sukumsirichart, Pantoja, Lightner, & Flegel, 2001; Sukumsirichart et al., 1999). Since there are major differences in the nucleotide sequence among HPV isolates, primers used to amplify a 350-bp amplicon from HPV isolate infecting P. chinensis (Pantoja & Lightner, 2000) do not amplify HPV isolates infecting P. monodon (Phromjai et al., 2001). Likewise, primers used to amplify HPV isolates from Thailand and India do not amplify the HPV isolate from Korea, suggesting that the HPV isolates from India and Thailand are more closely related to each other than to the Korean isolate (Safeena, Tyagi, Rai, Karunasagar, & Karunasagar, 2010; Umesha et al., 2003). In addition to PCR, mAbs have been developed for detection of HPV (Rukpratanporn et al., 2005). Such mAbs may be valuable in determining the serological relationship among different geographical isolates of HPV. Both in wild and commercial farming, shrimp are often infected with more than one virus. Multiplex RT-PCR methods are now available to simultaneously detect up to six viruses of penaeid shrimp including both DNA (IHHNV, HPV, monodon baculovirus, MBV, and WSSV) and RNA viruses (TSV and yellow head virus (YHV)) (Khawsak et al., 2008). However, the sensitivity of detection for all but one virus, WSSV, was lower than single-step PCR reported for those viruses. For example, the sensitivity of HPV detection by multiplex PCR was 1000-fold less than single PCR reported by Sukumsirichart et al. (1999). This suggests that further optimization is needed before multiplex PCR could be used for routine diagnostic purposes in HPV and, for that matter, other shrimp viruses. Recently, a LAMP assay has been developed for the detection of HPV (Nimitphak, Kiatpathomchai, & Flegel, 2008). The LAMP method combined with the detection of the amplicon by chromatographic LFDs allowed


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rapid and highly sensitive detection of HPV that was 10-fold more sensitive than one-step PCR (Sukumsirichart et al., 2002). With the availability of different PCR-based assays, as well as LAMP methods, rapid and sensitive detection of HPV in broodstock, hatcheries, and grow-out ponds is likely to become more common as shrimp farmers worldwide move toward biosecure production systems.

2.3. Spawner-isolated mortality virus In addition to IHHNV and HPV, there are two other lesser characterized LPVs that have been reported to infect shrimp. These include SMV and lymphoidal LPV. SMV was first reported in P. monodon at a research facility in Townsville, Queensland, Australia in 1993 (Fraser & Owens, 1996). The virus was found to be associated with mortalities in broodstock of P. monodon and is similar or possibly identical to mid-crop mortality syndrome of P. monodon in growout ponds (Owens, Haqshenas, McElena, & Coelen, 1998; Owens, McElena, Snape, Harris, & Smith, 2003). In laboratory challenge experiments, SMV was lethal to P. monodon, P. esculentus, P. japonicus, P. merguiensis, and Metapenaeus ensis, with mortality reaching 100%. Natural infections of the red-claw crayfish (Cheax quadricarinatus) with SMV have been recorded in Australia, although it is not known whether the virus is transferred from shrimp to crayfish or vice versa (Owens & McElena, 2000). In situ hybridization using an SMV-specific probe has been developed for the detection of SMV (Owens et al., 1998). Using DNA hybridization, the virus could be detected in endodermal tissues, including the distal ends of hepatopancreatic tubules, midgut and hindgut caecae, midgut, and hindgut folds. In animals displaying hemocytic enteritis, some hemocytes in the affected midgut showed viral infections. In experimentally infected shrimp with acute infection, SMV could be detected in the male reproductive tract, specifically in the terminal ampoule and the medial vas deferens as well as in the ovary and in both the strornal matrix and spheroid cells of the lymphoid organ. Using transmission electron microscopy, nonenveloped, icosahedral particles measuring 20 nm in diameter were observed in gut cells. The viral genome contains single-stranded DNA but the genome organization and relationship of SMV to other shrimp parvoviruses are not known (Fraser & Owens, 1996).

2.4. Lymphoidal LPV LPV was first detected in cultured P. monodon, P. merguiensis, P. esculentus, and hybrid P. monodon P. esculentus in Australia (Owens, DeBeer, &

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Smith, 1991). In the hybrid animals of P. monodon and P. esculentus, low mortality was reported from the time the shrimp were 3–4 g body weight (Munday & Owens, 1998). In LPV-infected shrimp, the lymphoid organ is hypertrophied and multinucleated “giant cells” are formed (Owens et al., 1991). These giant cells display mild nuclear hypertrophy and marginated chromatin. Often fibrocyte encapsulated spherical structures, identical to the lymphoid organ spheroids, are observed in these cells. Basophilic intranuclear inclusion bodies are commonly found in giant cells (Owens et al., 1991). The intranuclear inclusions observed in LPV-infected cells are somewhat similar to IHHNV intranuclear inclusions. While LPV inclusion bodies are distinctly spherical in morphology, the IHHNV inclusions are highly irregular in shape and often contain darkly basophilic chromatin processes. In heavily infected animals, paracrystalline arrays of LPV particles, measuring 18–20 nm in diameter, could be seen on the edge of the large electron-dense intranuclear inclusions in lymphoid organ tissues.

3. BIOPHYSICAL PROPERTIES, GENOME ORGANIZATION, AND GENE EXPRESSION 3.1. Virus morphology IHHNV is a small DNA virus with nonenveloped particles, 22 nm in diameter that contains a single-stranded linear DNA genome of 4.1 kb (Bonami, Trumper, Mari, Brehelin, & Lightner, 1990). This is analogous to other members of the densovirus group of parvoviruses where positive or negative DNA strands may be encapsidated, but not in the same virus particle (Kelly, Barwise, & Walker, 1977). Based on morphological and biochemical characteristics, IHHNV was tentatively classified as a member of the family Parvoviridae (Bonami et al., 1990). Among all the parvoviruses known to infect marine shrimp, the biophysical properties of IHHNV only have been studied in the most detail. The crystal structure of the IHHNV capsid (CP) protein expressed using a baculovirus expression system has been determined (Kaufmann et al., 2010). The 20 nm size virus-like particles (VLPs) purified from baculovirus-infected insect cells revealed that each VLP contains 60 copies of a 37.5 kDa protein that has b-barrel “jelly-roll” motifs similar to those of many other icosahedral viruses including parvoviruses. The N-terminal portion of the peptide has a “domain-swapped” conformation similar to that of the parvovirus of the insect Galleria mellonella but most of the surface loops show no similarity to structures of any other parvovirus (Kaufmann et al., 2010). The IHHNV CP protein is the smallest parvovirus CP protein


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known so far. These authors (Kaufmann et al., 2010) suggested that the small dimension of the IHHNV CP protein potentially provides an attractive platform for antigen presentation and for the delivery of double-stranded (ds) RNA or other immune stimulatory molecules. Like IHHNV, HPV particles are nonenveloped with icosahedral morphology measuring 22–24 nm in diameter.

3.2. Genome organization Parvoviruses are single-stranded DNA viruses known to contain two large open reading frames (ORFs), both on the same DNA strand. The first ORF that is located on the 50 -end of the genome encodes two nonstructural (NS) proteins, NS1 and NS2 via alternative splicing events (Muzy & Berns, 2001). NS1 is specifically required for replication. The second large ORF, located on the 30 -end of the viral DNA, codes for coat proteins. Three co-aminoterminal coat proteins have been detected in parvoviruses. NS1 is required for viral replication and has a role in the transactivation of viral promoters (Afanasiev, Galyov, Buchatsky, & Kozlov, 1991; Afanasiev, Kozlov, Carlson, & Beaty, 1994). The six genera of the family Parvoviridae are divided into two subfamilies, Parvovirinae and Densovirinae. Parvovirinae contains the genera Parvovirus, Erythrovirus, Dependovirus, Amdovirus, and Bocavirus. Among these genera, the Dependoviruses are replication defective and solely depend on helper Adenoviruses for replication. Densovirinae contains Densovirus, Iteravirus, Brevidensovirus, and Pefudensovirus. The members of the subfamily Parvovirinae infect vertebrate hosts, while viruses belonging to the subfamily Densovirinae infect arthropods. The physiochemical properties of the invertebrate parvoviruses are similar to those of vertebrate parvoviruses, although the former group shares very little sequence homology with the later. In vertebrate parvoviruses and densoviruses of the genera Iteravirus and Brevidensovirus, the coding sequences of all viral proteins are located on one strand of the viral genome, which by convention is designated the viral minus strand (Ward, Kimmick, Afanasiev, & Carlson, 2001). On the other hand, genus Densovirus of Densovirinae has a unique genome organization characterized by its ambisense structure (Muzy & Berns, 2001). Densoviruses have single-stranded genomes of 4–6 kb. Brevidensoviruses, which include IHHNV and Aedes aegypti densonucleosis virus (AaeDNV), Aedes albopictus densovirus (AalDNV), and Culex pipiens pallens densovirus (CppDNV), are viruses with approximately 4 kb monosense genomes with unmatched terminal hairpins.

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3.2.1 IHHNV IHHNV was the first shrimp virus for which nearly the entire genome was sequenced except for the highly structured regions at the ends of the linear genome (Shike et al., 2000). The genome organization of IHHNV is closely related to the densoviruses in the genus Brevidensovirus in the family Parvoviridae (Shike et al., 2000) and is shown in Fig. 3.6. Due to the similarity of genome organization of IHHNV to brevidensoviruses, it was proposed that the IHHNV should be renamed as P. stylirostris densovirus (PstDNV). Since then both names, IHHNV and PstDNV have been used in the published literature, and different isolates of PstDNV have been named based on their geographical origin. For example, PstDNV isolate from the Gulf of California is referred as PstDNV-GOC, PstDNV from India as PstDNVIndia, and so forth. In the following section, we have stuck to the conventional usage of IHHNV but provide parenthetically the name provided by the cited source. IHHNV genome contains three major ORFs: left, middle, and right (Shike et al., 2000). The left ORF (referred as NS1-b in Fig. 3.6) encodes a polypeptide of 666 amino acids with a molecular mass of 75.77 kDa and contains a conserved replication initiation motif, NTP-binding and helicase domain. This polyprotein is similar to the NS1 polyprotein of mosquito densoviruses. At the 50 -end of the left ORF, there are three putative acceptor sites (A1, A2, and A3). Upstream of the left ORF, there is a small ORF (referred as NS1-a in Fig. 3.6) that contains one putative 50 donor site (D1). The NS1 transcript undergoes splicing at D1 position of NS1-a and A1 position of NS1-b to generate a mature NS1 transcript (Dhar, Kaizer, & Lakshman, 2010). The middle ORF completely overlaps with the left ORF, encodes a protein of 363 amino acids (42.11 kDa), and shows no similarity with any other protein in the database. The function of the middle ORF is not known at present. The 50 -end of the right ORF overlaps with the left ORF, and this ORF encodes a protein-containing 329 amino acids (37.5 kDa). The right ORF is presumed to encode structural polypeptides for IHHNV, as of now considered the smallest reported parvovirus CP protein. Unlike other viruses in the family Parvoviridae where CP proteins are generally reported to contain two or more coat protein variants, IHHNV (PstDNV-GOC) codes for only a single type of CP protein (Shike et al., 2000). A stretch of 11 amino acids in the N-terminal region of the CP protein (17-DAHNEDEEHAE-27) is reminiscent of the phospholipase A2 (PLA2) catalytic site (Shike et al., 2000), but it lacks important conserved

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) 5

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Figure 3.6 A schematic representation of the genome organization of IHHNV, HPV, and representative members of other parvoviruses. The open reading frames (ORFs) are represented by open box, +, ORF in sense orientation; , ORF in antisense orientation; NS, nonstructural protein; VP, viral (capsid) protein. The numbers on the right-hand side of each virus represent the number of nucleotides sequenced for the corresponding virus. In IHHNV, the ORF representing NS1-a and the following ORF representing NS1-b are joined upon splicing of the intron located between the two ORFs (Dhar et al., 2010).

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motifs of PLA2s. A phospholipase A2 (PLA2) activity in the unique N-terminal extension of the largest minor CP protein plays a crucial role during parvovirus host cell infection (Canaan et al., 2004). Consequently, IHHNV does not have the enzymatic activity that has previously been described as a requirement for parvovirus infectivity. The middle ORF and the right ORF (coat protein) are coded in the same frame, whereas the left ORF (NS1) is coded in a different frame. In addition to three large ORFs, the IHHNV genome contains a small ORF upstream of the left ORF (referred as NS1-a in Fig. 3.6). The small ORF contains one putative 50 donor site (D1) and three putative acceptor sites (A1, A2, and A3) at the 50 -end of the left ORF. In addition to these ORFs, there is a potential ORF of 714 nt long in the minus strand of unknown function. The terminal hairpins of densoviruses range from 110 to 160 nts (Bergoin & Tijssen, 2000). Recently, the genome sequence of an Indian isolate of IHHNV (PstDNV-India) has been reported (Rai, Safeena, Karunasagar, & Karunasagar, 2011). The Indian isolate IHHNV (PstDNV-India) slightly differs from that of the IHHNV (PstDNV-GOC) isolate in the start of ORF2. Whereas the ORF1 and ORF2 of both isolates are located in different reading frames, the start of ORF1 and ORF2 differs between the two isolates. In IHHNV (PstDNV-GOC), the ORF2 completely overlaps with the ORF1, while in PstDNV-India, ORF2 originates 50 -upstream of ORF1 (Rai et al., 2011). It should be mentioned, however, unlike in IHHNV (PstDNV-GOC) (Dhar et al., 2010), the transcription initiation of the respective ORFs has not been mapped for IHHNV (PstDNVGOC) (Rai et al., 2011). Therefore, the authenticity of transcription initiation predicted based on the translation of ORF2 of IHHNV (PstDNVIndia) genomic sequence needs to be further validated. 3.2.2 HPV Like IHHNV, in the published literature HPV has been referred as P. monodon densovirus (PmDNV) and different isolates of PmDNV have been named based on their geographical origin and host, such as PmDNVIndia, PmDNV-Thailand, PmDNV-Korea, and PmergDNV. PmDNVThailand is a 6321 nucleotide long, negative-sense, single-stranded DNA virus, first isolated from infected P. monodon in Thailand (Sukhumsirichart, Attasart, Boonsaeng, & Panyim, 2006). The 50 - and 30 ends of the viral genome contain hairpin-like structures of approximately 222 and 215 bp, respectively. There are three large ORFs. The left ORF (ORF1), mid-ORF (ORF2), and right ORF (ORF3) on the plus


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(complementary) strand potentially code for three proteins of 428, 579, and 818 amino acids, equivalent to 50, 68, and 92 kDa, respectively. ORF1 encodes the putative NS2 of unknown function. ORF2 contains replication initiator motif and NTP-binding and helicase domains similar to NS1 of parvoviruses. There is a small overlap of 13–15 bases among the two ORF, though in different reading frames. NS1 and the coat protein are coded in the same frame whereas the NS2 is coded in a different frame (Sukhumsirichart et al., 2006). The ORF3 of the HPV genome encodes a capsid protein (VP) of approximately 92 kDa. This may be later cleaved residue to produce a 57-kDa structural protein. In addition to a larger genome size, the overall genomic organization of HPV is quite different from IHHNV, indicating diversity among the brevidensoviruses infecting shrimp (Fig. 3.6). Recently, the genomic sequences from isolates of an Australian (PmergDNV; La Fauce, Elliman, & Owens, 2007), an Indian (PmDNVIndia; Safeena et al., 2010), and a Korean (PmDNV-Korea; Jeeva et al., 2012) strain of HPV have been reported. The PmDNV-India has ORFs coding 426, 577, and 819 amino acids, respectively and shows closest homology at the nucleotide level (88%) with PmDNV-Thailand. PmDNVIndia showed 33, 32, and 91 amino acid substitutions compared to the PmDNV-Thailand in the NS2, NS1, and VP, respectively (Safeena et al., 2010). The Australian HPV isolate (PmergDNV) shows major differences with the Indian and Thai isolates. For example, the NS2 of PmergDNV is 255 bases and 261 bases shorter than the homologous genes of the Indian and Thai isolates of PmDNV, respectively (La Fauce, Elliman, et al., 2007; La Fauce, Layton, & Owens, 2007; Safeena et al., 2010). Like the Indian and the Australian strains, PmDNV-Korea also encodes three proteins, NS1, NS2, and VP. The NS1 protein showed highest homology to other reported HPV strains followed by the NS2 protein and the VP protein ( Jeeva et al., 2012). It has been proposed that HPV isolates for which the entire genomes have been sequenced should be assigned to a new genus “Hepanvirus” (Gangnonngiw et al., 2009).

3.3. Virus gene expression Gene expression in mammalian parvoviruses is regulated through a combination of several mechanisms, such as transcriptional initiation site usage, transcript splicing, translation initiation site usage, protein processing, leaky ribosomal scanning, temporal gene expression, and transactivation

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(Cotmore & Tattersall, 2006). However, much less is known about brevidensovirus gene expression. The type species, AaeDNV genome encodes two overlapping ORFs encoding the nonstructural proteins, NS1 and NS2 (Afanasiev et al., 1991). NS1 protein transactivates the structural gene promoter (Afanasiev et al., 1994). It is not known whether NS1 and NS2 originate from alternative splicing events as in another arthropod densovirus, Periplaneta fuliginosa densovirus (Yamagishi, Hu, Zheng, & Bando, 1999), or from initiation at an alternative AUG codon (Bergoin & Tijssen, 2000; Kimmick, Afanasiev, Beaty, & Carlson, 1998). Among parvoviruses-infecting shrimp, comprehensive gene expression information is only available for PstDNV-GOC isolate. Therefore, the following section discusses the gene expression information pertaining to this isolate and its comparison to some other representatives of Brevidensoviruses. 3.3.1 In silico characterization of IHHNV coding regions and promoters A promoter is a region of DNA that generally is located upstream of a gene and facilitates and regulates its transcription. A core promoter is the minimal portion of the promoter required to properly initiate transcription. In eukaryotes, the core promoter is the region of DNA that directs the initiation of transcription by RNA polymerase II and it generally spans from about nucleotide 40 to +40 relative to the transcription start site ( Juven-Gershon & Kadonaga, 2010). Transcription factor IID (TFIID)mediated transcription requires several precise sequences, termed core promoter elements that mediate the recruitment of TFIID and other basal transcription factors to the DNA template. These core promoter elements include the TATA box, the initiator (Inr), the motif 10 element (MTE), the downstream promoter element (DPE), the TFIIB recognition elements (BREu and BREd), the downstream core element, and the X core promoter element 1 (XCPE1) ( Juven-Gershon & Kadonaga, 2010). The MTE functions cooperatively with the Inr in a strict spatial manner. There is synergism between the MTE and the TATA box as well as between the MTE and the DPE ( Juven-Gershon, Cheng, & Kadonaga, 2006). There are three promoters (P2, P11, and P61) located upstream of the left, middle, and right ORFs in the IHHNV genome. The P2 and P61 promoters are presumed to regulate the expression of NS (1a and 1b) (encoded by left ORF) and VP (encoded by right ORF) genes, respectively. The P2 promoter region possesses the canonical TATA box (TATATAA) (Fig. 3.7), a GC-rich sequence GCGAGCGCT and a palindromic sequence ACCTATGACGTCATAGGT located downstream of the GC-rich


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Figure 3.7 Transient expression of firefly luciferase gene (fluc) in Sf9 insect cell cultures under the control of PstDNV promoters P2, P11, and P61 and their deletion versions. Different sequence motifs present in these promoters are summarized. The fluc expression was normalized to Renila luciferase (rLuc) expression and the levels of fLuc/rLuc are presented as horizontal bars with an arbitrary scale. The error bars represent the S.D. of replicate assays. The relative luciferase values within any promoter deletion series (P2, P11, or P61) with different letters are significantly different. The relative luciferase values for the full-length promoters P2, P11, and P61 are significantly different to each other as indicated by X, Y, and Z.

activator region but upstream of the TATA box. An initiation of transcription motif (Inr) CAGT is located 24 nucleotides downstream of the TATA box. The Inr sequence has been found to play an important role in the expression of many mammalian and arthropod promoters of both TATAcontaining and TATA-less types (Blissard, Kogan, Wei, & Rohrmann, 1992; Cherbas & Cherbas, 1993; Smale & Baltimore, 1989). The CAGT sequence motif is known to interact with cellular transcription factors, such as TFIID (Smale & Baltimore, 1989). In addition to TATA and Inr motifs, there is a G residue at position +24 from the transcription initiation site and a putative DPE ATCC, starting at the +28 nucleotide position. The G residue at position +24 of Drosophila core promoters was reported to result in a two to threefold higher level of basal transcription (Kutach & Kadonaga, 2000).

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There is an AP1 transcription factor-binding site four nucleotides downstream of the TATA box. The P61 promoter region does not have a canonical TATA box (Shike et al., 2000). However, several lines of evidence led the authors to conclude that this region could potentially serve as the promoter for the IHHNV right ORF. There is an AT-rich site (AAATAAAA), an Inr box (CAGT) 24 nucleotides downstream of the AT-rich site, a G nucleotide at +24 position, and a DPE starting at the +28 position (AGATC). In three Drosophila TATA-less promoters and in human TATA-less IRF-1 promoter, the GA/ TCGDPE motif that was located about 30 nucleotides downstream of the transcription start site has essential promoter function and is known to bind the cellular transcription factor TFIID (Burke & Kadonaga, 1996). Similarly, the Inr motif CAGT is known to interact with TFIID (Antonucci, Wen, & Rutter, 1989). In fact, the TATA sequence upstream of the Inr box was dispensable for efficient gene expression of AaeDNV, while mutation in the Inr motif reduced protein expression by 93% (Ward et al., 2001). Similarly to the structural P61 promoter, the P11 promoter driving the middle ORF does not have a canonical TATA box (Dhar, Lakshman, Natarajan, Allnutt, & van Beek, 2007; Shike et al., 2000). The P11 promoter has two tandem repeat CTTTC elements, a downstream TATA-like box (AAATATCG), an initiation of transcription signal (Inr or TIS) CATT, three G’s at position +22 to +24 relative to A of the Inr and a downstream promoter element (DPE) AGACC (Fig. 3.7), all of which conform to the rules of a eukaryotic promoter region (Kutach & Kadonaga, 2000). The AT-rich site (AAATATCG) being proximal to the Inr box most likely substitutes for the TATA element in the middle ORF promoter. The role of the tandem repeat elements CTTTC is not known at present. The functionality of Inr element is strengthened by the presence of G nucleotides at +22 to +24 and the DPE. In support of the in silico analysis, the subsequent transcription mapping confirmed that middle ORF transcription starts both at the C and the A of the Inr element (Dhar et al., 2010). When transcription starts at nucleotide C, the second G residues among the three downstream G’s become +24 (Dhar et al., 2010). 3.3.2 Functional characterization of IHHNV/PstDNV promoters Identification of the key transcriptional regulators is important in understanding the IHHNV/PstDNV pathogenesis in shrimp. To compare the functional activities, the three IHHNV/PstDNV promoters (i.e., P2, P11, and P61) were cloned upstream of a firefly luciferase gene in a promoter


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assay vector, pGL3-Basic or pGL3-Enhancer (Promega, Madison, WI) into nonhost insect (Spodoptera frugiperda cell line Sf9) cells and fish Epithelioma papulosum cyprinid cells (Dhar et al., 2007). It has been demonstrated that IHHNV/PstDNV promoters are pantropic in nature and are functional not only in shrimp but also in bacteria and in insect, and fish cell lines (Dhar et al., 2010, 2007; Dhar, van Beek, Moss, Bullis, & Allnutt, 2008). Using an in vitro assay in insect cell lines, the P2 promoter was found to be the strongest promoter followed by the P11 and P61. Since there is no immortal cell line of shrimp, the functionalities of shrimp viral promoters in nonhost cells opens up the possibility of determining the role of regulatory sequences in IHHNV/PstDNV gene expression. It remains to be determined if promoter activities vary depending on the host, for example, P. vannamei versus P. stylirostris, and what are the implications of differential gene expression on the manifestation of clinical signs in different hosts. Using a series of deletion constructs, Dhar and colleagues delineated the regulatory roles of individual motifs on promoter activity in IHHNV/ PstDNV (Dhar, Kaizer, Betz, Harvey, & Lakshman, 2011). The role of individual regulatory motif seems to vary depending on the IHHNV/PstDNV promoter. For example, in the P2 promoter, the deletion of the inverted repeat, DPE, and GC-rich regions had the highest negative impact on the reporter gene expression. In the P11 promoter, the deletions of DPE, G at the +24, and ASL box had the highest negative impact, while in the P61 promoter, DPE and G at +24 were the two key regulators of transcriptional activity (Fig. 3.7). This information is valuable in constructing shrimp viral promoter-based vectors for protein expression in insect cell cultures and in living shrimp. 3.3.3 Expression of IHHNV/PstDNV transcripts in virus-infected shrimp At least five classes of IHHNV/PstDNV transcripts (4.1, 2.6, 1.9, 1.3, and 0.9 kb) were observed on a Northern blot of total RNA from infected shrimp and hybridized with a probe representing the entire IHHNV/ PstDNV genome (Fig. 3.8A; Dhar et al., 2010). Based on the predicted size, the 2.6, 1.9, and 1.3 kb bands corresponded to the transcripts encoded by the left, middle, and the right ORFs, respectively. The origin of the 0.9 kb transcript is unknown so far but it might represent the small ORF (714 nt predicted size; Shike et al., 2000) present in the minus strand of the IHHNV/PstDNV genome, suggesting that the IHHNV/PstDNV genome could be ambisense like other densoviruses (Tijssen et al., 2003; Wang et al., 2005).

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A

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Figure 3.8 (A) Detection of IHHNV transcripts in Penaeus vannamei shrimp by Northern blot hybridization. The transcript sizes are indicated by arrows. Lane 1, total RNA from healthy P. vannamei; lane 2, total RNA from IHHNV-infected shrimp. (B) The quantification of PstDNV transcripts represented by the left (NS1), middle, and right (capsid) ORFs in experimentally challenged Penaeus vannamei shrimp by real-time RT-PCR. Each bar diagram represents the average copy number of the corresponding transcript in infected shrimp.

Since the genome size of IHHNV/PstDNV is 4.1 kb, the largest RNA transcript (4.1 kb) presumably represents the full-length transcript of the virus (Dhar et al., 2010). Even though its origin is unknown at present, it is likely to be transcribed by the P2 promoter and may represent an


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unprocessed IHHNV/PstDNV transcript. A similar full-length transcript (4.1 kb) has been detected in Aleutian mink disease parvovirus (AMDV), which is also an autonomous parvovirus belonging to the genus Amdovirus (Qui, Cheng, Burger, & Pintel, 2006). In AMDV, the full-length (4.1 kb) transcript is processed to translate both NS and structural proteins, whereas in IHHNV/PstDNV, in addition to the full-length transcript, different promoters drive the expression of viral-encoded genes. 3.3.4 Transcription initiation The origins of three transcripts represented by the three ORFs in IHHNV/ PstDNV were mapped (Dhar et al., 2010). The initiation of transcription for all three transcripts occurs at more than one site (at nucleotide 98 (A) and 101 (C) for NS1 gene, at nucleotide 466 (C) and 467 (A) for the gene representing middle ORF, and at nucleotide 2441 (C) and 2442 (A) for the CP gene, AF272215). The transcription of the middle ORF, which is located completely within the NS1 ORF, is not driven by the NS1 promoter (P2) instead it is driven by its own promoter, P11 (Dhar et al., 2010). 3.3.5 Transcription termination Unlike in prokaryotes, the 30 -ends of mature eukaryotic transcripts are not processed as precisely as the 50 -ends. Also, heterogeneity of 30 -ends is common in eukaryotes (Gilmartin, 2009; Zhao, Hyman, & Moore, 1999). In general, there are at least two signals required for eukaryotic transcription termination. The mammalian poly-A signal typically consists of a nearly 45-nt long core sequence that may be flanked by diverse auxiliary sequences that enhance cleavage and polyadenylation efficiency (Kim & Martinson, 2003). However, the core sequence consists of a highly conserved upstream positioning element (AAUAAA) that is recognized by a cleavage and polyadenylation-specific factor and a poorly defined downstream region rich in U or G/U. Downstream G/U or U-rich sequences are known to influence temporal patterns of gene expression (i.e., early vs. late transcription) in SV40 (Connelly & Manley, 1988). The poly (A) cleavage site, usually the dinucleotide CA, is generally located somewhere between these two elements (Guo & Sherman, 1996). As exceptions, the hexameric poly-A signal AAUAAA is not utilized in yeast and is not strictly conserved in some human genes (Beaudoing, Freier, Wyatt, Claverie, & Gautheret, 2000) and in human pathogenic parvovirus B19 (Ozawa et al., 1987). The mature transcripts of the IHHNV/PstDNV NS1 gene (left ORF) were found to be polyadenylated in two different sites, creating two size

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classes differentiated by 132–136 bases at the 30 -UTR. There is an AAUAAA element upstream of the polyadenylation region for the longer transcripts. Although there is no canonical AAUAAA signal upstream of the shorter left ORF transcripts, there is an AACAAA element in the corresponding site. A-rich sequences are present immediately downstream of both transcript sizes in the IHHNV/PstDNV DNA (Dhar et al., 2010). Unlike the left ORF, there is only one AAUAAA element upstream of both transcripts representing the middle ORF. The longer transcript also contains two UAUAAA elements downstream of AAUAAA, whereas the shorter transcript contains an UAUAAA and an UACAAA element downstream of AAUAAA. The CP gene transcript (right ORF) terminates in a single site. There is an AAUAAA element in the proximity of the polyadenylation site, and there are two more hexanucleotide elements (AACAAA and ACUAAA) upstream of the AAUAAA element (Dhar et al., 2010). The diversity in the transcript size classes and the presence or absence of canonical polyadenylation sites in IHHNV/PstDNV transcripts may reflect general evolutionary diversities of transcription termination in eukaryotic hosts (Gilmartin, 2009). It remains to be seen if the diversity of IHHNV/PstDNV transcripts is associated with the clinical manifestation and the host susceptibility (e.g., P. vannamei vs. P. stylirostris) in shrimp. 3.3.6 Transcript abundance assay In laboratory bioassays using P. vannamei shrimp, the abundance of transcripts representing NS1, CP, and middle ORF were measured by real-time RT-PCR (Dhar et al., 2010). There was no significant difference in the copy numbers of transcripts from the NS1 and CP genes. However, the transcript copy numbers of both NS and CP genes were significantly higher than the transcript copy number of the gene representing the middle ORF (Fig. 3.8B). Although the IHHNV/PstDNV transcript quantification data using luciferase as a reporter gene showed that both the NS and the CP genes are expressed at equivalent levels in IHHNV/PstDNV-infected shrimp (Dhar et al., 2010), the luciferase activity driven by the NS1 promoter, P2, in insect cell culture was significantly higher than the promoter activity of CP gene, P61, of IHHNV/PstDNV (Dhar et al., 2007; 2011). This suggests that the P61 promoter is likely to be transactivated in vivo by IHHNV/ PstDNV NS1 protein in shrimp. In mammalian parvoviruses, such as the minute virus of mice and the rodent parvovirus H-1, there is a temporal order of expression from the structural and NS gene promoters. In these


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viruses, the NS protein is expressed first, and this, in turn, transactivates the promoter for the structural gene (Doerig, Hirt, Beard, & Antonietti, 1988). In another mammalian parvovirus, AMDV, it has been suggested that weakness of the internal promoter may contribute to persistence of the virus in CRFK cells (Storgaard, Oleksiewicz, Bloom, Ching, & Alexandersen, 1997). Transactivation of structural gene promoters by viral NS proteins has also been reported in the A. aegypti and Junonia coenia densonucleosis viruses (Afanasiev et al., 1991, 1994; Giraud, Devauchelle, & Bergoin, 1992). It remains to be determined how the dynamics of IHHNV/PstDNV transcript abundances modulate viral pathogenesis and if the persistence of IHHNV/PstDNV infection in shrimp is dependent on the abundance of CP transcript. 3.3.7 Initiation of translation The Kozak consensus sequence plays a major role in the initiation of the translation process. The Kozak consensus sequence for initiation of translation in vertebrates is (GCC) GCCRCCATGG, where R is a purine (A or G) (Kozak, 2002). It has been proposed that some nucleotides of the Kozak sequence are more important than others, particularly the 3 and the +4 nucleotides. In a strongly expressed gene, the 3 (R) and the +4 (G) nucleotides should match exactly. In an adequately expressed gene, only one of those two sites must conform to consensus. In the absence of those two matches, a gene is considered as poorly expressed (Sakai et al., 2001). Analysis of the IHHNV/PstDNV genes demonstrated that only the left ORF follows the perfect consensus Kozak rule, whereas the middle ORF and the right ORF deviate slightly from the consensus (Dhar et al., 2010). Thus, in Kozak context (RNNAUGG), the left ORF could potentially be a strongly expressed gene (Kozak, 2002). However, only six of the seven nucleotides at the predicted translation initiation site of right ORF match with the consensus Kozak sequence, indicating the ORF as an “adequately expressed” gene (Kozak, 2002). In contrast, only five of the seven nucleotides near translation initiation of the middle ORF matched with the Kozak consensus sequence (Kozak, 2002), indicating that it is a poorly expressed gene (Sakai et al., 2001). Similar observations have been made in HPV of P. monodon (Sukhumsirichart et al., 2006), an iteravirusinfecting Dendrolimus punctatus (Wang et al., 2005), a human pathogenic B19 parvovirus (Ozawa et al., 1987) and a simian parvovirus (Vashisht, Faaberg, Aber, Brown, & O’Sullivan, 2004). A weak Kozak sequence content might not necessarily indicate a weakly translated gene, but that some

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other means such as leaky scanning or reinitiation/internal initiation of translation may play a greater role under such circumstances (Peri & Pandey, 2001). 3.3.8 Comparative gene expression of brevidensoviruses It is relevant to note a few reported similarities and differences in gene expression profiles among the three brevidensoviruses: IHHNV/PstDNV, AaeDNV (Afanasiev et al., 1991), and the C6/C36 DNV (AalDNV-2) of A. albopictus (Chen et al., 2004). Both AaeDNV and AalDNV-2 have only two promoters, namely, the NS and structural gene promoters that contain TATA motifs (Chen et al., 2004; Ward et al., 2001). However, IHHNV/ PstDNV contains three ORFs regulated by three distinct promoters, P2, P11, and P61 with only the P2 promoter containing the consensus TATA box motif (Dhar et al., 2010, 2011). Unlike the P61 structural promoter of IHHNV/PstDNV, no A-rich site or negative transcription regulator motif has been reported upstream of the TATA elements of the AaeDNV and AalDNV-2 structural genes. The structural gene expression of AaeDNV was shown to be induced in the presence of NS1. The exact site of NS1 recognition has not been elucidated even though the presence of the two genome termini of AaeDNV seems to enhance structural gene expression by the NS1 protein (Ward et al., 2001). The Inr/TIS motif of the structural gene is CAGT for AaeDNV and CATG for AalDNV-2, but CAGC for PstDNV. It remains to be seen if the IHHNV/PstDNV genome termini modulate the activity of P61 promoter.

3.4. Integration of IHHNV DNA in the host genome and implication in virus detection and disease resistance Integration of viral genome fragments into the host genome commonly occurs in arthropods, including insects, with persistent viral infections (Crochu et al., 2004; Lin et al., 1999). Insertion of WSSV-like sequences has been found in P. monodon from Australia (de la Vega, 2006). The phenomenon has been proposed to be involved in WSSV infectivity (Huang et al., 2008; Koyama et al., 2010). Evidence for insertion of IHHNV genome fragments into the shrimp genome have been reported in captured P. monodon from East Africa and Australia (Krabsetsve, Cullen, & Owens, 2004; Tang & Lightner, 2006). As a result, PCR-based screening of broodstock and postlarvae using primers to amplify a segment of the viral genome, which may otherwise be inserted in the host genome, fails to distinguish the infectious form of the virus from genome-integrated virus, and


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thus gives rise to false-positive results. Therefore, a PCR detection method using the primers IHHNV309F/R was developed to amplify IHHNV genomic regions that are not integrated to host genome (Tang et al., 2007). Random insertions of IHHNV genomic fragments in the P. monodon genome have been identified by PCR analysis from Thailand (Saksmerprome et al., 2011). Genome walking and PCR analysis with chimeric shrimp/virus primer sets indicated that viral inserts in shrimp genome DNA are in association with a host microsatellite-like sequence. The findings have implications in shrimp disease diagnosis because shrimp with some viral inserts may give false-positive results using the current standard methods including the commercially available IQ2000™ IHHNV (nested) (Farming IntelliGene Tech. Corp., Taiwan) method and the OIErecommended method that employs the primers IHHNV309F/R. This could potentially have a negative impact on international seafood trade, if products from uninfected shrimp with inserts are rejected due to positive PCR test results. Therefore, the detection methods for the infectious IHHNV need to be further improved to reduce the possibility of falsepositive results to correct for the random occurrences of various lengths of IHHNV inserts in the shrimp genome. Multiplex PCR employing primers sets that target the whole genome might be developed to overcome this problem. For example, the whole 4 kb IHHNV genome can be amplified by single-step or two-step PCR (V. Saksmerprome, unpublished). In addition, the concept of viral genome insertion could be important for the development of disease tolerant domesticated shrimp stocks via endogenous RNA interference (RNAi) (see Section 5.2). To test the hypothesis, offspring arising from uninfected parental shrimp with IHHNV inserts could be challenged with IHHNV to determine whether various types of viral inserts can provide protection against infection and/or disease caused by IHHNV. Such protection would suggest the possibility of heritable antiviral immunity in shrimp.

4. EVOLUTION OF SHRIMP PARVOVIRUSES 4.1. Genetic diversity of IHHNV IHHNV isolates have been classified into three genotypes (see in this section). While Genotypes I and II are infectious, Genotype III is noninfectious and has been shown to be integrated into the host genome (Krabsetsve et al., 2004; Tang & Lightner, 2006; Tang et al., 2007, 2003). Genotype I is distributed in the Americas and East Asia, while Genotype II is prevalent in

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South-East Asia; both are infectious to P. vannamei and P. monodon shrimp species. Genotype III is comprised of IHHNV-related sequences that have been found inserted in the genome of P. monodon and are considered not infectious (Krabsetsve et al., 2004; Tang & Lightner, 2006; Tang et al., 2003). Genotype III is divided in two subtypes: type IIIA is found in the Indo-Pacific region that includes Madagascar, Mauritius, and Tanzania, while type IIIB is mostly found in East Africa, India, and Australia (Tang et al., 2003; Tang et al., 2007; Fig. 3.9). At the nucleotide level, the genetic diversity of IHHNV has traditionally been considered limited. Based on the CP gene of 14 IHHNV isolates, Tang and colleagues found only 1.3% segregating sites. These findings led the authors to suggest that IHHNV was a slowly evolving stable genome (Tang et al., 2003). However, in a recent study using a much larger sample size, it was found that IHHNV has almost five times higher genetic diversity (6.9%) than previously reported. This increased nucleotide variation resulted in an increased IHHNV haplotype diversity and allowed the detection of a population structure among IHHNV isolates that was previously unnoticed AY102034 Thailand_Pm AY355307 Taiwan_Pm GU138651 Phillipines_Pv

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Figure 3.9 Phylogenetic relationship among different geographical isolates of IHHNV as determined by Bayesian analysis. The GenBank accession number, country of origin, and the abbreviated name of the host (Pm for Penaeus monodon, Pv for P. vannamei, Ps for P. stylirostris) are given for all the sequences taken for phylogenetic analysis.


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in the region (Robles-Sikisaka et al., 2010). To determine whether high levels of genetic diversity in IHHNV occur in other geographic regions, a more thorough sampling and sequencing is necessary.

4.2. Evolutionary mechanisms of IHHNV In an attempt to understand the drivers of molecular evolution of IHHNV several mechanisms have been studied. These include as follows. 4.2.1 Recombination Single-stranded DNA viruses, including parvoviruses, are thought to undergo extensive recombination (Martin et al., 2011). Although recombination studies on IHHNV have been limited, recombination signals on IHHNV isolates were recently assessed using different algorithms and no evidence of recombination events was found in the IHHNV CP gene sequences (Robles-Sikisaka et al., 2010). However, it remains to be determined whether recombination occurs at other sites in the IHHNV genome. 4.2.2 Positive selection The ratio of nonsynonymous substitution (amino acid altering substitutions) and synonymous substitution (substitutions that do not alter amino acids) (dN/dS) has been extensively used as an indicator of selection pressure. As with recombination, this mechanism has not been addressed extensively in IHHNV. However, signals of positive selection were discovered in several IHHNV lineages, in which the rates of amino acid replacements exceeded those of synonymous substitutions when compared to neutral expectations for genetic drift and mutation (Robles-Sikisaka et al., 2010). It is hypothesized that positive selection contributes to the higher genetic diversity observed in IHHNV. 4.2.3 Rates of nucleotide substitution Generally, it is assumed that DNA viruses evolve slowly, to a rate similar to that of their host because of their dependence on the host’s cellular machinery for replication (Duffy, Shackelton, & Holmes, 2008; Shackelton & Holmes, 2006). However, recently, using dated isolates a high rate of nucleotide substitution for IHHNV of 8.70 105 substitution/site/year was determined (Robles-Sikisaka et al., 2010). This rate is comparable to that of RNA viruses, which are known to evolve at a rate of 10–3 to 10–5 substitutions/site/year ( Jenkins, Rambaut, Pybus, & Holmes, 2002; Scholtissek, 1995). A recent study by Kim and colleagues independently corroborated this high rate of

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evolution for IHHNV (Kim, Kim, et al., 2011). Interestingly, other parvoviruses have been also found to evolve at rates similar to that of IHHNV. These include canine parvovirus (1.7 104), feline panleukopenia parvovirus (9.4 105) (Shackelton, Parrish, Truyen, & Holmes, 2005), human B19 erythrovirus (1 104) (Shackelton & Holmes, 2006), and the ssDNAcontaining begomovirus, tomato yellow leaf curl virus (2.88 104) (Duffy & Holmes, 2008). To our knowledge, IHHNV is the first marine invertebrate parvovirus where such high levels of nucleotide substitution have been estimated and may play a significant role in its genetic diversity. 4.2.4 IHHNV phylogeny The evolutionary relationships among the different IHHNV isolates have been assessed in several studies. Most of the studies performed to date used a small number of isolates from geographically distant sites. This limits the ability to observe relationships at a fine scale among IHHNV genotypes. Recently, the phylogenetic relationships from 61 IHHNV genotypes were studied, 14 of which were from different countries and isolated at different years and the rest from Mexico isolated in the same year (Robles-Sikisaka et al., 2010). This approach allowed studying the genetic structure of IHHNV isolates in more detail. This analysis showed all Mexican isolates formed a highly supported monophyletic group (Robles-Sikisaka et al., 2010). Three main clusters were observed (1) one with only Mexican southern haplotypes; (2) a second containing western and central Mexican haplotypes, which also contained Asian haplotypes illustrating the ancestral relationships of Mexican and Asian IHHNV isolates; and (3) a third clade contained a mixture of all geographic isolates, suggesting the genetic flow of both host and IHHNV (Robles-Sikisaka et al., 2010). However, the lack of in-depth sequencing of IHHNV isolates precludes the thorough analysis of the phylogenetic relationships of IHHNV in other geographic regions. For the purpose of this review, we analyzed the evolutionary relationships using the IHHNV VP1 CP gene from all representative geographic isolates available in the GenBank database. A Bayesian inference phylogenetic analysis was conducted using Mr. Bayes (Ronquist & Huelsenbeck, 2003) and an alignment of 25 IHHNV nucleotide VP1 CP sequences accessible from GenBank database. Sequences were aligned using Clustal X (Thompson, Gibson, Plewniak, Jeanmougin, & Higgins, 1997) incorporating the best-fit model of nucleotide substitution, TVM + G, selected using the program jModeltest (Posada, 2008). The topology of the phylogenetic tree shows three well-defined clades (Fig. 3.9), these clades correspond roughly to the three previously suggested


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genotypes (Tang & Lightner, 2006; Tang et al., 2007). The first clade is mostly formed by Southeast Asian isolates from Thailand, Philippines, and Vietnam from P. monodon hosts. The composition of this clade closely corresponds to that suggested for IHHNV Genotype 1 (Tang & Lightner, 2006). Additionally, the basal position of the branches in this clade suggests that these isolates are the oldest and that IHHNV strains occurring globally may have derived from an Asian P. monodon ancestor. This hypothesis has been proposed previously by other authors (Lightner et al., 2012; Tang et al., 2003). The second clade is highly supported with a posterior probability of 1 and is composed by a mixture of isolates from the Americas and East Asia from different host species P. monodon, P. vannamei, and P. stylirostris. This geographically and host-species diverse group suggests that these IHHNV isolates occurring in these regions are genetically similar and share close evolutionary relationships despite the geographic distance. This diverse clade illustrates the effects of reintroductions due to shrimp stocks movement into geographically distant regions for farming purposes and it also highlights the ability of IHHNV to adapt to different host species and subsequently evolve independently. Similar to clade 1, this clade also resembles the composition suggested for Genotype 2 (Tang & Lightner, 2006). The third clade contains isolates from the Indo-Pacific biogeographic region. However, this clade not only comprises IHHN-related noninfectious sequences incorporated in P. monodon genome (Tanzania AY124937, Madagascar DQ228358, and Australia EU675312) but also includes newly characterized infectious isolates from India and Australia. The basal position of these infectious strains in the clade suggests that that the noninfectious IHHNV-related sequences may have derived from an older infectious IHHNV isolate.

4.3. Genetic diversity and phylogeny of HPV The sequence analysis of HPV isolates from different geographic areas have shown consistently and relatively high levels of sequence similarities at the amino acid and nucleotide levels of the HPV genome ( Jeeva et al., 2012; La Fauce, Elliman, et al., 2007; Safeena et al., 2010). The highest level of sequence similarity has been found in the amino acid sequence of ORF2 that codes for the NS1 (97.1–99.8%) ( Jeeva et al., 2012). Levels of genetic variation have been estimated for full genome HPV sequences from Korea, Thailand, Australia, and Tanzania (Tang, Pantoja, & Lightner, 2008), showing that genetic diversity at the nucleotide is substantial among isolates, ranging from 12% to 21% (Tang et al., 2008). At the amino acid level, genetic

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variation is highest in ORF3 (coding for VP1 protein) with a mean genetic distance 24% and lowest in ORF2 (coding for NS1 protein) with a mean genetic distance of 7% (Tang et al., 2008). Both NS1 and VP1 amino acid sequences have been used to assess the phylogenetic relationships among geographically different HPV isolates. Phylogenetic clustering suggests the existence of three distinct HPV genotypes ( Jeeva et al., 2012; Tang et al., 2008). Type I include isolates from Korea, Madagascar, and Tanzania; Type II includes isolates from Thailand and Indonesia, while Type III contains HPV isolates from Australia and New Caledonia ( Jeeva et al., 2012; Tang et al., 2008). To study the evolutionary relationships of HPV isolates with other shrimp- and insect-infecting parvoviruses, we performed a Bayesian phylogenetic analysis using an alignment of VP1 nucleotide sequences and a bestfit model of nucleotide substitution GTR + G as selected by jModeltest (Posada, 2008), in Mr. Bayes (Ronquist & Huelsenbeck, 2003). The phylogenetic analysis shows all HPV isolates forming a highly supported monophyletic clade, while IHHNV isolates forming a separate sister clade, suggesting a common ancestral origin of both shrimp parvoviruses (Fig. 3.10). This distinction between IHHNV and HPV sequences has been observed in previous studies (La Fauce, Elliman, et al., 2007, Roeckring HQ699073 IHHNV S. Korea 1

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Figure 3.10 Evolutionary relationship of IHHNV and HPV to insect brevidensoviruses, as determined by Bayesian phylogenetic analysis. The GenBank accession number and the country of origin of the IHHNV and HPV isolates are indicated.


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et al., 2002; Safeena et al., 2010). A third highly supported separate clade was formed by all insect parvoviruses. The same basic topology that separates HPV, IHHNV, and insect parvoviruses has been observed previously (La Fauce, Elliman, et al., 2007, Roeckring et al., 2002; Safeena et al., 2010). A closer look of the HPV clade shows the different isolates arranged in four subclusters as opposed to the three previously suggested ( Jeeva et al., 2012; Tang et al., 2008). Previously described Genotypes II and III can be clearly identified; however, Genotype I is formed by the Tanzania and Madagascar HPV isolates excluding the Korean isolate (Fig. 3.10). The HPV Korean isolate forms a relatively highly supported clade with recently characterized Fenneropenaeus chinensis HPV South Korean isolate (GenBank accession JN082231) and F. chinensis HPV Chinese isolate (GenBank accession GU371276). These differences are possibly due to increased number of HPV isolates incorporated in the phylogenetic analysis that allows a better resolution of the evolutionary relationships among isolates that was not possible to detect previously with a smaller sample size in the previously published papers.

5. MANAGEMENT OF PARVOVIRUS INFECTION 5.1. Virus prevention There has been major progress in identifying and characterizing the etiologic agents as well as in developing diagnostic tools for diseases caused by parvoviruses, particularly those caused by IHHNV and HPV, in marine shrimp. However, the management of these diseases still primarily relies on preventative approaches. Avoidance of pathogens via the use of SPF broodstock and exclusion of carrier animals in culture systems are still considered as the cornerstone to mitigate the losses caused by parvoviruses in shrimp aquaculture. The application of PCR prescreening of wild- or pond-reared broodstock and/or their spawned eggs/nauplii and discarding those that test positive for the virus has shown to be effective in reducing the prevalence of the virus in culture conditions (Fegan & Clifford, 2001). Often, culture conditions such as high stocking density and poor water quality contribute to disease introduction and spread. It has been estimated that only a small portion of the nutrient input (generally less than 20% of nitrogen and 10% of phosphorus) is incorporated into animals and the rest ends up in the environment (Funge-Smith & Briggs, 1998). The increase in the levels of nitrogen- and phosphorus-containing waste in pond sediment coupled with the reduction of beneficial bacteria that recycle those nutrients

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lead to the deterioration of water quality (Rao, Karunasagar, Otta, & Karunasagar, 2000). Therefore, it has been suggested that supplementation of bacteria capable of oxidizing toxic wastes (bioaugmentation) could be useful in improving water quality in shrimp culture ponds. There is also a growing interest in using probiotics in shrimp aquaculture. Enhancement of general health conditions in shrimp via the incorporation of microbially derived components, such as beta-1, 3-glucans, peptidoglycans, and polysaccharides, into shrimp feed has been shown to stimulate the nonspecific immune mechanisms in shrimp. These general husbandry practices may help prevent parvovirus and other viral infections in shrimp aquaculture. Since the IHHNV outbreak occurred in P. stylirostris shrimp in 1980s, efforts were made to develop IHHNV-resistant lines of shrimp. Selected lines of P. stylirostris were developed that were found to be refractory to IHHNV infection. Subsequently, genetic markers and random amplified polymorphic DNA markers were identified for IHHNV-resistant P. stylirostris shrimp (Hizer, Dhar, Klimpel, & Garcia, 2002). However, these IHHNV-resistant stocks do not show resistance to diseases, such as white spot syndrome disease caused by the WSSV, and hence, their use has been limited. It is noteworthy that P. vannamei with high viral loads of IHHNV are reported to be resistant to infection by WSSV (Bonnichon, Bonami, & Lightner, 2006). In contrary to P. stylirostris, P. vannamei is relatively resistant to IHHNV, and this is considered to be among the principal factors that led to the adoption of P. vannamei as the principal shrimp species globally (Lightner, 2005).

5.2. Therapeutic approach: Viral inhibition by RNAi Currently, there is no therapeutic approach available to control parvovirus infection in shrimp aquaculture. But efforts are underway to develop antiviral therapy against parvovirus infection in shrimp. These efforts are primarily based on dsRNA-mediated gene silencing or the RNAi mechanism. RNA interference has been reported to efficiently protect shrimp against a number of highly pathogenic viruses including WSSV, YHV, and TSV (Ongvarrasopone, Saejia, Chanasakulniyom, & Panyim, 2011; Saksmerprome, Charoonnart, Gangnonngiw, & Withyachumnarnkul, 2009; Tirasophon, Roshorm, & Panyim, 2005; Tirasophon, Yodmuang, Chinnirunvong, Plongthongkum, & Panyim, 2007; Westenberg, Heinhuis, Zuidema, & Vlak, 2005; Yodmuang, Tirasophon, Roshorm, Chinnirunvong, & Panyim, 2006), and more recently IHHNV (Attasart, Kaewkhaw, Chimwai, Kongphom, & Panyim, 2011). The efficiency of RNAi in inhibiting IHHNV replication was


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demonstrated in P. vannamei in both preventive and therapeutic manners (Ho, Yasri, Panyim, & Udomkit, 2011). In the later study, dsRNA was designed to target two different regions of the IHHNV genome—one for the left and middle ORFs (ORF1–2) and another for the right ORF (ORF3). For prevention, significant inhibition was observed in groups treated with the two IHHNVspecific dsRNAs when compared to shrimp injected with nonspecific dsRNA. A therapeutic effect was also observed when specific dsRNA was administered within 24-h postchallenge with IHHNV. Notably, the ORF3 dsRNA was less potent than ORF1–2 dsRNA, indicating that abundance of a viral transcript might affect the efficiency of its corresponding dsRNA. As previously reported by Dhar et al. (2010), the promoter upstream of ORF1 is stronger than the one located upstream of ORF3, and this could lead to more abundant viral transcripts for ORF1 than for ORF3. It is possible that abundance of viral mRNA is not the sole parameter in determining the efficacy of dsRNA on viral inhibition. Other factorsincludingprimarysequenceandsecondarystructuralpropertiesofboththe dsRNA and the viral mRNA should be taken into account for optimizing dsRNA-mediated gene silencing. To date, intramuscular injection of dsRNA appears to be the main delivery method for successful shrimp viral inhibition in lab-scale trials. However, delivery of dsRNA via injection is not feasible for farm application but could have some application for broodstock protection. Therefore, development of an oral delivery method is necessary to make RNAimediated protective/therapeutic strategy for shrimp farming. Endogenous RNAi-based immunity in shrimp has been proposed to explain coexistence of viral fragments and putative reverse-transcriptase (RT) and integrase (IN) sequences in the shrimp genome (Flegel, 2009). According to this hypothesis, both RT and IN are responsible for random integration of viral genome fragments into the host genome. Subsequently, these viral inserts can be transcribed to antisense, immunospecific mRNA (imRNA) that can bind specifically with viral mRNA to produce dsRNA to stimulate the RNAi pathway, which lead to a reduction in viral replication. Whether or not RNAi triggered by imRNA is involved in an antiviral defense in shrimp is yet to be tested. If the inserted viral fragments are indeed involved in resistance to viral infection, such a mechanism would be useful for the production of pathogen-specific resistant shrimp.

6. CONCLUSION Shrimp aquaculture provides jobs to millions of people around the world directly or indirectly and is a major force of socioeconomic development of poor rural and coastal communities in Asia and South America. The

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rise of intensive shrimp aquaculture over the past few decades has also had a negative impact in aquatic ecosystems (Walker & Mohan, 2009). For example, anthropogenic stress created to the aquatic ecosystems by intensive farming has led to the emergence of an array of diseases in shrimp aquaculture (Walker & Winton, 2010). Often, these new diseases have spread even before the identification of the etiologic agent, the development of diagnostic tools, or knowledge about the disease epidemiology(such as virus replication cycle, mode of transmission, reservoirs, virulence, etc.) that favor the spread of these diseases. Emergence and global spread of IHHNV in shrimp aquaculture is one such example. Since the initial report of IHHNV in early 1980s (Lightner, Redman, & Bell, 1983), considerable efforts have been made to identify and characterize the etiologic agent and developing biological, serological, and molecular diagnostic tools for detection of IHHNV and other viral pathogens (Flegel, 2006). In fact, a number of novel findings have came out from research involving IHHNV that can be considered as landmarks in shrimp virology. For example, IHHNV was the first shrimp viral pathogen for which almost the entire genome sequence was determined (Shike et al., 2000) and for which diagnostic methods based on gene probes (Mari et al., 1993) and real-time PCR-based assays were developed (Dhar et al., 2001; Tang & Lightner, 2001). The phenomenon of crossprotection in a shrimp virus was first demonstrated with IHHNV, since P. stylirostris shrimp persistently infected with IHHNV were found to exhibit markedly reduced mortality upon subsequent challenge with WSSV (Tang et al., 2003). Integration of viral genomic sequences in the shrimp genome was also first demonstrated with IHHNV (Saksmerprome et al., 2011; Tang & Lightner, 2006). The role of such genome-integrated viral sequences in preventing lethal infections with a cognate virus and potentially with a different virus remains to be determined. It has been suggested that shrimp and perhaps all arthropods may have the ability to accommodate single or multiple viral infections in an adaptive manner without displaying any gross signs of disease (Flegel, 2007; Flegel & Pasharawipas, 1998). IHHNV can often occur as heavy infections in P. monodon without any significant lesions or measurable negative impact (Chayaburakul et al., 2004; Flegel et al., 2004). Therefore, understanding the molecular mechanisms that enable shrimp to carry single or multiple viral infection(s) without negative impact may also lead to the development of antiviral therapy in shrimp. The ability of IHHNV, a single-stranded DNA virus, to mutate at a rate similar to many RNA viruses (Robles-Sikisaka et al., 2010) emphasizes the


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need to regularly monitor IHHNV and other parvoviruses in shrimp. This will help in determining the emergence of any potentially virulent isolates and developing strategies to contain any inadvertent spread of a virulent virus within and across geographic boundaries. Due to the lack of availability of any therapeutic measures, prevention will continue to remain the cornerstone for the management of parvoviruses as well as other viral pathogens in shrimp. Use of SPF postlarvae to stock ponds is becoming more common as P. vannamei SPF domesticated lines are readily available. However, there remains a need to develop antiviral therapies as the shrimp aquaculture moves toward long-term sustainability. Although RNAi-based therapeutic approaches have shown promise against parvoviruses and other shrimp viruses at the experimental level, application of such measures at the farm remains a considerable challenge. In order for any therapeutic measure to be acceptable in shrimp farming, an oral delivery method has to be developed. There are major hurdles to production of therapies based on oral delivery. Foremost of these hurdles are the stability of the antiviral agent during feed preparation, feeding in an aquatic milieu, and in the gut, successful transmission to the hemolymph and successful induction of a strong humoral response. This is an area of research that will certainly gain much attention in the years to come as antiviral therapies at the experimental level show promising results.

ACKNOWLEDGMENTS The authors would like to thank Dr. Timothy W. Flegel, Mahidol University, Bangkok, Thailand, and Dr. F. C. Thomas Allnutt, BrioBiotech, Glenelg, Maryland, USA for providing valuable comments on the manuscript. The authors also thank Dr. Flegel for providing the figures 3.1, 3.2, 3.3, 3.4, and 3.5.

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