key: cord-1054224-ag5y781z
authors: Cowley, J.A.
title: Nidoviruses of Fish and Crustaceans
date: 2016-09-09
journal: Aquaculture Virology
DOI: 10.1016/b978-0-12-801573-5.00032-2
sha: aa0734b03303376ba9fbd690867137c23fe442cc
doc_id: 1054224
cord_uid: ag5y781z

Viruses with diverse virion architectures demarcated into four families in the order Nidovirales have been discovered in vertebrate mammalian and fish species, as well as in invertebrate crustacean and mosquito species. The order is unified by nidoviruses sharing intermediate (12.7 kb) to very long (31.7 kb) (+) ssRNA genomes, each possessing a long 5′-terminal gene encoding overlapping ORF1a and ORF1b reading frames that contain a diversity of functionally related enzymes and that are translated in toto using a −1 ribosomal frameshift mechanism, as well as by semiconserved strategies for transcribing a nested set of 3′-coterminal subgenomic mRNAs that translate the viral proteins. The nidovirus that is most important to an aquaculture species is yellow head virus (YHV), which causes disease in shrimp farmed throughout the Eastern Hemisphere and is classified in the genus Okavirus, family Roniviridae. Fathead minnow nidovirus, genus Bafinivirus, subfamily Torovirinae, family Coronaviridae, also causes disease in minnows grown for the baitfish industry in the United States. Virions similar in morphology to okaviruses and bafiniviruses have also been detected in several crab species. Of these, however, only Eriocheir sinensis ronivirus, which causes disease in the Chinese mitten crab, an important freshwater aquaculture species in China, has been shown to possess a ~22 kb ssRNA genome that supports its being a nidovirus, but its taxonomic classification awaits genome sequence analysis. This chapter provides an overview of the structure, replication and biology of these viruses with a particular focus on YHV disease characteristics, diagnostic methods and disease prevention strategies.

The order Nidovirales comprises four families of viruses containing long RNA genomes (de Groot et al., 2012b; Gorbalenya, 2008; Gorbalenya et al., 2006) , the Coronaviridae (de Groot et al., 2012a) , Arteriviridae (Siddell and Snijder, 2008; Faaberg et al., 2012) , Roniviridae (Mayo, 2002; Cowley et al., 2012) and Mesoniviridae (Nga et al., 2011; Zirkel et al., 2011; Lauber et al., 2012) . Viruses in the Coronaviridae are segregated into two subfamilies. The Coronavirinae comprises four genera (Alpha-, Beta-, Gamma-and Deltacoronavirus) of viruses infecting diverse mammals or birds and the Torovirinae comprises viruses in the Torovirus genus that infect horses, cattle and humans and in the Bafinivirus genus that infect fish (http:// ictvonline.org/virusTaxonomy.asp, de Groot et al., 2012a) . Viruses in the single Arterivirus genus of the Arteriviridae have been isolated from diverse mammalian species such as mice, monkeys, horses and pigs (Balasuriya and Snijder, 2008) . Viruses in the single Alphamesonivirus 1 genus of the Mesoniviridae have only been detected in mosquitoes (Lauber et al., 2012) and viruses in the single Okavirus genus of the Roniviridae have only been detected in crustaceans (Cowley et al., 2000a (Cowley et al., , 2012 Sittidilokratna et al., 2008) .

Depending on the family and genus, nidoviruses have diverse virion morphologies ranging from spherical to bacilliform, but despite this, they share common genome organization and transcription features (de Groot et al., 2012a) . All possess a genome comprised of an infectious linear positive-sense (+) ssRNA, which for coronaviruses (26.4-31.7 kb) is the longest of any known RNA virus (de Groot et al., 2012b) . While toroviruses (28.0-28.5 kb) (Snijder et al., 2013) , bafiniviruses (26.6-27.3 kb) (Schütze et al., 2006; Batts et al., 2012) and roniviruses (26.2-26.6 kb) (Cowley et al., 2000a; Cowley and Walker, 2002; Sittidilokratna et al., 2002 Sittidilokratna et al., , 2008 possess similarly long genomes, those of mesoniviruses (20.2 kb) (Nga et al., 2011; Zirkel et al., 2011 Zirkel et al., , 2013 Lauber et al., 2012) and arteriviruses (12.7-15.7 kb) (Snijder et al., 2013) are more compact. Despite being diverse in length and gene number, the 5′-genome region of all nidoviruses comprises two long genes encoding overlapping open reading frames (ORF1a and ORF1b) that can constitute up to three-fourths of the genome length (Cowley et al., 2000a; Cowley and Walker, 2002; Sittidilokratna et al., 2002 Sittidilokratna et al., , 2008 . At the ORF1a/1b gene overlap, all nidovirus genomes possess a "slippery" heptanucleotide sequence, which in conjunction with a nearby downstream sequence capable of folding into a pseudoknot structure, functions as a translational −1 ribosomal frameshift (RFS) element (Brierley, 1995; Brierley et al., 1989 Brierley et al., , 1992 Cowley et al., 2002a; ten Dam et al., 1990) . This −1 RFS element functions at between 20% and 30% efficiency in pausing polyprotein 1a (pp1a) translation from the ORF1a gene and then slipping the translational machinery back a single nucleotide to be in-frame with the ORF1b gene-coding sequence, thus allowing translation of pp1a to be continued though ORF1b to generate a pp1ab polyprotein. As the ORF1a and ORF1b genes encode nonstructural proteins (nsps) utilized in genome replication and mRNA transcription, the −1 RFS element appears to have been preserved in the evolution of nidoviruses by a need to regulate the abundance of proteins encoded in the ORF1b gene relative to those encoded in the ORF1a gene.

The pp1a polyprotein contains numerous functional motifs, including an ADP-ribose 1-phosphatase, papain-like proteases and a chymotrypsin-like serine protease (3C-like or main proteinase) involved in the site-specific cleavage the pp1a and pp1ab polyproteins into their function units, and the pp1ab polyprotein contains these enzymes in addition to ORF1b gene-encoded motifs, including a RNA-dependent RNA polymerase (RdRp), helicase, ribose methyltransferase, exoribonuclease and endoribonuclease involved in viral RNA transcription and genome replication (de Groot et al., 2012a; Ziebuhr et al., 2000 Ziebuhr et al., , 2003 Ziebuhr, 2005 Ziebuhr, , 2006 Cowley et al., 2000a; Sittidilokratna et al., 2002) . Positioned downstream of the ORF1a/1b gene are as few as 2 genes for roniviruses (Cowley et al., 2002a; Cowley and Walker, 2002; Sittidilokratna et al., 2008) to as many as 12 genes for coronaviruses that encode either the virion structural proteins or accessory proteins with various family and genus-specific functions (de Groot et al., 2012a,b) .

All nidoviruses also transcribe a nested set of 3′-coterminal capped and polyadenylated subgenomic (sg)mRNAs that encode the virus structural and accessory proteins (Pasternak et al., 2006; Cowley et al., 2002a Cowley et al., , 2004a Jitrapakdee et al., 2003; Sittidilokratna et al., 2006 Sittidilokratna et al., , 2008 . The mechanism by which these sgmRNAs are transcribed appears to have adopted complexity as nidovirus genomes have acquired greater numbers of genes, presumably to accommodate replication in more highly evolved hosts (Pasternak et al., 2006; Sawicki et al., 2007) . In okaviruses, which replicate in crustacean hosts with ancient origins (Siveter et al., 2001; and which have a simple genome organization comprising only the ORF1a/1b replicase genes and two genes (ORF2 and ORF3) encoding virion structural proteins, the genomic RNA and sgmRNAs used to translate these ORFs each begin with 5′-AC termini, with these sgmRNA termini mapping to a central position of highly conserved transcriptional regulatory sequences (TRSs) residing within the intergenic regions upstream of ORF2 and ORF3 (Cowley et al., 2002a; Sittidilokratna et al., 2008) . Similarly to all but the longest sgmRNA of toroviruses (Snijder et al., 1990; van Vliet et al., 2002; Smits et al., 2005) , these TRS elements appear to have roles in terminating negative-strand (−) RNA transcription in a lossy fashion that allows some transcripts to continue through to upstream TRSs or to the (+) RNA genome 5′-terminus. As evidenced by the presence of genomic-length and sgmRNA-length replicativeintermediate double-stranded (ds)RNAs in shrimp cells infected with gill-associated virus (GAV) (Cowley et al., 2002a) , the type species okavirus (Cowley et al., 2012) , it is speculated that transcription termination of the antisense RNAs might occur at precise positions, resulting in common 3′-termini, and that these then act directly as promoters for transcription initiation of the genomic and sgmRNAs. In all other nidoviruses, however, and for the longest of the sgmRNAs transcribed by toroviruses, the (−) and (+) strand sgmRNAs are transcribed using a far more complex discontinuous process involving the splicing of a common "anti-leader" sequence derived from the genome 5′-terminus to each (−) strand sgmRNA that then acts as a universal promoter for transcribing each (+) strand sgmRNA (Pasternak et al., 2006; Sawicki et al., 2007; Smits et al., 2005; van Vliet et al., 2002) .

Nidoviruses of aquatic species include the rod-shaped okaviruses GAV and yellow head virus (YHV) that primarily infect Penaeid shrimp (Longyant et al., 2005; Flegel, 2012; Flegel et al., 1997a; Cowley et al., 2000a Cowley et al., , 2002a Cowley and Walker, 2002; Sittidilokratna et al., 2008) and a morphologically similar virus with a ~22 kb ssRNA genome detected in diseased Chinese mitten crabs (Zhang and Bonami, 2007) . They also include the rod-shaped bafiniviruses white bream virus (WBV) detected in white bream in Germany (Granzow et al., 2001; Schütze et al., 2006; Ulferts et al., 2011) and fathead minnow nidovirus (FHMNV) detected in diseased fathead minnows in the United States (Iwanowicz and Goodwin, 2002; Batts et al., 2012) . A virus with a spherical coronavirus-like particle morphology has also been detected in color carp in Japan (Miyazaki et al., 2000 (Miyazaki et al., , 2001 , but it has not yet been demonstrated to possess a long (+) ssRNA genome with features characteristic of nidoviruses.

To aid clarity, the fish bafiniviruses and crustacean okaviruses will be described separately together with viruses not yet assigned formally as nidoviruses, but which share common structural features.

Rod-shaped nidoviruses classified in the genus Bafinivirus, subfamily Torovirinae, family Coronaviridae (de Groot et al., 2012a) have been discovered in wild white bream (Blicca bjoerkna L.) in Germany (Granzow et al., 2001) and in diseased fathead minnows (Pimephales promelas) cultured as baitfish in the United States (Iwanowicz and Goodwin, 2002; Table 32 .1 and Fig. 32.1 ). White bream support small wild freshwater fisheries in Europe and fathead minnows support a substantial freshwater baitfish culture industry in the United States (Meronek et al., 1997; McCann, 2012) . WBV and

The capped and polyadenylated (+) ssRNA genomes of WBV (Schütze et al., 2006) and FHMNV (Batts et al., 2012) each possess five genes encoding ORFs (ORF1a, 1b, 2, 3 and 4). The ORF1a/1b gene encodes pp1a and pp1ab polyproteins containing proteinase, polymerase and other replicase enzymes shared across all nidoviruses and ORF2, ORF3 and ORF4 encode a spike (S) glycoprotein, membrane (M) protein and nucleocapsid (N) proteins, respectively. Both viruses utilize −1 RFS elements comprised of an UUUAAAC "slippery" heptanucleotide sequence and a downstream RNA pseudoknot structure similar in predicted structure to facilitate translational read-through of the ORF1a/1b gene overlap, and possess intergenic nonanucleotide TRS elements near identical in sequence (Schütze et al., 2006; Batts et al., 2012) . Subgenomic mRNAs transcribed for each gene possess 5′-genome leader sequences upstream of the conserved TRS, indicating the use of a discontinuous transcription strategy comparable to those used by nidoviruses other than roniviruses and for the longest sgmRNA of toroviruses (Pasternak et al., 2006; Sawicki et al., 2007; van Vliet et al., 2002; Smits et al., 2005; Snijder et al., 1988) .

WBV (virus isolate DF 24/00) was discovered in 2000 in fish cell cultures inoculated with a mixed tissue homogenate of white bream (Blicca bjoerkna L.) as part of a virus monitoring program of wild fish populations in Germany (Granzow et al., 2001) . Although highly cytopathic for Epithelioma papulosum cyprini (EPC) cell cultures derived from carp (Cyprinus carpio) epidermal herpes virus-induced hyperplastic lesions, the health status of the white bream from which WBV was isolated was not reported, and there is no information on its pathogenic potential for this or other fish species.

FHMNV was isolated in 1997 from moribund wild-type and rosy red fathead minnows (Pimephales promelas) being reared as baitfish in ponds in central Arkansas (Iwanowicz and Goodwin, 2002) . The minnows were a mixture of wild and farmed fish from various unspecified US locations and were experiencing chronic mortality associated with eye and skin hemorrhages. Like WBV, FHMNV is highly cytopathic for EPC cells characterized initially by cell rounding, with multifocal syncytia (fused multinucleated cells) then forming as the cell monolayer degrades. Among several others examined, rainbow trout gonad (RTG-2) and fathead minnow (FHM) cell lines were also highly susceptible to FHMNV-induced cytopathology (Iwanowicz and Goodwin, 2002) . In FHMNV-infected EPC cells, histopathology is characterized by pyknotic nuclei displaying marginated chromatin, and in moribund FHMs, by lesions in the anterior kidney, liver and spleen, as well as severe multifocal necrosis of hepatocytes (Iwanowicz and Goodwin, 2002) .

In FHMs challenged with FHMNV, morbidity becomes evident by behavioral changes characterized by fish initially swimming erratically in circles before becoming listless either at the tank bottom or with their head orientated upward at the water surface (Iwanowicz and Goodwin, 2002) . In 19°C water, mortalities accumulate rapidly from 3 days post-challenge, but 17°C water delays mortality onset to 13 days post-challenge. Clinical signs consistent with the index case develop that include petechial hemorrhages in the eyes and skin (Iwanowicz and Goodwin, 2002) . Also evident can be hemorrhaging of muscle, generally localized to the epaxial musculature anterior to the vent, and below the epidermis at the cranium apex. The kidneys in many fish also become visible due to musculature edema and darkening of the trunk kidney. Productive infections do not establish in challenged channel catfish, rainbow trout or goldfish or in golden shiners cohabitated with challenged FHMs, suggesting that FHMNV is highly host-specific (Iwanowicz and Goodwin, 2002) .

FHMNV infection and disease can be diagnosed tentatively by behavioral changes and characteristic gross signs. Development of cytopathic effects in EPC and other fish cell lines that include syncytia formation can also assist diagnosis of FHMNV and WBV, with detection of rod-shaped virions by transmission electron microscopy (TEM) providing additional diagnostic confidence. Opportunities exist to develop specific and sensitive diagnostic reverse transcription-polymerase chain reaction (RT-PCR) tests because complete genome sequences are available for FHMNV (Batts et al., 2012) and WBV (Schütze et al., 2006) . For FHMNV, PCR tests have been described that amplify either a relatively conserved region in the ORF1b gene helicase motif or an ORF2 gene sequence (Batts et al., 2012) . Sequence analysis of the ORF2 gene region amplified by PCR has provided a means of differentiating FHMNV strains isolated in cell culture from FHMs being reared in Minnesota, Wisconsin and Illinois as well as present in jack pike (Esox masquinongy) fed live FHMs in Nebraska and a batch of creek chub (Semotilus atromaculatus) (Batts et al., 2012; McCann, 2012) . A constant-temperature RT loop-mediated isothermal amplification (RT-LAMP) test with a sensitivity of ~5 RNA copies has also been developed for FHMNV diagnosis and surveillance (Zhang et al., 2014) . While no molecular tests have been reported specifically for WBV diagnosis, immunoblotting using a polyclonal rabbit antiserum to a fusion protein to the pp1a main proteinase (Mpro) motif expressed in bacteria has been used to detect predicted Mpro cleavage products of pp1a and pp1ab in WBVinfected fish cells (Ulferts et al., 2011) . Opportunities thus exist to employ this or other WBV protein-specific antibodies in diagnostic immunotests.

There has been little impetus to control WBV, presumably because it has only been isolated once and not been identified to be pathogenic, because white bream is not an aquaculture species and as the wild fishery in Europe is small (Granzow et al., 2001) . This contrasts to FHMNV, which, due to its pathogenicity for FHMs, poses a significant threat to the multi-million dollar baitfish industry in the midwestern United States (Meronek et al., 1997; McCann, 2012) . Due to the industry value to this region and because FHMs are distributed widely in brooks, ponds and small lakes, the potential for virus dispersal via movements of live baitfish is currently regulated by state import policies and health certifications, as well as an overarching US Federal law called the Lacey Act (McCann, 2012) . While these regulations rely primarily on observing fish for disease symptoms, a survey of the three primary baitfish species farmed in Wisconsin employing cell culture in conjunction with PCR testing has proved useful in isolating, identifying and determining the prevalence and species distribution of FHMNV (McCann, 2012) . In Arkansas, it has been reported that destroying the farmed FHM population affected by FHMNV in 1997 in conjunction with high summer water temperatures might have been effective at eradicating the virus from this state (Batts et al., 2012) . As for any pathogen of aquaculture species, FHMNV control will rely on robust surveillance, translocation regulations and farm biosecurity, including stock destruction and water decontamination protocols.

The sequence and organization of the 26.2-26.6 kb (+) ssRNA genomes of GAV (Cowley et al., 2000a (Cowley et al., , 2004a Cowley and Walker, 2002) and YHV (Sittidilokratna et al., 2002 Jitrapakdee et al., 2003) , together with the transcription of a nested set of 3′-coterminal sgmRNAs used in protein translation (Cowley et al., 2002a; Sittidilokratna et al., 2008) , has identified them to be related distantly to diverse vertebrate and invertebrate viruses classified in the order Nidovirales (Cowley et al., 2012; de Groot et al., 2012a) . Due to observations of the viruses replicating and causing pathology in the lymphoid or "Oka" organ of shrimp (Oka, 1969; Spann et al., 1995) , GAV was assigned as the type species of a genus called Okavirus in a family named Roniviridae (sigla, rod-shaped nidovirus) based on the evolutionary divergence of GAV and YHV from other nidoviruses (Gorbalenya, 2008; Gorbalenya et al., 2002 Gorbalenya et al., , 2006 and on their rod-shaped enveloped virions (Spann et al., 1995 Boonyaratpalin et al., 1993; Chantanachookin et al., 1993; Nadala et al., 1997b) being distinct from the spherical enveloped virions formed by viruses in either the Coronaviridae or the Arteriviridae families of the Nidovirales known to exist at that time (de Groot et al., 2012a) .

In addition to GAV and YHV that have caused widespread disease in shrimp farmed throughout Eastern Hemisphere countries, a few viruses forming okavirus-like rod-shaped virions have been detected in crab species. Of these, Eriocheir sinensis ronivirus (EsRNV), which causes disease in the Chinese mitten crab, has been shown to possess a ~22 kb ssRNA genome (Zhang and Bonami, 2007) supportive of it being related to okaviruses, but no genome sequence information is yet available to confirm this relationship. The molecular and biological characteristics of GAV and YHV and the management of virus transmission and disease in farmed shrimp have been the subject of several comprehensive reviews (Dhar et al., 2004; Cowley and Walker, 2007; Lightner, 2011; Munro and Owens, 2007b; OIE, 2014; Walker and Sittidilokratna, 2008) . For completeness, information available on okaviruses (Table 32 .2) has also been reviewed and updated in the next sections, including more recent developments in diagnostic methods and potential antiviral therapies as well as brief descriptions of the crab viruses with virion morphologies suggestive of them being related to okaviruses.

YHD was first reported as the cause mass mortality in Giant Tiger shrimp (Penaeus monodon) being cultured intensively in Thailand in the early 1990s (Limsuwan, 1991) . The disease generally affected juvenile to subadult P. monodon 50-70 days into culture, and its pending onset was typically announced by a rapid abnormal increase in feed consumption followed 2-4 days later by an equally rapid cessation in feeding. Within 3-5 days of feeding cessation, moribund shrimp would amass at pond edges and die, often leading to almost complete pond losses. The name YHD was conceived because the bodies of many diseased shrimp had a bleached appearance often with a yellowed cephalothorax due to the gills developing a brownish color and the hepatopancreas becoming swollen and yellowed rather than dark brown (Limsuwan, 1991; Flegel et al., 1995b; Fig. 32 .3). Transmission trials, histology and TEM undertaken to identify the cause of YHD identified a rod-shaped enveloped virus (40-50 nm × 150-200 nm) speculated at the time to be an atypical baculovirus and accordingly given the name yellow-head baculovirus Chantanachookin et al., 1993) . However, when purified virions were identified to contain a genome comprised of a long (>22 kb) ssRNA, its name was revised to YHV (Wongteerasupaya et al., 1995b; Nadala et al., 1997a,b) . While thought to have emerged in Thailand, a review of histopathology and the morphology and morphogenesis of a virus cultured in primary shrimp lymphoid organ (LO) cells inoculated with a tissue extract of diseased P. monodon investigated at the time when shrimp aquaculture collapsed in Taiwan in the late 1980s suggests that YHV was the likely cause of that collapse (Chen and Kou, 1989) .

Soon after YHV was identified in Thailand, virions indistinguishable in morphology were detected in cells with hypertrophied nuclei sequestered within LO spheroids of overtly healthy P. monodon being farmed in eastern Australia (Spann et al., 1995) . The virus was named lymphoid organ virus (LOV) because it was not associated with disease and virus particles were only evident in LO spheroids; in addition no genome sequence information was available to determine its relationship to YHV (Spann et al., 1995; . Around the same time LOV was discovered in 1994, mass mortalities of subadult P. monodon were reported at four farms in North Queensland, Australia (Anderson and Owens, 2001; Spann et al., 1995 Walker et al., 1998; Munro et al., 2011) . As with YHD, the disease was accompanied by rapid feeding increases and cessation, shrimp swimming erratically at the water surface and as morbidity became acute, shrimp congregating in large numbers at pond edges before death and mortality rates of up to 80% at pond harvest. As more widespread disease outbreaks occurred during the 1996/97 season generally around 90-120 days into grow-out, the name 'mid-crop mortality syndrome' (MCMS) was adopted to describe the disease (Owens et al., 1998; Anderson and Owens, 2001; Walker et al., 2001; Cullen and Owens, 2004) . While gills of affected shrimp would typically become pink to brownish in color like YHD, disease gross signs were typified by a generalized reddened of the body and particularly at pereiopod, pleopod and tail fin extremities of experimentally challenged shrimp .

While up to four virus particle types were detected in MCMS-affected shrimp (Owens et al., 1998) , a rod-shaped enveloped virus particle morphologically indistinguishable from LOV and YHV was demonstrated to be the primary disease agent Oanh et al., 2011) . Due to the lack of genome sequence data to confirm their evolutionary relatedness, and as gill cells of diseased P. monodon possessed large numbers of virus particles in addition to LO cells, the virus was named GAV . However, following subsequent genome sequence comparisons of LOV and GAV (Cowley et al., 2000b) together with shrimp challenge data showing that an inoculum prepared from LO tissue of LOV-infected healthy shrimp could induce disease and mortality typical of GAV and that infection in the absence of disease could be induced using a diluted GAV inoculum , LOV and GAV were considered the same virus and the name LOV was abandoned. Gill-associated virus GAV Penaeus monodon Spann et al. (1995 , 1997 ) Cowley et al. (1999 , 2000a , 2002a Cowley and Walker (2002) Eriocheir sinensis ronivirus a EsRNV Chinese mitten crab (Eriocheir sinensis) Zhang and Bonami (2007) a Virion morphology and ~22 kb ssRNA genome consistent with okaviruses but its genome not yet sequenced to support inclusion in this genus.

In acute-stage infections associated with disease, YHV and GAV can invade all tissues of mesodermal and ectodermal origin. These include the LO, circulating hemocytes as well as developing hemocytes in hemopoietic tissues and fixed phagocytes in the heart, gill lamellae and epithelial pillar cells, and spongy connective tissues of various organs including the heart, stomach subcuticulum, antennal gland, gonads and nerve tissues, including neural ganglia, nerve fibers, neurosecretory and glial cells Chantanachookin et al., 1993; Lightner, 1996; Spann et al., , 2000 Tang and Lightner, 1999; Soowannayan et al., 2002) , as well as the fasciculate zone of eye (Smith, 2000; . Histopathology evident in hematoxylin and eosin (H&E) stained cephalothorax tissue sections is typified by severe necrosis in which cells display prominent nuclear pyknosis and karyorrhesis, as well as intense basophilic perinuclear inclusions in the cytoplasm resulting from nuclear phagocytosis and virus inclusions Chantanachookin et al., 1993; Lightner, 1996; Lu et al., 1994; Nash et al., 1992; OIE, 2014; Spann et al., , 2000 Wang and Chang, 2000) . Typical histopathology seen in the LO and gill lamellae of P. monodon with acute YHD are shown in Fig. 32 .2.

Both YHV and GAV can also persist over the life of wild shrimp in a subclinical or chronic infection state (Spann et al., 1995 (Spann et al., , 2003 Cowley et al., 2000b; Walker et al., 2001; Pasharawipas et al., 1997) . Infection can also persist in P. monodon and Pacific whiteleg shrimp (Litopenaeus vannamei) that survive experimental YHV challenge (Longyant et al., 2005 (Longyant et al., , 2006 Anantasomboon et al., 2007) and in farmed L. vannamei that survive a YHD outbreak (Senapin et al., 2010) . The only histopathology observed consistently in asymptomatic shrimp is LO spheroids comprised of partitioned aggregations of cells with hypertrophied nuclei (Spann et al., 1995; Anantasomboon et al., 2007) . Cells within these spheroids are often apoptotic and there is evidence for them being granular hemocytes exocytosed due to them having phagocytosed virusinfected cells or cell debris recognized to be foreign (Anggraeni and Owens, 2000; van de Braak et al., 2002; Duangsuwan et al., 2008) .

In ultrathin sections of infected shrimp tissue, mature virions of YHV appear bacilliform in shape (40-60 nm × 150-200 nm) and possess a lipid envelope decorated with regularly spaced diffuse surface projections (8 nm dia. × 11 nm long) Chantanachookin et al., 1993; Wang and Chang, 2000; Wongteerasupaya et al., 1995a,b) . The morphology of negatively stained virions purified from shrimp hemolymph by sucrose density-gradient ultracentrifugation has been reported to be similar (Wang and Chang, 2000; Wongteerasupaya et al., 1995a,b; Fig. 32 .3), except for the presence of narrowed envelope appendages extending from particle ends that can join the ends of curled particles to form donut-like structures (Nadala et al., 1997b) . The origin and purpose of the envelope extensions are unknown. However, the donut-shaped YHV particle structures quite closely resemble the virions (120-140 nm dia.) containing curled nucleocapsids formed by viruses classified within the Torovirus genus of the Torovirinae (de Groot et al., 2012b) , with the rod-shaped YHV particles being similar in dimensions and morphology to those of the fish viruses classified within the Bafinivirus genus of the Coronavirinae (Granzow et al., 2001; Iwanowicz and Goodwin, 2002) . FIGURE 32.2 Histopathology evident in tissue sections stained with H&E. Juvenile Penaeus monodon with acute YHD displaying marked cellular necrosis in the lymphoid organ, (A) 525×, (B) 1700× magnification, characterized by affected cells displaying pyknotic and karyorrhectic nuclei, with single or multiple perinuclear inclusion bodies ranging from pale to darkly basophilic (light to dark gray) apparent in some cells (arrows) as well as in (C) gill lamellae cells (1000×) displaying similar pyknotic and karyorrhectic nuclei (arrows) with some enlarged and generally spherical cells possibly representing immature hemocytes released in response to disease displaying a basophilic cytoplasm (Lightner, 1996) .

The nucleocapsid component of YHV virions appears as a coiled filament (16-30 nm dia.) with a 5-7 nm periodicity Chantanachookin et al., 1993; Nadala et al., 1997b; Wang and Chang, 2000) . Free nucleocapsid precursors (15-nm diameter) that occur abundantly in the cytoplasm of infected cells appear as flexible helical filaments up to 450 nm in length. During virion morphogenesis, nucleocapsids generally bud internally through membranes of the Golgiendoplasmic reticulum (ER) complex to generate enveloped virions, which sometimes are observed abutted end-to-end, as if formed consecutively from multiple linked nucleocapsids that appear to comprise the long filaments (Chantanachookin et al., 1993; . This consecutive mode of nucleocapsid and virion morphogenesis is also supported by purified virions being identified joined end-to-end (Wongteerasupaya et al., 1995a,b) . Virions often occur in the vicinity of nucleocapsid filaments between the outer and inner membranes of the nuclear envelope (Chantanachookin et al., 1993; Wang and Chang, 2000) , suggesting that the outer nuclear membrane can serve as an alternative to Golgi-ER membranes for virion maturation. Virions also bud infrequently from the cell plasma membrane Spann et al., 1995 . Nucleocapsid precursors and virions accumulate throughout the cytoplasm, with virions commonly seen congregated and sometimes densely packed within cytoplasmic vesicles Chantanachookin et al., 1993) . Exocytosis of these vesicles during cell degeneration appears to be the primary mode of virion release.

Initial analyses of the YHV genome showed it to be long (~22 kb), sensitive to RNase A, but not DNase 1 digestion (Wongteerasupaya et al., 1995b) and while preliminary in vitro translation analysis suggested it might be negative in polarity (Nadala et al., 1997b) , in situ hybridization (ISH) using RNA probes of either polarity confirmed it had positive polarity (Tang and Lightner, 1999) . Further characterization of the (+) ssRNA genomes of GAV (Cowley et al., 2000a Cowley and Walker, 2002) and YHV (Sittidilokratna et al., 2002 Jitrapakdee et al., 2003) showed them to be 26,235 and 26,662 nt in length, respectively, to possess a 5′-7-methylguanosine cap structure and a 3′-poly(A) tail and to be organized similarly to coronaviruses and arteriviruses ( Fig. 32.4) .

Downstream of a short 5′-terminal untranslated sequence, the genome of okaviruses contains a ~20 kb ORF1a/ORF1b replicase gene proceeded by an ORF2 gene that encodes the p20 nucleoprotein, an ORF3 gene that encodes a pp3 polyprotein from which the gp116 and gp64 envelope glycoproteins are derived and, in the case of GAV, a short ORF4 gene encoding a protein of unknown function that is not translated in abundance and that is truncated in YHV (Cowley et al., 2012) . Similarly to other nidoviruses, a slippery heptanucleotide (AAAUUUU) in conjunction with a downstream 131-nt sequence predicted to fold into a complex pseudoknot structure resides in the ORF1a/ORF1b gene overlap and functions as −1 RFS FIGURE 32.3 (A) Schematic structure of an Okavirus particle showing the positions of the envelope glycoproteins (gp116, gp64) and the nucleoprotein (p20) and electron micrographs of negatively stained rod-shaped virions of (B) YHV (Nadala et al., 1997a,b) and (C) GAV (Spann et al., 1995) showing the helical nucleocapsid and diffuse regularly spaced envelope projections as well as (D) GAV particles clustered tightly within a cytoplasmic vesicle surrounded by filamentous nucleocapsid precursors (arrow) in a lymphoid organ cell ) (Bars = 150 nm). (E) Photograph of Giant tiger shrimp (Penaeus monodon) showing yellow-brown (bleached) discoloration of the cephalothorax and gills (left side) as observed in early disease outbreaks in Thailand caused by YHV (Lightner, 1996) . element to facilitate read-through translation of the ORF1a gene pp1a polyprotein into the ORF1b gene at approximately 23% efficiency to generate a pp1ab polyprotein (Cowley et al., 2000a (Cowley et al., , 2012 de Groot et al., 2012a; Sittidilokratna et al., 2002) .

The ORF1a gene coding sequence contains four hydrophobic domains (HD1-HD4), each predicted to comprise multiple transmembrane (TM) sequences (Cowley et al., 2000a , Sittidilokratna et al., 2002 . A chymotrypsin-like cysteine proteinase (3CLpro) flanked by HD3 and HD4 and identified tentatively to use a VxHE↓(L,V) consensus cleavage-site motif functions in autocatalytic processing of the pp1a/pp1ab polyproteins into functional nsp units (Ziebuhr et al., 2003) . However, the okavirus 3CLpro appears to combine the Cys-His catalytic dyad of the coronavirus 3CLpro with a substrate-binding pocket representative of the 3C-like proteinase of plant potyviruses, thus distinguishing it from the cognate enzymes of other nidoviruses (Ziebuhr et al., 2003) . ORF1a also contains two putative papain-like proteinase motifs (PLP1 and PLPx) that lack a Zn 2+ -binding finger, as in the papain-like proteases (PLpro) of toroviruses, and share only remote structural similarity to PLpro motifs present in similar ORF1a gene positions of other nidoviruses (Sittidilokratna et al., 2002) . While shown to cleave consensus sites immediately to the N-terminal side of the 3CLpro domain and near the pp1ab C-terminus (Ziebuhr et al., 2003) , the number and nature of okavirus nsp units cleaved from the pp1a/pp1ab polyproteins by the 3CLpro and PLPs have yet to be determined.

The okavirus ORF1b gene-coding sequence component of the pp1ab replicase polyprotein contains homologs of a RdRp polymerase with a "SDD" active site motif characteristic of nidoviruses (Koonin, 1991; Gorbalenya et al., 2002) , as well as putative multinuclear Zn 2+ -binding finger-like domains preceding a 5′→3′ helicase domain (Zn-HEL), a 3′→5′exoribonuclease (ExoN), a Nidoviral endoribonuclease specific for Uridylate (NendoU) and a ribose-2′-O-methyltransferase (O-MT) present in the cognate pp1ab polyproteins of other nidoviruses (de Groot et al., 2012a,b; Faaberg et al., 2012; Cowley et al., 2000a Cowley et al., , 2012 Lauber et al., 2012; Sittidilokratna et al., 2002; Ziebuhr, 2005 Ziebuhr, , 2006 Ziebuhr, , 2008 Ziebuhr et al., 2000 Ziebuhr et al., , 2003 Gorbalenya, 2008; Gorbalenya et al., 2006) .

Unlike vertebrate nidoviruses in which the viral nucleoprotein gene resides close to the 3′-end of the genome, and more like the mosquito mesoniviruses in which the nucleoprotein appears to be encoded in an alternative reading frame in the N-terminal signal peptidase cleavage product of the ORF2a polyprotein from which the mature spike (S) envelope glycoprotein is also cleaved (Zirkel et al., 2013; Vasilakis et al., 2014) , the okavirus p20 nucleoprotein gene (ORF2) resides upstream of the 5 kb ORF3 gene, encoding the gp116 and gp64 envelope glycoproteins (Cowley et al., 2004a; Jitrapakdee et al., 2003; Sittidilokratna et al., 2006) . Positioning of the nucleoprotein gene at the 3′-genome end ensures its translation in high abundance because the 3′-coterminal sgmRNA transcription process that is utilized by vertebrate nidoviruses containing larger numbers of genes results in their abundance decreasing relative to their length (de Groot et al., 2012a) . However, as ~6 and ~5.5 kb sgmRNAs from which the p20 nucleoprotein and gp116/gp64 envelope glycoproteins are translated, respectively, are transcribed in similar relative abundance (Cowley et al., 2002a; Sittidilokratna et al., 2008) , okaviruses appear to have no need to order these genes similarly to coronaviruses or arteriviruses (de Groot et al., 2012a,b; FIGURE 32 .4 Schematic representation of the 26,662 nt (+) ssRNA genome of YHV and (subgenomic) sgmRNA1 and sgmRNA2 that translate ORF2 and ORF3, respectively. Each RNA contains a 5′-7-methylguanosine cap and a 3′-poly(A) tail. Functional domains identified in ORF1a include four hydrophobic regions (HD1-HD4), a 3C-like protease (3CLpro), a papain-like protease (PL1) and a domain with homology to PL1, but lacking the canonical α + β fold of papain-like proteases (PLX) and in ORF1b include a RNA-dependent RNA polymerase (RdRp), cysteine-and histidine-rich domain (C/H) Zn fingers, a helicase (HEL), an exoribonuclease (ExoN), a uridylatespecific endoribonuclease (N) and a ribose-O-methyl transferase (MT). The −1 ribosomal frame-shift (RFS) element allows translational read-through of ORF1a polyprotein pp1a into ORF1b to generate polyprotein pp1ab. ORF2 encodes the nucleoprotein (p20). ORF3 encodes a polyprotein (pp3) containing six transmembrane (TM) domains that undergoes posttranslational cleavage at the C-terminal side of TM3 and TM5 to generate the envelope glycoproteins gp116 and gp64, respectively, as well as a 22 kDa N-terminal triple-membrane-spanning fragment of unknown function. Known (▼) and likely (∇) sites of proteolytic cleavage in pp1a, pp1ab and pp3 are indicated . Faaberg et al., 2012) . The mesoniviruses have accommodated this need by having the nucleoprotein and S glycoprotein translated from the same sgmRNA (Lauber et al., 2012) . Similar to the mosquito mesoniviruses, the okaviruses also have no gene-encoding a membrane (M) protein integral to the virion structures formed by the vertebrate nidoviruses. Moreover, the presence of only two major genes (ORF2 and ORF3) in addition to the ORF1a/1b gene appears to have allowed okaviruses to preserve use of a sgmRNA transcription mechanism like that used for all but the longest sgmRNA of toroviruses (Snijder et al., 1990; Smits et al., 2005; Van Vliet et al., 2002) and less complex than that used by viruses in the Coronaviridae and Arteriviridae, and appear to have acquired greater gene numbers to accommodate replication in more highly evolved hosts (de Groot et al., 2012a,b; Faaberg et al., 2012) . In addition, YHV particles comprise three structural proteins, a p20 (20-22 kDa) nucleoprotein that binds viral RNA to form the nucleocapsid and two envelope glycoproteins, gp116 (110-135 kDa) and gp64 (63-67 kDa), that form the envelope spikes visible on virions and are utilized in cell attachment and entry (Cowley and Walker, 2002; Cowley et al., 2004a; Nadala et al., 1997b; Jitrapakdee et al., 2003; Sittidilokratna et al., 2006; Soowannayan et al., 2003 Soowannayan et al., , 2011 Fig. 32.4 ). In addition to p20-specific antibodies binding to precursor and virion nucleocapsids (Cowley et al., 2004a; Sittidilokratna et al., 2006) , RNA binding analyses using recombinant proteins expressed to various portions of the p20 coding sequence as well as to a synthetic p20 peptide sequence have localized its RNA binding domain to an N-terminal proline/arginine-rich sequence (Met 11 -Ile 29 ) (Soowannayan et al., 2011) . The mature gp116 and gp64 glycoproteins originate from posttranslational cleavage of the pp3 polyprotein encoded by the ORF3 gene (Cowley and Walker, 2002; Jitrapakdee et al., 2003; Soowannayan et al., 2011) . Of the six predicted TM domains in pp3, TM3 and TM5 appear to act as signal peptidase type 1-like sites ( Fig. 32.4) . Cleavage at the C-terminal side of these sites generates the N-terminus of gp116 anchored to the lipid membrane by TM4 and TM5, and the N-terminus of gp64 anchored by TM6. The fate and function of the 25.2 kDa N-terminal pp3 fragment, which contains three TM domains and a N-linked glycosylation site in its predicted ectodomain, have yet to be identified. Of the potential N-linked glycosylation sites in gp116 and gp64, six in gp116 and three in gp64 have been identified to possess mannose-rich glycans, with those in gp116 also possessing N-acetylgalactosamine and N-acetylglucosamine terminal-type sugars (Soowannayan et al., 2011) . Injection of shrimp with tunicamycin to inhibit N-linked glycosylation prior to YHV challenge results in reduced viral RNA and protein levels in hemolymph, numbers of mature virions in cells and mortality rate, confirming that the gp116 and gp64 glycosylation process is critical to YHV replication and thus disease (Soowannayan et al., 2013) . In variants of YHV1 (YHV1b) as well as a potential recombinant virus derived from ancestral YHV1b and YHV5 genotypes, a 54 amino acid deletion occurs at the N-terminus of gp116 just downstream of the pp3 TM3 cleavage site, but this deletion appears to have little if any impact on the pathogenic potential of these variants relative to YHV1a Gangnonngiw et al., 2009; Senapin et al., 2010) .

Initial comparisons of three ORF1b gene sequences of GAV from Australia with those reported or determined for YHV samples from Thailand identified 83.0% identity in a 135-nt region (Wongteerasupaya et al., 1997) , 80.9% identity in an adjoining 1068-nt region (Tang and Lightner, 1999) and 85.1% identity in a downstream 577-nt region including the C-terminus of the Zn-HEL motif (Cowley et al., 1999 (Cowley et al., , 2000b . These data showed YHV and GAV to be genotypic variants of a YHV complex and were assigned to genotypes YHV1 and YHV2, respectively Wijegoonawardane et al., 2008a) . Moreover, recent reanalysis of the 577-nt YHV sequence originated from healthy P. monodon broodstock sampled from a hatchery in Thailand (Cowley et al., 1999; J.A. Cowley, unpublished) has revealed it to be related mostly closely to YHV genotype 5 (YHV5) strains (Wijegoonawardane et al., 2008a; Gangnonngiw et al., 2009 ) than the virulent YHV1 genotype that was only type known to exist at the time. Based on a 423-nt ORF1b gene sequence amplified using a commercial YHV/GAV IQ2000 PCR test (Tang and Lightner, 1999; Cowley et al., 2004b) displaying 92.7% and 79.0% nt identity, respectively, to the reference GAV and YHV genome sequences, a third unique YHV genotype (YHV3) was identified in healthy wild P. monodon broodstock originating from the Andaman Sea in southern Thailand .

To identify whether other YHV genotypes might exist in geographically isolated populations of P. monodon, a consensus nested (n)PCR was used to amplify an ORF1b gene sequence from large numbers of juveniles, broodstock and postlarvae (PL) sampled during the early 2000s either from YHD outbreaks or from hatcheries in countries distributed widely across the natural Indo-Pacific distribution range of this species from Mozambique in eastern Africa to Fiji in the South Pacific (Wijegoonawardane et al., 2008a) . Phylogenetic analysis of the 671-nt sequence determined for 57 strains, including the reference YHV1 and GAV (=YHV2) strains, clustered the viruses into six discrete clades designated as genotypes 1-6 (YHV1-YHV6) (Fig. 32.5 ). YHV1 strains were associated solely with YHD episodes in Thailand and YHV2, YHV4 and YHV6 were the only genotypes detected in P. monodon from eastern Australia, India and Mozambique, respectively. Together with some country-specific inter-genotype branches also being evident, the genotype-country associations support the hypothesis of YHV existing as a complex of different genotypes that evolved in isolated shrimp populations, with intensive aquaculture and loosely regulated movements of wild P. monodon broodstock in Southeast Asia likely contributing to the mixed genotypes detected in countries like Vietnam Wijegoonawardane et al., 2008a) .

In support of the YHV complex hypothesis, sequence comparisons of ORF1b gene regions have confirmed the existence of a unique seventh genotype (YHV7) in P. monodon originating from the remote Joseph Bonaparte Gulf in northern Australia (Mohr et al., 2015, Fig. 32.5) , and based on another ORF1b gene sequence being found to be most closely related (87.6% nt identify) to reference YHV1 sequences (Tang and Lightner, 1999; Sittidilokratna et al., 2002) , a unique eighth genotype (YHV8) has been identified in Penaeus chinensis farmed in China's Hebei Province (Liu et al., 2014; Mohr et al., 2015) .

In addition to YHV existing as diverse genotypes, phylogenetic comparisons of ORF1b and ORF3 gene sequences identified 10 putative recombinant viruses among 28 strains selected to be representative of the YHV-YHV6 genotypes from the different Indo-Pacific regions . The recombinants originated from Vietnam, Taiwan, Indonesia, Malaysia and the Philippines and represented ORF1b:ORF3 genotype permutations 3:2, 3:5, 5:2 and 5:3, with the 3:2 permutation detected in 6 of the 10 viruses (Fig. 32.6 ). Maximum-likelihood breakpoint analysis of the ∼4.6 kb sequence joining the ORF1b-ORF3 gene regions of representative recombinant viruses from Indonesia (3:2), Vietnam (3:5) and the Philippines (5:2) localized the crossover sites to an identical position just upstream of the ORF1a gene-stop codon in the Indonesian and Vietnamese recombinants and to a position 12-nt further upstream in the recombinant from the Philippines . The ability of virus recombinants to be generated at high frequency at this crossover hotspot was confirmed by PCR analysis of progeny viruses produced in shrimp co-infected with YHV1 and GAV . These findings together with reports of other naturally occurring recombinant YHV strains indicate that genetic exchange amongst genotypes is occurring commonly in P. monodon in southeast Asia, which has profound implications for genotype assignment, disease diagnosis and the potential generation of viruses with increased pathogenic potential.

Within a decade of YHD being discovered in Thailand (Limsuwan, 1991; Boonyaratpalin et al., 1993; Chantanachookin et al., 1993) , disease symptoms consistent with acute YHV infection had been reported in most Asian countries with substantial P. monodon aquaculture industries, including China, Indonesia, Malaysia, the Philippines, Taiwan, Vietnam, India and Sri Lanka (Albaladejo et al., 1998; Natividad et al., 1999 Natividad et al., , 2002 Mohan et al., 1998; Wang and Chang, 2000; Walker et al., 2001; Siriwardena, 2000; Subasinghe et al., 2000; Rukyani, 2000; Khoa et al., 2000; Yang et al., 2000) . Viruses morphologically indistinguishable from YHV were also detected in Kuruma shrimp (Penaeus japonicus) farmed in Taiwan (Wang et al., 1996) and, although described as GAV, in healthy and diseased P. monodon farmed in Australia (Spann et al., 1995 Cowley et al., 1999; Munro et al., 2011) as well as in wild-caught P. monodon broodstock sampled from hatcheries (Cowley et al., 2000b; Walker et al., 2001) .

In southeast Asia, YHV has been detected in brackish water crustacean species, such as mysid shrimp (Palaemon styliferus) and krill (Ascetes sp.), as well as shrimp species such as the white banana prawn (Penaeus merguiensis) and red endeavor prawn (Metapenaeus ensis) that commonly co-inhabit aquaculture ponds growing P. monodon (Flegel et al., 1995a (Flegel et al., , 1997a . Although P. merguiensis and M. ensis can develop disease and die following experimental challenge (Chantanachookin et al., 1993; Flegel et al., 1995a) , these species appear more resilient to developing acute YHV infection in commercial ponds of P. monodon dying due to YHD. Similarly in Australia, GAV has been detected at low infection loads in Brown tiger shrimp (Penaeus esculentus) inhabiting P. monodon ponds affected by GAV disease . However, when challenged experimentally, P. esculentus as well as P. merguiensis and P. japonicus are susceptible to GAV disease and mortality, although in P. japonicus, disease susceptibility was lower in >20 g shrimp compared to 6-13 g shrimp (Spann et al., 2000 (Spann et al., , 2003 . Following injection challenge, Barred estuarine shrimp (Palaemon serrifer), Pacific blue prawn (Palaemon styliferus) and the freshwater prawn Macrobrachium sintangense are also susceptible to developing mild YHV symptoms that do not necessarily progress to cause mortality, while Macrobrachium rosenbergii and Macrobrachium lanchesteri are highly refractive to infection, the reasons for which remain to be determined (Longyant et al., 2005) . YHV infection in the absence of disease has similarly been induced experimentally in Jinga shrimp (Metapenaeus affinis) and the Yellow prawn (Metapenaeus brevicornis), but not in any of 16 different crab species challenged by injection (Longyant et al., 2006) . Australian red claw crayfish (Cherax quadricarinatus) also appear capable of supporting low-level YHV infection following experimental challenge (Soowannayan et al., 2015) .

Among shrimp species indigenous to the Americas, such as L. vannamei and blue shrimp (Penaeus stylirostris) Lu et al., 1994 Lu et al., , 1997 as well as white shrimp (Litopenaeus setiferus), brown shrimp (Penaeus aztecus) and northern pink shrimp (Penaeus duorarum) , juveniles but not PL stages up to PL20 develop systematic lethal YHV infections when fed carcasses of diseased shrimp (Lightner, 1996) . As in these species, P. monodon only become susceptible to YHD beyond approximately PL15 (Khongpradit et al., 1995) . Injection challenge of Daggerblade grass shrimp (Palaemonetes pugio) and the blue crab Callinectes sapidus have shown these species to also be susceptible to YHV infection .

Based on the potential susceptibility of western hemisphere shrimp species, the discovery of infectious YHV together with white-spot syndrome virus (WSSV) in frozen, uncooked commodity shrimp imported into the United States from Asia (Nunan et al., 1998; Durand et al., 2000) , and the occurrence of severe LO histopathology thought at the time to be exclusively indicative of YHD in farmed shrimp sampled during outbreaks of white-spot disease (WSD) in the late 1990s, it was speculated that YHV had been introduced into US shrimp farms together with WSSV (Lightner, 1996; . However, its presence could not be substantiated by other molecular diagnostic methods, and it was discovered subsequently that in shrimp with severe WSD, LO histopathology can be confused for that caused by acute YHV infection (Pantoja and Lightner, 2003; Lightner, 2011) .

Despite the erroneous early reports of YHV occurring in US shrimp farms, a virus consistent in genome sequence to YHV1 was identified subsequently in L. vannamei and P. stylirostris farmed in northwest Mexico from at least 1999 ( While the introduction of P. monodon into non-native regions might explain how YHV has been introduced into the Gulf of Mexico, it has only been identified and become established as an invasive species in Atlantic coastal regions of the Americas, including the Caribbean (Gómez-Lemos and Campos, 2008), Venezuela (Aguado and Sayegh, 2007) and Brazil (Coelho et al., 2001; Cintra et al., 2011) and in the South Atlantic Bight and Gulf of Mexico regions of the United States (Fuller et al., 2014) .

Unlike in Mexico, YHV has been established as a problematic disease of L. vannamei since its introduction in the early-2000s and subsequent rapid expansion to become the primary aquaculture species in Thailand and other countries in southeast Asia (Anantasomboon et al., 2007; Senapin et al., 2010) . To evaluate the potential for YHV to be translocated and become established in different regions, a comprehensive list of crustacean species capable of carrying infection has been compiled from a meta-analysis of available literature (Stentiford et al., 2009 ).

YHV infection can be transmitted horizontally to susceptible crustacean species by several natural challenge routes. These include cannibalization of moribund live shrimp, carcasses or small pieces of flesh from diseased shrimp, cohabitation with infected shrimp and exposure to waterborne virus particles (Flegel et al., 1995a; Lightner, 1996; Lu et al., 1997; Walker et al., 2001; Hamano et al., 2015; Soowannayan et al., 2015) . Shrimp ingestion of carrier species such as Acetes sp. and Palaemon styliferus can also transmit YHV infection and disease (Flegel et al., 1995b (Flegel et al., , 1997a . However, such oral transmission routes only appear to be effective in establishing productive infections and disease in shrimp from late PL life stages (PL17-PL20) onward (Flegel et al., 1995b; Khongpradit et al., 1995; . Muscle injection of hemolymph or filtered cephalothorax tissue homogenates from diseased shrimp is also very effective at transmitting infection at a defined dose and thus has been used extensively as an experimental challenge method Longyant et al., 2006) .

YHV transmission from broodstock to progeny in hatcheries was speculated early on to be the source of infection leading to widespread disease in P. monodon being farmed in Thailand (Chantanachookin et al., 1993; Pasharawipas et al., 1997) . Initially this assumption was poorly supported by data indicating a low prevalence of YHV infection in wild-caught broodstock sampled from Thai hatcheries (Flegel et al., 1997b) and difficulty in detecting YHV in broodstock reproductive organs. However, accumulated subsequent data now strongly supports vertical transmission being a primary route through which infection is perpetuated in wild populations and enters aquaculture ponds. Such data includes the widespread detection of YHV at high prevalence among P. monodon broodstock and PL sampled from hatcheries (Phan, 2001; Cowley et al., 2000b Cowley et al., , 2002b Spann et al., 1995; Walker et al., 2001; Wijegoonawardane et al., 2008a) as well as juveniles sampled from farm ponds (Spann et al., 1995; Pasharawipas et al., 1997) .

ISH and PCR data have also provided evidence for GAV occurring in male P. monodon spermatophores and spermatophore secretions as well as female ovaries . The presence of mature virions in spermatophore seminal fluid but not in sperm per se has been substantiated by TEM and nested (n)PCR and nPCR has also confirmed the presence of low GAV RNA amounts associated with unwashed eggs, but not newly hatched nauplii (Cowley et al., 2002a; Fig. 32 .7). In P. monodon, egg activation and fertilization requires a multitude of egg surface alterations that initiate immediately upon contact with seawater (Pongtippatee-Taweepreda et al., 2004) . Although the mechanism by which GAV/YHV are transmitted vertically remains unknown, opportunities for virus entry into eggs might present themselves during the extraordinary egg surface ultrastructural changes that occur as a result of the cortical reaction and egg-sperm interaction processes activated within the first few seconds after release from the female (Pongtippatee-Taweepreda et al., 2004) .

To directly examine the capacity of either gender of P. monodon to transmit GAV infection vertically, female broodstock captured from one location in north Queensland were inseminated artificially with the spermatophores of males captured at a disparate location. GAV strains present as a subclinical infection in each parent as well as in progeny batches reared to an early PL stage could thus be identified clearly by sequence analysis of a more variable region of the ORF3 gene (Cowley et al., 2002a) . GAV strain sequences identified in progeny demonstrated that virus could be transmitted from the male or female parent or both, with the predominant strain detected in progeny generally correlating with that existent in either parent at the highest infection load.

Gross signs such as erratic swimming and shrimp congregating at pond edges with a yellowed cephalothorax, bleaching/ reddening general body appearance and pink discolored gills and appendage extremities can alert to disease caused by YHV. For rapid presumptive diagnosis of YHD, gill squashes or hemocyte smears from moribund shrimp can be observed either directly by phase-contrast microscopy or by normal light microscopy after Wright-Giemsa staining (Nash et al., 1992; Lightner, 1996; FAO Fisheries Technical Paper 402/2 2001) . Gill filaments or subcuticular tissue fixed in Davidson's FIGURE 32.7 Transmission electron micrograph showing GAV particles within the seminal fluid of a spermatophore sampled from an adult male Penaeus monodon (Cowley et al., 2002b) . Bar = 200 nm.

AFA fixative and stained with H&E can also be observed by light microscopy without wax embedding and tissue sectioning (Lightner, 1996; OIE, 2014) . Pathologies predictive of YHD include evidence of nuclear pyknosis and karyorrhexis as well as medium-to-large numbers of evenly stained and deeply basophilic spherical cytoplasmic inclusions ≤2 mm in diameter in cells of mesodermal and ectodermal origins (Lightner, 1996; Flegel et al., 1997a) . Histology on wax-embedded cephalothorax tissue sections stained with H&E will typically delineate the nature of the cellular pathologies more precisely as well as assist in determining YHD severity based on the nature and numbers of LO spheroids (Chantanachookin et al., 1993; Boonyaratpalin et al., 1993; Nash et al., 1992; Flegel et al., 1995b Flegel et al., , 1997a OIE, 2014; Fig. 32.2) .

Despite the diagnostic value of histology, care must be taken when interpreting data because pathology evident in the LO of shrimp with severe WSD can be confused for that caused by YHV (Pantoja and Lightner, 2003; Lightner, 2011) . Histology alone is thus insufficient to diagnose YHV infection or disease with confidence unless supported by other methods. TEM on LO or gill tissue can also be used to confirm the presence of variable length (80-450 nm), filamentous nucleocapsid precursors and rod-shaped enveloped particles characteristic of YHV/GAV (Chantanachookin et al., 1993; Boonyaratpalin et al., 1993; Spann et al., 1995 Fig. 32 .3). Like histology, however, TEM is time consuming, requires specialized equipment and, for surveillance and diagnostic purposes, has generally been superseded by more rapid molecular diagnostic methods.

Primary cultures of shrimp LO cells support YHV replication (Chen and Kou, 1989; Chen and Wang, 1999) , and assays based on cytopathic effects and cell monolayer disintegration have been used to quantify virus infectivity (Lu et al., 1995a,b; Assavalapsakul et al., 2003) . However, because no continuous cell lines are yet available to easily culture YHV, bioassay in naïve shrimp remains the most common means of confirming the presence of infectious YHV Longyant et al., 2006; Lu et al., 1995b; McColl et al., 2004) . While culture methods are useful in amplifying infectious YHV, definitive diagnosis relies on TEM to detect virus nucleocapsids and/or virions or on molecular methods to detect YHV protein or RNA (OIE, 2014) .

Hemagglutination (HA) of chicken erythrocytes by YHV (Nadala et al., 1997b) or GAV particles Owens, 2005, 2006) in titrations of shrimp hemolymph or gill tissue extracts has also been investigated as a potential pond-side diagnostic method. While the HA method is not particularly sensitive and provides no specificity, there are circumstances in which it could be applied to guide disease management decisions at hatcheries and farms.

To diagnose YHV infection unequivocally, various molecular methods have been developed to detect either viral proteins or viral RNA. Methods to detect YHV proteins have employed polyclonal antiserum prepared to purified whole virus (Lu et al., 1996; Munro and Owens, 2007a) , monoclonal antibodies (MAbs) generated to purified whole virus and selected for specificity to the gp116 and gp64 envelope glycoproteins or the p20 nucleoprotein (Sithigorngul et al., 2000 Munro and Owens, 2007a; Sittidilokratna et al., 2006) , monospecific antiserum prepared to a p20 peptide sequence or to a p20-fusion protein expressed in Escherichia coli (Cowley et al., 2004a) and a recombinant single-chain variable fragment antibody to gp116 expressed in E. coli and constructed by PCR from a hybridoma clone generated to purified YHV particles (Intorasoot et al., 2007) . The range of antibody-based methods described for detecting YHV or GAV proteins includes dot-blot nitrocellulose enzyme immunoassays (DB-NC-EIA) (Intorasoot et al., 2007; Lu et al., 1996; Loh et al., 1998; Nadala and Loh, 2000; Sithigorngul et al., 2000) and Western blots (Cowley et al., 2004a; Intorasoot et al., 2007; Nadala et al., 1997a; Sithigorngul et al., 2000; Soowannayan et al., 2003; OIE, 2014) , a solid-phase noncompetitive enzyme-linked immunosorbent assay (ELISA) (Intorasoot et al., 2007) , as well as a sandwich ELISA employing polyclonal chicken antibodies raised against GAV proteins for protein capture and a p20 nucleoprotein-specified MAb for GAV protein detection Owens, 2006, 2007a) , immunohistochemistry (IHC) for detecting infected cells and tissues in histological sections (Sithigorngul et al., 2000; Soowannayan et al., 2003) and immunoelectron microscopy for detecting viral nucleocapsids and virions in ultrathin tissue sections or protein components of purified and negatively stained virions (Cowley et al., 2004a; Soowannayan et al., 2003) .

Of these methods, the DB-NC-EIA protocol, using gill tissue homogenates, provides a rapid and simple potential method to diagnose YHV infection (Nadala and Loh, 2000) . However, an even more simple-to-implement lateral-flow immunochromatographic test strip (ICTS) method has been developed for the pond-side diagnosis that employs either shrimp hemolymph or gill/appendage tissue extracts as a protein source, a polyclonal rabbit antiserum prepared to a recombinant YHV p20 protein to capture this viral protein and a p20-specific MAb (Y19) conjugated to colloidal gold as a detector antibody (Sithigorngul et al., 2007; Chaivisuthangkura et al., 2014 ). An ICTS method to co-detect YHV and WSSV in the same strip has also been described (Sithigorngul et al., 2011, Fig. 32.8) .

Antibody-based diagnostic methods can offer advantages in speed, simplicity and cost in detecting GAV/YHV compared with molecular methods to detect viral RNA such as PCR (Munro and Owens, 2006; Sithigorngul et al., 2007) . However, their analytical sensitivity is generally lower. For example, the lateral-flow ICTS developed to detect YHV is approximately 500-fold less sensitive in detecting YHV p20 protein compared with a one-step PCR test designed to detect YHV RNA (Wongteerasupaya et al., 1997; Sithigorngul et al., 2007) . YHV detection sensitivity was reduced further in an ICTS designed to co-detect YHV and WSSV (Sithigorngul et al., 2011) . Antibody specificity also needs to be considered. For example, while polyclonal antiserum to recombinant GAV p20 protein cross-detects the YHV p20 protein (Cowley et al., 2004a) and MAbs to the YHV p20 nucleoprotein (Y19) and gp64 glycoprotein (Y18) cross-detect the cognate GAV proteins, MAb V-3-2B specific to the YHV gp116 glycoprotein does not detect gp116 of GAV (Sithigorngul et al., 2000 (Sithigorngul et al., , 2007 Soowannayan et al., 2003) . Despite these limitations, protein-based virus detection methods provide practical and relatively cheap diagnostic options for managing YHV infection and disease risks at shrimp hatcheries and farms.

Molecular methods designed to detect YHV RNA generally provide the highest analytical sensitivity. ISH using digoxigenin (DIG)-labeled DNA or RNA probes is useful, like the IHC method, in associating YHV with specific tissue types and histopathology and thus in diagnosing infection as well as assessing disease severity (Spann et al., 2003; Tang and Lightner, 1999; Tang et al., 2002; OIE, 2014) . However, due to its complexity, ISH has been used primarily as a research tool to investigate YHV infection processes and disease progression.

Numerous PCR tests have been reported for either YHV and GAV diagnosis or research (Cowley et al., 2000b (Cowley et al., , 2004b Wongteerasupaya et al., 1997; Wijegoonawardane et al., 2008b Wijegoonawardane et al., , 2010 Dhar et al., 2002; Mouillesseaux et al., 2003; de la Vega et al., 2004; Tang and Lightner, 1999; Aranguren et al., 2012; de la Rosa-Vélez et al., 2006) . Of these, the one-step RT-PCR first reported for YHV (Wongteerasupaya et al., 1997) , a two-step multiplexed nPCR utilizing PCR primers to amplify both YHV and GAV and multiplexed virus-specific nPCR primers to amplify and differentiate YHV from GAV based on amplicon size (Cowley et al., 2004b ) and a two-step multiplexed PCR test utilizing combined degenerate primers designed to have broad specificity for YHV genotypes 1-6 and to assist in detecting other genotypic variants that might exist (Wijegoonawardane et al., 2008b) are endorsed for infection/disease diagnosis in the Yellowhead Disease chapter of the OIE Manual of Diagnostic Tests for Aquatic Animals 2014 (OIE, 2014) . Each of these tests targets regions in the okavirus ORF1b gene, and are likely to now require revision or caveats to their applicability as new YHV genotypes are discovered such as YHV7 (Mohr et al., 2015) and YHV 8 (Liu et al., 2014) because they were designed to sequences of YHV genotypes known at the time.

The inclusion of a nPCR step can improve YHV/GAV RNA detection efficiency up to 100-fold or more compared with one-step PCR (Cowley et al., 2000b (Cowley et al., , 2004b . Various real-time RT-PCR tests that provide viral RNA detection sensitivities further improved compared with nPCR as well as improved throughput capacity and linear dynamic range over which viral RNA loads and thus infection severity can be quantified accurately, have been described for detecting YHV, GAV or YHV genotypes 1-6 that utilize either SYBR-Green chemistry (Dhar et al., 2002; Mouillesseaux et al., 2003) or a TaqMan probe to generate fluorescence (de la Vega et al., 2004; Aranguren et al., 2012; Panichareon et al., 2011; Wijegoonawardane et al., 2010) . The detection limits of these real-time PCR tests can approach a single RNA copy. A modified version of the nPCR designed to differentiate YHV from GAV (Cowley et al., 2004b) is also available commercially (IQ2000 YHV/GAV PCR test) together with commercial real-time SYBR-Green PCR and RT-LAMP tests for YHV (GeneReach Biotechnology Corp.) .

In addition to the PCR designed to detect YHV and/or GAV (Cowley et al., 2004b) , a test employing multiplexed PCR primer sets to detect any of six different shrimp viruses and a TaqMan real-time PCR employing multiplexed PCR primers and probes labeled with different fluorophores to co-detect and quantify YHV and Taura syndrome virus (TSV) have been described (Khawsak et al., 2008; Aranguren et al., 2012) . As an alternative to gel electrophoresis to detect and confirm the expected size of PCR amplicons, hybridization to biotin-labeled virus-specific oligonucleotides followed by avidin capture and detection using anti-DIG-peroxidase has been used for ELISA-based visualization of DNA amplified in the presence of DIG-dUTP (Khawsak et al., 2008) . Hemolymph spotted onto specialized filter papers such as IsoCode (Schleicher and Schuell Inc.) has proved useful for field collection and preservation of YHV RNA for subsequent RT-PCR analysis (Kiatpathomchai et al., 2004) . The method avoids the complexities of tissue dissection and collection into liquidbased preservatives, and/or the need to refrigerate or freeze tissues, and can maintain RNA integrity in samples stored for periods of up to 9 months at ambient temperature (Kiatpathomchai et al., 2004) . Hemolymph stored as a liquid at either 4°C of 25°C for up to 5 days has also been found show little sensitivity drop-off in real-time PCR detection of YHV RNA (Ma et al., 2008) .

While RNA detection by PCR methods can provide exquisite sensitivity, most tests employ primers designed to genome sequences determined for a single strain/genotype and with no regard for them targeting gene regions constrained by their requirement to encode more highly conserved functional domains within YHV proteins. Based on these shortcomings in combination with YHCVs existing as a complex of genotypes that appear to have evolved in geographically isolated populations of P. monodon Soowannayan et al., 2003; Wijegoonawardane et al., 2008a) , extreme care is needed in interpreting data generated by any one particular PCR test. For example, the original RT-PCR test described to detect YHV (Wongteerasupaya et al., 1997) has retained good specificity for the YHV1 genotype due to the 3′-terminus of 10R PCR primer serendipitously targeting a sequence that has diverged in other identified genotypes (Wijegoonawardane et al., 2008b) . However, other tests, including the RT-nPCR designed to differentiate YHV1 from GAV (Cowley et al., 2004b) and the TaqMan real-time PCR to detect GAV (de la Vega et al., 2004) , have been identified to cross-detect other genotypes at variable efficiency depending on their sequence relatedness to GAV in the genome regions targeted by the PCR primers (Wijegoonawardane et al., 2008b; J.A. Cowley et al., unpublished) .

To detect YHV genotypes 1-6 as well as other genotypes that might exist, consensus RT-nPCR and TaqMan real-time RT-PCR tests have been described that use degenerate PCR primers designed to accommodate genotype sequence variations identified in an ORF1b gene region (Wijegoonawardane et al., 2008a (Wijegoonawardane et al., ,b, 2010 . Phylogenetic analyses of sequences amplified from this and another ORF1b gene region have proved useful in assigning YHV genotypes and in identifying new genotypes such as YHV7 found recently in P. monodon originating from the previously unstudied Joseph Bonaparte Gulf region of northern Australia (Mohr et al., 2015) .

While phylogenetic comparisons of ORF1b gene sequences have proved useful in classifying YHV genotypes, PCR tests designed to amplify sequences within the ORF2/ORF3 genes encoding the virus structural proteins have proved useful in identifying YHV1 subtypes YHV1a and YHV1b Wijegoonawardane et al., 2008a) as well as viruses representing recombinants Gangnonngiw et al., 2009) . Based on the high frequency at which recombinants comprising various permutations of the 5′-ORF1a/1b and the ORF2-3 3′-genome regions of parental YHV genotype 2, 3 and 5 viruses have been detected in P. monodon collected from various countries in southeast Asia , genotype assignments based on ORF1a/1b gene sequences alone need to be viewed with caution, particularly in regions where hatcheries are densely colocated and/or commonly source wild broodstock from diverse locations. In cases when a YHV genotype needs to be defined definitively, it is recommended that regions in both the ORF1a/1b and ORF2/ORF3 genes be amplified, sequenced and compared, as was done to identify the nature of a YHV genotype when first detected in shrimp farmed in Mexico (de la Rosa-Vélez et al., 2006) .

As an alternative to PCR, RT-LAMP methods have been described to detect YHV (Mekata et al., 2006 (Mekata et al., , 2009 Jaroenram et al., 2012; Khunthong et al., 2013) . A standard RT-LAMP test employing agarose gel electrophoresis to detect the amplified DNA ladder was found to be about 10-fold less sensitive than a commercial IQ2000 YHV/GAV nPCR test (Mekata et al., 2006) . In contrast, a real-time RT-LAMP test employing a photometer to quantify turbidity increases in the magnesium pyrophosphate by-product of DNA amplification was found to be about 10-fold more sensitive than the same commercial nPCR test (Mekata et al., 2009) . To avoid the need for either gel analysis or a photometer to detect amplified DNA, a RT-LAMP method modified to amplify biotin-labeled DNA ladder products has also been developed to detect YHV (Khunthong et al., 2013) . By hybridizing amplified DNA products to a fluorescein isothiocyanate-labeled DNA oligonucleotide, a simple lateral-flow dip (LFD) stick system was employed to trap and visualize the DNA duplexes as a dark-colored band (Khunthong et al., 2013) . A similarly simple tube-based colorimetric method has been described that utilizes gold nanoparticles (AuNP) to bind and detect YHV DNA ladder products amplified by RT-LAMP (Jaroenram et al., 2012) . These rapid, specific and highly sensitive methods that only require a constant temperature apparatus and simple LFD stick or tube-based colorimetric detection systems, such as LAMP, offer substantial potential for field diagnosis of YHV. However, due to the complexity of primers required for LAMP, the specificity they provide is likely to limit the applicability of any test to a single genotype.

No commercial treatments are yet available to protect farmed shrimp against YHD. Disease prevention thus relies on the regulatory oversight and surveillance by government quarantine and agricultural agencies (AQIS, 2000; Stentiford et al., 2009; Lightner et al., 1997; McColl et al., 2004) , on hatcheries and farms employing robust biosecurity systems that exclude virus entry via water, carrier species, equipment or personnel, as well as infection surveillance in ponds, pond bottom and pond water disinfection systems and farm management practices that avoid exposing shrimp to adverse stressors, such as rapid changes in water pH, pH levels >9 or prolonged periods of low (<2 ppm) dissolved oxygen (Limsuwan, 1996; Lightner, 1999 Lightner, , 2003 Lightner, , 2005 Walker and Mohan, 2009 ).

Since wild shrimp often carry a subclinical YHV or GAV infection (Spann et al., 1995; Pasharawipas et al., 1997; Cowley et al., 2000b; Anantasomboon et al., 2007; Castro-Longoria et al., 2008) and the virus can be transmitted vertically from both male and female broodstock (Cowley et al., 2002b) , infected progeny represent a major risk factor in farm disease. PCR screening of hemolymph, gill or pleopod biopsies collected nonsacrificially can provide a means of selecting broodstock that are either free of infection or infected at low levels, posing a lowered risk of virus transmission (Nadala et al., 1997a,b; Cowley et al., 2002b; Pasharawipas et al., 1997) . While less ideal, PL batches can also be screened and discarded if deemed to pose an unacceptable disease risk.

Progress made in domesticating and selectively breeding shrimp species of aquaculture importance that are specific pathogen-free (SPF) for YHV and other problematic viruses (Wyban, 1992; Wyban et al., 1992; Lotz, 1992 Lotz, , 1997 Lightner, 2003 Lightner, , 2005 Lightner et al., 2009) . However, unlike for infectious hypodermal and hematopoietic necrosis virus (IHHNV) and TSV, in which genetic lines of shrimp have been repeatedly challenged and selected to be specific pathogen-resistant (SPR) of disease caused by these viruses , as yet only shrimp breeding stocks SPF for YHV are available commercially (Lightner, 2005 (Lightner, , 2011 . While the farming of virus-free seed stock spawned from SPF L. vannamei has assisted in reducing YHV disease impacts in Asian regions that previously farmed P. monodon or P. chinensis (Lightner, 2011; Lightner et al., 2009 ), it has not provided a panacea because shrimp remain highly susceptible to YHD and other diseases originating from exogenous sources (Senapin et al., 2010) .

In Australia, rearing of domesticated P. monodon broodstock over several generations has resulted in profound improvements in production yields due to increased growth rates and survival (Preston et al., 2007 (Preston et al., , 2010 . A strategy employing early rearing of P. monodon broodstock in dedicated farm ponds, rather than solely in biosecure facilities, and PCR screening to remove individuals infected with GAV at high loads prior to each breeding cycle has proved useful in establishing breeding populations in which subclinical GAV infection is prevalent, but in which tolerance against GAV-induced disease has developed (J.A. Cowley and M.J. Sellars, unpublished) . Disease tolerance among P. monodon that have developed a capacity to accommodate YHV/GAV infection in a subclinical state is similar to that observed to have occurred within a few years of YHD emerging in Thailand (Pasharawipas et al., 1997) , with some "viral accommodation" process associated with the suppression of cell apoptosis (Flegel and Pasharawipas, 1998; Flegel, 2007; Khanobdee et al., 2002) and/or suppression of YHV gp116 envelope glycoprotein synthesis speculated to be involved. As mortalities in L. vannamei exposed per os to TSV before challenge with YHV also accumulate more slowly, preexisting infection by some viruses appears able to interfere or compete with YHV infection (Aranguren et al., 2012) . In the Americas where subclinical TSV infection occurs commonly in farmed L. vannamei, it has been speculated that such infections might have helped prevent YHV infection and disease from becoming established in this region (Aranguren et al., 2012) .

Extracts of various plants, herbs and seaweeds with immunostimulant or antimicrobial activities have been investigated as feed supplements to protect shrimp against viruses including WSSV and YHV (Sivasankar et al., 2015) . For example, a feed produced to contain a leaf ethanol extract of the Thai medicinal plant Clinacanthus nutans decreased mortalities in shrimp challenged with YHV by water immersion (Direkbusarakom et al., 1998) . Leaf extracts of the medicinal herbs Phyllanthus amarus and Phyllanthus urinaria also neutralize YHV infectivity, but their protective efficacy as a feed supplement was not assessed (Direkbusarakom et al., 1995) . An ethanol leaf extract of Guava (Psidillm guajava) also neutralizes YHV infectivity, but its addition to feed provides no protection against YHV challenge (Direkbusarakom et al., 1997) . While the feeding of such medicinal plant extracts has thus shown promise in protecting shrimp against YHD and other virus diseases, there appears to have been little interest from shrimp feed manufacturers in including such extracts in commercial feed pellets.

When processed and added to shrimp feed pellets, an ingredient produced from flocks of microalgae and bacteria grown in seawater ponds seeded with low-value carbon by-products, such as sugarcane bagasse or rice husks, and patented as Novacq can improve the growth performance of farmed P. monodon and L. vannamei by 30% and 50%, respectively (Glencross et al., 2012 (Glencross et al., , 2014 . Juvenile P. monodon fed on pellets containing Novacq have also displayed lowered mortality rates following GAV challenge (Sellars et al., 2015) . Thus ingredients like Novacq that will be included in commercial feeds for the primary reason of improving shrimp growth rates and general robustness might provide secondary benefits in reducing farm disease impacts by enhancing shrimp resilience against disease caused by GAV/YHV and other viruses.

RNA interference (RNAi) strategies based on the use of short interfering (si)RNA or either long dsRNA or hairpin dsRNA are being explored as a means of preventing diseases caused by most viruses problematic to shrimp aquaculture (Shekhar and Lu, 2009; Sagi et al., 2013) . Long dsRNA was identified early to direct a RNAi response more specific than siRNA (Robalino et al., 2004 (Robalino et al., , 2005 Westenberg et al., 2005) , presumably through mobilizing the Dicer RNase III dsRNA cleavage enzyme (Chen et al., 2011; Li et al., 2013; Su et al., 2008; Yao et al., 2010) as well as the Argonaut enzyme and other minor components of the RNA-induced silencing complex that mediates RNAi-based gene silencing in shrimp and other organisms (Unajak et al., 2006; Dechklar et al., 2008; Chen et al., 2011) . Gene silencing in shrimp mediated by long dsRNA is thus being investigated intensively as a potential therapeutic to combat YHD.

In an initial study using cultures of shrimp LO cells, transfection of in vitro transcribed dsRNA targeted to the YHV ORF1b gene and thus the YHV genomic and antigenomic length RNAs inhibited YHV replication more effectively than dsRNAs targeted to ORF3 gene sequences encoding the gp116 or gp64 glycoproteins (Tirasophon et al., 2005) . As okaviruses transcribe 3′-coterminal ORF2 and ORF3 sgmRNAs in greater abundance than genomic-length RNA (Cowley et al., 2002a; Cowley and Walker, 2002; Sittidilokratna et al., 2006) , dsRNA targeted to the ORF3 gene would interact with all three RNAs. It is not unexpected, therefore, that dsRNAs targeting the ORF1a/1b gene region of the low-abundance genomic and antigenomic RNAs are more effective in inhibiting YHV replication, and particularly because the genomiclength RNA is responsible for translating the pp1a and pp1ab polyproteins that contain the various enzymes critical for viral RNA transcription and replication.

In P. monodon challenged with YHV, an ORF1a/1b gene dsRNA inhibited virus replication specifically when injected up to 12 h post-challenge, and also reduced shrimp mortality when injected earliest at 3 h post-challenge (Tirasophon et al., 2007) . Using different methodology, the same ORF1a/1b gene dsRNA injected into hemolymph of P. monodon just prior to YHV challenge completed protected the shrimp for 10 days, at which time 90% of noninjected shrimp had died (Yodmuang et al., 2006) . Similarly, injection of a hairpin YHV ORF1a/1b gene dsRNA expressed and purified from bacteria into the hemolymph of L. vannamei as a therapeutic agent prior to YHV challenge inhibited mortalities over a 16-day period, with no evidence of YHV replication evident by one-step PCR analysis (Assavalapsakul et al., 2009) . Consistent with earlier findings, when dsRNA was injected as a prophylactic agent at different times post-challenge, YHV replication levels were suppressed most significantly at the earliest dsRNA injection time points (Assavalapsakul et al., 2009) . Injection of an ORF1a/1b gene hairpin dsRNA expressed in bacteria and purified using a cheap and simple process involving phenol:chloroform:isoamyl alcohol (25:24:1) extraction followed by RNase A and DNase digestion also suppresses YHV replication and mortalities after L. vannamei challenge with YHV (Saksmerprome et al., 2009 ). Injection of P. monodon with a pool of five dsRNAs targeted to different ORF1a/1b gene regions and expressed similarly in bacteria but purified differently, has also proved highly protective against subsequent GAV challenge (Sellars et al., 2011, Fig. 32.9 ). Moreover, delayed and slowed mortality rates in P. monodon challenged by GAV injection has also been provided by injection immediately after challenge of either of two in vitro-transcribed dsRNAs targeted to different ORF1b gene sequences (Oanh et al., 2011) .

In addition to virus-specific dsRNA, dsRNA-mediated RNAi of a P. monodon gene (PmYRP65) expressing a protein identified to be a cell-surface receptor for YHV has also been identified to suppress YHV replication in primary LO cell cultures (Assavalapsakul et al., 2006) . The same dsRNA injected into shrimp also completely abrogates mortalities following experimental YHV challenge (Assavalapsakul et al., 2014) . Muscle injection of shrimp with a dsRNA targeted to another endogenous P. monodon gene (PmRab7) encoding a small GTPase has similarly been found to inhibit replication of YHV and WSSV following challenge (Ongvarrasopone et al., 2008) . Suppression of its expression might interfere generally with virus entry or the transport of viral proteins into the cytosol because the PmRab7 protein binds to the VP28 envelop glycoprotein of WSSV (Sritunyalucksana et al., 2006) and has been suggested to function in endosomal membrane trafficking (Ongvarrasopone et al., 2008) .

The oral delivery of dsRNA expressed in E. coli following brief fixation of the bacteria in 0.5% formaldehyde before incorporation into feeds has also been examined for its ability to induce RNAi responses projective against GAV (Sellars et al., 2011) or YHV (Sanitt et al., 2014) . When processed into a wet feed, a pool of five dsRNA targeted diverse regions of the GAV ORF1a/1b gene in an attempt to amplify its efficacy did not reduce virus replication or morality rates in shrimp challenged by muscle injection of a minimal lethal dose of GAV (Sellars et al., 2011) . In contrast, fixed bacteria containing expressed dsRNA targeted to the Rab7 gene and/or the YHV ORF1a/1b gene and concentrated into small agarose blocks suitable as a feed for juvenile (250-300 mg) L. vannamei reduced mortalities following YHV challenge by water immersion, with feeding of the Rab7 dsRNA generating the highest mortality reductions followed by the Rab7 + YHV dsRNA and the YHV dsRNA (Sanitt et al., 2014) . Inexpensive and biodegradable polymer nanoparticles derived from chitosan and its quarternized derivative, QCH4, have also been assessed for their ability to bind and deliver YHV ORF1a/1b gene hairpin dsRNA expressed in and purified cheaply from E. coli (Saksmerprome et al., 2009; Theerawanitchpan et al., 2012) . Based on reduced YHV replication occurring in treated caterpillar (Spodoptera frugiperda, Sf9) cells, it was speculated that nanoparticle binding or encapsulation of dsRNA might offer advantages in dsRNA stability and/or delivery efficiency for feed-based dsRNA delivered via the shrimp gut (Theerawanitchpan et al., 2012) . Despite these promising data, serious investment in feed-based delivery of dsRNA to prevent YHD via commercial feed pellets seems improbable.

Injection of GAV-infected juvenile P. monodon with single or pooled dsRNAs targeted to five different ORF1a/1b gene regions has been identified to reduce GAV infection levels (Sellars et al., 2014) . Of the single dsRNAs tested, one targeted to the extreme 5′-end of the GAV genome reduced infection loads most profoundly followed by the dsRNA pool. Injection of the dsRNA pool into GAV-infected female P. monodon broodstock has confirmed its ability to reduce virus infection loads without impairing female reproductive capacity (M.J. Sellars et al., unpublished) . Thus, if dsRNA injection can be optimized to reduce viral infection loads to levels below which vertical transmission is unlikely to occur, this RNAi strategy might find application in hatcheries reliant on wild P. monodon broodstock captured from regions in which GAV or YHV are endemic or in cases when it might be desirable to eliminate viral infection from valuable domesticated breeding populations. FIGURE 32.9 High survival over time of juvenile Penaeus monodon injected with a pool of five dsRNAs targeted to the GAV ORF1b gene, but not with either a nonspecific luciferase dsRNA or saline immediately prior to injection challenge with GAV. Significant differences (p < 0.001) are indicated by different letters (Sellars et al., 2011). 

The freshwater Chinese mitten crab (Eriocheir sinensis) has become an important aquaculture species in many inland provinces in China (Zeng et al., 2012; Fig. 32.10) . With the increasingly intensive aquaculture of E. sinensis, several diseases have emerged. Among these, black gill syndrome (BGS) emerged in 1996 as a cause of substantial production losses (Zhang and Bonami, 2007) . As other symptoms associated with BGS are generalized, such as sluggishness and anorexia, and as affected crabs display respiratory difficulties manifest by them audibly sounding to "sigh" in the quiet of night as they respire bubbles, the disease is also referred to colloquially as "sighs" disease (SD) (Zhang and Bonami, 2007) .

In investigating the cause of SD in crabs collected from a farm in Hubei Province, filamentous viral nucleocapsid precursors (16-18 nm × 150-250 nm) up to 400 nm in length, sometimes closely aligned and amassed adjacent to ER membranes, and enveloped rod-shaped virions (24-42 nm × 60-170 nm) either scattered throughout the cell cytoplasm or packed densely within cytoplasmic vesicles, were identified by TEM in cells of connective tissues (Zhang and Bonami, 2007; Fig. 32.10) . Negative staining of virions purified from hemolymph identified regularly spaced surface projections.

In challenge trials, injection of naïve E. sinensis with ultra-filtered hemolymph from a disease crab resulted in deaths accumulating rapidly from day 13 to reach 100% by day 17 (Zhang and Bonami, 2007) . SD symptoms developed in approximately 30% crabs and none developed BGS suggested that BGS might be a secondary condition caused by crabs becoming moribund in pond culture environments (Zhang and Bonami, 2007) . Histopathology in diseased crabs was characterized by clusters (1-5 µm dia.) of pale to deeply basophilic necrotic cells displaying hypertrophied nuclei and nuclear karyorrhesis or pyknosis, and by cytoplasmic inclusion bodies (200-800 nm diameter) filled with enveloped virions. Necrotic cells were prominent in the crab LO and in connective tissues of various organs, including the gills, hepatopancreas, heart, gut and testes. In gills, basophilic inclusion bodies were replaced by eosinophilic foci in necrotic cells and eosinophilic areas in connective tissues (Zhang and Bonami, 2007) . (Zhang and Bonami, 2007). Due to the tissue tropism and histopathology closely resembling those manifest in shrimp infected with okaviruses, filamentous nucleocapsid precursors and enveloped rod-shaped virions sharing striking morphological similarities, except for virions appearing somewhat shorter than those described for YHV (40-50 nm × 150-200 nm, Boonyaratpalin et al., 1993; Chantanachookin et al., 1993) and GAV (34-42 nm × 183-200 nm, Spann et al., 1995 , 1997 , and a viral genome comprising a long (~22 kb) ssRNA, the virus was named EsRNV (Zhang and Bonami, 2007; Bonami and Zhang, 2011) . While the properties of EsRNV suggest a close evolutionary relationship to okaviruses, genome sequencing is needed to determine whether it should be classified within this or a new genus of the Roniviridae (Cowley et al., 2012) .

In the 1970s, viruses forming bacilliform (20-30 nm × 110-170 nm) particles together with long flexible particles up to 600 nm in length, similar in appearance to the virions and filamentous helical nucleocapsid precursors of okaviruses and bafiniviruses, were detected in the Blue crab (Callinectes sapidus) (Jahromi, 1977; Clark, 1978, 1979; Johnson, 1983) . Callinectes sapidus is indigenous to the US Atlantic coast and the Gulf of Mexico and while not an aquaculture species, supports an important wild fishery in this region. For the virus particle (20-30 nm × 110-170 nm) named tentatively as rhabdolike-virus A (RhVA, Jahromi, 1977; Johnson, 1983 ) based on morphological similarities to plant rhabdoviruses, virions were detected in Schwann cells in parallel arrays localized at ER and nuclear cistern membranes, and less frequently at plasma membranes. For the virus particle (25-30 nm × 100-150 nm) named ecdysial gland virus 2 (EGV-2, Clark, 1978, 1979) due to its detection in ecdysial (Y organ or mandibular) glands of eyestalk-ablated blue crabs, virions also matured as arrays aligned perpendicularly to the outer nuclear membrane, as well as at tubular ER membranes and the plasma membrane (Yudin and Clark, 1979) . EGV-2 occurred in association with a larger-diameter enveloped bacilliform particle (50-70 nm × 110-170 nm) decorated with surface projections that was named EGV-1 (Yudin and Clark, 1978) . EGV-1 and EGV-2 became evident after crab eyestalk ablation, suggesting that the infections were preexisting and escalated in response to stress (Yudin and Clark, 1978) . Similarities in virion morphology and morphogenesis suggest that RhVA and EGV-2 are the same virus. However, genome sequence is required to confirm their evolutionary relationships because the diameter of the bacilliform RhVA and EGV-2 virions is somewhat smaller than those described for okaviruses (Cowley et al., 2012) and bafiniviruses (de Groot et al., 2012a) .

Presencia del camarón tigre gigante Penaeus monodon (Crustacea, Penaeidae) en la costas del Estado Anzoátegui

A new virus isolated from cultured grass carp Ctenopharyngodon idella

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Low viral loads and lymphoid organ spheroids are associated with yellow head virus (YHV) tolerance in whiteleg shrimp Penaeus vannamei

The Diagnosis and Prevention of the Mid-Crop Mortality Syndrome of Pond-Reared Black Tiger Prawns (Penaeus monodon). Fisheries Research and Development Corporation Project

The haemocytic origin of lymphoid organ spheroid cells in the penaeid prawn Penaeus monodon

Import Risk Analysis, Prawns and Prawn Products. Draft Import Risk Analysis Paper

Protection from yellow head virus (YHV) infection in Penaeus vannamei pre-infected with Taura syndrome virus (TSV)

Propagation of infectious yellow head virus particles prior to cytopathic effect in primary lymphoid cell cultures of Penaeus monodon

Identification and characterisation of a Penaeus monodon lymphoid cell-expressed receptor for yellow head virus

Application of YHV-protease dsRNA for protection and therapeutic treatment against yellow head virus infection in Litopenaeus vannamei

Silencing of PmYPR65 receptor prevents yellow head virus infection in Penaeus monodon

Arteriviruses

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Viral diseases in commercially exploited crabs: a review

Non-occluded baculo-like virus, the causative agent of yellow-head disease in the black tiger shrimp (Penaeus monodon)

Ribosomal frameshifting on viral RNAs

Characterization of an efficient coronavirus ribosomal frameshifting signal, requirement for an RNA pseudoknot

Mutational analysis of the "slippery-sequence" component of a coronavirus ribosomal frameshifting signal

Fatal, virus-associated peripheral neuropathy and retinopathy in farmed Penaeus monodon in eastern Australia. II. Outbreak descriptions

Fatal, virus-associated peripheral neuropathy and retinopathy in farmed Penaeus monodon in eastern

Detection of the yellow-head virus (YHV) in wild blue shrimp, Penaeus stylirostris, from the Gulf of California and its experimental transmission to the Pacific white shrimp, Penaeus vannamei

Gene expression kinetics of the yellow head virus in experimentally infected Litopenaeus vannamei

Preferential suppression of yellow head virus (YHV) envelope protein gp116 in shrimp that survive challenge with YHV

Immunological-based assays for specific detection of shrimp viruses immunological-based assays for specific detection of shrimp viruses

Histology and ultrastructure reveal a new granulosis-like virus in Penaeus monodon affected by yellow-head disease

Infection of cultured cells from the lymphoid organ of Penaeus monodon Fabricius by monodon-type baculovirus (MBV)

Establishment of cell culture systems from penaeid shrimp and their susceptibility to white spot disease and yellow head viruses

Identification and functional characterization of Dicer2 and five single VWC domain proteins of Litopenaeus vannamei

Presence of Penaeus monodon in the continental shelf of the State of Para, northern Brazil (Crustacea, Decapoda, Penaeidae)

Ocorrência de Penaeus monodon Fabricius, 1798 no litoral dos estados de Pernambuco e Alagoas (Crustacea, Decapoda, Penaeidae)

The complete genome sequence of gill-associated virus of Penaeus monodon prawns indicates a gene organisation unique among nidoviruses

Molecular biology and pathogenesis of roniviruses

Yellow head virus from Thailand and gill-associated virus from Australia are closely related but distinct prawn viruses

Gill-associated virus of Penaeus monodon prawns, an invertebrate virus with ORF1a and ORF1b genes related to arteri-and coronaviruses

Detection of Australian gill-associated virus (GAV) and lymphoid organ virus (LOV) of Penaeus monodon by RT-nested PCR

Gill-associated virus of Penaeus monodon prawns; molecular evidence for the first invertebrate nidovirus

Gill-associated nidovirus of Penaeus monodon prawns transcribes 3′-coterminal subgenomic mRNAs that do not possess 5′-leader sequences

Vertical transmission of gill-associated virus (GAV) in the black tiger prawn Penaeus monodon

The gene encoding the nucleocapsid protein of gill-associated nidovirus of Penaeus monodon prawns is located upstream of the glycoprotein gene

Differential detection of gill-associated virus (GAV) from Australia and yellow head virus (YHV) from Thailand by multiplex RT-nested PCR

Family Roniviridae

Mid-crop mortality syndrome in Australian prawn farming, a case study

Family Coronaviridae

Order Nidovirales

Presumptive detection of yellow head virus by reverse transcriptase-polymerase chain reaction and dot-blot hybridization in Litopenaeus vannamei and L. stylirostris cultured on the Northwest coast of Mexico

Quantitative real-time RT-PCR demonstrates that handling stress can lead to rapid increases of gill-associated virus (GAV) infections levels in Penaeus monodon

Characterization of Argonaute cDNA from Penaeus monodon and implication of its role in RNA interference

Quantitative assay for measuring the Taura syndrome virus and yellow head virus load in shrimp by real-time RT-PCR using SYBR Green chemistry

Genomic organization, biology and diagnosis of Taura syndrome virus and yellow head virus of penaeid shrimp

Effect of Phyllanthus spp. against yellow-head baculovirus infection in black tiger shrimp, Penaeus monodon

Efficacy of guava (Psidium guajava) extracta gainst some fish and shrimp pathogenic agents

Protective efficacy of Clinacanthus nutans on yellow-head disease in black tiger shrimp (Penaeus monodon)

Changes in the histological organization and spheroid formation in lymphoid organ of Penaeus monodon infected with yellow head virus

Frozen commodity shrimp, potential avenue for introduction of white spot syndrome virus and yellow head virus

The Arteriviridae

Yellow head disease (YHC)

Update on viral accommodation, a model for host-viral interaction in shrimp and other arthropods

Historic emergence, impact and current status of shrimp pathogens in Asia

Active viral accommodation, a new concept for crustaceans response to viral pathogens

Environmental control of infectious shrimp diseases in Thailand

Progress in characterization and control of yellow-head virus of Penaeus monodon

Current status of research on yellow-head virus and white-spot virus in Thailand

Penaeus monodon captured broodstock surveyed for yellowhead virus and other pathogens by electron microscopy

Invasion of Asian tiger shrimp, Penaeus monodon Fabricius, 1798, in the western north Atlantic and Gulf of Mexico

Non-virulence of a recombinant shrimp nidovirus is associated with its non structural gene sequence and not a large structural gene deletion

Using novel bioactive substances to enhance the production of farmed shrimp

Effective use of microbial biomass products to facilitate the complete replacement of fishery resources in diets for the black tiger shrimp, Penaeus monodon

Presencia de Penaeus monodon Fabricius (Crustacea: Decapoda: Penaeidae) en aguas de la Guajira colombiana

Genomics and evolution of the Nidovirales

The palm subdomain-based active site is internally permuted in viral RNA-dependent RNA polymerases of an ancient lineage

Nidovirales, evolving the largest RNA virus genome

Identification and ultrastructural characterization of a novel virus from fish

Waterborne and cannibalism-mediated transmission of the Yellow head virus in Penaeus monodon

Characterization and diagnostic use of a recombinant single-chain antibody specific for the gp116 envelop glycoprotein of Yellow head virus

A new bacilliform fathead minnow rhabdovirus that produces syncytia in tissue culture

Occurrence of rhabdovirus-like particles in the blue crab, Callinectes sapidus

Rapid and sensitive detection of shrimp yellow head virus using loop-mediated isothermal amplification and a colorogenic nanogold hybridization probe

Identification and analysis of gp116 and gp64 structural glycoproteins of yellow head nidovirus of Penaeus monodon shrimp

Diseases caused by viruses, rickettsiae, bacteria, and fungi

Evidence for apoptotic correlated with mortality in the giant black tiger shrimp Penaeus monodon infected with yellow head virus

Multiplex RT-PCR assay for simultaneous detection of six viruses of penaeid shrimp

Vietnam

Susceptibility of the postlarval stages of black tiger shrimp (Penaeus monodon) to yellow-head baculovirus (YBV)

Rapid and sensitive detection of shrimp yellow head virus by loop-mediated isothermal amplification combined with a lateral flow dipstick

RT-PCR detection of yellow head virus (YHV) infection in Penaeus monodon using dried haemolymph spots

The phylogeny of RNA-dependent RNA polymerases of positive-strand RNA viruses

Mesoniviridae, a proposed new family in the order Nidovirales formed by a single species of mosquito-borne viruses

Identification and expression analysis of Dicer2 in black tiger shrimp (Penaeus monodon) responses to immune challenges

Handbook of Pathology and Diagnostic Procedures for Diseases of Penaeid Shrimp

The penaeid shrimp viruses TSV, IHHNV, WSSV, and YHV, current status in the Americas, available diagnostic methods and management strategies

Exclusion of specific pathogens for disease prevention in a penaeid shrimp biosecurity program

Biosecurity in shrimp farming, pathogen exclusion through the use of SPF stock and routine surveillance

Status of shrimp diseases and advances in shrimp health management

Shrimp diseases and current diagnostic methods

Risk of spread of penaeid shrimp viruses in the Americas by the international movement of live and frozen shrimp

Experimental infection of Western Hemisphere Penaeid shrimp with Asian white spot syndrome virus and Asian yellow head virus

Specific pathogen-free shrimp stocks in shrimp farming facilities as a novel method for disease control in crustaceans

Handbook for Cultivation of Black Tiger Prawns

Intensive shrimp pond management in Asia World Aquaculture '96, Book of Abstracts

Detection of a new genotype of yellow-head virus in farmed shrimp suspicious of EMS/AHPNS infection

Recent developments in immunologically-based and cell culture protocols for the specific detection of shrimp viral pathogens

Differences in the susceptibility of palaemonid shrimp species to yellow head virus (YHV) infection

Experimental infection of some penaeid shrimps and crabs by yellow head virus (YHV)

Developing specific pathogen-free (SPF) animal populations for aquaculture: a case study for IHHN virus of penaeid shrimp

Special topic review: viruses, biosecurity and specific pathogen-free stocks in shrimp aquaculture

Infection of the yellow head baculo-like virus (YBV) in two species of penaeid shrimp, Penaeus stylirostris (Stimpson) and Penaeus vannamei (Boone)

Development of a quantal assay in primary shrimp cell culture for yellow head baculovirus (YBV) of penaeid shrimp

Distribution of yellow-head virus in selected tissues and organs of penaeid shrimp Penaeus vannamei

Development of a nitrocellulose-enzyme immunoassay for the detection of yellow-head virus from penaeid shrimp

Infectivity of yellow head virus (YHV) and the Chinese baculo-like virus (CBV) in two species of penaeid shrimp Penaeus stylirostris (Stimpson) and Penaeus vannamei (Boone)

Fishes of Wisconsin E-Book. Wisconsin Department of Natural Resources

Stable yellowhead virus (YHV) RNA detection by qRT-PCR during six-day storage

Daggerblade grass shrimp (Palaemonetes pugio), a reservoir host for yellow-head virus (YHV)

A summary of taxonomic changes approved by ICTV

Viral Survey of Fathead Minnows, Golden Shiners, and White Suckers from Baitfish Dealers in Wisconsin

Detection of white spot syndrome virus and yellowhead virus in prawns imported into Australia

Detection of yellow head virus in shrimp by loop-mediated isothermal amplification (LAMP)

Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of yellow head virus in shrimp

A survey of the bait industry in the north-central region of the United States. North Am

Viremia-associated ana-aki-byo, A new viral disease in color carp Cyprinus carpio in Japan

Histopathology of viremia-associated ana-aki-byo in combination with Aeromonas hydrophila in color carp Cyprinus carpio in Japan

Histopathology of cultured shrimp showing gross signs of yellow head syndrome and white spot syndrome during 1994 Indian epizootics

Discovery of a new genotype of yellow head virus (YHV7) in giant tiger shrimp (Penaeus monodon) indigenous to northern Australia

Improvement in the specificity and sensitivity of detection for the Taura syndrome virus and yellow head virus of penaeid shrimp by increasing the amplicon size in SYBR Green real-time RT-PCR

Haemagglutination as a low cost detection method for gill-associated virus and by inference, yellowhead virus in Penaeus monodon Fabricius, 1798

Sensitivity and specificity of current diagnostic tests for Gill-associated virus in Penaeus monodon

Production of polyclonal and monoclonal antibodies against gill-associated virus and the development of an ELISA

Yellow head-like viruses affecting the penaeid aquaculture industry: a review

Gill-associated virus and its association with decreased production of Penaeus monodon in Australian prawn farms

Dot-blot nitrocellulose enzyme immunoassays for the detection of white-spot virus and yellow-head virus of penaeid shrimp

Detection of yellowhead virus and Chinese baculovirus in penaeid shrimp by the Western blot technique

Yellow-head virus, a rhabdovirus-like pathogen of penaeid shrimp

Routine and rapid diagnosis of Yellow-head disease in Penaeus monodon

Evidence of yellow head virus in cultured black tiger shrimp (Penaeus monodon Fabricius) from selected shrimp farms in the Philippines

Prevalence of yellow head virus in cultured black tiger shrimp (Penaeus monodon Fabricius) from selected shrimp farms in the Philippines

Infection and mortality of Penaeus vannamei at extreme salinities when challenged with Mexican yellow head virus

Discovery of the first insect nidovirus, a missing evolutionary link in the emergence of the largest RNA virus genomes

The detection of white spot syndrome virus (WSSV) and yellow head virus (YHV) in imported commodity shrimp

Pathogenicity of gill-associated virus and Mourilyan virus during mixed infections of black tiger shrimp (Penaeus monodon)

Chapter 2.2.8, Yellowhead disease Manual of Diagnostic Tests for Aquatic Animals

Studies on Penaeus orientalis Kishinouye. VIII. Structure of newly found lymphoid organ

Suppression of PmRab7 by dsRNA inhibits WSSV or YHV infection in shrimp

Putative spawner-isolated mortality virus associated with mid-crop mortality syndrome in farmed Penaeus monodon from northern Australia

Multiplex real-time PCR and high-resolution melting analysis for detection of white spot syndrome virus, yellow-head virus, and Penaeus monodon densovirus in penaeid shrimp

Similarity between the histopathology of white spot syndrome virus and yellow head syndrome virus and its relevance to diagnosis of YHV disease in the Americas

Latent yellow-head infections in Penaeus monodon and implications regarding disease resistance or tolerance

Nidovirus transcription, how to make sense…?

Prevalence and Co-Prevalence of White Spot Syndrome Virus (WSSV) and Yellow Head Complex Viruses (YHV-Complex) in Cultured Giant Tiger Prawn (Penaeus monodon) in Vietnam

Egg activation in the black tiger shrimp Penaeus monodon

Genetic improvement/shrimp breeding-knowledge of biological requirements critical to successful broodstock rearing

Black tiger breeding program yields record shrimp harvests in Australia

Induction of antiviral immunity by double-stranded RNA in a marine invertebrate

Double-stranded RNA induces sequence-specific antiviral silencing in addition to nonspecific immunity in a marine shrimp, convergence of RNA interference and innate immunity in the invertebrate antiviral response?

Indonesia

Gene silencing in crustaceans: from basic research to biotechnologies

A novel and inexpensive application of RNAi technology to protect shrimp from viral disease

Detection of yellow-head disease in intensive freshwater production systems of Litopenaeus vannamei

Protection of yellow head virus infection in shrimp by feeding of bacteria expressing dsRNAs

A contemporary view of coronavirus transcription

Characterization of white bream virus reveals a novel genetic cluster of nidoviruses

Penaeus monodon is protected against gill-associated virus by muscle injection but not oral delivery of bacterially expressed dsRNAs

Reduced loads of pre-existing gill-associated virus (GAV) infection in juvenile Penaeus monodon injected with single or multiple GAV-specific dsRNAs

Feed containing Novacq™ improves resilience of black tiger shrimp to gill-associated virus-induced mortality

Impact of yellow head virus outbreaks in the whiteleg shrimp

Application of nucleic-acid-based therapeutics for viral infections in shrimp aquaculture

An introduction to nidoviruses

Sri Lanka

Development of a monoclonal antibody specific to yellow head virus (YHV) from Penaeus monodon

Monoclonal antibodies specific to yellowhead virus (YHV) of Penaeus monodon

A convenient immunochromatographic test strip for rapid diagnosis of yellow head virus infection in shrimp

Simultaneous and rapid detection of white spot syndrome virus and yellow head virus infection in shrimp with a dual immunochromatographic strip test

Complete ORF1b-gene sequence indicates yellow head virus is an invertebrate nidovirus

Structural and antigenic analysis of the yellow head virus nucleocapsid protein p20

RNA transcription analysis and completion of the genome sequence of yellow head nidovirus

A virulent isolate of yellow head nidovirus contains a deformed envelope glycoprotein gp116

A review on plants and herbal extracts against viral diseases in aquaculture

A phosphatocopid crustacean with appendages from the Lower Cambrian

Torovirus non-discontinuous transcription, mutational analysis of a subgenomic mRNA promoter

Characterization of Berne virus genomic and messenger RNAs

A 3′-coterminal nested set of independently transcribed mRNAs is generated during Berne virus replication

Arterivirus molecular biology and pathogenesis

Use of a specific monoclonal antibody to determine tissue tropism of yellow head virus (YHV) of Penaeus monodon by in situ immunocytochemistry

Detection and differentiation of yellow head complex viruses using monoclonal antibodies

RNA-binding domain in the nucleocapsid protein of gill-associated nidovirus of penaeid shrimp

N-linked glycosylation is essential for the yellow head virus replication cycle

Australian red claw crayfish (Cherax quadricarinatus) is susceptible to yellow head virus (YHV) infection and can transmit it to the black tiger shrimp (Penaeus monodon)

Special topic review: viral disease of penaeid shrimp with particular reference to four viruses recently found in shrimp from Queensland

Lymphoid organ virus of Penaeus monodon from Australia

A yellow-head-like virus from Penaeus monodon cultured in Australia

Differences in susceptibility of some penaeid prawn species to gill-associated virus (GAV) infection

Detection of gill-associated virus (GAV) by in situ hybridisation during acute and chronic infections in Penaeus monodon and Penaeus esculentus shrimp

PmRab7 is a VP28-binding protein involved in white spot syndrome virus infection in shrimp

A critical review of susceptibility of crustaceans to Taura syndrome, yellowhead disease and white spot disease and implications of inclusion of these diseases in European legislation

A key gene of the RNA interference pathway in the black tiger shrimp, Penaeus monodon, identification and functional characterisation of Dicer-1

Thermatic Review on Management Strategies for Major Diseases in Shrimp Aquaculture

A yellow head virus gene probe, nucleotide sequence and application for in situ hybridization

Postlarvae and juveniles of a selected line of Penaeus stylirostris are resistant to infectious hypodermal and hematopoietic necrosis virus infection

In situ detection of Australian gill-associated virus with a yellow head virus gene probe

RNA pseudoknots, translational frameshifting and read-through on viral RNAs

Chitosan and its quaternized derivative as effective long dsRNA carriers targeting shrimp virus in Spodoptera frugiperda 9 cells

Silencing of yellow head virus replication in penaeid shrimp cells by dsRNA

Therapeutic inhibition of yellow head virus multiplication in infected shrimps by YHV-protease dsRNA

Characterization of bafinivirus main protease autoprocessing activities

Isolation and characterization of cDNA encoding Argonaute, a component of RNA silencing in shrimp (Penaeus monodon)

The roles of haemocytes and the lymphoid organ in the clearance of injected Vibrio bacteria in Penaeus monodon shrimp

Discontinuous and non-discontinuous subgenomic RNA transcription in a nidovirus

Mesoniviruses are mosquito-specific viruses with extensive geographic distribution and host range

Australia

Viral disease emergence in shrimp aquaculture, origins, impacts and the effectiveness of health management strategies

Yellow head virus, third Edition

The emergence of yellow head-related viruses in Australia

Yellow head complex viruses, transmission cycles and topographical distribution in the Asia-Pacific region

Yellow head virus infection in the giant tiger prawns Penaeus monodon cultured in Taiwan

Yellow head disease-like virus infection in the Kuruma shrimp Penaeus japonicus cultured in Taiwan

siRNA injection induces sequence-independent protection in Penaeus monodon against white spot syndrome virus

Genetic diversity in the yellow head nidovirus complex

Consensus RT-nested PCR to detect yellow head virus genotypes in penaeid shrimp

Homologous genetic recombination in the yellow head complex of nidoviruses infecting Penaeus monodon shrimp

A consensus real-time RT-PCR for detection of all genotypic variants of yellow head virus of penaeid shrimp

A non-occluded, systemic baculovirus that occurs in cells of ectodermal and mesodermal origin and causes high mortality in the black tiger prawn Penaeus monodon

Yellow-head virus of Penaeus monodon is an RNA virus

Detection of yellow-head virus (YHV) of Penaeus monodon by RT-PCR amplification

Selective breeding of specific pathogen-free (SPF) shrimp for high health and increased growth

Development and commercial performance of high health shrimp from SPF broodstock Penaeus vannamei

Thematic Review on Management Strategies for Major Diseases in Shrimp Aquaculture

A Dicer-1 gene from white shrimp Litopenaeus vannamei: expression pattern in the processes of immune response and larval development

YHV-protease dsRNA inhibits YHV replication in Penaeus monodon and prevents mortality

Two virus-like particles found in the ecdysial gland of the blue crab, Callinectes sapidus

A description of rhabdovirus-like particles in the mandibular gland of the blue crab, Callinectes sapidus

Aquaculture: Farming Aquatic Animals and Plants, second Edition

A roni-like virus associated with mortalities of the freshwater crab, Eriocheir sinensis Milne Edwards, cultured in China, exhibiting "sighs disease" and black gill syndrome

An epipodite-bearing crown-group crustacean from the Lower Cambrian

Development and evaluation of reverse transcription loop-mediated isothermal amplification assay for the detection of the fathead minnow nidovirus

The coronavirus replicase

The coronavirus replicase, insights into a sophisticated enzyme machinery

Coronavirus replicative proteins

Virus-encoded proteinases and proteolytic processing in the Nidovirales

The 3C-like proteinase of an invertebrate nidovirus links coronavirus and potyvirus homologs

An insect nidovirus emerging from a primary tropical rainforest

Identification and characterization of genetically divergent members of the newly established family Mesoniviridae