Penaeus Stylirostris Densovirus Research Articles

Simultaneous Production of a Virus-Like Particle Linked to dsRNA to Enhance dsRNA Delivery for Yellow Head Virus Inhibition

A co-expressed Penaeus stylirostris densovirus (PstDNV) capsid and dsRNA specific to the yellow head virus (YHV) protease (CoEx cpPstDNV/dspro) has been shown to suppress YHV replication in the Pacific white-legged shrimp (Litopenaeus vannamei). However, maintaining two plasmids in a single bacterial cell is not desirable; therefore, a single plasmid harboring both the PstDNV capsid and the dsRNA-YHV-pro gene was constructed under the regulation of a single T7 promoter, designated pET28a-Linked cpPstDNV-dspro. Following induction, this novel construct expressed an approximately 37-kDa recombinant protein associated with a roughly 400-bp dsRNA (Linked cpPstDNV-dspro). Under a transmission electron microscope, the virus-like particles (VLP; Linked PstDNV VLPs-dspro) obtained were seen to be monodispersed, similar to the native PstDNV virion. A nuclease digestion assay indicated dsRNA molecules were both encapsulated and present outside the Linked PstDNV VLPs-dspro. In addition, the amount of dsRNA produced from this strategy was higher than that obtained with a co-expression strategy. In a YHV infection challenge, the Linked PstDNV VLPs-dspro was more effective in delaying and reducing mortality than other constructs tested. Lastly, the linked construct provides protection for the dsRNA cargo from nucleolytic enzymes present in the shrimp hemolymph. This is the first report of a VLP carrying virus-inhibiting dsRNA that could be produced without disassembly and reassembly to control virus infection in shrimp.

Penaeus stylirostris densovirus proteins CP and NS1 interact with peritrophin of Litopenaeus vannamei

The Penaeus stylirostris densovirus (PstDNV) is a major virus of shrimps that severely harms the shrimp farming industry. Peritrophin is a peritrophic membrane protein with chitin binding activity. To examine the roles of peritrophin in viral infection, we used yeast two-hybrid to analyze the interaction between the Pacific white shrimp (Litopenaeus vannamei) peritrophin and PstDNV proteins (CP, NS1 and NS2). The yeast two-hybrid results showed that NS1 and peritrophin had an interaction, CP and peritrophin had an interaction as well, and NS2 had no interaction with peritrophin. We validated the interactions with GST pull-down assays. We then conducted RNA interference and qRT-PCR. The results showed that when pre-injection of dsRNA-peritrophin, the quantity of PstDNV in the shrimps injected with viruses was significantly lower than in the control group (P < 0.01), indicating the viral infection was decreased when the peritrophin gene expression was inhibited. The results indicated that peritrophin of L. vannamei participated in the PstDNV infection.

Double-Loop-Mediated Isothermal Amplification (D-LAMP) using colourimetric gold nanoparticle probe for rapid detection of infectious Penaeus stylirostris densovirus (PstDNV) with reduced false-positive results from endogenous viral elements

Endogenous viral elements (EVEs) of Penaeus stylirostris densovirus (PstDNV) in shrimp genome have been reported to occur randomly, and may cause false positive in diagnosis, because most of conventional detection methods only target a small nucleotide sequence of PstDNV. Since PstDNV is listed by the World Organization for Animal Health (OIE) as a reportable virus in crustacean, several countries require shrimp stocks imported for aquaculture to be screened for this virus. The rapidly and highly sensitive detection methods with reduced false positive from EVEs are necessary to guarantee and maintain absence of infectious PstDNV in traded stocks. To overcome problem posed by the viral insertions to shrimp genome, two set of primers were designed for LAMP combined with colourimetric gold nanoparticles (AuNPs), so-called D-LAMP, to detect two regions of PstDNV genome. The first target region targeted OIE-recommended nucleotide sequence of 1740–1968 (GenBank Accession no 273215) from our previous report. The second region targeted at the 3′end of the PstDNV genome that has been reported least likely to give rise to EVE in the shrimp genome. Detection sensitivity is approximately 100 copies. Results from LAMP combined with colourimetric AuNPs and conventional PCR methods are in agreement for detecting the infectious PstDNV. D-LAMP is also applicable to detect infectious type of PstDNV in P. stylirostris tissue sections (i.e. in situ DIG-labelled LAMP) and reaffirms its high specificity for detecting PstDNV infection.

The Penaeus stylirostris densovirus capsid protein interacts with the Litopenaeus vannamei BCCIP protein

Viral capsid proteins play an important role in the viral infection process. To identify the cellular proteins in shrimp that interact with the Penaeus stylirostris densovirus capsid protein (PstDNV-CP), we constructed a yeast two-hybrid (Y2H) cDNA library of the muscle tissue of Litopenaeus vannamei, and hybridized the bait vector pGBKT7-CP with this library. Cloning and sequencing showed that the shrimp protein interacting with PstDNV-CP was a homolog of BRCA2 and CDKN1A(p21)-interacting protein (BCCIP). We named this protein L. vannamei BCCIP (LvBCCIP). Further analysis showed that LvBCCIP interacted with L. vannamei calmodulin (LvCaM). We validated the interactions between PstDNV-CP and LvBCCIP, and between LvBCCIP and LvCaM, with GST pulldown assays. The gene expression of LvBCCIP increased significantly after PstDNV challenge. In addition, the PstDNV titer of PstDNV-challenged shrimp was significantly reduced after LvBCCIP expression was inhibited using double-stranded RNA (dsRNA) interference. These results indicated that LvBCCIP is critical to PstDNV pathogenesis in L. vannamei. Interestingly, the growth rate of L. vannamei was significantly reduced when LvBCCIP gene expression was silenced, indicating that LvBCCIP may also be associated with growth regulation in L. vannamei. Thus, the interaction between PstDNV-CP and LvBCCIP might explain why PstDNV infection leads to runt-deformity syndrome in shrimp.

Genomic analysis of a decapod Penstylidensovirus 1 isolated from Penaeus semisulcatus

The purpose of this study was to identify the genomic characteristics of Penaeus stylirostris densovirus (PstDNV 1) affected farming of the green tiger shrimp Penaeus semisulcatus. Shrimp samples tested positive for PstDNV 1 by PCR and showing pathognomonic lesions were taken for cloning. Genomic DNA from infected shrimp was digested with restriction enzymes and plasmid libraries made before taking recombinant clones for shotgun sequencing. Altogether, 11 PstDNV1 contigs 3,530 to 3,912 nucleotides (nt) long were generated from P. semisulcatus. Each sequence contained ORFs encoding nonstructural (NS) proteins NS1 and NS2 and coat protein. The genome length of the isolates ranged from 3,598 to 3,912 bases. The GC content of the isolates varied from 42.5 % to 43.2%. These eleven PstDNV 1 isolates shared a pairwise identity of 96.4%. The phylogenetic analysis of the eleven PstDNV 1 isolates and the previously reported sequences showed the formation of 5 groups. Group 1 contains isolates from China, Brazil, California, Hawaii and 4 sequences from Egypt (KT316255.1, KT316260.1, KT316256.1 and KT316256.1). The second group contains sequences from Australia and 4 sequences from Egypt (KT316253.1, KT316252.1, KT316251.1 and KT316250.1). The third group clusters sequences from Thailand, Vietnam, Taiwan and two sequences from Egypt (KT316257.1 and KT316259.1). The appearance of PstDNV 1 in Egypt occurred after the introduction of Litopenaeus vannamei and Fenneropenaeus indicus. The complete genomes characterized in this study suggest that PstDNV was introduced to Egypt via movement of infected animals from distinct parts of the world. These finding serve as evidence that stricter monitoring protocols for the movement of shrimp species are fundamental as biosecurity measures to avoid the spread of pathogens that could potentially compromise the growth of the shrimp industry in Egypt.

The Penaeus stylirostris densovirus capsid interacts with Litopenaeus vannamei troponin I

The Penaeus stylirostris densovirus (PstDNV) (also known as infectious hypodermal and hematopoietic necrosis virus, IHHNV), a very small DNA virus, is a major shrimp pathogen. The PstDNV genome encodes only two nonstructural proteins and one capsid protein. This virus is thus an ideal, simple model for the investigation of virus-host interactions. To explore the role of the PstDNV capsid in viral infections, a yeast two-hybrid (Y2H) cDNA library was constructed based on Pacific white shrimp, Litopenaeus vannamei mRNA. The Y2H library was then screened, using the PstDNV capsid protein as bait. We identified a host protein that interacted strongly with the PstDNV capsid as L. vannamei troponin I (LvTnI). An in vitro co-immunoprecipitation experiment further supported this interaction. In addition, an in vivo neutralization experiment showed that the vaccination with anti-LvTnI significantly reduced PstDNV copies in PstDNV-challenged shrimp, indicating that the interaction between the PstDNV capsid and cellular LvTnI is essential for PstDNV infection. This result has important implications for our understanding of the mechanisms by which PstDNV infects shrimp.

Complete Genome Sequences of Four Major Viruses Infecting Marine Shrimp in Egypt.

The genome sequences of four economically important shrimp viruses, Penaeus stylirostris densovirus 1, hepatopancreatic parvovirus, yellow head virus, and gill-associated virus, are reported here. Genome data are fundamental for epidemiological studies in determining the origins of these viruses detected for the first time in Egypt and in developing disease management strategies.

Administration of co-expressed Penaeus stylirostris densovirus-like particles and dsRNA-YHV-Pro provide protection against yellow head virus in shrimp

The activation of the innate RNA interference pathway through double-stranded RNAs (dsRNAs) is one of the approaches to protecting shrimp from viruses. Previous studies have shown that injection of specific dsRNAs can successfully inhibit viral infection in shrimp. However, inhibition requires high levels of dsRNA and dsRNA stability in shrimp is limited. Virus-like particles (VLPs) have been applied to deliver nucleic acids into host cells because of the protection of dsRNAs from host endonucleases as well as the target specificity provided by VLPs. Therefore, this study aimed to develop Penaeus stylirostris densovirus (PstDNV) VLPs for dsRNA deliver to shrimp. The PstDNV capsid protein was expressed and can be self-assembled to form PstDNV VLPs. Co-expression of dsRNA-YHV-Pro and PstDNV capsid protein was achieved in the same bacterial cells, whose structure was displayed as the aggregation of VLPs by TEM. Tested for their inhibiting yellow head virus (YHV) from infecting shrimp, the dsRNA-YHV-Pro-PstDNV VLPs gave higher levels of YHV suppression and a greater reduction in shrimp mortality than the delivery of naked dsRNA-YHV-Pro. Therefore, PstDNV-VLPs are a promising vehicle for dsRNA delivery that maintains the anti-virus activity of dsRNA in shrimp over a longer period of time as compared to native dsRNAs.

Prevalence, diversity and co-occurrence of the white spot syndrome virus, monodon baculovirus and Penaeus stylirostris densovirus in wild populations of Penaeus monodon in the Philippines

The farming of the black tiger shrimp Penaeus monodon in the Philippines relies on wild broodstock. PCR was thus used to determine the prevalence of white spot syndrome virus (WSSV), monodon baculovirus (MBV) and Penaeus stylirostris densovirus (PstDV) in a total of 178 shrimp from 6 geographically disparate locations where broodstock are captured for use in hatcheries. PCR amplicons were also sequenced to identify phylogenetic relationships of the virus haplotypes detected. Shrimp from southeastern Luzon (Camarines Norte) had the highest prevalence of each of the 3 viruses and were frequently co-infected with 2 or more viruses. No viruses were detected in shrimp from northwestern Luzon (Pangasinan). MBV was most prevalent and PstDV strains displayed the most genetic diversity. WSSV was detected at 3 sites, and a VP28 gene sequence examined was invariant and consistent with strains found in many countries, including Thailand, China, Japan, Korea, Indonesia, Iran, Brazil and Mexico. WSSV open reading frame 94 gene sequence analysis identified location-specific repeat types. MBV sequences were dissimilar to haplotypes detected in India. PstDV sequences were diverse and included 2 lineages detected either in Australia or in the United States, Ecuador, Taiwan, China and Vietnam. The PCR data confirmed that WSSV, MBV and PstDV are endemic in P. monodon in the Philippines but that populations at some locations might remain free of infection.

Variation in virome diversity in wild populations of Penaeus monodon (Fabricius 1798) with emphasis on pathogenic viruses

Marine animals typically harbor a community of viruses, a number of which are known to cause diseases. In shrimp aquaculture, viral pathogens are the principal causes of major economic losses. However, the composition of the viral load of shrimps in wild population is poorly known. In this study, we explored the viral diversity in the microbiome of wild Penaeus monodon collected from six sites in the Philippines, with a view to detecting pathogenic forms. We employed a metagenomic approach via particle-associated nucleic acid isolation, sequence-independent single primer amplification, and pyrosequencing. Virome analysis of shrimp samples from different sites revealed distinct virome profiles, and hence significant differences in diversity, among the various sites based on number of OTUs, Shannon-Weaver Index, and Inverse Simpson Index. Sequences of key shrimp pathogens were detected such as the white spot syndrome virus (WSSV), and Penaeus stylirostris densovirus (PstDV). However, the patterns of distribution of the pathogenic viruses varied; whereas WSSV was found only in three out of six sites and PstDV were found in all but one site. The results also revealed shrimp-associated viruses that have not yet been observed in P. monodon such as avian virus-like, insect virus-like, plankton virus-like and bacteriophage-like sequences. Despite the diverse array of viruses detected in the study, a large proportion remains unidentified (i.e., similarity to sequences in the database was lower than the threshold required for definitive identification), and therefore could represent unexplored virus sequences and viral genomes in the environment.

In situ DIG-labeling, loop-mediated DNA Amplification (ISDL) for highly sensitive detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV)

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is also known as Penaeus stylirostris densovirus (PstDNV). Since it is listed as a reportable crustacean disease by the World Organization for Animal Health (OIE), many countries require testing for IHHNV in shrimp stocks imported for aquaculture, necessitating rapid and highly sensitive detection methods to guarantee and maintain absence of IHHNV in traded stocks. Here we describe a new, highly sensitive method for detection of IHHNV in shrimp tissues. It involves in situ loop-mediated DNA amplification (IS-LAMP) employing digoxigenin-11-dUTP (DIG-dUTP) as part of the deoxynucleotide triphosphate (dNTP) mix. This resulted in DIG-labeling of the DNA amplicons (i.e., in situ, DIG-labeling LAMP or ISDL) allowing direct in situ, immuno-histochemical detection of IHHNV using an anti-DIG antibody conjugated with alkaline phosphatase, and eliminating the lengthy hybridization step of conventional ISH. The target region for the method is at the 3'end of the IHHNV genome that has been reported least likely to give rise to endogenous viral elements (EVE) in the shrimp genome. When compared to the results for IHHN detection by conventional in situ hybridization (ISH) using a DIG-labeled oligonucleotide probe, signals from the ISDL method were stronger and also gave positive results with some tissues unreactive by the conventional ISH method. Additional comparison of the two methods revealed that the proteinase-K digestion step needed for normal ISH was unnecessary for ISDL. This further shortened the assay time and improved the tissue integrity necessary to clearly indicate the location of positive hybridization signals. The ISDL method gave no cross- reactions with tissues of shrimp infected with other common pathogens, and the total time needed to carry out ISDL was one-third of that needed for conventional ISH. Statement of relevanceThis work describes an in situ, DIG-labeling LAMP (ISDL) method, as a new, highly sensitive method for detection of IHHNV in shrimp tissues. The developed method can be used as a confirmatory test carried out with tissue sections to confirm the results from molecular assays, especially when immune-histochemical methods are not available to do so. ISDL is especially important for molecular detection from a new or unexpected location and for molecular detection in newly suspected carriers to determine whether they are actually infected or just mechanical carriers.

One base pair deletion and high rate of evolution: Keys to viral accommodation of Australian Penaeus stylirostris densovirus

Abstract Penaeus stylirostris densovirus ( Pst DV) has caused large economic losses to shrimp farming worldwide. Here, a 1-bp deletion within the nuclear localization signal-encoding sequence of open reading frame 1 (ORF1; NS1) of Australian Pst DV was identified as a cause of 199-amino acid shortened NS1 protein and production of a second protein, C-terminal NS1. This mutation is believed to reduce virulence as it strongly modifies the characteristics of NS1, which is responsible for the majority of enzymatic activities in Pst DV. This finding supports a hypothesis regarding accommodation of Pst DV in Australian prawns in relation to viral genetics. However, a high degree of evolution (1.55 × 10 − 3 substitutions/site/year) and genetic variation for the virus was attributable to the viral recombination observed with 10 potential genomic breakpoints identified. With this finding, we suggest that awareness of the emergence of new virulent strains should be increased as a preventative measure against future outbreaks of Pst DV in the Australian Indo-Pacific.

An improved validated SYBR green-based real-time quantitative PCR assay for the detection of the Penaeus stylirostris densovirus in penaeid shrimp

The Penaeus stylirostris densovirus (PstDV) (also known as infectious hypodermal and hematopoietic necrosis virus, IHHNV), one of the major shrimp pathogens, has a worldwide distribution in farmed and wild shrimp populations. Outbreaks of IHHNV have been associated with substantial economic losses which are accompanied by a negative social impact. Current diagnostic PCR tests may result in false-positive results as several parts of PstDV genome may be endogenized in the nuclear genome of the shrimp P. stylirostris. A one-step qPCR SYBR-Green based quantitative real-time polymerase chain reaction (qPCR) assay to detect different isolates of the IHHNV in shrimp samples was developed. The detection limit of the assay was 81 viral copies of targeted DNA per reaction. The specificity of the assay was evaluated by melting curve analysis, which showed that the IHHNV product generated a single melt peak at 81.4±0.044°C. The assay was more sensitive than conventional PCR. The standardized PCR was shown to be highly sensible, specific, robust, and reproducible, which makes it an economical and powerful tool for both diagnostic applications and general research of IHHNV.

Prevalence of the infectious hypodermal and hematopoietic necrosis virus in shrimp (Penaeus vannamei) broodstock in northwestern Mexico

The Penaeus stylirostris densovirus (PstDNV or IHHNV) is the smallest of the known shrimp viruses. It causes severe mortalities in juveniles and sub-adults of the blue shrimp Penaeus stylirostris, while specimens of the white shrimp Penaeus vannamei infected by this virus exhibit reduced growth rates and negative effects on the feed-conversion rate (FCR). To date, no descriptive epidemiological surveys on the prevalence of this virus in shrimp broodstock have been performed. In this study, the prevalence of IHHNV in broodstock of the white shrimp P. vannamei from hatcheries on the northwest of Mexico region was estimated. Prevalence vary across different regions from high (63%) to low (6%) in shrimp broodstock. Several factors, as transport of pathogens by human activities, or the absence or implementation of ineffective biosecurity measures, may explain the observed differences. To the best of our knowledge, the present study is the first to examine the prevalence of IHHNV on broodstock.

Separation of endogenous viral elements from infectious Penaeus stylirostris densovirus using recombinase polymerase amplification

Non-infectious Penaeus stylirostris densovirus (PstDV)-related sequences in the shrimp genome cause false positive results with current PCR protocols. Here, we examined and mapped PstDV insertion profile in the genome of Australian Penaeus monodon. A DNA sequence which is likely to represent infectious PstDV was also identified and used as a target sequence for recombinase polymerase amplification (RPA)-based approach, developed for specifically detecting PstDV. The RPA protocol at 37 °C for 30 min showed no cross-reaction with other shrimp viruses, and was 10 times more sensitive than the 309F/R PCR protocol currently recommended by the World Organization for Animal Health (OIE) for PstDV diagnosis. These features, together with the simplicity of the protocol, requiring only a heating block for the reaction, offer opportunities for rapid and efficient detection of PstDV.

Immunological-based assays for specific detection of shrimp viruses

Among shrimp viral pathogens, white spot syndrome virus (WSSV) and yellow head virus (YHV) are the most lethal agents, causing serious problems for both the whiteleg shrimp, Penaeus (Litopenaeus) vannamei, and the black tiger shrimp, Penaeus (Penaeus) monodon. Another important virus that infects P. vannamei is infectious myonecrosis virus (IMNV), which induces the white discoloration of affected muscle. In the cases of taura syndrome virus and Penaeus stylirostris densovirus (PstDNV; formerly known as infectious hypodermal and hematopoietic necrosis virus), their impacts were greatly diminished after the introduction of tolerant stocks of P. vannamei. Less important viruses are Penaeus monodon densovirus (PmDNV; formerly called hepatopancreatic parvovirus), and Penaeus monodon nucleopolyhedrovirus (PemoNPV; previously called monodon baculovirus). For freshwater prawn, Macrobrachium rosenbergii nodavirus and extra small virus are considered important viral pathogens. Monoclonal antibodies (MAbs) specific to the shrimp viruses described above have been generated and used as an alternative tool in various immunoassays such as enzyme-linked immunosorbent assay, dot blotting, Western blotting and immunohistochemistry. Some of these MAbs were further developed into immunochromatographic strip tests for the detection of WSSV, YHV, IMNV and PemoNPV and into a dual strip test for the simultaneous detection of WSSV/YHV. The strip test has the advantages of speed, as the result can be obtained within 15 min, and simplicity, as laboratory equipment and specialized skills are not required. Therefore, strip tests can be used by shrimp farmers for the pond-side monitoring of viral infection.

Bioinformatical analysis of nuclear localisation sequences in penaeid densoviruses

Nuclear location sequences (NLSs) link proteins to importation molecules for transportation into the nucleus. A bioinformatical search of the penaeid parvoviruses was undertaken to look for NLS. All three ORFs of Penaeus merguiensis densovirus (PmergDNV) have functional NLS whilst only the two non-structural proteins of Penaeus stylirostris densovirus (PstDNV) appear to. In PmergDNV, NS1 has a NLS similar to DNA helicase Q1, NS2 is similar to Dorsal and VP1 is similar to SV40 T-antigen signal. In PstDNV, NS2 has a NLS that is an unrecognised pattern unless it is a monopartite Chelsky signal whilst NS1 has both a Dorsal and minute virus of mouse signals. The capsid protein NLS of PstDNV is likely to be inefficient. Spawner isolated mortality virus has a NLS like DNA helicase Q1. These NLSs affect the nature of inclusion bodies seen with light microscopy, basophilic in PmergDNV; eosinophilic in PstDNV and the site of encapsidation, nuclear in PmergDNV; cytoplasmic in PstDNV as seen with TEM. Many possible NLSs in penaeid parvoviruses are homologues to those in eukaryotic organisms and need to be tested experimentally.

Endogenous Brevidensovirus-like elements in Cherax quadricarinatus: Friend or foe?

Nine endogenous Brevidensovirus-like elements (EBreVE) were identified in redclaw freshwater crayfish Cherax quadricarinatus from different sources suggesting that these elements are widespread in redclaw in northern Queensland, Australia. These endogenous virus-like elements shared nucleotide identities (70–100%) and amino acid similarities (34–100%) with infectious hypodermal and haematopoietic necrosis virus (IHHNV) scientifically classified as Penaeus stylirostris densovirus (PstDNV). They may not have originated from IHHNV genomes, but could be derived from another uncharacterised member of the genus Brevidensovirus that share nucleotide similarities with IHHNV. The most striking feature of EBreVEs was that in each case, the segment (portion) of viral sequences inserted into the host genomes was from the same region of the viral genome and most likely derived from non-structural protein regions of ancestral virus, but they cannot be assembled into one consensus sequence. The EBreVEs may be inserted into the redclaw genomes following chronic or persistent infection by a corresponding virus that may have occurred as multiple independent integration events years ago leading to the accumulation of several integrated elements in their genomes. Histological examination and polymerase chain reaction (PCR) suggested that these insertions may have a protective function to their host.

Persistence of Penaeus stylirostris densovirus delays mortality caused by white spot syndrome virus infection in black tiger shrimp (Penaeus monodon)

BackgroundPersistent infection of Penaeus stylirostris densovirus (PstDNV) (also called IHHNV) and its non-infectious inserts in the black tiger shrimp, Penaeus monodon (P. monodon) genome are commonly found without apparent disease. Here, we introduced the method of multiplex PCR in order to differentiate shrimp with viral inserts from ones with the infectious virus. The method allowed us to study the effect of pre-infection of IHHNV, in comparison to IHHNV inserts, on WSSV resistance in P. monodon.ResultsA multiplex PCR system was developed to amplify the entire IHHNV genome, ensuring the accurate diagnosis. Field samples containing IHHNV DNA templates as low as 20 pg or equivalent 150 viral copies can be detected by this method. By challenging the two groups of diagnosed shrimp with WSSV, we found that shrimp with IHHNV infection and those with viral inserts responded to WSSV differently. Considering cumulative mortality, average time to death of shrimp in IHHNV-infected group (day 14) was significantly delayed relative to that (day 10) of IHHNV-inserted group. Real-time PCR analysis of WSSV copy number indicated the lower amount of WSSV in the IHHNV-infected group than the virus-inserted group. The ratio of IHHNV: WSSV copy number in all determined IHHNV-infected samples ranged from approximately 4 to 300-fold.ConclusionThe multiplex PCR assay developed herein proved optimal for convenient differentiation of shrimp specimens with real IHHNV infection and those with insert types. Diagnosed shrimp were also found to exhibit different WSSV tolerance. After exposed to WSSV, the naturally pre-infected IHHNV P. monodon were less susceptible to WSSV and, consequently, survived longer than the IHHNV-inserted shrimp.

Genomics, Molecular Epidemiology and Diagnostics of Infectious hypodermal and hematopoietic necrosis virus

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is one of the major viral pathogens of penaeid shrimps worldwide, which has resulted in severe mortalities of up to 90% in cultured Penaeus (Litopenaeus) stylirostris from Hawaii and hence designated Penaeus stylirostris densovirus (PstDNV). IHHNV is distributed in shrimp culture facilities worldwide. It causes large economic loss to the shrimp farming industry. Our knowledge about the natural reservoirs of IHHNV is still scarce. Recent studies suggest that there is sufficient sequence variation among the isolates from different locations in Asia, suggesting multiple geographical strains of the virus. Four complete genomes and several partial sequences of the virus are available in the GenBank. Complete genome information would be useful for assessing the specificity of diagnostics for viruses from different geographical areas. Comparisons of complete genome sequences will help us gain insights into point mutations that can affect virulence of the virus. In addition, because of unavailability of shrimp cell lines for culturing IHHNV in vitro, quantification of virus is difficult. The recent progress in research regarding clinical signs, geographical distribution, complete genome sequence and genetic variation, transmission has made it possible to obtain information on IHHNV. A comprehensive understanding of IHHNV infection process, pathogenesis, structural proteins and replication is essential for developing prevention measures. To date, no effective prophylactic measure for IHHNV infection is available for shrimp to reduce its impact. This review provides an overview of key issues regarding IHHNV infection and disease in commercially important shrimp species.