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Yellow head virus (YHV) is a pathogen which causes high mortality in penaeid shrimp. Previous studies suggested that YHV enters shrimp cells via clathrin-mediated endocytosis. This research investigated the roles of clathrin adaptor protein 2 subunit β (AP-2β) from Penaeus monodon during YHV infection. PmAP2-β was continuously up-regulated more than twofold during 6-36 hpi. Suppression of PmAP2-β significantly reduced YHV copy numbers and delayed shrimp mortality. Quantitative RT-PCR revealed that knockdown of PmAP2-β significantly enhanced the expression level of PmSpätzle, a signaling ligand in the Toll pathway, by 30-fold at 6 and 12 hpi. Moreover, the expression levels of gene components in the Imd and JAK/STAT signaling pathways under the suppression of PmAP2-β during YHV infection were also investigated. Interestingly, anti-lipopolysaccharide factor isoform 3 (ALFPm3) was up-regulated by 40-fold in PmAP2-β knockdown shrimp upon YHV infection. In addition, silencing of PmAP2-β dramatically enhanced crustinPm1 expression in YHV-infected shrimp. Knockdown of ALFPm3 and crustinPm1 significantly reduced shrimp survival rate. Taken together, this work suggested that PmAP2-β-deficiency promoted the Toll pathway signalings, resulting in elevated levels of ALFPm3 and crustinPm1, the crucial antimicrobial peptides in defence against YHV.

In 2013, a unique seventh yellow head virus genotype (YHV7) was detected in Black Tiger shrimp (Penaeus monodon) broodstock that suffered high mortality following their capture from Joseph Bonaparte Gulf (JBG) in northern Australia. To assist with its diagnosis and assessment of its distribution, prevalence and pathogenicity, YHV7-specific TaqMan real-time qPCR and conventional nested PCR primer sets were designed to ORF1b gene sequences divergent from the other YHV genotypes. Using high (≥10 8 ) copies of plasmid (p)DNA controls containing ORF1b gene inserts of representative strains of YHV genotypes 1-7, both PCR tests displayed specificity for YHV7. Amplifications of serial 10-fold dilutions of quantified YHV7 pDNA and synthetic ssRNA showed that both tests could reliably detect 10 genome copies. Pleopods/gills from wild P. monodon sourced from locations in geographically disparate regions across northern Australia as well as 96 juveniles (48 either appearing normal or displaying signs of morbidity) from a commercial pond experiencing mortalities were screened to partially validate the diagnostic capacity of the qPCR test. Based on these data and PCR primer/probe sequence mismatches with other newly identified YHV genotypes, both YHV7-specific PCR tests should prove useful in the sensitive detection and discrimination of this genotype from YHV 2 (gill-associated virus) and YHV6 that can occur in Australian P. monodon, as well as from YHV genotypes currently listed as exotic to Australia., (Crown Copyright © 2019. Published by Elsevier B.V. All rights reserved.)

Genotype 8 of yellow head virus (YHV-8) was identified recently, but the complete genome sequence of this new genotype has not been reported. In this study, the complete genome of YHV-8 isolate 20120706 collected from Hebei Province of China in 2012 was sequenced. It was found to be 26,769 nucleotides (nt) in length, including a 20,060-nt open reading frame 1 (ORF1), a 435-nt ORF2, and a 4971-nt ORF3. Isolate 20120706 shared 79.7-83.9% nucleotide sequence identity with all seven of the complete genome sequences of YHV that have been reported so far. The topology of a phylogenetic tree constructed based on the ORF1b region clearly showed that strain 20120706, together with five other YHV-8 strains isolated in China, represents a new genotype of YHV. This is the first report of the complete genome sequence of a YHV-8 isolate, and the 20120706 isolate will be useful for further analysis of the epidemiology and evolution of YHV-8.

Unlabelled: A new genotype of yellow head virus (YHV), designated as YHV-8, was found in farmed shrimp Fenneropenaeus chinensis suffering suspectedly from EMS/AHPNS (early mortality disease/acute hepatopancreatic necrosis disease) in China in 2012. In this study, a one-step, real-time reverse-transcription loop-mediated isothermal amplification (rRT-LAMP) assay was developed for better detection of both genotypes of YHV-1 and YHV-8. A set of six specific primers was successfully designed targeting a conserved region of the YHV genome. The LAMP reaction was optimized to contain 8 mmol l(-1) Mg(2+) and 1·4 mmol l(-1) dNTPs, and to be performed at 58°C for 60 min. The detection sensitivity of the rRT-LAMP method was as low as 7 × 10(0)  copies per reaction. The specificity of the method was validated by the absence of any cross-reaction with the RNA samples extracted from other shrimp viruses (Taura syndrome virus, white spot syndrome virus, infectious hypodermal and haematopoietic necrosis virus, hepatopancreatic parvovirus) and specific pathogen-free (SPF) shrimp. The resulting standard curves showed high correlation coefficient values. Furthermore, the test of farm samples showed that YHV was detected in three of 111 Litopenaeus vannamei, six of eight Fenneropenaeus chinensis, five of 19 Macrobrachium rosenbergii and none of the nine Marsupenaeus japonicus. These results suggest that this assay is applicable widely as a new rapid and sensitive detection method in the research of YHV., Significance and Impact of the Study: In this study, we designate a new genotype of yellow head virus (YHV) as YHV genotype 8 (YHV-8) which was detected in diseased shrimp in China. A rapid, sensitive and specific rRT-LAMP detecting method for both YHV-8 and YHV-1 has been established. It is anticipated that this novel assay will be instrumental for diagnosis and surveillance of the virulent genotypes of YHV., (© 2016 The Society for Applied Microbiology.)

The synthesis of two comb-like dextran surfactant polymers, that are different in their dextran molecular weight (MW) distribution and the presence of carboxylic groups, and their characterization are reported. A bimodal carboxylic dextran surfactant polymer consists of poly(vinyl amine) (PVAm) backbone with carboxyl higher MW dextran, non-functionalized lower MW dextran and hydrophobic hexyl branches; while a monomodal dextran surfactant polymer is PVAm grafted with non-functionalized lower MW dextran and hexyl branches. Layer formation of non-covalently attached dextran chains with bimodal MW distributions on a surface plasmon resonance (SPR) chip was investigated from the perspective of mixed physisorption of the bimodal and monomodal surfactant polymers. Separation distances between the carboxylic longer dextran side chains within the bimodal surfactant polymer and between the whole bimodal surfactant molecules on the chip surface could be well-controlled. SPR analysis of shrimp yellow head virus using our mixed surfactant chips showed dependence on synergetic adjustment of these separation distances., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Yellow head virus (YHV) is a highly virulent pathogen that has caused severe mortality in cultivated shrimp (Penaeus monodon and Penaeus vannamei) in Thailand. There are several technologies that are applied to detect YHV for further control of the disease. RT-PCR is currently widely used in the laboratory, but it has some disadvantages related to cost, time-consuming and complexity. An alternative assay combines RT with loop-mediated isothermal amplification (LAMP) that not only provides high specificity, sensitivity and rapidity, but is also cheaper and more suitable for field applications in shrimp aquaculture than the RT-PCR. RT-LAMP is performed under isothermal conditions with a set of four to six primers designed to recognize six to eight distinct target sequences, and it has been combined with a chromatographic lateral-flow dipstick (LFD) to detect LAMP amplified product, which avoids the use of gel electrophoresis. In this study, RT-LAMP for the detection of YHV was developed by isothermal amplification at 65 °C for 45 min, followed by hybridization with an FITC-labeled DNA probe for 5 min and detected by LFD within 5 min (time required approximately 55 min, excluding RNA extraction and preparation time). The detection limit of RT-LAMP-LFD was 0.1 pg RNA extracted from shrimp infected with YHV equivalent to the nested RT-PCR, and no cross reaction was observed with other common shrimp viral pathogens. The LAMP method described in this study showed a rapid, high sensitivity and specificity and it is recommended as user-friendly for diagnosis of YHV in the field., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Salt-induced self-aggregation of gold nanoparticles (AuNP) carrying unisense ssDNA probes can be prevented specifically by complementary DNA. Loop-mediated isothermal amplification (LAMP) can amplify DNA rapidly. Here, the two techniques were combined to detect yellow head virus (YHV). The LAMP-AuNP method required 60 min for LAMP and 5 min for hybridization of LAMP products to an AuNP-labeled ssDNA probe followed by salt induced probe-particle aggregation to visualize color development. The detection sensitivity of the method was comparable to that of the commercial IQ2000™ nested RT-PCR but only required ~65 min to produce a result, and did not cross-detect other shrimp viruses. As the LAMP-AuNP protocol only requires a heating block, it offers opportunities for rapid detection of YHV., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A multiplex real-time PCR and high-resolution melting (HRM) analysis was developed to detect simultaneously three of the major viruses of penaeid shrimp including white spot syndrome virus (WSSV), yellow-head virus (YHV), and Penaeus monodon densovirus (PmDNV). Plasmids containing DNA/cDNA fragments of WSSV and YHV, and genomic DNAs of PmDNV and normal shrimp were used to test sensitivity of the procedure. Without the need of any probe, the products were identified by HRM analysis after real-time PCR amplification using three sets of viral specific primers. The results showed DNA melting curves that were specific for individual virus. No positive result was detected with nucleic acids from shrimp, Penaeus monodon nucleopolyhedrovirus (PemoNPV), Penaeus stylirostris densovirus (PstDNV), or Taura syndrome virus (TSV). The detection limit for PmDNV, YHV and WSSV DNAs were 40fg, 50fg, and 500fg, respectively, which was 10 times more sensitive than multiplex real-time PCR analyzed by agarose gel electrophoresis. In viral nucleic acid mixtures, HRM analysis clearly identified each virus in dual and triple infection. To test the capability to use this method in field, forty-one of field samples were examined by HRM analysis in comparison with agarose gel electrophoresis. For HRM analysis, 11 (26.83%), 9 (21.95%), and 4 (9.76%) were infected with WSSV, PmDNV, and YHV, respectively. Agarose gel electrophoresis detected lesser number of PmDNV infection which may due to the limit of sensitivity. No multiple infection was found in these samples. This method provides a rapid, sensitive, specific, and simultaneous detection of three major viruses making it as a useful tool for diagnosis and epidemiological studies of these viruses in shrimp and carriers., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A strip test for the dual detection of white spot syndrome virus (WSSV) and yellow head virus (YHV) was developed using monoclonal antibodies (MAbs) specific to the WSSV major envelope protein VP28 (W1 and W30) and the YHV nucleocapsid protein p20 (Y19 and Y21). The MAbs W30 and Y19 were conjugated with colloidal gold and sprayed onto a glass fiber pad that was placed adjacent to a sample chamber. The MAbs W1 and Y21 and the goat anti-mouse immunoglobulin G (GAM) antibody were sprayed onto a nitrocellulose membrane in strips at positions designated W, Y and C, respectively. These test strips were placed in plastic cases and stored desiccated in a plastic bag. The test strips were assessed for their ability to detect WSSV and YHV simultaneously using pleopods sampled from shrimp. A pleopod homogenate in application buffer 100μl was applied to the sample chamber to flow through the nitrocellulose membrane strip, and antibody-protein complexes could be observed within 15min. In sample from shrimp infected with WSSV and/or YHV, viral protein bound to the colloidal gold-conjugated MAbs. These complexes were captured by the MAbs at the W and/or Y test lines, resulting in the appearance of reddish-purple coloured bands. Any unbound colloidal gold-conjugated MAbs migrated pass the W and Y lines would be captured by the GAM antibody, forming a band at position C. When samples not containing WSSV and YHV proteins or containing viral proteins at below the detection limit of the test, only the band at position C was observed. The sensitivity of the test was comparable to dot blot tests using single MAbs, and ∼500-fold less sensitive than a 1-step PCR test for WSSV and 1000-fold less sensitive than an RT-PCR test for YHV. Despite this lower sensitivity, the dual strip test has advantages in speed and simplicity in not requiring sophisticated equipment or specialized skills. The ability to co-detect WSSV and YHV provides simultaneously cost savings., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Gill-associated virus (GAV) and Mourilyan virus (MoV) can occur at very high prevalence in healthy black tiger shrimp (Penaeus monodon) in eastern Australia, and both have been detected in moribund shrimp collected from mid-crop mortality syndrome (MCMS) outbreaks. Experimental evidence presented here indicates that GAV, but not MoV, is the cause of MCMS. Firstly, in healthy P. monodon used for experimental infections, pre-existing MoV genetic loads were very high (mean >10(9) viral RNA copies μg(-1) total RNA) and did not increase significantly following lethal challenge with an inoculum containing both GAV and MoV. In contrast, GAV genetic loads prior to challenge were low (mean ∼10(5) RNA copies μg(-1) total RNA) and increased >10(4)-fold in moribund shrimp. Secondly, dsRNAs targeted to the GAV RNA-dependent RNA polymerase (RdRp) or helicase gene regions reduced GAV genetic loads, delayed the onset of mortalities and improved survival following challenge. In contrast, dsRNA targeted to the MoV RdRp gene (L RNA) was highly effective in reducing MoV genetic loads, but mortality rates were unaffected. Targeting of the MoV S2 RNA, encoding a small non-structural protein (NSs2), a putative supressor of RNA interference, did not reduce the MoV genetic loads or enhance knockdown of GAV when administered simultaneously with dsRNA targeted to the GAV helicase gene. Overall, the data show that P. monodon can tolerate a high-level MoV infection and that mortalities are associated with GAV infection.

Gill-associated virus (GAV) was found to be associated with decreased prawn, Penaeus monodon, production when prawns from three farms (n = 45 ponds, 1800 prawns) were monitored for GAV over the production season using a graded RT-nPCR. The grading system used was a visualization of either the outer or inner nested PCR products. Prevalence and loading of GAV were associated with disease severity. Ponds with a higher initial prevalence and a larger increase in GAV load over the production period suffered disease outbreaks. Ponds with low initial prevalence of GAV but a larger increase in prevalence and large increase in load over the production period suffered chronic disease with no disease outbreak identified, yet low production. However, the ponds with moderate to low initial prevalence of GAV with a low increase in prevalence and load of GAV over the production period incurred no disease outbreak and comparatively high production. Ponds with GAV prevalence greater than 75% at 1 month post-stocking should be considered for termination as they have a high probability (95%) of having a disease outbreak. Emergency harvest when a disease outbreak occurs will significantly limit mortality losses., (© 2010 Blackwell Publishing Ltd.)

Viruses and viral diseases of crabs were observed and investigated earlier than the first observation of viruses in shrimp. In fact, crabs were used as biological models to investigate crustacean virology at the beginning of shrimp aquaculture development. More than 30 viruses have been reported in crabs, including those related to the known virus families Reoviridae, Bunyaviridae, Roniviridae and a group of Bacilliform enveloped nuclear viruses. This review reports data on several important viral diseases of crabs, particularly those associated with pathology of organs and tissues of commercially and ecologically significant host species., (Copyright © 2010. Published by Elsevier Inc.)

A real-time quantitative (q)RT-PCR employing consensus degenerate PCR primers was developed to detect all six genotypes known currently to comprise the yellow head virus (YHV) complex and found commonly in Penaeus monodon shrimp. The test primers possess only limited (eight-fold) degeneracy and target ORF1b gene sequences identified to be highly conserved amongst 57 strains of the six genotypes detected in P. monodon sourced from various regions of the Indo-Pacific. The qRT-PCR amplifies a 147bp sequence and analysis of dilutions of synthetic genotype 2 RNA showed it to be 99.8% efficient and capable of detecting as few as 2.5 RNA copies reliably. As the test detects all six YH-complex genotypes, is extremely sensitive, capable of quantifying infection loads, and amenable to high-throughput application, it should prove useful for managing infections in P. monodon broodstock and seedstock used for aquaculture., (Crown Copyright 2010. Published by Elsevier B.V. All rights reserved.)

A real-time reverse transcription loop-mediated isothermal amplification (real-time RT-LAMP) method was applied for detecting the replicase polyprotein-encoding gene of yellow head virus (YHV) in shrimp, Penaeus monodon. It is a novel, gene-specific assay that amplifies nucleic acid with high specificity, sensitivity and rapidity under isothermal conditions using a set of six specially designed primers that recognize eight distinct sequences of the target gene. This method works with even low copies of DNA and is based on magnesium pyrophosphate turbidity detection by an inexpensive photometer for quantitative analysis. A user-friendly protocol was developed with optimal conditions standardized at 63 degrees C for 60 min. With this protocol, the assay sensitivity was 10 times higher than the widely used YHV nested RT-PCR system. Cross-reactivity analysis using other shrimp virus DNA/cDNA and YHV-negative shrimp demonstrated high specificity of the assay. The real-time RT-LAMP method was performed also for an internal control gene, EF-1alpha, to compare with the expressions of the YHV gene in different organs of infected shrimp, and the resulting standard curves showed high correlation coefficient values. These results suggest that this assay is applicable widely as a new quantitative detection method in the pursuit of YHV-free shrimp culture.

Objective: To identify whether black tiger prawns (Penaeus monodon) in the Weipa region of the Gulf of Carpentaria, Queensland, are free of gill-associated virus (GAV) and Mourilyan virus (MoV), which are endemic in P. monodon along the east coast of Queensland., Procedure: Preliminary screening suggested that Weipa might be a source of P. monodon that are free of GAV and MoV. To assess this, more than 150 prawns captured near Weipa were maintained locally in tanks for 2 weeks and bled three times as a stressor to promote higher-level infections. The existence of GAV and MoV in lymphoid organ tissue was then determined using real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Some prawns were maintained in tanks for an additional 75 days before being tested., Results: Real-time qRT-PCR did not detect GAV in any of 33 pools of RNA isolated from the 166 prawns tested. MoV was detected in five pools of RNA at extremely low viral RNA copy numbers close to the sensitivity threshold of the test. MoV was also detected at a similar low copy number in one of nine pearl-oyster mantle samples used as negative controls., Conclusions: GAV infection is either absent or, like MoV, potentially present at a very low prevalence in juvenile P. monodon inhabiting the inshore waters at Weipa. This region can thus be recommended as a good source of P. monodon certifiable as specific pathogen-free for GAV and MoV, which is desirable for domestication and selective breeding programs in Australia.

Yellow-head virus (YHV) is a major pathogen in penaeid shrimps. We surveyed 13 crustacean species in eight families from two orders that are commonly found in the Mississippi coastal area and freshwater environments as potential reservoir or carrier hosts of YHV. Using semi-nested polymerase chain reaction (PCR) on relatively small sample sizes, we did not detect any natural infection. However, when the daggerblade grass shrimp, Palaemonetes pugio, and the blue crab, Callinectes sapidus, were exposed to YHV by injection and per os, YHV was detected in the tissue of P. pugio and in the hemolymph of C. sapidus when tested by semi-nested reverse transcription PCR (RT-PCR) and real time quantitative reverse transcription PCR (qRT-PCR). YHV replicated in P. pugio, causing 8% mortality (9/112) after injection, with the viral titer reaching a peak at 14days post-inoculation (dpi) and remaining detectable at 36dpi. The number of infected animals and viral load, however, were relatively low but the virus still remained infectious to penaeids when administered by feeding. When YHV was injected into P. pugio, in situ hybridization detected a positive response to it at 7dpi in connective tissue of hepatopancreas, muscle, and midgut. Viral RNA in injected C. sapidus remained at a low level for 3days, and it was not detected from 7dpi onwards. In fed C. sapidus, the viral RNA reached a peak at 3dpi and still detectable at 7dpi, but it became undetectable at 14 and 21dpi. These data suggest that P. pugio under some conditions could act as a reservoir host for YHV but that the blue crab could serve as a poor, short term carrier host only.

Yellow head virus (YHV) is a highly virulent pathogen of penaeid shrimp. An isolate obtained from Penaeus vannamei during a yellow head disease outbreak in February 2006 in Ratchaburi Province, Thailand was purified following passage in experimentally infected shrimp. SDS-PAGE of purified virions indicated that envelope glycoprotein gp116 in the Ratchaburi/2006 isolate was smaller and relatively less abundant than in the Chachoengsao/1998 YHV reference strain. The variant gp116 reacted poorly in immunoblots with a gp116 mouse monoclonal antibody and a rabbit anti-serum to a baculovirus-expressed, C-terminally truncated, [His](6)-tagged gp116 that reacted strongly with gp116 of the homologous Chachoengsao/1998 strain. The anti-gp116 polyclonal serum also failed to neutralise the infectivity of the Ratchaburi/2006 isolate in in-vivo assays conducted in P. vannamei, but effectively neutralised the infectivity of the reference strain. Sequence analysis of the approximately 6.0 kb structural protein gene region and 3'UTR of the Ratchaburi/2006 isolate indicated >99.9% overall nucleotide identity with the Chachoengsao/1998 strain. However, in Ratchaburi/2006 a deletion in ORF3, corresponding to 54 amino acids near the N-terminal signal peptidase cleavage site of gp116, resulted in the loss of six conserved cysteine residues and two predicted N-glycosylation sites. Analysis of this ORF3 region in 25 viruses representing each of the six genotypes in the yellow head complex identified this modified form of gp116 in two other virulent YHV isolates classified as genotype 1b. The data indicate that, although the deletion causes a significant structural deformation of gp116 which reduces its incorporation into virions and eliminates the major neutralisation sites, the virus remains highly infectious, virulent and fit for survival.

A consensus RT-nested (n)PCR is described that detects the six distinct genotypic variants in the yellow head virus (YHV) complex. The PCR primers targeted ORF1b gene regions more highly conserved amongst the reference strains of YHV (genotype 1) and gill-associated virus (GAV, genotype 2) and a set of 57 field isolates containing multiple representatives of each genotype. The test employed short PCR (359 bp) and nPCR (147 bp) amplicons to minimise the effects of RNA degradation. To ensure < or = 8-primer degeneracy, two primers were designed to each site, one accommodating sequence variations amongst genotype 1 isolates and the other variations amongst isolates of the other genotypes. The analytical sensitivity limits of the PCR and nPCR were estimated to be approximately 1250 and approximately 1.25 RNA copies, respectively. The superior group-specificity of the consensus RT-nPCR compared to other OIE-recommended PCR tests for YHV/GAV was demonstrated using RNA from 17 Penaeus monodon shrimp infected with representatives of each of the six genotypes. Phylogenetic analysis using the 94 nt ORF1b gene sequence spanned by the nPCR primers generated genotype assignments that were consistent with those obtained using the extended 671 nt sequence used for the initial identification of genotypes.

Penaeus monodon shrimp collected from across the Indo-Pacific region during 1997-2004 were screened for the presence of yellow head-related viruses. Phylogenetic analyses of amplified ORF1b gene segments identified at least six distinct genetic lineages (genotypes). Genotype 1 (YHV) was detected only in shrimp with yellow head disease. Genotype 2 (GAV) was detected in diseased shrimp with the less severe condition described as mid-crop mortality syndrome and in healthy shrimp from Australia, Thailand and Vietnam. Other genotypes occurred commonly in healthy shrimp. Sequence comparisons of structural protein genes (ORF2 and ORF3), intergenic regions (IGRs) and the long 3'-UTR supported the delineation of genotypes and identified both conserved and variant structural features. In putative transcription regulating sequences (TRSs) encompassing the sub-genomic mRNA 5'-termini, a core motif (5'-GUCAAUUACAAC-3') is absolutely conserved. A small (83 nt) open reading frame (ORF4) in the 3'-UTR of GAV is variously truncated in all other genotypes and a TRS-like element preceding ORF4 is invariably corrupted by a A>G/U substitution in the central core motif (5'-UU(G/U)CAAC-3'). The data support previous evidence that ORF4 is a non-functional gene under construction or deconstruction. The 3'-UTRs also contain predicted 3'-terminal hairpin-loop structures that are preserved in all genotypes by compensatory nucleotide substitutions, suggesting a role in polymerase recognition for minus-strand RNA synthesis.

A simple yellow head virus (YHV) "strip test" was developed using monoclonal antibody Y19 (against the p20 structural protein) conjugated with colloidal gold as the detector antibody. Rabbit anti-recombinant p20 (rp20) protein antibody was used as a capture antibody at the test line (T) and goat anti-mouse IgG antibody (GAM) was used as the capture antibody at the control line (C). The ready-to-use strip was housed in a plastic case for convenient application and stored in the desiccated plastic bag. A sample volume of 100 microl of either haemolymph or gill or appendage homogenates in application buffer was applied to the sample chamber at one end of the strip and allowed to flow by chromatography through the nitrocellulose membrane to the other end. In test samples containing YHV, the virus would bind to colloidal gold conjugated monoclonal antibody and the resulting complex would be captured by the rabbit anti-rp20 antibody at the test line to give a reddish-purple band. Any unbound monoclonal antibody conjugated with colloidal gold moved across the test line to be captured by the GAM to form a band at the control line (C). In the sample without YHV or below the limit of detection for the kit, only the control line was demonstrated. This method was about 500 times less sensitive than that of one-step RT-PCR, but slightly more sensitive than dot blotting. Therefore, it could be used for primary screening of individual shrimp or pooled shrimp samples to confirm high levels of YHV infection or YHV disease outbreaks. This kit can be used to detect gill associated virus (GAV) infection as well since the monoclonal antibody used in this kit cross-reacted well with GAV. The beneficial features of this kit are that simple, convenient, and rapid results that can be obtained without the requirement of sophisticated tools or special skills.

In order to assess the presence of yellow head virus (YHV) in shrimp farms along the Pacific coast of Mexico, 39 samples from 26 randomly chosen farms were analysed by means of reverse transcriptase-polymerase chain reaction (RT-PCR) and dot-blot hybridization. Eleven samples were positive for YHV. The disease was reproduced by means of an infectivity bioassay performed with an extract of pleopods from the positive samples. Cumulative mortality reached 50% in 14 days. Four pairs of primers which amplified several YHV genome regions were designed and used to test dead and surviving shrimp from the bioassay by RT-PCR, resulting in positive results for every expected amplicon. The results of this study provide presumptive evidence of the presence of YHV in Mexican shrimp farms at least during 1999-2000.

This study reports the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy between a reverse transcriptase-nested polymerase chain reaction (RT-nPCR) and an enzyme-linked immunosorbent assay (ELISA) for the detection of gill-associated virus (GAV) from a sample of 120 Penaeus monodon. Subsequently, the same comparisons were applied to the ELISA and haemagglutination (HA) assays for detection of GAV from a second 120 prawns. The optical density (OD) or dilution cut-off point had a direct influence on the tested parameters. The cut-off OD of 0.5-0.6 with the ELISA produced a sensitivity of 98% compared with RT-nPCR. However, these OD produced the lowest accuracy (85.8% and 86.7%, respectively). The OD cut off of 0.75 resulted in the highest accuracy (91.7%) and NPV (81.3%) while it had the second highest sensitivity (97%) and PPV (93.3%). However, the OD cut off of 0.9 had the highest specificity (80%). With regards to HA, the titre cut off at 8 resulted in the highest sensitivity, specificity and NPV (94%, 100% and 100%, respectively) compared with the ELISA, while the HA titre of 16 gave the highest accuracy (73%) and the second highest specificity (75%). A HA titre of 64 gave the highest PPV (81%). Using the RT-nPCR as the gold standard, the ELISA had an accuracy of 91.7% when using a cut off >0.75 as a positive result. When compared with the ELISA, the HA had an accuracy of 73% when using an HA titre cut off greater than 16 as a positive result. These results indicate that alternative tests for GAV (ELISA and HA) can be used to explore multiple questions about the disease status of P. monodon stocks in a cost-effective manner.