Detection of white spot syndrome virus in seafood samples using a magnetosome-based impedimetric biosensor.

White spot syndrome virus (WSSV) is a significant threat to the aquaculture sector, causing mortality among crabs and shrimps. Currently available diagnostic tests for WSSV are not rapid or cost-effective, and a new detection method is therefore needed. This study demonstrates the development of a biosensor by functionalization of magnetosomes with VP28-specific antibodies to detect WSSV in seafood. The magnetosomes (1 and 2 mg/ml) were conjugated with VP28 antibody (0.025-10 ng/µl), as confirmed by spectroscopy. The magnetosome-antibody conjugate was used to detect the VP28 antigen. The binding of antigen to the magnetosome-antibody complex resulted in a change in absorbance. The magnetosome-antibody-antigen complex was then concentrated and brought near a screen-printed carbon electrode by applying an external magnetic field, and the antigen concentration was determined using impedance measurements. The VP28 antigen (0.025 ng/µl) bound more efficiently to the magnetosome-VP28 antibody complex (0.025 ng/µl) than to the VP28 antibody (0.1 ng/µl) alone. The same assay was repeated to detect the VP28 antigen (0.01 ng/µl) in WSSV-infected seafood samples using the magnetosome-VP28 antibody complex (0.025 ng/µl). The WSSV in the seafood sample was also drawn toward the electrode due to the action of magnetosomes controlled by the external magnetic field and detected using impedance measurement. The presence of WSSV in seafood samples was verified by Western blot and RT-PCR. Cross-reactivity assays with other viruses confirmed the specificity of the magnetosome-based biosensor. The results indicate that the use of the magnetosome-based biosensor is a sensitive, specific, and rapid way to detect WSSV in seafood samples.

Molecular identification of white spot syndrome virus (WSSV) and associated risk factors for white spot disease (WSD) prevalence in shrimp (Penaeus monodon) aquaculture in Bangladesh.

White spot disease has caused significant economic losses in the shrimp farming industry of Bangladesh over the last two decades. The responsible virus, WSSV, may show severe disease with significant mortality depending on farm management and environmental and seasonal changes. Data on farm management and environmental parameters were collected from the southwest region of Bangladesh in 2018, and WSSV infection was confirmed by the species-specific gene VP28 using conventional PCR, real-time PCR and sequencing. Through bivariate analysis, nine significant risk factors for WSD were identified, viz. farm age, presence of nursery pond, reservoir of PL, weed in farm area, control of weed, stocking density, stocking frequency, ammonia and oxygen concentration. This study detected 46 WSSV-infected shrimp farms by conventional PCR, whereas real-time PCR identified 47 WSSV-positive out of 49 farms. WSSV prevalence was highest in the Khulna region, with 100% positivity in all seasons. WSSV loads ranged from 5.62 × 109 to 2.01 × 1015 copies/g of shrimp tissue. The VP28 gene sequence confirmed that 15 representative samples were 100% identical to the 2018 WSSV strain of India. The relationships among risk factors, prevalence and severity of disease, and origin of WSSV strains could be impactful for WSD management.

Simultaneous detection of multiple bacterial and viral aquatic pathogens using a fluorogenic loop-mediated isothermal amplification-based dual-sample microfluidic chip.

Rapid and user-friendly diagnostic tests are necessary for early diagnosis and immediate detection of diseases, particularly for on-site screening of pathogenic microorganisms in aquaculture. In this study, we developed a dual-sample microfluidic chip integrated with a real-time fluorogenic loop-mediated isothermal amplification assay (dual-sample on-chip LAMP) to simultaneously detect 10 pathogenic microorganisms, that is Aeromonas hydrophila, Edwardsiella tarda, Vibrio harveyi, V. alginolyticus, V. anguillarum, V. parahaemolyticus, V. vulnificus, infectious hypodermal and haematopoietic necrosis virus, infectious spleen and kidney necrosis virus, and white spot syndrome virus. This on-chip LAMP provided a nearly automated protocol that can analyse two samples simultaneously, and the tests achieved limits of detection (LOD) ranging from 100 to 10-1  pg/µl for genomic DNA of tested bacteria and 10-4 to 10-5  pg/µl for recombinant plasmid DNA of tested viruses, with run times averaging less than 30 min. The coefficient of variation for the time-to-positive value was less than 10%, reflecting a robust reproducibility. The clinical sensitivity and specificity were 93.52% and 85.53%, respectively, compared to conventional microbiological or clinical methods. The on-chip LAMP assay provides an effective dual-sample and multiple pathogen analysis, and thus would be applicable to on-site detection and routine monitoring of multiple pathogens in aquaculture.

Detection of infectious hypodermal and hematopoietic necrosis virus and white spot syndrome virus in whiteleg shrimp (Penaeus vannamei) imported from Vietnam to South Korea.

In this study, whiteleg shrimp (Penaeus vannamei) imported from Vietnam were collected from South Korean markets, and examined for 2 viruses: infectious hypodermal and hematopoietic necrosis virus (IHHNV, recently classified as decapod penstyldensovirus-1), and white spot syndrome virus (WSSV). Among 58 samples, we detected IHHNV in 23 samples and WSSV in 2 samples, using polymerase chain reaction and sequencing analyses. This is the first report of IHHNV and WSSV detection in imported shrimp, suggesting that greater awareness and stricter quarantine policies regarding viruses infecting shrimp imported to South Korea are required.

Rapid, CRISPR-Based, Field-Deployable Detection Of White Spot Syndrome Virus In Shrimp.

Rapid, sensitive, point-of-care diagnostics are critical for managing infectious diseases. Here we adapt the CRISPR-based SHERLOCK method to develop a rapid, accurate, single copy detection assay for White Spot Syndrome Virus, the most devastating virus impacting global shrimp aquaculture. Further, we combine paper matrix nucleic acid extraction and lateral flow colorimetric reporting to create a fully field-deployable, next-generation diagnostic with potential to transform veterinary pathology, disease ecology, and animal production.

First detection of white spot syndrome virus (WSSV) in the mud shrimp Austinogebia edulis in Taiwan.

The white spot syndrome virus (WSSV) causes mass mortalities in the aquaculture of shrimps worldwide. The mud shrimp Austinogebia edulis (Ngoc-Ho & Chan, 1992) is an economically important sea food item occurring along the west coast of Taiwan. While the population of A. edulis began to decrease with some fluctuations in the last decade, the current study aims to discover the causes for such sporadic population decline. This study explores the effects of microbial pathogens and innate immunity on the populations of A. edulis. Here, we report firstly about WSSV infection of A. edulis from the coastal zone of western Taiwan which is one of the possible causes of population decrease of A. edulis in Shengang. However, WSSV infection is not the only reason for its population decrease because a similar infection rate of WSSV was found in Wangong. Population changes may be related to both environmental pollution stress and WSSV. Both factors likely caused a massive reduction of hemocytes and an abnormal increase of phenoloxidase and superoxide dismutase activity, which were spectrophotometrically measured. Since there is no effective way to treat WSSV infection, improving the coastal environment appears the most effective way to increase the population size of feral shrimps.

Establishment of pyrosequencing technology to detect White Spot Syndrome Virus (WSSV) in cultured aquatic animals.

This study aimed to establish pyrosequencing methods to detect white spot syndrome virus (WSSV). One pair of polymerase chain reaction (PCR) primers, and one pyrosequencing primer, were designed for WSSV. The pyrosequencing reaction system and conditions were optimized and a pyrosequencing method for detecting WSSV was successfully established. This method was able to specifically detect WSSV in eight viruses, with high sensitivity. The minimum detectable limit for nucleic acid was 23 copies/µL. The method was verified by detecting WSSV in 1881 batches of samples collected from domestic and imported shrimps. The detection results were more sensitive than conventional PCR. This research has therefore provided a new detection method for monitoring, and controlling aquatic animal virus diseases.

Medium optimization and characterization of cell culture system from Penaeus vannamei for adaptation of white spot syndrome virus (WSSV).

The lack of shrimp cell lines and difficulty in establishing shrimp cell culture systems, with an appropriate medium is a major concern in the aquaculture sector. The present study attempts to address this issue by developing an in vitro cell culture system from various tissues (hemocytes, heart, lymphoid tissue, hepatopancreas, gill, eye stalk, and muscle) of Penaeus vannamei (P.vannamei) using commercially available L-15 medium. The cell culture medium was formulated using five different media such as HBSCM-1, HBSCM-2, HBSCM-3, HBSCM-4, and HBSCM-5 containing L-proline and glucose with fetal bovine serum (FBS) supplements. Among the different media used, the HBSCM-5 medium with supplements showed good attachment and proliferation of cells with fibroblast-like, epithelioid, round, and adherent cell morphology in hemocyte culture. The same medium was further screened using different tissues to enhance the cell growth. The hemocytes, heart, and lymphoid tissue cells were passaged five times and maintained up to 20 days. Hepatopancreas and gill cells initially showed good morphological features and survived for more than ten days following subculture cells. Eye stalks and muscle cells perished within five days and did not show any unique morphology. The primary hemocyte cells were subjected to species identification, using cytochrome oxidase subunit I (COI) gene. To assess the primary hemocyte cell culture, cells were used for in vitro propagation of white spot syndrome virus (WSSV) and confirmed by the conventional polymerase chain reaction (PCR). Similarly, the primary cells were treated with bacterial extracellular products (ECPs) from Vibrio parahaemolyticus and Vibrio harveyi, to evaluate the cytotoxicity.

Multiple infections caused by white spot syndrome virus and Enterocytozoon hepatopenaei in pond-reared Penaeus vannamei in India and multiplex PCR for their simultaneous detection.

White leg shrimp, Penaeus vannamei, were collected on a monthly basis from grow-out ponds located at Tamil Nadu and Andhra Pradesh states along the east coast of India for screening of viral and other pathogens. Totally 240 shrimp samples randomly collected from 92 farms were screened for white spot syndrome virus (WSSV), infectious hypodermal and haematopoietic necrosis virus (IHHNV), infectious myonecrosis virus (IMNV) and Enterocytozoon hepatopenaei (EHP). The number of shrimp collected from shrimp farms ranged from 6 to 20 based on the body weight of the shrimp. All the shrimp collected from one farm were pooled together for screening for pathogens by PCR assay. Among the samples screened, 28 samples were WSSV-positive, one positive for IHHNV and 30 samples positive for EHP. Among the positive samples, four samples were found to be positive for both WSSV and EHP, which indicated that the shrimp had multiple infections with WSSV and EHP. This is the first report on the occurrence of multiple infections caused by WSSV and EHP. Multiplex PCR (m-PCR) protocol was standardized to detect both pathogens simultaneously in single reaction instead of carrying out separate PCR for both pathogens. Using m-PCR assay, naturally infected shrimp samples collected from field showed two prominent bands of 615 and 510 bp for WSSV and EHP, respectively.

Molecular variability and genetic structure of white spot syndrome virus strains from northwest Mexico based on the analysis of genomes.

White spot syndrome virus (WSSV) has a ∼300 kb double-stranded DNA genome. It originated in China, spread rapidly through shrimp farms in Asia, and subsequently to America. This study determined complete genome sequences for nine historic WSSV strains isolated from Pacific white shrimp (Litopenaeus vannamei) captured in farm ponds in northwest Mexico (Sinaloa and Nayarit). Genomic DNA was captured by an amplification method using overlapping long-range PCR and sequencing by Ion Torrent-PGM. Complete genome sequences were assembled (length range 255-290 kb) and comparative genome analysis with WSSV strains revealed substantial deletions (3 and 10 kb in two regions) in seven strains, with two strains differing from the rest. Phylogenetic analysis identified that the WSSV strains from the northern area of the state of Sinaloa clustered with strains from China (LC1, LC10, DVI) and Korea (ACF2, ACF4), while those from the southern region of the state of Nayarit (AC1 and JP) differed from both of those and from strains found in Taiwan and Thailand. Our data offer insights into the diversity of the WSSV genome in one country and their divergent origin, suggest that it entered Mexico via multiple routes and that specific genome regions can accommodate substantial deletions without compromising viability.

Silencing of HIF-1 in WSSV-infected white shrimp: Effect on viral load and antioxidant enzymes.

Hypoxia inducible factor-1 (HIF-1) is a transcriptional factor that induces genes involved in glucose metabolism. HIF-1 is formed by a regulatory α-subunit (HIF-1α) and a constitutive ß-subunit (HIF-1ß). The white spot syndrome virus (WSSV) induces a shift in glucose metabolism and oxidative stress. HIF-1α is associated with the induction of metabolic changes in tissues of WSSV-infected shrimp. However, the contributions of HIF-1 to viral load and antioxidant responses in WSSV-infected shrimp have been not examined. In this study, the effect of HIF-1 silencing on viral load and the expression and activity of antioxidant enzymes (superoxide dismutase-SOD, glutathione S-transferase-GST, and catalase) along with oxidative damage (lipid peroxidation and protein carbonyl) in tissues of white shrimp infected with the WSSV were studied. The viral load increased in hepatopancreas and muscle after WSSV infection, and the accumulative mortality was of 100% at 72 h post-infection. The expression and activity of SOD, catalase, and GST decreased in each tissue evaluated after WSSV infection. Protein carbonyl concentrations increased in each tissue after WSSV infection, while lipid peroxidation increased in hepatopancreas, but not in muscle. Silencing of HIF-1α decreased the WSSV viral load in hepatopancreas and muscle of infected shrimp along with shrimp mortality. Silencing of HIF-1α ameliorated the antioxidant response in a tissue-specific manner, which translated to a decrease in oxidative damage. These results suggest that HIF-1 is essential for restoring the antioxidant response, which counters the oxidative injury associated with WSSV infection.

Evaluation of the Sensitivity of the Flow Through Assay for detection of White Spot Syndrome Virus (WSSV) using a cocktail of monoclonal antibodies.

A panel of four monoclonal antibodies (C-05, C-14, C-38 and C-56) specific to VP28 of White spot syndrome virus (WSSV) were evaluated individually and in cocktail to increase sensitivity of the Flow Through Assay (FTA) for detection of the virus. Recombinant VP28 and semi purified WSSV was used as antigen for evaluation. Out of the total 11 cocktails and four individual of MAbs, 2 MAb cocktails C-05 + C-56 and C-14 + C-56 exhibited highest sensitivity in the FTA. The two MAb cocktail were 100 times more sensitive than 1-step PCR and nearly equivalent to 2-step PCR for the detection of WSSV. The detection limit of WSSV by MAb cocktail increased by two fold compared to the single MAb C-05 currently being used in (FTA).

Semiquantitative dot-blot immunogold assay for specific detection of white spot syndrome virus.

A dot-blot immunogold assay (DBIA) was developed to detect white spot syndrome virus (WSSV) using the polyclonal antibody VP26 (anti-VP26). The anti-VP26 was immobilized on gold nanoparticles (Ab-AuNPs), and a nitrocellulose membrane was used as a detection pad. When the target WSSV bound to the Ab-AuNPs a reddish dot appeared on the surface of the membrane used within 2-5 Min, which could be seen with the naked eye. The test was able to detect WSSV at concentrations as low as 105 copies µL-1 of WSSV. The DBIA developed had good specificity, and the colloidal gold probe can be applied within 2-3 days when stored at 4 °C. For real sample analysis, the DBIA was applied to samples of seawater used for shrimp cultivation without sample preparation. The results indicate that sample 1 showed a positive result, whereas samples 2 and 3 produced negative results. Then, samples 2 and 3 were spiked with WSSV for method validation. To confirm the performance of the DBIA developed, polymerase chain reaction (PCR) was conducted and the PCR results were the same as those found by the DBIA. Therefore, the DBIA developed could be applied for WSSV detection in real water samples.

Direct sequencing of the white spot syndrome virus from Brazil: Genome assembly and new insights on phylogeny.

White spot syndrome virus (WSSV) has been the cause of great economic losses in world shrimp farming. In this work the genome of a Brazilian WSSV isolate was determined from direct sequencing of total DNA extracted from an infected whiteleg shrimp, and assembled based on a chimera template approach. Comparisons between WSSV-BR and other isolates revealed that the Brazilian virus has a relatively small genome, and is very similar to isolates from Thailand and Mexico. A phylogenetic relationship using different approaches has demonstrated that these isolates share a common evolutionary history. An analysis of conflicting phylogenetic signals also considering genomes of other isolates revealed that the evolutionary history of WSSV may be related to recombination events. We observed that these events can also be traced at some level by analyzing the homologous regions in the WSSV genome. The existence of recombination events introduces a new point of view that must be considered in the evolutionary history of WSSV.

An optimized double-antibody sandwich ELISA for quantitative detection of WSSV in artificially infected crayfish.

Developing a rapid, accurate and quantitative method for detecting white spot syndrome virus (WSSV) is extremely urgent and critical for reducing the risk of white spot disease outbreaks. In the present work, an optimized double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for quantitative detection of WSSV. The method employed rabbit polyclonal antibodies against WSSV as the capture antibody and previously produced anti-WSSV monoclonal antibodies as the detector antibody. A standard curve of the log concentration of WSSV versus OD value was established, which was linear in the concentration range of 120-7680ng/mL, and the linear regression equation was y=0.166x-0.151. Viral proteins in different tissues of crayfish (Procambarus clarkia) post artificial infection with WSSV were quantitatively measured using the DAS-ELISA. WSSV proliferated quickly within 60h post infection and gradually slowed down afterwards. According to the linear regression relationship, the viral proteins in hemolymph, gut and gonad were firstly able to be quantified at 24h post infection with the concentrations of 186, 158 and 128ng/mL, respectively. These three tissues also contained higher viral proteins than the gill, heart, hepatopancreas and muscle during the entire infection period. The viral protein concentration in gut reached the highest level of 6220ng/mL at 72h post infection. Real time quantitative PCR was also used to detect the dynamic change of viral copies in crayfish hemolymph post WSSV infection, with similar results for both assays. The developed DAS-ELISA could detect WSSV propagation from initial to moribund stage in infected crayfish and demonstrated potential application for diagnosis of WSSV.

Quantitative risk assessment of WSSV transmission through partial harvesting and transport practices for shrimp aquaculture in Mexico.

This quantitative risk assessment provided an analytical framework to estimate white spot syndrome virus (WSSV) transmission risks in the following different scenarios: (1) partial harvest from rearing ponds and (2) post-harvest transportation, assuming that the introduction of contaminated water with viral particles into shrimp culture ponds is the main source of viral transmission risk. Probabilities of infecting shrimp with waterborne WSSV were obtained by approaching the functional form that best fits (likelihood ratio test) published data on the dose-response relationship for WSSV orally inoculated through water into shrimp. Expert opinion defined the ranges for the following uncertain factors: (1) the concentrations of WSSV in the water spilled from the vehicles transporting the infected shrimp, (2) the total volume of these spills, and (3) the dilution into culture ponds. Multiple scenarios were analysed, starting with a viral load (VL) of 1×102mL-1 in the contaminated water spilled that reached the culture pond, whose probability of infection of an individual shrimp (Pi) was negligible (1.7×10-7). Increasing the VL to 1×104.5mL-1 and 1×107mL-1 yielded results into very low (Pi=5.3×10-5) and high risk (Pi=1.6×10-2) categories, respectively. Furthermore, different pond stocking density (SD) scenarios (20 and 30 post-larvae [PL]/m2) were evaluated, and the probability of infection of at least one out of the total number of shrimp exposed (PN) was derived; for the scenarios with a low VL (1×102mL-1), the PN remained at a negligible risk level (PN, 2.4×10-7 to 1.8×10-6). For most of the scenarios with the moderate VL (1×104.5mL-1), the PN scaled up to a low risk category (PN, 1.1×10-4 to 5.6×10-4), whereas for the scenarios with a high VL (1×107mL-1), the risk levels were high (PN, 2.3×10-2 to 3.5×10-2) or very high (PN, 1.1×10-1 to 1.6×10-1) depending on the volume of contaminated water spilled in the culture pond (VCWSCP, 4 or 20L). In the sensitivity analysis, for a SD of 30 PL/m2, it was shown that starting with a VL of 1×105mL-1 and a VCWSCP of 12L, the PN was moderate (1.05×10-3). This was the threshold for greater risks, given the increase in either the VCWSCP or VL. These findings supported recommendations to prevent WSSV spread through more controlled transportation and partial harvesting practices.

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.

An Elegant Analysis of White Spot Syndrome Virus Using a Graphene Oxide/Methylene Blue based Electrochemical Immunosensor Platform.

White spot syndrome virus (WSSV) is a major devastating virus in aquaculture industry. A sensitive and selective diagnostic method for WSSV is a pressing need for the early detection and protection of the aquaculture farms. Herein, we first report, a simple electrochemical immunosensor based on methylene blue dye (MB) immobilized graphene oxide modified glassy carbon electrode (GCE/GO@MB) for selective, quick (35 ± 5 mins) and raw sample analysis of WSSV. The immunosensor was prepared by sequential modification of primary antibody, blocking agent (bovine serum album), antigen (as vp28 protein), secondary antibody coupled with horseradish peroxidase (Ab2-HRP) on the GCE/GO@MB. The modified electrode showed a well-defined redox peak at an equilibrium potential (E1/2), -0.4 V vs Ag/AgCl and mediated H2O2 reduction reaction without any false positive result and dissolved oxygen interferences in pH 7 phosphate buffer solution. Under an optimal condition, constructed calibration plot was linear in a range of 1.36 × 10-3 to 1.36 × 107 copies µL-1 of vp28. It is about four orders higher sensitive than that of the values observed with polymerase chain reaction (PCR) and western blot based WSSV detection techniques. Direct electrochemical immunosensing of WSSV in raw tissue samples were successfully demonstrated as a real sample system.

Lateral flow assay for rapid detection of white spot syndrome virus (WSSV) using a phage-displayed peptide as bio-recognition probe.

White spot disease caused by the white spot syndrome virus (WSSV) has a major socio-economic impact on shrimp farming in India. It has been realized that a field-usable diagnostic capable of rapid detection of WSSV can prevent huge economic losses in disease outbreaks. In this work, we explored the possibility of using a peptide as bio-recognition probe in a field-usable device for the detection of WSSV from infected shrimps and prawns. A commercially available random phage-display library was screened against rVP28 (a major structural protein of WSSV, expressed as a recombinant protein in Escherichia coli). A bacteriophage clone VP28-4L was obtained, and its binding to purified rVP28 protein as well as WSSV from infected shrimp Litopaeneus vannamei tissue was confirmed by ELISA and western blot. The apparent equilibrium dissociation constant (Kd,app) was calculated to be 810 nM. VP28-4L did not show cross-reactivity with any other shrimp viruses. A 12-mer peptide (pep28, with the sequence 'TFQAFDLSPFPS') displayed on the VP28-4L was synthesized, and its diagnostic potential was evaluated in a lateral flow assay (LFA). Visual detection of WSSV could be achieved using biotinylated-pep28 and streptavidin-conjugated gold nanoparticles. In LFA, 12.5 µg/mL of the virus could be detected from L. vannamei gill tissue homogenate within 20 min. Pep28 thus becomes an attractive candidate in bio-recognition of WSSV in field-usable diagnostic platforms benefitting the aquaculture sector.