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.

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.

Prevalence and genomic analysis of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in Litopenaeus vannamei shrimp farmed in Shanghai, China.

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is prevalent among farmed shrimp and results in significant reductions in shrimp production. In order to gain a better understanding of the prevalence of IHHNV in the Litopenaeus vannamei shrimp population of Shanghai, China, samples were collected during two cultivation seasons and subjected to diagnostic PCR. The results of this study showed that 167 out of 200 shrimp were positive for IHHNV, indicating a high viral prevalence (83.5 %) in farmed shrimp populations. Our results also indicated that there was a moderate correlation between IHHNV prevalence and water temperature, salinity and pH and only a slight correlation with the concentration of dissolved oxygen (DO). A mathematical model was developed in order to predict the relationship between these four characteristics of water quality and IHHNV prevalence, ultimately resulting in an estimate of the best water quality criteria (IHHNV prevalence = 0) where T = 30 °C pH = 8.0, DO = 18.3 mg/L, and salinity = 1.5 ‰. Additionally, two IHHNV genotypes were identified, the sequencing of which revealed a high similarity to the known IHHNV genotypes based on a comparison of their nucleotide and amino acid sequences. Two types of repetitive sequences were detected at both the 5' and 3' ends of the non-coding regions, which are commonly found in other IHHNV genomic sequences. Phylogenetic analysis indicated that the IHHNV Shanghai genotypes were closely related to strains from Ganyu and Sheyang, but not to strains originating from Fujian, China. This finding suggests that IHHNVs have emerged independently several times in China.

High Occurrence of the Decapod Penstyldensovirus (PstDV1) Detected in Postlarvae of Penaeus vannamei Produced in Commercial Hatcheries of Mexico.

The decapod penstyldensovirus (PstDV1) is a widely spread shrimp pathogen that causes high mortalities in the shrimp Penaeus stylirostris, while in P. vannamei, it has been associated with induction of the runt deformity syndrome. Using shrimp post-larvae (PL, stages PL13-PL21) collected from 16 commercial hatcheries from Mexico, and a sensitive PCR protocol for its detection, a survey of the PstDV1 prevalence in larvae was undertaken. A high overall prevalence of PstDV1 (49.5 %) in shrimp PL from the studied hatcheries was found. This study reveals that PstDV1 occurs persistently in PL populations, which may have significant implications for its dispersal.

First complete genome of an Ambidensovirus; Cherax quadricarinatus densovirus, from freshwater crayfish Cherax quadricarinatus.

In 1999, the causative agent of an epizootic in Cherax quadricarinatus was described, and given the provisional name Cherax quadricarinatus parvovirus-like. Sequencing of the 6334 nt genome identified three open-reading frames on the top strand coding NS3 (35.55 kDa), NS1 (67.36 kDa) and NS2 (35.18 kDa) and on the bottom strand a single open reading frame which most likely encodes 4 structural proteins. Motifs characteristic of the Densovirinae were found in the ORFs. Phylogenetic analysis of the amino acids in NS1 places the genome in the genus Ambidensovirus, most closely related to the marine sea star densovirus (75%, E=0.0) and distantly related to Acheta domestica densovirus (44.1%). The virus name is proposed as species Decapod ambidensovirus, variant Cherax quadricarinatus densovirus. This is the first Ambidensovirus to be found in decapod crustaceans and the first of the subfamily Densovirinae to be sequenced from a freshwater crayfish. Cherax quadricarinatus densovirus and sea star densovirus are the first highly related Densovirinae to infect phylogenetically disparate hosts and are thus far, unique among the Densovirinae.

Development of an overlapping PCR method to clone the full genome of infectious hypodermal and hematopoietic necrosis virus (IHHNV).

Decapod Penstyldensovirus 1, previously named as infectious hypodermal and hematopoietic necrosis virus (IHHNV), is an economically important pathogen that causes shrimp diseases worldwide. However, a rapid method for cloning full-length IHHNV genome sequences is still lacking, which makes it difficult to study the genomics and molecular epidemiology of IHHNV. Here, a novel and rapid PCR technique was developed to determine the complete genomic sequences of IHHNV. The IHHNV genome was amplified in two overlapping fragments which each yielded a 2kb PCR product covering the first half or the second half of IHHNV genome, respectively. Using this method, six complete genomic sequences of IHHNV, which were collected from different regions of Zhejiang province in China, were cloned and sequenced successfully. The new cloning method will greatly facilitate the study on the genomics and molecular epidemiology of IHHNV.

Rapid detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) by real-time, isothermal recombinase polymerase amplification assay.

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) causes mortality or runt deformity syndrome in penaeid shrimps and is responsible for significant economic losses in the shrimp aquaculture industry. Here, we describe a novel real-time isothermal recombinase polymerase amplification (RPA) assay developed for IHHNV detection. Using IHHNV plasmid standards and DNA samples from a variety of organisms, we evaluated the ability of the IHHNV-RPA assay to detect IHHNV based on analysis of its sensitivity, specificity, rapidity, and reproducibility. Probit analysis of eight independent experimental replicates indicated satisfactory performance of the RPA assay, which is sufficiently sensitive to detect as few as 4 copies of the IHHNV genome within 7 min at 39 °C with 95 % reliability. Therefore, this rapid RPA method has great potential for applications, either in field use or as a point of care diagnostic technique.

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.

Development of duplex real-time PCR for the detection of WSSV and PstDV1 in cultivated shrimp.

BACKGROUND: The White spot syndrome virus (WSSV) and Penaeus stylirostris penstyldensovirus 1 (previously named Infectious hypodermal and hematopoietic necrosis virus-IHHNV) are two of the most important viral pathogens of penaeid shrimp. Different methods have been applied for diagnosis of these viruses, including Real-time PCR (qPCR) assays. A duplex qPCR method allows the simultaneous detection of two viruses in the same sample, which is more cost-effective than assaying for each virus separately. Currently, an assay for the simultaneous detection of the WSSV and the PstDV1 in shrimp is unavailable. The aim of this study was to develop and standardize a duplex qPCR assay for the simultaneous detection of the WSSV and the PstDV1 in clinical samples of diseased L. vannamei. In addition, to evaluate the performance of two qPCR master mixes with regard to the clinical sensitivity of the qPCR assay, as well as, different methods for qPCR results evaluation. RESULTS: The duplex qPCR assay for detecting WSSV and PstDV1 in clinical samples was successfully standardized. No difference in the amplification of the standard curves was observed between the duplex and singleplex assays. Specificities and sensitivities similar to those of the singleplex assays were obtained using the optimized duplex qPCR. The analytical sensitivities of duplex qPCR were two copies of WSSV control plasmid and 20 copies of PstDV1 control plasmid. The standardized duplex qPCR confirmed the presence of viral DNA in 28 from 43 samples tested. There was no difference for WSSV detection using the two kits and the distinct methods for qPCR results evaluation. High clinical sensitivity for PstDV1 was obtained with TaqMan Universal Master Mix associated with relative threshold evaluation. Three cases of simultaneous infection by the WSSV and the PstDV1 were identified with duplex qPCR. CONCLUSION: The standardized duplex qPCR was shown to be a robust, highly sensitive, and feasible diagnostic tool for the simultaneous detection of the WSSV and the PstDV1 in whiteleg shrimp. The use of the TaqMan Universal Master Mix and the relative threshold method of data analysis in our duplex qPCR method provided optimal levels of sensitivity and specificity.

Infectious hypodermal and hematopoietic necrosis virus from Brazil: Sequencing, comparative analysis and PCR detection.

A 3739 nucleotide fragment of Infectious hypodermal and hematopoietic necrosis virus (IHHNV) from Brazil was amplified and sequenced. This fragment contains the entire coding sequences of viral proteins, the full 3' untranslated region (3'UTR) and a partial sequence of 5' untranslated region (5'UTR). The genome organization of IHHNV revealed the three typical major coding domains: a left ORF1 of 2001 bp that codes NS1, a left ORF2 (NS2) of 1091 bp that codes NS2 and a right ORF3 of 990 bp that codes VP. Nucleotide and amino acid sequences of the three viral proteins were compared with putative amino acid sequences of viruses reported from different regions. Comparisons among genomes from different geographic locations reveal 31 nucleotide regions that are 100% similar, distributed throughout the genome. An analysis of secondary structure of UTR regions, revealed regions with high probability to form hairpins, that may be involved in mechanisms of viral replication. Additionally, a maximum likelihood analysis indicates that Brazilian IHHNV belongs to lineage III, in the infectious IHHNV group, and is clustered with IHHNV isolates from Hawaii, China, Taiwan, Vietnam and South Korea. A new nested PCR targeting conserved nucleotide regions is proposed to detect IHHNV.

Prevalence of viral pathogens WSSV and IHHNV in wild organisms at the Pacific Coast of Mexico.

This study investigated whether white spot syndrome virus and Infectious hypodermal and hematopoietic necrosis virus, can survive in wild invertebrates and vertebrates in the environment surrounding shrimp farms along the Pacific coast of Mexico. The evidences imply that both viruses have a potential of persisting in crabs, blue, white and brown shrimps. The most prevalent virus, IHHNV was present in 19.5% (344/1736) followed by WSSV in 3.6% (65/1736). Coinfection of WSSV and IHHNV was also detected in crabs, blue and white shrimps. This is the first prevalence report of WSSV and IHHNV associated with wildlife species in Mexico.

Natural occurrence of White spot syndrome virus and Infectious hypodermal and hematopoietic necrosis virus in Neohelice granulata crab.

White spot syndrome virus (WSSV) and Infectious hypodermal and hematopoietic necrosis virus (IHHNV) are two infectious agents associated to economic losses in shrimp aquaculture. As virus spread occurs through vectors and hosts, this study sought to verify the presence of WSSV and IHHNV in Neohelice granulata crab from Lagoa dos Patos estuary in Brazil and nearby shrimp farms. DNA extractions were performed with phenol/chloroform protocol. Molecular diagnosis was carried out by nested PCR for WSSV and one-step PCR for IHHNV. Results showed the presence of WSSV on crabs of both Lagoa dos Patos and farms, while IHHNV was found only on crabs collected in estuary. This is the first study to report IHHNV presence in N. granulata. Moreover, as analyzed crabs had no clinical symptoms or showed in situ mortality, we suggest its use as a bioindicator for virus occurrence in aquatic environments.

Characterization of the promoter elements of Bombyx mori bidensovirus nonstructural gene 1.

Bombyx mori Bidensovirus (BmBDV), a bipartite virus possesses two single-stranded linear DNAs (VD1 and VD2) and shows high pathogenic ability to Bombyx mori. Previous research found that the genes of nonstructural protein ns1 and ns2 were in the same transcript. To investigate the mechanism of transcriptional regulation of ns1 and ns2 genes, the 5'-flanking sequence (289 nt) of ns1 gene, encompasses the regions of the common terminal sequence (CTS) and the predicted P5 promoter from the 5'-terminus of the viral genome to the transcription initiation site of the ns1 gene was cloned and fused to the upstream of the luciferase reporter gene. The luciferase reporter assay showed that the 53 nt CTS of VD1 and VD2 can downregulate the activity of P5 by 13.3 %. The comparison in different cell lines showed that P5 possessed high promoter activity in BmN and Hi5 cell lines. Interestingly, P5 also had high activity in Hela cells, a kind of cancer cell of human. Subsequent truncated promoter analysis showed that the 31 nt (-236 to -206 nt) sequence is very important to P5 for the activity down to 36.5 % after deletion of it. While the activity also remained 26.5 % after the deletion of the TATA box, suggesting that the promoter is TATA independent. Moreover, in order to further understand the activity intensity of P5, a comparison with other three promoters, B. mori actin3 (Bm-actin3), B. mori nuclear polyhedrosis virus (BmNPV) immediate early 1 gene promoter (BmNPV-ie-1), and a synthetic promoter (3xP3) was carried out, the result indicated that the activity of P5 was weaker than that of anyone of them.

Organization of the ambisense genome of the Helicoverpa armigera Densovirus.

A natural densovirus (DNV) of a serious phytophagous pest, Helicoverpa armigera, was isolated. The genome of HaDNV contained 6,039 nucleotides (nt) and included inverted terminal repeats (ITRs) of 545 nt with terminal Y-shaped hairpins of 126 nt. Its DNA sequence and ambisense organization with four typical open reading frames (ORFs) demonstrated that it belonged to the genus Densovirus in the subfamily Densovirinae of the family Parvoviridae.

Complete nucleotide sequence analysis of a Korean strain of hepatopancreatic parvovirus (HPV) from Fenneropenaeus chinensis.

Hepatopancreatic parvovirus (HPV) of shrimp is distributed worldwide and the entire genome of Thailand and Indian strains (PmDNV) and one Australian strain (PmergDNV) have now been reported. The complete nucleotide sequence of a HPV strain isolated from the fleshy prawn Fenneropenaeus chinensis in Korea (FcDNV) was determined and compared to previously reported sequences. The entire genome of FcDNV contains 6,336 nucleotides, with 40% G+C content, which is the biggest of the known HPV strains. The HPV genome has three open reading frames (ORFs) with a slight overlap between the first and second ORFs. The three ORFs encode the NS2 and NS1 proteins and VP that consist of 425, 578, and 820 amino acids, respectively. Among the three proteins, the NS1 protein shows the highest sequence similarity to the NS1 protein of other known HPV strains, followed by the NS2 protein and the VP protein. Phylogenetic analyses showed that HPV can be grouped into three genotypes, as previously reported, and FcDNV can be grouped as genotype I, with HPV strains isolated in Madagascar and Tanzania. The nucleotide sequences of the noncoding regions at the 5'- and 3'-ends of the plus-strand genome showed a Y-shaped hairpin structure and simple hairpin structure, respectively.

Genomic sequence of infectious hypodermal and hematopoietic necrosis virus (IHHNV) KLV-2010-01 originating from the first Korean outbreak in cultured Litopenaeus vannamei.

Due to the need to track and monitor genetic diversity, the genome of the infectious hypodermal and hematopoietic necrosis virus (IHHNV) strain KLV-2010-01 in cultured Litopenaeus vannamei shrimp that originated from the first Korean outbreak in 2010 was sequenced and analyzed. The genome, with a length of 3914 nucleotides, was sequenced from the Korean IHHNV. The genome encoded three large and overlapping open reading frames: ORF1 (NS-1) of 2001 bp, ORF2 (NS-2) of 1092 bp and ORF3 (capsid protein) of 990 bp. The overall organization, size and predicted amino acid sequence of the three ORFs in Korean IHHNV were highly similar to those of members of the infectious IHHNV group, and the most closely related strains were IHHNVs described from Ecuador and Hawaii. Additionally, phylogenetic analysis showed that the Korean IHHNV was clustered with lineage III in the infectious IHHNV group and was most similar to IHHNV isolates from Ecuador, China and Taiwan.

Additional random, single to multiple genome fragments of Penaeus stylirostris densovirus in the giant tiger shrimp genome have implications for viral disease diagnosis.

Scattered reports of viral inserts in shrimp and insect genomes led to the hypothesis that random, autonomous insertion of such sequences occurs in these organisms and leads to specific, heritable immunity. To test the prediction regarding random insertion of viral sequences into the shrimp genome, we examined the giant tiger shrimp for random genomic insertions of Penaeus stylirostris densovirus (also called IHHNV). By PCR analysis using a set of 7 overlapping primer pairs to cover the whole IHHNV genome (4 kb), PCR failure with some pairs indicated sequence gaps that revealed a random pattern of putative viral inserts in the genomes of individual shrimp. Targeting a putative insert from one arbitrarily selected specimen, we used genome walking to reveal a viral insert linked to a host microsattelite-like fragment. This differed from 2 previously reported inserted fragments of IHHNV in P. monodon. In one specimen, 2 slightly different inserts were revealed, probably on paired chromosomes. By design and use of chimeric shrimp/virus primer pairs we proved that similar insertions occurred in several shrimp specimens, including those infected with IHHNV but showing no signs of disease. For the infected specimens, the inserts gave false positive PCR test results using 309F/R primers and a new IQ2000 test protocol currently recommended for detection of infectious IHHNV. This is the first experimental support for the hypothesis-based prediction that a random number and length of sequence fragments from a single virus genome may occur in the shrimp genome. Since some inserts can give false positive results for infectious IHHNV with the recommended methods above, they may have a negative effect on international seafood trade. In addition, discard of domesticated shrimp breeding stocks based on such false positive results might have negative consequences, if such inserts are related to shrimp viral disease tolerance, as also hypothesized.