Polymeric enteral diets as primary treatment of active Crohn's disease: a prospective steroid controlled trial.

Some insects use endogenous reverse transcriptase (RT) to make variable viral copy DNA (vcDNA) fragments from viral RNA in linear (lvcDNA) and circular (cvcDNA) forms. The latter form is easy to extract selectively. The vcDNA produces small interfering RNA (siRNA) variants that inhibit viral replication via the RNA interference (RNAi) pathway. The vcDNA is also autonomously inserted into the host genome as endogenous viral elements (EVE) that can also result in RNAi. We hypothesized that similar mechanisms occurred in shrimp. We used the insect methods to extract circular viral copy DNA (cvcDNA) from the giant tiger shrimp (Penaeus monodon) infected with a virus originally named infectious hypodermal and hematopoietic necrosis virus (IHHNV). Simultaneous injection of the extracted cvcDNA plus IHHNV into whiteleg shrimp (Penaeus vannamei) resulted in a significant reduction in IHHNV replication when compared to shrimp injected with IHHNV only. Next generation sequencing (NGS) revealed that the extract contained a mixture of two general IHHNV-cvcDNA types. One showed 98 to 99% sequence identity to GenBank record AF218266 from an extant type of infectious IHHNV. The other type showed 98% sequence identity to GenBank record DQ228358, an EVE formerly called non-infectious IHHNV. The startling discovery that EVE could also give rise to cvcDNA revealed that cvcDNA provided an easy means to identify and characterize EVE in shrimp and perhaps other organisms. These studies open the way for identification, characterization and use of protective cvcDNA as a potential shrimp vaccine and as a tool to identify, characterize and select naturally protective EVE to improve shrimp tolerance to homologous viruses in breeding programs.

A novel dual CRISPR-Cas assay for detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in penaeid shrimp without false positives from its endogenous viral elements (EVEs)

BackgroundShrimp have the ability to accommodate viruses in long term, persistent infections without signs of disease. Endogenous viral elements (EVE) play a role in this process probably via production of negative-sense Piwi-interacting RNA (piRNA)-like fragments. These bind with Piwi proteins to dampen viral replication via the RNA interference (RNAi) pathway. We searched a genome sequence (GenBank record JABERT000000000) of the giant tiger shrimp (Penaeus monodon for the presence of EVE related to a shrimp parvovirus originally named infectious hypodermal and hematopoietic necrosis virus (IHHNV).ResultsThe shrimp genome sequence contained three piRNA-like gene clusters containing scrambled IHHNV EVE. Two clusters were located distant from one another in pseudochromosome 35 (PC35). Both PC35 clusters contained multiple sequences with high homology (99%) to GenBank records DQ228358 and EU675312 that were both called “non-infectious IHHNV Type A” (IHHNV-A) when originally discovered. However, our results and those from a recent Australian P. monodon genome assembly indicate that the relevant GenBank records for IHHNV-A are sequence-assembly artifacts derived from scrambled and fragmental IHHNV-EVE. Although the EVE in the two PC35 clusters showed high homology only to IHHNV-A, the clusters were separate and distinct with respect to the arrangement (i.e., order and reading direction) and proportional content of the IHHNV-A GenBank records. We conjecture that these 2 clusters may constitute independent allele-like clusters on a pair of homologous chromosomes. The third EVE cluster was found in pseudochromosome 7 (PC7). It contained EVE with high homology (99%) only to GenBank record AF218266 with the potential to protect shrimp against current types of infectious IHHNV. One disadvantage was that some EVE in PC7 can give false positive PCR test results for infectious IHHNV.ConclusionsOur results suggested the possibility of viral-type specificity in EVE clusters. Specificity is important because whole EVE clusters for one viral type would be transmitted to offspring as collective hereditary units. This would be advantageous if one or more of the EVE within the cluster were protective against the disease caused by the cognate virus. It would also facilitate gene editing for removal of non-protective EVE clusters or for transfer of protective EVE clusters to genetically improve existing shrimp breeding stocks that might lack them.

Infection with infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a crustacean disease that caused large-scale mortality in Penaeus stylirostris, deformity and growth retardation in Penaeus vannamei and Penaeus monodon. We surveyed the presence of IHHNV in three major shrimp-producing regions in Ecuador, namely Guayas, El Oro, and Esmeralda. The data show that IHHNV is endemic (3.3–100% prevalence) to shrimp farms in these regions. The whole genome sequences of representative circulating IHHNV genotypes in Ecuador and Peru showed that these genotypes formed a separate cluster within the Type II genotypes and were divergent from other geographical isolates of IHHNV originating in Asia, Africa, Australia, and Brazil. In experimental bioassays using specific pathogen-free (SPF) P. vannamei, P. monodon, and P. stylirostris and representative IHHNV isolates from Ecuador and Peru, the virus did not cause any mortality or induce clinical signs in any of the three penaeid species. Although IHHNV-specific Cowdry type A inclusion bodies were histologically detected in experimentally challenged P. vannamei and P. monodon and confirmed by in situ hybridization, no such inclusions were observed in P. stylirostris. Moreover, P. vannamei had the highest viral load, followed by P. monodon and P. stylirostris. Based on IHHNV surveillance data, we conclude that the currently farmed P. vannamei lines in Ecuador are tolerant to circulating IHHNV genotypes. The genome sequence and experimental bioassay data showed that, although the currently circulating genotypes are infectious, they do not induce clinical lesions in the three commercially important penaeid species. These findings suggest a potentially evolving virus-host relationship where circulating genotypes of IHHNV co-exist in equilibrium with P. vannamei raised in Peru and Ecuador.

The immune response of Caspase-3 gene in infectious hypodermal and hematopoietic necrosis virus (IHHNV) infection of Litopenaeus vannamei

Over the past four decades, shrimp aquaculture has turned into a major industry providing jobs for millions of people worldwide especially in countries with large coastal boundaries. While the shrimp industry continues to expand, the sustainability of shrimp aquaculture has been threatened by the emergence of diseases. Diseases caused by single-stranded DNA containing viruses, such as infectious hypodermal and hematopoietic necrosis virus (IHHNV) and hepatopancreatic parvovirus (HPV), have caused immense losses in shrimp aquaculture since the early 1980s. In fact, the disease outbreak in the blue shrimp (Penaeus stylirostris) caused by IHHNV in early 1980s ultimately led to the captive breeding program in shrimp being shifted from P. stylirostris to the white shrimp (Penaeus vannamei), and today P. vannamei is the preferred cultured shrimp species globally. To date, four single-stranded DNA viruses are known to affect shrimp; these include IHHNV, HPV, spawner-isolated mortality virus (SMV) and lymphoidal parvo-like virus (LPV). Due to the economic losses caused by IHHNV and HPV, most studies have focused on these two viruses, and only IHHNV is included in the OIE list of Crustacean Diseases. Hence this review will focus on IHHNV and HPV. IHHNV and HPV virions are icosahedral in morphology measuring 20-22nm in size and contain a single-stranded DNA (ssDNA) of 4-6kb in size. Both IHHNV and HPV are classified into the sub-order Brevidensoviruses, family Densovirinae. The genome architecture of both viruses are quite similar as they contain two completely (as in IHHNV) or partially overlapping (as in HPV) non-structural and one structural gene. Histopathology and polymerase chain reaction (PCR)-based methods are available for both viruses. Currently, there is no anti-viral therapy for any viral diseases in shrimp. Therefore, biosecurity and the use of genetically resistant lines remains as the corner stone in the management of viral diseases. In recent years, gene silencing using the RNA interference (RNAi) approach has been reported for both IHHNV and HPV via injection. However, the delivery of RNAi molecules via oral route remains a challenge, and the utility of RNAi-based therapy has yet to be materialized in shrimp aquaculture.

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is one of the most important pathogens infecting penaeid shrimp and causes huge economic losses in the shrimp culture industry worldwide. Because no commercial vaccine is yet available, the most effective way of containing the disease is by the routine screening of juveniles and adults for the presence or absence of the virus. Therefore, our goal is to establish a simple and rapid examination system for infectious hypodermal and hematopoietic necrosis virus in places such as shrimp ponds. Two detection methods, real-time PCR and loop-mediated isothermal amplification (LAMP), were developed for IHHNV diagnosis, and then their specificity and sensitivity were compared in the study. Using the real-time PCR method, the assay had a detection limit of six copies of DNA template of IHHNV, and had a correlation coefficient of 0.99521 between template concentration and threshold cycle value at the template concentration of 6.038×104 to 6.038×109cps/mL. Furthermore, the approach had no signal response to genomic DNA of white spot syndrome virus and shrimp. The result revealed that the detection method had high specificity and sensitivity for IHHNV detection. However, the costly real-time thermal cycler and technically demanding were deemed to be not appropriate for IHHNV detection in field conditions. LAMP was a novel, sensitive and rapid detection technique and could be applied for disease diagnosis in aquaculture. Here, a set of four primers was designed using PrimerExplorer V4 software by targeting the IHHNV genome DNA, and used to develop the LAMP method for IHHNV detection. Using the detection system, target DNA was amplified and visualized on agarose gel within 60min under isothermal condition at 64℃. Also, the LAMP amplicon was observed directly in the reaction tube by addition of SYBR Green I for a naked-eye inspection. Sensitivity assay showed that the method also had a detection limit of six copies of DNA template of IHHNV. Moreover, genomic DNA of white spot syndrome virus and shrimp were unable to be detected within 60min using the LAMP method. Overall, these data revealed that the LAMP method had an equivalent to the real-time PCR method in specificity and sensitivity for IHHNV detection. Considering that the LAMP method had great advantage in its performance and low cost, this technique was more suitable for IHHNV detection in field conditions. Therefore, the LAMP method could be applied routinely to check the shrimp in hatcheries and growout ponds, so that virus carrying shrimp could be found during the early infection stages and counter measures could be devised before the infection becomes epizootic.

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) can cause mass mortalities in western blue shrimp Penaeus stylirostris, runt deformity syndrome in Pacific white shrimp P. vannamei and scalloped abdominal shell deformities in black tiger shrimp P. monodon. In P. monodon, however, PCR-based diagnosis of IHHNV can be complicated by the presence of a chromosome-integrated, non-replicating endogenous viral element (EVE). To facilitate high-throughput screening of P. monodon for IHHNV infection and/or EVE sequences, here we report real-time PCR tests designed to specifically detect IHHNV Lineage I, II and III but not EVE Type A sequences and vice versa. Using 108 dsDNA copies of plasmid (p)DNA controls containing either IHHNV or EVE-Type A sequences, both tests displayed absolute specificity. The IHHNV-q309 PCR reliably detected down to ≤10 copies of pDNA, at which levels a 309F/R PCR amplicon was just detectable, and the presence of an IHHNV-EVE sequence did not significantly impact its sensitivity. The IHHNV-qEVE PCR was similarly sensitive. Testing of batches of P. monodon clinical samples from Vietnam/Malaysia and Australia identified good diagnostic concordance between the IHHNV-q309 and 309F/R PCR tests. As expected for a sequence integrated into host chromosomal DNA, IHHNV-qEVE PCR Ct values were highly uniform among samples from shrimp in which an EVE was present. The highly specific and sensitive IHHNV-q309 and IHHNV-qEVE real-time PCR tests described here should prove useful for selecting broodstock free of IHHNV infection and in maintaining breeding populations of P. monodon specific pathogen free for IHHNV, and if desired, also free of IHHNV-EVE sequences.

Nucleotide sequence variations of a 2.9 kb fragment of infectious hypodermal and hematopoietic necrosis virus (IHHNV) isolated from samples of Penaeus monodon were determined and compared with an isolate from Hawaii. The infection characteristics of these isolates were examined by histology, in situ hybridization, and laboratory challenge studies with P. vannamei. Isolates of IHHNV were obtained from samples collected from the SE Asia region (the Philippines, Thailand, and Taiwan). Isolates of putative IHHNV were obtained from African samples (Tanzania, Madagascar, and Mauritius). The Philippine isolate had a very high nucleotide sequence identity (99.8%) to Hawaii IHHNV. The Thailand isolate showed a slightly lower identity (96.2%). The putative IHHNV sequences collected from Tanzania and Madagascar showed greater divergence from Hawaii IHHNV, 8.2% difference for Tanzania and 14.1% difference for Madagascar. A phylogenetic analysis showed that the Philippine IHHNV clustered with IHHNV found in the western hemisphere. This supports the theory that the Philippines was the origin of IHHNV that was first detected in Hawaii. In the laboratory infection study, both the Philippine and Thailand IHHNV were passed into P. vannamei, and the infected shrimp did not suffer any mortalities. In another laboratory infection, P. vannamei injected with a tissue homogenate of P. monodon from Madagascar, which tested positive for IHHNV by PCR, did not demonstrate IHHNV infection, suggesting that this putative IHHNV is not infectious to P. vannamei.

We developed a PCR assay that can detect infectious hypodermal and hematopoietic necrosis virus (IHHNV) but that does not react with IHHNV-related sequences in the genome of Penaeus monodon from Africa and Australia. IHHNV is a single-stranded DNA virus that has caused severe mortality and stunted growth in penaeid shrimp. Recently, IHHNV-related sequences were found in the genome of some stocks of P. monodon from Africa and Australia. These virus-related sequences have a high degree of similarity (86 and 92% identities in nucleotide sequence) to the viral genome, which has often generated false-positive reactions during PCR screening of these stocks. For this assay, a pair of IHHNV primers (IHHNV309F/R) was selected. The sequences of these primers match (100% of nucleotides) the target sequence in IHHNV, but mismatch 9 or 12 nucleotides of the genomic IHHNV-related sequences. This PCR assay was tested with various IHHNV isolates and with a number of samples of shrimp DNA that contained IHHNV-related sequences. This assay can reliably distinguish IHHNV DNA from shrimp DNA: it only detects IHHNV. Also, this pair of primers was included in a duplex PCR to detect IHHNV and simultaneously determine the presence of an IHHNV-related sequence. Using these primers, the PCR assay has a sensitivity equivalent to a PCR assay commonly used for detecting IHHNV in Litopenaeus vannamei, and can be used for routine detection.

Prevalence of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in Penaeus vannamei cultured in northeastern Brazil

The main purpose of this report is to provide hard evidence that the shrimp parvovirus, infectious hypodermal and hematopoietic necrosis virus (IHHNV), has not resulted “in significant consequences, for example, production losses, morbidity or mortality at a zone or country level” in Thailand since at least 2010. It also reveals that no single polymerase chain reaction (PCR) test is sufficient to identify IHHNV‐infected shrimp. It presents historical evidence and new evidence from 11 commercial ponds cultivating the giant tiger shrimp Penaeus monodon in Thailand. These ponds were selected because they were the ponds that gave positive PCR test results for IHHNV using two methods recommended for IHHNV diagnosis by World Organization for Animal Health (WOAH) (IHHNV‐309 and IHHNV‐389). However, an additional in‐house “IHHNV Long‐amp method” (IHHNV‐LA) was also used to amplify 90% of the 4‐kb IHHNV genome sequence, and it also gave false‐positive test results with 2 of the 11 ponds (IHHNV‐LA positive, but histological tests negative). Further tests using normal histopathological analysis for the presence of pathognomonic Cowdry A type inclusions (CAI), in situ hybridization (ISH) and immunohistochemistry (IHC) could confirm IHHNV infections in only two of the three ponds PCR‐positive using all three PCR methods. In addition, positive detection of CAI alone was equivalent to ISH or IHC in confirming IHHNV infection after a positive test with any of the PCR methods used. In summary, the recommended WOAH PCR methods gave false‐positive test results for IHHNV infection with 9/11 ponds (82%). All 11 ponds gave profitable harvests despite the confirmation of IHHNV in two ponds, where it was accompanied by various additional pathogens. Unfortunately, according to current practice, positive PCR test results with the WOAH methods alone sometimes leads to rejection of traded shrimp products without assurance that the test results are not false‐positive results that may arise from endogenous viral elements (EVE).

Polychaetes (Perinereis helleri) reared in sand beds filtering nutrients from shrimp (Penaeus monodon) culture ponds can transiently carry IHHNV

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.

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.