Co-Infection of Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) and White Spot Syndrome Virus (WSSV) in the Wild Crustaceans of Andaman and Nicobar Archipelago, India.

The present study was intended to screen the wild crustaceans for co-infection with Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) and White Spot Syndrome Virus (WSSV) in Andaman and Nicobar Archipelago, India. We screened a total of 607 shrimp and 110 crab samples using a specific polymerase chain reaction, and out of them, 82 shrimps (13.5%) and 5 (4.5%) crabs were found positive for co-infection of IHHNV and WSSV. A higher rate of co-infection was observed in Penaeus monodon and Scylla serrata than other shrimp and crab species. The nucleotide sequences of IHHNV and WSSV obtained from crab in this present study exhibited very high sequence identity with their counterparts retrieved from various countries. Histopathological analysis of the infected shrimp gill sections further confirmed the eosinophilic intra-nuclear cowdry type A inclusion bodies and basophilic intra-nuclear inclusion bodies characteristics of IHHNV and WSSV infections, respectively. The present study serves as the first report on co-infection of WSSV and IHHNV in Andaman and Nicobar Archipelago, India and accentuates the critical need for continuous monitoring of wild crustaceans and appropriate biosecurity measures for brackishwater aquaculture.

Endogenous piRNA-guided slicing triggers responder and trailer piRNA production from viral RNA in Aedes aegypti mosquitoes.

In the germline of animals, PIWI interacting (pi)RNAs protect the genome against the detrimental effects of transposon mobilization. In Drosophila, piRNA-mediated cleavage of transposon RNA triggers the production of responder piRNAs via ping-pong amplification. Responder piRNA 3' end formation by the nuclease Zucchini is coupled to the production of downstream trailer piRNAs, expanding the repertoire of transposon piRNA sequences. In Aedes aegypti mosquitoes, piRNAs are generated from viral RNA, yet, it is unknown how viral piRNA 3' ends are formed and whether viral RNA cleavage gives rise to trailer piRNA production. Here we report that in Ae. aegypti, virus- and transposon-derived piRNAs have sharp 3' ends, and are biased for downstream uridine residues, features reminiscent of Zucchini cleavage of precursor piRNAs in Drosophila. We designed a reporter system to study viral piRNA 3' end formation and found that targeting viral RNA by abundant endogenous piRNAs triggers the production of responder and trailer piRNAs. Using this reporter, we identified the Ae. aegypti orthologs of Zucchini and Nibbler, two nucleases involved in piRNA 3' end formation. Our results furthermore suggest that autonomous piRNA production from viral RNA can be triggered and expanded by an initial cleavage event guided by genome-encoded piRNAs.

Research progress on hosts and carriers, prevalence, virulence of infectious hypodermal and hematopoietic necrosis virus (IHHNV).

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is one of the major viral pathogens of penaeid shrimp and it has spread worldwide. IHHNV causes substantial economic loss to the shrimp farming industry and has been listed as a notifiable crustacean disease pathogen by the World Organization for Animal Health (OIE). In this paper, we reviewed studies on the hosts and carriers, prevalence, genotypes and virulence of IHHNV. The pathogenesis mechanisms of IHHNV and the viral interference between IHHNV and white spot syndrome virus (WSSV) were also discussed. The mechanism of IHHNV infection and its virulence difference in different hosts and different developmental stages have not been fully studied yet. The mechanisms underlying viral interference between IHHNV and WSSV are not yet fully understood. Further studies are needed to elucidate the precise molecular mechanisms underlying IHHNV infection and to apply the insights gained from such studies for the effective control and prevention of IHHNV disease.

A fast and visual method for duplex shrimp pathogens detection with high specificity using rapid PCR and molecular beacon.

Molecular diagnosis of genome is one of the major methods for pathogens detection. The commonly used PCR method can realize an exponential amplification of the target gene but is time-consuming. In this work, we proposed a duplex and visual method using rapid PCR combined with molecular beacons to specifically detect two kinds of shrimp pathogens in one reaction tube. We only need to observe the fluorescence change of the reaction tube with naked eye to determine the result. A home-made automatic transfer equipment allows reaction tubes shuttling quickly between two water baths to achieve rapid PCR amplification. A simple device was also designed to present the detection results easily determined with naked eye. This duplex and visual detection method is fast, low-cost and of high specificity. From DNA extraction to results judgment, only 15 min was enough. Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) and Vibrio parahaemolyticus (VP) are two common shrimp pathogens which were chosen as our detection objects. This method may give a possibility to conduct end-point visual duplex detection, which may make a positive influence on the pathogen prevention.

Public Health Response to Aedes aegypti and Ae. albopictus Mosquitoes Invading California, USA.

Aedes aegypti and Ae. albopictus mosquitoes, primary vectors of dengue and chikungunya viruses, were recently detected in California, USA. The threat of potential local transmission of these viruses increases as more infected travelers arrive from affected areas. Public health response has included enhanced human and mosquito surveillance, education, and intensive mosquito control.

The infectious hypodermal and haematopoietic necrosis virus: a brief review of what we do and do not know.

Given its high prevalence, its wide distribution and its remarkable capacity to cause severe mortality in shrimp, the infectious hypodermal and haematopoietic necrosis virus (IHHNV) may deserve far more attention than it has received, as it remains considered as one of the most serious problems plaguing the global shrimp farming industry. Furthermore, its real measurable impact over wild shrimp populations remains unknown. Undeniably, the progress that we have reached today on the knowledge of its geographical distribution, clinical signs, genetic diversity, transmission and virulence may help to identify and understand important aspects of its biology and pathogenesis. However, the information regarding the molecular events that occur during the infection process is scarce. Thus, it may not be surprising to find that there are no therapeutic options available for the prophylaxis or treatments to reduce the deleterious impact of this viral pathogen to date. The aim of this review is to integrate and discuss the current state of knowledge concerning several aspects of the biology of IHHNV and to highlight potential future directions for this area of research.

Comparative proteome analysis of silkworm in its susceptibility and resistance responses to Bombyx mori densonucleosis virus.

Bombyx mori densonucleosis virus (BmDNV) is one of the most disastrous viruses in cocoon production. Silkworm resistance to BmDNV has been examined previously using a number of traditional biochemical and molecular techniques. In this study, a near isogenic line, BC(6), was constructed to eliminate the difference in inherited background, which has 99.9% identity with the susceptible strain but carries a resistant gene. We utilized a proteomic approach involving two-dimensional differential gel electrophoresis and mass spectrometry to examine changes in the midgut proteins from the susceptible and resistant silkworm larvae infected with BmDNV. The protein profiles were compared and 9 differentially expressed proteins were identified by mass spectrometry. In the resistant strains, the heat-shock 70-kDa protein cognate, cytochrome P450, vacuolar ATP synthase subunit B, arginine kinase, vacuolar ATP synthase subunit D and glutathione S-transferase sigma were strongly upregulated and α-tubulin was downregulated. Our results imply that these upregulated genes and the downregulated genes might be involved in B. mori immune responses against BmDNV-Z infection.

Potential for the Anopheles gambiae densonucleosis virus to act as an "evolution-proof" biopesticide.

"Evolution-proof" or "late-life-acting" insecticides (LLAIs) preferentially kill older adult mosquitoes and are of extreme interest to control vector-borne diseases such as malaria. We used quantitative PCR to assess whether the Anopheles gambiae densonucleosis virus (AgDNV) had potential as an LLAI. After infection, AgDNV titers increased modestly during larval development but replicated slower than the host cells, resulting in a significant decrease in the normalized virus titer during larval and pupal development. Normalized virus titers dramatically increased after adult emergence, peaking in 7- to 10-day-old adults. Unlike other DNVs, AgDNV does not significantly replicate in preadult mosquitoes but rather preferentially replicates in older adults. The natural dynamics of AgDNV make it ideal for expression of insect-specific toxin genes as a biological LLAI.

A large scale laboratory cage trial of Aedes densonucleosis virus (AeDNV).

Aedes aegypti (L.) (Diptera: Culicidae) the primary vector of dengue viruses (DENV1-4), oviposit in and around human dwellings, including sites difficult to locate, making control of this mosquito challenging. We explored the efficacy and sustainability of Aedes Densonucleosis Virus (AeDNV) as a biocontrol agent for Ae. aegypti in and among oviposition sites in large laboratory cages (> 92 m3) as a prelude to field trials. Select cages were seeded with AeDNV in a single oviposition site (OPS) with unseeded OPSs established at varied distances. Quantitative real-time polymerase chain reaction was used to track dispersal and accumulation of AeDNV among OPSs. All eggs were collected weekly from each cage and counted. We asked: (1) Is AeDNV dispersed over varying distances and can it accumulate and persist in novel OPSs? (2) Are egg densities reduced in AeDNV treated populations? AeDNV was dispersed to and sustained in novel OPSs. Virus accumulation in OPSs was positively correlated with egg densities and proximity to the initial infection source affected the timing of dispersal and maintenance of viral titers. AeDNV did not significantly reduce Ae. aegypti egg densities. The current study documents that adult female Ae. aegypti oviposition behavior leads to successful viral dispersal from treated to novel containers in large-scale cages; however, the AeDNV titers reached were not sufficient to reduce egg densities.

Induction of apoptosis in densovirus infected Aedes aegypti mosquitoes.

The mechanism of death in densovirus infected mosquitoes remains unexplored. This study investigated the cellular consequences of densovirus infection in Aedes aegypti mosquitoes after a second generation challenge with a densovirus isolated from adult Aedes albopictus mosquitoes in Thailand (AThDNV). Specimens were analyzed by TUNEL assay, fluorescent in situ hybridization (FISH) and a calorimic assay to detect activation of caspase 3-like activity. After challenge, moribund mosquitoes showed considerable evidence of TUNEL positive cells. The caspase 3-like activity assay showed that the presence of TUNEL positive cells was associated with increased levels of activated caspase 3-like activity in AThDNV infected mosquitoes.

Prevention of Dengue fever through plant based mosquito repellent Clausena dentata (Willd.) M. Roem (Family: Rutaceae) essential oil against Aedes aegypti l. (Diptera: Culicidae) mosquito.

BACKGROUND: Plant based repellent against mosquito borne diseases are used recently because synthetic repellents cause side effects like breathing problem, eye irritation, head ache, cough, etc. The use of natural products for dengue control would protect the environment, reduce dependence on expensive synthetic repellents and also generate local employment. MATERIAL AND METHODS: Essential oil was isolated by steam distillation which was used against the bites of Aedes aegypti and duration of protection period was assessed. Skin-irritant potential test was also conducted on 25 healthy volunteers by using four-point scale. RESULTS: The increase in the concentrations of essential oil increased the mean protection time against the bites of Aedes aegypti. The lowest mean protection time was 180.0 min for 2.5% and highest time of 255.0 min for 10%. The mean score of zero for skin-irritant potential test for all the concentrations indicated that the essential oil did not cause irritation to human skin. CONCLUSIONS: Results indicated that the use of plant based repellent for the control of dengue fever would replace the currently used synthetic repellents which causes many side effects.

Evaluation of mosquito densoviruses for controlling Aedes aegypti (Diptera: Culicidae): variation in efficiency due to virus strain and geographic origin of mosquitoes.

Four mosquito densovirus strains were assayed for mortality and infectivity against Aedes aegypti larvae from different geographic regions. The viral titers were quantified by real-time PCR using TaqMan technology. Firstinstar larvae were exposed to the same titer of each densovirus strain for 48 hours. All strains of densoviruses exhibited larvicidal activity and caused more than 80% mortality and infectivity in the three mosquito strains. AalDNV-exposed larvae had the highest mortality rate. The mean time to death of AalDNV-exposed larvae was shorter than other DNVs-exposed larvae. We can conclude that different densovirus strains exhibit some variations in their pathogenicity to different populations of Ae. aegypti mosquitoes. A few mosquitoes from Chachoengsao and Bangkok exposed to AeDNV and AThDNV survived to the adult stage to lay eggs and showed 22% to 50% vertical transmission in the F1 generation. Phylogenetic analysis of four densovirus strains indicated that mosquito densoviruses are separated into two distinct clades.

Detección del virus de la necrosis infecciosa hipodérmica y hematopoyética (IHHNV) en camarones blancos cultivados asintomáticos, Litopenaeus vannamei (BOONE) en Venezuela / Detection of the infectious hypodermal and hematopoietic necrosis virus (IHHNV) in asymtomatic cultured white shrimp, Litopenaeus vannamei (Boone) in Venezuela

El Virus de la Necrosis Infecciosa Hipodérmica y Hematopoyética (IHHNV) es un virus que causa altas mortalidades en Litopenaeus stylirostris y el síndrome de la deformidad del rostro (RDS) en L. vannamei. Con el fin de determinar la presencia del IHHNV en camarones cultivados asintomáticos, se analizaron muestras de camarones cultivados L. vannamei de cinco granjas camaroneras localizadas en el oriente y occidente de Venezuela. Se analizaron un total de 90 muestras por granja, de tres tallas: PL8-PL15, juveniles de 5-6 g y de 12 a 15gde peso. El ADN total fue extraído de muestras homogeneizadas de pleópodos, mediante el uso de kit comerciales. La detección del IHHNV fue realizado, tanto por hibridación mediante “Dot Blot” como por PCR utilizando los kits de Diagxotics ShrimpProbe y ShrimPCaRe Simplex, respectivamente. Se detectó un total de siete muestras positivas, provenientes de cuatro (B, C, D y E) de las cinco granjas camaroneras estudiadas, variando las prevalencias entre 1,1 y 3,3 por ciento por granja. Todas las muestras positivas correspondieron a individuos de 5 a 6 g de peso. La técnica diagnóstica por PCR fue más sensible para la detección del IHHNV que la hibridación por “Dot Blot”. La presencia del IHHNV en camarones asintomáticosy a bajos niveles de prevalencia puede implicar que las poblaciones de camarones utilizadas para cultivo en Venezuela son resistentes o en todo caso tolerantes a este virus.

The Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) is a pathogen that may cause high mortalities in Litopenaeus stylirostris and the Runt Deformity Syndrome (RDS) in L. vannamei. In order to detect the presence of IHHNV in asymptomatic, cultivated shrimp, it were analyzed shrimp samples of cultivated L. vannamei from 5 farms located in the east and west costs of Venezuela. A total of 90 samples per farm were analyzed, using three sizes: PL8-PL15, juveniles of 5-6 g and of 12-15 g of weight. The DNA was extracted from homogenized samples of pleopods, using commercial kits. The detection of IHHNV was carried out by both dot blot hybridization and PCR using the Diagxotics kits ShrimpProbe and ShrimPCaRe Simplex, respectively. A total of 7 positive samples from 4 (B, C, D and E) of the 5 shrimp farms studied were detected. The prevalence in the farms ranged from 1.1 to 3.3%. All positive samples corresponded to individuals of 5 to 6 g of weight. PCR was a more sensitive technique than the dot blot hybridization. The presence of IHHNV in asymptomatic shrimp at low values of prevalence could imply that the shrimp populations used for culture in Venezuela are resistant or at least tolerant to this viral pathogen.

Simian parvoviruses: biology and implications for research.

The simian parvoviruses (SPVs) are in the genus Erythrovirus in the family Parvoviridae and are most closely related to the human virus B19. SPV has been identified in cynomolgus, rhesus, and pigtailed macaques. All of the primate erythroviruses have a predilection for erythroid precursors. Infection, which is common in macaques, is usually clinically silent. Disease from SPV is associated with immunosuppression due to infection with various retroviruses (SIV, simian retrovirus, and simian-human immunodeficiency virus), surgery, drug toxicity studies, and posttransplantation immunosuppressive treatment and therefore is of concern in studies that use parvovirus-positive macaques.

PCR assay for discriminating between infectious hypodermal and hematopoietic necrosis virus (IHHNV) and virus-related sequences in the genome of Penaeus monodon.

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.

Viral interference between infectious hypodermal and hematopoietic necrosis virus and white spot syndrome virus in Litopenaeus vannamei.

White spot syndrome virus (WSSV) is highly virulent and has caused significant production losses to the shrimp culture industry over the last decade. Infectious hypodermal and hematopoietic necrosis virus (IHHNV) also infects penaeid shrimp and, while being less important than WSSV, remains a major cause of significant production losses in Litopenaeus vannamei (also called Penaeus vannamei) and L. stylirostris (also called Penaeus stylirostris). These 2 viruses and their interactions were previously investigated in L. stylirostris. We report here laboratory challenge studies carried out to determine if viral interference between IHHNV and WSSV also occurs in L. vannamei, and it was found that experimental infection with IHHNV induced a significant delay in mortality following WSSV challenge. L. vannamei infected per os with IHHNV were challenged with WSSV at 0, 10, 20, 30, 40 and 50 d post-infection. Groups of naïve shrimp infected with WSSV alone died in 3 d whereas shrimp pre-infected with IHHNV for 30, 40 or 50 d died in 5 d. Real-time PCR analysis showed that the delay correlated to the IHHNV load and that WSSV challenge induced a decrease in IHHNV load, indicating some form of competition between the 2 viruses.

Low impact of infectious hypodermal and hematopoietic necrosis virus (IHHNV) on growth and reproductive performance of Penaeus monodon.

No controlled studies on the effect of infectous hypodermal and necrosis virus (IHHNV) on Penaeus monodon have been previously reported. Here we describe domesticated P. monodon that became positive for IHHNV and other viruses at variable levels of prevalence during cultivation in 16 open-air, earthen ponds. These were stocked with domesticated postlarvae (PL) that tested negative for 7 shrimp viruses including IHHNV at 6% prevalence in 3 checks using polymerase chain reaction (PCR) methods. These PL were derived from domesticated female broodstock that individually tested negative for the same viruses. At 4 mo of culture, the shrimp in some ponds without obvious mortality tested positive by PCR methods for IHHNV and 3 other viruses at variable levels of maximum estimated prevalence (MEP). Stained tissue sections showed no lesions typical of IHHNV, but in situ hybridization tests with an IHHNV-specific DNA probe were positive. There was no significant difference in mean body weight (i.e. ca. 25 g) between shrimp groups positive or negative for IHHNV. Similar results were obtained with IHHNV negative and positive adults at 1 yr. Adults that individually tested negative for all 7 viruses and some that tested lightly positive for IHHNV were bred for the next generation. There were no significant differences in the number of eggs (> 600 000) and nauplii (ca. 300,000) produced by females negative and positive for IHHNV. From these females, 11/49 (22%) IHHNV PCR-positive PL batches were obtained from PCR-negative spawners, while 8/11 (73%) were obtained from IHHNV PCR-positive spawners. The results suggested that IHHNV infection can be transmitted vertically but does not seriously retard growth of P. monodon or affect fecundity of lightly infected broodstock.

A web-based multimedia spatial information system to document Aedes aegypti breeding sites and dengue fever risk along the US-Mexico border.

This paper describes a web-based multimedia spatial information system used to support a study of the re-invasion of Aedes aegypti, the mosquito vector for dengue fever, in the deserts of the southwest United States/northwest Mexico. The system was developed applying Open Geospatial Consortium and World Wide Web Consortium Open Specifications and using Open Source Software. The system creates a sensory-rich environment, one which allows users to interact with the system to explore connections among data (maps, remotely sensed images, text, graphs, 360 degree panoramas and photos), visualize information, formulate their own interpretations, generate hypotheses and reach their own conclusions.

Mosquito densonucleosis viruses cause dramatically different infection phenotypes in the C6/36 Aedes albopictus cell line.

Mosquito densoviruses generally establish persistent infections in mosquito cell lines including the C6/36 Aedes albopictus cell line. In contrast, the closely related Haemagogus equinus densovirus (HeDNV) causes dramatic cytopathic effects in the C6/36 Aedes albopictus cell line. Infection of C6/36 cells by HeDNV causes internucleosomal fragmentation of host chromosomal DNA, changes in cellular morphology (membrane budding, apoptotic bodies), caspase activation and exposure of phosphatidylserine on the cellular membrane. This is accompanied by a higher rate of infection and more vigorous production of virus in these cells. These observations are consistent with the induction of apoptosis during infection. In contrast, expression of AeDNV proteins in C6/36 cells does not cause obvious cytopathic effects although NS1 expression causes accumulation of cells in G2 phase. C6/36 cells persistently infected with AeDNV were not protected from superinfection with HeDNV. Thus, there does not seem to be an antiviral state induced by AeDNV persistent infection.