Biology subjects

Flegel, T. W.

Publications and source records attributed to Flegel, T. W..

10 recordsLinked to original sources

bioRxiv2023

White spot syndrome virus endogenous viral elements (EVE) revealed by circular viral copy DNA (cvcDNA) in shrimp

Circular, viral copy DNA (cvcDNA) can reveal the existence of endogenous viral elements (EVE) in shrimp genomic DNA. Here we describe isolation and sequencing of cvcDNA from a breeding stock of the whiteleg shrimp Penaeus vannamei. The stock was developed by onward breeding and selection for white spot syndrome virus (WSSV)-free individual survivors of white spot disease outbreaks. The stock exhibits high tolerance to WSSV. A pool of DNA extracted from 10 shrimp from this stock was subjected to cvcDNA isolation and amplification followed by high throughput sequencing. This revealed DNA fragments corresponding to locations covering much of the 300,000 bp WSSV genome. However, high frequency-read-fragments (HFRF) mapped to a surprisingly small region of approximately 1,400 bp. We hypothesized that the HFRF reflected their selection due to provision of tolerance to WSSV. Four PCR primer sets were designed to cover the 1,365 bp mapped region. One pair (Set 1) targeted the whole 1,365 bp mapped region, while another 3 primer sets (Set 2-4), targeted regions within the 1,365 bp target. All 4 primer sets were used with DNA samples from each of 36 shrimp from the same breeding stock (including the 10 used for cvcDNA preparation). Individual positive PCR results varied widely from shrimp to shrimp, ranging from only 1 primer set up to 4 primer sets. Only 1 specimen gave an amplicon of 1,365 bp, while others gave single to multiple positive amplicons in both continuous and discontinuous regions. This indicated that the amplicons did not arise from contiguous targets and might vary in insertion length. The results also confirmed that none of the shrimp were infected with WSSV. Sequencing of the PCR amplicons of expected sizes revealed sequence identity to extant WSSV genomes. This data will be used to screen the breeding stock for EVE that provide WSSV tolerance.

zoology↗

bioRxiv2023

Circular viral copy DNA of Dengue virus (DENV-2) isolated from infected mosquito cell cultures

Dengue virus serotype 2 (DENV-2) is a mosquito-borne disease in the family Flaviviridae. It has been previously shown that DENV-2 can infect C6/36 mosquito cells and cause initial cytopathic effects that dissipate upon serial split-passage to yield persistently infected cultures with normal growth and morphology. In other words, the cell line accommodated persistent DENV-2 infections. It has recently been found that insect viral infections induce the production of viral copy DNA (vcDNA) fragments via host reverse transcriptase (RT). The vcDNA occurs in both linear (lvcDNA) and circular (cvcDNA) forms and produces small interfering RNA (siRNA) transcripts that can result in an immediate protective RNA interference (RNAi) response. The vcDNA can lead to the host acquiring endogenous viral elements (EVE) in genomic DNA. Thus, we hypothesized that DENV-2 cvcDNA and DENV-2-EVE would arise in C6/36 insect cells challenged with DENV-2 virus in vitro. Here we describe the successful isolation and characterization of cvcDNA constructs homologous to DENV-2 from laboratory challenges with C6/36 cells. At least 1 of these appeared to arise from a DENV-2-EVE. We also show that a cvcDNA preparation derived from the DENV-2 infected in C6/36 insect cells and subjected to rolling circle amplification (RCA) significantly reduced DENV-2 replication when applied to naive C6/36 cells prior to the DENV-2 challenge. This preliminary work lays the groundwork for further studies using the C6/36 cell model to screen and characterize protective EVE against insect and shrimp viruses.

microbiology↗

bioRxiv2022

A novel, ssDNA Bidnavirus in the giant freshwater prawn Macrobrachium rosenbergii

A purported parvovirus producing circular, eosinophilic inclusions in the nuclei of tubule epithelial cells of the hepatopancreas (HP) of the giant freshwater prawn Macrobrachium rosenbergii (Mr) was first reported from Malaysia in 1994. In 2009, similar inclusions in Mr from Thailand were confirmed to contain single-stranded DNA (ssDNA). In 2021, Mr samples showing similar HP inclusions were reported to give positive PCR test results for decapod Hepanhamaparvovirus (DHPV) using universal DHPV primers designed from many viral isolates of 3 penaeid shrimp species. However, the matching DNA probe revealed positive in situ hybridization (ISH) results with host HP nuclei only, and not with the intranuclear inclusions. At the time, the negative ISH result with the intranuclear inclusions has not been further elucidated. Here we describe metagenomic analysis and de novo assembly from 2016 samples of Mr with similar intranuclear inclusions that showed negative PCR test results using the DHPV universal primers. Genomes identified through metagenomics have revealed a novel ssDNA genome sequence of 6,723 bases that was confirmed by a single PCR amplicon. ISH using specific probes derived from this genome sequence demonstrated the ssDNA presented in both grossly normal nuclei and in the intranuclear inclusions. Tests with archived DNA from the samples used in the 2021 publication yielded positive PCR results for both DHPV and the novel sequence, suggesting that the shrimp had dual infections. Phylogenetic analysis of the novel sequence revealed no relationship to any known DHPV type. Instead, the sequence most closely matched the polymerase B gene in the V1 fragment from Bombyx mori bidensovirus 2 (BmBDV2) is characterized by 2 ssDNA genome fragments and has been classified in the family Bidnaviridae. The 2 BmBDV2 fragments V1 and V2 each produce their own independent capsid protein genes and virions. In contrast, the new Mr virus is independent with a single 6,723 base ssDNA genome, and we propose the name Macrobrachium hepatopancreatic bidnavirus (MHBV).

microbiology↗

bioRxiv2022

Wenzhou shrimp virus 8 (WZV8) diagnosis by unique inclusions in shrimp hepatopancreatic E-cells and by RT-PCR

The genome sequence of Wenzhou shrimp virus 8 (WzSV8) (GenBank record KX883984.1) was described in 2015 from wide screening for RNA viruses in aquatic animals. A closely related sequence (GenBank record OK662577.1) from the whiteleg shrimp Penaeus vannamei was deposited in 2021 under the name Penaeus vannamei picornavirus (PvPV). In 2022 another closely related sequence (GenBank accession: OP265432) was submitted under the name Penaeus vannamei solinvivirus (PvSV). In 2021, prior to the publication of PvPV and PvSV, we used an RT-PCR method devised from the sequence of KX883984.1 (described herein) to screen for WzSV8 in specimens of cultivated shrimp. Samples that gave positive RT-PCR results were subsequently tested by in situ hybridization (ISH) analysis to identify virus target tissues. Several tissues gave positive ISH results within morphologically normal nuclei. Thus, these tissues were of no use for diagnosis of WzSV8 by normal histological analysis. However, unique basophilic, cytoplasmic inclusions within vacuoles in the hepatopancreatic E-cells were also found in the same WzSV8-positive shrimp specimens, sometimes accompanied by a smaller eosinophilic inclusion. We call these Lightner double Inclusions (LDI) that can be considered pathognomonic for diagnosis of WzSV8 infection when detected using the light microscope. Although no current proof of WzSV8 is the cause of disease, it is important to investigate new viruses and related tissue anomalies, even from normal cultivated shrimp, to determine whether they may have any relationship to significant negative effects on the production of cultivated shrimp.

zoology↗

bioRxiv2021

Shrimp draft genome contains independent clusters of ancient and current endogenous viral elements (EVE) of the parvovirus IHHNV

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 draft genome of the giant tiger shrimp (Penaeus monodon)(GenBank record JABERT000000000) for the presence of EVE related to a shrimp parvovirus originally named infectious hypodermal and hematopoietic necrosis virus (IHHNV). ResultsThe shrimp draft genome contained 3 piRNA-like gene clusters containing scrambled IHHNV EVE. Two clusters were located distant from one another in linkage group 35 (LG35). Both LG35 clusters contained multiple DNA fragments 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 LG35 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 linkage group 7 (LG7). It contained EVE with high homology (99%) only to GenBank record AF218266 with the potential to protect shrimp against 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 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.

genetics↗

bioRxiv2021

Shrimp parvovirus circular DNA fragments arise from both endogenous viral elements (EVE) and the infecting virus

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.

molecular biology↗

bioRxiv2021

A potential prokaryotic and microsporidian pathobiome that may cause shrimp white feces syndrome (WFS)

White feces syndrome (WFS) in shrimp cultivation ponds is characterized by the occurrence of shrimp with abnormal, white intestines (midguts) combined with large floating mats of white, shrimp fecal strings. The etiology for WFS is complex, similar to diarrhea in humans. EHP-WFS is a type of WFS characterized by massive quantities of spores from the microsporidian parasite Enterocytozoon hepatopenaei (EHP) together with mixed, unidentified bacteria in the shrimp hepatopancreas, midgut and fecal strings. However, WFS does not always develop in shrimp with severe EHP infections in controlled laboratory challenges. Further, in EHP-WFS outbreak ponds, some shrimp show white midguts (WG) while others in the same pond show grossly normal midguts (NG). We hypothesized that comparison of the microbial flora between WG and NG from the same EHP-WFS pond would reveal probable combinations of microbes significantly associated with EHP-WFS. To test this hypothesis, we selected a pond exhibiting a severe EHP-WFS outbreak in cultivated Penaeus vannamei and used a combination of microscopic and microbial profiling analyses to compare WG and NG samples. By histology, EHP plasmodia and spores were confirmed in the hepatopancreas (HP) and midgut of WG and NG shrimp, but pathological severity and spore quantity was higher in the WG shrimp. In addition, intestinal microbiomes in WG shrimp were less diverse and had higher abundance of bacteria from the genera Vibrio and Propionigenium. Propionigenium quantity in the HP of WG shrimp was significantly higher (P = 1.08e-5) than in NG shrimp (4,506 vs. 3 copies /100 ng DNA, respectively). These findings supported our hypothesis by revealing two candidate bacterial genera that should be tested in combination with EHP as a potential eukaryote-prokaryote pathobiome that causes EHP-WFS in P. vannamei. HighlightsO_LIWhite feces syndrome (WFS) shrimp often harbor the microsporidian Enterocytozoon hepatopenaei (EHP) C_LIO_LIThe hepatopancreas (HP) and midgut of EHP-WFS shrimp had more EHP copies and spores than EHP-non.-WFS shrimp C_LIO_LIVibrio spp., Propionigenium sp. and EHP dominated in HP microbiomes of EHP-WFS shrimp C_LIO_LIPropionigenium copy numbers were uniquely high in the HP of EHP-WFS shrimp C_LIO_LIEHP-WFS shrimp also showed intestinal microbiomes of reduced diversity but more heterogeneity C_LI

molecular biology↗

bioRxiv2020

Infectious myonecrosis virus (IMNV) and decapod iridescent virus 1 (DIV1) detected in Penaeus monodonfrom the Indian Ocean

Infectious myonecrosis virus (IMNV) was first discovered in the Americas in 2004 as a new lethal pathogen of cultivated whiteleg shrimp Penaeus vannamei, but infections were not lethal for the giant tiger shrimp Penaeus monodon. In 2007, it was reported in diseased P. vannamei cultivated in Indonesia but, until recently, not from other countries in Asia. Decapod iridescent virus (DIV1) was first reported from China in 2016 and is lethal for the crayfish Cherax quadricarinatus and Procambarus clarkii, for the penaeid shrimp P. vannamei and P. chinensis and for the palaemonid shrimp Macrobrachium rosenbergii and Exopalaemon carinicauda. It has not yet been reported from other Asian countries. Here we describe the occurrence of positive test results for IMNV and DIV1 using polymerase chain reaction (PCR) technology during screening of grossly normal, broodstock-size, wild P. monodon captured from the Indian Ocean and held in a biosecurity facility for screening. Amplicons for each virus were obtained from two widely separated targets in the relevant viral genomes listed at GenBank, and sequencing revealed 99-100% identity to the targets for each virus. Based on these results, the captured specimens were destroyed. The results raised the possibility that grossly normal, captured P. monodon might serve as potential vehicles for introduction of IMNV and/or DIV1 to shrimp hatcheries and farms. Thus, we recommend that appropriate precautions be taken to avoid this possibility.

zoology↗

bioRxiv2020

Potential universal PCR method to detect decapod hepanhamaparvovirus (DHPV) in crustaceans

Parvoviruses that infect the hepatopancreas (HP) of the penaeid shrimp Penaeus chinensis, P. monodon, and P. merquiensis were previously called hepatopancreatic parvoviruses (HPV). They are now classified in the family Parvoviridae, sub-family Hamaparvovirinae as members of the same genus called Hepanhamaparvovirus and referred to as decapod hepanhamaparvovirus, designated here as DHPV. However, a virus that causes similar lesions in the HP of the giant river prawn Macrobrachium rosenbergii resembles hepanhamaparvoviruses by microscopy and histochemistry. Unfortunately, no genome information is yet available and PCR detection methods that work for DHPV in P. monodon do not work with M. rosenbergii. For hatchery samples of M. rosenbergii in Thailand with DHPV-like lesions, we hypothesized it might be possible to design primer pairs from 8 full DHPV genome sequences at GenBank for use in PCR detection of DHPV in M. rosenbergii. Using this strategy, we successfully designed a new set of primers and a PCR protocol called the DHPV-U method that gave an amplicon with DNA extracts from larvae of M. rosenberigii samples that showed DHPV-like lesions, while extracts from normal larvae gave none. DNA extracts from P. monodon infected with DHPV also gave amplicons. At the same time, the normal PCR method for DHPV in P. monodon gave no amplicon with the M. rosenbergii DNA extracts. The DHPV-U amplicons from P. monodon and M. rosenbergii shared 99% sequence identity, and in situ hybridization (ISH) assays using the DIG-labeled amplicon gave positive histochemical results in the HP tissue of both P. monodon and M. rosenbergii. The DHPV-U method is now being used in Thailand for detection of DHPV in both P. monodon and M. rosenbergii. Overall, the results support the proposal that the HP virus in M. rosenbergii is also a hepanhamaparvovirus. Based on 100% sequence identity of the target region in the currently published DHPV sequences at GenBank, the DHPV-U method may also work for detection of other DHPV isolates.

molecular biology↗

bioRxiv2020

Full genome characterization of Laem Singh virus (LSNV) in shrimp Penaeus monodon

Laem Singh virus (LSNV) was discovered in 2006 and proposed as a necessary but insufficient cause of retarded growth in the giant tiger shrimp Penaeus monodon. Its closest relatives were plant viruses including an unassigned Sobemovirus and viruses in the family Luteoviridae. During succeeding years, attempts to obtain the full LSNV genome sequence by genome walking failed. However, recent publication of the full sequence of Wenzhou shrimp virus 9 (WZSV 9) at GenBank revealed that LSNV sequences in our database shared 99% sequence identity with it. Thus, we hypothesized that LSNV and WZSV 9 were different isolates of the same virus species. Here we confirm that hypothesis by cloning and sequencing of the full genome of LSNV from P. monodon and by showing that it consists of two fragments each with 99% identity to the matching fragments of WZSV.

microbiology↗