Acute hepatopancreatic necrosis disease (AHPND) is a severe, newly emergent penaeid shrimp disease caused by Vibrio parahaemolyticus that has already led to tremendous losses in the cultured shrimp industry. Until now, its disease-causing mechanism has remained unclear. Here we show that an AHPND-causing strain of V. parahaemolyticus contains a 70-kbp plasmid (pVA1) with a postsegregational killing system, and that the ability to cause disease is abolished by the natural absence or experimental deletion of the plasmid-encoded homologs of the Photorhabdus insect-related (Pir) toxins PirA and PirB. We determined the crystal structure of the V. parahaemolyticus PirA and PirB (PirAvp and PirBvp) proteins and found that the overall structural topology of PirAvp/PirBvp is very similar to that of the Bacillus Cry insecticidal toxin-like proteins, despite the low sequence identity (<10%). This structural similarity suggests that the putative PirABvp heterodimer might emulate the functional domains of the Cry protein, and in particular its pore-forming activity. The gene organization of pVA1 further suggested that pirABvp may be lost or acquired by horizontal gene transfer via transposition or homologous recombination.

In this study, we used a systems biology approach to investigate changes in the proteome and metabolome of shrimp hemocytes infected by the invertebrate virus WSSV (white spot syndrome virus) at the viral genome replication stage (12 hpi) and the late stage (24 hpi). At 12 hpi, but not at 24 hpi, there was significant up-regulation of the markers of several metabolic pathways associated with the vertebrate Warburg effect (or aerobic glycolysis), including glycolysis, the pentose phosphate pathway, nucleotide biosynthesis, glutaminolysis and amino acid biosynthesis. We show that the PI3K-Akt-mTOR pathway was of central importance in triggering this WSSV-induced Warburg effect. Although dsRNA silencing of the mTORC1 activator Rheb had only a relatively minor impact on WSSV replication, in vivo chemical inhibition of Akt, mTORC1 and mTORC2 suppressed the WSSV-induced Warburg effect and reduced both WSSV gene expression and viral genome replication. When the Warburg effect was suppressed by pretreatment with the mTOR inhibitor Torin 1, even the subsequent up-regulation of the TCA cycle was insufficient to satisfy the virus's requirements for energy and macromolecular precursors. The WSSV-induced Warburg effect therefore appears to be essential for successful viral replication.

White spot syndrome virus (WSSV) virions were purified from the hemolymph of experimentally infected crayfish Procambarus clarkii, and their proteins were separated by 8 to 18% gradient sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) to give a protein profile. The visible bands were then excised from the gel, and following trypsin digestion of the reduced and alkylated WSSV proteins in the bands, the peptide sequence of each fragment was determined by liquid chromatography-nano-electrospray ionization tandem mass spectrometry (LC-nanoESI-MS/MS) using a quadrupole/time-of-flight mass spectrometer. Comparison of the resulting peptide sequence data against the nonredundant database at the National Center for Biotechnology Information identified 33 WSSV structural genes, 20 of which are reported here for the first time. Since there were six other known WSSV structural proteins that could not be identified from the SDS-PAGE bands, there must therefore be a total of at least 39 (33 ؉ 6) WSSV structural protein genes. Only 61.5% of the WSSV structural genes have a polyadenylation signal, and preliminary analysis by 3 rapid amplification of cDNA ends suggested that some structural protein genes produced mRNA without a poly(A) tail. Microarray analysis showed that gene expression started at 2, 6, 8, 12, 18, 24, and 36 hpi for 7, 1, 4, 12, 9, 5, and 1 of the genes, respectively. Based on similarities in their time course expression patterns, a clustering algorithm was used to group the WSSV structural genes into four clusters. Genes that putatively had common or similar roles in the viral infection cycle tended to appear in the same cluster.

The protein components of the white spot syndrome virus (WSSV) virion have been well established by proteomic methods, and at least 39 structural proteins are currently known. However, several details of the virus structure and assembly remain controversial, including the role of one of the major structural proteins, VP26. In this study, Triton X-100 was used in combination with various concentrations of NaCl to separate intact WSSV virions into distinct fractions such that each fraction contained envelope and tegument proteins, tegument and nucleocapsid proteins, or nucleocapsid proteins only. From the protein profiles and Western blotting results, VP26, VP36A, VP39A, and VP95 were all identified as tegument proteins distinct from the envelope proteins (VP19, VP28, VP31, VP36B, VP38A, VP51B, VP53A) and nucleocapsid proteins (VP664, VP51C, VP60B, VP15). We also found that VP15 dissociated from the nucleocapsid at high salt concentrations, even though DNA was still present. These results were confirmed by CsCl isopycnic centrifugation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry, by a trypsin sensitivity assay, and by an immunogold assay. Finally, we propose an assembly process for the WSSV virion.White spot syndrome virus (WSSV) is a widespread and disastrous viral pathogen of cultured shrimp that also attacks crabs and crayfish as well as many other crustaceans (3,10,16,22). The WSSV virion is an enveloped particle of approximately 275 by 120 nm with an olivaceous-to-bacilliform shape, and it has a nucleocapsid (300 by 70 nm) with periodic striations (22,25). The most obvious feature of WSSV is the presence of a thread-like extension at one end of the virion (2, 25), which gives this virus the family name Nimaviridae (13).A virion is a complex assembly of macromolecules exquisitely suited for the protection and delivery of viral genomes. Its structural proteins are particularly important, since these proteins are the first molecules to interact with the host, and they therefore play critical roles in cell targeting as well as in the triggering of host defenses. Recently, thanks to the introduction of proteomic methods, the total number of known WSSV structural proteins increased to 39 (5, 17).Immunogold electron microscopy (IEM) has been used to identify VP28, VP26, VP31, VP51C, VP36B, VP41A, VP12B, and VP180 as envelope proteins (4,5,8,9,(28)(29)(30) and VP664 as a nucleocapsid protein (7). Other studies (1,6,(18)(19)(20) that combined detergent treatment and Western blotting confirmed and expanded most of these results (VP28, VP19, and VP73 as envelope proteins; VP24, VP15, and VP35 as nucleocapsid proteins) but also identified VP26 as a nucleocapsid protein.Here for the first time we embark on a systematic study of the structural proteins of WSSV that not only allows us to resolve the question of VP26's location but also reveals the existence of a previously unreported component of the WSSV virion. This co...

A growing awareness of the diversity and ubiquity of microbes (eukaryotes, prokaryotes, and viruses) associated with larger 'host' organisms has led to the realisation that many diseases thought to be caused by one primary agent are the result of interactions between multiple taxa and the host. Even where a primary agent can be identified, its effect is often moderated by other symbionts. Therefore, the one pathogen-one disease paradigm is shifting towards the pathobiome concept, integrating the interaction of multiple symbionts, host, and environment in a new understanding of disease aetiology. Taxonomically, pathobiomes are variable across host species, ecology, tissue type, and time. Therefore, a more functionally driven understanding of pathobiotic systems is necessary, based on gene expression, metabolic interactions, and ecological processes. Disease in a Microbe-Dominated World The pathobiome (see Glossary) concept arose from human studies in which disruption of a healthpromoting and ecologically stable gut microbiome resulted in dysbiosis: a microbiome community of low-diversity and modified metabolic state, exposing the gut to invasion by, and proliferation of, pathogenic agents [1,2]. Dysbiotic communities can subvert the immune system and lead to further deleterious effects [3]. This concept is being adopted for research into the pathology of other animals and plants because attempts to explain syndromic conditions by identifying a single pathogenic agent are often incomplete (i.e., the one pathogen-one disease paradigm is often insufficient to explain many diseases [4-6]). Pathobiomes differ from those assemblages representing healthy or 'normal' states. What is 'normal' likely encompasses a range of assemblages that need to be understood before a pathobiome can be reliably distinguished from them. There is a lack of consistency in defining 'pathobiome' in the literature, ranging from a single pathogenic agent interacting with its biotic and abiotic environments (e.g., [5]) to the effects of interacting communities of microbes on host health [7]. Our synthesis (Box 1) is based on the effects of multiple symbionts, across all domains of life, on host health. The term 'microbiome' generally excludes eukaryotes; therefore, in this review, we use the term 'symbiome' to describe the whole assemblage of associated organisms excluding the host, and 'symbiont' for individual taxa within that assemblage. This definition is concordant with an inclusive scheme of symbiosis acknowledged in [9], which ranges from neutralism (neutral effect on both partners) to mutual beneficial effects and mutual antagonistic effects, and all other possible combinations of neutral, beneficial, and antagonistic effects. The duration of the association need not necessarily be long-term, as interactions can be effective on even short timescales; great variability in duration of association is both possible and likely. This inclusive definition is not inconsistent with some previous usages of the term, and is required by the large div...

The Warburg effect is an abnormal glycolysis response that is associated with cancer cells. Here we present evidence that metabolic changes resembling the Warburg effect are induced by a nonmammalian virus. When shrimp were infected with white spot syndrome virus (WSSV), changes were induced in several metabolic pathways related to the mitochondria. At the viral genome replication stage (12 h postinfection [hpi]), glucose consumption and plasma lactate concentration were both increased in WSSV-infected shrimp, and the key enzyme of the pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G6PDH), showed increased activity. We also found that at 12 hpi there was no alteration in the ADP/ATP ratio and that oxidative stress was lower than that in uninfected controls. All of these results are characteristic of the Warburg effect as it is present in mammals. There was also a significant decrease in triglyceride concentration starting at 12 hpi. At the late stage of the infection cycle (24 hpi), hemocytes of WSSV-infected shrimp showed several changes associated with cell death. These included the induction of mitochondrial membrane permeabilization (MMP), increased oxidative stress, decreased glucose consumption, and disrupted energy production. A previous study showed that WSSV infection led to upregulation of the voltage-dependent anion channel (VDAC), which is known to be involved in both the Warburg effect and MMP. Here we show that double-stranded RNA (dsRNA) silencing of the VDAC reduces WSSV-induced mortality and virion copy number. For these results, we hypothesize a model depicting the metabolic changes in host cells at the early and late stages of WSSV infection.During the early stage of most lytic viral infections, when the virus genome is being replicated, viral products will modulate host cell metabolic homeostasis to boost activities such as glycolysis, the pentose phosphate pathway (PPP), and fatty acid metabolism in order to favor pathogen biosynthesis and fulfill the pathogen's energy requirements. Subsequently, at the late stage of infection, when virion maturation is complete, damage to the cell's metabolism can lead to cell death, which in turn allows the new virions to be released. Using cell culture systems and state-of-the-art techniques, systems-level metabolic flux profilings have monitored these events in several mammalian viruses, including human cytomegalovirus (HCMV) and hepatitis C virus (HCV) (6,20,21). In some viruses, such as HCV and Kaposi's sarcoma herpesvirus (KSHV), these metabolic changes are characteristic of the Warburg effect (5, 6). The Warburg effect, which is an abnormal glycolytic response that is associated with cancer cells and tumors, involves the mitochondria and is partly mediated by the voltage-dependent anion channel (VDAC) (19). Interestingly, the VDAC also plays a critical role in cell death via its involvement in mitochondrial membrane permeabilization (MMP) (27,30).In a global study of the changes in protein expression levels in the stomachs of shrimp in...

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Acute hepatopancreatic necrosis disease (AHPND) (formerly, early mortality syndrome) is a high-mortality-rate shrimp disease prevalent in shrimp farming areas. Although AHPND is known to be caused by pathogenic Vibrio parahaemolyticus hosting the plasmid-related PirABvp toxin gene, the effects of disturbances in microbiome have not yet been studied. We took 62 samples from a grow-out pond during an AHPND developing period from Days 23 to 37 after stocking white postlarvae shrimp and sequenced the 16S rRNA genes with Illumina sequencing technology. The microbiomes of pond seawater and shrimp stomachs underwent varied dynamic succession during the period. Despite copies of PirABvp, principal co-ordinates analysis revealed two distinctive stages of change in stomach microbiomes associated with AHPND. AHPND markedly changed the bacterial diversity in the stomachs; it decreased the Shannon index by 53.6% within approximately 7 days, shifted the microbiome with Vibrio and Candidatus Bacilloplasma as predominant populations, and altered the species-to-species connectivity and complexity of the interaction network. The AHPND-causing Vibrio species were predicted to develop a co-occurrence pattern with several resident and transit members within Candidatus Bacilloplasma and Cyanobacteria. This study’s insights into microbiome dynamics during AHPND infection can be valuable for minimising this disease in shrimp farming ponds.