cDNA Segment Research Articles

Three Homologous ArfGAPs Participate in Coat Protein I-mediated Transport

ArfGAP1 is a prototype of GTPase-activating proteins for ADP-ribosylation factors (ARFs) and has been proposed to be involved in retrograde transport from the Golgi apparatus to the endoplasmic reticulum (ER) by regulating the uncoating of coat protein I (COPI)-coated vesicles. Depletion of ArfGAP1 by RNA interference, however, causes neither a discernible phenotypic change in the COPI localization nor a change in the Golgi-to-ER retrograde transport. Therefore, we also examined ArfGAP2 and ArfGAP3, closely related homologues of ArfGAP1. Cells in which ArfGAP1, ArfGAP2, and ArfGAP3 are simultaneously knocked down show an increase in the GTP-bound ARF level. Furthermore, in these cells proteins resident in or cycling through the cis-Golgi, including ERGIC-53, beta-COP, and GM130, accumulate in the ER-Golgi intermediate compartment, and Golgi-to-ER retrograde transport is blocked. The phenotypes observed in the triple ArfGAP knockdown cells are similar to those seen in beta-COP-depleted cells. Both the triple ArfGAP- and beta-COP-depleted cells accumulate characteristic vacuolar structures that are visible under electron microscope. Furthermore, COPI is concentrated at rims of the vacuolar structures in the ArfGAP-depleted cells. On the basis of these observations, we conclude that ArfGAP1, ArfGAP2, and ArfGAP3 have overlapping roles in regulating COPI function in Golgi-to-ER retrograde transport.

The Role of Zinc Finger Protein 521/Early Hematopoietic Zinc Finger Protein in Erythroid Cell Differentiation

ZNF521 (zinc finger protein 521) is a transcription factor with an N-terminal transcriptional repressor motif and 30 zinc finger domains. Although a high expression level of ZNF521 in human CD34+ progenitors and hematopoietic malignancies has been demonstrated, the functional role of ZNF521 in hematopoietic cell differentiation has not been clarified. In this study, we analyzed the role of ZNF521 in erythroid cell differentiation using the short hairpin RNA (shRNA)-mediated gene silencing method. Down-regulation of ZNF521 mediated by transient expression of shRNA for ZNF521 resulted in increased synthesis of hemoglobin in K562 and HEL cell lines as compared with control cells. K562-derived clones in which ZNF521 was constitutively silenced by shRNA also showed marked synthesis of hemoglobin and an increased expression level of glycophorin A. Since GATA-1 is the key regulator of erythroid differentiation, the effect of ZNF521 on transcription activity of GATA-1 was analyzed using a luciferase assay. GATA-1 activity was markedly inhibited by ZNF521 in a dose-dependent manner. Deletion analysis of ZNF521 showed that the repressive effect requires an N-terminal repression motif. Furthermore, the direct interaction of ZNF521 with GATA-1 was demonstrated. These results indicate that ZNF521 modulates erythroid cell differentiation through direct binding with GATA-1.

Fn14-TRAIL, a Chimeric Intercellular Signal Exchanger, Attenuates Experimental Autoimmune Encephalomyelitis

Hallmarks of the pathogenesis of autoimmune encephalomyelitis include perivascular infiltration of inflammatory cells into the central nervous system, multifocal demyelination in the brain and spinal cord, and focal neuronal degeneration. Optimal treatment of this complex disease will ultimately call for agents that target the spectrum of underlying pathogenic processes. In the present study, Fn14-TRAIL is introduced as a unique immunotherapeutic fusion protein that is designed to exchange and redirect intercellular signals within inflammatory cell networks, and, in so doing, to impact multiple pathogenic events and yield a net anti-inflammatory effect. In this soluble protein product, a Fn14 receptor component (capable of blocking the pro-inflammatory TWEAK ligand) is fused to a TRAIL ligand (capable of inhibiting activated, pathogenic T cells). Sustained Fn14-TRAIL expression was obtained in vivo using a transposon-based eukaryotic expression vector. Fn14-TRAIL expression effectively prevented chronic, nonremitting, paralytic disease in myelin oligodendrocyte glycoprotein-challenged C57BL/6 mice. Disease suppression in this model was reflected by decreases in the clinical score, disease incidence, nervous tissue inflammation, and Th1, Th2, and Th17 cytokine responses. Significantly, the therapeutic efficacy of Fn14-TRAIL could not be recapitulated simply by administering its component parts (Fn14 and TRAIL) as soluble agents, either alone or in combination. Its functional pleiotropism was manifest in its additional ability to attenuate the enhanced permeability of the blood-brain barrier that typically accompanies autoimmune encephalomyelitis.

Regulation of Cardiac Specific nkx2.5 Gene Activity by Small Ubiquitin-like Modifier

The cardiac specific homeobox gene nkx2.5, a member of the nk-2 class family, plays a central role in cardiogenesis and is a target of the small ubiquitin-like modifier (SUMO). Nkx2.5 was modified by SUMO on its 51st amino acid, a lysine residue conserved across species but absent in other nk-2 members. Conversion of this lysine to an arginine (K51R) substantially reduced Nkx2.5 DNA binding and also its transcriptional activity. Unexpectedly, mutant K51R was targeted by ubiquitin. E3 ligase PIAS proteins PIAS1, PIASx, and PIASy, but not PIAS3, enhanced SUMO-1 attachment to Nkx2.5 on the primary SUMO acceptor site. SUMO-2 linkage to Nkx2.5 was catalyzed only by PIASx and not by other PIAS proteins. SUMO conjugation stabilized the formation of Nkx2.5-containing complexes that led to robust transcriptional activation. Thus, SUMO modification serves as a positive regulator for Nkx2.5 transcriptional activity.

Pyrosequence analysis of expressed sequence tags for Manduca sexta hemolymph proteins involved in immune responses

The tobacco hornworm Manduca sexta is widely used as a model organism to investigate the biochemical basis of insect physiological processes but little transcriptome information is available. To get a broad view of the larval hemolymph proteins, particularly those related to immunity, we synthesized and sequenced cDNA fragments from a mixture of eight total RNA samples: fat body and hemocytes from larvae injected with killed bacteria, fat body, hemocytes, integument and trachea from naïve larvae, and fat body and hemocytes from wandering larvae. Using massively parallel pyrosequencing, we obtained 95,458 M. sexta expressed sequence tags (ESTs) at an average size of 185 bp per read. A majority of the sequences (69,429 reads) could be assembled into 7231 contigs with an average size of 300 bp, 1178 of which had significant similarity with Drosophila genes from various functional groups. Only ∼8% (606) of the contigs matched known M. sexta cDNA sequences, representing 186 of the 375 unique NCBI entries. The remaining 6625 contigs represented newly discovered cDNA segments from this well studied biochemical model insect. A search of the 7231 contigs using Tribolium castaneum, Drosophila melanogaster, and Bombyx mori immunity-related sequences revealed 424 cDNA contigs with significant similarity ( E-value <1×10 −5). These included 218 previously unknown M. sexta sequences coding for putative defense molecules such as pattern recognition receptors, serine proteinases, serpins, Spätzle, Toll-like receptors, intracellular signaling molecules, and antimicrobial peptides.

Pyruvate Dehydrogenase Complex Deficiency Caused by Ubiquitination and Proteasome-mediated Degradation of the E1β Subunit

Congenital deficiencies of the human pyruvate dehydrogenase (PDH) complex are considered to be due to loss of function mutations in one of the component enzymes. Here we describe a case of PDH deficiency associated with the PDH E1beta subunit (PDHB) gene. The clinical phenotype of the patient was consistent with reported cases of PDH deficiency. Cultured skin fibroblasts demonstrated a 55% reduction in PDH activity and markedly decreased immunoreactivity for PDHB protein, compared with healthy controls. Surprisingly, nucleotide sequence analyses of cDNAs corresponding to the patient PDH E1alpha (PDHA1) and PDHB genes revealed no pathological mutations. Moreover, the relative expression level of PDHB mRNA and the rates of transcription and translation of the PDHB gene were normal. However, PDC activity could be restored in cells from this patient following treatment with MG132, a specific proteasome inhibitor, and normal levels of E1beta could be detected in MG132-treated cells. Similar results were obtained following treatment with Tyr-phostin 23 (Tyr23), a specific inhibitor of epidermal growth factor receptor-protein-tyrosine kinase (EGFR-PTK), which also restored E1beta protein levels to those in cells from healthy subjects or from patients with PDHA1 deficiency. The index patient's cells contained a high basal level of EGFR-PTK activity that correlated with the high level of ubiquitination of cellular proteins, although the total EGFR protein levels were similar to those in cells from Elalpha-deficient subjects and healthy subjects. These data indicate that PDH deficiency in our patient involves a post-translational modification in which EGFR-PTK-mediated tyrosine phosphorylation of the E1beta protein leads to enhanced ubiquitination followed by proteasome-mediated degradation. They also provide a novel mechanism accounting for congenital deficiency of the PDH complex and perhaps other inborn errors of metabolism.

Tula and Puumala hantavirus NSs ORFs are functional and the products inhibit activation of the interferon‐beta promoter

The S RNA genome segment of hantaviruses carried by Arvicolinae and Sigmodontinae rodents encodes the nucleocapsid (N) protein and has an overlapping (+1) open reading frame (ORF) for a putative nonstructural protein (NSs). The aim of this study was to determine whether the ORF is functional. A protein corresponding to the predicted size of Tula virus (TULV) NSs was detected using coupled in vitro transcription and translation from a cloned S segment cDNA, and a protein corresponding to the predicted size of Puumala virus (PUUV) NSs was detected in infected cells by Western blotting with an anti-peptide serum. The activities of the interferon beta (IFN-beta) promoter, and nuclear factor kappa B (NF-kappaB)- and interferon regulatory factor-3 (IRF-3) responsive promoters, were inhibited in COS-7 cells transiently expressing TULV or PUUV NSs. Also IFN-beta mRNA levels in IFN-competent MRC5 cells either infected with TULV or transiently expressing NSs were decreased. These data demonstrate that Tula and Puumala hantaviruses have a functional NSs ORF. The findings may explain why the NSs ORF has been preserved in the genome of most hantaviruses during their long evolution and why hantavirus-infected cells secrete relatively low levels of IFNs.

The Alloantigenic Sites of α3α4α5(IV) Collagen: PATHOGENIC X-LINKED ALPORT ALLOANTIBODIES TARGET TWO ACCESSIBLE CONFORMATIONAL EPITOPES IN THE α5NC1 DOMAIN

The Alloantigenic Sites of α3α4α5(IV) Collagen: PATHOGENIC X-LINKED ALPORT ALLOANTIBODIES TARGET TWO ACCESSIBLE CONFORMATIONAL EPITOPES IN THE α5NC1 DOMAIN

Role of DNA Methylation in Stable Gene Repression

A large fraction of the animal genome is maintained in a transcriptionally repressed state throughout development. By generating viable Dnmt1(-)(/)(-) mouse cells we have been able to study the effect of DNA methylation on both gene expression and chromatin structure. Our results confirm that the underlying methylation pattern has a profound effect on histone acetylation and is the major effector of me-H3(K4) in the animal genome. We demonstrate that many methylated genes are subject to additional repression mechanisms that also impact on histone acetylation, and the data suggest that late replication timing may play an important role in this process.

Salicylates Trigger Protein Synthesis Inhibition in a Protein Kinase R-like Endoplasmic Reticulum Kinase-dependent Manner

The non-steroidal anti-inflammatory drug aspirin and its metabolite, sodium salicylate, have profound effects on cellular protein synthesis and cell physiology. However, the underlying mechanism by which they cause these responses remains unclear. We show here that salicylates induce phosphorylation of the alpha-subunit of eukaryotic translation initiation factor 2 (eIF2alpha), resulting in the inhibition of mRNA translation in cells. Exposure of cells to acetyl salicylic acid resulted in strong activation of eIF2alpha stress-activated protein kinase R-like endoplasmic reticulum kinase (PERK). Analysis of fibroblasts with a targeted deletion of the perk gene revealed that PERK is indispensable for triggering the phosphorylation of eIF2alpha as well as the inhibition of protein synthesis induced by salicylates. Although salicylate treatment did not trigger activation of inositol-requiring enzyme 1, there was an increased expression of the pro-apoptotic transcription factor CHOP-(gadd153), a downstream event to eIF2alpha phosphorylation known to mediate endoplasmic reticulum stress-mediated responses. Thus, salicylates selectively trigger an endoplasmic reticulum stress-responsive signaling pathway initiated through activation of PERK to induce their cellular effects.

Cloning and characterization of a stearoyl-ACP desaturase gene from Jatropha curcas

Using degenerate primers and RT-PCR, RACE techniques, a 1491 bp cDNA segment of stearoyl-acyl carrier protein desaturase (SAD) is cloned from developing seeds of Jatropha curcas L. The segment contains a 1191 bp of complete open reading frame (ORF). Analysis in the BLAST on NCBl shows that Jatropha curcas SAD (JSAD) gene encodes a protein precursor composed of a signal peptide of 33 amino acids and a mature peptide of 363 amino acids. The homological analysis shows that JSAD has high level of homology both in nucleotide sequence and in amino acid sequence to other plants SADs. The nucleotide and peptide identity of JSAD to Ricinus communis SAD (RSAD) is up to 89% and 96.2% respectively. Molecular modeling of JSAD indicates that its three-dimensional structure strongly resembled the crystal structure of RSAD.

Molecular cloning and characterization of Duck CD25

The IL-2Rα chain (CD25, Tac) is an essential component of high affinity IL-2Rs, playing critical role for the immune specificity of antigen-activated T-cell clonal expansion. Up to now, no duck cytokine receptor has been described. Here, the cDNA segment of a duck cytokine receptor (duCD25), encoding a 226 aa precursor protein with a 20 aa signal peptide, was isolated. Then a novel mouse monoclonal antibody (mAb) was generated using the prokaryotically expressed duCD25 protein as immunogen. Using this mAb, the endogenous duCD25 molecule was localized on the surface of duck lymphocytes, and the duck IL-2-induced lymphocyte proliferation was further inhibited. Furthermore, flow cytometry analysis showed that duCD25 positive cells were upregulated in ducks infected with avian influenza virus (H9N2). Our findings confirm that duCD25 is a receptor of duck interleukin-2, and duCD25 positive cells play a potential role in H9N2 virus infection.

Construction of rat calcineurin A α cDNA recombinant adenovirus vector and its identification

Rat calcineurin (CaN) A alpha isoform (Ppp3ca) cDNA recombinant adenovirus vector was constructed in order to explore the effect of CaN on the myocardium apoptosis induced by ischemia-reperfusion injury. Total RNA was isolated from the heart of the adult Wistar rat, and Ppp3ca CDS segment of approximate 1.59 kb size was amplified by reverse transcriptional PCR method. Ppp3ca cDNA segment was cloned into pMD18-T Simple vector for sequencing, and the right clone was named T-Ppp3ca. Ppp3ca cDNA segment obtained from T-Ppp3ca was ligated with pShuttle2-IRES-EGFP to construct a recombinant plasmid pShuttle2-Ppp3ca-IRES-EGFP. Ppp3ca-IRES-EGFP expression cassette containing CMV, Ppp3ca-IRES-EGFP and SV40 polyA DNA fragment (3.97 kb) obtained from pShuttle2-Ppp3ca-IRES-EGFP was connected with pAdeno-X backbone sequence to construct a recombinant plasmid pAdeno-Ppp3ca. After being identified by PCR and enzyme digestion, recombinant plasmid pAdeno-Ppp3ca was packaged in HEK293 cells. Supernatant of adenovirus from HEK293 cells was collected after a visible cytopathic effect (CPE) appeared. The DNA of the recombinant adenovirus was extracted with the standard method. The presence of the recombinant adenovirus was verified by PCR. The results showed that sequencing results verified that the PCR product of Ppp3ca gene was identical to GenBank. Agarose electrophoresis showed the bands of recombined plasmid pAdeno-Ppp3ca and the recombinant adenovirus identified by enzyme digestion and PCR were in the right range corresponding with expectation. It was concluded that the recombinant adenovirus carrying rat calcineurin A alpha (Ppp3ca) cDNA as well as a report gene-enhancer green fluorescent protein gene was successfully constructed in this experiment.

Synthesis, Processing, and Composition of the Virion-associated HTLV-1 Reverse Transcriptase

It is not known whether the low infectivity and low virion-associated polymerase activity of human T-cell lymphotropic virus type-1 (HTLV-1) are due to the quantity or quality of the reverse transcriptase (RT), because the protein has not yet been fully characterized. We have developed anti-RT antibodies and constructed HTLV-1 expression plasmids that express truncated or hemagglutinin-tagged Pol polyproteins to examine the maturation and composition of HTLV-1 RT. We detected virion-associated proteins corresponding to RT-integrase (IN) (pr98) and RT (p62) as well as smaller proteins containing the polymerase (p49) or RNase H domains. We have identified the amino acid sequences in the Pol polyprotein that are cleaved by HTLV-1 protease to yield RT and IN. We have also identified the cleavage sites within RT that give rise to the p49 polymerase fragment. Immunoprecipitation of an epitope-tagged p62 subunit coprecipitated p49, indicating that the HTLV-1 RT complex can exist as a p62/p49 heterodimer analogous to the RT of HIV-1 (p66/p51).

Reactive Oxygen Species Induce Tyrosine Phosphorylation of and Src Kinase Recruitment to NO-sensitive Guanylyl Cyclase

Soluble guanylyl cyclase (sGC) is the major cytosolic receptor for nitric oxide (NO) that converts GTP into the second messenger cGMP in a NO-dependent manner. Other factors controlling this key enzyme are intracellular proteins such as Hsp90 and PSD95, which bind to sGC and modulate its activity, stability, and localization. To date little is known about the effects of posttranslational modifications of sGC, although circumstantial evidence suggests that reversible phosphorylation may contribute to sGC regulation. Here we demonstrate that inhibitors of protein-tyrosine phosphatases such as pervanadate and bisperoxo(1,10-phenanthroline)oxovanadate(V) as well as reactive oxygen species such as H2O2 induce specific tyrosine phosphorylation of the beta1 but not of the alpha1 subunit of sGC. Tyrosine phosphorylation of sGCbeta1 is also inducible by pervanadate and H2O2 in intact PC12 cells, rat aortic smooth muscle cells, and in rat aortic tissues, indicating that tyrosine phosphorylation of sGC may also occur in vivo. We have mapped the major tyrosine phosphorylation site to position 192 of beta1, where it forms part of a highly acidic phospho-acceptor site for Src-like kinases. In the phosphorylated state Tyr(P)-192 exposes a docking site for SH2 domains and efficiently recruits Src and Fyn to sGCbeta1, thereby promoting multiple phosphorylation of the enzyme. Our results demonstrate that sGC is subject to tyrosine phosphorylation and interaction with Src-like kinases, revealing an unexpected cross-talk between the NO/cGMP and tyrosine kinase signaling pathways at the level of sGC.

Partial nucleotide sequences and expression patterns of frog ( Rana pipiens) ephrin-A2 and ephrin-A5 mRNA

We have generated 362 bp and 547 bp partial sequences for Rana pipiens ephrin-A2 and ephrin-A5 mRNA, respectively. Translation homologies for the comparable segments of cDNA of chicken, mouse and human are 90.8, 86.9 and 84.4% for the ephrin-A2 sequence and 85.7, 85.0 and 85.0% for the ephrin-A5 sequence. Digoxigenin-labeled riboprobes were prepared and applied by means of in situ hybridization to whole-mounts of the brains of mature adults and expression patterns in tadpoles were also explored. The RNA probes revealed similar posterior (high) to anterior (low) expression gradients in the adult tectum, demonstrating that both ephrin-As are expressed in the adult Ranid frog tectum. Only the ephrin-A2 probe was tested on tadpole brain, yielding an appropriately graded expression pattern similar to the adult.

Molecular analysis of two strains of hantavirus isolated from Zhejiang province

目的 浙江新分离的汉坦病毒ZJ4、ZJ7毒株,与20年前浙江分离的汉坦病毒Z10疫苗株的M片段核苷酸序列进行比较,揭示它们之间的差异.方法提取汉坦病毒RNA,利用逆转录-聚合酶链反应扩增病毒部分M基因片段,克隆入质粒载体,进行核苷酸序列测定及分析.结果汉坦病毒浙江新分离株ZJ4、ZJ7均为 HTN型病毒,ZJ4、ZJ7株与Z10株部分M片段核苷酸的同源性分别为88.3%和92.3%.结论新分离株ZJ4、ZJ7株与疫苗株Z10株分离时间跨度有20多年,其相互间变异不大,证实了HTN型汉坦病毒基因的稳定性。

DTL, the Drosophila Homolog of PIMT/Tgs1 Nuclear Receptor Coactivator-interacting Protein/RNA Methyltransferase, Has an Essential Role in Development

We describe a novel Drosophila gene, dtl (Drosophila Tat-like), which encodes a 60-kDa protein with RNA binding activity and a methyltransferase (MTase) domain. Dtl has an essential role in Drosophila development. The homologs of DTL recently described include PIMT (peroxisome proliferator-activated receptor-interacting protein with a methyltransferase domain), an RNA-binding protein that interacts with and enhances the nuclear receptor coactivator function, and TGS1, the methyltransferase involved in the formation of the 2,2,7-trimethylguanosine (m3G) cap of non-coding small RNAs. DTL is expressed throughout all of the developmental stages of Drosophila. The dtl mRNA has two ORFs (uORF and dORF). The product of dORF is the 60-kDa PIMT/TGS1 homolog protein that is translated from an internal AUG located 538 bp downstream from the 5' end of the message. This product of dtl is responsible for the formation of the m3G cap of small RNAs of Drosophila. Trimethylguanosine synthase activity is essential in Drosophila. The deletion in the dORF or point mutation in the putative MTase active site results in a reduced pool of m3G cap-containing RNAs and lethality in the early pupa stage. The 5' region of the dtl message also has the coding capacity (uORF) for a 178 amino acid protein. For complete rescue of the lethal phenotype of dtl mutants, the presence of the entire dtl transcription unit is required. Transgenes that carry mutations within the uORF restore the MTase activity but result in only partial rescue of the lethal phenotype. Interestingly, two transgenes bearing a mutation in uORF or dORF in trans can result in complete rescue.

Type I, II and X Collagen Gene Expression Pattern in Bone Formation Using rh-BMP2

Bone morphogenic protein (BMP) induces ectopic bone. In that process, there are some tissue-specific collagens (type I, II and X) in osteocartilagenous matrix. And these types of collagen are also observed in the process of ossification in condylar cartilage. We searched for the localization of collagen gene expression in ectopic bone induction using rh-BMP and condylar cartilage ossification. BMP-type I collagen complex were implanted into the subcutaneous tissue of the dorsal region of ICR mice. Then we made specimens 3 days, 1week, and 2weeks after implanting. For condylar cartilage, we pick out condylar region from ICR mice, and made specimens. The probes was made from mouse type I, II and X collagen cDNA segment (kikindly provided by Dr. Ninomiya of Okayama Universy). The process of ectopic bone induction using rh-BMP was the process through the intermediary of rapidly formed cartilagenoid tissue. And there are some difference of type I, II and X collagen gene expressions between the process using rh-BMP and that of condylar cartilage. Those gene expressions imply that the ectopic hard tissue formative cells induced in early stage will take on the characters of osteoblasts and chondrocytes, and that the cells in growing cell layer in cartilagenoid tissue can differentiate into both osteoblasts and chondrocytes.

RT-nested PCR detection of Mourilyan virus in Australian Penaeus monodon and its tissue distribution in healthy and moribund prawns

Mourilyan virus (MoV) is a newly identified virus of Penaeus monodon prawns that is genetically related to the Uukuniemi virus and other phleboviruses of the Bunyaviridae. This paper describes an RT-nested PCR test that can reliably detect between 2 and 6 copies of a synthetic MoV RNA. Total RNA isolated from the lymphoid organ, gills and haemocytes of P. monodon with moderate infections gave comparable amplicon yields in the RT-PCR step of the test. However, in prawns with extremely low-level infections, haemocytes and gill tissue proved slightly more reliable in detecting MoV RNA following nested PCR. The distribution of MoV in tissues of healthy and moribund P. monodon was examined by in situ hybridisation (ISH) using a digoxigenin-labelled DNA probe to a approximately 0.8 kb M RNA segment cDNA insert in clone pMoV4.1. The DNA probe targeted a region in the MoV M RNA segment containing a coding sequence with homology to the C-terminus of the G2 glycoprotein of phleboviruses. In healthy prawns harbouring an unapparent MoV infection, ISH signal primarily occurred in the lymphoid organ, where it was more prominent in hypertrophied cells of 'spheroids' than within cells of normal tubules. ISH signal was also sometimes detected in cells of cuticular epithelium, segmental nerve ganglion and the antennal and tegmental glands. MoV was distributed widely throughout these and other cephalothoracic tissues of mesodermal and ectodermal origin in moribund P. monodon following experimental infection or collected from farm pond edges during disease episodes. Transmission electron microscopy of gill of moribund, captive-reared P. monodon identified spherical (approximately 85 nm diameter) to ovoid MoV particles (approximately 85 x 100 nm) in and around highly necrotic cells in which the nucleus and other organelles had disintegrated. MoV virions co-existed with rod-shaped virions of gill-associated virus and were often seen clustered within cytoplasmic vacuoles or associated with the outer rim of concentric ring-shaped structures comprised of endoplasmic membranes likely to represent degenerated Golgi.