Wayne L. Gray

Effect of handling stress on susceptibility of channel catfish Ictalurus punctatus to Ichthyophthirius multifiliis and channel catfish virus infection

A quantitative bioassay employing immersion exposure was developed for the infection of channel catfish Ictalurus punctatus with the protozoan parasite Ichthyophthirius multifiliis, commonly referred to as ich. This bioassay as well as waterborne challenge of channel catfish with channel catfish virus (CCV) was used to investigate the effect of confinement stress on the sensitivity of the fish to exposure of these pathogens. Infestation by ich was shown to be proportional to the density of infective theronts in the exposure tank and low-water crowding stress was shown to increase susceptibility of catfish to infection. Mortality from CCV was related to the virus exposure dose; however, low-water crowding stress did not affect mortality. Increased susceptibility, due to crowding stress of naive channel catfish to I. multifiliis but not to CCV, suggests a difference in the defence mechanisms. Stress-induced increased susceptibility to I. multifiliis may be due to a suppression of an innate protection mechanism. The lack of effect of stress on CCV mortality may be due to protection afforded by an inducible system which was not affected by the stressor, or the lethal effects of the virus were too fast for the stress to change susceptibility in fish exposed to CCV for the first time.

The simian varicella virus and varicella zoster virus genomes are similar in size and structure.

Simian varicella virus (SVV) DNA was purified from viral nucleocapsids and the molecular structure of the SVV genome was determined. SVV DNA was analyzed by agarose gel electrophoresis of BamHI, BglII, EcoRI, and PstI restriction endonuclease digests. SVV and varicella zoster virus (VZV) DNAs were demonstrated to have distinct restriction endonuclease profiles. Summation of the sizes of individual restriction endonuclease fragments indicate the size of SVV DNA is congruent to 121 kilobase pairs (kbp) or congruent to 76.8 megadaltons (Md). Electron microscopy, lambda exonuclease analysis, and Southern blot DNA hybridizations were utilized to determine the molecular structure of the SVV genome and to construct restriction endonuclease maps. The results indicate that SVV DNA consists of a long component (L, congruent to 100 kbp) covalently linked to a short component (S, congruent to 20 kbp) which is composed of a unique short sequence (Us, 5.3 +/- 0.7 kbp) bracketed by inverted repeat sequences (TRs and IRs, congruent to 7.2 kbp). The presence of 0.5 M PstI restriction endonuclease fragments indicates that the S component may invert relative to the L component and that the genome exists in two major isomeric forms. The findings demonstrate that the SVV and VZV genomes are similar in size and structure.

Effect of dietary cortisol on resistance of channel catfish to infection by Ichthyopthirius multifiliis and channel catfish virus disease

Many diseases of fish are more likely to occur after a period of stress. A number of physiological changes occur in fish during stress including the secretion of cortisol. Cortisol has several effects including the induction of gluconeogenesis and immunosuppression. The latter activity of cortisol is thought to be the reason stress is often followed by a disease outbreak. These experiments were done to determine the role of cortisol, in the absence of stress, in affecting the susceptibility of channel catfish to Ichthyopthirius multifiliis (ich) and channel catfish virus (CCV). Cortisol mixed in the food resulted in reduced liver size and abolished the increase of plasma cortisol usually induced by confinement stress. Dietary cortisol provided at 200-mg/kg feed increased the susceptibility of channel catfish to infection in an immersion challenge with ich theronts. The severity of infection in fish provided cortisol at 100-mg/kg feed was not different than controls. Dietary cortisol at either 100 or 200 mg/kg did not affect mortality due to CCV exposure. These data suggest that increased cortisol suppressed the protective mechanism against ich but not against CCV.

Detection of channel catfish virus DNA in latently infected catfish.

Channel catfish virus (CCV) disease is an acute haemorrhagic disease in juvenile channel catfish (Ictalurus punctatus). While fish that survive primary CCV infection are suspected of being carriers of CCV, little is known concerning CCV latency. In this report, fingerling catfish were infected with CCV by experimental immersion challenge. Infected fish displayed clinical signs of CCV disease, but 22% of infected fish survived the acute disease. At 140 days post-infection, PCR analysis detected CCV DNA in the blood, brain, intestines, kidney, liver and peripheral blood leukocytes of latently infected fish. Further analysis indicated the CCV genome may exist as circular or concatemeric DNA during virus latency. This study, employing an experimental model of CCV disease, confirms that CCV establishes a latent infection of channel catfish.

Simian varicella virus: characterization of virion and infected cell polypeptides and the antigenic cross-reactivity with varicella-zoster virus

Simian varicella virus (SVV) causes a varicella-like disease in non-human primates. In this study, SVV virions were purified from SVV-infected BSC-1 cells by zonal and differential gradient centrifugation and the virion polypeptide composition was analysed by SDS-PAGE. SVV virions had a buoyant density of 1.21 g/ml, identical to the value obtained for varicella-zoster virus (VZV) virions purified by the same method. Electron microscopy of the concentrated SVV virions revealed characteristic herpesvirus morphology. SVV virions consisted of at least 30 polypeptide species ranging from 16K to greater than 200K. The electrophoretic profiles of radiolabelled SVV and VZV virion polypeptides were very similar. Immunoprecipitations of solubilized SVV-infected cell preparations using SVV immune sera revealed at least 18 viral polypeptides with an Mr range of 12K to 142K and six glycoproteins ranging from 46K to 115K. In addition, extensive cross-reactivity between SVV and VZV proteins and glycoproteins was demonstrated by immunoprecipitation with heterologous immune sera. The high degree of antigenic relatedness between SVV and VZV provides further support for simian varicella as a model for VZV infections.

The genomes of simian varicella virus and varicella zoster virus are colinear

Simian varicella virus (SVV) causes an exanthematous disease in non-human primates which is clinically similar to varicella zoster virus (VZV) infection of humans. In this study, the genetic relatedness of SVV and VZV was confirmed and the location of SVV DNA sequences homologous to VZV restriction endonuclease (RE) fragments and viral genes was determined. VZV DNA RE fragments representing 98.3% of the VZV genome were 32P-labeled and hybridized to RE digested, immobilized SVV DNA. Homologous sequences were located throughout the viral DNAs in similar map positions, indicating a colinear relationship between the VZV and SVV genomes. 32P-labeled VZV glycoprotein (gp I, II, III, and IV) and gene 62 DNA probes also hybridized to SVV DNA in a colinear manner. The results suggest that the location of specific SVV genes may be predicted from the known map positions of homologous VZV genes. This study provides further support for SVV infection of non-human primates as a model for VZV infection of humans.

Simian Varicella Virus Expresses a Latency-Associated Transcript That Is Antisense to Open Reading Frame 61 (ICP0) mRNA in Neural Ganglia of Latently Infected Monkeys

ABSTRACT Simian varicella virus (SVV) and varicella-zoster virus (VZV) are closely related alphaherpesviruses that cause varicella (chickenpox) in nonhuman primates and humans, respectively. After resolution of the primary disease, SVV and VZV establish latent infection of neural ganglia and may later reactivate to cause a secondary disease (herpes zoster). This study investigated SVV gene expression in neural ganglia derived from latently infected vervet monkeys. SVV transcripts were detected in neural ganglia, but not in liver or lung tissues, of latently infected animals. A transcript mapping to open reading frame (ORF) 61 (herpes simplex virus type 1 [HSV-1] ICP0 homolog) was consistently detected in latently infected trigeminal, cervical, and lumbar ganglia by reverse transcriptase PCR. Further analysis confirmed that this SVV latency-associated transcript (LAT) was oriented antisense to the gene 61 mRNA. SVV ORF 21 transcripts were also detected in 42% of neural ganglia during latency. In contrast, SVV ORF 28, 29, 31, 62, and 63 transcripts were not detected in ganglia, liver, or lung tissues of latently infected animals. The results demonstrate that viral gene expression is limited during SVV latency and that a LAT antisense to an ICP0 homolog is expressed. In this regard, SVV gene expression during latency is similar to that of HSV-1 and other neurotropic animal alphaherpesviruses but differs from that reported for VZV.

Transcriptional regulation of the channel catfish virus genome direct repeat region

Channel catfish virus (CCV), a member of the herpesvirus family, causes a severe haemorrhagic disease in juvenile channel catfish. In this report, we confirm that CCV gene expression is temporally regulated into immediate-early (IE), early and late phases, similar to that of other herpesviruses. The transcriptional regulation of the 14 genes within the direct repeat region of the CCV genome was determined by Northern hybridization analysis of RNA isolated from infected cells in the presence or absence of metabolic inhibitors. Two CCV genes within the direct repeat, ORFs 1 and 3, expressed IE transcripts. Early RNAs were encoded by ORFs 2-9 and 11-14. ORFs 4, 7 and 10-13 expressed late transcripts after the onset of viral DNA replication. A time-course study conducted without metabolic inhibitors confirmed that CCV direct repeat transcription is temporally regulated. The characterization of CCV transcription during cytolytic infection in vitro will provide a foundation for the analysis of CCV gene expression in tissues of acutely and latently infected catfish.

EXPERIMENTAL SIMIAN VARICELLA VIRUS INFECTION OF ST. KITTS VERVET MONKEYS

Experimental simian varicella virus (SVV) infection of St. Kitts vervet monkeys was evaluated as an animal model to investigate human varicella‐zoster virus (VZV) infections. During the incubation period, viremia disseminated infectious virus throughout the body via infected peripheral blood lymphocytes (PBLs). A vesicular skin rash in the inguinal area, and on the abdomen, extremities, and face appeared on day 7–10 postinfection. Necrosis and hemorrhage in lung and liver tissues from acutely infected monkeys were evident upon histologic analysis. Recovery from simian varicella was accompanied by a rise in the serum neutralizing antibody response to the virus. SVV latency was established in trigeminal ganglia of monkeys which resolved the acute infection. This study indicates that experimental SVV infection of St. Kitts vervets is a useful animal model to investigate SVV and VZV pathogenesis and to evaluate potential antiviral agents and vaccines.

Cloning the simian varicella virus genome in E. coli as an infectious bacterial artificial chromosome

Simian varicella virus (SVV) is closely related to human varicella-zoster virus and causes varicella and zoster-like disease in nonhuman primates. In this study, a mini-F replicon was inserted into a SVV cosmid, and infectious SVV was generated by co-transfection of Vero cells with overlapping SVV cosmids. The entire SVV genome, cloned as a bacterial artificial chromosome (BAC), was stably propagated upon serial passage in E. coli. Transfection of pSVV-BAC DNA into Vero cells yielded infectious SVV (rSVV-BAC). The mini-F vector sequences flanked by loxP sites were removed by co-infection of Vero cells with rSVV-BAC and adenovirus expressing Cre-recombinase. Recombinant SVV generated using the SVV-BAC genetic system has similar molecular and in vitro replication properties as wild-type SVV. To demonstrate the utility of this approach, a SVV ORF 10 deletion mutant was created using two-step Red-mediated recombination. The results indicate that SVV ORF 10, which encodes a homolog of the HSV-1 virion VP-16 transactivator protein, is not essential for in vitro replication but is required for optimal replication in cell culture.