Channel Catfish Virus Disease Research Articles

Kaempferol is a novel antiviral agent against channel catfish virus infection through blocking viral attachment and penetration in vitro.

Channel catfish virus (CCV, Ictalurid herpesvirus 1) is the causative pathogen of channel catfish virus disease, which has caused high mortality and substantial economic losses in the catfish aquaculture industry. Due to the lack of licensed prophylactic vaccines and therapeutic drugs, the prevention and control of CCV infection seem to remain stagnant. Active compounds from medicinal plants offer eligible sources of pharmaceuticals and lead drugs to fight against endemic and pandemic diseases and exhibit excellent effect against viral infection. In this study, we evaluated the antiviral ability of 12 natural compounds against CCV with cell models in vitro and found kaempferol exhibited the strongest inhibitory compound against CCV infection among all the tested compounds. Correspondingly, kaempferol decreased transcription levels of viral genes and the synthesis of viral proteins, as well as reduced proliferation and release of viral progeny, the severity of the CPE induced by CCV in a dose-dependent manner, based on quantitative real-time PCR (RT-qPCR), western blotting, viral cytopathic effects (CPE) and viral titer assessment. Moreover, time-of-drug-addition assays, virus attachment, and penetration assays revealed that kaempferol exerted anti-CCV activity probably by blocking attachment and internalization of the viral entry process. Altogether, the present results indicated that kaempferol may be a promising candidate antiviral agent against CCV infection, which shed light on the development of a novel and potent treatment for fish herpesvirus infection.

Channel catfish virus entry into host cells via clathrin-mediated endocytosis

Channel catfish virus (CCV), an important member of the family Alloherpesviridae, causes a lethal infection in channel catfish. As with most animal viruses, the initial step of infection by CCV is entry into host cells, which is also a promising antiviral target for CCV disease. This study investigated the mechanism of host cell invasion by CCV using a series of biochemical inhibitor assays in channel catfish cells. CCV infection in host cells was does-dependently inhibited when cells were treated with endosomal acidification inhibitors (5 μM chloroquine, 50 nM bafilomycin A1, and 1 mM ammonium chloride) and hypertonic medium (50 mM sucrose) , which suggests that CCV invades host cells in a manner dependent on low-pH and the endocytic pathway. Moreover, when the cells were pretreated with inhibitors of clathrin-mediated endocytosis, including chlorpromazine (2 μM) and dynasore (50 μM), the CCV infection in the host cells was strongly inhibited. In contrast, the destruction of cellular cholesterol by methyl-β-cyclodextrin and nystatin and inhibition of macropinocytosis had no effect on viral entry. Altogether, these findings indicate that CCV infects host cells via clathrin-mediated endocytosis in a low-pH-dependent manner, suggesting that this CCV entry pathway offers an antiviral target against CCV disease.

Virulence and immunogenicity of blue catfish alloherpesvirus in channel, blue and blue×channel hybrid catfish.

Blue catfish alloherpesvirus (BCAHV) is a novel virus isolated from the blue catfish (Ictalurus furcatus). To date, the ultrastructure, virulence and immunogenicity of BCAHV have not been reported. Given the importance of blue catfish in producing channel ♀ (I. punctatus)×♂ blue (I.furcatus) catfish hybrids and the increasing demand for hybrid catfish in the US catfish industry, the susceptibility of blue, channel and hybrid catfish to BCAHV was assessed. Further, the cross-protective potential of BCAHV against Ictalurid herpesvirus 1 (IcHV1) was investigated in channel and hybrid catfish that survive BCAHV exposure. Neutralization assays revealed BCAHV is refractive (neutralization index [NI]=0) to anti-IcHV1 monoclonal antibody Mab 95, compared to IcHV1 (NI=1.8). Exposure of blue catfish fingerling to 1.3×105 TCID50 /L BCAHV produced cumulative mortality of 51.67±0.70% and pathologic changes similar to those of channel catfish virus disease. No mortality was observed in channel or hybrid catfish. Twenty-eight days post-challenge, surviving channel and hybrid catfish were exposed to 9.4×104 TCID50 /L IcHV1 (LC50 dose), resulting in 100% relative per cent survival compared to naïve cohorts. These data provide baseline information for BCAHV and lay the groundwork for future studies. Data also identify BCAHV as a potential vaccine candidate against IcHV1. Based on host range and immunogenicity evaluations, in addition to genome sequence data from previous studies, BCAHV should be given consideration as a new species of Ictalurivirus.

Development of a Colloidal Gold Immunochromatographic Strip for the Rapid Detection of Channel Catfish Virus.

Channel catfish virus disease (CCVD) has resulted in great economic losses and has restricted the development of fisheries. There is therefore, a need for rapid and efficient diagnostic methods to control the spread of CCVD. A colloidal gold immunochromatographic strip has been developed for the detection of CCVD. In this study, a colloidal gold immunochromatographic strip for channel catfish virus (CCV) detection was developed using the monoclonal antibody 8B6 conjugated with colloidal gold as the detector antibody. A rabbit anti-CCV antibody was used as the capture complex at the test line, and a goat anti-mouse IgG antibody was used as the capture antibody at the control line. The strip was characterized in its specificity, sensitivity, and stability. In addition, an infection experiment was performed to test the applicability of the test strip. The strip was able to detect concentrations of the virus (104 tissue culture infective dose (TCID50)/mL) and showed analytical specificity when tested against other viral pathogens. The strips were still usable after 30 days of storage at 60°C. It was possible to detect CCV experimentally in infected fish within 10-15 min of using the strip. The strip can be used as a rapid and convenient tool for on-site diagnosis to control outbreaks and the spread of CCVD. The immunochromatographic strip was the first to be developed and applied for the detection of CCVD.

Comparison of susceptibility of various fish species to experimental infection with channel catfish virus

Channel catfish virus (CCV) disease is an acute haemorrhagic disease in juvenile channel catfish (Ictalurus punctatus). To date channel catfish is the only species affected by natural outbreaks of the CCV but juvenile large mouth bass (Micropterus salmoides) and silurus (Silurus meriaionalis) have suffered high mortalities in recent years in China. Histopathological phenomenon of sick fish is similar to CCV disease, and the identified virus was CCV. In this report, the pathogenicity of infectious CCV was examined by infection trials on the first known host species, the channel catfish and other teleosts. Our results indicated that there were higher detection rates of CCV from large mouth bass and silurus fish. Channel catfish virus did not induce mortality in other cypriniformes, but histopathological studies revealed that carp might be infected by both bathing and intraperitoneal infection. No deaths, clinical or histopathological signs, were found in the six other species exposed by immersion or injection. Experimental infection studies confirm that CCV infect not only channel catfish but also other species (large mouth bass, silutus and carp). The outbreaks of CCV disease only occurred when the cultured temperature was above 25 °C.

Differential susceptibility of blue catfish, Ictalurus furcatus (Valenciennes), channel catfish, I. punctatus (Rafinesque), and blue × channel catfish hybrids to channel catfish virus

Channel catfish virus (CCV), also known as ictalurid herpesvirus-1 (IHV-1), primarily affects juvenile channel catfish, Ictalurus punctatus (Rafinesque), that are less than 6 months old and was first reported by Fijan (1968). CCV outbreaks can be sporadic, and are usually associated with fry and fingerlings when the water temperature is above 25 C (Plumb 1978). The virus has been reported to be transmitted vertically (Wise, Harrell, Busch & Boyle 1988) and horizontally (reviewed in Plumb 1978). The external signs of CCV disease (CCVD) include exophthalmia, distended abdomen and haemorrhages at the bases of fins. The trunk kidney may exhibit oedema and necrosis, and this tissue is commonly used to confirm the presence of the virus using a tissue culture assay (reviewed in Wolf 1988). In addition, the virus appears to maintain a latent state in leucocytes (Bowser, Munson, Jarboe, Francis-Floyd & Waterstrat 1985), which raises the possibility that latent CCV infection may alter the immune response to other pathogens. It has been over 30 years since it was determined that different strains of catfish exhibited differential resistance to CCV when the virus was mixed with their feed (Plumb, Green, Smitherman & Pardue 1975). A subsequent study by Plumb & Chappell (1978) examined the relative susceptibility of blue catfish, I. furcatus (Valenciennes), and reciprocal blue · channel hybrids to CCV. Since Plumb s (1978) study, there have been no reports on the relative susceptibility of blue catfish or hybrids to CCV. The present study was conducted to determine the relative susceptibility of four different groups of fish: blue catfish, a blue · channel hybrid, a group of channel catfish obtained from 10 farms in the Mississippi Delta, hereafter referred to as the industry pool (IP), and a new strain of catfish produced by the Catfish Genetics Research Unit of USDA at Stoneville, MS (USDA 102 · 103). For each strain of fish, nine replicate tanks were stocked with 40 fish per tank; eight tanks were used for virus challenge while the remaining tank was used as an uninfected control. Fish were placed in 38 L tanks that were filled to 11 L and had a flowthrough rate of 1.8 L min and allowed to acclimatize for 8 days. Fish were fed to satiation twice per day beginning the day after stocking and feeding continued throughout the course of the study. The blue catfish (avg. wt. 3.45 0.18 g) used in this study were of the D and B strain. Fry from seven different spawns were pooled and raised communally in tanks until used in the challenge. The hybrid catfish (avg. wt. 4.10 0.21 g) were produced by crossing female USDA 103 strain channel catfish and D and B strain blue catfish. Twelve hybrid spawns were pooled and used in this Journal of Fish Diseases 2008, 31, 77–79

Susceptibility of channel catfish fry to Channel Catfish Virus (CCV) challenge increases with age

Susceptibility of channel catfish to Channel Catfish Virus Disease (CCVD) has been generally considered to be inversely related to age. However, in experimental immersion challenges, we found that channel catfish fry, 3 to 8 d post hatch (dph), are most resistant to CCV and susceptibility increases with age. Initial studies involved 2 spawns that had high CCV carrier percentage. To determine if the resistance seen in the fry was related to the CCV carrier status of the parents, we selected 4 spawns from CCV negative parents and 2 spawns from CCV positive parents and immersion challenged them at 8, 23, 36 and 60 dph with 0, 2.5 x 10(4) or 2.5 x 10(6) plaque forming units (PFU) of CCV l(-1). Survivors of the low-dose exposed groups were rechallenged at 120 dph with 2.5 x 10(6) PFU CCV l(-1). Each brood demonstrated increasing susceptibility to CCVD with age and only the fish that were initially exposed at 60 dph developed protective immunity. Time course assays evaluating tissue levels of virus in channel catfish exposed to CCV at 7, 21 and 42 dph suggested that the resistance was an early event in the infection process. The resistance in fry was most pronounced in fish from CCV positive spawns and was correlated to neutralizing antibody titers in the maternal parent in the 8 dph challenge. However, other factors may be involved because all groups displayed the initial resistance and subsequent susceptibility to CCVD. The age effect may be an important influence on the progression of CCVD outbreaks and indicates the need to consider age for experimental challenges. Additionally, we documented the level of vertical transmission of CCV. Fry from the 4 positive spawns had a CCV prevalence of 40 to 75 %.

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.

Detection of channel catfish virus DNA in acutely infected channel catfish,Ictalurus punctatus(Rafinesque), using the polymerase chain reaction

Channel catfish virus (CCV) causes an acute haemorrhagic disease in channel catfish,Ictalurus punctatus(Rafinesque), fry and fingerlings. The present study describes a polymerase chain reaction (PCR)‐based assay for detection of CCV DNA in the tissues of acutely infected juvenile catfish. The assay is rapid, sensitive and specifically detects CCV DNA derived from epidemiologically distinct viral isolates. The use of two independent PCR primers sets, each specific for particular CCV genes (open reading frames 8 and 59), provides a means to confirm the results and minimize false‐positive results. The method identifies CCV DNA in several tissues of acutely infected fish, including the brain, blood, intestine, kidney and liver. The CCV PCR assay is useful for the diagnosis of acute CCV disease and for studies to investigate the molecular basis of CCV pathogenesis.

Preliminary Studies of a Newly Developed Subunit Vaccine for Channel Catfish Virus Disease

Abstract The soluble channel catfish virus (CCV) envelope was harvested and used as a vaccine for channel catfish virus disease. Three- to four-day-old eggs of channel catfish Ictalurus punctatus and 1-week-old fry were vaccinated by immersion. A booster was given to subgroups of fry 2 weeks after vaccination. Vaccinated and nonvaccinated control groups were challenged with viable CCV 8 weeks after vaccination. All challenged nonvaccinated control fry died during the first experiment, and 56% died in the second experiment. Survival offish vaccinated as eggs or fry was 31 and 82%, respectively; survival of groups given a booster dose was 81 and 89%, respectively.

Histopathology, electron microscopy and isolation of channel catfish virus in experimentally infected European catfish, Silurus glanis L.

Abstract Two groups of European catfish, Silurus glanis L., fingerlings were infected with channel catfish virus (CCV) by either intraperitoneal injection with 105 TCID50 of CCV, or bathing in water containing 105 TCID50 of CCV per 1·0 ml. The virus was isolated from spleen, intestine and brain of CCV‐injected fish at day 1 and the titres ranged from 102·1 to 103·3 TCID50/g. However, the tissue distribution of CCV was irregular and no virus was isolated after day 3 post‐exposure. In CCV‐bathed fish, the virus was isolated only from the liver of one specimen at day 3 post‐exposure. No clinical signs of CCV disease developed in any of the fish. Specimens in each regime from all sampling periods showed some minor histopathological changes, but there were no differences between treatments. Lesions included oedema and focal haemorrhage in the liver and the spleen was congested. Electron micrographs of tissue samples showed the presence of a few virus particles around the nuclei of kidney, spleen and intestinal cells, and in or around a myelinated nerve within the optic lobes of infected fish during the first 4 days of infection.

Passive Transfer of Sera with Antivirus Neutralizing Activity from Adult Channel Catfish Protects Juveniles from Channel Catfish Virus Disease

Juvenile channel catfish Ictalurus punctatus were found to be protected from experimental infections of channel catfish virus (CCV) after passive transfer of adult sera with anti-CCV neutralizing activity. Juveniles in control groups that received injections of normal serum or saline and were then challenged with CCV experienced average cumulative mortalities of 66 and 71%, respectively. In contrast, juveniles receiving serum with anti-CCV activity (neutralization index of 102.6) via intraperitoneal injections had an average cumulative mortality of 1%. Furthermore, fish in the control groups showed typical signs of channel catfish virus disease and contained virus concentrations of up to 107˙TCID50 (50% tissue culture infective dose endpoint) per gram of tissue. In comparison, no signs of disease were observed among fish receiving serum containing anti-CCV neutralizing activity. These studies demonstrated that serum with anti-CCV neutralizing activity (presumably due to anti-CCV antibody) obtained from adult channel catfish can protect juveniles from lethal challenges with CCV. However, the role of antivirus antibodies in recovery from primary infections and in virus expression during latent infections remains unknown.

Seasonal Variation in Channel Catfish Virus Antibody Titers in Adult Channel Catfish

Abstract Serum antibody titers for channel catfish virus (CCV) were measured in adult channel catfish (Ictalurus punctatus) that had survived an epizootic of channel catfish virus disease (CCVD) as fingerlings. Serum antibody titer data were grouped into four categories for statistical analysis based on the following titer levels: 30, high. Significantly lower antibody titers were observed in January and April than in July and October. The increase in the number of high titers concurrent with elevated water temperature may indicate the existence of carriers persisting 4 years after the original epizootic. Based on the findings of this study, success in isolating CCV from channel catfish during colder months of the year may be enhanced, in part, by the low immune status of the fish at this time.

Comparison of Various Procedures to Detect Neutralizing Antibody to the Channel Catfish Virus in California Brood Channel Catfish

Abstract The first outbreak of channel catfish virus disease (CCVD) since 1973 occurred in California in the fall of 1981 in channel catfish (Ictalurus punctatus). In order to determine the possible spread of CCV to other farms, brood catfish were tested for serum neutralizing (SN) antibody to CCV. Procedures that are commonly used to detect SN antibody in catfish sera gave conflicting results, and tests were conducted to resolve the discrepancy. The lack of standardization caused the variance in results because the brown bullhead (BB) cell line was significantly less (P > .05) sensitive to CCV than the channel catfish ovary (CCO) cell line, less virus could be detected in preformed CCO cell monolayers as compared with the drop technique, and the ability to detect SN was affected by the dilution of serum. The procedure adopted for detecting SN antibody to CCV was to mix equal volumes of CCV solution containing 100 infective doses-50% with sera diluted 1:25 for 1 hr at 25 C, then inoculate BB cells into 6 wells by the drop technique on a 96-well microtiter plate. Using this procedure, SN antibody to CCV was detected in 7 of 17 major brood populations located in southern California and CCV was subsequently isolated from fingerlings at two of the farms.

Histopathological Changes in Channel Catfish (Ictalurus punctatus) Experimentally and Naturally Infected with Channel Catfish Virus Disease

Two groups of fingerling channel catfish (Ictalurus punctatus), one experimentally infected with channel catfish virus disease by transmission through water, and the other collected during an active epizootic, were examined histologically. Previously unreported pathology of pancreatic and brain tissue is described. The virus elicits a histopathological response similar to that reported for the salmonid viruses.

Effects of Temperature on Mortality of Fingerling Channel Catfish (Ictalurus punctatus) Experimentally Infected with Channel Catfish Virus

When water temperature was reduced from 28 to 19 C for channel catfish (Ictalurus punctatus) fingerlings 24 hr after injection with channel catfish virus (CCV), 24% mortality occurred. In comparison, fish held at a constant temperature of 28 C had a mortality of 94% and those held in 19 C water had 14% mortality. Reducing the temperature to 19 C when the first deaths occurred at 28 C (64–72 hr after injection) resulted in 78% mortality. Insignificant mortality occurred in fish injected with Hanks’ balanced salt solution. Results of temperature change are discussed as to their potential value in reducing the effects of CCV disease under cultural conditions.

Fish Viruses: Histopathologic Changes Associated with Experimental Channel Catfish Virus Disease

A histologic examination was carried out on fry with experimentally induced channel catfish virus disease (CCVD). The infection produced a hemorrhagic and necrotic disease with the target organs being the kidneys, liver, digestive tract, and skeletal muscle. The results agree with symptoms of the disease and with published data on quantifications of virus in specific organs.