Channel Catfish Virus Infection 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.

Heparan sulfate is the attachment factor associated with channel catfish virus infection on host cells.

Channel catfish virus (CCV; family Alloherpesviridae) infects channel catfish, causing great harm to aquaculture fisheries and economic development. Attachment is the first step in viral infection and relies on the interaction of virions with components of the extracellular matrix (ECM). The present study aimed to explored the role of the main three ECM components in CCV attachment. Western blotting and quantitative real-time PCR analysis showed that neither collagen nor hyaluronic acid treatments had significant effects on CCV attachment. When exogenous heparin was used as a competitive inhibitor, the adhesion of heparin sodium salt to CCV was dose-dependent. When the concentration of heparin sodium salt was 10 mg/mL, the inhibitory effect on CCV infection of channel catfish ovary (CCO/BB) cells was more than 90%. Heparinase I could significantly prevent CCV attachment by digesting heparan sulfate on the cell surface, and both heparin sodium salt and heparinase I could dose-dependently reduce CCV titers, suggesting that heparin plays an important role in CCV attachment. In addition, the binding experiments between heparin-agarose beads and virions showed that CCV virions could specifically bind to heparin in a dose-dependent manner. The above results suggested that heparan sulfate might be an attachment factor involved in CCV infection of CCO/BB cells. These results increase our understand of the attachment mechanism of CCV and lay the foundation for further research on antiviral drugs.

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.

Channel catfish virus ORF25 and ORF63 genes are essential for viral replication in vitro.

The channel catfish virus (CCV) is a lethal pathogen to aquatic animals that can provoke severe haemorrhagic disease in juvenile channel catfish. Although the CCV genome has been fully sequenced, the molecular mechanisms of CCV infection and pathogenesis are less well known. Genomic DNA replication is a necessary and key event for the CCV life cycle. In this study, the impacts of the putative helicase and primase encoded by viral ORF25 and ORF63 on CCV genome replication and infection were evaluated in channel catfish ovary (CCO) cells. The results showed that the number of CCV genome copies was decreased significantly in virus-infected CCO cells after knockdown of ORF25 and ORF63 using RNA interference. In contrast, the overexpression of ORF25 and ORF63 led to slight increase in the number of virus genome copies. Consistent with the above results, the present results also showed that the expressions of CCV true-late genes which strictly depend on viral DNA replication, were significantly increased or repressed by overexpression or RNA interference targeting viral ORF25 and ORF63 genes in virus-infected CCO cells. In addition, knockdown of ORF25 and ORF63 remarkably inhibited CCV-induced cytopathic effects and decreased progeny virus titres in CCO cells. Moreover, transmission electron microscopy observation of CCO cells infected with CCV accompanied by siRNA targeting the viral ORF25 and ORF63 genes showed that the number of virus particles was remarkably reduced. Taken together, these results indicated that ORF25 and ORF63 are essential for regulating CCV genome replication and CCV-induced infection. Our findings will provide an understanding of the replication mechanisms of CCV and contribute to the development of antiviral strategies for controlling CCV infection in channel catfish culture.

Acyclovir inhibits channel catfish virus replication and protects channel catfish ovary cells from apoptosis

The channel catfish virus (CCV) can cause lethal hemorrhagic infection in channel catfish, resulting in significant economic losses in the fish industry. Effective drugs for the virus are still lacking. Acyclovir is known as a potent antiviral agent against human herpes viruses and some animal DNA viruses. The present study was undertaken to explore the antiviral response and mechanism of acyclovir against CCV in channel catfish ovary (CCO) cells. Acyclovir was able to significantly inhibit the expression of viral genes related to CCV viral DNA synthesis and suppress viral replication at a safe concentration. Furthermore, acyclovir blocked the cytopathic effects and apoptosis induced by CCV, thereby maintaining the normal cellular morphological structure, as shown by the protection of CCO cells from the formation of apoptotic bodies or nuclear fragmentation. Moreover, reverse transcript quantitative polymerase chain reaction (RT-qPCR) demonstrated that acyclovir suppressed the expression of caspase 3, caspase 8 and caspase 9, while there was no significant impact on the expression of the apoptosis-inhibiting gene bcl-2 in CCV-infected cells. In addition, acyclovir did not promote the expression of immune-related genes such as MyD88, Mx1, IRF3, IRF7, IFN-I, NF-kB and IL-1β, suggesting that the antiviral activity of acyclovir to CCV infection is not achieved by facilitating the expression of immune-related genes in CCO cells. Taken together, the results from this study suggest that acyclovir could effectively regulate CCV-induced infection, and thus is a promising therapeutic agent against CCV. Our results will aid our understanding of the pharmacological mechanisms of antiviral agents.

Transcriptomic analysis reveals differentially expressed genes and a unique apoptosis pathway in channel catfish ovary cells after infection with the channel catfish virus

The channel catfish virus (CCV) can cause lethal hemorrhagic infection in juvenile channel catfish, thereby resulting in a huge economic loss to the fish industry. The genome of the CCV has been fully sequenced, and its prevalence is well documented. However, less is known about the molecular mechanisms and pathogenesis of the CCV. Herein, the channel catfish ovary cells (CCO) were infected with CCV and their transcriptomic sketches were analyzed using an RNA sequencing technique. In total, 72,686,438 clean reads were obtained from 73,231,128 sequence reads, which were further grouped into 747,168 contigs. These contigs were assembled into 49,119 unigenes, of which 20,912 and 18,333 unigenes were found in Nr and SwissProt databases and matched 15,911 and 14,625 distinctive proteins, respectively. From these, 3641 differentially expressed genes (DEGs), comprising 260 up-regulated and 3381 down-regulated genes, were found compared with the control (non-infected) cells. For verification, 16 DEGs were analyzed using qRT-PCR. The analysis of the DEGs and their related cellular signaling pathways revealed a substantial number of DEGs that were involved in the apoptosis pathway induced by CCV infection. The apoptosis pathways were further elucidated using standard apoptosis assays. The results showed that CCV could induce extrinsic apoptosis pathway (instead of a mitochondrial intrinsic apoptosis pathway) in CCO cells. This study helps our understanding of the pathogenesis of CCV and contributes to the prevention of CCV infection in channel catfish.

Identification of envelope protein ORF10 of channel catfish herpesvirus

Channel catfish virus (CCV) is a viral pathogen of fry and fingerling channel catfish and can cause significant commercial loss. Previous studies have shown that the CCV virion contains at least 25 predicted structural proteins, including viral protein 10, which is encoded by the orf10 gene of the CCV. In this paper, the orf10 gene was expressed in Escherichia coli and used to produce a specific antibody. Western blot analysis confirmed that open reading frame 10 is an envelope protein. A viral neutralization assay demonstrated that open reading frame 10 antiserum was able to inhibit CCV infection of channel catfish ovary cells, suggesting that viral protein 10 is likely to play an important role in the CCV infection of channel catfish ovary cells.

Generation and Characterization of Monoclonal Antibodies Against Channel Catfish Virus

Three monoclonal antibodies (MAbs) against channel catfish virus (CCV) were generated from mice immunized with purified CCV. Western blot analysis revealed that the MAb 3G12 reacted with three CCV proteins of 94 kDa, 130 kDa, and 170 kDa; the MAb 4C4 reacted with two CCV proteins of 130 kDa and 170 kDa; and the MAb 4D4 reacted with two CCV proteins of 94 kDa and 98 kDa. Indirect immunofluorescence assay showed intense fluorescence in the CCV-infected channel catfish ovary (CCO) cells in areas corresponding to the location of granular structures. In addition, the three MAbs could completely neutralize CCV at a dilution of 1:500. This study demonstrated that these MAbs could recognize CCV specifically and will be useful in the development of diagnostic methods for the detection of fish CCV infection.

Antibody response of channel catfish after channel catfish virus infection and following dexamethasone treatment

Channel catfish virus (CCV, Ictalurid herpesvirus 1) and CCV disease have been extensively studied. Yet, little is known about CCV-host interaction after resolution of the primary infection. In order to determine potential recrudescence of CCV from latency, we established latency by exposing channel catfish juveniles with CCV or a thymidine kinase-negative recombinant (CCVlacZ) at a dose that caused less than 20% mortality. Then, we evaluated antibody response by serially sampling the same fish at 0 (pre-infection), 30, 60 and 90 d post challenge (DPC). We then attempted to induce viral recrudescence by intramuscular administration of dexamethasone and sampled the fish at 2, 4, 7, or 10 d post treatment. Recrudescence was evaluated by leukocyte co-cultivation and cell culture of tissue homogenates but no virus was detected. Western blot data demonstrated the highest number of seropositive fish by 30 DPC and a secondary antibody induction after dexamethasone treatment. The antigen specificity of the secondary response corresponded to viral proteins with molecular masses similar to those recognized by the same fish by 30 DPC. The recognized proteins were predominantly large, ranging from approximately 90 to >200 kDa. Expression analysis of selected virus genes at 90 DPC and following dexamethasone treatment demonstrated occasional immediate-early virus gene expression in peripheral blood leukocytes. Early and late gene expression was rarely detected. The combined data suggest restricted re-activation of CCV in our experimental system. Primary and secondary responses and virus gene expression were demonstrated in CCVlacZ-exposed fish but were less frequent than in CCV-exposed fish.

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

Channel catfish virus gene expression in experimentally infected channel catfish, Ictalurus punctatus (Rafinesque).

Channel catfish virus (CCV) produces an acute haemorrhagic disease in fingerling channel catfish and establishes latent infection in fish that survive the primary infection. This study investigated CCV gene expression in tissues of experimentally infected fish. Reverse transcriptase polymerase chain reaction assays were developed for detection of transcripts expressed by each of the CCV direct repeat region genes in CCV-infected channel catfish ovary cells and in tissues of infected fish. Immediate-early, early and late gene transcripts were detected in the blood, brain, kidney and liver tissues of acutely infected catfish demonstrating active viral replication in multiple tissues during the early stages of CCV infection. However, there was no evidence for viral replication by 24 days post-infection in tissues of fish that survived the acute disease. Viral latency-associated transcripts encoded by CCV direct repeat genes were not detected in latently infected catfish. The results of this study provide a foundation for further studies to investigate the molecular basis of CCV pathogenesis and latency.

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 naı̈ve 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.

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.