Antiviral activities of Clinacanthus nutans (Burm.f.) Lindau extract against Cyprinid herpesvirus 3 in koi (Cyprinus carpio koi)

Cyprinid herpesvirus 3 (CyHV‐3) or koi herpesvirus (KHV) is a virulent viral infection in common carp and koi. The disease has caused global epizootic and economic loss in fish aquaculture and in the wild. Clinacanthus nutans (Burm. f.) Lindau is a well‐known medicinal plant used in Thai traditional medicine. Virucidal effects of the plant extract against human herpes simplex virus have been reported. In this study, C. nutans crude extract was tested for antiviral activities against CyHV‐3 in koi carp. Results showed effective antiviral activity against CyHV‐3 pre‐ and post‐infection. The 50% lethal concentration (LC₅₀) of extract was higher than 5 mg/ml. The 50% effective dose (ED₅₀) was 0.99 mg/ml, 0.78 mg/ml, 0.75 mg/ml and 0.71 mg/ml at 1, 2, 3 and 4 hr pre‐infection, respectively. The ED₅₀ from post‐infection tests was 2.05 mg/ml and 2.34 mg/ml at 0 and 24 hr, respectively. These results demonstrated that crude extract expressed antiviral activity against CyHV‐3 and can be applied as a therapeutic agent in common carp and koi aquaculture.     

Detection methods of Cyprinid herpesvirus 2 infection in silver crucian carp (Carassius auratus gibelio) via a pORF72 monoclonal antibody

Cyprinid herpesvirus 2 (CyHV‐2) is the main pathogen responsible for causing haematopoietic necrosis disease in Carassius auratus gibelio. Although many nucleic acid‐based diagnostic methods have been applied, no stable and sensitive immunological diagnostic approaches have been reported. In this study, to detect CyHV‐2 in clinical samples using immunological methods, recombinant ORF72 protein (pORF72), encoded by the CyHV‐2 ORF72 gene, was used as a capture antigen to identify blood and tissues infected with CyHV‐2. First, ORF72 gene was amplified from the CyHV‐2 genome and cloned into a PGEX‐4t‐3 expression vector to produce pORF72 in Escherichia coli. The purified pORF72 was used as an immunogen to prepare monoclonal antibodies. The Western blotting assays revealed that the monoclonal antibody could specifically identify the pORF72. Furthermore, an immunohistochemical protocol and a blood smear method were established to detect CyHV‐2 in carps. The results indicate that the monoclonal antibody against pORF72 could be utilized as an effective detection tool for haematopoietic necrosis disease in Carassius auratus gibelio.     

Ultrastructural analysis of sequential cyprinid herpesvirus 3 morphogenesis in vitro

Cyprinid herpesvirus 3 (CyHV‐3) is an alloherpesvirus, and it is the aetiological agent of koi herpesvirus disease. Although the complex morphogenic stages of the replication cycle of CyHV‐3 were shown to resemble that of other members of the Herpesvirales, detailed analysis of the sequence and timing of these events was not definitively determined. This study describes these features through a time course using cyprinid cell cultures (KF‐1 and CCB) infected with CyHV‐3 (KHV isolate, H361) and analysed by transmission electron microscopy. Rapid viral entry was noted, with high levels of intracellular virus within 1–4 h post‐infection (hpi). Intranuclear capsid assembly, paracrystalline array formation and primary envelopment of capsids occurred within 4 hpi. Between 1 and 3 days post‐infection (dpi), intracytoplasmic secondary envelopment occurred, as well as budding of infectious virions at the plasma membrane. At 5–7 dpi, the cytoplasm contained cytopathic vacuoles, enveloped virions within vesicles, and abundant non‐enveloped capsids; also there was frequent nuclear deformation. Several morphological features are suggestive of inefficient viral assembly, with production of non‐infectious particles, particularly in KF‐1 cells. The timing of this alloherpesvirus morphogenesis is similar to other members of the Herpesvirales, but there may be possible implications of using different cell lines for CyHV‐3 propagation.     

Validation of a serum neutralization test for detection of antibodies specific to cyprinid herpesvirus 3 in infected common and koi carp (Cyprinus carpio)

Cyprinid herpesvirus 3 (CyHV‐3) is the aetiological agent of a serious infective, notifiable disease affecting common carp and varieties. In survivors, infection is generally characterized by a subclinical latency phase with restricted viral replication. The CyHV‐3 genome is difficult to detect in such carrier fish that represent a potential source of dissemination if viral reactivation occurs. In this study, the analytical and diagnostic performance of an alternative serum neutralization (SN) method based on the detection of CyHV‐3‐specific antibodies was assessed using 151 serum or plasma samples from healthy and naturally or experimentally CyHV‐3‐infected carp. French CyHV‐3 isolate 07/108b was neutralized efficiently by sera from carp infected with European, American and Taiwanese CyHV‐3 isolates, but no neutralization was observed using sera specific to other aquatic herpesviruses. Diagnostic sensitivity, diagnostic specificity and repeatability of 95.9%, 99.0% and 99.3%, respectively, were obtained, as well as a compliance rate of 89.9% in reproducibility testing. Neutralizing antibodies were steadily detected in infected carp subjected to restrictive or permissive temperature variations over more than 25 months post‐infection. The results suggest that this non‐lethal diagnostic test could be used in the future to improve the epidemiological surveillance and control of CyHV‐3 disease.     

Identification of a novel cyprinid herpesvirus 3 genotype detected in koi from the East Asian and South‐East Asian Regions

Cyprinid herpesvirus 3 (CyHV‐3) is a highly contagious virus that causes significant morbidity and mortality in common carp Cyprinus carpio L. and considered to be one of the most important pathogens of koi and common carp worldwide. Cyprinid herpesvirus 3 infected consignments imported from East Asian and South‐East Asian regions were identified during quarantine period in Singapore, and virus from a 2005 consignment was successfully isolated in koi fin cells. A combination of sequence analyses and duplex PCR were used to characterize 15 CyHV‐3 isolates detected in koi consignments between 2005 and 2011. Sequence analyses of the enlarged 9/5, SphI‐5 and TK gene regions identified both the Asian 1 (n = 11) and European 4 (n = 4) genotypes. Duplex PCR analysis of two variable marker regions between ORF29 and ORF30 (marker I) as well as ORF133 and its upstream region (marker II) revealed viruses of genotypes J (I⁺⁺II⁺), U/I (I⁻⁻II⁻), an intermediate genotype (I⁺⁺II⁻) and a novel genotype, I⁺⁺II^+Δ, which was identified in viruses from seven different consignments. This novel genotype has a 13‐bp deletion in marker II, while maintaining the I⁺⁺ allele of marker I. The I⁺⁺II^+Δ genotype may have emerged from East Asian and South‐East Asian regions in recent years.     

Evaluation of zebrafish (Danio rerio) as an animal model for the viral infections of fish

Zebrafish (Danio rerio) is a laboratory model organism used in different areas of biological research including studies of immune response and host–pathogen interactions. Thanks to many biological tools available, zebrafish becomes also an important model in aquaculture research since several fish viral infection models have been developed for zebrafish. Here, we have evaluated the possible use of zebrafish to study infections with fish viruses that have not yet been tested on this model organism. In vitro studies demonstrated that chum salmon reovirus (CSV; aquareovirus A) and two alloherpesviruses cyprinid herpesvirus 1 (CyHV‐1) and cyprinid herpesvirus 3 (CyHV‐3) are able to replicate in zebrafish cell lines ZF4 and SJD.1. Moreover, CSV induced a clear cytopathic effect and up‐regulated the expression of antiviral genes vig‐1 and mxa in both cell lines. In vivo studies demonstrated that both CSV and CyHV‐3 induce up‐regulation of vig‐1 and mxa expression in kidney and spleen of adult zebrafish after infection by i.p. injection but not in larvae after infection by immersion. CyHV‐3 is eliminated quickly from fish; therefore, virus clearing process could be evaluated, and in CSV‐infected fish, a prolonged confrontation of the host with the pathogen could be studied.     

鲤疱疹病毒3型ORF136基因编码蛋白多克隆抗体的制备与鉴定

为了对鲤疱疹病毒3型(Cyprinid herpesvirus 3,CyHV-3)ORF136基因编码蛋白进行功能研究和血清学诊断,本实验通过对ORF136基因推导的第31~157位氨基酸序列进行PCR扩增,并与原核载体pET-32a(+)连接,转化至大肠杆菌Rosetta(DE3)感受态后进行IPTG诱导表达,将纯化后的重组蛋白免疫新西兰白兔(Oryctolagus cuniculus)以制备ORF136多克隆抗体,运用Western blot和间接免疫荧光技术对抗体进行鉴定。结果表明,重组融合表达蛋白大小与预期一致,约为35 kD,且主要分布在包涵体中。Western blot分析显示,免疫兔后获得的纯化ORF136多克隆抗体能特异性识别纯化的CyHV-3和感染CyHV-3的KS细胞;间接免疫荧光分析进一步表明ORF136多抗能识别感染CyHV-3的KS细胞。ORF136多克隆抗体的制备为ORF136蛋白功能研究和CyHV-3血清学诊断方法的建立提供了重要基础。     

Establishment of a new cell line susceptible to Cyprinid herpesvirus 3 (CyHV‐3) and possible latency of CyHV‐3 by temperature shift in the cells

A new cell line named CCF‐K104 predominantly consisting of fibroblastic cells showed optimal growth at temperatures from 25 °C to 30 °C. Serial morphological changes in the cells induced by Cyprinid herpesvirus 3 (CyHV‐3) included cytoplasmic vacuolar formation, cell rounding and detachment. Mature virions were purified from CyHV‐3‐infected CCF‐K104 cells by sucrose gradient ultracentrifugation and had a typical herpesvirus structure on electron microscopy. Infectious CyHV‐3 was produced stably in CCF‐K104 cells over 30 viral passages. Our findings showed that CCF‐K104 is a useful cell line for isolation and productive replication of CyHV‐3. A temperature shift from 25 °C to 15 °C or 35 °C did not allow serial morphological changes as observed at 25 °C for 14 days. Under the same conditions, real‐time PCR showed that CyHV‐3 was present with low viral DNA loads, suggesting that CyHV‐3 may establish latent infection in CCF‐K104 cells. Amplification of the left and right terminal repeat sequences of the CyHV‐3 genome arranged in a head‐to‐tail manner was detected by nested PCR following an upshift in temperature from 25 °C to 35 °C. The PCR results suggested that the circular genome may represent a latent form of CyHV‐3.     

Rapid visual detection of cyprinid herpesvirus 2 by recombinase polymerase amplification combined with a lateral flow dipstick

Herpesviral haematopoietic necrosis (HVHN), caused by cyprinid herpesvirus 2 (CyHV‐2), causes significant losses in crucian carp (Carassius carassius) aquaculture. Rapid and convenient DNA assay detection of CyHV‐2 is useful for field diagnosis. Recombinase polymerase amplification (RPA) is a novel isothermal DNA amplification and detection technology that can amplify DNA within 30 min at ~37°C by simulating in vivo DNA recombination. Herein, a rapid and convenient detection assay based on RPA with a lateral flow dipstick (LFD) was developed for detecting CyHV‐2. The highly conserved ORF72 of CyHV‐2 was targeted by specific and sensitive primers and probes. The optimized assay takes only 15 min at 38°C using a water bath, with analysis of products by 2% agarose gel electrophoresis within 30 min. A simple lateral flow strip based on the unique probe in reaction buffer was developed for visualization. The entire RPA‐LFD assay takes 50 min less than the routine PCR method, is 100 times more sensitive and displays no cross‐reaction with other aquatic viruses. The combined isothermal RPA and lateral flow assay (RPA‐LFD) provides a simple, rapid, reliable method that could improve field diagnosis of CyHV‐2 when resources are limited.     

Susceptibility of koi × crucian carp and koi × goldfish hybrids to koi herpesvirus (KHV) and the development of KHV disease (KHVD)

Hybrids of koi, Cyprinus carpio × crucian carp, Carassius carassius and koi × goldfish, Carassius auratus, proved to be susceptible to koi herpesvirus (KHV, syn. CyHV‐3) and developed KHV disease (KHVD). While hybrids of koi × goldfish were partly resistant to mortality following infection by immersion, most koi × crucian carp hybrids died after bath infection. KHV DNA was detected in dead fish but also in all surviving animals by different polymerase chain reactions (PCRs). According to these results, hybrid crossbreeding does not seem to prevent severe losses associated with KHV in terms of inducing KHVD. The present study showed severe losses after a waterborne KHV infection of between 35% and 100% in koi × goldfish and koi × crucian carp hybrids as well as in SPF carp.     

Pathogenesis of acute and chronic diseases caused by cyprinid herpesvirus‐3

The pathogenesis of cyprinid herpesvirus‐3 (CyHV‐3) was studied using different lineages of carp/koi. After exposure to the virus, infected cells were first found in the skin by histopathology and by in situ hybridization. The epidermis of the skin was most severely damaged and often sloughed off in the fish sampled on days 5 through 8, and the fish that were highly sensitive to the virus died within 8 or 10 days after infection. Serum osmolality of the infected fish, particularly just before death, was significantly lower, suggesting that the osmotic shock consequent on the damage to the skin was the direct cause of the acute deaths. On the other hand, clinical and histopathological observations indicate that the carp of a less sensitive lineage most probably died of viral encephalitis around 3 weeks after infection. For these fish, the largest number of infected cells was found in the central nervous system (CNS) sampled on day 12. A substantial amount of viral genome was found in the CNS of carp surviving more than 1 year after the infection. Thus, the CNS is probably a major target for CyHV‐3, and the virus can persistently infect the CNS, presumably establishing latency.     

Validation of a KHV antibody enzyme‐linked immunosorbent assay (ELISA)

Koi herpesvirus (KHV) causes KHV disease (KHVD). The virus is highly contagious in carp or koi and can induce a high mortality. Latency and, in some cases, a lack of signs presents a challenge for virus detection. Appropriate immunological detection methods for anti‐KHV antibodies have not yet been fully validated for KHV. Therefore, it was developed and validated an enzyme‐linked immunosorbent assay (ELISA) to detect KHV antibodies. The assay was optimized with respect to plates, buffers, antigens and assay conditions. It demonstrated high diagnostic and analytical sensitivity and specificity and was particularly useful at the pond or farm levels. Considering the scale of the carp and koi industry worldwide, this assay represents an important practical tool for the indirect detection of KHV, also in the absence of clinical signs.     

Cyprinid herpesvirus 3 as a potential biological control agent for carp (Cyprinus carpio) in Australia: susceptibility of non‐target species

Carp (Cyprinus carpio L.) is a pest species in Australian waterways, and cyprinid herpesvirus 3 (CyHV‐3) is being considered as a potential biological control (biocontrol) agent. An important consideration for any such agent is its target specificity. In this study, the susceptibility to CyHV‐3 of a range of non‐target species (NTS) was tested. The NTS were as follows: 13 native Australian, and one introduced, fish species; a lamprey species; a crustacean; two native amphibian species (tadpole and mature stages); two native reptilian species; chickens; and laboratory mice. Animals were exposed to 100–1000 times the approximate minimum amount of CyHV‐3 required to cause disease in carp by intraperitoneal and/or bath challenge, and then examined clinically each day over the course of 28 days post‐challenge. There were no clinical signs, mortalities or histological evidence consistent with a viral infection in a wide taxonomic range of NTS. Furthermore, there was no molecular evidence of infection with CyHV‐3, and, in particular, all RT‐PCRs for viral mRNA were negative. As a consequence, the results encourage further investigation of CyHV‐3 as a potential biocontrol agent that is specific for carp.     

Analysing codon usage bias of cyprinid herpesvirus 3 and adaptation of this virus to the hosts

The codon usage patterns of open reading frames (ORFs) in cyprinid herpesvirus 3 (CyHV‐3) have been investigated in this study. The high correlation between GC₁₂% and GC₃% suggests that mutational pressure rather than natural selection is the main factor that determines the codon usage and base component in the CyHV‐3, while mutational pressure effect results from the high correlation between GC₃% and the first principal axis of principle component analysis (Axis 1) on the relative synonymous codon usage (RSCU) value of the viral functional genes. However, the interaction between the absolute codon usage bias and GC₃% suggests that other selections take part in the formation of codon usage, except for the mutational pressure. It is noted that the similarity degree of codon usage between the CyHV‐3 and goldfish, Carassius auratus (L.), is higher than that between the virus and common carp, Cyprinus carpio L., suggesting that the goldfish plays a more important role than the common carp in codon usage pattern of the CyHV‐3. The study of codon usage in CyHV‐3 can provide some evidence about the molecular evolution of the virus. It can also enrich our understanding about the relationship between the CyHV‐3 and its hosts by analysing their codon usage patterns.     

New detection of Cyprinid herpesvirus 2 associated with mass mortality in colour crucian carp (Carassius auratus), in China

During October 2016, a mass mortality of colour crucian carp (Carassius auratus), which the affected fish were lethargic, inappetence and anoxic, was occurred in a fish farm located in Chengdu, Sichuan province, China. To elucidate the aetiology of this outbreak, histological and electron microscope examination, molecular investigation were conducted. Pathologic examination revealed multi foci necrosis on haematopoietic organs, gills, hearts and pancreas. Transmission electron microscopy observations exhibited sphere herpesvirus‐like particles distributed amongst the tissues of gill, spleen and kidney. Molecular analysis is verified that the causative agent of this outbreak was Cyprinid herpesvirus 2 (CyHV‐2). This report first report CyHV‐2 in colour crucian carp, which increases the concern about damage of CyHV‐2 and its potential role in species.     

Susceptibility of isogeneic ginbuna Carassius auratus langsdorfii Temminck et Schlegel to cyprinid herpesvirus‐2 (CyHV‐2) as a model species

Herpesviral haematopoietic necrosis (HVHN), caused by cyprinid herpesvirus‐2 (CyHV‐2), has affected the commercial production of the goldfish Carassius auratus and gibelio carp Carassius auratus gibelio. High water temperature treatments are reported to reduce the mortality rate of infected goldfish and elicit immunity in the survivors. To define the mechanism by which this intervention induces resistance, clonal ginbuna Carassius auratus langsdorfii, which is closely related to both species and has been used in fish immunology, may represent a promising model species. In this study, we investigated the susceptibility of clonal ginbuna strains to CyHV‐2 and the effect of high water temperature treatment on infected ginbuna and goldfish. Experimental intraperitoneal infection with CyHV‐2 at 25 °C caused 100% mortality in ginbuna strains, which was accompanied by histopathological changes typical of HVHN. Both infected ginbuna S3n strain and goldfish, exposed to high temperature for 6 days [shifting from 25 °C (permissive) to 34 °C (non‐permissive)], showed reduced mortalities after the 1st inoculation, and subsequent 2nd virus challenge to 0%, indicating induction of immunity. It was concluded that ginbuna showed a similar susceptibility and disease development in CyHV‐2 infection compared to goldfish, suggesting that ginbuna can be a useful fish model for the study of CyHV‐2 infection and immunity.     

New cell lines for efficient propagation of koi herpesvirus and infectious salmon anaemia virus

The production of piscine viruses, in particular of koi herpesvirus (KHV, CyHV‐3) and infectious salmon anaemia virus (ISAV), is still challenging due to the limited susceptibility of available cell lines to these viruses. A number of cell lines from different fish species were compared to standard diagnostic cell lines for KHV and ISAV regarding their capability to exhibit a cytopathic effect (CPE) and to accumulate virus. Two cell lines, so far undescribed, appeared to be useful for diagnostic purposes. Fr994, a cell line derived from ovaries of rainbow trout (Oncorhynchus mykiss), produced constantly high ISA virus (ISAV) titres and developed a pronounced CPE even at high cell passage numbers, while standard cell lines are reported to gradually loose these properties upon propagation. Another cell line isolated from the head kidney of common carp (Cyprinus carpio), KoK, showed a KHV induced CPE earlier than the standard cell line used for diagnostics. A third cell line, named Fin‐4, established from the fin epithelium of rainbow trout did not promote efficient replication of tested viruses, but showed antigen sampling properties and might be useful as an in vitro model for virus uptake or phagocytosis.     

First detection of Cyprinid Herpesvirus 2 (CyHV‐2) in goldfish (Carassius auratus) in France

Massive mortalities of Carassius auratus (L.) occurred in a farm in France during summer 2014. Fish presented anorexia, loss of scales and large amounts of mucus on the gills. Necrosis of the distal tip of the filament and the lamellae, combined with fusion of the lamellae, was observed, as well as necrosis in the hematopoietic organs and in the digestive tract. The histological examination led to hypothesize the implication of a virus in the mortality. The presence of cyprinid herpesvirus 2 (CyHV‐2) in dead fish was demonstrated by amplification and sequencing of portions of the DNA polymerase and helicase genes, both sequences exhibiting 100% identity with CyHV‐2 from Japan. In an attempt to find genetic markers of variation, two regions containing tandem repeats in the Japanese genome were amplified from a virus‐positive sample from the present outbreak. A first region (mB) was fully identical to the Japanese isolate. However, the second region (mA) exhibited a range of deletions and substitutions compared to CyHV‐2 from Japan. This is the first report of CyHV‐2 in France in association with mortality of goldfish and the first identification of a molecular marker for its tracing.