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.     

Detection of koi herpesvirus DNA in tissues of infected fish

A newly recognized herpesvirus, koi herpesvirus or KHV, causes a lethal disease in common carp, Cyprinus carpio , and its colourful strain known as koi or fancy carp. In this study, we report new outbreaks of the disease, present initial characterization of the KHV genome, and describe assays for detection of KHV DNA in infected cells and tissues of infected fish. Restriction endonuclease (RE) profiles of viral DNA derived from two epidemiologically distinct KHV isolates were identical to each other. Cloned KHV BamHI and SphI DNA probes specifically hybridized to KHV DNA, but not to DNAs derived from a variety of other fish herpesviruses. The KHV DNA probes detected KHV DNA in tissues of experimentally infected koi fish by DNA hybridization. The KHV specific polymerase chain assays (PCR) were developed for rapid detection and confirmation of KHV DNA in tissues of infected fish.     

Antiviral activity of exopolysaccharides from Arthrospira platensis against koi herpesvirus

Although koi herpesvirus (KHV) has a history of causing severe economic losses in common carp and koi farms, there are still no treatments available on the market. Thus, the aim of this study was to test exopolysaccharides (EPS) for its antiviral activity against KHV, by monitoring inhibition and cytotoxic effects in common carp brain cells. These substances can be easily extracted from extracellular algae supernatant and were identified as groups of sulphated polysaccharides. In order to reach this aim, Arthrospira platensis, which is well known for its antiviral activity of intra‐ and extracellular compounds towards mammalian herpesviruses, was investigated as standard organism and compared to commercial antiviral drug, ganciclovir, which inhibits the viral DNA polymerization. The antiviral activity of polysaccharides of A. platensis against KHV was confirmed in vitro using qualitative assessment of KHV life cycle genes, and it was found by RT‐PCR that EPS, applied at a concentration of >18 μg mL^?1 and a multiplicity of infection (MOI) of 0.45 of KHV, suppressed the viral replication in common carp brain (CCB) cells even after 22 days post‐infection, entirely. Further, this study presents first data indicating an enormous potential using polysaccharides as an additive for aquacultures to lower or hinder the spread of the KHV and koi herpesvirus disease (KHVD) in future.     

Koi herpesvirus epizootic in cultured carp and koi, Cyprinus carpio L., in Taiwan

Koi herpesvirus (KHV) poses a significant threat to cultured koi and common carp, both Cyprinus carpio L. Since the first reported case in Israel in 1998, KHV has rapidly spread worldwide. This study investigates the spread of KHV to Taiwan by collecting 49 cases of suspected common carp and koi infections from 2003 to 2005 for analysis. Clinical signs included lethargy, anorexia, increased respiratory movements and uncoordinated swimming. Hyperaemia, haemorrhage on body surface and necrotic gill filaments were recorded. Gill epithelial hyperplasia, necrosis and eosinophilic intranuclear inclusion bodies were observed by histological examination, while virions were detected using transmission electron microscopy. By detecting the presence of the KHV thymidine kinase (TK) gene and the KHV 9/5 gene using polymerase chain reaction (PCR), 37 cases were identified as KHV-positive, and the cumulative mortality of infected fish was 70-100%. Positive cases showed identical sequences for the genes analysed, implying that they were of the same origin. For the KHV 9/5 gene sequence, these cases exhibited 100% identity with the Japanese strain (TUMST1, accession number AP008984) and 99% identity with the Israeli (KHV-I, DQ177346) and US (KHV-U, DQ657948) strains. Additionally, a loop-mediated isothermal amplification (LAMP) assay was performed and found to be more sensitive than PCR tests, suggesting its potential use as a rapid diagnostic method for KHV. This is the first epidemiological study of KHV infection in cultured common carp and koi in Taiwan.     

Studies of the ultrastructure and morphogenesis of fish pathogenic viruses grown in cell culture

A birnavirus (infectious pancreatic necrosis virus, IPNV), three rhabdoviruses (viral haemorrhagic septicaemia virus, VHSV; infectious haematopoietic necrosis virus, IHNV; and spring viraemia of carp virus, SVCV) and an iridovirus (isolate from a sheatfish) were investigated with regard to their morphogenetic interactions with cells in culture. In cells infected with birnavirus, a granular viromatrix, single virions randomly distributed in the cytoplasm, viral particles aggregated in pseudocrystals and cytoplasmic tubuli similar in diameter to that of the virus were found. Rhabdoviruses entered the cells by viropexis and replicated within the cytoplasm. Maturation occurred predominantly at the cell membrane and sporadically at membranes of the Golgi cisternae. Inclusion bodies were found partially consisting of viral nucleocapsids. After budding, new virions were found adsorbed to the cell membrane. Viral haemorrhagic septicaemia virus, known to exhibit an atypical shape because of preparative procedures, could be identified by immunostaining using two monoclonal antibodies directed against G- and N-proteins and colloidal gold. Iridoviruses entered the cells by viropexis. Viral particles were found in coated vesicles. Subsequently, vesicles without a clathrin coat were detected. Replication occurred within prominent cytoplasmic inclusion bodies. Isometric viral nucleocapsids were transported in an unknown manner to the cell membrane and matured by budding.     

锦鲤疱疹病毒囊膜蛋白ORF59的克隆、分析及其主要B细胞表位区的原核表达

从感染锦鲤疱疹病毒（Koi herpesvirus,KHV）的锦鲤（Cyprinus carpiokoi）肾脏组织中提取DNA,通过PCR扩增了KHVORF59基因。该基因全长411bp,所编码的蛋白包含136个氨基酸,分子量14.3kDa,等电点（PI）6.91,有12个潜在的O糖基化位点。此研究克隆的KHVORF59基因第130位碱基由G突变为A,使其编码的第44位氨基酸由Ala突变为Thr。采用DNAStar程序,在综合分析二级结构柔性区、蛋白的亲水性、表面可能性和抗原性指数的基础上,预测了KHVORF59蛋白主要B细胞表位,并将其区段的编码序列与KHVORF59完整编码序列分别克隆入原核表达载体pET-32a（＋）,构建重组质粒pET32a-ORF59S和pET32a-ORF59C,转入大肠杆菌Rosetta菌株,IPTG诱导表达。SDS-PAGE及WesternBlot分析显示,pET32a-ORF59S可以高效表达,表达的截短KHVORF59蛋白主要以可溶性形式存在,采用HisBindResin填料,层析纯化了该截短蛋白。     

Case report: concurrent herpesviral and presumptive iridoviral infection associated with disease in cultured shortnose sturgeon,Acipenser brevirostrum (L.), from the Atlantic coast of Canada

Approximately 8 weeks after a chlorine insult associated with the city water supply, shortnose sturgeon, Acipenser brevirostrum (L.), from one group presented with small (3–4 mm) irregular foci of cutaneous pallor that involved the dorsocranial integument with progressive ulceration of the nascent lesions. Various bacterial organisms were isolated from the cutaneous lesions, but not from the internal viscera. Histologically, the nuclei of the intralesional and perilesional epidermal cells often exhibited margination of the chromatin that resulted in a homogenous, pale, amphophilic, tinctorial quality of the nucleoplasm consistent with a herpesvirus infection. In addition, rare lamellar epithelial cells were prominently enlarged due to an abundant, dense, basophilic cytoplasm characteristic of an iridovirus infection. Inoculation of cutaneous lesion and kidney, spleen, liver sample pools from affected shortnose sturgeon onto white sturgeon spleen (WSS‐2) cell line induced cytopathic effect characterized by syncytia formation. Ultrastructural analysis of infected WSS‐2 cells revealed viral particles with a characteristic herpesvirus morphology. Intranuclear hexagonal capsids had a diameter of 95–108 nm, and enveloped particles present in the cytoplasm of infected cells had a diameter of 176–196 nm. This is the first report of a herpesvirus and a possible iridovirus‐like infection in shortnose sturgeon.     

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.     

Koi herpesvirus and carp oedema virus: Infections and coinfections during mortality events of wild common carp in the United States

Koi herpesvirus (KHV; cyprinid herpesvirus‐3) and carp oedema virus (CEV) are important viruses of common and koi carp (Cyprinus carpio); however, the distribution of these viruses in wild common carp in North America is largely unknown. During the summers of 2017 and 2018, 27 mass mortalities of common carp were reported from four states in the USA (Minnesota, Iowa, Pennsylvania and Wisconsin), the majority of which were distributed across eight major watersheds in southern Minnesota. Samples from 22 of these mortality events and from five clinically healthy nearby carp populations were screened for KHV, CEV and SVCV using real‐time polymerase chain reaction (qPCR). KHV was confirmed in 13 mortality events, CEV in two mortality events and coinfections of KHV/CEV in four mortality events. Nucleotide sequence analysis revealed that the KHV and CEV detected here are closely related to European lineages of these viruses. While molecular detection alone cannot conclusively link either virus with disease, the cases described here expand the known range of two important viruses. This is also the first reported detection of KHV and CEV coinfections in wild carp populations.