Detection of novel strains of cyprinid herpesvirus closely related to koi herpesvirus

Abstract

Cyprinid herpesvirus 3 (CyHV-3) or koi herpesvirus (KHV) is a devastating virus of carp. Using generic primers for the DNA polymerase and the major capsid protein genes of cyprinid herpesviruses, nucleotide sequences divergent from previously described CyHV-3 were obtained. At least 3 novel groups of putative CyHV-3-like viruses were identified, sharing 95 to 98% nucleotide identity with CyHV-3 strains. Carp carrying the CyHV-3 variants did not show clinical signs consistent with CyHV-3 infection and originated from locations with no actual CyHV-3 outbreaks. These strains might represent low-or non-pathogenic variants of CyHV-3.KEY WORDS: Carp · Alloherpesviridae · Phylogeny · CyHV-3 · Variant strains Resale or republication not permitted without written consent of the publisherDis Aquat Org 107: [113][114][115][116][117][118][119][120] 2013 third member of the genus Cyprinivirus, the haema topoietic necrosis herpes virus of goldfish or cyprinid herpesvirus 2 (CyHV-2), which specifically infects goldfish Carassius auratus (Jung & Miyazaki 1995, Davison et al. 2013.The complete genome sequences of 3 CyHV-3 isolates (KHV-U, KHV-I and TUMST1) originating from different geographical locations show only limited differences among each other (Aoki et al. 2007) and share more than 99% DNA sequence identity over the complete genomes (Avarre et al. 2011). Recent papers have described some degree of genetic variability between CyHV-3 isolates based upon nucleotide polymorphisms in specific regions of the genome and variation in the variable number of tandem repeats (VNTRs; Bigarré et al. 2009, Kurita et al. 2009, Avarre et al. 2011. In general, the variability between CyHV-3 strains, particularly in the welldefined open reading frames, is limited, whereas the VNTRs could prove to be valuable markers for epidemiological studies aimed at identifying the source of disease outbreaks.In the present study, novel CyHV DNA sequences were detected in carp and koi tissues by application of generic PCR primers capable of detecting a wide range of cyprinid herpesviruses. The CyHV sequences suggest that infections have occurred with viruses closely related to CyHV-3 but yet genetically distinguishable. A preliminary molecular characterisation of the novel cyprinid herpesviruses was carried out, and the sequences were compared with other herpesviruses from fish. MATERIALS AND METHODSThe material used in this study was taken as part of a surveillance programme or routine diagnostic testing of diseased carp and koi in the UK, Austria, Italy and the Netherlands from 2006 to 2012. Gill and kidney tissues were sampled from common carp/koi carp and stored in absolute ethanol. Total DNA was extracted from approximately 20 mg of tissue using the QIAamp DNA Mini Kit (Qiagen) or 50 µl of a clarified 10% tissue homogenate prepared for virus culture using the QIAamp virus mini Kit (Qiagen) following the manufacturer's protocol. The DNA was eluted in a 50 and a 60 µl volume, respectively. Purified DNA samples were stored at −20°C until...

Is There Any Species Specificity in Infections with Aquatic Animal Herpesviruses?–The Koi Herpesvirus (KHV): An Alloherpesvirus Model

“…The rainbow trout were divided into four groups including fish kept at 15°C (n=10) and 20°C (n=10) water temperature for the infection trial and two additional tanks containing fish (n=5, respectively) kept as negative controls at the same temperatures. The fish were confirmed free from notifiable pathogens like viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) by RT-PCR [29] and additionally with the conventional PCR and nested PCR [30] according to Engelsma et al…”

“…Carp (n=15, 80-100 g) were obtained from a commercial farm in German federal state Thuringia and tested free for CyHV-1, -2 and -3 by PCR [24,30] for 10 years. They were divided into three groups (2×n=5, for the infection trial at 15°C and 20°C, respectively) and as a negative control (n=5).…”

“…Koi herpesvirus isolate KHV-E (D 132), kindly provided by Dr. Keith Way (CEFAS, UK), was allowed to replicate in CCB cells [30,31]. The virus was characterized at the protein level by immunofluorescence assays [24], at the genomic level for quantification by qPCR [32] modified according [31] and at the infectious dose level by titrations onto CCB cells according to Spearman and Kärber [33].…”

Contribution of Next-Generation Sequencing to Aquatic and Fish Virology

“…For instance, the polymerase chain reaction (PCR) is the most widely used method for viral detection [36,37,38]. One of its main strengths is its high sensitivity, although false negatives can occur when: (i) the viral load is below the threshold level, which is often the case in healthy carriers; (ii) the biological samples contain chemical or biological substances that inhibit the enzymatic amplification process; (iii) there is genetic divergence between the sequence of the screened agent and the primers used for the amplification of a targeted area, which is often the case for poorly characterized viruses such as aquatic ones [39], or (iv) the virus evolves rapidly, as is the case for RNA viruses, which undergo reassortment mechanisms. PCR generally targets one or at most a few species and variants.…”

“…However, the complete sequencing of a series of variants of CyHV-3, possibly less pathogenic than the typical virulent strains and for which only short sequences are available, would certainly help formulate hypotheses on the function of some genes involved in virulence [39]. …”