Establishment of a new cell line susceptible to Cyprinid herpesvirus 3 (CyHV‐3) and possible latency

Volume 3: viral, bacterial and fungal infections., 177-244; 19 pp of ref. However, antibody-producing immune cells take time to become activated, and over time, if a fish is no longer sick, anti-KHV antibody production may slow down or stop. Een KHV uitbraak in een dergelijk systeem kan leiden tot sterfte van vele aanwezige karpers waardoor dat specifieke ecosysteem met de daarbij behorende dieren en planten helemaal uit balans zal raken. Temperature shift assay. Taken together, the results presented above suggest that ORF134 deletion does not affect CyHV-3 pathogenicity in common carp and the protective immune response developed by surviving fish. Specific RACE products (see Table S2) were isolated by agarose gel electrophoresis, and the inserts in plasmid clones were capillary sequenced. The expected sizes of the PCR products for the spliced ORF6 are indicated above the bars for primers ORF6s-F291 and -R291 or primers ORF6s-F216 and -R216.

Researchers in Germany were among the earliest to describe KHVD in koi (Bretzinger et al. The absence of cross-contamination between tanks was controlled using the approach described in Figure 9 (data not shown). There were no amplification products detected with eel herpesvirus (AngHV-1), koi herpesvirus (KHV, CyHV-3), or Chinese giant salamander iridovirus (GSIV) genomic DNA (Figure 3). Affinity-purified polyclonal rabbit IgG anti-cIl10 was produced by immunization of rabbits with purified recombinant cIl10 according to a 3 mo standard protocol (Eurogentec). Aquat. 1). Products were visualized on agarose gels stained with ethidium bromide and either photographed under shortwave UV illumination or excised under longwave UV illumination.

Next, an NheI restriction site was inserted into the intergenic region of ORF 136 and ORF 137 (between nucleotides 231835 and 231836 of the KHV genome) using a site-directed mutagenesis kit (Stratagene), resulting in pGEMT-136NheI. Then, the ∆CT of individual samples relative to that of the calibrator sample was calculated as ∆∆CT = ∆CT(sample) – ∆CT(calibrator), and the relative gene expression level was determined as . However, these differences resulted in no alteration of the amino acid sequence (GenBank accession JQ815364), but only one amino acid alteration (GenBank accession EU349287). Generally herpesviruses that infect fishes show latent infection and reactivation (6, 14). Five CyHV-3 genes and a common carp housekeeping gene, β-actin, were detected using TaqMan real-time PCR. Biological invasions: economic and environmental cost of alien plant, animal and microbe species. The second most abundant AngHV-1 envelope protein, encoded by ORF95, was previously shown to be related to the haemagglutinin-esterase protein of infectious salmon anaemia virus (ISAV) (E-value = 10-16) [15].

The reaction mixture (25μL) contained 1.6μM each of inner primer (JBIP and JFIP), 0.2μM each of outer primer (JF3 and JB3), 1mM of dNTP mix, 0.5M betaine (Sigma, USA), 6mMMgCl2, 8U Bst DNA polymerase large fragment (New England Biolabs, USA), 1× the supplied buffer, and 5μL template DNA that was heated at 95°C for 5min. The LAMP reactions were incubated in a heating block set at 64°C for 60min, containing 0, 2, 4, 6, 8, and 10mMMgCl2, respectively. CCBs were used for the culture of two separate CyHV-3 isolates, namely H361 and N076 (which were KHV-U/I and KHV-U/I/TUMST1 strain intermediates, respectively). DNA sequencing.The complete genome sequences were determined commercially (Hitachi Instruments Service Co., Ltd., Tokyo, Japan) by standard shotgun sequencing. 36(10): 823-830 CrossRef, Medline. The infected cultures were exposed to two freeze/thaw cycles at −70 °C, and then centrifuged at 3800 × g (Eppendorf 5804 R). Outbreaks are triggered by handling stress, transportation, and variations in water temperature.(1) The three cyprinid herpesviruses are closely related to anguillid herpesvirus 1; all 4 viruses are grouped within the genus Cyprinivirus.

Thickened and distorted secondary lamellae are lined with plump, sometimes vacuolated epithelium that piles up into confluent sheets with admixed inflammation, particularly at the base of the filaments. Sexually matured common carp (>c.350 mm) form groups in breeding habitats for mating when females spawn (Barus et al., 2002). Alloherpesviruses are distinct and highly diverged from both the Herpesviridae and Malacoherpesviridae. Multiple requests from the same IP address are counted as one view. The maximum likelihood tree was rooted with two mammalian herpesviruses (HHV-1 and HHV-8). However, the cost of the PCR equipment and extensive reaction time have slowed the implementation of PCR-based testing in diagnostic laboratories. ‘They are incredibly dangerous to other organisms, because they raise sediment by sucking detritus from the bottom and filtering it, which eliminates sunlight, and cuts out macrophytes or aquatic plants.

Results—The CyHV3 vaccine provided substantial protective immunity against challenge exposure. This was most probably caused by the progressive deterioration of the branchial epithelium, which in teleosts plays a prominent role in osmoregulation, and which was seen concomitantly with decreasing electrolyte levels in the serum of carp under CyHV-3 infection. The quantification of CyHV-3 DNA in natural sediment using the manual method and external standard virus revealed that its concentration was 1.2×10(4) to 3.3×10(5) copies DNA/kg. The fact that anti-virus antibodies neutralize in vitro the pathogenic effects of the virus emphasizes the central role probably played by the antibodies in anti-CyHV-3 protection in vivo.

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