Development of real-time recombinase polymerase amplification (RPA) and RPA combined with lateral flow dipstick (LFD) assays for the rapid and sensitive detection of cyprinid herpesvirus 3.

In this issue, we established rapid, cost-effective, and simple detection methods including recombines polymerase amplification with lateral flow dipstick (RPA-LFD) and real-time RPA for cyprinid herpesvirus 3(CyHV-3), and evaluated their sensitivity, specificity, and applicability, the real-time RPA method could achieve sensitive diagnosis of CyHV-3 within 1.3 copies per reaction, respectively. The real-time RPA method is 10-fold more sensitive than RPA-LFD method. The exact number of CyHV-3 can be calculated in each sample by real-time RPA. The sera from koi also can be tested in these methods. In addition, no cross-reaction was observed with other related pathogens, including carp oedema virus (CEV), spring viraemia of carp virus (SVCV), cyprinid herpesvirus 1(CyHV-1), cyprinid herpesvirus 2(CyHV-2), type I grass carp reovirus (GCRV-I), type II GCRV (GCRV-II), type III GCRV (GCRV-III), and Aeromonas hydrophila.

Disentangling genetic effects on transcriptional and post-transcriptional gene regulation through integrating exon and intron expression QTLs.

Expression quantitative trait loci (eQTL) studies typically consider exon expression of genes and discard intronic RNA sequencing reads despite their information on RNA metabolism. Here, we quantify genetic effects on exon and intron levels of genes and their ratio in lymphoblastoid cell lines, revealing thousands of cis-QTLs of each type. While genetic effects are often shared between cis-QTL types, 7814 (47%) are not detected as top cis-QTLs at exon levels. We show that exon levels preferentially capture genetic effects on transcriptional regulation, while exon-intron-ratios better detect those on co- and post-transcriptional processes. Considering all cis-QTL types substantially increases (by 71%) the number of colocalizing variants identified by genome-wide association studies (GWAS). It further allows dissecting the potential gene regulatory processes underlying GWAS associations, suggesting comparable contributions by transcriptional (50%) and co- and post-transcriptional regulation (46%) to complex traits. Overall, integrating intronic RNA sequencing reads in eQTL studies expands our understanding of genetic effects on gene regulatory processes.

Assessing tropism and genetic traits of carp oedema virus isolates to enhance detection strategies.

Introduction: Carp oedema virus (CEV) is a relatively understudied poxvirus. It exhibits an affinity for gill and skin epithelial cells. Investigations were conducted into selected aspects of CEV biology, with a focus on determining cell and tissue tropism of CEV, acquiring gene sequences and updating CEV tests in fish tissues. Material and Methods: A total of 238 common carp tissue samples from nine aquaculture farms were analysed. The study evaluated the efficacy of intermediate detection of CEV by real-time PCR and in situ hybridisation. The genes encoding protein P4a were sequenced, analysed and aligned in a phylogenetic tree using a molecular evolution model. Results: In situ hybridisation revealed the necessity to validate the Centre for Environment, Fisheries and Aquaculture Science protocols for sampling for CEV detection and to use the tissues for which the virus has the highest tropism, namely the skin and kidneys, rather than solely the gills. The level of genetic variability was determined, and it was shown that CEV mutates systematically. The creation of two distinct phylogenetic clades confirms certain strains' description as Polish isolates. Conclusion: Determining the localisation of CEV genetic material in organs and tissues is pivotal for shaping the World Organisation for Animal Health guidelines. The utility of molecular diagnostics has been demonstrated in the skin and kidney of carp, in addition to the gills, impelling their inclusion in diagnostic protocols. The clusters identified in the phylogenetic tree offer valuable insights for developing the current PCR primers. The prevalence of CEV infection in aquaculture, juxtaposed with its notably lower detection in wild fish, underscores the significance of mandatory molecular diagnostic testing for CEV in carp farming.

Occurrence of herpesvirus in fish.

Introduction: Herpesviruses are common agents in animals of the aquatic environment. They infect many species of fish but only lead to disease in one or two species. Nevertheless, infected fish without clinical symptoms can actively transfer infectious agents to disease-susceptible species. The aim of the study was to identify and prove the natural presence of different herpesviruses. Material and Methods: Koi, Nile tilapia, grass carp, goldfish and crucian carp were infected with a herpesvirus isolate 99% identical to goldfish herpesvirus (GHV) or cyprinid herpesvirus 2 (CyHV-2) obtained from crucian carp. Before and after infection, samples were collected non-lethally at different time points from all five fish species to identify and evaluate the replication of viruses naturally infecting the fish as well as the CyHV-2 experimentally infecting them. Gill swabs and separated leukocytes were subjected to PCR and the results compared. Results: These samples yielded DNA of koi herpesvirus (KHV, also referred to as CyHV-3), GHV and a new herpesvirus. While Asian-lineage CyHV-3 DNA was detected in samples from crucian carp and goldfish, CyHV-2 DNA was found in samples from koi and tilapia. A new, hitherto unknown herpesvirus was identified in samples from grass carp, and was confirmed by nested PCR and sequence analysis. The survival rates were 5% for grass carp, 30% for tilapia, 55% for crucian carp, 70% for koi and 100% for goldfish at 20 days post infection. Evolutionary analyses were conducted and five clusters were visible: CyHV-1 (carp pox virus), CyHV-2 with sequences from koi and tilapia, CyHV-3 with sequences from crucian carp and goldfish, probable CyHV-4 from sichel and a newly discovered herpesvirus - CyHV-5 - from grass carp. Conclusion: The results obtained with the molecular tools as well as from the animal experiment demonstrated the pluripotency of aquatic herpesviruses to infect different fish species with and without visible clinical signs or mortality.