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.

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.

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.

Comparative transcriptional analysis between virulent isolate HN1307 and avirulent isolate GD1108 of grass carp reovirus genotype II.

As a widespread epidemic virus, genotype II of the grass carp reovirus poses a significant threat to the grass carp farming industry in China. Different genotype II isolates cause different degrees of virulence, although the underlying pathogenic mechanisms remain largely unknown. In this work, infections of grass carp with the virulent isolate grass carp reovirus (GCRV)-HN1307 and the avirulent isolate GCRV-GD1108 were performed to reveal a possible mutual transcriptional discrepancy. More differentially expressed genes (DEGs) were identified in the HN1307-infected group, which defined a grossly similar gene ontology (GO) pattern and different pathway landscape as the GD1108-infected group. Gene set enrichment analysis revealed that pathways related to innate immunity and metabolism were reciprocally activated and suppressed, respectively, following infection withHN1307, compared with GD1108. The trend analysis further indicated that immune-related pathways were involved in one of the four statistically significant profiles. Network analysis of transcription factor-gene interactions and protein-protein interactions on the immune-related profile suggested that among the core transcriptional factors (TFs) (UBTF, HCFC1, MAZ, MAX, and NRF1) and the hub proteins (Tlr3, Tlr7, Tlr9, Irf3, and Irf7), the latter were highly enriched in the toll-like receptor signaling pathway. Real-time quantitative PCR performed on the selected mRNAs validated the relative expression. This work will provide insights into the distinct transcriptional signatures from avirulent and virulent isolates of GCRV, which may contribute to the development of products for prevention.

Genome-wide Association Studies of Retinal Vessel Tortuosity Identify Numerous Novel Loci Revealing Genes and Pathways Associated With Ocular and Cardiometabolic Diseases.

Purpose: To identify novel susceptibility loci for retinal vascular tortuosity, to better understand the molecular mechanisms modulating this trait, and reveal causal relationships with diseases and their risk factors. Design: Genome-wide Association Studies (GWAS) of vascular tortuosity of retinal arteries and veins followed by replication meta-analysis and Mendelian randomization (MR). Participants: We analyzed 116 639 fundus images of suitable quality from 63 662 participants from 3 cohorts, namely the UK Biobank (n = 62 751), the Swiss Kidney Project on Genes in Hypertension (n = 397), and OphtalmoLaus (n = 512). Methods: Using a fully automated retina image processing pipeline to annotate vessels and a deep learning algorithm to determine the vessel type, we computed the median arterial, venous and combined vessel tortuosity measured by the distance factor (the length of a vessel segment over its chord length), as well as by 6 alternative measures that integrate over vessel curvature. We then performed the largest GWAS of these traits to date and assessed gene set enrichment using the novel high-precision statistical method PascalX. Main Outcome Measure: We evaluated the genetic association of retinal tortuosity, measured by the distance factor. Results: Higher retinal tortuosity was significantly associated with higher incidence of angina, myocardial infarction, stroke, deep vein thrombosis, and hypertension. We identified 175 significantly associated genetic loci in the UK Biobank; 173 of these were novel and 4 replicated in our second, much smaller, metacohort. We estimated heritability at ∼25% using linkage disequilibrium score regression. Vessel type specific GWAS revealed 116 loci for arteries and 63 for veins. Genes with significant association signals included COL4A2, ACTN4, LGALS4, LGALS7, LGALS7B, TNS1, MAP4K1, EIF3K, CAPN12, ECH1, and SYNPO2. These tortuosity genes were overexpressed in arteries and heart muscle and linked to pathways related to the structural properties of the vasculature. We demonstrated that retinal tortuosity loci served pleiotropic functions as cardiometabolic disease variants and risk factors. Concordantly, MR revealed causal effects between tortuosity, body mass index, and low-density lipoprotein. Conclusions: Several alleles associated with retinal vessel tortuosity suggest a common genetic architecture of this trait with ocular diseases (glaucoma, myopia), cardiovascular diseases, and metabolic syndrome. Our results shed new light on the genetics of vascular diseases and their pathomechanisms and highlight how GWASs and heritability can be used to improve phenotype extraction from high-dimensional data, such as images. Financial Disclosures: The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Purification and concentration of infectious koi herpesvirus using steric exclusion chromatography.

Koi herpesvirus (KHV) is the causative agent of a koi herpesvirus disease (KHVD) inducing high mortality rates in common carp and koi (Cyprinus carpio). No widespread effective vaccination strategy has been implemented yet, which is partly due to side effects of the immunized fish. In this study, we present an evaluation of the purification of infectious KHV from host cell protein and DNA, using the steric exclusion chromatography. The method is related to conventional polyethylene glycol (PEG) precipitation implemented in a chromatographic set-up and has been applied for infectious virus particle purification with high recoveries and impurity removal. Here, we achieved a yield of up to 55% of infectious KHV by using 12% PEG (molecular weight of 6 kDa) at pH 7.0. The recoveries were higher when using chromatographic cellulose membranes with 3-5 µm pores in diameter instead of 1 µm. The losses were assumed to originate from dense KHV precipitates retained on the membranes. Additionally, the use of >0.6 M NaCl was shown to inactivate infectious KHV. In summary, we propose a first step towards a purification procedure for infectious KHV with a possible implementation in fish vaccine manufacturing.

PascalX: a Python library for GWAS gene and pathway enrichment tests.

SUMMARY: 'PascalX' is a Python library providing fast and accurate tools for mapping SNP-wise GWAS summary statistics. Specifically, it allows for scoring genes and annotated gene sets for enrichment signals based on data from, both, single GWAS and pairs of GWAS. The gene scores take into account the correlation pattern between SNPs. They are based on the cumulative density function of a linear combination of χ2 distributed random variables, which can be calculated either approximately or exactly to high precision. Acceleration via multithreading and GPU is supported. The code of PascalX is fully open source and well suited as a base for method development in the GWAS enrichment test context. AVAILABILITY AND IMPLEMENTATION: The source code is available at https://github.com/BergmannLab/PascalX and archived under doi://10.5281/zenodo.4429922. A user manual with usage examples is available at https://bergmannlab.github.io/PascalX/.

Genome-wide analysis identifies genetic effects on reproductive success and ongoing natural selection at the FADS locus.

Identifying genetic determinants of reproductive success may highlight mechanisms underlying fertility and identify alleles under present-day selection. Using data in 785,604 individuals of European ancestry, we identified 43 genomic loci associated with either number of children ever born (NEB) or childlessness. These loci span diverse aspects of reproductive biology, including puberty timing, age at first birth, sex hormone regulation, endometriosis and age at menopause. Missense variants in ARHGAP27 were associated with higher NEB but shorter reproductive lifespan, suggesting a trade-off at this locus between reproductive ageing and intensity. Other genes implicated by coding variants include PIK3IP1, ZFP82 and LRP4, and our results suggest a new role for the melanocortin 1 receptor (MC1R) in reproductive biology. As NEB is one component of evolutionary fitness, our identified associations indicate loci under present-day natural selection. Integration with data from historical selection scans highlighted an allele in the FADS1/2 gene locus that has been under selection for thousands of years and remains so today. Collectively, our findings demonstrate that a broad range of biological mechanisms contribute to reproductive success.

Development of a rapid and sensitive reverse transcription real-time quantitative PCR assay for detection and quantification of grass carp reovirus II.

Hemorrhagic disease of grass carp, which is induced by grass carp reovirus II (GCRV-II), leads to mass mortality in grass carp culture and causes enormous economic loss. However, there is currently no quantitative analysis method for the detection of GCRV-II, which is greatly restricted the etiological and epidemiological study of the disease. In this study a reverse transcription TaqMan PCR (RT-qPCR) assay was developed for the quantitative detection of GCRV-II. The probe and primers targeted location is the segment 6 (S6) region of the GCRV-II genome which is highly conserved. Standard curves were drawn and criteria were confirmed after the determination of the optimum reaction conditions. The species-specific assay showed that the method is highly specific and has no cross reactions with other pathogens. The assay was sufficiently sensitive to detect as low as 10 copies of virus RNA. Moreover, the method has a very good repeatability for batches and inter-batches sample detection. Then the method was applied to detect the virus in tissue samples from clinically infected grass carp, compared with conventional RT-seminested PCR, the RT-qPCR represents a specific value for detection rate of positive samples. In summary, the RT-qPCR was applied and achieved high sensitivity and specificity for GCRV-II detection.

New Insights into Lymphocystis Disease Virus Genome Diversity.

Lymphocystis disease viruses (LCDVs) are viruses that infect bony fish which has been found in different locations across the globe. Four virus species have been classified by the International Committee on Taxonomy of Viruses (ICTV), despite remarkable discrepancies in genome size. Whole genome sequencing and phylogenetic analysis of LCDVs from wild fish from the North Sea and partial sequences from gilthead sea bream of an aquafarm located in the Aegean Sea in Turkey confirm that the LCDV1 genome at 100 kb is approximately half the size of the genomes of LCDV2-4. Since the fish species, of which LCDV1 was isolated, differ taxonomically at the order level, co-speciation can be excluded as the driver of the adaptation of the genome of this nucleocytoplasmic large DNA virus, but may represent an adaptation to the lifestyle of this demersal fish in the northeast Atlantic.

Development of an attenuated vaccine against Koi Herpesvirus Disease (KHVD) suitable for oral administration and immersion.

Since the end of the1990ies, Cyprinid herpesvirus 3 (also known as koi herpesvirus, KHV) has caused mass mortality events of koi and common carp all over the globe. This induced a high economic impact, since the KHV disease cannot be cured up to now, but only prevented by vaccination. Unfortunately, there is only one commercial vaccine available which is not approved in most countries. Therefore, there is an urgent need for new, safe and available vaccines. In this study, a live attenuated vaccine virus was generated by cell culture passages of virulent KHV, and shown to protect carp or koi after immersion or oral application against wild type challenge. An advantage of boost immunization was demonstrated, especially after oral application. Vaccination induced no or mild clinical signs and protecting antibodies have been measured. Additionally, the vaccine virus allowed differentiation of infected from vaccinated animals (DIVA) by PCR. The attenuation of the newly generated vaccine was tracked down to a partial deletion of open reading frame 150. This was confirmed by the generation of engineered ORF150 deletion mutants of wild-type KHV which exhibited a similar attenuation in vivo.

Cross-GWAS coherence test at the gene and pathway level.

Proximal genetic variants are frequently correlated, implying that the corresponding effect sizes detected by genome-wide association studies (GWAS) are also not independent. Methods already exist to account for this when aggregating effects from a single GWAS across genes or pathways. Here we present a rigorous yet fast method for detecting genes with coherent association signals for two traits, facilitating cross-GWAS analyses. To this end, we devised a new significance test for the covariance of datapoints not drawn independently but with a known inter-sample covariance structure. We show that the distribution of its test statistic is a linear combination of χ2 distributions with positive and negative coefficients. The corresponding cumulative distribution function can be efficiently calculated with Davies' algorithm at high precision. We apply this general framework to test for dependence between SNP-wise effect sizes of two GWAS at the gene level. We extend this test to detect also gene-wise causal links. We demonstrate the utility of our method by uncovering potential shared genetic links between the severity of COVID-19 and (1) being prescribed class M05B medication (drugs affecting bone structure and mineralization), (2) rheumatoid arthritis, (3) vitamin D (25OHD), and (4) serum calcium concentrations. Our method detects a potential role played by chemokine receptor genes linked to TH1 versus TH2 immune response, a gene related to integrin beta-1 cell surface expression, and other genes potentially impacting the severity of COVID-19. Our approach will be useful for similar analyses involving datapoints with known auto-correlation structures.

Simultaneous Isolation and Identification of Largemouth Bass Virus and Rhabdovirus from Moribund Largemouth Bass (Micropterus salmoides).

Largemouth bass is an important commercially farmed fish in China, but the rapid expansion of its breeding has resulted in increased incidence of diseases caused by bacteria, viruses and parasites. In this study, moribund largemouth bass containing ulcer foci on body surfaces indicated the most likely pathogens were iridovirus and rhabdovirus members and this was confirmed using a combination of immunohistochemistry, cell culture, electron microscopy and conserved gene sequence analysis. We identified that these fish had been co-infected with these viruses. We observed bullet-shaped virions (100−140 nm long and 50−100 nm in diameter) along with hexagonal virions with 140 nm diameters in cell culture inoculated with tissue homogenates. The viruses were plaque purified and a comparison of the highly conserved regions of the genome of these viruses indicated that they are most similar to largemouth bass virus (LMBV) and hybrid snakehead rhabdovirus (HSHRV), respectively. Regression infection experiments indicated fish mortalities for LMBV-FS2021 and HSHRV-MS2021 were 86.7 and 11.1%, respectively. While co-infection resulted in 93.3% mortality that was significantly (p < 0.05) higher than the single infections even though the viral loads differed by >100-fold. Overall, we simultaneously isolated and identified LMBV and a HSHRV-like virus from diseased largemouth bass, and our results can provide novel ideas for the prevention and treatment of combined virus infection especially in largemouth bass.

Recombinant Lactococcus lactis Expressing Grass Carp Reovirus VP6 Induces Mucosal Immunity Against Grass Carp Reovirus Infection.

Grass carp haemorrhagic disease caused by grass carp reovirus II is a serious disease of the aquaculture industry and vaccination is the only effective method of GCRV protection. In this study, Lactococcus lactis was used as oral vaccine delivery to express the GCRV II VP6 protein. We evaluated the protective efficacy of the live vaccine strain to induce mucosal immune protection. After oral administration, the recombinant strains remained in the hindgut for antigen presentation and increased the survival rate 46.7% and the relative percent survival 42.9%, respectively versus control vaccination. Though L. lactis alone can induce the inflammatory response by stimulating the mucosal immune system, the recombinant L. lactis expressing VP6 greatly enhanced nonspecific immune responses via expression of immune related genes of the fish. Furthermore, both systemic and mucosal immunity was elicited following oral immunization with the recombinant strain and this strain also elicited an inflammatory response and cellular immunity to enhance the protective effect. L. lactis can therefore be utilized as a mucosal immune vector to trigger high levels of immune protection in fish at both the systemic and mucosal levels. L. lactis is a promising candidate for oral vaccine delivery.

Estimating RNA dynamics using one time point for one sample in a single-pulse metabolic labeling experiment.

BACKGROUND: Over the past decade, experimental procedures such as metabolic labeling for determining RNA turnover rates at the transcriptome-wide scale have been widely adopted and are now turning to single cell measurements. Several computational methods to estimate RNA synthesis, processing and degradation rates from such experiments have been suggested, but they all require several RNA sequencing samples. Here we present a method that can estimate those three rates from a single sample. METHODS: Our method relies on the analytical solution to the Zeisel model of RNA dynamics. It was validated on metabolic labeling experiments performed on mouse embryonic stem cells. Resulting degradation rates were compared both to previously published rates on the same system and to a state-of-the-art method applied to the same data. RESULTS: Our method is computationally efficient and outputs rates that correlate well with previously published data sets. Using it on a single sample, we were able to reproduce the observation that dynamic biological processes tend to involve genes with higher metabolic rates, while stable processes involve genes with lower rates. This supports the hypothesis that cells control not only the mRNA steady-state abundance, but also its responsiveness, i.e., how fast steady state is reached. Moreover, degradation rates obtained with our method compare favourably with the other tested method. CONCLUSIONS: In addition to saving experimental work and computational time, estimating rates for a single sample has several advantages. It does not require an error-prone normalization across samples and enables the use of replicates to estimate uncertainty and assess sample quality. Finally the method and theoretical results described here are general enough to be useful in other contexts such as nucleotide conversion methods and single cell metabolic labeling experiments.

Susceptibilities of ten fish cell lines to infection with Tilapia lake virus.

Tilapia lake virus disease (TiLVD) caused by Tilapia lake virus (TiLV) is a great threat to the global tilapia culture industry. Effective prevention and control strategies have not been developed due to limited basic research of pathogenesis of TiLVD. Cell lines from different fish species have been found to be permissive to TiLV infection. In the current study, we comprehensively analyzed TiLV susceptibilities to 10 permanent growing fish cell lines. We found that the highest viral titers were generated onto TiB cells originated from the tilapia species Oreochromis mossambicus, MSF from the largemouth bass Micropterus salmoides, CAMK from the hybrid snakehead Channa argus × Channa maculata and SS derived from the perch species Siniperca chuatsi. Viral copy numbers from these four cell lines ranged from 4 × 107 copies/µL to 4.6 × 108 copies/µL. Confocal immunofluorescent microscopy also indicated that all 10 cell lines can support varying degrees of viral infection and replication. TiLV particles can be observed in cells from randomly selected three fish species using electron microscope. This study will assist in research and development of prevention and control of TiLVD.

Establishment and evaluation of qPCR and real-time recombinase-aided amplification assays for detection of largemouth bass ranavirus.

Largemouth bass ranavirus disease (LMBVD) caused by largemouth bass ranavirus (LMBV) has resulted in severe economic losses in the largemouth bass (Micropterus salmoides) farming industry in China. Early and accurate diagnosis is the key measure for the prevention and control of LMBVD. In this study, a quantitative polymerase chain reaction (qPCR) and a real-time recombinase-aided amplification (real-time RAA) assay were established for the detection of LMBV. The sensitivity and specificity of these two methods, and the efficacy for detection of LMBV from clinical samples were also evaluated. Results showed that the real-time RAA reaction was completed in <30 min at 39℃ with a detection limit of 58.3 copies, while qPCR reaction required 60 min with a detection limit of 5.8 copies. Both methods were specific for LMBV, where no cross-reactions observed with the other tested fish pathogens. Comparing the amplification results of both assays to the results obtained by virus isolation using 53 clinical tissue samples, results showed that the clinical sensitivity of real-time RAA and qPCR were 93.75% and 100% respectively, and the clinical specificity of both were 100%. Our results showed that qPCR is more suitable for quantitative analysis and accurate detection of LMBV in the laboratory, while real-time RAA is more suitable as a point-of-care diagnostic tool for on-site detection and screening of LMBV under farm conditions and in poorly equipped laboratories.

Meta-GWAS Reveals Novel Genetic Variants Associated with Urinary Excretion of Uromodulin.

BACKGROUND: Uromodulin, the most abundant protein excreted in normal urine, plays major roles in kidney physiology and disease. The mechanisms regulating the urinary excretion of uromodulin remain essentially unknown. METHODS: We conducted a meta-analysis of genome-wide association studies for raw (uUMOD) and indexed to creatinine (uUCR) urinary levels of uromodulin in 29,315 individuals of European ancestry from 13 cohorts. We tested the distribution of candidate genes in kidney segments and investigated the effects of keratin-40 (KRT40) on uromodulin processing. RESULTS: Two genome-wide significant signals were identified for uUMOD: a novel locus (P 1.24E-08) over the KRT40 gene coding for KRT40, a type 1 keratin expressed in the kidney, and the UMOD-PDILT locus (P 2.17E-88), with two independent sets of single nucleotide polymorphisms spread over UMOD and PDILT. Two genome-wide significant signals for uUCR were identified at the UMOD-PDILT locus and at the novel WDR72 locus previously associated with kidney function. The effect sizes for rs8067385, the index single nucleotide polymorphism in the KRT40 locus, were similar for both uUMOD and uUCR. KRT40 colocalized with uromodulin and modulating its expression in thick ascending limb (TAL) cells affected uromodulin processing and excretion. CONCLUSIONS: Common variants in KRT40, WDR72, UMOD, and PDILT associate with the levels of uromodulin in urine. The expression of KRT40 affects uromodulin processing in TAL cells. These results, although limited by lack of replication, provide insights into the biology of uromodulin, the role of keratins in the kidney, and the influence of the UMOD-PDILT locus on kidney function.

It is everywhere-A survey on the presence of carp edema virus in carp populations in Germany.

Carp edema virus (CEV) is the causative agent of koi sleepy disease (KSD), a serious gill disease affecting common carp, Cyprinus carpio, and its ornamental variety, koi. After recent detections of the virus in various countries around the world, KSD has emerged as a new global disease in carp. However, the prevalence of the infection in carp populations in a given geographical region has not been studied thoroughly. The present communication reports an investigation into the presence of CEV in carp and koi populations in Germany. For this purpose, gill samples collected from carp and koi populations suffering from gill diseases or collected for a routine examination of their health status were tested for the presence of CEV by PCR. In total, 651 fish samples from 401 carp or koi cases were examined in 2015 and 2016, additional 118 samples from previous studies were included in the examination. CEV was detected in archive samples from carp dating back to 2007, and in koi samples dating back to 2009. From 2015 to 2016, CEV was detected in 69% of cases from carp populations examined from the main carp-producing areas in Germany, and in 41% of the examined cases from koi populations from all over Germany. Clinical KSD occurred mainly from April to June in carp populations at water temperatures ranging from 8 to 12°C and in koi populations at water temperatures ranging from 18 to 22°C. Most fish from clinically affected carp or koi populations harboured high virus loads of above 10,000 copies of CEV-specific DNA per 250 ng DNA, while gills from fish of other fish species from the ponds, including goldfish, grass carp and European perch were found CEV negative or harboured a low virus load. A phylogenetic analysis revealed the presence of multiple CEV variants from genogroup I in carp and genogroup II in koi populations in Germany. Genetically identical genogroup I isolates were detected in carp from different geographical locations in Germany and in other European carp populations. Some German genogroup II variants were identical to variants previously recorded from koi in Asian and other European countries. The data presented here show that CEV is highly prevalent in German common carp and koi populations and implies the spreading of this virus by intense trading of common carp and koi without necessary risk mitigating measures. As infections with this virus may induce serious disease, CEV diagnostic should be included in health surveillance and disease monitoring programmes.