Emergence of epizootic hemorrhagic disease in Europe.

Epizootic hemorrhagic disease (EHD) is a non-contagious arthropod-borne disease transmitted by blood-sucking midges of the genus Culicoides. It affects domestic and wild ruminants, mainly white-tailed deer and cattle. At the end of October and in November 2022, outbreaks of EHD were confirmed in several cattle farms in Sardinia and Sicily. This is the first detection of EHD in Europe. The loss of free status and the lack of effective prophylactic measures could have significant economic consequences for infected countries.

[Emergence of epizootic hemorrhagic disease in Europe]. / Émergence de la maladie hémorragique épizootique en Europe.

Epizootic hemorrhagic disease (EHD) is a non-contagious arthropod-borne disease transmitted by blood-sucking midges of the genus Culicoides. It affects domestic and wild ruminants, mainly white-tailed deer and cattle. At the end of October and in November 2022, outbreaks of EHD were confirmed in several cattle farms in Sardinia and Sicily. This is the first detection of EHD in Europe. The loss of free status and the lack of effective prophylactic measures could have significant economic consequences for infected countries.

Development and evaluation of recombinase polymerase amplification combined with lateral flow dipstick assays for co-detection of epizootic haemorrhagic disease virus and the Palyam serogroup virus.

BACKGROUND: Epizootic haemorrhagic disease virus (EHDV) and the Palyam serogroup viruses (PALV) have led to significant economic losses associated with livestock production globally. A rapid, sensitive and specific method for the detection of EHDV and PALV is critical for virus detection, monitoring, and successful control and elimination of related diseases. RESULTS: In the present study, a recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD) assay for the co-detection of genome segment 1 (Seg-1) of EHDV and PALV was developed and evaluated. The analytical sensitivities of the established RPA-LFD assay in the detection of EHDV and PALV were 7.1 copies/µL and 6.8 copies/µL, respectively. No cross-reaction with other members of the genus Orbivirus, including African horse sickness virus, bluetongue virus, Guangxi orbivirus, Tibet orbivirus and Yunnan orbivirus was observed. The established RPA-LFD assay accurately detected 39 EHDV strains belonging to 5 serotypes and 29 PALV strains belonging to 3 serotypes. The trace back results of quantitative real-time polymerase chain reaction (qRT-PCR) and the established RPA-LFD assay on sentinel cattle were consistent. The coincidence rates of qRT-PCR and the established RPA-LFD assay in 56 blood samples from which EHDV or PALV had been isolated and 96 blood samples collected from cattle farms were more than 94.8 %. The results demonstrated that the established RPR-LFD assay is specific, sensitive and reliable, and could be applied in early clinical diagnosis of EHDV and PALV. CONCLUSIONS: This study highlights the development and application of the RPA-LFD assay in the co-detection of EHDV and PALV for the first time. The assay could be used as a potential optional rapid, reliable, sensitive and low-cost method for field diagnosis of EHDV and PALV.

Generation and characterization of aptamers against grass carp reovirus infection for the development of rapid detection assay.

Grass carp reovirus (GCRV) causes devastating viral haemorrhagic disease in farmed grass carp (Ctenopharyngon idellus). As novel molecular probes, aptamers have been widely applied in rapid diagnosis and efficient therapies against virus or diseases. In this study, three single-stranded DNA (ssDNA) aptamers were selected against GCRV-infected CIK cells via SELEX (systematic evolution of ligands by exponential enrichment technology). Secondary structures predicted by MFOLD indicated that aptamers formed stem-loop structures, and GVI-11 had the lowest ΔG value of -30.84 KJ/mol. Three aptamers could specifically recognize GCRV-infected CIK cells, with calculated dissociation constants (Kd) of 220.86, 176.63 and 278.66 nM for aptamers GVI-1, GVI-7 and GVI-11, respectively, which indicated that they could serve as specific delivery system for antiviral therapies. The targets of aptamers GVI-1, GVI-7 and GVI-11 on the surface of GCRV-infected cells could be membrane proteins, which were trypsin-sensitive. Furthermore, FAM-labelled aptamer GVI-7 could be applied to detect GCRV infection in vivo. It is the first time to generate and characterize aptamers against GCRV-infected cells. These aptamers have great potentials in development of rapid diagnosis technology and antiviral agents against GCRV infection in aquaculture.

A TaqMan-based real-time PCR assay for specific detection of novel duck reovirus in China.

BACKGROUND: In China, Newly emerging duck reovirus (NDRV) variants have been causing major disease problems in cherry valley ducks. NDRV has the potential to cause high morbidity and 5-50% mortality rates. Severe hemorrhagic-necrosis in the liver and spleen were commonly seen in NDRV affected ducks. The availability of upgraded methods for rapid diagnosis of newly emerging DRV variants is crucial for successful DRV infection control and prevention. RESULTS: In this study, we present a TaqMan-based real-time PCR assay (RT-qPCR) for the detection of NDRV infection. Using the conserved regions within the NDRV genome, we designed the specific primers and probe. The lower limit of detection for NDRV infection was 10 copies/µL (Ct values: 38.3) after the optimization of the RT-qPCR conditions. By cross-checking with other duck viral pathogens, no cross-reactivity was observed confirming the assay was highly specific for the detection of NDRV. Reproducibility of the RT-qPCR was confirmed by intra- and inter-assay variability was less than 2.91%(Intra-assay variability of Ct values: 0.07-1.48%; Interassay variability of Ct values: 0.49-2.91%). This RT-qPCR and conventional PCR (cPCR) detected one hundred and twenty samples of NDRV infection from different regions. The result shows that the positive rates were 94.17 and 84.17% respectively. The detection rate of RT-qPCR rapid detection assay was 10% higher than that of the cPCR method. CONCLUSION: This research developed a highly sensitive, specific, reproducible and versatile of RT-qPCR for quantitatively detecting NDRV. It can be used to study the pathogenesis and epidemiology investigation of NDRV.

Development of indirect immunofluorescence assay for TCID50 measurement of grass carp reovirus genotype II without cytopathic effect onto cells.

Grass carp reovirus (GCRV) caused severe hemorrhagic disease with significant losses of fingerling and yearling grass carp, Cyenopharyngodon idellus, in southeast Asian. It was first isolated in 1983 in China, and clade analysis of the different GCRV isolates indicates there are at least three different genotypes I, II, and III. In recent years, GCRV genotype II has been determined as a dominant virus type which cause severe obvious clinical signs in fish but no cytopathic effect onto presently available cell culture. TCID50 is one of standard method to quantity infectious virus particles. In the present study, an indirect immunofluorescence assay (IFA) was developed using antibody against a protein encoded by segment 10 of GCRV genotype II. Moreover, the specific assay to differentitate GCRV of different genotypes and a sensitive assay for determination of GCRV genotype II were developed respectively. The results showed the IFA only can recognize genotype II virus at the lowest initial concentration of 550 genomic copies/ml. Furthermore, comparison of results obtained from qPCR and the TCID50 assay combined IFA was conducted. The results indicated that TCID50 of GCRV isolates JX0901 and HZ08 differs with 2 log steps reduction in the numbers of viruses compared with the number of genome copies detected by qPCR. The immunofluorescence assay developed is sensitive, specific, and the TCID50 combined with IFA will be a standardizable technique for the quantitation and detection of infectious GCRV in cell culture without cytolysis.

Detection of mammalian orthoreovirus type-3 (Reo-3) infections in mice based on serotype-specific hemagglutination protein sigma-1.

BACKGROUND: Reovirus type-3 infections cause severe pathologies in young mice and thus influence animal experiments in many ways. Therefore, the Federation of Laboratory Animal Science Associations (FELASA) recommends an annual screening in laboratory mice as part of a thorough health monitoring program. Based on the high protein sequence homology among the different reovirus serotypes, immunofluorescence antibody assay and other indirect methods relying on the whole virus are presumably cross-reactive to antibodies triggered by mammalian orthoreovirus infections independent of the serotype. METHODS: The serotype-specific protein σ-1 was expressed in Escherichia coli with an N-terminal Strep-tag and a C-terminal His-tag. The purified Strep-rσ-1-His-construct was used to develop an indirect ELISA by testing defined positive and negative sera obtained by experimental infection of mice as well as field sera. RESULTS: The Strep-rσ-1-His-ELISA provided high sensitivity and specificity during validation. Notably, a high selectivity was also observed for sera positively tested for other relevant FELASA-listed pathogens. Screening of field samples indicated that a commercial reovirus type-3-based ELISA might be cross-reactive to other murine reovirus serotypes and thus produces false-positive results. CONCLUSIONS: The prevalence of reovirus type-3 might be overestimated in German animal facilities and most likely in other countries as well. The occurrence of other reovirus serotypes, however, raises the question if murine health monitoring programs should be extended to these pathogens.

A multiplex xTAG assay for the simultaneous detection of five chicken immunosuppressive viruses.

BACKGROUND: Chicken anemia virus (CAV), avian reovirus (ARV), infectious bursal disease virus (IBDV), Marek's disease virus (MDV) and reticuloendotheliosis virus (REV) all cause immunosuppressive disease in birds through vertical or horizontal transmission. Mixed infections with these immunosuppressive pathogens lead to atypical clinical signs and obstruct accurate diagnoses and epidemiological investigations. Therefore, it is essential to develop a high-throughput assay for the simultaneous detection of these immunosuppressive viruses with high specificity and sensitivity. The aim of this study was to establish a novel method using a RT-PCR assay combined with fluorescence labeled polystyrene bead microarray (multiplex xTAG assay) to detect single or mixed viral infections. RESULTS: The results showed that the established xTAG assay had no nonspecific reactions with avian influenza virus (AIV), infectious bronchitis virus (IBV), newcastle disease virus (NDV), infectious laryngotracheitis virus (ILTV), Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS). The limit of detection was 1.0 × 103 copies/µL for IBDV and 1.0 × 102copies/µL for the other four viruses. Ninety field samples were tested and the results were confirmed using conventional RT-PCR methods. The detection results of these two methods were 100% consistent. The established multiplex xTAG assay allows a high throughput and simultaneous detection of five chicken immunosuppressive viruses. CONCLUSION: The multiplex xTAG assay has been showed to be an additional tool for molecular epidemiology studies of five chicken immunosuppressive viruses in the poultry industry.

Development of a VP38 recombinant protein-based indirect ELISA for detection of antibodies against grass carp reovirus genotype II (iELISA for detection of antibodies against GCRV II).

Currently, serological assays for grass carp reovirus genotype II (GCRV-II) diagnosis are not available. In this study, an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against GCRV-II was developed. The structural protein VP38 of GCRV-II was used as the coating antigen. Monoclonal antibodies (mAb) against IgM of grass carp labelled with HRP were used as a secondary antibody. The antigen concentration and serum dilution were optimized using chess board titration. Furthermore, the specificity of indirect ELISA assay was confirmed by cross check with sera positive for other grass carp pathogens. In comparison with results obtained from indirect immunofluorescence assay (IFA) and Western blot by testing of 60 serum samples to evaluate the sensitivity and specificity of the ELISA, agreement between 90% and 96.7% was reached, respectively. A serological survey was performed using the assay with grass carp field serum samples. The seropositive rate of the 242 serum samples was 69.8%. In conclusion, the developed indirect ELISA is a very specific and sensitive test that will be useful for large-scale serological surveys to detect indirectly GCRV II infections as well as to monitor the changes of antibody level after immunization.

A double-stranded probe coupled with isothermal amplification for qualitative and quantitative detection of avian reovirus.

We applied a probe-based real-time loop-mediated isothermal amplification (Cy5-RTqLAMP) technique targeting the avian reovirus (ARV) S3 gene to develop a rapid, sensitive, and specific method for virus detection and quantification. This test specifically detected the presence of ARV, but not other viruses or bacteria present in clinical or artificially spiked samples, including Newcastle disease virus, infectious bursal disease virus, fowl adenovirus, Marek's disease virus, Escherichia coli, and Salmonella spp. This test can detect ARV in less than one hour with an analytical sensitivity of 10 viral gene copies and 1 fg of total cDNA. The Cy5-RTqLAMP does not yield false positive results and is 100 times more sensitive than conventional PCR. This test was shown to be able to detect the presence of ARV in clinical samples. A similar strategy may be used for detection of other important human and animal viral pathogens.

Serological evidence of human infection with Pteropine orthoreovirus in Central Vietnam.

Pteropine orthoreovirus, potentially of bat origin, has been reported to cause respiratory tract infections among human beings in Southeast Asia. Twelve IgG ELISA-positive cases with antibodies against Pteropine orthoreovirus were detected among 272 human serum samples collected between March and June 2014 from in and around Hue City, Central Vietnam. These 12 cases were IgM ELISA negative. Neutralizing antibodies were also detected among six of these cases with the highest titer of 1:1,280 in 2 cases (both female, 32 and 68 years old, respectively). This is the first report of human infection with Pteropine orthoreovirus in Central Vietnam. These findings indicate the need for surveillance on Pteropine orthoreovirus infections in Southeast Asia to enable prevention and control strategies to be developed should a change in virulence occur.

Pteropine orthoreovirus infection among out-patients with acute upper respiratory tract infection in Malaysia.

This study aims to assess the incidence rate of Pteropine orthreovirus (PRV) infection in patients with acute upper respiratory tract infection (URTI) in a suburban setting in Malaysia, where bats are known to be present in the neighborhood. Using molecular detection of PRVs directly from oropharyngeal swabs, our study demonstrates that PRV is among one of the common causative agents of acute URTI with cough and sore throat as the commonest presenting clinical features. Phylogenetic analysis on partial major outer and inner capsid proteins shows that these PRV strains are closely related to Melaka and Kampar viruses previously isolated in Malaysia. Further study is required to determine the public health significance of PRV infection in Southeast Asia, especially in cases where co-infection with other pathogens may potentially lead to different clinical outcomes.

Development and application of molecular methods (PCR) for detection of Tasmanian Atlantic salmon reovirus.

Molecular (PCR) diagnostic tests for the detection and identification of aquareovirus in general, and Tasmanian Atlantic salmon reovirus (TSRV) specifically, were developed, and their diagnostic sensitivity and specificity were determined and compared with virus isolation in cell culture. Intralaboratory and interlaboratory comparison of PCR (conventional hemi-nested RT-PCR & RT-qPCR) and virus isolation in cell culture using finfish cell lines, CHSE-214 and EPC, was carried out for the detection and identification of TSRV using field samples of farmed Atlantic salmon Salmo salar, L. from various aquaculture sites around Tasmania. The interlaboratory comparison of diagnostic methods was carried out between two laboratories, AAHL-CSIRO and DPIPWE-Tasmania. A total of 144 fish from nine sites (12-33 fish per site) were sampled from two regions of Tasmania (Tamar River estuary in the north and Huon River estuary in the south-east) during late spring to early summer of 2009, and the data were analysed using different statistical approaches. The prevalence of TSRV ranged from 6% to 22% in both regions. All the diagnostic methods (data from both laboratories) had high specificity, while the estimated sensitivity varied between tests with RT-qPCR being the most sensitive (95.2%) method followed by virus isolation and then conventional hemi-nested RT-PCR.

Detection of grass carp reovirus (GCRV) with monoclonal antibodies.

Grass carp reovirus (GCRV) is a pathogen that causes hemorrhagic disease of grass carp. It is the most serious infectious disease of carp and causes serious losses of fingerlings of grass carp and black carp. In this study, a recombinant VP4, one of the viral core proteins, was constructed with a histidine tag and expressed at a high level in E. coli, and the expressed protein was mainly found in the form of inclusion bodies. The expressed VP4 protein was recognized by an anti-His-tag monoclonal antibody and goat anti-GCRV serum. Four monoclonal antibodies (16B7, 39E12, 13C3 and 14D1) against the recombinant VP4 protein were produced. These MAbs did not react with any of the tested viruses or fish cells lines in the ELISA tests except GCRV. In western blotting analysis, a protein band was observed when the recombinant VP4 protein of GCRV was used as an antigen, but a 68-kDa band was observed when natural capsid proteins of GCRV were used as antigens. Furthermore, a sandwich ELISA was developed for detection of GCRV. The detection limit of the test was 105 TCID50 of GCRV per mL.

Use of IgY antibody to recombinant avian reovirus σC protein in the virus diagnostics.

Avian reovirus (ARV) is an important agent of several diseases causing considerable losses in poultry farming. An outer capsid protein (σC) of ARV, is known as a virus-cell attachment protein essential for virus infectivity. In this study, the σC gene of ARV was cloned and expressed in Escherichia coli. The expressed recombinant protein was used as immunogen for raising a specific IgY antibody in laying hens. At 14 weeks post immunization, the antibody titers in serum and egg yolk reached 302,000 and 355,000, respectively. The IgY antibody was capable to neutralize ARV in BHK-21 cells and it strongly reacted in ELISA with ARV but not with heterologous viruses. The IgY antibody detected ARV in field samples of infected animal tissues in dot blot assay. These results suggest that an efficient, economic and rapid diagnostics of ARV can be performed routinely using the IgY antibody against a recombinant ARV σC protein.

Epizootic hemorrhagic disease in a yak.

Epizootic hemorrhagic disease virus (EHDV) infection was diagnosed in a 3-year-old yak. The yak had signs of intermittent tremors, dysphagia, oral ulcerative lesions, hemorrhagic enteritis, tachypnea, and thrombocytopenia. Postmortem diagnostics confirmed EHDV (serotype 2) using reverse-transcriptase polymerase chain reaction (RT-PCR). Gross and histopathological results were consistent with EHDV reported in other species.

Épizootie hémorragique chez un yack. Une infection par le virus de la maladie épizootique hémorragique du cerf a été diagnostiquée chez un yack âgé de 3 ans. Le yack présentait des signes de tremblements intermittents, de dysphagie, de lésions ulcératives buccales, d'entérite hémorragique, de tachypnée et de thrombocytopénie. Le diagnostic postmortem a confirmé l'épizootie hémorragique (sérotype 2) à l'aide de la technique RT-PC. Les résultats bruts et histopathologiques étaient conformes à l'épizootie hémorragique signalée chez d'autres espèces.(Traduit par Isabelle Vallières).

Virus Neutralization Test for Detecting and Quantifying Serum-Neutralizing Antibodies to Epizootic Hemorrhagic Disease Virus (EHDV) (Serotypes 1, 2, and 4-8).

The virus neutralization test (VNT) is a functional immunoassay which detects the presence and quantity of neutralizing antibodies. It is a highly sensitive and specific test. As with most neutralization assays, the EHDV VNT does not react with all virus-targeting antibodies, but specifically with those antibodies that bind to VP2, the outermost capsid structural protein of the virus. The interaction between VP2 and neutralizing antibodies can block EHDV cell binding, neutralizing its infectivity. The detection and quantification of neutralizing antibodies are indicative of how protected an animal is against reinfection. The EHD VNT can therefore be a useful tool to monitor the efficacy of a vaccination campaign. VP2 is also the main determinant of EHDV serotype specificity, and so EHDV-neutralizing antibodies which target VP2 are also serotype-specific. Throughdetecting and quantifying neutralizing antibodies, the VNT can discriminate the EHDV serotype responsible for an infection and provides insights into the time of infection. It is considered the gold standard test for identifying and quantifying antibodies against EHDV serotypes present in test serum samples. The assay is performed in vitro and is based on inhibition of virus infectivity in the presence of neutralizing antibodies. A neutralizing antibody titer is determined through the presence or absence of cytopathic effect in a cell monolayer. The VNT is a relatively inexpensive assay using standard laboratory equipment; however, to perform the assay, cell cultures, significant time, intensive labor, and technical skill are required.

Detection of EHDV Antibodies Using Enzyme-Linked Immunosorbent Assay (ELISA).

Enzyme-linked immunosorbent assay (ELISA) is a relatively inexpensive, rapid, and high-throughput diagnostic tool to detect antibodies raised against epizootic hemorrhagic disease virus (EHDV) in ruminant serum. While the presence of EHDV antibodies only confirms prior exposure to the virus, it does not conclusively determine infection status. The c-ELISA can be used in conjunction with other diagnostic tests (e.g., real-time PCR) to reinforce diagnosis of infection or as a surveillance tool to support disease control. The EHDV competition ELISA (c-ELISA) described here is a commercial diagnostic assay, recommended by the World Organisation for Animal Health (WOAH), that detects ruminant antibodies against the highly conserved EHDV structural protein, VP7.

End-Point RT-PCR Assays for Detection and Typing of Epizootic Hemorrhagic Disease Virus.

The emergence of EHDV in Europe during the autumn of 2022 reinforces the need for molecular tools (RT-PCR) for rapid detection of animals infected with this virus. Viral genome testing can be performed on whole blood under anticoagulant, spleen, and bloody organ homogenates from ruminants. It can also be performed on cell culture following viral isolation tests. Various so-called classical or end-point RT-PCRs will be described, which permit the amplification of a part of the viral genome (targeting segment 7) allowing the detection of EHDV whatever the serotype (pan-RT-PCR) and also to amplify a portion of the gene coding the viral protein (VP) 2 enabling serotyping. The PCR amplification products are visualized by agarose gel electrophoresis. Sequencing of the type-specific RT-PCR amplification products allows for the serotype of the virus to be determined.

Real-Time RT-PCR for the Diagnosis of Epizootic Hemorrhagic Disease Virus.

Real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) has become an essential tool in rapid and reliable detection of animal diseases such as epizootic hemorrhagic disease (EHD). Here we provide a protocol for the detection of epizootic hemorrhagic disease virus (EHDV) genetic material in blood and tissue samples, using a real-time RT-PCR that targets a conserved region in segment 9 of the EHDV genome. This protocol can be used to detect up to approximately 90 samples in a single run and can be completed in less than 4 h.