Quantification of Marek's disease virus in chicken lymphocytes using the polymerase chain reaction with fluorescence detection

doi:10.1016/S0166-0934(96)02172-6

Journal of Virological Methods

Volume 65, Issue 1, April 1997, Pages 75-81

https://doi.org/10.1016/S0166-0934(96)02172-6 Get rights and content

Abstract

A quantitative assay was developed for Marek's disease virus (MDV). The assay determines the numbers of viral genomes present in samples by polymerase chain reaction (PCR) amplification of a portion of the viral genome for a restricted number of cycles. Fluorescent-tagged primers are used for the PCR amplification which allows quantification of the fluorescent product. Previously, quantitation of Marek's disease virus has required plaque assays, which are laborious and potentially error-prone, and this has limited quantitative comparisons. The PCR assay is rapid, less laborious and can be applied to high levels of accuracy, since replicate assays can be carried out relatively easily. The PCR-based assay assesses the number of viral genomes present in the sample, rather than the numbers of infected cells measured in the plaque assay, however correlation between the two assays is high, suggesting viral copy number per cell may be rather uniform. In crosses between genetically resistant and susceptible animals the PCR-based assay was correlated significantly with subsequent development of disease, and was a better predictor than the plaque assay of the likelihood of development of pathological disease in the birds studied.

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The amount of virus in each group was measured, and samples were collected according to the physical and behavioural conditions of the shrimp. There is a high correlation between the viral genome copy number and the number of infective virions (Bumstead et al., 1997). We can thus use quantitative real-time PCR to quantitate the viral load over a wide dynamic range in the form of the genome copy number (Dhar et al., 2001).
White spot syndrome virus (WSSV) proliferates rapidly and causes a high fatality rate in shrimp. Additionally, there is a lack of effective prevention and treatment methods for this disease. Although WSSV pathology is well-studied by molecular biology techniques, little research has have focused on the clinical signs of this disease based on individual shrimp performances. In this study, we recorded the clinical signs of diseased shrimp by observing their physiological and behavioural states. The physiological state of shrimp, including their heart rate (HR), heart rate variability (HRV), and ventilatory rate (VR), was recorded with a high-speed camera, which was used for crustacean disease diagnosis for the first time. The behavioural state of the shrimp consisted of their behaviour in the pond before sampling and the action demonstrated by the shrimp while falling into the water. Quantitative of WSSV in the shrimp was performed after measuring their physiological and behavioural characteristics. The virus was detected in both shrimp with and without white spots. The amount of virus in shrimp with or without white spots in the sand was relatively low and that of shrimp with white spots swimming in the water was high. The HR and VR both first increased and then decreased as the amount of virus increased gradually. There was a significant negative correlation between HRV and virus quantity in diseased shrimp. The swimming ability of shrimp with high virus quantities was weaker than that of shrimp with low virus quantities. Based on the data, we infer that the burrowing behaviour of shrimp is a disease resistance strategy; the slow swimming or knocking at the wall are symptoms of shrimp suffering from severe infection stage; WSSV destroys the heart, gill and muscle tissue of shrimp, causing damage to their basic motor function. Using the HRV index, we uncovered a fast and effective method with which to determine the virus quantity in diseased shrimp. On the basis of the above symptoms, WSSV is demonstrated as a shrimp disease not only related to the immune system, but also to the motor nervous system and respiratory circulatory systems.
Loop-mediated isothermal amplification assay for detection of four immunosuppressive viruses in chicken
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Loop-mediated isothermal amplification (LAMP) methods to detect chicken infectious anemia virus (CIAV), reticuloendotheliosis virus (REV), and Marek’s disease virus (MDV), and a reverse transcription (RT)-LAMP assay to detect infectious bursal disease virus (IBDV), were developed. The CIAV-LAMP, REV-LAMP, MDV-LAMP, and IBDV-RT-LAMP methods were performed using four sets of six primers targeting the VP1 gene of CIAV, the gp90 gene of REV, the Meq gene of MDV, and the VP2 gene of IBDV. The results (a change in color) were observed visually. The methods showed high specificity and sensitivity. The detection limits were 50 genomic copies of CIAV, 16 genomic copies of REV, 20 genomic copies of MDV, and 250 genomic copies of IBDV. When used to test clinical samples, the results of the LAMP assays were in 100% agreement with a previously described PCR. Therefore, the LAMP assays are simple, rapid, highly sensitive, and specific methods for detecting four immune-suppressive viruses.
Virus and host genomic, molecular, and cellular interactions during Marek's disease pathogenesis and oncogenesis
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Advances in chicken genomics and genomic technology (sequencing, association studies, etc.) have allowed researchers to comb the entire genome for genes and pathways influencing MD resistance. QTL mapping (Bumstead et al., 1997; Vallejo et al., 1998; Yonash et al., 1999) and subsequent comparative genomics for loci identification (Burt et al., 1999) as well as DNA microarrays (Heidari et al., 2010; Hu et al., 2015) have all been employed to identify loci relevant to MD. Vaccination and selective breeding approaches for chicken immunization and disease resistance are beginning to exploit data on avian immune responses, the MHC, and cytokines as well as genomic data to optimize efficiency (i.e., innate immunity enhancement and modification) and scope (i.e., optimize number of strains and/or pathogens protected against) (Kaiser, 2010).
Marek's Disease Virus (MDV) is a chicken alphaherpesvirus that causes paralysis, chronic wasting, blindness, and fatal lymphoma development in infected, susceptible host birds. This disease and its protective vaccines are highly relevant research targets, given their enormous impact within the poultry industry. Further, Marek's disease (MD) serves as a valuable model for the investigation of oncogenic viruses and herpesvirus patterns of viral latency and persistence—as pertinent to human health as to poultry health. The objectives of this article are to review MDV interactions with its host from a variety of genomic, molecular, and cellular perspectives. In particular, we focus on cytogenetic studies, which precisely assess the physical status of the MDV genome in the context of the chicken host genome. Combined, the cytogenetic and genomic research indicates that MDV-host genome interactions, specifically integration of the virus into the host telomeres, is a key feature of the virus life cycle, contributing to the viral achievement of latency, transformation, and reactivation of lytic replication. We present a model that outlines the variety of virus-host interactions, at the multiple levels, and with regard to the disease states.
Detection of specific UL49 sequences of Marek's disease virus CVI988/Rispens strain using loop-mediated isothermal amplification
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In spite of the advantages of IFA it is still a cell culture-based assay and requires a fluorescence microscope. A huge step forward in detection and differentiation of MDV strain was achieved by the application of PCR and real-time PCR assays (Becker et al., 1992; Silva, 1992; Bumstead et al., 1997; Islam et al., 2004; Baigent et al., 2011). Using PCR-based techniques it was possible to differentiate all three MDV serotypes and also the attenuated CVI988/Rispens strain (Silva, 1992; Król et al., 2007; Baigent et al., 2011; Renz et al., 2013; Gimeno et al., 2014).
Marek's disease (MD) is a tumoral disease of chickens that can be controlled by vaccines based on non-pathogenic strains of turkey herpesvirus (HVT), SB-1 strain belonging to serotype 2, or the attenuated CVI988/Rispens strain belonging to serotype 1 of Marek's disease virus (MDV). Currently, the ‘gold standard’ in MD prophylaxis is the Rispens strain-based vaccine which protects against very virulent MDV and disease onset. Previous studies have shown that loop-mediated isothermal amplification (LAMP) is a rapid alternative to polymerase chain reaction (PCR) for detection and differentiation of HVT, SB-1 and virulent MDV strains. The aim of this study was to develop and evaluate a novel LAMP assay for the detection of the U_L49 Rispens-specific region. This assay was validated using material from infected chicken embryo fibroblasts (CEFs) and tissue samples from vaccinated chickens. The analytical sensitivity of the assay was 10-times higher than PCR and reliably amplified 0.1 log₁₀ TCID_50/ml. The MDV Rispens was also detected at 18 h after infection of CEFs. The results showed LAMP to be selective and a sensitive method to detect Rispens as early as 3 d.p.v. in all internal organs of chickens. Furthermore, the method was also capable to detect Rispens in 5 out of 26 chickens originating from different flocks. A mismatch amplification mutation assay (MAMA-PCR) confirmed the presence of Rispens strain in all LAMP-positive chickens. This is the first report of the specific visual detection of Rispens in vitro and in vivo using LAMP. The method may be useful for monitoring of successful chicken vaccination as well as in vitro studies in infected cell cultures.
Analysis on the dynamic changes of the amount of WSSV in Chinese shrimp Fenneropenaeus chinensis during infection
2013, Aquaculture
Citation Excerpt :
Quantitative real-time PCR can accurately quantify the virus load over a wide dynamic range in the form of its genome copy number (Dhar et al., 2001). And it has been proved that high association exists between the virus genome copy number and the number of infective virion (Bumstead et al., 1997). The pleopod had high WSSV detection rate (Lo et al., 1997).
White spot syndrome virus (WSSV) is the most severe viral pathogen to the shrimp culture industry worldwide. The temporal and spatial dynamic changes of WSSV in Chinese shrimp Fenneropenaeus chinensis maintained at 22 ± 1 °C during infection via injection were studied through the quantitative real-time PCR method. The pleopods were selected for virus quantification at 0, 6, 12, 18, 24, 27, 30, 33, 36, 39, 42, 45 and 48 hours post-injection (hpi). Besides the pleopod, different tissues including the hepatopancreas, nerve, heart, eyestalk, muscle, lymphoid organ, gut, stomach, gill and skin were also selected for viral load analysis at 0, 12, 27 and 30 hpi as well as the moribund stage after 40 hpi. The curve of viral load change in vivo assumed a very similar shape with the typical viral one-step growth curve. The infection process was classified into three stages: the light infection stage (0 to 18 hpi) when the viral load in the pleopod was at the level of no more than 10³/ng DNA and no mortality appeared; the moderate infection stage (18 to 45 hpi), corresponding to the logarithmic phase of WSSV replication, when the viral load in the pleopod ranged from 10³ to 10⁵/ng DNA and a low mortality rate was observed; and the heavy infection stage when WSSV propagation went into the plateau phage and massive mortality happened. A turning point from chronic infection to acute infection was suggested from 27 to 30 hpi when the viral load in most tissues increased substantially. The progress rate of WSSV in different tissues during the infection was different and the most favorite replication tissue of WSSV in early infection is the stomach, followed by the gill. The heaviest infected tissues are the skin, stomach and gill, explaining the symptoms of skin color changes, reduction of food consumption and gathering at the water surface due to dyspnea. The hepatopancreas, muscle and nerve were not the main replication sites in advanced infection. Also, the expression profiles of two anti-virus genes FcALF and FcAV were analyzed. This study might provide important data to study the virus infection mechanism.
Comparative efficacy of BAC-derived recombinant SB-1 vaccine and the parent wild type strain in preventing replication, shedding and disease induced by virulent Marek's disease virus
2010, Research in Veterinary Science
A widely used vaccine against Marek’s disease (MD) in poultry is the virus SB-1, which is antigenically-related to the causative agent, Marek’s disease herpesvirus. We recently cloned the SB-1 genome as an infectious bacterial artificial chromosome, BAC, (pSB-1). The protective efficacies and replication kinetics of pSB-1 and the parent strain (SB-1) were compared in an experimental model of MD induced by a virulent strain, RB-1B. Although vaccine virus replication and shedding was lower for pSB-1 than for SB-1, both vaccines reduced replication and shedding of RB-1B, and were equally effective in protecting chickens against MD. With the cloning of pSB-1, we have now generated full length genomic clones of MD vaccine virus strains belonging to each of the three serotypes. Vaccine viruses derived from each of these clones demonstrated protective efficacies at levels similar to those produced by the respective parent viruses, demonstrating their suitability to be used as vaccine candidates.

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Research paperQuantification of Marek's disease virus in chicken lymphocytes using the polymerase chain reaction with fluorescence detection

Abstract

Virology

The genetic basis of disease resistance in chickens

Marek's Disease—a Model for Herpesvirus Oncology

Feather follicle epithelium: A source of enveloped and infectious cell-free herpesvirus from Marek's disease of chickens

J. Natl. Cancer Inst.

Herpes-type virus isolated in cells cultures from tumours of chickens with Marek's disease. I. Studies in cell culture

J. Natl. Cancer Inst.

Use of the polymerase chain reaction for diagnosis of natural infection of chickens and turkeys with Marek's disease virus and reticuloendotheliosis virus

Avian Pathol.

Research paper
Quantification of Marek's disease virus in chicken lymphocytes using the polymerase chain reaction with fluorescence detection