Development and evaluation of a multiplex reverse-transcription real-time PCR assay for detection of equine respiratory disease viruses

Ghoniem, Shimaa M.; Deeb, Ayman Hany El; Aggour, Mohammed G; Hussein, Hussein Awad

doi:10.1177/1040638718799388

Cited by 4 publications

(5 citation statements)

References 17 publications

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“…The genomic DNA of EHV-4 was detected in horse fetal liver and spleen isolates by successful amplification of a specific 580 pb fragment of virus gB using the n-PCR second cycle, confirming the circulation of EHV-4 and playing a critical role in recurrent abortion outbreaks in Egyptian horses as reported in previous occasions in Egypt (Al-Shammari et al, 2016;Amer et al, 2011;Ghoniem et al, 2018), the finding also prove the superiority of n-nested PCR as a molecular test over the other serological assays. n-PCR could be used as a test of choice in EHV-4 diagnostic due to its accuracy and higher sensitivity up to one viral DNA copy, which overcomes the negativity of serological assays in recently or latently infected horses and reasonable cost with higher sensitivity, especially in low resources countries and where rt-PCR not available, many assays were developed and used by (Borchers and Slater, 1993;Varrasso et al, 2001;Wang et al, 2007).…”

Section: Advances In Animal and Veterinary Sciencessupporting

confidence: 62%

“…Several PCRs using unique primers were intended to identify EHV-1 and EHV-4 in clinical samples, tissues, or cell lines that had been injected with the virus (Dynon et al, 2001;Lawrence et al, 1994;O'Keefe et al, 1994;Wagner et al, 1992). The multiplex PCR and rt-PCR can simultaneously detect and discriminate EHV-1 and EHV-4 from each other (Ataseven et al, 2009;Diallo et al, 2007;Ghoniem et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

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Equine Herpes Virus 4 (EHV4) Investigation in Aborted Egyptian Mares; Molecular Detection, Isolation, and Phylogeny for Viral Glycoprotein B

Khattab¹,

Abdelmegeed²,

Mashaly³

et al. 2022

AAVS

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An abortion outbreak was reported in horse studs, Egypt, in 2019. Investigating the role of EHV-4 in this abortion outbreak in mares using various molecular detection techniques were done using the tentative diagnosis by rt-PCR. The presence of 16 liver and spleen samples out of 20 (80%) from aborted fetuses were positive for EHV-4, but all were negative for EHV-1. Virus isolation trial for EHV-4 were done, eleven samples out of 20 (55%) on the CAM of ECE were positive. The virus glycoprotein B (gB) fragment (580pb) was amplified in selected isolates using the nested PCR (n-PCR), sanger sequencing of gB from three isolates with phylogenetic analysis reveals the full identity between the Egyptian isolates from the outbreaks and other EHV-4 strains available in databases proving wide distance with other EHV-8 and EHV-1. The study recommends rt-PCR as a screening test for the tentative diagnosis of EHV-4 in epidemiological studies and n-PCR as a sensitive differential test for the virus detection. The virological studies and molecular assays confirm the involvement of EHV-4 in equine abortion, suggests the possibility that latent virus reactivation in mares could result in abortion due to certain factors, including stress more epidemiological investigation. Whole-genome sequencing is required to address any genetic recombination in EHV-4 associated with abortion cases.

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Section: Advances In Animal and Veterinary Sciencessupporting

confidence: 62%

Section: Introductionmentioning

confidence: 99%

Equine Herpes Virus 4 (EHV4) Investigation in Aborted Egyptian Mares; Molecular Detection, Isolation, and Phylogeny for Viral Glycoprotein B

Khattab¹,

Abdelmegeed²,

Mashaly³

et al. 2022

AAVS

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“…Ghoniem et al . [ 32 ] evaluated the effectiveness of the developed RT-PCR assay by examining 152 samples obtained from sick horses. In another study by Aeschbacher et al .…”

Section: Discussionmentioning

confidence: 99%

“…Equine influenza virus RNA was detected in six out of ten samples. Ghoniem et al [32] evaluated the effectiveness of the developed RT-PCR assay by examining 152 samples obtained from sick horses. In another study by Aeschbacher et al [31], 20 horse samples, 11 pig samples, and two bird samples were used to determine the diagnostic value of the PCR.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of a novel real-time polymerase chain reaction assay for identifying H3 equine influenza virus in Kazakhstan

Sandybayev,

Strochkov,

Beloussov

et al. 2023

Vet World

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Background and Aim: Equine influenza (EI) is a highly contagious disease that causes fever and upper respiratory tract inflammation. It is caused by influenza virus A, belonging to the Orthomyxoviridae family, with subtypes H3N8 and H7N7. This study presents data on the development of a real-time polymerase chain reaction (RT-PCR) assay using TaqMan probes to detect the H3 subtype of EI virus (EIV). Materials and Methods: The evaluation of the developed RT-PCR assay involved five strains of EIV as positive controls and ten nasopharyngeal swab samples collected from horses. RNA was isolated using the GeneJet Viral DNA and RNA Purification Kit, and primers and probes were designed using the Integrated DNA Technology PrimerQuest Tool. The assay was optimized by investigating the annealing temperature, primer and probes concentrations, sensitivity, and specificity. Sequencing was performed using the Thermo Fisher 3130 Genetic Analyzer, and the evolutionary history was inferred using the Neighbor-Joining method. Results: The designed primers and probes, targeting the H3 gene, were found to be specific to the EIV. The RT-PCR assay was capable of detecting as low as 50 femtogram (f) or 3 × 103 copies of genomic RNA. No cross-reactions were observed with other respiratory viral and bacterial pathogens, indicating the high specificity of the assay. To evaluate its effectiveness, ten nasopharyngeal swab samples collected from farms in North Kazakhstan regions during disease monitoring were analyzed. The accuracy of the analysis was confirmed by comparing the results with those obtained from a commercial RT-PCR assay for EI identification. The developed RT-PCR assay exhibited high sensitivity and specificity for detecting the EIV. Conclusion: The results demonstrate that the developed RT-PCR assay is suitable for diagnosing EI. This simple, highly sensitive, and specific assay for detecting H3 EIV can be a reliable tool for diagnosing and surveilling EI. Implementing this RT-PCR assay in veterinary practice will enhance and expedite the timely response to potential outbreaks of EI, thus positively impacting the overall epizootic well-being of EI in Kazakhstan. Keywords: equine influenza, hemagglutinin, horses, primers, probe, real-time polymerase chain reaction assay, virus.

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“…Real-time PCR for the detection of EHV-1 and EHV-4 was performed using the Qiagen QuantiTect Multiplex PCR Mastermix kit (Qiagen, Hilden, Germany). Samples were tested using a multiplex PCR capable of distinguishing between EHV-1 and EHV-4 strains [ 26 , 27 ] and a national animal health laboratory network (NAHLN) real-time PCR for influenza virus matrix gene [ 28 ] on an Applied Biosystems 7500 Fast Real-Time PCR System (Life Technologies, Carlsbad, CA, USA). The PCR used to detect the presence of the equine viruses detected in this study used the following thermocycler conditions: initial denaturation 95 °C for 5 min, denaturation for 30 s at 95 °C, annealing at 57 °C for 30 s, extension at 72 °C for 60 s for 45 cycles, final extension for 10 min, hold at 4 °C.…”

Section: Methodsmentioning

confidence: 99%

New Parvoviruses and Picornavirus in Tissues and Feces of Foals with Interstitial Pneumonia

Altan

Hui

et al. 2021

Viruses

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Six foals with interstitial pneumonia of undetermined etiology from Southern California were analyzed by viral metagenomics. Spleen, lung, and colon content samples obtained during necropsy from each animal were pooled, and nucleic acids from virus-like particles enriched for deep sequencing. The recently described equine copiparvovirus named eqcopivirus, as well as three previously uncharacterized viruses, were identified. The complete ORFs genomes of two closely related protoparvoviruses, and of a bocaparvovirus, plus the partial genome of a picornavirus were assembled. The parvoviruses were classified as members of new ungulate protoparvovirus and bocaparvovirus species in the Parvoviridae family. The picornavirus was classified as a new species in the Salivirus genus of the Picornaviridae family. Spleen, lung, and colon content samples from each foal were then tested for these viral genomes by nested PCR and RT-PCR. When present, parvoviruses were detected in both feces and spleen. The picornavirus, protoparvovirus, and eqcopivirus genomes were detected in the lungs of one animal each. Three foals were co-infected with the picornavirus and either a protoparvovirus, bocaparvovirus, or eqcopivirus. Two other foals were infected with a protoparvovirus only. No viral infection was detected in one animal. The complete ORFs of the first equine protoparvoviruses and bocaparvovirus, the partial ORF of the third equine picornavirus, and their detection in tissues of foals with interstitial pneumonia are described here. Testing the involvement of these viruses in fatal interstitial pneumonia or other equine diseases will require larger epidemiological and/or inoculation studies.

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Development and evaluation of a multiplex reverse-transcription real-time PCR assay for detection of equine respiratory disease viruses

Cited by 4 publications

References 17 publications

Equine Herpes Virus 4 (EHV4) Investigation in Aborted Egyptian Mares; Molecular Detection, Isolation, and Phylogeny for Viral Glycoprotein B

Equine Herpes Virus 4 (EHV4) Investigation in Aborted Egyptian Mares; Molecular Detection, Isolation, and Phylogeny for Viral Glycoprotein B

Evaluation of a novel real-time polymerase chain reaction assay for identifying H3 equine influenza virus in Kazakhstan

New Parvoviruses and Picornavirus in Tissues and Feces of Foals with Interstitial Pneumonia

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