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Development and validation of a portable, point-of-care canine distemper virus qPCR test.

ABSTRACT: Canine distemper virus (CDV) is a multi-host pathogen that can cause significant mortality in domestic, wild terrestrial and marine mammals. It is a major conservation threat in some endangered species. Infection can result in severe respiratory disease and fatal encephalitis. Diagnosis and disease monitoring in wildlife, and differentiation of CDV from rabies (a life-threatening zoonotic disease that can produce similar neurologic signs), would benefit from the availability of a portable, point-of-care (POC) diagnostic test. We therefore developed a quantitative RT-PCR assay for CDV using shelf-stable, lyophilized reagents and target-specific primers and probes for use with the handheld Biomeme two3™ qPCR thermocycler. Biomeme's extraction methodology, lyophilized reagents, and thermocycler were compared to our standard laboratory-based methods to assess sensitivity, efficiency and overall test performance. Results using a positive control plasmid for CDV showed comparable sensitivity (detection of 50 copies) and PCR efficiency between the two platforms, and CDV detection was similar between platforms when tested using a modified live CDV vaccine. Significantly higher Ct values (average Ct = 5.1 cycles) were observed using the Biomeme platform on known CDV positive animal samples. CDV detection using the Biomeme platform was similar in 25 of 26 samples from suspect CDV cases when compared to standard virology laboratory testing. One false positive was observed that was negative upon retest. The Biomeme methodology can be adapted for detection of specific targets, and this portable technology saves time by eliminating the need for local or international sample transport for laboratory-based diagnostics. However, results of our testing suggest that decreased diagnostic sensitivity (higher Ct values) relative to laboratory-based methods was observed using animal samples, so careful validation and optimization are essential. Portable qPCR platforms can empower biologists and wildlife health professionals in remote and low-resource settings, which will greatly improve our understanding of CDV disease ecology and associated conservation threats in wildlife.

SUBMITTER: Tomaszewicz Brown A

PROVIDER: S-EPMC7176111 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Development and validation of a portable, point-of-care canine distemper virus qPCR test.

Tomaszewicz Brown Ania A McAloose Denise D Calle Paul P PP Auer Angelika A Posautz Annika A Slavinski Sally S Brennan Robin R Walzer Chris C Seimon Tracie A TA

PloS one 20200422 4

Canine distemper virus (CDV) is a multi-host pathogen that can cause significant mortality in domestic, wild terrestrial and marine mammals. It is a major conservation threat in some endangered species. Infection can result in severe respiratory disease and fatal encephalitis. Diagnosis and disease monitoring in wildlife, and differentiation of CDV from rabies (a life-threatening zoonotic disease that can produce similar neurologic signs), would benefit from the availability of a portable, point ...[more]

PMID: 32320441

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Project description:To achieve proper diagnosis of dogs based on acute clinical symptoms and poorly preserved field samples taken from animals that died due to canine distemper (CD), a new differential diagnostic test has been developed based on polymerase chain reaction (PCR). In this study, more than 150 samples collected from dogs showing respiratory, gastrointestinal and neurological signs suggesting canine distemper virus (CDV) infection were examined. The samples consisted of urine, blood and nasal swabs collected from clinically ill patients, sent to our laboratory by clinicians from various veterinary clinics throughout Hungary. Various organs collected during the necropsy of dogs with pathological changes that suggested CDV infection were also included. Three distinct PCRs were designed. For diagnostic purposes, a primer pair specific to a 409 bases-long segment within the conservative part of the large polymerase region (L) of the CDV genome was designed. Using this test, out of the 150 analyzed samples, 46 (30.66%) proved to be positive for CDV, indicating that CDV still represents a high risk to the canine population in Hungary. For the phylogenetical analysis, a primer pair that completely encompasses the hemagglutinin (H) gene of the CDV genome was designed. The amplicons of this region were sequenced in both directions using the appropriate primers. Our results indicate that several different CDV genotypes are currently present in Hungary. Nine of the analyzed Hungarian strains turned out to belong to the so-called Arctic group of CDVs, and were most closely related to non-European strains from North America, China and Greenland, as well as to the phocine distemper virus 2 (PDV-2) isolated from Baikal seals (Phoca sibirica). One of the Hungarian strains showed high similarity to other European isolates from Denmark, Germany, Italy and Turkey, as well as to other isolates from geographically more distant regions, such as the USA. Three Hungarian strains seem to join a new cluster that is formed by only a couple of strains, one isolated from a mink in Denmark, and another from a dog in North America. Using a third set of primers, a restriction fragment length polymorphism (RFLP) assay has also been designed for the fast and reliable differentiation of the wild-type CDVs from the vaccine strains.

Project description:Canine distemper (CD) is a highly contagious viral disease worldwide. Although live attenuated vaccine is available as a preventive measure against the disease, cases of vaccination failure highlight the importance of potential alternative agent against canine distemper virus (CDV). CDV infects cells mainly by binding signaling lymphocyte activation molecule (SLAM) and Nectin-4 receptor. Here, to develop a new and safe antiviral biological agent for CD, we constructed and expressed CDV receptor proteins fused with Fc region of canine IgG-B, namely, SLAM-Fc, Nectin-Fc and SLAM-Nectin-Fc in HEK293T cells, and antiviral activity of these receptor-Fc proteins was subsequently evaluated. The results showed that the receptor-Fc proteins efficiently bound to receptor binding domain (RBD) of CDV-H, meanwhile, these receptor-Fc proteins competitively inhibited the binding of His-tagged receptor proteins (SLAM-His or Nectin-His) to CDV-H-RBD-Flag protein. Importantly, receptor-Fc proteins exhibited potent anti-CDV activity in vitro. Treatment with receptor-Fc proteins at the pre-entry stage dramatically suppressed CDV infectivity in Vero cells stably expressing canine SLAM. The minimum effective concentration (MEC) of SLAM-Fc, Nectin-Fc and SLAM-Nectin-Fc was 0.2 μg/mL, 0.2 μg/mL, 0.02 μg/mL. The 50% inhibition concentration (IC50) of three proteins was 0.58 μg/mL, 0.32 μg/mL and 0.18 μg/mL, respectively. Moreover, treatment with receptor-Fc proteins post viral infection can also inhibit CDV reproduction, the MEC of SLAM-Fc, Nectin-Fc and SLAM-Nectin-Fc was same as pre-treatment, and the IC50 of receptor-Fc proteins was 1.10 μg/mL, 0.99 μg/mL and 0.32 μg/mL, respectively. The results suggested that the receptor-Fc proteins were more effective for pre-entry treatment than post-infection treatment, furthermore, SLAM-Nectin-Fc was more effective than SLAM-Fc and Nectin-Fc. These findings revealed the receptor-Fc proteins were promising candidates as inhibitor against CDV.

Dataset Information

Development and validation of a portable, point-of-care canine distemper virus qPCR test.

Publications

Development and validation of a portable, point-of-care canine distemper virus qPCR test.

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