VP2 Gene Of Canine Parvovirus Research Articles

Prognostication of haemato-biochemical, electrolyte and blood gas parameters in canine parvoviral enteritis

Canine parvovirus (CPV) infection remains as a major threat to canine practitioners as well as pet owners as it requires intensive care for at least seven days and is associated with a high rate of fatality. This study conducted on 40 dogs with diarrhoea revealed a prevalence of 35 per cent (14/40) for CPV infection in Thissur, Kerala. Aetiology was confirmed by polymerase chain reaction (PCR) targeting the VP2 gene of CPV to get amplicon of approximately 583 bp length. Alterations in various blood parameters like blood gases, electrolytes, metabolites, haematological parameters and serum biochemical parameters are analysed and accessed as prognostic markers. Notable variations could be detected in the levels of pCO2, pO2, pSO2, HCO3-, base deficit, per cent of granulocytes, total erythrocyte count (TEC), haemoglobin (Hb), volume of packed red cells (VPRC), total protein, albumin, blood urea nitrogen (BUN) and alkaline phosphate (ALP). Mortality was associated with major decrease in the levels of HCO3- and a greater base deficit as evidenced from the blood gas analysis values. No correlation could be obtained for hypokalaemia and elevated levels of lactate in relation with mortality.

Expression of recombinant 35 kDa fragment of VP2 protein of canine parvovirus using Escherichia coli expression system.

Background and Aim:Canine parvovirus (CPV) is one of the most common viral infections in dogs, causing acute hemorrhagic gastroenteritis and high mortality. Vaccination effectively prevents CPV infection. However, the currently available CPV vaccines have concerns such as maternal immunity interference, shedding of virus vaccine, and false-positive result based on polymerase chain reaction after vaccination. A subunit vaccine can overcome these problems. This study aimed to express the recombinant 35 kDa fragment of the VP2 protein (consisting of epitopes 1-7) and the recombinant full-length VP2 protein (consisting of epitopes 1-10) and to study the ability of these two recombinant proteins to react with rabbit anti-CPV polyclonal antibodies.Materials and Methods:The full length and 35 kDa fragment of VP2 gene of CPV were cloned into the pBAD202 Directional TOPO™ expression vector and expressed in E. coli. The recombinant full-length and the recombinant 35 kDa fragment proteins of VP2 were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting.Results:The recombinant full-length and the recombinant 35 kDa fragment VP2 genes were successfully cloned and expressed. The optimum concentrations of arabinose and induction time for the recombinant full-length and the recombinant 35 kDa fragment VP2 proteins were 0.2% for 6 h and 0.02% for 6 h, respectively. The recombinant full-length and the recombinant 35 kDa fragment VP2 protein molecular weights were approximately 81 and 51 kDa, respectively. The recombinant full-length and the recombinant 35 kDa fragment VP2 proteins specifically interacted with rabbit anti-CPV polyclonal antibodies.Conclusion:These results suggest that the recombinant 35 kDa fragment and the recombinant full-length VP2 proteins may be useful in developing a CPV diagnostic test or vaccine.

An Improved Polymerase Cross-Linking Spiral Reaction Assay for Rapid Diagnostic of Canine Parvovirus 2 Infection.

With increasing complications of canine parvovirus infection cases, disease diagnosis and treatment have become more difficult. In this study, specificity primers for the conserved region of the VP2 gene of canine parvovirus 2 (CPV-2) were synthesized and evaluated. An improved polymerase cross-linking spiral reaction (PCLSR) method for early and rapid diagnosis of CPV-2 was established. The results showed that the amplification reaction was optimal when run at 62°C for 50 min and could be used to detect CPV-2 without any cross-reactions with other pathogens of canine infectious diseases. Reaction results were directly judged by the naked eyes, with the positive amplification tube shown as luminous yellow and the negative tube as bright purple. Compared with the previously reported polymerase spiral reaction (PSR) method for CPV-2 detection, this reaction was performed using improved primer pairs and a better dye identification method (using an indicator comprising phenol red and cresol red). The detection limit of PCLSR was 3.9 × 101 copies using gel electrophoresis or a visible dye. The positive rate of 132 clinical samples was 42.42%, which was identically the same as that of the PSR method and slightly higher than that of the colloidal gold strip method (39.39%). The newly developed CPV-PCLSR assay shows the advantage of rapid visualization of results and offers a convenient and rapid method for early CPV-2 diagnosis with higher sensitivity and specificity than the established methods.

Cloning, Expression and Purification of Native VP2 Mature Peptide of Canine Parvovirus in Prokaryotic Expression System

Canine Parvovirus (CPV-2) is a major cause of gastroenteritis in dogs and wild carnivores worldwide. There are three variants of CPV-2 (CPV2a, CPV2b and CPV2c) circulating worldwide and generated due to mutations at VP2 capsid protein. VP2 is the major immuno dominant capsid protein that determines host specificity and diversity of the virus. In the present study, VP2 gene of Canine parvovirus was cloned and purified in E.coli (DE3) expression system. In-silico analysis of transformed pET22b-VP2 sequences were completely matched with amino acids sequences of the selected mature peptide. Purification of the r-VP2 protein was carried out in native condition by Ni-NTA sepharose column chromatography method. The purified protein can be used for development of diagnostics and prophylaxis for Canine Parvovirus (CPV-2). How to cite this article: Tuteja D, Mondal B. Cloning, Expression and Purification of Native VP2 Mature Peptide of Canine Parvovirus in Prokaryotic Expression System. J Adv Res Med Sci Tech 2019; 6(3&4): 4-7. DOI: https://doi.org/10.24321/2394.6539.201905

Isolation and sequence analysis of the complete VP2 gene of canine parvovirus from Chinese domestic pets and determination of the pathogenesis of these circulating strains in beagles

Canine parvovirus (CPV) causes acute gastroenteritis in domestic dogs, cats, and several wild carnivore species. In this study, the full-length VP2 gene of 36 CPV isolates from dogs and cats infected between 2016 and 2017 in Beijing was sequenced and analyzed. The results showed that, in dogs, the new CPV-2a strain was the predominant variant (n = 18; 50%), followed by the new CPV-2b (n = 6; 16.7%) and CPV-2c (n = 3; 8.3%) strains, whereas, among cats, the predominant strain was still CPV-2 (n = 9; 25%). One new CPV-2a strain, 20170320-BJ-11, and two CPV-2c strains, 20160810-BJ-81 and 20170322-BJ-26, were isolated and used to perform experimental infections. Multiple organs of beagles that died tested PCR positive for CPV, and characteristic histopathological lesions were observed in organs, including the liver, spleen, lungs, kidneys, small intestines, and lymph nodes. Experimental infections showed that the isolates from the epidemic caused high morbidity in beagles, indicating their virulence in animals and suggesting the need to further monitor evolution of CPV in China.

ABSENCE OF PARVOVIRUS SHEDDING IN FECES OF THREATENED CARNIVORES FROM MISIONES, ARGENTINA.

Since its emergence in the 1970s, canine parvovirus (CPV) has spread worldwide and infects a wide variety of mammalian hosts, including domestic and nondomestic carnivores. Today it is one of the most important pathogenic viruses associated with high morbidity and mortality in domestic dogs ( Canis familiaris). In South America, the range of wild hosts has been scarcely studied and the epidemiology of CPV in wildlife is still unclear. In 2011, feces from five wild carnivores (bush dog [ Speothos venaticus] , jaguar [ Panthera onca], puma [ Puma concolor], oncilla [ Leopardus guttulus], and ocelot [ Leopardus pardalis]) were collected in Misiones, Argentina, using a detection dog. Of the 289 feces collected, 209 (72.3%) had sufficient sample remaining to be used in this study and the majority of these were genetically confirmed to individual (81.3%) and sex (78.4%) level. In fact, these samples represent a minimum of 115 individuals (10 jaguars, 13 pumas, 33 ocelots, 38 oncillas, and 21 bush dogs). Through polymerase chain reaction, a 583-bp fragment in the VP2 gene of CPV was amplified in these samples. While no samples showed evidence of infection, this does not exclude the occurrence of CPV in wild carnivores in the area, as intermittent viral shedding could condition the diagnosis of CPV in feces of infected wild mammals. Locally, it is recommended that long-term monitoring of parvovirus be continued in wildlife and expanded to domestic carnivores. Internationally, this study provides a useful contribution to the approach to the sylvatic cycle of parvovirus in wild carnivores.

Evolutionary and genetic analysis of the VP2 gene of canine parvovirus

BackgroundCanine parvovirus (CPV) type 2 emerged in 1978 in the USA and quickly spread among dog populations all over the world with high morbidity. Although CPV is a DNA virus, its genomic substitution rate is similar to some RNA viruses. Therefore, it is important to trace the evolution of CPV to monitor the appearance of mutations that might affect vaccine effectiveness.ResultsOur analysis shows that the VP2 genes of CPV isolated from 1979 to 2016 are divided into six groups: GI, GII, GIII, GIV, GV, and GVI. Amino acid mutation analysis revealed several undiscovered important mutation sites: F267Y, Y324I, and T440A. Of note, the evolutionary rate of the CPV VP2 gene from Asia and Europe decreased. Codon usage analysis showed that the VP2 gene of CPV exhibits high bias with an ENC ranging from 34.93 to 36.7. Furthermore, we demonstrate that natural selection plays a major role compared to mutation pressure driving CPV evolution.ConclusionsThere are few studies on the codon usage of CPV. Here, we comprehensively studied the genetic evolution, codon usage pattern, and evolutionary characterization of the VP2 gene of CPV. The novel findings revealing the evolutionary process of CPV will greatly serve future CPV research.

Isolation of Canine parvovirus with a view to identify the prevalent serotype on the basis of partial sequence analysis.

Aim:The aim of this study was to isolate Canine parvovirus (CPV) from suspected dogs on madin darby canine kidney (MDCK) cell line and its confirmation by polymerase chain reaction (PCR) and nested PCR (NPCR). Further, VP2 gene of the CPV isolates was amplified and sequenced to determine prevailing antigenic type.Materials and Methods:A total of 60 rectal swabs were collected from dogs showing signs of gastroenteritis, processed and subjected to isolation in MDCK cell line. The samples showing cytopathic effects (CPE) were confirmed by PCR and NPCR. These samples were subjected to PCR for amplification of VP2 gene of CPV, sequenced and analyzed to study the prevailing antigenic types of CPV.Results:Out of the 60 samples subjected to isolation in MDCK cell line five samples showed CPE in the form of rounding of cells, clumping of cells and finally detachment of the cells. When these samples and the two commercially available vaccines were subjected to PCR for amplification of VP2 gene, a 1710 bp product was amplified. The sequence analysis revealed that the vaccines belonged to the CPV-2 type and the samples were of CPV-2b type.Conclusion:It can be concluded from the present study that out of a total of 60 samples 5 samples exhibited CPE as observed in MDCK cell line. Sequence analysis of the VP2 gene among the samples and vaccine strains revealed that samples belonged to CPV-2b type and vaccines belonging to CPV-2.

Phylogenetic Analysis of Canine Parvovirus VP2 Gene in China.

In this study, a total of 37 samples (58.0%) were found through PCR assay to be positive for canine parvovirus (CPV) of 66 suspected faecal samples of dogs collected from various cities throughout China. Eight CPV isolates could be obtained in the CRFK cell line. The sequencing of the VP2 gene of CPV identified the predominant CPV strain as CPV-2a (Ser297Ala), with two CPV-2b (Ser297Ala). Sequence comparison revealed homologies of 99.3-99.9%, 99.9% and 99.3-99.7% within the CPV 2a isolates, within the CPV 2b isolates and between the CPV 2a and 2b isolates, respectively. In addition, several non-synonymous and synonymous mutations were also recorded. The phylogenetic tree revealed that most of the CPV strains from different areas in China were located in the formation of a large branch, which were grouped together along with the KU143-09 strain from Thailand and followed the same evolution. In this study, we provide an updated molecular characterization of CPV 2 circulation in China.

Visual Detection of Canine Parvovirus Based on Loop-Mediated Isothermal Amplification Combined with Enzyme-Linked Immunosorbent Assay and with Lateral Flow Dipstick

ABSTRACTLoop-mediated isothermal amplification (LAMP) combined with enzyme-linked immunosorbent assay (LAMP–ELISA) and with lateral flow dipstick (LAMP–LFD) are rapid, sensitive and specific methods for the visual detection of clinical pathogens. In this study, LAMP–ELISA and LAMP–LFD were developed for the visual detection of canine parvovirus (CPV). For LAMP, a set of four primers (biotin-labeled forward inner primers) was designed to specifically amplify a region of the VP2 gene of CPV. The optimum time and temperature for LAMP were 60 min and 65°C, respectively. The specific capture oligonucleotide probes, biotin-labeled CPV probe for LAMP–ELISA and fluorescein isothiocyanate-labeled CPV probe for LAMP–LFD were also designed for hybridization with LAMP amplicons on streptavidin-coated wells and LFD strips, respectively. For the comparison of detection sensitivity, conventional PCR and LAMP for CPV detection were also performed. The CPV detection limits by PCR, PCR–ELISA, LAMP, LAMP–ELISA and LAMP–LFD were 102, 102, 10−1, 10−1 and 10−1 TCID50/ml, respectively. In tests using artificially contaminated dog fecal samples, the samples with CPV inoculation levels of ≥1 TCID50/ml gave positive results by both LAMP–ELISA and LAMP–LFD. Our data indicated that both LAMP–ELISA and LAMP–LFD are promising as rapid, sensitive and specific methods for an efficient diagnosis of CPV infection.

Phylogenetic analysis of canine parvovirus partial VP2 gene in India

A total of 85 samples (58.0 %) were found to be positive for Canine parvovirus (CPV) by PCR assay (Hfor/Hrev primers) out of 158 suspected faecal samples of dogs collected from various states/union territories of India. Nine CPV isolates could be obtained in A-72 cell line. The sequencing of the partial VP2 gene of CPV identified the predominant CPV strain as CPV-2a (Ser297Ala) with one CPV-2b (Ser297Ala) and another CPV-2a variant strain (Ser297Gly). Several non-synonymous and synonymous mutations were also recorded in this study. The phylogenetic tree revealed that most of the CPV sequences from Tamil Nadu (Southern India) and Maharashtra (Western India) obtained during 2011 and few sequences from Northern India obtained during 2012 were grouped together along with CPV-2a (Ser297Ala) strains from China and India and followed the same evolution; although there was definitive indication of separate lineages too by few other sequences.

Development and evaluation of loop-mediated isothermal amplification assay for rapid and sensitive detection of canine parvovirus DNA directly in faecal specimens

Aims: To develop a specific and highly sensitive loop‐mediated isothermal amplification (LAMP) technique for the rapid detection of canine parvovirus (CPV) DNA directly in suspected faecal samples of dogs by employing a simple method of template preparation. Methods and Results: LAMP reaction was developed by designing two sets of outer and inner primers, which target a total of six distinct regions on VP2 gene of CPV. The template DNA was prepared by a simple boiling and chilling method. Of the 140 faecal samples screened by the developed LAMP and the conventional PCR assays, 104 samples (74·28%) were found positive by LAMP, whereas 81 samples (57·85%) were found positive by PCR. The specificity of the LAMP assay was tested by cross‐examination of common pathogens of dogs and further confirmed by sequencing. The detection limit of the LAMP was 0·0001 TCID50 ml−1, whereas the detection limit of the PCR was 1000 TCID50 ml−1. Conclusions: The developed LAMP assay detects CPV DNA in faecal specimens directly within an hour by following a simple and rapid boiling and chilling method of template preparation. The result also shows that the developed LAMP assay is specific and highly sensitive in detecting CPV. Significance and Impact of the Study: The result indicates the potential usefulness of LAMP which is a simple, rapid, specific, highly sensitive and cost‐effective field‐based method for direct detection of CPV from the suspected faecal samples of dogs.

Immunogenicity of a DNA-launched replicon-based canine parvovirus DNA vaccine expressing VP2 antigen in dogs

A replicon-based DNA vaccine encoding VP2 gene of canine parvovirus (CPV) was developed by cloning CPV-VP2 gene into a replicon-based DNA vaccine vector (pAlpha). The characteristics of a replicon-based DNA vaccine like, self-amplification of transcripts and induction of apoptosis were analyzed in transfected mammalian cells. When the pAlpha-CPV-VP2 was injected intradermal as DNA-launched replicon-based DNA vaccine in dogs, it induced CPV-specific humoral and cell mediated immune responses. The virus neutralization antibody and lymphocyte proliferative responses were higher than conventional CPV DNA vaccine and commercial CPV vaccine. These results indicated that DNA-launched replicon-based CPV DNA vaccine was effective in inducing both CPV-specific humoral and cellular immune responses and can be considered as effective alternative to conventional CPV DNA vaccine and commercial CPV vaccine.

An oral Sindbis virus replicon-based DNA vaccine containing VP2 gene of canine parvovirus delivered by Escherichia coli elicits immune responses in dogs

A Sindbis virus replicon-based DNA vaccine containing VP2 gene of canine parvovirus (CPV) was delivered by Escherichia coli to elicit immune responses. The orally immunized dogs developed CPV-specific serum IgG and virus neutralizing antibody responses. The cellular immune responses analyzed using lymphocyte proliferation test and flow cytometry indicated CPV-specific sensitization of both CD3+CD4+ and CD3+CD8+ lymphocytes. This study demonstrated that the oral CPV DNA vaccine delivered by E. coli can be considered as a promising approach for vaccination of dogs against CPV.

Sequence analysis of VP2 gene of canine parvovirus isolated from domestic dogs in Italy in 1994–2010

Sequence analysis of VP2 gene of canine parvovirus isolated from domestic dogs in Italy in 1994–2010

Genetic Analysis of VP2 Gene of Canine Parvovirus Isolates in Korea

The purpose of this study was to genetically characterize CPV isolates from Korea. The VP2 gene of 31 isolates was characterized by DNA sequencing and their phylogeny. Among the 31 field CPV isolates, 28 isolates were classified as type 2a and other 3 isolates as type 2b. The isolates in 2a-I, II and III subclusters have unique mutations. The isolates in 2a-IV and V subclusters had similar amino acid sequences to type 2a isolates from other parts of the world. The isolates in type 2b had similar amino acid sequences to type 2b isolates from Asia, Italy, and U.S.A. The molecular analysis of VP2 gene of CPV provided the useful information for the identification of CPV types and the understanding of their genetic relationship.

Sequence analysis of VP2 gene of canine parvovirus isolated from domestic dogs in Japan in 1999 and 2000

Seven strains of canine parvovirus (CPV) were isolated from affected dogs in Japan between 1999 and 2000, and their VP2 genes were genetically analyzed. Comparison of the predicted amino acid sequences of VP2 suggested that three field isolates corresponded to CPV type 2a, while the other four to CPV type 2b. The phylogenetic tree constructed from the VP2 genes showed that the newly isolated strains are classified into the cluster consisting of recent Japanese and Taiwanese field isolates, which are distinct from Vietnamese isolates, United States Isolates, or classical CPV type 2. These results suggest that the CPV transmission occurred between Japan and Taiwan in 1990s, and the offspring are still circulating in both countries.

Rapid method utilizing the polymerase chain reaction for detection of canine parvovirus in feces of diarrheic dogs

By using primers based on the sequence of the VP2 gene of canine parvovirus (CPV), we established a rapid and specific assay for identification of the virus from fecal specimens based on the polymerase chain reaction (PCR). By use of a pair of primers, a specific 226-bp sequence was amplified by the PCR. All strains of CPV tested gave a specific amplification product by the PCR, while neither porcine parvovirus nor host cell did so. The PCR assay can detect fewer particles of CPV than the conventional methods, being able to detect CPV from fecal specimens in a rapid manner, provided that gel filtration of the samples through a spun column was done to remove inhibitory substances from the fecal specimens. These results suggest that the PCR assay can detect the presence of CPV in dogs early enough to prevent secondary infection by CPV in veterinary hospitals.