ABSTRACT
Here, we report the discovery of two viruses associated with a disease characterized by severe diarrhea on a large-scale goat farm in Jilin province. Electron Microscopy observations revealed two kinds of virus particles with the sizes of 150-210 nm and 20-30 nm, respectively. Detection of 276 fecal specimens from the diseased herds showed the extensive infection of peste des petits ruminants virus (63.77%, 176/276) and caprine enterovirus (76.81%, 212/276), with a co-infection rate of 57.97% (160/276). These results were partially validated with RT-PCR, where all five PPRV-positive and CEV-positive specimens yielded the expected size of fragments, respectively, while no fragments were amplified from PPRV-negative and CEV-negative specimens. Moreover, corresponding PPRV and CEV fragments were amplified in PPRV and CEV double-positive specimens. Histopathological examinations revealed severe microscopic lesions such as degeneration, necrosis, and detachment of epithelial cells in the bronchioles and intestine. An immunohistochemistry assay detected PPRV antigens in bronchioles, cartilage tissue, intestine, and lymph nodes. Simultaneously, caprine enterovirus antigens were detected in lung, kidney, and intestinal tissues from the goats infected by the peste des petits ruminants virus. These results demonstrated the co-infection of peste des petits ruminants virus with caprine enterovirus in goats, revealing the tissue tropism for these two viruses, thus laying a basis for the future diagnosis, prevention, and epidemiological survey for these two virus infections.
Subject(s)
Coinfection , Diarrhea , Enterovirus Infections , Goat Diseases , Goats , Peste-des-Petits-Ruminants , Peste-des-petits-ruminants virus , Animals , Peste-des-Petits-Ruminants/virology , Peste-des-Petits-Ruminants/epidemiology , Peste-des-Petits-Ruminants/pathology , Peste-des-petits-ruminants virus/isolation & purification , Peste-des-petits-ruminants virus/genetics , Goat Diseases/virology , Goat Diseases/epidemiology , China/epidemiology , Coinfection/veterinary , Coinfection/virology , Coinfection/epidemiology , Enterovirus Infections/veterinary , Enterovirus Infections/virology , Enterovirus Infections/epidemiology , Diarrhea/virology , Diarrhea/veterinary , Diarrhea/epidemiology , Enterovirus/isolation & purification , Enterovirus/genetics , Enterovirus/classification , Feces/virology , PhylogenyABSTRACT
Peste des petis ruminants (PPR) is an acute, highly contagious fatal disease affecting both domestic and wild small ruminants, caused by Morbillivirus caprinae (also known as peste des petis ruminants virus (PPRV)). Herein, a rapid method based on recombinase aided amplification-clustered regularly interspaced short palindromic repeats-Cas12a (RAA-CRISPR Cas12a) to detect PPRV was developed. CRISPR RNAs and RAA primers for PPRV-N (nucleocapsid) and PPRV-M (matrix) fragments were designed. The reaction system was constructed following screening and optimization. Detection could be completed within in 50â¯minutes at 37°C. Detection of gradient dilutions of plasmids carrying of PPRV N and M gene fragments indicated a minimum limit of detection of 10 copies/µL. There were no cross-reactions with related viruses and all tested lineages of PPRV were detected successfully. The method also showed good repeatability. The detection of clinical samples (previously detected using reverse transcription polymerase chain reaction (RT-PCR)) indicated good consistency between the RAA-CRISPR Cas12a method and RT-PCR. Thus, the RAA-CRISPR Cas12a method for rapid PPRV diagnosis has strong specificity, high sensitivity, and stable repeatability. Moreover, the results can be observed visually under blue or UV light or using lateral flow strips without complex instruments.
Subject(s)
CRISPR-Cas Systems , Goats , Peste-des-Petits-Ruminants , Peste-des-petits-ruminants virus , Sensitivity and Specificity , Peste-des-petits-ruminants virus/genetics , Peste-des-petits-ruminants virus/isolation & purification , Animals , Peste-des-Petits-Ruminants/diagnosis , Peste-des-Petits-Ruminants/virology , Nucleocapsid Proteins/genetics , Viral Matrix Proteins/genetics , Goat Diseases/diagnosis , Goat Diseases/virology , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA, Viral/genetics , Sheep , Bacterial Proteins , Endodeoxyribonucleases , CRISPR-Associated ProteinsABSTRACT
The aim of the present study was to investigate the frequency, genetic variability, and phylogeny of the peste des petits ruminants virus (PPRV) in ovine and caprine fetuses. During 2014 and 2017, a total of 1054 embryos/fetuses were collected in Turkey. A real-time RT-PCR assay was used for the detection of the PPRV RNA. Genetic characterization and phylogenetic analysis of the PPRV field isolates were conducted by sequencing fusion (F) protein and nucleoprotein (N) gene segments. Samples were also collected from ewes (n = 83) and nanny goats (n = 3) that had aborted and whose embryos/fetuses were found to be PPRV positive. PPRV positive embryos/fetuses were also tested for the presence of Listeria monocytogenes, Campylobacter spp., Coxiella burnetii, Chlamydophila abortus, Brucella spp., akabane virus, aino virus, bluetongue virus, border disease virus, bovine viral diarrhea virus, Cache Valley virus, and Schmallenberg virus. PPRV RNA was detected in 123 (11.7â¯%) of the 1054 embryos/fetuses, 78 of the 83 (94â¯%) ewes and 3 (100â¯%) nanny goats. Border disease virus RNA and Chlamydophila abortus DNA were detected in 7 and 12 PPRV positive sheep fetuses, respectively, while other bacterial and viral agents were not detected. Phylogenetically, the field isolates in this study belong to lineage IV, and compared to other strains of lineage IV considered in this study, they showed 1 and 5 new amino acid substitutions in the F and N gene sequences, respectively. The results of the study suggest that PPRV plays an important role in abortion. Therefore, PPRV needs to be taken into consideration in sheep and goats abortions.
Subject(s)
Abortion, Veterinary , Goat Diseases , Goats , Peste-des-Petits-Ruminants , Peste-des-petits-ruminants virus , Phylogeny , Sheep Diseases , Animals , Peste-des-petits-ruminants virus/genetics , Peste-des-petits-ruminants virus/isolation & purification , Sheep , Abortion, Veterinary/virology , Abortion, Veterinary/microbiology , Peste-des-Petits-Ruminants/virology , Female , Goat Diseases/virology , Goat Diseases/microbiology , Sheep Diseases/virology , Sheep Diseases/microbiology , Pregnancy , Turkey/epidemiology , Aborted Fetus/virology , Aborted Fetus/microbiology , RNA, Viral/geneticsABSTRACT
The Peste des petits ruminant virus (PPRV) is a member of the Paramyxoviridae family and is classified into the genus Measles virus. PPRV predominantly infects small ruminants, leading to mortality rates of nearly 100%, which have caused significant economic losses in developing countries. Host proteins are important in virus replication, but the PPRV nucleocapsid (N) protein-host interacting partners for regulating PPRV replication remain unclear. The present study confirmed the interaction between PPRV-N and the host protein vimentin by co-immunoprecipitation and co-localization experiments. Overexpression of vimentin suppressed PPRV replication, whereas vimentin knockdown had the opposite effect. Mechanistically, N was subjected to degradation via the ubiquitin/proteasome pathway, where vimentin recruits the E3 ubiquitin ligase NEDD4L to fulfill N-ubiquitination, resulting in the degradation of the N protein. These findings suggest that the host protein vimentin and E3 ubiquitin ligase NEDD4L have an anti-PPRV effect.
Subject(s)
Nucleocapsid Proteins , Peste-des-petits-ruminants virus , Vimentin , Virus Replication , Nucleocapsid Proteins/metabolism , Nucleocapsid Proteins/genetics , Vimentin/metabolism , Vimentin/genetics , Animals , Peste-des-petits-ruminants virus/physiology , Peste-des-petits-ruminants virus/genetics , Peste-des-petits-ruminants virus/metabolism , Humans , Ubiquitination , Host-Pathogen Interactions , HEK293 Cells , Nedd4 Ubiquitin Protein Ligases/metabolism , Nedd4 Ubiquitin Protein Ligases/genetics , Cell Line , Peste-des-Petits-Ruminants/virology , Peste-des-Petits-Ruminants/metabolism , Protein BindingABSTRACT
Peste des petits ruminants (PPR) is an acute, contact infectious disease caused by the small ruminant morbillivirus (SRMV), and its morbidity in goats and sheep can be up to 100% with significant mortality. Nanobody generated from camelid animals such as alpaca has attracted wide attention because of its unique advantages compared with conventional antibodies. The main objective of this study was to produce specific nanobodies against SRMV and identify its characteristics. To obtain the coding gene of SRMV-specific nanobodies, we first constructed an immune phage-displayed library from the VHH repertoire of alpaca that was immunized with SRMV-F and -H proteins. By using phage display technology, the target antigen-specific VHHs can be obtained after four consecutive rounds of biopanning. Results showed that the size of this VHH library was 2.26 × 1010 CFU/mL and the SRMV-F and -H specific phage particles were greatly enriched after four rounds of biopanning. The positive phage clones were selected and sequenced, and total of five independent different sequences of SRMV-specific nanobodies were identified. Subsequently, the DNA fragments of the five nanobodies were cloned into E. coli BL21(DE3), respectively, and three of them were successfully expressed and purified. Specificity and affinity towards inactivated SRMV of these purified nanobodies were then evaluated using the ELISA method. Results demonstrated that NbSRMV-1-1, NbSRMV-2-10, and NbSRMV-1-21 showed no cross-reactivity with other antigens, such as inactivated BTV, inactivated FMDV, His-tag labeled protein, and BSA. The ELISA titer of these three nanobodies against inactivated SRMV was up to 1:1000. However, only NbSRMV-1-21 displayed SRMV neutralizing activity at a maximum dilution of 1:4. The results indicate that the nanobodies against SRMV generated in this study could be useful in future applications. This study provided a novel antibody tool and laid a foundation for the treatment and detection of SRMV.
Subject(s)
Bacteriophages , Camelids, New World , Peste-des-Petits-Ruminants , Peste-des-petits-ruminants virus , Single-Domain Antibodies , Animals , Sheep , Single-Domain Antibodies/genetics , Escherichia coli/genetics , Peste-des-petits-ruminants virus/genetics , Peste-des-Petits-Ruminants/prevention & control , Antibodies , Antigens , GoatsABSTRACT
This study aimed at investigating the genetic lineages of peste des petits ruminants virus (PPRV) currently circulating in Burkina Faso. As part of PPR surveillance in 2021 and 2022, suspected outbreaks in different regions were investigated. A risk map was produced to determine high-risk areas for PPR transmission. Based on alerts, samples were obtained from three regions and all sampled localities were confirmed to fall within PPR high risk areas. We collected swab samples from the eyes, mouth, and nose of sick goats. Some tissue samples were also collected from dead animals suspected to be infected by PPRV. In total, samples from 28 goats were analysed. Virus confirmation was performed with RT-PCR amplification targeting the nucleocapsid (N) gene. Partial N gene sequencing (350 bp) was carried out using the RT-PCR products of positives samples to characterise the circulating lineages. Eleven sequences, including ten new sequences, have been obtained. Our study identified the presence of the PPRV lineage IV in the three studied regions in Burkina Faso with a genetic heterogeneity recorded for the sequences analysed. Previously published data and results of this study suggest that PPRV lineage IV seems to be replacing lineage II in Burkina Faso.
Subject(s)
Goat Diseases , Peste-des-Petits-Ruminants , Peste-des-petits-ruminants virus , Animals , Peste-des-petits-ruminants virus/genetics , Peste-des-Petits-Ruminants/epidemiology , Burkina Faso/epidemiology , Molecular Epidemiology , Goat Diseases/epidemiology , Phylogeny , Ruminants , GoatsABSTRACT
Peste des petits ruminants (PPR) is a highly contagious viral disease and one of the deadliest affecting wild goats, sheep, and small ruminants; however, goats are generally more sensitive. The causative agent is the Peste des Petits Ruminants virus (PPRV), which is a single-stranded RNA virus of negative polarity belonging to the Paramyxoviridae family. In February 2020, an active outbreak of PPR was reported in a herd of a transhumant farmer in the village of Gainth Pathé (department of Kounguel, Kaffrine region, Senegal). Of the ten swabs collected from the goats, eight returned a positive result through a quantitative real-time PCR. The sample that yielded the strongest signal from the quantitative real-time PCR was further analyzed with a conventional PCR amplification and direct amplicon sequencing. A phylogenetic analysis showed that the sequence of the PPR virus obtained belonged to lineage IV. These results confirm those found in the countries bordering Senegal and reinforce the hypothesis of the importance of animal mobility between these neighboring countries in the control of PPRV. In perspective, following the discovery of this lineage IV in Senegal, a study on its dispersion is underway throughout the national territory. The results that will emerge from this study, associated with detailed data on animal movements and epidemiological data, will provide appropriate and effective information to improve PPR surveillance and control strategies with a view to its eradication.
Subject(s)
Goat Diseases , Peste-des-Petits-Ruminants , Peste-des-petits-ruminants virus , Sheep Diseases , Sheep , Animals , Peste-des-petits-ruminants virus/genetics , Peste-des-Petits-Ruminants/epidemiology , Senegal/epidemiology , Phylogeny , Goat Diseases/epidemiology , Ruminants , Goats , Sheep Diseases/epidemiologyABSTRACT
BACKGROUND: Peste des petits ruminants virus (PPRV) is a highly contagious pathogen that strongly influences the productivity of small ruminants worldwide. Acetylation is an important post-translational modification involved in regulation of multiple biological functions. However, the extent and function of acetylation in host cells during PPRV infection remains unknown. METHODS: Dimethylation-labeling-based quantitative proteomic analysis of the acetylome of PPRV-infected Vero cells was performed. RESULTS: In total, 1068 proteins with 2641 modification sites were detected in response to PPRV infection, of which 304 differentially acetylated proteins (DAcPs) with 410 acetylated sites were identified (fold change < 0.83 or > 1.2 and P < 0.05), including 109 up-regulated and 195 down-regulated proteins. Gene Ontology (GO) classification indicated that DAcPs were mostly located in the cytoplasm (43%) and participated in cellular and metabolic processes related to binding and catalytic activity. Functional enrichment indicated that the DAcPs were involved in the minichromosome maintenance complex, unfolded protein binding, helicase activity. Only protein processing in endoplasmic reticulum pathway was enriched. A protein-protein interaction (PPI) network of the identified proteins further indicated that a various chaperone and ribosome processes were modulated by acetylation. CONCLUSIONS: To the best of our knowledge, this is the first study on acetylome in PPRV-infected host cell. Our findings establish an important baseline for future study on the roles of acetylation in the host response to PPRV replication and provide novel insights for understanding the molecular pathological mechanism of PPRV infection.
Subject(s)
Peste-des-Petits-Ruminants , Peste-des-petits-ruminants virus , Animals , Chlorocebus aethiops , Peste-des-petits-ruminants virus/genetics , Vero Cells , Acetylation , Proteomics , Ruminants , Protein Processing, Post-Translational , GoatsABSTRACT
Peste des petits ruminants virus (PPRV) is known to induce transient immunosuppression in infected small ruminants by modulating several cellular pathways involved in the antiviral immune response. Our study shows that the PPRV-coded non-structural proteins C and V can interact with the cellular NF-κB p65 subunit. The PPRV-C protein interacts with the transactivation domain (TAD) while PPRV-V interacts with the Rel homology domain (RHD) of the NF-κB p65 subunit. Both viral proteins can suppress the NF-κB transcriptional activity and NF-κB-mediated transcription of cellular genes. PPRV-V protein expression can significantly inhibit the nuclear translocation of NF-κB p65 upon TNF-α stimulation, whereas PPRV-C does not affect it. The NF-κB-mediated pro-inflammatory cytokine gene expression is significantly downregulated in cells expressing PPRV-C or PPRV-V protein. Our study provides evidence suggesting a role of PPRV non-structural proteins V and C in the modulation of NF-κB signalling through interaction with the NF-κB p65 subunit.
Subject(s)
Peste-des-Petits-Ruminants , Peste-des-petits-ruminants virus , Animals , Humans , Peste-des-petits-ruminants virus/genetics , Peste-des-Petits-Ruminants/metabolism , Cytokines/genetics , Cytokines/metabolism , NF-kappa B/genetics , NF-kappa B/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism , Ruminants , Gene Expression , Goats/geneticsABSTRACT
In the southern region of Iraq, Peste des petits ruminants (PPR) has been identified and diagnosed. The study was done on (300) local sheep breeds of varying ages and sexes exhibiting PPR symptoms, while (25), healthy sheep breeds served as the control group. Additionally, the diagnosis of PPRV was confirmed by PCR. Infected sheep exhibit a variety of clinical symptoms. However, DNA sequencing was used to detect genetic links and genetic variation, and the results revealed a closed genetic relationship with the NCBI BLAST PPRV India isolate (GU014574.1) at total genetic variation (0.02-0.01%). Results indicate a large rise in PCV and ESR in conjunction with leukocytopenia and lymphocytopenia, a significant difference in clotting factor indices, and a significant increase in ALT, AST, and CK. In addition, there was a substantial variation in acute phase response. Postmortem examinations revealed various erosive lesions on the upper and lower gums, severe hemorrhagic enteritis, particularly of the small intestine, and obvious congestion of the lungs. Histopathological changes revealed an obvious flattening of the intestinal mucosa as well as an enlargement of the villi. In addition to a granuloma in the sub-mucosa, chronic inflammatory cells, primarily lymphocytes, were seen invading the mucosa. It has been determined that the sickness was circulating in the southern region of Iraq and severely afflicted sheep, which might result in significant economic losses owing to the detrimental effects of the virus that causes the disease on the various bodily parts.
Subject(s)
Goat Diseases , Peste-des-Petits-Ruminants , Peste-des-petits-ruminants virus , Sheep Diseases , Sheep , Animals , Peste-des-Petits-Ruminants/diagnosis , Peste-des-Petits-Ruminants/epidemiology , Peste-des-Petits-Ruminants/pathology , Peste-des-petits-ruminants virus/genetics , Iraq/epidemiology , Goats , Sheep Diseases/epidemiology , Goat Diseases/diagnosisABSTRACT
In this study, we recombinantly expressed the V protein of the peste des petits ruminants virus (PPRV) and evaluated its diagnostic value for PPRV infection using an indirect ELISA (i-ELISA). The optimal concentration of the coated antigen of V protein was 15 ng/well at a serum dilution of 1:400, and the optimal positive threshold value was 0.233. A cross-reactivity assay showed that the V protein-based i-ELISA was specific to PPRV with consistent reproducibility and showed a specificity of 82.6% and a sensitivity of 100% with a virus neutralization test. Using the recombinant V protein as an antigen in ELISA is useful for seroepidemiological studies of PPRV infections.
Subject(s)
Goat Diseases , Peste-des-Petits-Ruminants , Peste-des-petits-ruminants virus , Animals , Peste-des-petits-ruminants virus/genetics , Peste-des-Petits-Ruminants/diagnosis , Reproducibility of Results , Antibodies, Viral , Enzyme-Linked Immunosorbent Assay , Recombinant Proteins/genetics , Goats , Goat Diseases/diagnosisABSTRACT
The peste des petits ruminant (PPR) virus is a transboundary virus found in small domestic ruminants that causes high morbidity and mortality in naive herds. PPR can be effectively controlled and eradicated by vaccinating small domestic ruminants with a live-attenuated peste des petits ruminant virus (PPRV) vaccine, which provides long-lasting immunity. We studied the potency and safety of a live-attenuated vaccine in goats by detecting their cellular and humoral immune responses. Six goats were subcutaneously vaccinated with a live-attenuated PPRV vaccine according to the manufacturer's instructions, and two goats were kept in contact. Following vaccination, the goats were monitored daily, and we recorded their body temperature and clinical score. Heparinized blood and serum were collected for a serological analysis, and swab samples and EDTA blood were collected to detect the PPRV genome. The safety of the used PPRV vaccine was confirmed by the absence of PPR-related clinical signs, a negative pen-side test, a low virus genome load as detected with RT-qPCR on the vaccinated goats, and the lack horizontal transmission between the in-contact goats. The strong humoral and cellular immune responses detected in the vaccinated goats showed that the live-attenuated PPRV vaccine has a strong potency in goats. Therefore, live-attenuated vaccines against PPR can be used to control and eradicate PRR.
Subject(s)
Goat Diseases , Peste-des-Petits-Ruminants , Peste-des-petits-ruminants virus , Vaccines, Attenuated , Animals , Goat Diseases/diagnosis , Goats , Immunity, Humoral , Peste-des-petits-ruminants virus/genetics , Vaccines, Attenuated/adverse effectsABSTRACT
Colorectal cancer (CRC) is a common and highly malignant neoplasm, ranking as the fourth most frequent cause of cancer-related deaths worldwide. Recently, non-human oncolytic viruses such as Peste des petits ruminants virus (PPRV) are considered as a potent candidate in the viral therapy of cancer. In the current study, the apoptotic effects of matrix (M) protein of PPRV was investigated on SW480 CRC cells. The M gene was cloned into the pcDNA™3.1/Hygro(+) expression vector and transfected into the cancer cells. The cytotoxic effects of the M protein on SW480 cells were confirmed using MTT assay. Furthermore, flow cytometry results showed that the M protein induces apoptosis in 91 % of CRC cells. Interestingly, the expression of the M gene in SW480 cells led to the up-regulation of genes including Bax, p53, and Caspase-9, as well as an increase in the Bax/Bcl-2 ratio. By using bioinformatics modeling, we hypothesized that the M protein could interact with Bax factor through its BH3-like motif and could further activate the intrinsic apoptosis pathway. Ultimately, this study provided the first evidence of the pro-apoptotic activity of PPRV M protein indicating its possible development as a promising novel anti-cancer agent.
Subject(s)
Colorectal Neoplasms , Peste-des-Petits-Ruminants , Peste-des-petits-ruminants virus , Animals , Humans , Peste-des-petits-ruminants virus/genetics , bcl-2-Associated X Protein/genetics , Apoptosis , GoatsABSTRACT
The study describes the expression of recombinant truncated nucleocapsid protein (NP) of peste des petits ruminants (PPR) virus in the baculovirus system (PPRV-rBNP) and its potential application as a diagnostic antigen in ELISA for diagnosis of PPR in sheep and goats. The PPRV N-terminal immunogenic region (1-266 aa) of the NP coding sequence was amplified and cloned into the pFastBac HT A vector. The PPRV-rBNP with a molecular weight of â¼30 kDa was expressed in an insect cell system using generated recombinant baculovirus through Bac-to-Bac® Baculovirus Expression System. The crude PPRV-rBNP or Ni-NTA affinity-purified NP was characterized by SDS-PAGE and immunoblot using standard PPRV-specific sera. The PPRV-rBNP reacted well with PPRV anti-N specific monoclonal and polyclonal antibodies and PPRV-specific antiserum, suggesting that the expressed PPRV-rBNP is in its native form. The crude PPRV-rBNP as a diagnostic antigen was evaluated either as a coating antigen or standard positive control antigen in the Avidin-Biotin ELISA using the known standard panel reagents. The results showed that the expressed PPRV-rBNP can be an alternative diagnostic antigen to E. coli expressed recombinant PPRV-NPN and the utility of PPRV-rBNP avoids the need to use live PPRV antigen in the diagnostic ELISA. Hence, this allows scope in the future for large-scale field application of the recombinant antigen-based assays for diagnosis/surveillance and monitoring of PPR at the eradication as well as post-eradication phases in endemic countries or PPR non-endemic countries.
Subject(s)
Peste-des-Petits-Ruminants , Peste-des-petits-ruminants virus , Animals , Sheep , Peste-des-petits-ruminants virus/genetics , Nucleocapsid Proteins/genetics , Baculoviridae/genetics , Escherichia coli/genetics , Peste-des-Petits-Ruminants/diagnosis , Peste-des-Petits-Ruminants/epidemiology , Enzyme-Linked Immunosorbent Assay/methods , Antibodies , GoatsABSTRACT
Phage display is a well-established technique used for selecting novel ligands having affinity to a plethora of targets including proteins, viruses, whole bacterial and mammalian cells as well as lipid targets. In the present study, phage display technology was used to identify peptides having affinity to PPRV. The binding capacity of these peptides was characterized through various formats of ELISA using phage clones, linear and multiple antigenic peptides. The whole PPRV was used as an immobilized target in a surface biopanning process using a 12-mer phage display random peptide library. After five rounds of biopanning, forty colonies were picked and amplified followed by DNA isolation and amplification for sequencing. Sequencing suggested 12 different clones expressing different peptide sequence Phage-ELISA was performed using all 12 phage clones. Results indicated that four phage clones i.e., P4, P8, P9 and P12 had a specific binding activity to PPR virus. Linear peptides displayed by all 12 clones were synthesized using solid phase peptide synthesis and subjected to virus capture ELISA. No significant binding of the linear peptides with PPRV was evident which may be due to loss of conformation of linear peptide after coating. When the four selected phage clones displayed peptide sequences were synthesized in Multiple antigenic peptide (MAP) format and used in virus capture ELISA, the results indicated significant binding of PPRV to the MAPs. It may be due to increased avidity and/or better projection of binding residues in 4-armed MAPs as compared to linear peptides. MAP-peptides were also conjugated on gold nanoparticles (AuNPs). Visual colour change from wine red to purple was observed on addition of PPRV in MAP-conjugated AuNPs solution. This colour change may be attributable to the networking of PPRV with MAP -conjugated AuNPs resulting in aggregation of AuNPs. All these results supported the hypothesis that the phage display selected peptides were capable of binding to the PPRV. The potential of these peptides to develop novel diagnostic or therapeutic agents remains to be investigated.
Subject(s)
Bacteriophages , Metal Nanoparticles , Peste-des-Petits-Ruminants , Peste-des-petits-ruminants virus , Animals , Peste-des-petits-ruminants virus/genetics , Peste-des-Petits-Ruminants/diagnosis , Gold , Peptides/metabolism , Bacteriophages/genetics , GoatsABSTRACT
This study was conducted to develop a cell culture based PPR virus vaccine candidate using recent Bangladeshi strain of peste des petits ruminant's (PPR) virus. PPR virus was isolated from field outbreaks, confirmed by RT-PCR and used as viral inoculum for serial passaging in Vero cells for adaptation and attenuation. 60th serial passage had completed and RT-PCR and real time RT-PCR were done in every 5 passages for confirmation of PPR virus in tissue culture fluid (TCF). To assess the adaptation and attenuation cytopathology, virus titration, sequencing of both F and N genes and live animal experimentation were done. Different cellular alterations produced by PPR virus in infected Vero cells including syncytia formation, development of both intranuclear and intra cytoplasmic inclusion bodies and finally cell degradation are the indications of adaptation. The virus titre was found 2.5, 3.31, 3.55, 4.44, 4.71 and 6.5 Log10 TCID50/ml at 10th, 20th, 30th, 40th, 50th and 60th passages level respectively. In F gene sequence analysis it has been observed that few nucleotide (nt) and mino acid (aa) has been substituted as the effects of serial passaging of PPR virus in Vero cells. TCF at 60th passage level was found effective to produced protective antibody (Ab) titre in live animal experimentation. It is concluded that serially passaged and Vero cells adapted PPR virus TCF could be used as a vaccine candidate for further use to develop a potent & effective vaccine against PPR diseases.
Subject(s)
Goat Diseases , Peste-des-Petits-Ruminants , Peste-des-petits-ruminants virus , Viral Vaccines , Animals , Chlorocebus aethiops , Peste-des-Petits-Ruminants/prevention & control , Vero Cells , Peste-des-petits-ruminants virus/genetics , Cell Culture Techniques , GoatsABSTRACT
Peste des petits ruminants virus (PPRV) is a highly contagious morbillivirus related to measles and canine distemper virus, mostly affecting small ruminants. The corresponding PPR disease has a high clinical impact in goats and is characterized by fever, oral and nasal erosions, diarrhoea and pneumonia. In addition, massive infection of lymphoid tissues causes lymphopaenia and immune suppression. This results in increased susceptibility to secondary bacterial infections, explaining the observed high mortality in some outbreaks. We studied the pathogenesis of PPR by experimental inoculation of Dutch domestic goats with a recombinant virulent PPRV strain modified to express EGFP and compared it to an EGFP-expressing vaccine strain of PPRV. After intratracheal inoculation with virulent PPRV, animals developed fever, viraemia and leucopaenia, and shed virus from the respiratory and gastro-intestinal tracts. Macroscopic evaluation of fluorescence at the peak of infection 7 days post-inoculation (dpi) showed prominent PPRV infection of the respiratory tract, lymphoid tissues, gastro-intestinal tract, mucosae and skin. Flow cytometry of PBMCs collected over time demonstrated a cell-associated viraemia mediated by infected lymphocytes. At 14 dpi, pathognomonic zebra stripes were detected in the mucosa of the large intestine. In contrast, vaccine strain-inoculated goats remained largely macroscopically fluorescence negative and did not present clinical signs. A low-level viraemia was detected by flow cytometry, but at necropsy no histological lesions were observed. Animals from both groups seroconverted as early as 7 dpi and sera efficiently neutralized virulent PPRV in vitro. Combined, this work presents a study of the pathogenesis of wild type- and vaccine-based PPRV in its natural host. This study shows the strength of recombinant EGFP-expressing viruses in fluorescence-guided pathogenesis studies.
Subject(s)
Goat Diseases , Peste-des-Petits-Ruminants , Peste-des-petits-ruminants virus , Viral Vaccines , Animals , Peste-des-petits-ruminants virus/genetics , Peste-des-Petits-Ruminants/prevention & control , Viremia/veterinary , Goats , Viral Vaccines/genetics , Goat Diseases/prevention & controlABSTRACT
BACKGROUND: Peste des petits ruminants (PPR), foot-and-mouth disease (FMD) and sheep pox and goat pox are three important infectious diseases that infect goats, sheep and other small ruminants. It is well-known that the prevention of three diseases rely mainly on their individual vaccines. However, the vaccines have a variety of different disadvantages, such as short duration of immunity, increasing the number of vaccinations, and poor thermal stability. The purpose of this study is to construct a recombinant goat pox virus (rGPV) capable of expressing the F gene of PPRV and the P12A3C gene of FMDV as a live vector vaccine. RESULTS: The IRES, FMDV P12A3C and PPRV F genes into the multi-cloning site of the universal transfer plasmid pTKfpgigp to construct a recombinant transfer plasmid pTKfpgigpFiP12A3C, and transfected GPV-infected lamb testis (LT) cells with liposomes and produced by homologous recombination Recombinant GPV (rGPV/PPRVF-FMDVP12A3C, rGPV). The rGPV was screened and purified by green florescence protein (GFP) and xanthine-guanine-phosphoribosyltransferase gene (gpt) of Escherichia coli as selective markers, and the expression of rGPV in LT cells was detected by RT-PCR and immunofluorescence techniques. The results showed that the virus strain rGPV/PPRVF-FMDVP12A3C containing FMDV P12A3C and PPRV F genes was obtained. The exogenous genes FMDV P12A3C and PPRV F contained in rGPV were normally transcribed and translated in LT cells, and the expression products could specifically react with PPRV and FMDV antiserum. Then, the rGPV was intradermally inoculated with goats, the animal experiments showed that rGPV/PPRVF-FMDVP12A3C could induce high levels of specific antibodies against GPV, PPRV and FMDV. CONCLUSIONS: The constructed rGPV induced high levels of specific antibodies against GPV, PPRV and FMDV. The study provides a reference for " one vaccine with multiple uses " of GPV live vector vaccine.
Subject(s)
Capripoxvirus , Foot-and-Mouth Disease Virus , Goat Diseases , Peste-des-Petits-Ruminants , Peste-des-petits-ruminants virus , Sheep Diseases , Viral Vaccines , Male , Sheep , Animals , Peste-des-petits-ruminants virus/genetics , Foot-and-Mouth Disease Virus/genetics , Capripoxvirus/genetics , Antibodies, Viral , Vaccines, Synthetic , Peste-des-Petits-Ruminants/prevention & control , GoatsABSTRACT
BACKGROUND: Peste des petits ruminants (PPR) disease is a cross-species infectious disease that severely affects small ruminants and causes great losses to livestock industries in various countries. Distinguishing vaccine-immunized animals from naturally infected animals is an important prerequisite for the eradication of PPR. At present PPRV are classified into lineages I through IV, and only one vaccination strain, Nigeria/75/1, belongs to lineage II, but all of the epidemic strains in China at present are from lineage IV. RESULTS: To achieve this goal, we developed an SYBR Green I real-time qRT-PCR method for rapid detection and identification of PPRV lineages II and IV by analyzing different melting curve analyses. The negative amplification of other commonly circulating viruses such as orf virus, goat poxvirus, and foot-and-mouth disease virus demonstrated that primers targeting the L gene of PPRV were extremely specific. The sensitivity of the assay was assessed based on plasmid DNA and the detection limit achieved was 100 copies of PPRV lineages II and IV. CONCLUSION: Since the method has high sensitivity, specificity, and reproducibility, it will be effectively differentiated PPRV lineages II from PPRV lineages IV in PPRV infected animals.
Subject(s)
Goat Diseases , Peste-des-Petits-Ruminants , Peste-des-petits-ruminants virus , Animals , Peste-des-petits-ruminants virus/genetics , Reverse Transcriptase Polymerase Chain Reaction/veterinary , Reproducibility of Results , Peste-des-Petits-Ruminants/epidemiology , Ruminants , Goats , Goat Diseases/epidemiologyABSTRACT
Six different biosample collection cards, often collectively referred to as FTA (Flinders Technology Associates) cards, were compared for their ability to inactivate viruses and stabilize viral nucleic acid for molecular testing. The cards were tested with bluetongue virus, foot-and-mouth disease virus (FMDV), small ruminant morbillivirus (peste des petits ruminants virus), and lumpy skin disease virus (LSDV), encompassing non-enveloped and enveloped representatives of viruses with double-stranded and single-stranded RNA genomes, as well as an enveloped DNA virus. The cards were loaded with virus-containing cell culture supernatant and tested after one day, one week, and one month. The inactivation of the RNA viruses was successful for the majority of the cards and filters. Most of them completely inactivated the viruses within one day or one week at the latest, but the inactivation of LSDV presented a greater challenge. Three of the six cards inactivated LSDV within one day, but the others did not achieve this even after an incubation period of 30 days. Differences between the cards were also evident in the stabilization of nucleic acid. The amount of detectable viral genome on the cards remained approximately constant for all viruses and cards over an incubation period of one month. With some cards, however, a bigger loss of detectable nucleic acid compared with a directly extracted sample was observed. Using FMDV, it was confirmed that the material applied to the cards was sufficiently conserved to allow detailed molecular characterization by sequencing. Furthermore, it was possible to successfully recover infectious FMDV by chemical transfection from some cards, confirming the preservation of full-length RNAs.