Universal peptide-based potential vaccine design against canine distemper virus (CDV) using a vaccinomic approach.

Canine distemper virus (CDV) affects many domestic and wild animals. Variations among CDV genome linages could lead to vaccination failure. To date, there are several vaccine alternatives, such as a modified live virus and a recombinant vaccine; however, most of these alternatives are based on the ancestral strain Onderstepoort, which has not been circulating for years. Vaccine failures and the need to update vaccines have been widely discussed, and the development of new vaccine candidates is necessary to reduce circulation and mortality. Current vaccination alternatives cannot be used in wildlife animals due to the lack of safety data for most of the species, in addition to the insufficient immune response against circulating strains worldwide in domestic species. Computational tools, including peptide-based therapies, have become essential for developing new-generation vaccines for diverse models. In this work, a peptide-based vaccine candidate with a peptide library derived from CDV H and F protein consensus sequences was constructed employing computational tools. The molecular docking and dynamics of the selected peptides with canine MHC-I and MHC-II and with TLR-2 and TLR-4 were evaluated. In silico safety was assayed through determination of antigenicity, allergenicity, toxicity potential, and homologous canine peptides. Additionally, in vitro safety was also evaluated through cytotoxicity in cell lines and canine peripheral blood mononuclear cells (cPBMCs) and through a hemolysis potential assay using canine red blood cells. A multiepitope CDV polypeptide was constructed, synthetized, and evaluated in silico and in vitro by employing the most promising peptides for comparison with single CDV immunogenic peptides. Our findings suggest that predicting immunogenic CDV peptides derived from most antigenic CDV proteins could aid in the development of new vaccine candidates, such as multiple single CDV peptides and multiepitope CDV polypeptides, that are safe in vitro and optimized in silico. In vivo studies are being conducted to validate potential vaccines that may be effective in preventing CDV infection in domestic and wild animals.

SEROLOGICAL RESPONSE TO CANINE DISTEMPER VACCINATION IN WILD CAUGHT RACCOONS (PROCYON LOTOR).

Canine distemper virus (CDV) is a well-known RNA virus that affects domestic dogs and all families of wild terrestrial carnivores. Spillover infections from wildlife to domestic animals are mitigated by preventive vaccination, but there is limited information on the off-label use of veterinary vaccines for wildlife like raccoons (Procyon lotor). Twenty wild-caught raccoons were inoculated with a commercial recombinant DNA canarypox-vectored CDV vaccine, applying a regimen of two serial doses by SC route with an interval of 25-28 days between doses. The CDV serum virus neutralizing antibody (VNA) baseline titers and the postvaccination titers were measured at fixed time points. Forty percent (8/20) of the wild-caught raccoons had CDV VNA titers of 1:8 or greater upon intake, and all but a single individual were juvenile animals. Approximately one month following the first vaccine dose, 8% (1/12) of raccoons seronegative at baseline had serum CDV VNA titers of 1:24 or greater. Approximately one month following the booster vaccine dose, 67% (8/12) of raccoons seronegative at baseline had serum CDV VNA titers of 1:24 or greater. Among raccoons with CDV VNA titers greater than or equal to 1:8 at baseline, 13% (1/8) demonstrated a fourfold or greater rise in titer one month after the first vaccine dose, whereas 38% (3/8) reached the same threshold one month after the booster dose. The presence of naturally acquired CDV VNA in juvenile raccoons at the time of vaccination may have interfered with the humoral VNA response. A regimen of at least two serially administered SC vaccine doses may be immunogenic for raccoons, but further investigation of alternative routes, regimens, and CDV vaccine products is also warranted for this species.

Rapid diagnosis of canine respiratory coronavirus, canine influenza virus, canine distemper virus and canine parainfluenza virus with a Taqman probe-based multiplex real-time PCR.

Canine Infectious Respiratory Disease Complex (CIRDC) is a highly infectious diseases. Canine respiratory coronavirus (CRCoV), Canine influenza virus (CIV), Canine distemper virus (CDV), and Canine parainfluenza virus (CPiV) are crucial pathogens causing CIRDC. Due to the similar clinical symptoms induced by these viruses, differential diagnosis based solely on symptoms can be challenging. In this study, a multiplex real-time PCR assay was developed for detecting the four RNA viruses of CIRDC. Specific primers and probes were designed to target M gene of CRCoV, M gene of CIV, N gene of CDV and NP gene of CPiV. The detection limit is 10 copies/µL for CIV or CRCoV, while the detection limit of CDV or CPiV is 100 copies/µL. Intra-group and inter-group repeatability coefficient of variation (CV) were both less than 2 %. A total of 341 clinical canine samples were analyzed, and the results indicated that the method developed in our study owns a good consistency and better specificity compared with the conventional reverse transcription PCR. This study provides a new method to enable the simultaneous detection of all four pathogens in a single reaction, improving the efficiency for monitoring the prevalence of four viruses in CIRDC, which benefits the control of CIRDC.

A Canine Distemper Outbreak in Urban Red Foxes (Vulpes vulpes) in Brussels, Belgium, 2020.

Canine distemper has been observed infrequently in Belgian wildlife, mainly stone martens (Martes foina) and red foxes (Vulpes vulpes). This report describes an outbreak in the Brussels urban red fox population, characterized by its high density. The identified virus matched those within a cluster of viruses found previously in red foxes in Germany. Different canine distemper virus (CDV) strains, found in Belgian wild carnivores, share relationships with viruses found farther east. This and other reports indicate an endemic distribution of CDV in wild carnivores in Europe whereby the complex interplay of population density, group immunity, and infection of metapopulations determines the pattern of spatiotemporally alternating outbreaks.

Development and characterization of a novel specific monoclonal antibody against mink enteritis virus and its antigen epitope analysis.

This study prepared a novel monoclonal antibody (MAb) against mink enteritis parvovirus (MEV) and identified its antigen epitope. The antibody subclass is identified as IgG1, the titers of the MAb is up to 1:1 × 106 and keeps stably after low-temperature storage for 9 months or 11 passages of the MAb cells. The MAb can specifically recognize MEV in the cells in IFA, but not Aleutian disease virus (ADV) or canine distemper virus (CDV). Its antigen epitope was identified as a polypeptide containing 5 key amino acids (378YAFGR382) and the homology in 20 MEV strains, 4 canine parvovirus strains, and 4 feline panleukopenia virus strains was 100%. This study supplies a biological material for developing new methods to detect MEV.

Epitope mapping and a candidate vaccine design from canine distemper virus.

Background: Canine distemper (CD) is a worldwide spread disease that has been described in 12 families of mammals, especially in the Carnivora order, being better studied in domestic canines where vaccination represents the best means of control. CD is controlled by vaccination, but many cases of the disease still occur in vaccinated animals. Aim: The aim of this work was to study antigen-specific epitopes that can subsidize the development of a new vaccine approach. Methods: Mapping of T cell reactive epitopes for CD virus (CDV) was carried out through enzyme-linked immunospot assays using 119 overlapped synthetic peptides from the viral hemagglutinin protein, grouped in 22 pools forming a matrix to test the immune response of 32 animals. Results: Evaluations using the criteria established to identify reactive pools, demonstrated that 26 animals presented at least one reactive pool, that one pool was not reactive to any animal, and six pools were the most frequent among the reactive peptides. The crisscrossing of the most reactive pools in the matrix revealed nine peptides considered potential candidate epitopes for T cell stimulation against the CDV and those were used to design an in-silico protein, containing also predicted epitopes for B cell stimulation, and further analyzed using immune epitope databases to ensure protein quality and stability. Conclusion: The final in silico optimized protein presents characteristics that qualify it to be used to develop a new prototype epitope-based anti-CDV vaccine.

Histopathological Analysis of Brains from Dogs Infected with Canine Distemper Virus.

We describe the use of conventional histology and immunohistochemistry against canine distemper virus (CDV) to examine the brains of domestic dogs with a confirmed diagnosis of CDV infection. Histologically, to identify the main typical lesions, we used conventional H&E stain; to evaluate the progressive demyelination, we used Luxol Fast Blue stain; and to identify the presence of viral particles in these affected regions, we used immunohistochemistry against CDV. We confirm that the histopathological analysis of brains of distemper-infected dogs is a powerful tool to evaluate the typical brain lesions and could be used as an interesting natural model to continue studying the pathogenesis of canine distemper in different species and/or other morbillivirus infections, like measles.

Canine Distemper Virus Pathogenesis in the Ferret Model.

Canine distemper virus (CDV) is a highly contagious pathogen within the morbillivirus genus infecting a wide range of different carnivore species. The virus shares most biological features with other closely related morbilliviruses, including clinical signs, tissue tropism, and replication cycle in the respective host organisms.In the laboratory environment, experimental infections of ferrets with CDV were established as a potent surrogate model for the analysis of several aspects of the biology of the human morbillivirus, measles virus (MeV). The animals are naturally susceptible to CDV and display severe clinical signs resembling the disease seen in patients infected with MeV. As seen with MeV, CDV infects immune cells and is thus associated with a strong transient immunosuppression. Here we describe several methods to evaluate viral load and parameters of immunosuppression in blood-circulating immune cells isolated from CDV-infected animals.

An improved system to generate recombinant canine distemper virus.

BACKGROUND: Canine distemper virus (CDV) is a pathogen with the capability of cross-species transmission. It has crossed the species barrier to infect many other species, and its host range is expanding. The reverse genetic platform, a useful tool for scientific research, allows the generation of recombinant viruses from genomic cDNA clones in vitro. METHODS: To improve the reverse genetic system of CDV, a plasmid containing three independent expression cassettes was constructed for co-expression of the N, P, and L genes and then transfected with a full-length cDNA clone of CDV into Vero cells. RESULTS: The results indicated that the established rescue system has the advantages of being more convenient, easy to control the transfection ratio, and high rescue efficiency compared with the conventional reverse genetics system. CONCLUSION: This method not only reduces the number of transfection plasmids, but also improves the rescue efficiency of CDV, which could provide a reference for the recovery of other morbilliviruses.

Snapshot study of canine distemper virus in Bangladesh with on-site PCR detection and nanopore sequencing.

Canine distemper virus (CDV) is a highly contagious virus that affects domestic and wild animals, causing severe illness with high mortality rates. Rapid monitoring and sequencing can provide valuable information about circulating CDV strains, which may foster effective vaccination strategies and the successful integration of these into conservation programs. During two site visits in Bangladesh in 2023, we tested a mobile, deployable genomic surveillance setup to explore the genetic diversity and phylogenetic patterns of locally circulating CDV strains. We collected and analysed 355 oral swab samples from stray dogs in Rajshahi and Chattogram cities, Bangladesh. CDV-specific real-time RT-PCR was performed to screen the samples. Out of the 355 samples, 7.4% (10/135) from Rajshahi city and 0.9% (2/220) from Chattogram city tested positive for CDV. We applied a real-time RT-PCR assay and a pan-genotype CDV-specific amplicon-based Nanopore sequencing technology to obtain the near-completes. Five near-complete genome sequences were generated, with phylogenetic relation to the India-1/Asia-5 lineage previously identified in India. This is the first study to provide genomic data on CDV in Bangladesh and the first demonstration of a mobile laboratory setup as a powerful tool in rapid genomic surveillance and risk assessment for CDV in low resource regions.

A Bacterium-like Particle Vaccine Displaying Envelope Proteins of Canine Distemper Virus Can Induce Immune Responses in Mice and Dogs.

Canine distemper virus (CDV) can cause fatal infections in giant pandas. Vaccination is crucial to prevent CDV infection in giant pandas. In this study, two bacterium-like particle vaccines F3-GEM and H4-GEM displaying the trimeric F protein or tetrameric H protein of CDV were constructed based on the Gram-positive enhanced-matrix protein anchor (GEM-PA) surface display system. Electron microscopy and Western blot results revealed that the F or H protein was successfully anchored on the surface of GEM particles. Furthermore, one more bacterium-like particle vaccine F3 and H4-GEM was also designed, a mixture consisting of F3-GEM and H4-GEM at a ratio of 1:1. To evaluate the effect of the three vaccines, mice were immunized with F3-GEM, H4-GEM or F3 and H4-GEM. It was found that the level of IgG-specific antibodies and neutralizing antibodies in the F3 and H4-GEM group was higher than the other two groups. Additionally, F3 and H4-GEM also increased the secretion of Th1-related and Th2-related cytokines. Moreover, F3 and H4-GEM induce IgG and neutralizing antibodies' response in dogs. Conclusions: In summary, F3 and H4-GEM can provoke better immune responses to CDV in mice and dogs. The bacterium-like particle vaccine F3 and H4-GEM might be a potential vaccine candidate for giant pandas against CDV infection.

Heritability and genome-wide association study of vaccine-induced immune response in Beagles: A pilot study.

Both genetic and non-genetic factors contribute to individual variation in the immune response to vaccination. Understanding how genetic background influences variation in both magnitude and persistence of vaccine-induced immunity is vital for improving vaccine development and identifying possible causes of vaccine failure. Dogs provide a relevant biomedical model for investigating mammalian vaccine genetics; canine breed structure and long linkage disequilibrium simplify genetic studies in this species compared to humans. The objective of this study was to estimate the heritability of the antibody response to vaccination against viral and bacterial pathogens, and to identify genes driving variation of the immune response to vaccination in Beagles. Sixty puppies were immunized following a standard vaccination schedule with an attenuated combination vaccine containing antigens for canine adenovirus type 2, canine distemper virus, canine parainfluenza virus, canine parvovirus, and four strains of Leptospira bacteria. Serum antibody measurements for each viral and bacterial component were measured at multiple time points. Heritability estimations and GWAS were conducted using SNP genotypes at 279,902 markers together with serum antibody titer phenotypes. The heritability estimates were: (1) to Leptospira antigens, ranging from 0.178 to 0.628; and (2) to viral antigens, ranging from 0.199 to 0.588. There was not a significant difference between overall heritability of vaccine-induced immune response to Leptospira antigens compared to viral antigens. Genetic architecture indicates that SNPs of low to high effect contribute to immune response to vaccination. GWAS identified two genetic markers associated with vaccine-induced immune response phenotypes. Collectively, these findings indicate that genetic regulation of the immune response to vaccination is antigen-specific and influenced by multiple genes of small effect.

Immunogenicity and protective efficacy of a novel bacterium-like particle-based vaccine displaying canine distemper virus antigens in mice and dogs.

Canine distemper virus (CDV) poses a severe threat to both domesticated and wild animals, including multiple carnivores. With the continued expansion of its host range, there is an urgent need for the development of a safer and more effective vaccine. In this study, we developed subunit vaccines based on a bacterium-like particle (BLP) delivery platform containing BLPs-F and BLPs-H, which display the CDV F and H glycoprotein antigens, respectively, using the antigen-protein anchor fusions produced by a recombinant baculovirus insect cell expression system. The combination of BLPs-F and BLPs-H (CDV-BLPs), formulated with colloidal manganese salt [Mn jelly (MnJ)] adjuvant, triggered robust CDV-specific antibody responses and a substantial increase in the number of interferon gamma (IFN-γ)-secreting CD4+ and CD8+ T cells in mice. Dogs immunized intramuscularly with this vaccine not only produced CDV-specific IgG but also displayed elevated concentrations of IFN-γ and interleukin 6 in their serum, along with an increase of the CD3+CD4+ and CD3+CD8+ T cell subsets. Consequently, this heightened immune response provided effective protection against disease development and reduced viral shedding levels following challenge with a virulent strain. These findings suggest that this BLP-based subunit vaccine has the potential to become a novel canine distemper vaccine. IMPORTANCE: Many sensitive species require a safe and effective distemper vaccine. Non-replicating vaccines are preferred. We constructed subunit particles displaying canine distemper virus (CDV) antigens based on a bacterium-like particle (BLP) delivery platform. The CDV-BLPs formulated with theMn jelly adjuvant induced robust humoral and cell-mediated immune responses to CDV in mice and dogs, thereby providing effective protection against a virulent virus challenge. This work is an important step in developing a CDV subunit vaccine.

Recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) designed for rapid detection of canine distemper virus.

In the present study, recombinase polymerase amplification (RPA) was combined with the colloidal gold lateral flow dipstick (LFD) method to establish a new, stable, and efficient assay for the detection of canine distemper virus (CDV). We designed a set of specific primers labeled with biotin and a specific probe labeled with dSpacer and C3 spacer, according to the conserved region in the N-terminal gene sequence of CDV. The reaction conditions and systems were then optimized, and the sensitivity and specificity were analyzed for potential clinical application. The results showed that the RPA-LFD assay for CDV detection was successfully established. We also found that the temperature in a closed fist (35°C) is optimal for the RPA reaction. The optimal ratio of primer to probe was 2:1. The minimum detection limit of the RPA-LFD assay was 1 × 101 the median tissue culture infective dose (TCID50)/mL. Using this assay with samples from experimentally infected dogs, CDV was detected in nasal secretions, eye secretions, and blood on the fourth day post infection. In summary, this novel RPA-LFD assay for CDV detection is simple to use, and preliminary findings indicate its high specificity and sensitivity.

Epidemiological survey of infectious agents in free-ranging maned wolves (Chrysocyon brachyurus) in Northeastern Brazil.

Infectious diseases are one of the most concerning threats to maned wolves (Chrysocyon brachyurus) due to the potential impact on free-ranging populations. The species is currently classified as vulnerable according to the national list of threatened species and occurs mainly in open habitats, such as the Cerrado, a tropical savannah, which comprises its main distribution area in Brazil. In the northeastern region, it occurs in the Cerrado of Bahia, Piauí, Maranhão, and Tocantins states. Therefore, this study aimed to investigate the occurrence of infectious agents in Chrysocyon brachyurus through an epidemiological assessment of free-ranging individuals in western Bahia, specifically in the Barreiras microregion, a Cerrado area intensely fragmented and anthropized by agricultural activity. Eleven specimens were evaluated for serological titration, antigen research, and genetic material research for canine distemper virus (CDV), canine parvovirus (CPV), adenovirus-canine-type 1 (CAdV-1), canine coronavirus (CCoV), Leptospira interrogans and Toxoplasma gondii from 2020 to 2022. In addition to maned wolves, domestic dogs were also evaluated and tested. All maned wolves (100%) evaluated by the dot-ELISA technique exhibited immunoglobulin M (IgM) and seven (64%) exhibited immunoglobulin G (IgG) against CDV and CPV, while 100% exhibited IgG against CDV when using the immunochromatographic technique. Regarding CAdV-1, 90% were seropositive for IgG, while 64% exhibited IgG against T. gondii. Nine dogs from the region were also sampled, and all (100%) exhibited IgM and IgG against CDV and CPV. For IgG against T. gondii and against CAdV-1, 90% of the animals were seropositive. Molecular evaluation yielded negative results for all maned wolves and dogs assessed for CAdV-1, CDV, and T. gondii, as well as the CCoV antigen. These data indicate the occurrence of viral agents and Toxoplasma gondii in maned wolves and dogs, suggesting circulation in both populations.

Therapeutic mitigation of measles-like immune amnesia and exacerbated disease after prior respiratory virus infections in ferrets.

Measles cases have surged pre-COVID-19 and the pandemic has aggravated the problem. Most measles-associated morbidity and mortality arises from destruction of pre-existing immune memory by measles virus (MeV), a paramyxovirus of the morbillivirus genus. Therapeutic measles vaccination lacks efficacy, but little is known about preserving immune memory through antivirals and the effect of respiratory disease history on measles severity. We use a canine distemper virus (CDV)-ferret model as surrogate for measles and employ an orally efficacious paramyxovirus polymerase inhibitor to address these questions. A receptor tropism-intact recombinant CDV with low lethality reveals an 8-day advantage of antiviral treatment versus therapeutic vaccination in maintaining immune memory. Infection of female ferrets with influenza A virus (IAV) A/CA/07/2009 (H1N1) or respiratory syncytial virus (RSV) four weeks pre-CDV causes fatal hemorrhagic pneumonia with lung onslaught by commensal bacteria. RNAseq identifies CDV-induced overexpression of trefoil factor (TFF) peptides in the respiratory tract, which is absent in animals pre-infected with IAV. Severe outcomes of consecutive IAV/CDV infections are mitigated by oral antivirals even when initiated late. These findings validate the morbillivirus immune amnesia hypothesis, define measles treatment paradigms, and identify priming of the TFF axis through prior respiratory infections as risk factor for exacerbated morbillivirus disease.

Canine distemper virus infection of vaccinal origin in a 14-week-old puppy.

The body of a 14-wk-old puppy (Canis familiaris) was submitted to the Animal Health Laboratory, University of Guelph, Ontario for postmortem examination following a history of intermittent anorexia and lethargy progressing to pyrexia, pruritic skin rash, mucoid nasal discharge, decreased mentation, dysphagia, muscle twitches, and focal seizures. Gross examination revealed rhinitis and pulmonary edema. Histologically, there was fibrinonecrotizing bronchopneumonia, tracheitis, and neutrophilic and lymphohistiocytic rhinitis; rarely within the cortical gray and white matter of the brain were small clusters of glial cells, with rare individual neutrophils in the choroid plexus. Although canine distemper was suspected, none of the usual supportive histologic lesions of distinct syncytial cells, viral inclusion bodies, or demyelinating leukoencephalitis were observed. Lung and brain tissues were PCR-positive for canine distemper virus (CDV), and CDV was detected immunohistochemically in the brain. The agent from the PCR-positive sample from the brain was genotyped and was a 99.9% match to the CDV Rockborn strain, indicating that the disease agent in our case was vaccinal in origin. Our unusual case highlights the possibility of reversion to virulence in a modified-live virus vaccine, and the occurrence of a disease in the absence of a full complement of the usual and compatible histologic lesions.

Rational attenuation of canine distemper virus (CDV) to develop a morbillivirus animal model that mimics measles in humans.

Morbilliviruses are members of the family Paramyxoviridae and are known for their ability to cause systemic disease in a variety of mammalian hosts. The prototypic morbillivirus, measles virus (MeV), infects humans and still causes morbidity and mortality in unvaccinated children and young adults. Experimental infection studies in non-human primates have contributed to the understanding of measles pathogenesis. However, ethical restrictions call for the development of new animal models. Canine distemper virus (CDV) infects a wide range of animals, including ferrets, and its pathogenesis shares many features with measles. However, wild-type CDV infection is almost always lethal, while MeV infection is usually self-limiting. Here, we made five recombinant CDVs, predicted to be attenuated, and compared their pathogenesis to the non-attenuated recombinant CDV in a ferret model. Three viruses were insufficiently attenuated based on clinical signs, fatality, and systemic infection, while one virus was too attenuated. The last candidate virus caused a self-limiting infection associated with transient viremia and viral dissemination to all lymphoid tissues, was shed transiently from the upper respiratory tract, and did not result in acute neurological signs. Additionally, an in-depth phenotyping of the infected white blood cells showed lower infection percentages in all lymphocyte subsets when compared to the non-attenuated CDV. In conclusion, infection models using this candidate virus mimic measles and can be used to study pathogenesis-related questions and to test interventions for morbilliviruses in a natural host species.IMPORTANCEMorbilliviruses are transmitted via the respiratory route but cause systemic disease. The viruses use two cellular receptors to infect myeloid, lymphoid, and epithelial cells. Measles virus (MeV) remains an important cause of morbidity and mortality in humans, requiring animal models to study pathogenesis or intervention strategies. Experimental MeV infections in non-human primates are restricted by ethical and practical constraints, and animal morbillivirus infections in natural host species have been considered as alternatives. Inoculation of ferrets with wild-type canine distemper virus (CDV) has been used for this purpose, but in most cases, the virus overwhelms the immune system and causes highly lethal disease. Introduction of an additional transcription unit and an additional attenuating point mutation in the polymerase yielded a candidate virus that caused self-limiting disease with transient viremia and virus shedding. This rationally attenuated CDV strain can be used for experimental morbillivirus infections in ferrets that reflect measles in humans.

A method for screening CDV microneutralization activity in microvolume samples.

OBJECTIVE: This study aims to establish a screening method for canine distemper virus (CDV) microneutralizing activity suitable for microvolume samples. METHODS: This method is based on the Indirect immunofluorescence assay (IFA) established on Vero-slam cells. First, by comparing the sensitivities of CDV neutralizing monoclonal antibody (1C42H11) and NP protein monoclonal antibody (CDV-NP) in IFA experiments, CDV-NP was selected as the primary antibody. Then, by detecting the infection rates of multi-concentrations of CDV neutralized with water, the minimum CDV concentration with an infection rate greater than 90% was defined as the minimum stable infection concentration, which was used as the neutralizing solution for this method. Finally, the CDV-positive neutralizing serum (neutralizing titer 1:708) was diluted into multiple dilution groups as test samples, and then neutralized in equal volumes with the neutralizing solution to detect the neutralizing activity detection rates of each dilution group and the lowest detection limit of this method. RESULTS: The results showed that the neutralizing activity of serum with a CDV neutralizing titer of 1:708 diluted 212 times was the lowest limit of detection, and the detection rate of microneutralizing activity was 63.54 ± 4.774%. CONCLUSION: This study established an economical, stable, and easy-to-operate CDV microneutralizing activity high-throughput screening method, laying a methodological foundation for the development of native CDV neutralizing antibodies based on single B cells.

Canine distemper virus (CDV)-neutralizing activities of an anti-CDV canine-derived single-chain variable antibody fragment 4-15 (scFv 4-15) screened by phage display technology.

Canine distemper virus (CDV) is a highly contagious pathogen that causes severe diarrhea, fever and vomiting in domestic dogs, posing a serious threat to the dog breeding industry. Currently, there are no effective therapeutic agents for emergency treatment despite the availability of vaccines against CDV infection. Single-chain fragment variable (scFv) antibody has been demonstrated to effectively inhibit virus infections, suggesting a potential candidate as a therapeutic agent for canine distemper. In this study, a phage-displayed scFv library was constructed from the peripheral blood lymphocytes of dog immunized intramuscularly with live-attenuated CDV vaccine, and was subjected to four rounds of pannings against CDV. Subsequent indirect enzyme-linked immunosorbent assay screening revealed high-affinity scFv antibodies specific to CDV, and indirect immunofluorescence assay screening revealed CDV-neutralizing activity of scFv antibodies. Our results showed that a scFv antibody 4-15 (scFv 4-15) with high-affinity binding to CDV and neutralizing activity against CDV was obtained, which displayed effective therapeutic potential in vivo for dogs challenged with a lethal dose of CDV. Conclusively, the scFv 4-15 with high-affinity binding and neutralizing activity to CDV that was obtained by phage display technology provides a promising candidate for the therapeutic agents against CDV infection.