Canine Distemper Virus Vaccine Research Articles

CHALLENGES IN DEVELOPING VACCINE FOR CANINE DISTEMPER

The Canine Distemper Virus (CDV) represents a considerable risk to domestic and wild carnivore populations globally, notwithstanding the existence of vaccinations. This study examines the significant obstacles in creating successful CDV vaccines, especially regarding virus evolution, immune evasion, cross-species transmission, and vaccine reluctance. This review aims to consolidate existing research on CDV vaccine development, emphasizing emerging technologies, such as nanoparticle and DNA vaccines, and their capacity to address present challenges in immunogenicity and cross-species protection. Through the analysis of recent findings, this review delineates significant gaps in the comprehension of CDV's genetic variety of CDVs, the sustainability of wildlife reservoirs, and the obstacles to attaining extensive vaccination coverage. This review evaluates the influence of public health infrastructure and socioeconomic factors on vaccination distribution and uptake constraints, particularly in developing areas. These findings highlight the significance of a One Health approach, which amalgamates veterinary, wildlife, and public health viewpoints to address CDV epidemics more efficiently. Future research should focus on augmenting vaccine efficacy in non-domestic species, increasing public awareness of immunization initiatives, and creating thermostable vaccines for improved distribution accessibility. This review offers critical insights into the intricacies of the CDV vaccination and presents a framework for enhancing worldwide initiatives to combat this destructive virus.

Persistence of Infectious Canine Distemper Virus in Murine Xenotransplants of Canine Histiocytic Sarcoma Cells after Intratumoral Application.

Oncolytic viruses and morbilliviruses in particular, represent an interesting therapeutic approach for tumors with a poor prognosis and frequent resistance to conventional therapies. Canine histiocytic sarcomas (HS) exemplify such a neoplasm in need for new curative approaches. Previous investigations demonstrated a limited success of an acute intratumoral application of canine distemper virus (CDV) on xenotransplanted canine histiocytic sarcoma cells (DH82 cells), while persistently CDV-infected DH82 cell transplants exhibited a complete spontaneous regression. Therefore, the present study focuses on an intratumoral application of persistently CDV vaccine strain Onderstepoort-infected DH82 (DH82 Ond p.i.) cells into non-infected subcutaneous DH82 cell transplants in a murine model. DH82 cell transplants that received 10 applications, two days apart, showed a transient growth retardation as well as larger areas of intratumoral necrosis, lower mitotic rates, and a decreased intratumoral vascularization compared to controls. Viral mRNA was detected in all neoplasms following application of DH82 Ond p.i. cells until 66 days after the last injection. Furthermore, infectious virus was present until 62 days after the last injection. Although complete regression was not achieved, the present application regimen provides promising results as a basis for further treatments, particularly with genetically modified viruses, to enhance the observed effects.

Safety and Immunogenicity of Morbillivirus canis Vaccines for Domestic and Wild Animals: A Scoping Review.

Morbillivirus canis (canine distemper virus (CDV)) is recognized as a multihost pathogen responsible for a transmissible disease affecting both domestic and wild animals. A considerable portion of wildlife populations remain unvaccinated due to a lack of safety and immunogenicity data on existing vaccines for the prevention of CDV infection in these species. This review aimed to assess the current state of CDV vaccination research for both domestic and wild animals and to explore novel vaccine candidates through in vivo studies. It also sought to synthesize the scattered information from the extensive scientific literature on CDV vaccine research, identify key researchers in the field, and highlight areas where research on CDV vaccination is lacking. A scoping review was conducted across four databases following the PRISMA-ScR protocol, with information analyzed using absolute and relative frequencies and 95% confidence intervals (CIs) for study number proportions. Among the 2321 articles retrieved, 68 met the inclusion criteria and focused on CDV vaccines in various animal species, such as dogs, ferrets, minks, and mice. Most of the scientific community involved in this research was in the USA, Canada, France, and Denmark. Various vaccine types, including MLV CDV, recombinant virus, DNA plasmids, inactivated CDV, and MLV measles virus (MeV), were identified, along with diverse immunization routes and schedules employed in experimental and commercial vaccines. Safety and efficacy data were summarized. Notably, 37 studies reported postimmunization CDV challenge, primarily in dogs, revealing the survival rates of vaccinated animals. In summary, CDV vaccines generally demonstrate an acceptable safety profile in dogs and show promise as a means of controlling CDV. However, significant gaps in vaccine research persist, particularly concerning wildlife reservoirs, indicating the need for further investigation.

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.

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.

DNA Vaccine Co-Expressing Hemagglutinin and IFN-γ Provides Partial Protection to Ferrets against Lethal Challenge with Canine Distemper Virus.

Canine distemper (CD), caused by canine distemper virus (CDV), is a highly contagious and lethal disease in domestic and wild carnivores. Although CDV live-attenuated vaccines have reduced the incidence of CD worldwide, low levels of protection are achieved in the presence of maternal antibodies in juvenile animals. Moreover, live-attenuated CDV vaccines may retain residual virulence in highly susceptible species and cause disease. Here, we generated several CDV DNA vaccine candidates based on the biscistronic vector (pIRES) co-expressing virus wild-type or codon-optimized hemagglutinin (H) and nucleocapsid (N) or ferret interferon (IFN)-γ, as a molecular adjuvant, respectively. Apparently, ferret (Mustela putorius furo)-specific codon optimization increased the expression of CDV H and N proteins. A ferret model of CDV was used to evaluate the protective immune response of the DNA vaccines. The results of the vaccinated ferrets showed that the DNA vaccine co-expressing the genes of codon-optimized H and ferret IFN-γ (poptiH-IRES-IFN) elicited the highest anti-CDV serum-neutralizing antibodies titer (1:14) and cytokine responses (upregulated TNF-α, IL-4, IL-2, and IFN-γ expression) after the third immunization. Following vaccination, the animals were challenged with a lethal CDV 5804Pe/H strain with a dose of 105.0 TCID50. Protective immune responses induced by the DNA vaccine alleviated clinical symptoms and pathological changes in CDV-infected ferrets. However, it cannot completely prevent virus replication and viremia in vivo as well as virus shedding due to the limited neutralizing antibody level, which eventually contributed to a survival rate of 75% (3/4) against CDV infection. Therefore, the improved strategies for the present DNA vaccines should be taken into consideration to develop more protective immunity, which includes increasing antigen expression or alternative delivery routes, such as gene gun injection.

Establishment and evaluation of a multiplex real-time RT-PCR for quantitative and differential detection of wild-type canine distemper virus from vaccine strains

This study sought to establish a real-time reverse transcription (RT)-PCR method to differentially detect canine distemper virus (CDV) wild-type and vaccine strains. To this end, a pair of CDV universal primers and two specific minor groove binder (MGB) probes, harboring a T/C substitution in the hemagglutinin (H) gene, were designed. Using a recombinant plasmid expressing the H gene of the CDV wild-type or vaccine strain as standards, a sensitive and specific multiplex real-time RT-PCR was established for quantitative and differential detection of CDV wild-type and vaccine strains. The limit of detection for this multiplex assay was 22.5 copies/μL and 2.98 copies/μL of viral RNA for wild-type and vaccine strains, respectively. Importantly, the wild-type and vaccine MGB probes specifically hybridized different genotypes of wild-type CDV circulating in China as well as globally administered vaccine viruses, respectively, with no cross-reactivity observed with non-CDV viruses. Moreover, this method was successfully applied for the quantitative detection of CDV RNA in tissue samples of experimentally infected breeding foxes, raccoon dogs, and minks. Additionally, the multiplex real-time RT-PCR was able to detect the viral RNA in the whole blood samples as early as 3 days post-infection, 3 to 4 days prior to the onset of clinical signs in these CDV infection animals. Hence, the established multiplex real-time RT-PCR method is useful for differentiating wild-type CDV and vaccine strains in China, and for conducting canine distemper early diagnosis as well as dynamic mechanism of CDV replication studies in vivo.

Development of a monoclonal antibody recognizing novel linear neutralizing epitope on H protein of canine distemper virus vaccine strains (America-1 genotype)

Canine distemper virus (CDV) is an economically important virus responsible for canine distemper (CD), a highly contagious disease that afflicts various animal species worldwide. The hemagglutinin (H) protein is the major neutralizing target of virus. Therefore, it is often considered as immunogen to prepare neutralizing antibodies. The accurate identification of neutralizing epitope will provide important antigenic information and extend the knowledge of mechanisms of virus neutralization. In this study, we generated a neutralizing monoclonal antibody (mAb) 4C6 against CDV H protein, and defined the minimal linear epitope 238DIEREFDT245, which was highly conserved in America-1 genotype of CDV strains (vaccines). The mAb 4C6 could not react with a CDV strain that had two substitutions of D238Y and R241G in the epitope, which appeared in most CDV strains of the other genotypes. Besides, a few different amino acid mutations in the epitope were also included. Collectively, the epitope 238DIEREFDT245 was variable in the other genotypes of CDV strains. The epitope 238DIEREFDT245 was exposed to the surface of CDV H protein, showing good antigenicity. These data will provide insights into structure, function and antigenicity of H protein and lay the foundation for the development of diagnostic technologies and vaccine design for CDV.

In-vitro Replication Competence of Three Modified-live Canine Distemper Vaccines

Over the past several years, there have been a number of severe canine distemper virus (CDV) outbreaks in shelters involving adult dogs that were considered fully vaccinated with modified live vaccine prior to exposure. Shelters vaccinating large numbers of animals during admission or high-volume public vaccine clinics may reconstitute modified live vaccines (MLV) hours ahead of use and store them under non-ideal cold chain conditions. There is little guidance regarding the durability of MLV potency, and viral components may degrade under storage conditions (both pre- and post-reconstitution) employed by shelters in the name of efficiency. This study aimed to generate a model for examining the effect of vaccine husbandry on the in-vitro replication competence of MLV CDV. Shelters were surveyed to determine the most common vaccine brands and the maximum duration between reconstitution and use of CDV MLV to guide the time/temperature conditions in our in vitro study. CDV-susceptible Madin-Darby canine kidney (MDCK) cells were grown in Dulbecco’s modified Eagle’s medium containing fetal bovine serum, penicillin/streptomycin, and l-glutamine, seeded into a 96-well plate, and inoculated with CDV MLV. There were 3 replicates per time/temperature condition. Viral replication was allowed to proceed for 72 hours. Fluorescently labeled anti-CDV antibodies were used to identify cells infected with vaccine-derived CDV via flow cytometry. A Kruskal-Wallis test was used to test differences in medians between the groups. Three quadrivalent CDV vaccine brands were assessed in our model system to examine virus replication competency in vitro. Two commercial vaccines, including the most reported brand in our survey, did not produce infection beyond baseline uninoculated controls, while a third showed robust infect rates. This brand was used for subsequent trials of different storage conditions. Immediate reconstitution infected a median of 14% (IQR 10, 18) of cells. In comparison, 10 min at room temperature (RT) pre-reconstitution (PreR), 10 min at RT post-reconstitution (PostR), 10 min at 4 °C PostR, 60 min at RT PreR, 60 min at RT PostR, and 60 min at °4C PostR infected a median of 15% (IQR 10,20), 14% (IQR 8,23) 14% (11, 24), 11% (8, 21), 13% (9, 26), and 10% (7, 20), respectively. Similarly, 120 min at RT PreR, 120 min at RT PostR, 120 min at 4 °C PostR, 120 min RT PreR followed by 60 min 4 °C, and 1440 min at RT PreR followed by 1440 min at RT PostR infected a median of 9% (8, 17), 10% (8, 20), (9% (8, 22), 8% (9, 22), and 9% (10, 23) of cells, respectively. There was no difference between conditions. Our in-vitro model demonstrated no significant difference in median cell infection rates between time/temperature conditions up to an extreme of 24 hours pre-reconstitution/24 hours post-reconstitution at room temperature.

A recent distemper virus outbreak in the growing canine populations of Galapagos Islands: a persistent threat for the endangered Galapagos Sea Lion.

Canine Distemper Virus (CDV) is a highly contagious virus that can cross mammalian species barriers and has widespread impacts on both domestic animals and wildlife populations. This study describes a recent outbreak of CDV in the Galapagos Islands in 2019. A total number of 125 dogs with clinical signs compatible with CDV were included in this study. Nasal swabs were taken and analyzed by RT-qPCR for the detection of CDV, resulting in a positivity rate of 74.4% (IC95%, 66-81%). Among the CDV positive dogs, 82.2% presented with respiratory signs, 48.8% neurological signs, and 28.9% gastrointestinal signs. CDV has been previously reported in the domestic canine population of the Galapagos Islands in 2001 and 2004. The current study shows how CDV is still a threat for the endemic and endangered Galapagos sea lion, despite recent policies for dog population control and CDV vaccination.

Biological and Genetic Characterization of Canine Distemper Virus Vaccine Candidate Named as CD1901-100

Biological and Genetic Characterization of Canine Distemper Virus Vaccine Candidate Named as CD1901-100

Establishment of Multiplex RT-PCR for Differentiating between the CD1901 and Lederle Strains of Canine Distemper Virus

Canine distemper virus (CDV) is a life-threatening pathogen in dogs. Clinical, pathological and molecular methods are required to diagnose CDV infection, and it is important to differentiate between the Korean CDV strain CD1901 and Lederle CDV vaccine strain. Therefore, in this study, we used multiplex reverse- transcription polymerase chain reaction (RT-PCR) to differentiate between the CD1901 and Lederle strains. A primer set was designed based on the CDV nucleoprotein gene and nucleotide sequence variation in the fusion (F) gene. First, 224-bp DNA bands were amplified from viral RNA of the CD1901 and Lederle strains. Then, 428- and 326-bp DNA bands were amplified in the CD1901 and Lederle strain, respectively. The multiplex RT-PCR detection limits were 2.53 and 0.8 median tissue culture infectious dose/reaction for the CD1901 and Lederle strains, respectively. No cross-reactions were detected in non-CDV reference viruses, including rabies virus, parvovirus, canine adenovirus types 1 and 2, and parainfluenza virus. The results indicate that our one-step multiplex RT-PCR is useful for differentiating between wildtype and vaccine CDV strains.

Transcriptome Analysis Reveals the Alternative Splicing Changes in the Immune-Related Genes of the Giant Panda (Ailuropoda melanoleuca), in Response to the Canine Distemper Vaccine.

Canine distemper virus (CDV) is a highly fatal virus to the giant panda (Ailuropoda melanoleuca). Although vaccination is a key preventative measure in captive giant pandas, the immune response of giant pandas after vaccination remains unclear. Therefore, this study focuses on differential alternative splicing (DAS) events of giant pandas before and after vaccination to investigate the role of alternative splicing in the immune response of giant pandas after CDV vaccination. In this study, we identified 1113 DAS genes, which had 1288 DAS events. The KEGG functional enrichment analysis of DAS genes showed enrichment of some DNA damage repair and immune-related pathways. In the combined analysis of DAS and differentially expressed genes (from our previous research), we identified 66 differentially expressed genes with a DAS event, and found that some important immune-related genes, such as IL15, IL18, IL18RAP, CHUK, IFI44, CD40, and CD46 underwent DAS events and were involved in the immune response of giant pandas after CDV vaccination. We describe here the alternative splicing events of giant pandas after CDV vaccination for the first time and show that the results indicated that alternative splicing has an important role in regulating the immune response of giant pandas after vaccination.

RECOMBITEK CANINE DISTEMPER VACCINE AS AN ALTERNATIVE FOR PUREVAX DISTEMPER VACCINE IN ENDANGERED BLACK-FOOTED FERRETS (MUSTELA NIGRIPES).

Black-footed ferrets (Mustela nigripes) are an endangered species in North America that are highly sensitive to canine distemper virus (CDV) infections and any exposure could be devastating to species recovery. The U.S. Fish and Wildlife Service recovery program has safely used a recombinant DNA (rDNA) canarypox-vectored CDV vaccine, Purevax® Ferret Distemper (PFD), to vaccinate black-footed ferrets. Because of a PFD shortage in 2015, an rDNA vaccine labeled for use in dogs, Recombitek® CDV (rCDV), was chosen to vaccinate black-footed ferrets. Our goal was to compare the serum neutralizing (SN) titers after vaccination of 17 captive and 18 wild black-footed ferrets with rCDV or PFD, respectively, considering ≥1:128 as a protective titer. Both vaccines produced comparable 1 yr postvaccination protective titers in captive and wild black-footed ferrets. In wild black-footed ferrets, one PFD vaccination produced SN titers similar to two PFD vaccinations at 1 yr postvaccination. One year after vaccination with rCDV, SN titers in captive black-footed ferrets were higher than in wild ferrets. These results indicate rCDV may be an effective alternative CDV vaccine in captive black-footed ferrets and PFD should be prioritized for wild ferrets because one dose was effective for animals that can be difficult to recapture.

Canine distemper virus induces early stages of apoptosis in in vitro culture of canine peripheral blood mononuclear cells

Canine distemper virus (CDV) has been described to induce apoptosis in lymphoid tissues, lymphoid cells, and the cerebellum of naturally infected dogs. The immunosuppression stage established from CDV infection is not always associated with virus detection in damaged cells. Examining the host response at the early stages of immunosuppression, especially the expression of activators and/or inhibitors of apoptosis processes, can provide an important understanding of how the immune responses to CDV are orchestrated. For this purpose, canine peripheral blood mononuclear cells (PBMC) collected from healthy dogs were cultured and infected by the CDV vaccine strain (Onderstepoort). After 24 h post-infection (p.i.), early events of apoptosis were detected by search for annexin V™ and ApoTrace™ markers. The expression of mRNA of Bax, BCl-2, caspase-3, 8 and 9, and also survivin genes was performed in infected and uninfected canine PBMC at 24 h p.i. by real-time polymerase chain reaction. The vaccine strain induced loss of PBMC viability with the expression of annexin™ V FITC and Apo-Trace™ FITC in more than 80% of infected cells compared to the control group (P < 0.001) at 24 h p.i. The initiation of early apoptosis after 24h post-infection contrasts with higher expression of mRNA of survivin and BCl-2, two major anti-apoptotic proteins (p < 0.0002). PBMC infected by CDV increased survivin and BCL-2 gene transcription, concomitant to a reduced level of Bax, caspase 3, 8 and 9. This is the first description of survivin expression, an anti-apoptotic mediator, induced by CDV infection on in vitro cultured canine PBMC.

Prevalence of Neutralizing Antibodies to Canine Distemper Virus and Response to Vaccination in Client-Owned Adult Healthy Dogs.

Re-vaccinations against canine distemper virus (CDV) are commonly performed in 3-year intervals. The study’s aims were to determine anti-CDV antibodies in healthy adult dogs within 28 days of vaccination against CDV, and to evaluate factors associated with the presence of pre-vaccination antibodies and with the antibody response to vaccination. Ninety-seven dogs, not vaccinated within 1 year before enrollment, were vaccinated with a modified live CDV vaccine. A measurement of the antibodies was performed before vaccination (day 0), on day 7, and 28 after the vaccination by virus neutralization. A response to vaccination was defined as a ≥4-fold titer increase by day 28. Fisher’s exact test was used to determine factors associated with a lack of antibodies and vaccination response. In total, 94.8% of the dogs (92/97; CI 95%: 88.2–98.1) had antibodies (≥10) prior to vaccination. A response to vaccination was not observed in any dog. Five dogs were considered humoral non-responders; these dogs neither had detectable antibodies before, nor developed antibodies after vaccination. Young age (<2 years) was significantly associated with a lack of pre-vaccination antibodies (p = 0.018; OR: 26.825; 95% CI: 1.216–1763.417). In conclusion, necessity of re-vaccination in adult healthy dogs should be debated and regular vaccinations should be replaced by antibody detection.

MODIFIED LIVE DISTEMPER VACCINES CARRY LOW MORTALITY RISK FOR CAPTIVE AFRICAN WILD DOGS, LYCAON PICTUS

Recently, canine distemper virus (CDV) has been linked to population declines in the endangered African wild dog (Lycaon pictus). As CDV appears able to persist in wildlife, threats to free-ranging wild dogs cannot be eliminated by vaccinating domestic dogs. Conservation managers may therefore consider CDV vaccination of wild dogs in highly threatened populations. For use in field conservation, the ideal CDV vaccine would be safe, immunogenic, and readily available in Africa. The CDV vaccine type most commonly used for domestic dogs (modified live vaccine) is available in Africa, and apparently immunogenic in wild dogs, but has been linked to fatal vaccine-induced distemper in captive wild dogs. However, alternatives are either ineffective (inactivated vaccine) or difficult to obtain in Africa (recombinant vaccine). Data from a questionnaire survey of zoo vaccination practices were therefore combined with studbook tracing to assess the safety of modified live CDV vaccine in captive African wild dogs. Among 135 wild dog pups given modified live CDV vaccine for the first time, there was a single, unconfirmed, case of potential vaccine-induced distemper. Pups given modified live vaccine survived better than those given inactivated vaccine or no vaccine. Although studbook tracing revealed higher overall pup survival at zoos which responded to the questionnaire than at zoos which did not, tracing of all pups born during a 20-yr period that lived long enough to be vaccinated (n = 698 pups in 155 litters) revealed no mortality events consistent with vaccine-induced distemper. Modified live CDV vaccine thus appears to carry low mortality risks for African wild dog pups in captivity, and may warrant trials in free-ranging populations.

Changes in the MicroRNA Profile of the Giant Panda After Canine Distemper Vaccination and the Integrated Analysis of MicroRNA-Messenger RNA.

Canine distemper (CD) is a significant threat to wild and captive giant panda populations. Captive giant pandas are inoculated with canine distemper virus (CDV) vaccination to prevent the infection with the CDV. As an important regulator, microRNA (miRNA) plays a crucial role in regulating gene expression, including in disease immunity. To understand the role of miRNA in immune response to CDV vaccination, we investigated the miRNA expression profile in five giant panda cubs after two inoculations, 21 days apart. A total of 187 conserved miRNAs and 96 novel miRNAs were identified. Among the 187 conserved miRNAs, 29 differentially expressed miRNAs were found postinoculation. The upregulation of miR-16, miR-182, miR-30b, and miR-101 indicated that the innate immune may be enhanced, whereas the upregulation of miR-142 and miR-19a are probably involved in the enhanced cellular immune response. However, the downregulated miR-155 and miR-181a might indicate the giant panda has weak ability to produce antibodies and memory B cells. Integrated analysis of miRNA-messenger RNA (mRNA) found 20 negatively regulated miRNA-mRNA pairs, where downregulated miR-204 might enhance giant panda cub innate immunity by increasing TLR6 expression, and downregulated miR-330 might activate macrophages and regulate the immune response by increasing TMEM106A expression. Our research provides key information for future development to enhance the immune response of giant pandas and potentially improve the survival of captive and wild giant panda populations threatened by CD.

Canine Distemper Outbreak by Natural Infection in a Group of Vaccinated Maned Wolves in Captivity.

Canine distemper virus (CDV) is one of the most significant infectious disease threats to the health and conservation of free-ranging and captive wild carnivores. CDV vaccination using recombinant canarypox-based vaccines has been recommended for maned wolf (Chrysocyon brachyurus) after the failure of modified live vaccines that induced disease in vaccinated animals. Here, we report a CDV outbreak in a captive population of maned wolves that were previously vaccinated. Five juveniles and one adult from a group of seven maned wolves housed in an outdoor exhibit died in April–May 2013 in a zoo in the Metropolitan Region, Chile. Clinical signs ranged from lethargy to digestive and respiratory signs. Diagnosis of CDV was confirmed by histopathology, antibody assays, and viral molecular detection and characterization. The phylogenetic analyses of the nucleotide sequence of the H gene of the CDV genome identified in the two positive samples suggest a close relation with the lineage Europe 1, commonly found in South America and Chile. CDV infections in maned wolves have not been previously characterized. To the authors’ best knowledge, this is the first report of the clinical presentation of CDV in a canine species previously immunized with a recombinant vaccine.

Isolation and Phylogenetic Analysis of the Canine Distemper Virus from a Naturally Infected Dog in China

Background: Canine distemper virus (CDV) can infect a variety of predators, such as Canidae, Mustelidae and Procyonidae family and cause high morbidity and mortality. Genetic/antigenic heterogeneity has been observed among various CDV strains, notably in the haemagglutinin (H) gene. Based on sequence analysis of the H gene, wild-type CDV strains can be divided into distinct genotypes. Methods: We used Vero/SLAM cells to isolate CDV strains from clinically naturally infected dog tissues. Here, the H gene sequence of the isolated strain was analyzed for the restriction enzyme polymorphism with RT-PCR-RFLP method and the method could be used to distinguish CDV vaccine strains from wild-type strains. Furthermore, the genetic and antigenic variation of the H gene was also analyzed.Result: Our results indicated that Vero cells expressing canine SLAM allow the efficient isolation of CDV wild-type strains. The isolated CDV was named Shandong strain. Phylogenetic analysis identified that Shandong strain was CDV wild-type strain, the Asian genotype I. Analysis of the potential glycosylation site of the H gene and determination of the neutralization titer PD50 showed that there were immunogenic differences between the Shandong strain and the vaccine strains.