Intratumoral Canine Distemper Virus Infection Inhibits Tumor Growth by Modulation of the Tumor Microenvironment in a Murine Xenograft Model of Canine Histiocytic Sarcoma.

Histiocytic sarcomas refer to highly aggressive tumors with a poor prognosis that respond poorly to conventional treatment approaches. Oncolytic viruses, which have gained significant traction as a cancer therapy in recent decades, represent a promising option for treating histiocytic sarcomas through their replication and/or by modulating the tumor microenvironment. The live attenuated canine distemper virus (CDV) vaccine strain Onderstepoort represents an attractive candidate for oncolytic viral therapy. In the present study, oncolytic virotherapy with CDV was used to investigate the impact of this virus infection on tumor cell growth through direct oncolytic effects or by virus-mediated modulation of the tumor microenvironment with special emphasis on angiogenesis, expression of selected MMPs and TIMP-1 and tumor-associated macrophages in a murine xenograft model of canine histiocytic sarcoma. Treatment of mice with xenotransplanted canine histiocytic sarcomas using CDV induced overt retardation in tumor progression accompanied by necrosis of neoplastic cells, increased numbers of intratumoral macrophages, reduced angiogenesis and modulation of the expression of MMPs and TIMP-1. The present data suggest that CDV inhibits tumor growth in a multifactorial way, including direct cell lysis and reduction of angiogenesis and modulation of MMPs and their inhibitor TIMP-1, providing further support for the concept of its role in oncolytic therapies.

Mesenchymal to epithelial transition driven by canine distemper virus infection of canine histiocytic sarcoma cells contributes to a reduced cell motility in vitro.

Sarcomas especially of histiocytic origin often possess a poor prognosis and response to conventional therapies. Interestingly, tumours undergoing mesenchymal to epithelial transition (MET) are often associated with a favourable clinical outcome. This process is characterized by an increased expression of epithelial markers leading to a decreased invasion and metastatic rate. Based on the failure of conventional therapies, viral oncolysis might represent a promising alternative with canine distemper virus (CDV) as a possible candidate. This study hypothesizes that a CDV infection of canine histiocytic sarcoma cells (DH82 cells) triggers the MET process leading to a decreased cellular motility. Immunofluorescence and immunoblotting were used to investigate the expression of epithelial and mesenchymal markers followed by scratch assay and an invasion assay as functional confirmation. Furthermore, microarray data were analysed for genes associated with the MET process, invasion and angiogenesis. CDV-infected cells exhibited an increased expression of epithelial markers such as E-cadherin and cytokeratin 8 compared to controls, indicating a MET process. This was accompanied by a reduced cell motility and invasiveness. Summarized, these results suggest that CDV infection of DH82 cells triggers the MET process by an increased expression of epithelial markers resulting in a decreased cell motility in vitro.

Oxidative Stress in Canine Histiocytic Sarcoma Cells Induced by an Infection with Canine Distemper Virus Led to a Dysregulation of HIF-1α Downstream Pathway Resulting in a Reduced Expression of VEGF-B in vitro.

Histiocytic sarcomas represent malignant tumors which require new treatment strategies. Canine distemper virus (CDV) is a promising candidate due to its oncolytic features reported in a canine histiocytic sarcoma cell line (DH82 cells). Interestingly, the underlying mechanism might include a dysregulation of angiogenesis. Based on these findings, the aim of the present study was to investigate the impact of a persistent CDV-infection on oxidative stress mediated changes in the expression of hypoxia-inducible factor (HIF)-1α and its angiogenic downstream pathway in DH82 cells in vitro. Microarray data analysis, immunofluorescence for 8-hydroxyguanosine, superoxide dismutase 2 and catalase, and flow cytometry for oxidative burst displayed an increased oxidative stress in persistently CDV-infected DH82 cells (DH82Ond pi) compared to controls. The HIF-1α expression in DH82Ond pi increased, as demonstrated by Western blot, and showed an unexpected, often sub-membranous distribution, as shown by immunofluorescence and immunoelectron microscopy. Furthermore, microarray data analysis and immunofluorescence confirmed a reduced expression of VEGF-B in DH82Ond pi compared to controls. In summary, these results suggest a reduced activation of the HIF-1α angiogenic downstream pathway in DH82Ond pi cells in vitro, most likely due to an excessive, unusually localized, and non-functional expression of HIF-1α triggered by a CDV-induced increased oxidative stress.

Seroprevalence of viral and vector-borne bacterial pathogens in domestic dogs (Canis familiaris) in northern Botswana.

BACKGROUND: Domestic dogs (Canis familiaris) have the potential to act as disease reservoirs for wildlife and are important sentinels for common circulating pathogens. Therefore, the infectious disease seroprevalence among domestic dogs in northern Botswana may be indicative of pathogen exposure of various wildlife species. The objective of this study was to assess the seroprevalence of Ehrlichia spp., Borrelia burgdorferi, Anaplasma spp., Dirofilaria immitis, canine adenovirus, canine parvovirus, and canine distemper virus in domestic dogs as proxies of disease prevalence in the local wildlife in the Okavango Delta region of Botswana. Statistical analysis assessed crude and factor-specific seroprevalence proportions in relation to age, sex, and geographical location as predictors of seropositivity. Logistic regression was used to identify adjusted predictors of seropositivity for each of the pathogens of interest. RESULTS: Samples from 233 dogs in a total of seven locations in Maun, Botswana, and surrounding villages were collected and serologically analyzed. No dogs were seropositive for B. burgdorferi, while low seroprevalence proportions were observed for Anaplasma spp. (2.2%) and D. immitis (0.9%). Higher seroprevalence proportions were observed for the tick-borne pathogen Ehrlichia spp. (21.0%), and 19.7% were seropositive for canine adenovirus (hepatitis). The highest seroprevalence proportions were for canine parvovirus (70.0%) and canine distemper virus (44.8%). The predictors of seropositivity revealed that adults were more likely to be seropositive for canine adenovirus, canine distemper virus, and canine parvovirus than juveniles, and location was a risk factor for canine adenovirus, canine distemper virus, canine parvovirus, and Ehrlichia spp. CONCLUSIONS: Results indicate that increasing tick control and vaccination campaigns for domestic dogs may improve the health of domestic animals, and potentially wildlife and humans in the Okavango Delta since viral and vector-borne bacterial pathogens can be transmitted between them.

Canine distemper virus increased the differentiation of CD4+CD8+ T cells and mRNA expression of inflammatory cytokines in peripheral blood lymphocyte from canine.

BACKGROUND: Canine distemper virus (CDV) can cause a highly contagious disease to canid. However, how CDV affects peripheral blood lymphocyte (PBL) remains unclear. METHODS: In this study, CDV infected PBL was cultured to investigate the effect of CDV on the differentiation of lymphocytes and the mRNA expression of inflammatory cytokines in PBL. RESULTS: The results showed that CDV changed the phenotype of lymphocytes and increased the percentage of CD4+CD8+ T cells. To explore the effect of immune response of lymphocytes to CDV, the mRNA expression of pro- and anti-inflammatory cytokines was examined. Interleukin (IL-6, IL-12B), and tumor necrosis factor (TNF)-α mRNA expression was significantly increased at 12-48 h after CDV infection. IL-10 mRNA expression was dramatically enhanced at 12-36 h after CDV infection. However, IL-4 and transforming growth factor (TGF-ß) were not response to CDV infection. These results indicated that PBL differentiated intoCD4+CD8+ T cells and improved the inflammatory response to CDV infection. CONCLUSIONS: After CDV infection, PBL differentiated into CD4+CD8+ T cells and initiated inflammatory response.

Oncolytic activity of canine distemper virus in canine mammary tubular adenocarcinoma cells.

Canine distemper virus (CDV), bearing a close resemblance to measles virus, represents a promising candidate for oncolytic therapy; however, its application and underlying oncolytic mechanisms in canine mammary carcinoma cells remain to be explored. Here, we found that an attenuated canine distemper vaccine strain, CDV-L, efficiently infected and inhibited the growth of canine mammary tubular adenocarcinoma CIPp cells but not MDCK cells in vitro. Transcriptomic analysis of CDV-L-infected CIPp cells revealed substantially differentially expressed genes in apoptotic and NF-κB signalling pathways. Subsequent validations confirmed that CDV-L-induced apoptosis of CIPp cells through the caspase-8 and caspase-3 pathway. Identification of phosphorylated-IκBα, phosphorylated-p65 and the nuclear translocation of p65 confirmed the activation of the NF-κB signalling pathway. Inhibition of the NF-κB pathway abrogated CDV-L-induced cleaved-caspase-3 and cleaved-PARP. In a CIPp subcutaneous xenograft mouse model, intratumoural injections of CDV-L significantly restricted tumour growth without apparent pathology, and virus remained localized within the tumour. Taken altogether, these findings indicate that CDV-L exerts an antitumour effect in CIPp cells, and that apoptosis and the NF-κB pathway play essential roles in this process.

Infectious Progression of Canine Distemper Virus from Circulating Cerebrospinal Fluid into the Central Nervous System.

UNLABELLED: In the current study, we generated recombinant chimeric canine distemper viruses (CDVs) by replacing the hemagglutinin (H) and/or phosphoprotein (P) gene in an avirulent strain expressing enhanced green fluorescent protein (EGFP) with those of a mouse-adapted neurovirulent strain. An in vitro experimental infection indicated that the chimeric CDVs possessing the H gene derived from the mouse-adapted CDV acquired infectivity for neural cells. These cells lack the CDV receptors that have been identified to date (SLAM and nectin-4), indicating that the H protein defines infectivity in various cell lines. The recombinant viruses were administered intracerebrally to 1-week-old mice. Fatal neurological signs of disease were observed only with a recombinant CDV that possessed both the H and P genes of the mouse-adapted strain, similar to the parental mouse-adapted strain, suggesting that both genes are important to drive virulence of CDV in mice. Using this recombinant CDV, we traced the intracerebral propagation of CDV by detecting EGFP. Widespread infection was observed in the cerebral hemispheres and brainstems of the infected mice. In addition, EGFP fluorescence in the brain slices demonstrated a sequential infectious progression in the central nervous system: CDV primarily infected the neuroependymal cells lining the ventricular wall and the neurons of the hippocampus and cortex adjacent to the ventricle, and it then progressed to an extensive infection of the brain surface, followed by the parenchyma and cortex. In the hippocampal formation, CDV spread in a unidirectional retrograde pattern along neuronal processes in the hippocampal formation from the CA1 region to the CA3 region and the dentate gyrus. Our mouse model demonstrated that the main target cells of CDV are neurons in the acute phase and that the virus spreads via neuronal transmission pathways in the hippocampal formation. IMPORTANCE: CDV is the etiological agent of distemper in dogs and other carnivores, and in many respects, the pathogenesis of CDV infection in animals resembles that of measles virus infection in humans. We successfully generated a recombinant CDV containing the H and P genes from a mouse-adapted neurovirulent strain and expressing EGFP. The recombinant CDV exhibited severe neurovirulence with high mortality, comparable to the parental mouse-adapted strain. The mouse-infectious model could become a useful tool for analyzing CDV infection of the central nervous system subsequent to passing through the blood-cerebrospinal fluid barrier and infectious progression in the target cells in acute disease.

Controversial results of therapy with mesenchymal stem cells in the acute phase of canine distemper disease.

Distemper disease is an infectious disease reported in several species of domestic and wild carnivores. The high mortality rate of animals infected with canine distemper virus (CDV) treated with currently available therapies has driven the study of new efficacious treatments. Mesenchymal stem cell (MSC)-based therapy is a promising therapeutic option for many degenerative, hereditary, and inflammatory diseases. Therefore, the aim of this study was to characterize stem cells derived from the canine fetal olfactory epithelium and to assess the systemic response of animals infected with CDV to symptomatic therapy and treatment with MSCs. Eight domestic mongrel dogs (N = 8) were divided into two groups: support group (SG) (N = 5) and support group + cell therapy (SGCT) (N = 3), which were monitored over 15 days. Blood samples were collected on days 0, 6, 9, 12, and 15 to assess blood count and serum biochemistry (urea, creatinine, alanine transferase, alkaline phosphatase, gamma-glutamyl transferase, total protein, albumin, and globulin), and urine samples were obtained on days 0 and 15 for urinary evaluation (urine I). The results showed a high mortality rate (SG = 4 and SGCT = 2), providing inadequate data on the clinical course of CDV infection. MSC therapy resulted in no significant improvement when administered during the acute phase of canine distemper disease, and a prevalence of animals with high mortality rate was found in both groups due to the severity of symptoms.

Pathogenesis of canine distemper virus in experimentally infected raccoon dogs, foxes, and minks.

Canine distemper virus (CDV) infects a broad range of carnivores and causes a highly contagious disease with severe immunosuppression. The disease severity markedly varies in different species. To investigate the pathogenesis of CDV in raccoon dog (Nyctereutes procyonoides), fox (Vulpes vulpes) and mink (Neovison vison) species, three groups of CDV sero-negative animals were infected with CDV strain LN(10)1. This CDV strain belongs to the Asia-1 genotype, which is epidemiologically predominant in carnivores in China. CDV infection provoked marked differences in virulence in the three species that were studied. Raccoon dogs developed fever, severe conjunctivitis, and pathological lesions, with 100% (5/5) mortality and with high viral RNA loads in organs within 15 days post infection (dpi). In infected foxes, the onset of the disease was delayed, with 40% (2/5) mortality by 21 dpi. Infected minks developed only mild clinical signs and pathological lesions, and mortality was not observed. Raccoon dogs and foxes showed more severe immune suppression (lymphopenia, decreased lymphocyte proliferation, viremia and low-level virus neutralizing antibodies) than minks. We also observed a distinct pattern of cytokine mRNA transcripts at different times after infection. Decreased IFN-γ and IL-4 mRNA responses were evident in the animals with fatal disease, while up-regulation of these cytokines was observed in the animals surviving the infection. Increased TNF-α response was detected in animals with mild or severe clinical signs. Based on the results, we could distinguish three different patterns of disease after experimental CDV infection, e.g. a mild form in minks, a moderate form in foxes and a severe disease in raccoon dogs. The observed differences in susceptibility to CDV could be related to distinct host cytokine profiles. Comparative evaluation of CDV pathogenesis in various animal species is pivotal to generate models suitable for the evaluation of CDV-host interactions and of vaccine response.

Morbillivirus control of the interferon response: relevance of STAT2 and mda5 but not STAT1 for canine distemper virus virulence in ferrets.

UNLABELLED: The V proteins of paramyxoviruses control the innate immune response. In particular, the V protein of the genus Morbillivirus interferes with the signal transducer and activator of transcription 1 (STAT1), STAT2, and melanoma differentiation-associated protein 5 (mda5) signaling pathways. To characterize the contributions of these pathways to canine distemper virus (CDV) pathogenesis, we took advantage of the knowledge about the mechanisms of interaction between the measles virus V protein with these key regulators of innate immunity. We generated recombinant CDVs with V proteins unable to properly interact with STAT1, STAT2, or mda5. A virus with combined STAT2 and mda5 deficiencies was also generated, and available wild-type and V-protein-knockout viruses were used as controls. Ferrets infected with wild-type and STAT1-blind viruses developed severe leukopenia and loss of lymphocyte proliferation activity and succumbed to the disease within 14 days. In contrast, animals infected with viruses with STAT2 or mda5 defect or both STAT2 and mda5 defects developed a mild self-limiting disease similar to that associated with the V-knockout virus. This study demonstrates the importance of interference with STAT2 and mda5 signaling for CDV immune evasion and provides a starting point for the development of morbillivirus vectors with reduced immunosuppressive properties. IMPORTANCE: The V proteins of paramyxoviruses interfere with the recognition of the virus by the immune system of the host. For morbilliviruses, the V protein is known to interact with the signal transducer and activator of transcription 1 (STAT1) and STAT2 and the melanoma differentiation-associated protein 5 (mda5), which are involved in interferon signaling. Here, we examined the contribution of each of these signaling pathways to the pathogenesis of the carnivore morbillivirus canine distemper virus. Using viruses selectively unable to interfere with the respective signaling pathway to infect ferrets, we found that inhibition of STAT2 and mda5 signaling was critical for lethal disease. Our findings provide new insights in the mechanisms of morbillivirus immune evasion and may lead to the development of new vaccines and oncolytic vectors.

Canine distemper virus associated with a lethal outbreak in monkeys can readily adapt to use human receptors.

A canine distemper virus (CDV) strain, CYN07-dV, associated with a lethal outbreak in monkeys, used human signaling lymphocyte activation molecule as a receptor only poorly but readily adapted to use it following a P541S substitution in the hemagglutinin protein. Since CYN07-dV had an intrinsic ability to use human nectin-4, the adapted virus became able to use both human immune and epithelial cell receptors, as well as monkey and canine ones, suggesting that CDV can potentially infect humans.

Recombinant canine distemper virus serves as bivalent live vaccine against rabies and canine distemper.

Effective, safe, and affordable rabies vaccines are still being sought. Attenuated live vaccine has been widely used to protect carnivores from canine distemper. In this study, we generated a recombinant canine distemper virus (CDV) vaccine strain, rCDV-RVG, expressing the rabies virus glycoprotein (RVG) by using reverse genetics. The recombinant virus rCDV-RVG retained growth properties similar to those of vector CDV in Vero cell culture. Animal studies demonstrated that rCDV-RVG was safe in mice and dogs. Mice inoculated intracerebrally or intramuscularly with rCDV-RVG showed no apparent signs of disease and developed a strong rabies virus (RABV) neutralizing antibody response, which completely protected mice from challenge with a lethal dose of street virus. Canine studies showed that vaccination with rCDV-RVG induced strong and long-lasting virus neutralizing antibody responses to RABV and CDV. This is the first study demonstrating that recombinant CDV has the potential to serve as bivalent live vaccine against rabies and canine distemper in animals.

Detecção molecular e análise filogenética do gene H de amostras do vírus da cinomose canina em circulação no município de Campinas, São Paulo / Molecular detection and phylogenetic analysis of the gene H from canine distemper virus isolates circulating at the municipality of Campinas, São Paulo

O vírus da cinomose canina (CDV), um Morbillivirus da família Paramyxoviridae, é o agente etiológico de doença neurológica e sistêmica em cães. O diagnóstico laboratorial da infecção requer o isolamento viral ou detecção do material genético do vírus em secreções ou tecidos de cães com suspeita clínica da doença. A diversidade genética entre os isolados de CDV pode ser aferida pelo sequenciamento efilogenia molecular do gene que codifica a hemaglutinina viral (gene H), havendo atualmente um especial interesse em comparar as amostras circulantes a campo com o genogrupo América-1, que abrange as cepas presentes nas vacinas disponíveis no mercado. No presente estudo, foi realizada a detecção molecular do gene H de CDV a partir de amostras biológicas colhidas ante- e post- -mortem de 15 cães com sinais clínicos sugestivos de cinomose na região metropolitana de Campinas, São Paulo. Dez dos 15 cães analisados tiveram ao menos um órgão positivo na detecção molecular e os amplicons obtidos foram submetidos ao sequenciamento nucleotídico seguido de análise filogenética molecular. De forma semelhante ao que já foi reportado para estudo analisando a diversidade do gene H em outros países, a reconstrução filogenética obtida para as amostras de casos de cinomose da região de Campinas demonstrou as mesmas foram agrupadas junto a amostras norte-americanas, europeias e japonesas recentes, em um grupo genético distinto do grupo de amostras clássicas de CDV, nomeado America-1, o qual engloba as estirpes vacinais Snyder Hill, Onderstepoort e Lederle.

Canine distemper virus (CDV), a Morbillivirus of the family Paramyxoviridae, is the etiological agent of neurological and systemic disease in dogs. The laboratory diagnosis of infection requires viral isolation or detection of genetic material of the virus in secretions or tissues of dogs with clinical suspicion of the disease. The genetic diversity among isolates of CDV can be assessed by sequencing and phylogenetic analysis of the gene that encodes the viral hemagglutinin (H gene), and there is currently a special interest in comparing the strains currently circulating in the field with the genogroup America-1, which comprises strains present in vaccines available in the market. In this study, the molecular detection of CDV gene H was performed from biological samples harvested ante-and post-mortem from 15 dogs with clinical signs suggestive of canine distemper in the metropolitan region of Campinas, São Paulo. Ten of the 15 dogs examined had at least one positive organ under molecular detection and the obtained amplicons were sequenced and further analyzed by molecular phylogenetic analysis. Similarly to what has already been reported on previous studies regarding the diversity of the gene H in other countries, the phylogenetic reconstruction obtained for the samples of cases of distemper from Campinas region showed they were grouped with the North American, European and Japanese newly described samples, a genetic group distinguished from classical samples of CDV, named America-1, which encompasses the vaccine strains Snyder Hill, Onderstepoort and Lederle.

[Morbilliviruses].

The genus Morbillivirus in the family Paramyxoviridae contains many pathogens, which are important for medicine or veterinary medicine. Because each morbillivirus has restricted host range and serologically monotypic, the virus infection and transmission is effectively controlled by vaccinations and surveillance. Rinderpest virus has been eradicated in 2011, and elimination of measles virus progresses worldwide. Recently, a new cell receptor for measles virus, nectin4 was identified. Both SLAM, a molecule expressing on immune cells, and nectin4, a molecule expressing on epithelial cells, are important to infectivity and pathogenicity of the virus.

Canine distemper virus induces apoptosis in cervical tumor derived cell lines.

Apoptosis can be induced or inhibited by viral proteins, it can form part of the host defense against virus infection, or it can be a mechanism for viral spread to neighboring cells. Canine distemper virus (CDV) induces apoptotic cells in lymphoid tissues and in the cerebellum of dogs naturally infected. CDV also produces a cytopathologic effect, leading to apoptosis in Vero cells in tissue culture. We tested canine distemper virus, a member of the Paramyxoviridae family, for the ability to trigger apoptosis in HeLa cells, derived from cervical cancer cells resistant to apoptosis. To study the effect of CDV infection in HeLa cells, we examined apoptotic markers 24 h post infection (pi), by flow cytometry assay for DNA fragmentation, real-time PCR assay for caspase-3 and caspase-8 mRNA expression, and by caspase-3 and -8 immunocytochemistry. Flow cytometry showed that DNA fragmentation was induced in HeLa cells infected by CDV, and immunocytochemistry revealed a significant increase in the levels of the cleaved active form of caspase-3 protein, but did not show any difference in expression of caspase-8, indicating an intrinsic apoptotic pathway. Confirming this observation, expression of caspase-3 mRNA was higher in CDV infected HeLa cells than control cells; however, there was no statistically significant change in caspase-8 mRNA expression profile. Our data suggest that canine distemper virus induced apoptosis in HeLa cells, triggering apoptosis by the intrinsic pathway, with no participation of the initiator caspase -8 from the extrinsic pathway. In conclusion, the cellular stress caused by CDV infection of HeLa cells, leading to apoptosis, can be used as a tool in future research for cervical cancer treatment and control.

Canine distemper virus matrix protein influences particle infectivity, particle composition, and envelope distribution in polarized epithelial cells and modulates virulence.

In paramyxoviruses, the matrix (M) protein mediates the interaction between the envelope and internal proteins during particle assembly and egress. In measles virus (MeV), M mutations, such as those found in subacute sclerosing panencephalitis (SSPE) strains, and differences in vaccine and wild-type M proteins can affect the strength of interaction with the envelope glycoproteins, assembly efficiency, and spread. However, the contribution of the M protein to the replication and pathogenesis of the closely related canine distemper virus (CDV) has not been characterized. To this end this, we generated a recombinant wild-type CDV carrying a vaccine strain M protein. The recombinant virus retained the parental growth phenotype in VerodogSLAMtag cells, but displayed an increased particle-to-infectivity ratio very similar to that of the vaccine strain, likely due to inefficient H protein incorporation. Even though infectious virus was released only from the apical surface, consistent with the release polarity of the wild-type CDV strain, envelope protein distribution in polarized epithelial cells reproduced the bipolar pattern seen in vaccine strain-infected cells. Most notably, the chimeric virus was completely attenuated in ferrets and caused only a mild and transient leukopenia, indicating that the differences in particle infectivity and envelope protein sorting mediated by the vaccine M protein contribute importantly to vaccine strain attenuation.

Lights and shades on an historical vaccine canine distemper virus, the Rockborn strain.

Both egg- and cell-adapted canine distemper virus (CDV) vaccines are suspected to retain residual virulence, especially if administered to immuno-suppressed animals, very young pups or to highly susceptible animal species. In the early 1980s, post-vaccine encephalitis was reported in dogs from various parts of Britain after administration of a particular batch of combined CDV Rockborn strain/canine adenovirus type-1 vaccine, although incrimination of the Rockborn strain was subsequently retracted. Notwithstanding, this, and other reports, led to the view that the Rockborn strain is less attenuated and less safe than other CDV vaccines, and the Rockborn strain was officially withdrawn from the markets in the mid 1990s. By sequencing the H gene of the strain Rockborn from the 46th laboratory passage, and a commercial vaccine (Candur(®) SH+P, Hoechst Rousell Vet GmbH), the virus was found to differ from the commonly used vaccine strain, Onderstepoort (93.0% nt and 91.7% aa), and to resemble more closely (99.6% nt and 99.3% aa) a CDV strain detected in China from a Lesser Panda (Ailurus fulgens). An additional four CDV strains matching (>99% nt identity) the Rockborn virus were identified in the sequence databases. Also, Rockborn-like strains were identified in two vaccines currently in the market. These findings indicate that Rockborn-like viruses may be recovered from dogs or other carnivores with distemper, suggesting cases of residual virulence of vaccines, or circulation of vaccine-derived Rockborn-like viruses in the field.

Detecção do vírus da cinomose canina em cães assintomáticos e não vacinados / Canine distemper virus detection in asymptomatic and non vaccinated dogs

A quantitative real time polymerase chain reaction (PCR) revealed canine distemper virus presence in peripheral blood samples from asymptomatic and non vaccinated dogs. Samples from eleven domestic dogs with no signs of canine distemper and not vaccinated at the month of collection were used. Canine distemper virus vaccine samples in VERO cells were used as positive controls. RNA was isolated with Trizol®, and treated with a TURBO DNA-free kit. Primers were designed for canine distemper virus nucleocapsid protein coding region fragment amplification (84 bp). Canine b-actin (93 bp) was utilized as the endogenous control for normalization. Quantitative results of real time PCR generated by ABI Prism 7000 SDS Software showed that 54.5 percent of dogs with asymptomatic canine distemper were positive for canine distemper virus. Dissociation curves confirmed the specificity of the real time PCR fragments. This technique could detect even a few copies of viral RNA and identificate subclinically infected dogs providing accurate diagnosis of this disease at an early stage.

A reação em cadeia da polimerase (PCR) em tempo real revelou a presença do vírus da cinomose canina em amostra de sangue de cães assintomáticos e não vacinados. Amostra de onze cães domésticos sem nenhum sinal clínico de cinomose e que não foram vacinados no mês da coleta de sangue foram utilizados para análise. Amostra vacinal do vírus da cinomose canina em células VERO foi utilizada como controle positivo. O RNA total foi isolado utilizando-se Trizol®, e tratadas com o Kit TURBO DNA-free. Os iniciadores foram desenhados para amplificar a região do nucleocapsídeo viral com 319pb e 84pb para a PCR convencional e PCR em tempo real, respectivamente. O fragmento alvo da b-actina canina com 93pb foi utilizado como controle endógeno e normalizador. Resultados quantitativos da PCR em tempo real gerados pelo programa ABI Prism 7000 SDS demonstraram que 54,5 por cento dos cães assintomáticos foram positivos para o vírus da cinomose canina. As curvas de dissociação confirmaram a especificidade dos fragmentos da PCR em tempo real. A detecção precoce do RNA viral é importante para a identificação de cães subclinicamente infectados e limitar a difusão da doença.

Viral obesity: fact or fiction?

The aetiology of obesity is multifactorial. An understanding of the contributions of various causal factors is essential for the proper management of obesity. Although it is primarily thought of as a condition brought on by lifestyle choices, recent evidence shows there is a link between obesity and viral infections. Numerous animal models have documented an increased body weight and a number of physiologic changes, including increased insulin sensitivity, increased glucose uptake and decreased leptin secretion that contribute to an increase in body fat in adenovirus-36 infection. Other viral agents associated with increasing obesity in animals included canine distemper virus, rous-associated virus 7, scrapie, Borna disease virus, SMAM-1 and other adenoviruses. This review attempted to determine if viral infection is a possible cause of obesity. Also, this paper discussed mechanisms by which viruses might produce obesity. Based on the evidence presented in this paper, it can be concluded that a link between obesity and viral infections cannot be ruled out. Further epidemiologic studies are needed to establish a causal link between the two, and determine if these results can be used in future management and prevention of obesity.

Conserved leucine residue in the head region of morbillivirus fusion protein regulates the large conformational change during fusion activity.

Paramyxovirus cell entry is controlled by the concerted action of two viral envelope glycoproteins, the fusion (F) and the receptor-binding (H) proteins, which together with a cell surface receptor mediate plasma membrane fusion activity. The paramyxovirus F protein belongs to class I viral fusion proteins which typically contain two heptad repeat regions (HR). Particular to paramyxovirus F proteins is a long intervening sequence (IS) located between both HR domains. To investigate the role of the IS domain in regulating fusogenicity, we mutated in the canine distemper virus (CDV) F protein IS domain a highly conserved leucine residue (L372) previously reported to cause a hyperfusogenic phenotype. Beside one F mutant, which elicited significant defects in processing, transport competence, and fusogenicity, all remaining mutants were characterized by enhanced fusion activity despite normal or slightly impaired processing and cell surface targeting. Using anti-CDV-F monoclonal antibodies, modified conformational F states were detected in F mutants compared to the parental protein. Despite these structural differences, coimmunoprecipitation assays did not reveal any drastic modulation in F/H avidity of interaction. However, we found that F mutants had significantly enhanced fusogenicity at low temperature only, suggesting that they folded into conformations requiring less energy to activate fusion. Together, these data provide strong biochemical and functional evidence that the conserved leucine 372 at the base of the HRA coiled-coil of F(wt) controls the stabilization of the prefusogenic state, restraining the conformational switch and thereby preventing extensive cell-cell fusion activity.