In-vitro antiviral efficacy of ribavirin and interferon-alpha against canine distemper virus

Canine distemper is a highly contagious disease with high incidence and lethality in the canine population. The objective of this study was to evaluate the efficacy of antiviral action with ribavirin (RBV), interferon-alpha (IFNα), and combinations of RBV and IFNα against canine distemper virus (CDV). Vero cells inoculated with CDV were treated with RBV, IFNα, and combinations of these drugs. The efficacy to inhibit viral replication was evaluated by adding the compounds at different times to determine which step of the viral replicative process was affected. Both drugs were effective against CDV in vitro. The IFNα was the most active compound, with an average IC₅₀ (50% inhibitory concentration) value lower than the IC₅₀ of the RBV. Ribavirin (RBV) was more selective than IFNα, however, and neither drug showed extracellular antiviral activity. The combination of RBV and IFNα exhibited antiviral activity for the intra- and extracellular stages of the replicative cycle of CDV, although the intracellular viral inhibition was higher. Both RBV and IFNα showed high antiviral efficacy against CDV, and furthermore, RBV + IFNα combinations have shown greater interference range in viral infectivity. These compounds could potentially be used to treat clinical disease associated with CDV infection.     

The antiviral activity of six South African plants traditionally used against infections in ethnoveterinary medicine

Viral infections remain a major threat to humans and animals and there is a crucial need for new antiviral agents especially with the development of resistant viruses. The hexane, dichloromethane, acetone and methanol extracts of six plant species selected for their traditional use against infections were tested for in vitro antiviral activity against canine distemper virus (CDV), canine parainfluenza virus-2 (CPIV-2), feline herpesvirus-1 (FHV-1) and lumpy skin disease virus (LSDV). All extracts were tested for their cytotoxicity using a colorimetric tetrazolium-based (MTT) assay and were tested for antiviral efficacy at concentrations below CC(50) values on the various cell types used in this study. The antiviral activity of extracts was tested using virucidal and attachment assays. In the virucidal assay, extracts were incubated with virus prior to infection. The most potent inhibition was observed with the acetone and methanol extracts of Podocarpus henkelii against CDV and LSDV, which inhibited replication of the viruses by >75% at 3μg/ml with selectivity index (SI) values ranging between 12 and 45. Excellent activity was also found with the hexane extracts of Plumbago zeylanica and Carissa edulis against CDV, with the extracts reducing viral-induced CPE by 50% and 75% respectively. The hexane extract of C. edulis had moderate activity against FHV-1 with EC(50)<70μg/ml and SI value <2. Only the acetone extract of P. henkelii moderately inhibited replication of LSD virus in the attachment assay, with low activity in other extracts. Of the four extracts with significant antiviral activity, two were prepared from P. henkelii. Therefore, future work will focus on isolating and characterizing the substance(s) responsible for bioactivity in extracts of this species.     

In vitro assessment of the antiviral potential of trans-cinnamic acid,quercetin and morin against equid herpesvirus 1

The antiviral activity of quercetin, morin and trans-cinnamic acid was evaluated in vitro against equid herpesvirus 1 (EHV-1) by determining the virucidal activity and using the time of addition assay to test inhibition of the viral replication cycle. The cytotoxicity of each substance was assessed using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. Quercetin showed virucidal action and inhibition of the viral replication cycle at 0 and 1 h. Morin showed potential virucidal and viral replication cycle inhibition at 0 h. Trans-cinnamic acid did not show virucidal activity but inhibited the viral replication cycle at −1 and 0 h. This study demonstrates the potential of these compounds as future antiviral candidates in relation to viruses of importance in veterinary medicine.     

Use of hydrophilic extra-viral domain of canine distemper virus H protein for enzyme-linked immunosorbent assay development

Simple methods for measuring the levels of serum antibody against canine distemper virus (CDV) would assist in the effective vaccination of dogs. To develop an enzyme-linked immunosorbent assay (ELISA) specific for CDV, we expressed hydrophilic extra-viral domain (HEVD) protein of the A75/17-CDV H gene in a pET 28a plasmid-based Escherichia (E.) coli vector system. Expression was confirmed by dot and Western blotting. We proposed that detection of E. coli-expressed H protein might be conformation-dependent because intensities of the reactions observed with these two methods varied. The H gene HEVD protein was further purified and used as an antigen for an ELISA. Samples from dogs with undetectable to high anti-CDV antibody titers were analyzed using this HEVD-specific ELISA and a commercial CDV antibody detection kit (ImmunoComb). Levels of HEVD antigenicity measured with the assays and immunochromatography correlated. These data indicated that the HEDV protein may be used as antigen to develop techniques for detecting antibodies against CDV.     

GC/MS analysis of high-performance liquid chromatography fractions from Sophora flavescens and Torilis japonica extracts and their in vitro anti-neosporal effects on Neospora caninum

We analyzed alcoholic extracts of herbs possessing anti-neosporal activity against Neospora (N.) caninum. To identify the chemical components of Sophora (S.) flavescens and Torilis (T.) japonica associated with anti-neosporal activity, specific fractions were isolated by high-performance liquid chromatography (HPLC). In vitro activity of the fractions against N. caninum was then assessed. Gas chromatography/mass spectrometry (GC/MS) was used to identify and quantify specific anti-neosporal molecules in the herbal extracts. Almost all HPLC fractions of S. flavescens and T. japonica had higher levels of anti-neosporal activity compared to the not treated control. Active constituents of the extracts were sophoridane, furosardonin A, and tetraisopropylidene-cyclobutane in S. flavescens; 5,17-β-dihydroxy-de-A-estra-5,7,9,14-tetraene, furanodiene, and 9,12-octadecadienoic acid (Z,Z)-(CAS,1) in T. japonica.     

In vitro effects of Musa x paradisiaca extracts on four developmental stages of Haemonchus contortus

This study was carried out to evaluate the in vitro effect of Musa x paradisiaca stem and leaf against the parasitic nematode of small ruminants Haemonchus contortus. Three extracts (aqueous, methanolic and/or dichloromethane) of Musa x paradisiaca stem and leaf were tested in vitro on four developmental stages of H. contortus using egg hatch assay (EHA), larval development assay (LDA), L3 migration inhibition assay (LMI) and adult worm motility assay (AWM). The highly significant (P < 0.0001) ability to stop larval development (inhibition >67% for each extract) and the negative effect of the dichloromethane extract of leaf on adult worm motility (43% of inhibition of motility after 24 h of incubation) compared to the negative controls, suggest anthelmintic properties of Musa x paradisiaca stem and leaf against H. contortus. The active principles responsible for the activity could be secondary metabolites such as terpenoid and flavonoid compounds present in the leaf and stem of the plant.     

In vitro assessment of the antiviral potential of trans-cinnamic acid, quercetin and morin against equid herpesvirus 1

The antiviral activity of quercetin, morin and trans-cinnamic acid was evaluated in vitro against equid herpesvirus 1 (EHV-1) by determining the virucidal activity and using the time of addition assay to test inhibition of the viral replication cycle. The cytotoxicity of each substance was assessed using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. Quercetin showed virucidal action and inhibition of the viral replication cycle at 0 and 1 h. Morin showed potential virucidal and viral replication cycle inhibition at 0 h. Trans-cinnamic acid did not show virucidal activity but inhibited the viral replication cycle at −1 and 0 h. This study demonstrates the potential of these compounds as future antiviral candidates in relation to viruses of importance in veterinary medicine.     

Inhibitory effects of ribavirin alone or combined with human alpha interferon on feline infectious peritonitis virus replication in vitro

The antiviral activities of ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide; virazole), either alone or in combination with recombinant human leukocyte (alpha) interferon (rHuIFN-alpha), were evaluated against feline infectious peritonitis virus (FIPV) in feline kidney-cell cultures. The 50% inhibitory dose (ID50) of ribavirin for uninfected, rapidly dividing cells was approximately 17 micrograms ml-1 whereas the ID50 for FIPV was 2.5 micrograms ml-1. The therapeutic index (TI) of ribavirin (i.e. the ratio of the minimum cell-toxic dose to minimum virus-inhibitory dose) was 6.8. Although a dose-dependent inhibition of viral infectivity occurred at non-toxic doses, maximum antiviral effects (greater than or equal to 4 log10 reduction in FIPV) occurred at cytotoxic doses. When low or moderate doses of ribavirin were combined with either 10 or 100 U of rHuIFN-alpha ml-1, the resulting antiviral effects were significantly greater than the sum of the observed effects from either ribavirin or rHuIFN-alpha alone. Significant synergistic interactions with rHuIFN-alpha occurred at ribavirin doses of 1, 5, 12.5 and 25 micrograms ml-1. Synergistic combinations of rHuIFN-alpha and ribavirin produced up to an 80-fold or a 200-fold relative increase in FIPV antiviral activities compared with that produced by equivalent doses, respectively, of ribavirin or rHuIFN-alpha alone. In cell growth studies, the addition of either 10 or 100 U of rHuIFN-alpha ml-1 to test doses of ribavirin did not increase the anticellular effect observed with ribavirin alone; seemingly, the potentiation of ribavirin antiviral activity by rHuIFN-alpha was independent of any additive cytotoxic effects. Potentially, synergistic combinations of the two antiviral agents in vivo may decrease the therapeutic dose of ribavirin required for inhibition of FIPV and thus reduce drug toxicity.     

Prevalence of antibodies against canine distemper virus and canine parvovirus among foxes and wolves from Spain

Viral diseases can influence the population dynamics of wild carnivores and can have effects on carnivore conservation. Hence, a serologic survey was conducted in an opportunistic sample of 137 foxes (Vulpes vulpes) and 37 wolves (Canis lupus) in Spain for 1997-2007 to detect antibodies against canine distemper virus (CDV) and against canine parvovirus (CPV) by indirect ELISA. Antibodies against CDV were detected in 18.7% of the analyzed animals and antibodies against CPV in 17.2%. There was no difference in antibody prevalence to CDV between both species, even in the same region (P>0.05), but there was a significant difference in antibody prevalence to CPV between foxes (5.1%) and wolves (62.2%) (P<0.05). In fox populations there was a significant difference in antibody prevalence to CDV between geographic areas (Aragón 26.4%, La Mancha 7.8%, P<0.05). In wolf populations there was significantly higher antibody prevalence against CPV (P<0.05) in Castilla y León (100%) than in the Cantabric region (53.3%). There was no significant sex or age-related difference in the antibody prevalence against CDV or CPV in foxes. These results indicate that contact with CDV is widespread among wild canid populations in Spain and that CPV is endemic in the Iberian wolf population. The implications of these results are briefly discussed.     

Immunophenotypical characterization of monocytes in canine distemper virus infection

Canine distemper virus (CDV) infection induces multifocal demyelination in the central nervous system (CNS). It is thought that the resident macrophages of the CNS, the microglia, as well as invading monocytes associated with the inflammatory reaction may play a central role in the demyelinating process. To evaluate changes in peripheral monocytes in CDV infection their immunophenotype was characterized by flow cytometry during the course of an experimental CDV infection in dogs. The highest number of CDV-infected monocytes was found in dogs developing demyelinating lesions. In CD18, CD45, CD44, and CD14 neither up- nor down-regulation was observed. Marked up-regulation occurred in a number of surface molecules including CD1c, B7-1 and B7-2, MHC I, and CD11b. Peak expression was found at 4-5 weeks post-infection (PI), regardless of clinical outcome. All these molecules play an important role in the host's immune response, notably antigen presentation and cell adhesion. These results demonstrate that CDV infection in vivo may enhance several macrophage functions. This could lead to more effective clearance of the virus but may also increase demyelination through a bystander effect in animals that accumulated significant amounts of CDV in the CNS.     

Hematological adverse effects and pharmacokinetics of ribavirin in pigs following intramuscular administration

Ribavirin (RBV) is a synthetic guanosine analog that is used as a drug against various viral diseases in humans. The in vitro antiviral effects of ribavirin against porcine viruses were demonstrated in several studies. The purposes of this study were to evaluate the adverse effects and pharmacokinetics of ribavirin following its intramuscular (IM) injection in pigs. Ribavirin was formulated as a double‐oil emulsion (RBV‐DOE) and gel (RBV‐Gel), which were injected into the pigs as single‐dose IM injections. After injection of RBV, all of the pigs were monitored. The collected serum and whole blood samples were analyzed by liquid chromatography–tandem mass spectrometry and complete blood count analysis, respectively. All of the ribavirin‐treated pigs showed significant decreases in body weight compared to the control groups. Severe clinical signs including dyspnea, anorexia, weakness, and depression were present in ribavirin‐treated pigs until 5 days postinjection (dpi). The ribavirin‐treated groups showed significant decrease in the number of red blood cells and hemoglobin concentration until 8 dpi. The mean half‐life of the RBV‐DOE and RBV‐Gel was 27.949 ± 2.783 h and 37.374 ± 3.502 h, respectively. The mean peak serum concentration (C_max) and area under the serum concentration–time curve from time zero to infinity (AUC_inf) of RBV‐DOE were 8340.000 ± 2562.577 ng/mL and 16 0095.430 ± 61 253.400 h·ng/mL, respectively. The C_max and AUC_inf of RBV‐Gel were 15 300.000 ± 3764.306 ng/mL and 207526.260 ± 63656.390 h·ng/mL, respectively. The results of this study provided the index of side effect and pharmacokinetics of ribavirin in pigs, which should be considered before clinical application.     

犬瘟热的诊断及其预防免疫的研究进展

本文对犬瘟热（CD）的诊断、预防免疫和免疫失败的影响因素及犬瘟热病毒（CDV）的宿主范围进行了综述。CDV不仅感染陆生食肉动物，而且也感染水生食肉动物，并且其宿主范围还在不断扩大。CDV感染主要采用病毒分离、特异性病毒抗原或特异性核酸检测等方法确诊。疫苗包括灭活的CDV疫苗、麻疹病毒（MV）异源苗及CDV弱毒活苗。疫苗接种犬的免疫反应主要取决于毒株特性及犬的应答能力，只有弱毒活苗能诱导产生持久而坚强的保护力。尽管多年来CDV弱毒活苗的使用控制了CD的发生，但最近免疫过的犬发生CD的病例并不少见。分析免疫失败的原因，主要是母源抗体干扰、疫苗质量差、其它病毒的免疫抑制以及CDV流行株可能发生了变异等因素的影响。     

犬IFN-α优化基因的真核表达及抗病毒活性的研究

本研究根据酵母系统密码子的偏嗜性,对成熟蛋白基因核苷酸序列进行密码子偏嗜性改造,利用毕赤酵母分泌型表达载体pPICZαC来表达犬IFN-α成熟蛋白基因。并采用非洲绿猴肾细胞(Vero) -水疱性口炎病毒(VSV)系统以细胞病变抑制法为基础来测定重组犬α干扰素的效价,同样方法测定了表达产物对PRV和CDV的抑制作用。本试验实现了犬α干扰素在酵母中的高表达,表达量高达58 mg/L,SDS-PAGE检测大小约为24 ku,比预测分子质量大,推测发生了糖基化。表达产物具有较强的种属特异性,在Vero细胞上对PRV具有较高的抗病毒活性,达到了3.6×10⁷ U/L,对CDV也有很好的抑制作用。本研究为犬IFN-α以后的临床应用研究奠定基础。     

抗CDV、CAV卵黄抗体的制备

本研究用CDV、CAV分别免疫产蛋鸡，收集鸡蛋，用海藻酸钠—硫酸铵法提取卵黄抗体(IgY)，并对提取的IgY采用了SDS-PAGE和低压层析检测纯度，结果表明用该法提取的IgY纯度较高。用间接BUSA测定提取的IgY活性，结果证明提取的抗CAV IgY的活性较高，而抗CDV IgY的活性损失较大。     

In vitro antiviral activity of chestnut and quebracho woods extracts against avian reovirus and metapneumovirus

Field evidences have suggested that a natural extract, containing tannins, could be effective against poultry enteric viral infections. Moreover previous studies have shown that vegetable tannins can have antiviral activity against human viruses. Based on this knowledge three different Chestnut (Castanea spp.) wood extracts and one Quebracho (Schinopsis spp.) wood extract, all containing tannins and currently used in the animal feed industry, were tested for in vitro antiviral activity against avian reovirus (ARV) and avian metapneumovirus (AMPV). The MTT assay was used to evaluate the 50% cytotoxic compounds concentration (CC₅₀) on Vero cells. The antiviral properties were tested before and after the adsorption of the viruses to Vero cells. Antiviral activities were expressed as IC₅₀ (concentration required to inhibit 50% of viral cytopathic effect). CC₅₀s of tested compounds were >200 μg/ml. All compounds had an extracellular antiviral effect against both ARV and AMPV with IC₅₀ values ranging from 25 to 66 μg/ml. Quebracho extract had also evident intracellular anti-ARV activity (IC₅₀ 24 μg/ml). These preliminary results suggest that the examined vegetable extracts might be good candidates in the control of some avian virus infections. Nevertheless further in vivo experiments are required to confirm these findings.     

Prevalence of positive antibody test results for canine parvovirus (CPV) and canine distemper virus (CDV) and response to modified live vaccination against CPV and CDV in dogs entering animal shelters

Canine parvovirus (CPV) and canine distemper virus (CDV) infections are relatively common in animal shelters and are important population management issues since the immune status of incoming dogs is usually unknown. This study aimed to determine the prevalence of positive antibody test results for CPV and CDV in incoming dogs aged ≥ 4 months and to measure antibody response over 2 weeks following vaccination with a modified live vaccine (MLV). Dogs aged 4-24 months entering an adoption-guarantee shelter (Shelter 1, n=51) and aged ≥ 4 months entering a limited admission shelter (Shelter 2; n=51) were enrolled. Dogs from Shelter 1 had been vaccinated with MLV at a municipal shelter 5 days before enrollment, whereas dogs from Shelter 2 had no known history of vaccination at enrollment. Sera were obtained on day 1, immediately prior to CPV/CDV MLV, and tested using an in-clinic ELISA kit to detect CPV/CDV antibodies. Dogs negative for CPV and/or CDV were retested at day 6-8 and those dogs still negative at day 6-8 were retested at day 13-15. Prior to CPV/CDV MLV on day 1, more dogs tested positive for CPV (Shelter 1 - 68.6%; Shelter 2 - 84.3%) than for CDV (Shelter 1 - 37.3%; Shelter 2 - 41.2%). On day 1, prior to MLV, all spayed/neutered animals tested CPV antibody-positive (n=17/102) and CPV antibody-positive dogs were older than serologically negative dogs (Shelter 1, P=0.0029; Shelter 2, P=0.0042). By day 13-15, almost all dogs were CPV antibody-positive (Shelter 1 - 97.9%; Shelter 2 - 100.0%) and CDV antibody-positive (Shelter 1 - 93.8%; Shelter 2 - 97.8%). MLV induces protective antibody titers against CPV/CDV in almost all dogs after 13-15 days.     

三种药物对PRRSV的体外抑制试验

选取干扰素、黄芪多糖、利巴韦林作为抗病毒药物,利用MTT法与细胞病变(CPE)抑制试验,通过PRRSV感染Marc-145细胞、病毒在细胞中复制、药物直接和病毒作用三个环节,对药物的作用效果进行评价。结果显示,3种药物体外对PRRSV的都具有抑制作用,干扰素、黄芪多糖体外对PRRSV具有明显的阻断作用,利巴韦林体外对PRRSV具有杀灭作用。因此,这3种药物在细胞水平上均有抑制病毒的作用,为防控PRRS提供了理论依据。     

Canine distemper virus in coyotes: a serologic survey

Serum samples from 228 coyotes were selected randomly from a serum bank assembled from Texas from 1975 to 1984 and were evaluated serologically for neutralizing antibodies against canine distemper virus (CDV). One hundred and twenty-eight (56%) of the 228 coyotes had antibody titers of greater than or equal to 1:5 against CDV (seropositive). The serologic prevalence (seroprevalence) of antibodies against CDV infection was higher in the spring (62%) than in the fall (40%). The seroprevalence of CDV in various age groups was different; 25 of 101 coyotes (25%) were seropositive at less than 1 year of age, 35 of 52 (67%) were positive between 1 and 2 years of age, and 68 of 75 (91%) were positive at greater than or equal to 2 years of age. The results indicated that CDV was enzootic in coyote populations of southern Texas, with an increasing number of seropositive coyotes noted annually. The sex of the coyote did not appear to be related to the seroprevalence against CDV.     

Clinical use of serum parvovirus and distemper virus antibody titers for determining revaccination strategies in healthy dogs

OBJECTIVE: To assess whether serum canine parvovirus (CPV) and canine distemper virus (CDV) antibody titers can be used to determine revaccination protocols in healthy dogs. DESIGN: Case series. ANIMALS: 1,441 dogs between 6 weeks and 17 years old. PROCEDURE: CPV and CDV antibody titers in serum samples submitted to a commercial diagnostic laboratory were measured by use of indirect fluorescent antibody (IFA) tests. On the basis of parallel measurements of CPV and CDV serum antibody titers in 61 paired serum samples determined by use of hemagglutination inhibition and serum neutralization methods, respectively, we considered titers > or = 1:5 (IFA test) indicative of an adequate antibody response. RESULTS: Age, breed, and sex were not significantly associated with adequate CPV- or CDV-specific antibody responses. Of 1,441 dogs, 1,370 (95.1%) had adequate and 71 (4.9%) had inadequate antibody responses to CPV, whereas 1,346 of 1,379 (97.6%) dogs had adequate and 33 (2.4%) had inadequate responses to CDV. Vaccination histories were available for 468 dogs (468 for CPV, 457 for CDV). Interval between last vaccination and antibody measurement was 1 to 2 years for the majority (281/468; 60.0%) of dogs and 2 to 7 years for 142 of 468 (30.3%) dogs. Interval was < 1 year in only 45 of 468 (9.6%) dogs. CONCLUSIONS AND CLINICAL RELEVANCE: The high prevalence of adequate antibody responses (CPV, 95.1%; CDV, 97.6%) in this large population of dogs suggests that annual revaccination against CPV and CDV may not be necessary.     

Staphylococcus aureus chronic intramammary infection modifies protein expression of transforming growth factor beta (TGF-β) subfamily components during active involution

The objectives of this study were to determine whether Staphylococcus aureus chronic intramammary infection (IMI) influences protein expression of TGF-β subfamily components and collagen I and to examine the histomorphometric changes that occur in mammary stroma and parenchyma during active mammary gland involution. Twenty-one Holstein non-pregnant cows in late lactation either uninfected or with chronic natural S. aureus IMI were included in this study. Cows were slaughtered at 7, 14 and 21 d after cessation of milking and samples for immunohistochemical and morphometric analysis were taken. Protein expression of TGF-β1, TGF-β2 and TGF-β3 was significantly higher in chronically infected quarters than in uninfected controls at the three involution stages studied. Immunostaining of TGF-βR1 and TGF-βR3 and collagen I was significantly higher in S. aureus-infected quarters than in uninfected controls at every involution time evaluated. The percentages of tissue area composed of parenchyma and intralobular stroma were significantly higher in S. aureus-infected than in uninfected quarters. Chronic S. aureus mastitis modifies protein expression of the three TGF-β isoforms and type 1 and 3 receptors, which was associated with changes directed to limit the scope of inflammation and injury to the host.