Effect of KLP-602 on virus replication in cell cultures

The effect of KLP-602 (active substance: lysozyme dimer) on the replication of two animal viruses: the TK900 strain of Aujeszky's disease virus and the Roakin strain of the Newcastle disease virus were investigated. The maximal tolerable dose of the drug was determined for two cell cultures (CECC and GMK) and the effect of the medicine on the titre range of infectious viruses and their adsorption was assayed. The direct impact of KLP-602 on the viral strains used was also determined. And finally the replication dynamics of viruses in the presence of KLP-602 preparation was estimated. KLP-602 showed no direct effect on either the viruses applied in the study or their adsorption. The drug, introduced into the culture 24 hours before its infection, did not affect the replication of the pseudorabies virus, but decreased the titre of the Newcastle disease virus. KLP-602 introduced simultaneously with the infection considerably lowered the final titres of both viruses. The medicine had the greatest inhibitory effect on the replication dynamics of both types of viruses in the CECC and of the pseudorabies virus in the GMK culture upon the maximal tolerable concentrations of drug and low infectious doses of viruses applied.     

Biological properties of Roakin strain of NDV and TK900 strain of ADV after serial passages in CECC in the presence of methisoprinol and KLP-602

Twenty serial passages of the TK900 strain of Aujeszky's disease virus (ADV) and the Roakin strain of Newcastle disease virus (NDV) were made in a chicken embryo cell culture (CECC), in the presence of two antiviral agents: Methisoprinol and KLP-602. The physicochemical properties of passaged viruses were determined. The results obtained suggest that Methisoprinol causes changes in the structure of viral proteins, whereas KLP-602 affects the envelope-dependent properties of the virus. It was also found that the alternations observed in passaged viruses were temporary phenotypic changes only, and not a consequence of permanent transformations of their genotypes.     

Effect of methisoprinol and KLP-602 on virus replication in chicken embryos

The aim of the present study was to examine the effects of two immunomodulators (KLP-602 and Methisoprinol) on the proliferation of two strains of Newcastle disease virus in chick embryos. The effect of the maximum tolerable doses of both drugs (Methisoprinol--6 mg/embryo, KLP-602--5 mg/embryo) on lymphocyte reactivity were determined prior to the experiment. Both drugs inhibited the replication of the Roakin strain of NDV in various experimental designs, but neither of them affected the proliferation of the LaSota strain of NDV.     

Influence of methisoprinol on the replication of rhabdoviruses isolated from carp (Cyprinus carpio) and catfish (Ictalurus melas): in vitro study

Rhabdoviruses constitute one of the most pathogenic viruses isolated from rainbow trout and carp culture. Several viruses were also isolated from other species of fish. These viruses are mostly associated with epizootics and heavy losses. Spring viraemia of carp virus (SVCV) and pike fry rhabdovirus (PFRV) have been the most extensively studied, due to their significant economic impact. Significant progress has been made towards controlling the major bacterial fish diseases using vaccines, but this approach has not yet been successful in preventing viral diseases in fish culture. However, for an effective therapeutic approach, specific drugs should be developed to selectively inhibit virus replication and/or stimulate antiviral protection. In this investigation we examined the in vitro influence of methisoprinol on the SVCV and virus isolated from catfish (Ictalurus melas) replication by measuring their RNA synthesis. The viruses were propagated in EPC cells and cell cultures containing methisoprinol were followed by infection with SVCV or catfish rhabdovirus suspension containing 10(7) TCID50/ml. Methisoprinol (Polfa, Poland) at concentrations of 0, 100, 200, 300, 400 and 500 microg/ml of medium (Glasgow MEM) was used in this study. The results of this study show the strong inhibition of incorporation (cpm) of [3H]-uridine into SVCV and catfish rhabdovirus RNA in cell culture exposed to methisoprinol at various concentrations. The highest percent of inhibition of viral RNA at 72 h after infection with two rhabdoviruses were observed in doses of 400 and 500 microg/ml of methisoprinol in medium. The results of this in vitro study showed that methisoprinol inhibits the rhabdoviruses isolated from carp and catfish.     

马传染性贫血病毒弱毒株LTR点突变型嵌合感染性克隆的构建

马传染性贫血病毒（Equine infectious anemia virus,EIAV）长末端重复序列（Long terminal repeat,LTR）是基因组中高度变异区之一,EIAV LTR的变异对于指导病毒的复制和病毒致病性具有重要的生物学意义。为了阐明我国马传染性贫血病毒弱毒疫苗毒力致弱的分子机制,由马传贫强毒至弱毒致弱过程中不同代次毒株LTR序列的分析,以驴胎皮肤弱毒株疫苗全长感染性克隆pLGFD3-8为父本,选取LTR R区的TAR起始碱基,poly（A）附加位点,采用反向遗传操作对其位点进行PCR体外定点突变,将弱毒序列构建的逆向点突变型全基因克隆转染到驴胎皮肤细胞（FDD）并在FDD上传代,通过逆转录酶活性（RT）检测、RT-PCR方法及real-time RT PCR检测并验证其感染性。检测结果为构建的逆向点突变型感染性克隆在FDD上被拯救,其衍生病毒感染的FDD上出现明显的细胞病变;细胞培养上清可检测到RT酶活性和RT-PCR阳性;电镜下可见大量典型的EIAV颗粒pLGFD-M点突变型嵌合克隆衍生病毒与其父本克隆衍生病毒pLGFD3-8复制水平相似。此结果为进一步深入研究LTR对马传染性贫血病毒复制水平和毒力的影响奠定了基础。     

Immunogenicity of infectious bursal disease viruses in chickens.

Cross-protective properties of infectious bursal disease viruses (IBDVs) were studied. Viruses represented different subtypes of serotype 1, including recently isolated viruses (variants), and a serotype 2 virus. Chickens were vaccinated at 3 weeks of age with inactivated vaccines containing 10(5), 10(6), 10(7), or 10(8) mean tissue-culture infectious dose of a given virus and challenged 2 weeks later using either 10(2) or 10(3.5) mean embryo infectious dose (EID50) of either a standard virus or a variant serotype 1 virus. Protection was evaluated at 5 and 10 days post-challenge, based on gross and microscopic lesions, body weight, and bursa/body-weight ratios. The serotype 2 virus did not confer protection on birds challenged with the serotype 1 viruses. Vaccines made of variant viruses at the low doses protected chickens challenged with the high or low doses of either the standard or the variant viruses. Vaccines made of the standard or variant strains at low doses protected against high or low challenge doses of the standard strain. Vaccines made of the high dose of any of the standard strains protected chickens against the variant virus when the low challenge dose (10(2) EID50) was used, but not when the high challenge dose (10(3.5) EID50) was used. The lowest dose of the standard viruses vaccines required to confer protection against the variant virus varied depending on the strain. Results indicated that protection depended on the strain and dose of both the vaccine and challenge viruses and that the variant strains and standard strains share a common protective antigen(s).     

Characterization of an infectious pancreatic necrosis (IPN) virus carrier cell culture with resistance to superinfection with heterologous viruses

A state of persistence of a non susceptible fish cell line with infectious pancreatic necrosis virus (IPNV) was established in vitro by experimental infection. The persistently infected culture showed sustained production of infectious virus and could be continuously passaged for months. A distinct feature of this culture is that only a very small fraction of the cells harbours virus replication, in contrast to other reported IPNV-persistently infected cells from salmonid fish, where nearly all the cells express viral antigens. In spite of the small number of detectable IPNV-infected cells, the carrier culture shows resistance to superinfection with homologous as well as heterologous viruses. Temperature shift-up experiments indicate that viral interference is due to continuous replication of IPNV in the culture. Quantitation of Mx gene expression suggested that the interference phenomenon could be mediated by the activation of the interferon (IFN) system. However, conditioned medium from the IPNV-infected cell cultures only marginally protected other cells against VHSV infection, indicating that other type I IFN-independent mechanism may be underlying the resistance of the persistently infected culture to infection with heterologous viruses. Our study defines a novel in vitro model of IPNV persistence and contributes to the understanding of the widespread distribution of aquabirnaviruses in marine and fresh water environments by establishing a carrier state in non susceptible fish species.     

Characterization of infectious laryngotracheitis viruses, antigenic comparison by kinetics of neutralization and immunization studies.

Five isolates of infectious laryngotracheitis virus were compared by pock formation on the chorioallantoic membrane of embryonated eggs, plaque size in chicken embryo kidney tissue culture, and antigenic relationship using reciprocal kinetics of neutralization. The A4557-5 strain of infectious laryngotracheitis virus, which causes mild respiratory disease, produced pocks with a zone of edema on the chorioallantoic membrane. A virulent virus (Virus 1), isolated from an outbreak of severe disease characterized by a diphtheritic laryngotracheitis, produced the largest plaques in chicken embryo kidney cell culture. Other virulent viruses (Viruses 2, 3 and V154) did not have unique growth characteristics when grown on the chorioallantoic membrane or in chicken embryo kidney cell culture. All viruses were closely related antigenically as shown by kinetics of neutralization but viruses 2 and 3 were not homogeneous with the other three viruses when neutralized by anti-V154 chicken serum. Following aerosol infection, chickens infected with the A4557-5 virus were immune to challenge with virulent V154 virus. However, in comparison to SA-2 virus, this virus was a less effective immunizing agent when administered by the vent or drinking water methods.     

Persistent infections of a field strain of rabies virus in murine neuroblastoma (NA-C1300) cell cultures.

Rabies virus from the brain of a striped skunk (Mephitis mephitis) from Ontario was inoculated into murine neuroblastoma (NA-C1300) cell cultures. These cultures were incubated and the cells were subcultured every three to four days. The presence of viral antigen in the cell cultures was monitored by direct immunofluorescent staining and in the culture fluids by titration in either baby hamster kidney (BHK/C13) or NA cells or in experimental mice. The virus-infected NA cultures evolved from an initial high viral concentration in supernatant fluid through a period of decreasing titers of infectious virus in the supernatant fluids to a final phase where no infectious virus has been found following cell culture and animal inoculation methods attempted although the persistently infected cells remained 95-100% viral nucleocapsid antigen-positive. Possible mechanisms involved in the perpetuation of this infection are discussed. This is the first report of a persistent infection of cell cultures by a field strain of rabies virus.     

Evaluation of 9-(2-phosphonylmethoxyethyl) adenine therapy for feline immunodeficiency virus using a quantitative polymerase chain reaction.

To determine the efficacy of 9-(2-phosphonylmethoxyethyl)adenine (PMEA) as a prophylactic chemotherapeutic agent for the treatment of lentivirus infections, three groups of specific pathogen free cats were treated with 0, 3, or 6 mg kg-1 twice daily doses of PMEA beginning 24 h prior to virus challenge with feline immunodeficiency virus Petaluma strain. Treatment was continued for 7 weeks post challenge. During this time cats were monitored for drug toxicity, virus specific antibody response, circulating viral antigen and infectious recoverable virus. To determine the long-term influence of PMEA therapy the cats were monitored for 1 year following the cessation of treatment. The low levels of infectious virus present in blood prompted the development of quantitative polymerase chain reaction assay to enumerate viral DNA burdens in the peripheral blood mononuclear cells of the infected cats and thereby assess drug efficacy. The results indicate that, although prophylactic PMEA did not prevent infection, it did substantially limit feline immunodeficiency virus replication. Furthermore, viral DNA levels remained low in the cats receiving drug a full year (the duration of the study) after cessation of treatment.     

Comparison of serological tests for the measurement of the primary immune response to avian infectious bronchitis virus vaccines

The immune response of individual chickens exposed in an aerosol apparatus to live commercial avian infectious bronchitis virus (IBV) vaccines was measured by serum neutralization (SN), haemagglutination inhibition (HI), complement fixation (CF) and agar gel precipitin (AGP) tests over a period of 14 weeks.The SN titres showed considerable variation for individual chickens and in a large number of birds were negative until 14 weeks after infection.Positive HI titres were recorded for most birds at one week after infection and persisted throughout the observation period. Some relationship was seen between HI and SN titres particularly in birds showing a high immune response.The results obtained with the AGP were transient, variable and did not compare well with results obtained by the other tests. The highest number of positive AGP reactors were seen two to three weeks after infection.Most birds showed positive titres by the CF test at some time after infection but titres were always low and did not correlate with results obtained by the other tests.Twenty-two weeks after vaccination 23 chickens were challenged by aerosol exposure to the Massachusetts 41 strain of IBV and four days later the trachea, kidneys and oviducts were removed from each bird for attempted virus isolation. Virus was recovered from only one kidney and 11 trachea samples. The mean pre-challenge HI and SN titres of birds from which no virus was recovered were significantly higher than the mash titres of vaccinated birds from which virus was isolated after challenge.     

鸡传染性法氏囊病超强毒Gx与疫苗毒Gt在鸡体内的复制研究

本试验利用鸡传染性法氏囊病超强毒Gx及其致弱疫苗毒Gt为对象,研究超强毒与弱毒株在鸡体主要免疫器官内的复制情况,以探讨两类毒株表现不同生物特性的原因。分别利用鸡胚半数致死量和鸡胚成纤维细胞半数感染量对超强毒Gx和疫苗株Gt进行病毒滴度的测定;再利用荧光定量RT-PCR对两类毒株进行病毒量的校准。以相同量的病毒对2周龄SPF鸡进行攻毒。攻毒试验表明超强毒Gx能造成47.5%的死亡,法氏囊、脾脏、胸腺等免疫器官均严重损伤;而疫苗毒Gt无致死,且未能造成任何病理可见的损伤。病毒的体内复制情况表明超强毒相对于疫苗毒复制更为迅速,病毒载量更高。     

Effect of acyclovir on the replication of turkey herpesvirus and Marek's disease virus

The toxicity of acyclovir for chick embryo fibroblasts and its effect on the replication of turkey herpesvirus (strain FC 126) and Marek's disease virus (strain HPRS 16) multiplied on fibroblast culture was studied. The influence of using acyclovir on the development of the tumour process in birds infected with a virulent Marek's disease virus was also determined. Acyclovir used in doses below 12.5 micrograms ml-1 proved to be nontoxic for chick embryo fibroblast culture. It inhibited in vitro replication of turkey herpesvirus and Marek's disease virus. It was also shown to diminish the development of tumours in birds infected with Marek's disease virus.     

鸡传染性支气管炎H120细胞活疫苗规模化生产的研究

通过近两年时间的试验,摸索出并掌握了利用转瓶培养鸡传支H120细胞弱毒的关键技术,利用转瓶机成功生产出鸡传支H120细胞弱毒活疫苗三批,依照<中华人民共和国兽用生物制品质量标准>,经物理性状、无菌、支原体、特异性、外源病毒及安全检验均合格,EID50平均值分别为6.93、6.71和6.82,免疫保护率均达87%以上.     

THE SEROLOGICAL RESPONSE OF CHICKENS TO AUSTRALIAN LENTOGENIC STRAINS OF NEWCASTLE DISEASE VIRUS

SUMMARY: Australian lentogenic Newcastle disease viruses were evaluated as uninactivated vaccines in Australian chickens, the response being evaluated by the production of haemagglutination-inhibition (HI) antibodies. Two viruses, V4 and PM9, induced high levels of antibody and were readily transmissible between chickens by contact exposure. Three other viruses were poorly immunogenic and poorly transmissible. Chickens vaccinated intramuscularly with the V4 strain produced higher HI antibody titres than chickens vaccinated by the orotracheal, intranasal and intraocular routes. HI antibody titres in chickens vaccinated with the V4 strain reached peak levels 3 to 5 weeks after vaccination and waned considerably during the next 2 to 4 weeks. However, low levels of HI antibody persisted for at least 36 weeks after vaccination. Intramuscular vaccination with the V4 strain of one-day-old chicks lacking maternal antibody to Newcastle disease virus resulted in 42–70% mortality and the survivors developed very high titres of HI antibody. Similar chickens inoculated orotracheally showed signs of depression and developed high titres of HI antibody, but there were no mortalities. Chickens 1-, 2-, 3- and 4-weeks-old and lacking maternally derived HI antibody to Newcastle disease virus suffered no adverse reaction to intramuscular or orotracheal vaccination. The antibody response of the 1-week-old chickens was considerably poorer than that of the older chickens. Following orotracheal vaccination with the V4 strain, chickens with low levels of maternally derived antibody responded with low levels of HI antibody. On the other hand, in the progeny of hens hyperimmunised with the V4 strain the production of active antibody following orotracheal vaccination was delayed until the level of passive antibody had declined considerably. There was no response to intramuscular vaccination in congenitally hyperimmune chickens. The minimum HI antibody inducing dose of V4 vaccine, when measured 3 weeks after vaccination of 6-weeks-old chickens, was 10^5.6 50% egg infectious doses.     

马传染性贫血病毒疫苗株S2基因不同变异位点逆向突变感染性克隆的体外复制特性分析

为研究EIAV弱毒疫苗株(EIAV_(FCCV15))S2基因发生的稳定性突变对疫苗株特性的作用以及S2基因的功能,以EIAVFDDVl5感染性克隆质粒pFDDV3-8为模板,根据疫苗研制过程中S2基因发生的4个主要稳定性变异位点,构建及拯救出S2基因不同差异位点逆向突变为强毒株相应氨基酸的6株感染性克隆衍生毒株.经实时定量PCR、逆转录酶活性和western blot等检测表明,疫苗株S2基因4个稳定性变异位点全部逆向突变的感染性克隆衍生毒、,pFDDVS2rl-3-4-5在体外培养靶细胞中的复制水平低于亲本疫苗株感染性克隆衍生病毒和其他组合的逆向突变的感染性克隆衍生病毒,提示ELAV疫苗株S2蛋白4个稳定突变位点的综合作用可能是决定疫苗株和强毒株特性差异的因素之一.以上结果为体内感染S2基因逆向突变感染性克隆衍生病毒,进一步揭示S2基因在ELAV弱毒疫苗致弱过程中的作用提供了依据.     

Sensitivity and specificity of the fluorescent antibody technique for detection of infectious laryngotracheitis virus.

The specificity of a fluorescent conjugate to infectious laryngotracheitis virus was examined using chick trachea organ culture or tissue sections infected with other avian viruses (adenovirus, infectious bronchitis, poxvirus, reovirus, Newcastle disease virus, Marek's disease virus, avian encephalomyelitis and infectious bursal agent) or Mycoplasma gallisepticum. Confirmation of virus replication in these preparations was obtained by either 1) demonstration of virus titre increase or 2) demonstration of fluorescence when using the homologous conjugate. Once either of these criteria had been satisfied, negative results with the infectious laryngotracheitis conjugate were taken to indicate that the conjugate would not present false positive results in differentiated cells infected with these heterologous viruses. The spectrum of reactivity of the infectious laryngotracheitis conjugate was then examined on organ cultures infected with several infectious laryngotracheitis isolates from across Canada. Finally, the conjugate was applied to experimental and natural cases of infectious laryngotracheitis and its efficiency was compared to routine virus isolation methods.     

Virus interference between H7N2 low pathogenic avian influenza virus and lentogenic Newcastle disease virus in experimental co-infections in chickens and turkeys

Low pathogenicity avian influenza virus (LPAIV) and lentogenic Newcastle disease virus (lNDV) are commonly reported causes of respiratory disease in poultry worldwide with similar clinical and pathobiological presentation. Co-infections do occur but are not easily detected, and the impact of co-infections on pathobiology is unknown. In this study chickens and turkeys were infected with a lNDV vaccine strain (LaSota) and a H7N2 LPAIV (A/turkey/VA/SEP-67/2002) simultaneously or sequentially three days apart. No clinical signs were observed in chickens co-infected with the lNDV and LPAIV or in chickens infected with the viruses individually. However, the pattern of virus shed was different with co-infected chickens, which excreted lower titers of lNDV and LPAIV at 2 and 3 days post inoculation (dpi) and higher titers at subsequent time points. All turkeys inoculated with the LPAIV, whether or not they were exposed to lNDV, presented mild clinical signs. Co-infection effects were more pronounced in turkeys than in chickens with reduction in the number of birds shedding virus and in virus titers, especially when LPAIV was followed by lNDV. In conclusion, co-infection of chickens or turkeys with lNDV and LPAIV affected the replication dynamics of these viruses but did not affect clinical signs. The effect on virus replication was different depending on the species and on the time of infection. These results suggest that infection with a heterologous virus may result in temporary competition for cell receptors or competent cells for replication, most likely interferon-mediated, which decreases with time.     

Response of mink, skunk, red fox and raccoon to inoculation with mink virus enteritis, feline panleukopenia and canine parvovirus and prevalence of antibody to parvovirus in wild carnivores in Ontario.

Mink virus enteritis, feline panleukopenia and canine parvovirus-2 were inoculated separately into groups of raccoon, mink, red fox and striped skunk. Raccoons were highly susceptible to mink virus enteritis and feline panleukopenia, with animals developing clinical illness, and several dying within six to ten days of inoculation with lesions typical of parvovirus infection. Both viruses were shed in high titre in the feces of infected raccoons, and high antibody titres were stimulated. Raccoons inoculated with canine parvovirus-2 showed no signs; shedding of virus was sporadic though moderate titres of antibody developed. Mink inoculated with mink virus enteritis and feline panleukopenia developed signs and lesions of early parvovirus infection. No signs or significant lesions followed canine parvovirus-2 inoculation. Shedding of virus was heavy (mink virus enteritis) or sporadic (feline panleukopenia and canine parvovirus-2), though good serological responses were elicited to all three viruses. Red fox showed no signs of infection, shed all three viruses only sporadically, and the serological response was strong only to feline panleukopenia. Skunks developed low antibody titres, but no signs, and did not shed virus. Antibody to parvovirus was found in 79.2% of 144 wild red foxes; 22.3% of 112 wild raccoons; 1.3% of 157 wild skunks and 6/7 coyotes in southern Ontario. The likely significance of these viruses to wild and captive individuals and populations of these carnivores is discussed.     

Growth kinetic studies of avian infectious bronchitis virus in tracheal organ cultures.

An egg-adapted vaccine strain (H120) and an organ culture-passaged field strain (HV-10) of avian infectious bronchitis (AIB) virus were propagated in tracheal organ cultures and their growth kinetics examined using nine-day-old embryonated fowl eggs and chick tracheal explants for virus assay. When the H120 strain was assayed in embryonated eggs, titres were approximately log10 2-0 ID50 (50 per cent infectious dose) per ml higher than when assays were performed in tracheal explants. The HV-10 strain, assayed in tracheal explants, yielded higher titres than did the H120 strain, but when assayed in embryonated eggs, yielded only minimal and variable virus titres.