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1.
Evidence of the widespread occurrence of reticuloendotheliosis virus (REV) sequence insertions in fowl poxvirus (FPV) genome of field isolates and vaccine strains has increased in recent years. However, only those strains carrying a near intact REV provirus are more likely to cause problems in the field. Detection of the intact provirus or REV protein expression from FPV stocks has proven to be technically difficult. The objective of the present study was to evaluate current and newly developed REV and FPV polymerase chain reaction (PCR) assays to detect the presence of REV provirus in FPV samples. The second objective was to characterize REV insertions among recent "variant" FPV field isolates and vaccine strains. With REV, FPV, and heterologous REV-FPV primers, five FPV field isolates and four commercial vaccines were analyzed by PCR and nucleotide sequence analysis. Intact and truncated REV 5' long terminal repeat (LTR) sequences were detected in all FPV field isolates and vaccine strains, indicating heterogeneous REV genome populations. However only truncated 3' LTR and envelope sequences were detected among field isolates and in one vaccine strain. Amplifications of the REV envelope and 3' LTR provided strong evidence to indicate that these isolates carry a near intact REV genome. Three of the four FPV vaccine strains analyzed carried a solo complete or truncated 5' LTR sequence, indicating that intact REV provirus was not present. Comparison of PCR assays indicated that assays amplifying REV envelope and REV 3' LTR sequences provided a more accurate assessment of REV provirus than PCR assays that amplify the REV 5' LTR region. Therefore, to differentiate FPV strains that carry intact REV provirus from those that carry solo 5' LTR sequences, positive PCR results with primers that amplify the 5' LTR should be confirmed with more specific PCR assays, such as the envelope, or the REV 3' LTR PCR. 相似文献
2.
The immune effects of fowlpox virus (FPV) field isolates and vaccine strains were evaluated in chickens infected at the age of 1 day and 6 weeks. The field isolates and the obsolete vaccine strain (FPV S) contained integrated reticuloendotheliosis virus (REV) provirus, while the current vaccine strain (FPVST) carries only REV LTR sequences. An indirect antibody ELISA was used to measure the FPV-specific antibody response. The non-specific humoral response was evaluated by injection of two T-cell-dependent antigens, sheep red blood cells (SRBC) and bovine serum albumin (BSA). There was no significant difference in the antibody response to FPV between chickens infected with FPV various isolates and strains at either age. In contrast, antibody responses to both SRBC and BSA were significantly lower in 1-day-old chickens inoculated with field isolates and FPV S at 2-3 weeks post-inoculation. Furthermore, cell-mediated immune (CMI) responses measured by in vitro lymphocyte proliferation assay and in vivo using a PHA-P skin test were significantly depressed in chickens inoculated with field isolates and FPV S at the same periods. In addition, thymus and bursal weights were lower in infected chickens. These immunosuppressive effects were not observed in chickens inoculated with the current vaccine strain, FPVST, at any time. The results of this study suggest that virulent field isolates and FPV S have immunosuppressive effects when inoculated into young chickens, which appeared in the first 3 weeks post infection. REV integrated in the FPV field isolates and FPV S may have played a central role in the development of immunosuppression. 相似文献
3.
Current strains of fowlpox virus (FWPV) carrying circulating reticuloendotheliosis virus (FWPV-REV) sequence are becoming more pathogenic to poultry. This is evidenced by the fact that vaccination with current available FWPV vaccines provides limited protection against them. To characterize REV insertions in a collection of both older and more recent field isolates, we developed three different types of adjacent oligoprobes and primer sets from specific genomic locations of FWPV and REV: REV-ENV (accession no. K02537, 1382-2260), FWPV-REV integration site (accession no. AF006064, 86-1328), FWPV (accession no. AF198100, 232461-232670), and REV-LTR (accession no. V01204, 305-496). The data indicated that the primers from the REV-ENV region and the TaqMan probes specifically targeted REV-ENV sequences of FWPV-REV strains. Furthermore, the strains were differentiated based on quantitative melting temperature (T(m)) of their amplified products using FRET-based probes. The amplified products were further characterized by sequencing and multiple sequence alignment analysis. The results suggest that integrated REV-ENV sequences are both common and mostly conserved in field isolates. However, the minor variations found within the short-targeted ENV sequence from FWPV-REV strains suggest that these strains could have either undergone periodic point mutational changes or integration with different REV-ENV subtypes. 相似文献
4.
通过向无外源病毒污染的鸡痘病毒活疫苗中添加不同剂量的禽网状内皮组织增生症病毒(REV),然后用间接免疫荧光法(IFA)进行检测,确定了该IFA方法的最低检出量为每500羽份疫苗中污染20 TCID_(50)的REV。使用该方法对国内16家企业生产的60批鸡痘病毒活疫苗进行了检验,结果显示2个企业生产的4批疫苗REV检测为阳性。随机选取5批IFA检测阴性样品和4批IFA检测阳性样品,按鸡检查法进行外源病毒检验,结果两种方法对REV污染的检测结果的符合率为100%。 相似文献
5.
Fowlpox virus (FWPV), an important pathogen of poultry, replicates very efficiently in the featherless areas of skin, and persists in dried and desiccated scabs for prolonged periods. Although the molecular mechanisms underlying the stability of the virus are not completely known, we recently identified the presence of a virus-encoded novel DNA repair enzyme, CPD-photolyase, in FWPV. This enzyme repairs the ultraviolet (UV)-induced pyrimidine dimers, converting them to monomers using photons from white light as a renewable source of energy. In this study, we examined the role of photolyase in the pathogenesis of fowlpox. A comparison of pathogenesis of fowlpox in chickens infected with parental FWPV with that in chickens infected with photolyase-deficient FWPV (Phr(-) FWPV) found no significant differences in terms of replication of virus or formation of secondary lesions. When the virions isolated from infected scabs were exposed to UV light, UV-damaged parental FWPV, unlike Phr(-) FWPV, were rescued through the CPD-photolyase-mediated photoreactivation pathway by at least 48%. However, the mutant virus triggered host's immune response and conferred complete protection against subsequent challenge with virus similar to that conferred by the parental virus. Since the mutant virus is less stable than the parental virus in the infected scabs but is as immunogenic, Phr(-) FWPV might be less persistent in the environment. Furthermore, this particular genetic locus can also be used to insert foreign genes for the development of FWPV recombinant vaccines. 相似文献
6.
Because of reticuloendotheliosis virus (REV) contamination in commercial poultry vaccines, polymerase chain reaction (PCR) assays have been described to increase the sensitivity of biological assays used to detect REV in vaccines. The PCR assay designed to amplify the long terminal repeat (LTR) region of REV identified REV LTRs in many of the commercial fowl poxvirus (FPV) vaccines evaluated. These commercial vaccines were not thought to be contaminated with replicating REV because of the lack of REV outbreaks, the lack of in vitro amplification, and lack of a serologic response to REV. As previously described, the FPV S vaccine strain is known to carry infectious integrated proviral REV, whereas FPV M vaccine strain and its derivatives carry integrated LTRs or remnants of REV proviral DNA inserted into the FPV genome. Another PCR assay designed to amplify the envelope gene of REV was used to verify that the envelope proviral gene was not present in REV LTR PCR-positive samples. Southern blot analysis with REV LTR probes hybridized to the 9-kb EcoRI genomic fragment of all FPV and pigeon poxviruses evaluated, whereas the envelope probe did not hybridize to any poxvirus genome. Sequence analysis of the 9-kb EcoRI fragment indicated that an integrated REV LTR exists in the 9-kb EcoRI of some poxvirus genomes. A new PCR assay designed to amplify integrated REV LTRs in the 9-kb EcoRI fragment identified complete and incomplete integrated REV LTRs in all FPV and pigeon poxvirus genomes evaluated. 相似文献
7.
本文利用聚合酶链式反应对来自澳大利亚昆士兰州、维多利亚州和新南维尔士州的４株禽痘病毒田间分离株及二疫苗株中网状内皮组织增殖病毒的ＬＴＲ片断、ｅｎｖ及ｒｅｌ基因因进行了检测。所有４株痘病毒分离株均为ＬＴＲ片断、ｅｎｖ阳性，曾被ＰＣＲ证实为ＬＴＲ阳性的一疫苗株在本实验中也为ＬＴＲ、ｅｎｖ阳性，另一疫苗株为ＬＴＲ、ｅｎｖ阴性，所有上述禽痘病毒株均为ｒｅｌ阴性。 相似文献
8.
为调查福建规模化养鸡场病鸡的死亡原因,试验以福建省送检的鸡冠及头部皮肤有大量结节状痘痂的病鸡病料为研究对象进行了PCR鉴定。初步鉴定为鸡痘病毒（FWPV）株后,通过接种鸡胚绒毛尿囊膜（CAM）分离病毒;用原代鸡胚成纤维细胞（CEF）和传代细胞DF-1细胞对病毒进行传代,观察该分离毒株在两种细胞上培养特性的异同;对被感染的CEF进行超薄切片观察病毒的分布及病毒粒子的形态;并针对该分离株的TK基因和FPV175基因序列进行同源性分析。结果显示,接种经抗生素处理后的病料匀浆液上清的CAM出现大面积单个白色隆起的痘斑,匀浆痘斑后同时接种CEF和DF-1细胞,两种细胞均能产生稳定可持续传代的细胞病变,但病变出现的时间及病变程度不同;透射电镜下观察到典型的FWPV粒子密集分布在CEF的胞浆中,清晰可见卵圆形外膜包裹着的两侧凹陷的核心。对其中23个病毒粒子进行统计测得病毒粒子的大小为（258~344） nm×（153~238） nm;针对FWPV TK基因和FPV175基因的PCR检测及测序结果与GenBank收录的FWPV（登录号：NC_002188.1）核苷酸序列同源性分别高达100%和99.8%。以上结果表明该分离毒株为FWPV,命名为FWPV-FJ01,为国内FWPV的防治提供了参考依据。 相似文献
9.
A vaccine strain of fowlpox virus (FPV) was genetically engineered to produce avian influenza virus hemagglutinin (HA). This was accomplished by inserting a cDNA copy of the avian influenza virus HA gene, which was regulated by a vaccinia virus promoter, into the FPV thymidine kinase (TK) gene. Two types of recombinant viruses, differing only in the orientation of the HA gene relative to an adjacent foreign gene (lacZ), were created. Following preliminary identification of FPV recombinants based on the generation of beta-galactosidase (lacZ gene product), correct insertion of the HA gene into the genomes of these viruses was verified by hybridization studies. Susceptible chickens vaccinated with these FPV recombinants produced specific hemagglutination-inhibiting antibodies against the HA antigen. In view of this immune response, these viruses may serve as vaccines against avian influenza virus. In this regard, they appeared to be less virulent than the parental virus. 相似文献
10.
表达H9亚型禽流感病毒HA基因重组鸡痘病毒疫苗的遗传稳定性 总被引：4，自引：0，他引：4
将表达H9亚型禽流感病毒HA基因重组鸡痘病毒疫苗连续传代至30代，取第10、20、30代重组病毒作为受检代次，进行外源基因片段的克隆与测序，以间接免疫荧光试验检验各代次的表达，并以第10、20、30代重组鸡痘病毒疫苗进行免疫保护效力试验。对各代次模板进行PCR反应，分别扩增出特异的1．7kb外源基因片段，酶切位点与原始代次相同；各代次外源基因序列与原始序列相比，仅有1个碱基发生变化，不涉及氨基酸的变化；免疫荧光试验显示各代次均有显著表达；各免疫组在免疫后第7、10、14、20天的HI抗体效价(log2)逐渐上升；攻毒后第5天各免疫组排毒率与对照组相比差异显著，各免疫组之间无显著差异。结果表明：此重组载体疫苗具有较好的遗传稳定性，经过30代的传代后外源插入基因及其表达未见变化，免疫保护效力亦与原代一致。 相似文献
11.
禽痘病毒感染对禽流感重组禽痘病毒疫苗免疫效力的影响 总被引：1，自引：0，他引：1
表达禽流感病毒 (AIV)HA和NA基因的重组禽痘病毒rFPV_HA_NA能够诱导鸡体产生 10 0 %抵抗高致病性禽流感病毒 (HPAIV)H5N1的攻击。而当鸡群已进行禽痘疫苗免疫或者感染了禽痘病毒的情况下 ,此重组疫苗的免疫效力如何 ?首先用禽痘病毒S_FPV_0 17人工感染SPF试验鸡 ,既而在感染后的不同间隔时间接种重组疫苗 ,免疫后检测鸡群的HI抗体水平 ,同时用 10 0LD50 的HPAIVH5N1进行攻击。结果重组疫苗免疫与禽痘病毒人工感染时间间隔在 4周 (或以上 )时 ,预先感染禽痘病毒对重组疫苗的免疫效力不构成影响 ,对禽流感的保护力为 10 0 % ,而间隔时间在 1、2、3周时 ,重组疫苗的免疫保护效力则受到不同程度的影响。 相似文献
12.
Isa G Pfister K Kaaden OR Czerny CP 《Journal of veterinary medicine. B, Infectious diseases and veterinary public health》2002,49(1):21-23
To provide a fast and easy method to detect antibodies against fowlpox virus (FWPV) particularly in high numbers of chicken sera we established a monoclonal blocking enzyme-linked immunosorbent assay (ELISA). We chose two different monoclonal antibodies (mAb), anti-FWPV 3D9/2B3 and anti-FWPV 8F3/2E11, which are both directed against the 39-kDa protein of FWPV strain HP-1. The blocking ELISA depends on the blocking of mAb binding to solid-phase antigen in the presence of positive serum. For an epidemiological study a total of 184 serum samples from Gambian chicken flocks were analysed against each of the mAbs. Four of the sera were shown to contain FWPV antibodies. These four sera showed a positive cut-off value of more than 50% inhibition exclusively in the test against the mAb anti-FWPV 8F3/2E11. This phenomenon can be explained by the binding of the mAbs to distinct epitopes on the same protein. 相似文献
13.
A concurrent infection of chickens with infectious laryngotracheitis virus (ILTV), a herpesvirus, and fowlpox virus (FWPV), an avipoxvirus, is described. Two techniques, an immunohistochemistry (IHC) technique and a multiplex polymerase chain reaction (PCR), were used to examine 11 tissue samples from chickens clinically diagnosed as FWPV-infected, but only IHC was used to examine six tissue-paraffin blocks prepared from turkeys suspected of having FWPV infection. By multiplex PCR, both FWPV and ILTV were detected from three chicken samples (FI-90, FI-93, and FI-94); both FWPV and ILTV were detected from only two samples (FI-93 and FI-94) by IHC. All chicken samples were positive for FWPV by both PCR and IHC. Viral DNA from these samples was further confirmed by restriction enzyme analysis. When turkey samples were analyzed by the double-stain IHC, all six samples showed the presence of FWPV antigens, but no ILTV antigens. The double IHC technique, using monoclonal antibodies against FWPV and ILTV, was successful in simultaneous demonstration of specific FWPV and ILTV antigens colocalized in infected tissue samples as well as within individual cells. This paper emphasizes the importance of reliable tests that detect specifically the presence of ILTV and FWPV in infected tissue samples. The multiplex PCR assay holds potential to be versatile, rapid, and more sensitive (100%) than IHC (67%) for the simultaneous detection of two different avian viruses. Furthermore, the presence of mixed infection should always be kept in mind in the virologic analysis of respiratory sickness of poultry. 相似文献
14.
M E Boursnell P F Green J I Campbell A Deuter R W Peters F M Tomley A C Samson P T Emmerson M M Binns 《Veterinary microbiology》1990,23(1-4):305-316
In this paper we report the development and testing of a fowlpox virus vector system. Insertion sites in non-essential regions within the terminal inverted repeats of the virus have been characterised. Foreign genes inserted into these sites are shown to be present in two copies in the resultant recombinant virus. To test the potential use of this vector as a live vaccine the fusion gene of Newcastle disease virus (NDV) has been inserted into a vaccine strain of fowlpox virus, and inoculated into chickens. The experiments demonstrate the ability of the recombinant to protect chickens against challenge by a virulent strain of NDV and to elicit the formation of anti-fusion protein antibody. 相似文献
15.
J Jieyuan P B Spradbrow 《Zentralblatt für Veterin?rmedizin. Reihe B. Journal of veterinary medicine. Series B》1992,39(5):388-390
Chickens were given various fowlpox vaccines on food pellets--a commercial vaccine (strain M), and the same strain after a single passage on chorio-allantoic membrane or in chicken embryo fibroblasts. All three oral vaccines induced antibodies at levels similar to those induced by commercial strain M administered to the wingweb. The oral vaccine derived from chorio-allantoic membrane gave protection similar to that obtained with vaccine administered by the wingweb, but this required a thousandfold more virus. 相似文献
16.
Use of restriction fragment length polymorphism, immunoblotting, and polymerase chain reaction in the differentiation of avian poxviruses. 总被引：3，自引：0，他引：3
Restriction deoxyribonucleic acid (DNA) fragment profile analysis coupled with immunogenic protein profile analysis has provided useful information in determining the differences between vaccine strains and field isolates of fowlpox virus (FPV). The DNA of strains examined in this study clearly fell into 3 minor groups of restriction patterns similar but distinct from one another: restriction patterns exhibited by the vaccine strains except 1 vaccine strain, Vac-82; restriction profiles indicated by Vac-82 and field isolates FI-38 and FI-42; and restriction patterns indicated by field isolates FI-43, FI-51, FI-54, and FI-56. Furthermore, when the strains were analyzed and compared by immunoblotting analysis, they showed group differences similar to the differences in restriction profiles. Both techniques provided high sensitivity in verifying differences between vaccine strains and field isolates of FPV. The disparity found in restriction fragments or immunogenic protein profile between vaccine strains and field isolates does not exclude the appreciable high degree of DNA sequence conservation and homology. However, the minor disparity observed in these strains suggests a molecular basis for why vaccinated commercial flocks could have continually been infected by variant strains of FPV. A rapid and sensitive polymerase chain reaction method, which amplified a product from the 4b core protein gene of the FPV genome, was developed for identification and differentiation of members of the genus Avipoxvirus. Whereas total DNA from either vaccine strains or field isolates was used as template for amplifying a predicted product of 578 or 1409 bp, only cleavage of the amplified product (1409 bp) represented an additional detection technique for species differentiation. An attempt to distinguish between strains on the basis of amplification product was partially successful. 相似文献
17.
Prukner-Radovcić E Lüschow D Ciglar Grozdanić I Tisljar M Mazija H Vranesić D Hafez HM 《Avian diseases》2006,50(3):440-444
In the last 3 yr, several outbreaks of avian poxviruses (APVs) have been observed in different parts of Croatia. Four strains of APVs, from chickens, a pigeon, and a turkey, were isolated from cutaneous lesions by inoculation onto the chorioallantoic membranes (CAM) of 12-day-old specific-pathogen-free chicken embryos. The resulting proliferative CAM lesions contained eosinophilic cytoplasmic inclusion bodies. The characteristic viral particles of poxvirus were detected in the infected CAM and also in the infected tissues by transmission electron microscopy. Further identification and differentiation of the four various APVs were carried out by the use of a polymerase chain reaction (PCR) combined with restriction enzyme analysis. Using one primer set, which framed a region within the APV 4b core protein gene, it was possible to detect APV-specific DNA from all four tested isolates. PCR results revealed no recognizable differences in size of amplified fragments between the different APVs from chickens, turkey, and pigeon. Restriction enzyme analysis of PCR products using NlaIII showed the same cleavage pattern for turkey and chicken isolates and a different one for the pigeon isolate. Multiplex PCR for direct detection of APV and reticuloendotheliosis virus (REV) was carried out to determine the possible integration of REV in the genome of isolated APVs. The obtained results revealed that REV was present in chicken and turkey strains of poxviruses, whereas the pigeon isolate was negative. It is not known whether the avipoxvirus vaccine strain used in Croatia is contaminated with REV or if the REV is naturally contaminating Croatian field strains of fowl poxvirus. The latter is indicated by the negative REV finding in the pigeon, which was not vaccinated. The results of the present study indicate the reemergence of fowlpox in Croatia, where infections have not been recorded since 1963 and never confirmed etiologically. 相似文献
18.
A novel pox virus, condorpox virus (CPV) isolated from the spleen of an Andean condor (Vultur gryphus) by inoculation of chorioallantoic membranes (CAM) of specific pathogen free (SPF) chicken embryos was compared biologically, antigenically and genetically with fowlpox virus (FPV), the type species of the genus Avipoxvirus. Susceptible chickens inoculated with CPV developed only mild localized lesions but were not protected against subsequent challenge with FPV. Based on Western blotting, in addition to the presence of cross-reacting antigens, distinct differences in antigenic profiles of CPV and FPV were observed. Sequence analysis of a 4.5 kb HindIII fragment of CPV genomic DNA revealed the presence of eight co-linear genes corresponding to FPV open reading frame (ORF)193-198, 201 and 203. Interestingly, reticuloendotheliosis virus (REV) sequences present in the genome of all FPV were absent in CPV. Although, the results of a phylogenic analysis suggested that CPV is a member of the genus Avipoxvirus, its unique antigenic, biologic and genetic characteristics distinguish it from FPV to be considered as a new member of this genus. 相似文献
19.
Safety and immunogenicity of ERA strain of rabies virus propagated in a BHK-21 cell line. 总被引：5，自引：2，他引：3 
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下载免费PDF全文 K F Lawson R Hertler K M Charlton J B Campbell A J Rhodes 《Canadian journal of veterinary research》1989,53(4):438-444
The ERA strain of rabies virus was propagated in a baby hamster kidney cell line (BHK-21/C13). The viral titer was 10(1.8) tissue culture infective doses (TCID) higher than that of commercial ERA vaccine. The ERA/BHK-21 vaccine in baits retained titers of 10(6.3) to 10(6.4), TCID when subjected to daily temperature fluctuations from 9 degrees C to 24 degrees C for 21 days. This titer, according to a dose response in laboratory foxes, was still capable of immunizing up to 100% of foxes consuming a bait. The ERA/BHK-21 vaccine, when presented in baits, produced antibodies in 80 to 100% of dogs consuming more than one bait. Duration of immunity in foxes, from feeding the ERA strain rabies virus in baits, as determined by resistance to challenge with virulent virus, was at least 48 months. The vaccine strain retained some pathogenicity for nontarget species. In tests carried out on foxes, raccoons, dogs, cats and cattle, the vaccine did not cause vaccine-induced rabies. One of 14 skunks which consumed four baits developed vaccine-induced rabies, but virus could not be isolated from the salivary glands of this animal. The vaccine, when presented in baits, caused vaccine-induced rabies in 37% of laboratory mice, 3.4% of Microtus and 2.6% of Peromyscus species. Rabies virus could not be isolated from the salivary glands of rodents with vaccine-induced rabies. It was concluded that ERA virus propagated in BHK-21/C13 cells and incorporated in an acceptable bait produced a high titer, stable, immunogenic and safe vaccine for foxes. 相似文献
20.
表达传染性法氏囊病病毒VP2基因的重组鸡痘病毒的构建及其免疫保护性研究 总被引：1，自引：0，他引：1
以脂质体转染技术构建了表达鸡传染性法氏囊病病毒（ＩＢＤＶ）ＶＰ２基因的重组鸡痘病毒ＦＰＶ－ＶＰ２，该病毒在鸡胚成纤维细胞及鸡体内均能稳定产生子代病毒，经翅皮下５×１０５ＰＦＵ／羽免疫１日龄ＳＰＦ鸡，免疫后４周以１００ＬＤ５０／羽ＩＢＤＶ超强毒株Ｇ株攻毒，获得了５／６的保护，但不能有效预防临床发病及法氏囊受损萎缩。实验结果证明了ＶＰ２是ＩＢＤＶ的宿主保护性抗原，提示Ｔ细胞介导的免疫可能在ＩＢＤＶ的免疫中起着较为重要的作用。本研究为ＩＢＤＶ重组病毒疫苗研制进行了有益探索。 相似文献