Reticuloendotheliosis virus integration in the fowl poxvirus genome: not a recent event

Integration of reticuloendotheliosis virus (REV) into the genome of fowl poxvirus (FPV) has been reported recently. With a view to determine whether this event had occurred in the past, we screened by polymerase chain reaction (PCR) for the presence of REV provirus in the DNAs of nine avian poxviruses, some of which had been lyophilized 50 yr ago. For REV, 5' long terminal repeat (LTR) and REV envelope sequences were amplified, whereas for FPV, the major envelope antigen gene and the region flanking REV sequences were amplified. In six of seven FPV strains examined, the specific PCR amplicons were obtained for both REV provirus and FPV sequences. One isolate in which presence of REV 5' LTR and envelope was not detected by PCR, a LTR remnant was detected by Southern hybridization. Interestingly, no REV sequence was detected in either canary poxvirus or pigeon poxvirus genome. These observations indicate that REV integration in the FPV genome is not a recent phenomenon but probably occurred prior to 1949.     

Reticuloendotheliosis virus (REV) long terminal repeats incorporated in the genomes of commercial fowl poxvirus vaccines and pigeon poxviruses without indication of the presence of infectious REV

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.     

Existence of variant strains Fowlpox virus integrated with Reticuloendotheliosis virus in its genome in field isolates in Tanzania

Fowlpox virus (FPV) is one example of poultry viruses which undergoes recombination with Reticuloendotheliosis virus (REV). Trepidation had been raised, and it was well established on augmented pathogenicity of the FPV upon integration of the full intact REV. In this study, we therefore intended at assessing the integration of REV into FPV genome of the field isolates obtained in samples collected from different regions of Tanzania. DNA extraction of 85 samples (scabs) was performed, and FPV-specific PCR was done by the amplification of the highly conserved P4b gene. Evaluation of FPV–REV recombination was done to FPV-specific PCR positively identified samples by amplifying the env gene and REV long terminal repeats (5′ LTR). A 578-bp PCR product was amplified from 43 samples. We are reporting for the first time in Tanzania the existence of variant stains of FPV integrated with REV in its genome as 65 % of FPV identified isolates were having full intact REV integration, 21 % had partial FPV–REV env gene integration and 5 % had partial 5′ LTR integration. Despite of the fact that FPV–REV integrated stains prevailed, FPV–REV-free isolates (9 %) also existed. In view of the fact that full intact REV integration is connected with increased pathogenicity of FPV, its existence in the FPV genome of most field isolates could have played a role in increased endemic, sporadic and recurring outbreaks in selected areas in Tanzania.     

应用聚合酶链式反应检测禽痘病毒田间分离株及疫苗株中网状内皮组织增殖病毒LTR片断,env及re1基因

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。     

Evaluation of immune effects of fowlpox vaccine strains and field isolates

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.     

Construction and characterization of a fowlpox virus field isolate whose genome lacks reticuloendotheliosis provirus nucleotide sequences

Fowlpox virus (FWPV) has been isolated from vaccinated chicken flocks during subsequent fowlpox outbreaks that were characterized by a high degree of mortality and significant economic losses. This inability of current vaccines to induce adequate immunity in poultry could be reflective of an antigenic and/or biologic distinctiveness of FWPV field isolates. In this regard, whereas an infectious reticuloendotheliosis virus (REV) provirus is present in the majority of the field viruses' genomes, only remnants of REV long terminal repeats (LTR) have been retained in the DNAs of each vaccine strain. Although it has not been demonstrated whether the partial LTRs can provide an avenue for FWPV to reacquire the REV provirus by homologous recombination, utilizing viruses of which genomes lack any known integrated retroviral sequences could resolve concern over this issue. Therefore, such an entity was created by genetically modifying a recently isolated field strain of FWPV. This selection, in lieu of a commercial vaccine virus, as the progenitor was based on the probability that a virus circulating in the environment would be more antigenically similar to others in this locale and thus might be a better candidate for vaccine development. A comparison in vivo of the pathogenic traits of the parental wild-type field isolate, its genetically modified progeny, and a rescue mutant in whose genome the REV provirus was inserted at its previous location, indicated that elimination of the provirus sequence correlated with reduced virulence. However, even with elimination of the parasitic REV, the modified FWPV was still slightly more invasive than a commercial vaccine virus. Interestingly, both types of attenuated FWPV elicited a similar degree of antibody production in inoculated chickens and afforded them protection against a subsequent challenge by a field virus, the origin of which was temporally and geographically distinct from that of the progenitor strain. Due to its antigenicity being retained despite a decrease in virulence, this REV-less FWPV could potentially be developed as a vaccine against fowlpox.     

Molecular characteristics of Polish field strains of Marek's disease herpesvirus isolated from vaccinated chickens

Background

Twenty-nine Marek''s disease virus (MDV) strains were isolated during a 3 year period (2007-2010) from vaccinated and infected chicken flocks in Poland. These strains had caused severe clinical symptoms and lesions. In spite of proper vaccination with mono- or bivalent vaccines against Marek''s disease (MD), the chickens developed symptoms of MD with paralysis.Because of this we decided to investigate possible changes and mutations in the field strains that could potentially increase their virulence. We supposed that such mutations may have been caused by recombination with retroviruses of poultry - especially reticuloendotheliosis virus (REV).

Methods

In order to detect the possible reasons of recent changes in virulence of MDV strains, polymerase chain reaction (PCR) analyses for meq oncogene and for long-terminal repeat (LTR) region of REV were conducted. The obtained PCR products were sequenced and compared with other MDV and REV strains isolated worldwide and accessible in the GeneBank database.

Results

Sequencing of the meq oncogene showed a 68 basepair insertion and frame shift within 12 of 24 field strains. Interestingly, the analyses also showed 0.78, 0.8, 0.82, 1.6 kb and other random LTR-REV insertions into the MDV genome in 28 of 29 of strains. These genetic inserts were present after passage in chicken embryo kidney cells suggesting LTR integration into a non-functional region of the MDV genome.

Conclusion

The results indicate the presence of a recombination between MDV and REV under field conditions in Polish chicken farms. The genetic changes within the MDV genome may influence the virus replication and its features in vivo. However, there is no evidence that meq alteration and REV insertions are related to the strains'' virulence.     

Use of restriction fragment length polymorphism, immunoblotting, and polymerase chain reaction in the differentiation of avian poxviruses.

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.     

Application of PCR assays to determine the genotype of Babesia bovis parasites isolated from cattle with clinical babesiosis soon after vaccination against tick fever

OBJECTIVE: To demonstrate the value of PCR assays to determine the genotypes of Babesia bovis in cattle with clinical signs of babesiosis within 3 weeks after vaccination against tick fever. DESIGN: Samples from 5 cases of babesiosis in cattle soon after vaccination against tick fever were analysed in two PCR assays. PROCEDURE: Parasite DNA was purified from blood taken from cattle with signs of babesiosis within 3 weeks of vaccination against tick fever. DNA was also prepared from the tissues of animals that died of babesiosis. Two PCR assays that amplify repeat sequences of DNA within the B bovis genes, Bv80 and BvVA1, were used to differentiate the genotypes of field isolates and vaccine strains of B bovis. RESULTS: One of the five cases of babesiosis was found to be caused by a vaccine strain, but PCR analyses showed that the predominant isolate in the other four cases was not the vaccine strain. CONCLUSIONS: PCR assays on the DNA of B bovis obtained from the blood or tissues of cattle clinically affected with tick fever within 3 weeks after vaccination are useful to distinguish between vaccine strains and field isolates as the source of infection.     

Isolation and molecular biological investigations of avian poxviruses from chickens, a turkey, and a pigeon in Croatia

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.     

马传染性贫血病毒第19、26代驴胎皮肤细胞弱毒前病毒DNA全基因序列分析

不同代次马传染性贫血驴胎皮肤细胞弱毒(Fetal donkey dermal virus,FDDV)的免疫保护效果各不相同,只有第10～15代驴胎皮肤细胞弱毒具有良好的免疫保护效果,可作为疫苗使用,继续传代则疫苗的保护率下降。为确定有、无免疫保护效果的FDDV在基因水平上的差异,本实验对无免疫保护效果的第19、26代驴胎皮肤细胞弱毒前病毒DNA进行了全基因序列测定,并与已测序的疫苗毒株进行序列比较。第19代和第26代FDDV全基因核苷酸序列同源性高达99.5%,与疫苗毒株全基因核苷酸序列的同源性分别为96.9%、96.7%。LTR是EIAV在细胞传代中变异最显著的区域,第19、26代FDDV的LTR与疫苗毒的LTR同源性仅为89.6%、89.3%。马传贫病毒的gag基因高度保守,第19、26代FDDV与疫苗毒株的gag基因推导氨基酸序列仅有2个氨基酸不同。第19、26代FDDV与疫苗毒株的pol基因、env基因的推导氨基酸序列的同源性分别为98.9%、98.8%、93.7%、93.6%。由序列比较结果可以推断,第19代、第26代FDDV不具有免疫保护效果的主要原因可能是由于LTR和env基因的变异,导致病毒复制能力下降或免疫原性丧失,不能诱导机体产生良好的免疫反应。     

马传染性贫血病毒自然感染马LTR序列分析

我国从20世纪70年代开始使用EIAV弱毒疫苗后,在全国范围内基本控制了马传贫的发生,我们从现地少数EIAV琼扩阳性马外周血中扩增并克隆了二株EIAV前病毒5'LTR序列,并与国内外毒株5'LTR序列进行了比较分析,发现中国EIAV LTR特有的特异性细胞转录因子结合基序,同时发现PEA2位点不是强毒特有的基序,在弱毒中亦发现该基序.     

鸡肿瘤病料中马立克氏病病毒和禽网状内皮组织增生症病毒共感染的研究

采用细胞培养、间接荧光抗体试验(IFA)、聚合酶链式反应(PCR)和斑点杂交(Dotblot)的方法从我国不同地区发生肿瘤的病料中同时进行MDV和REV的分离和鉴定。在分离到的13株MDV野毒株中，有4株培养物既能在IFA中与REV的单抗反应，又可以用PCR扩增出REV的LTR；另有4株培养物能扩增出REV的LTR，但在IFA中却不与REV的单抗反应。结果表明我国MD肿瘤中存在着REV的共感染，且我国MDV某些野毒株的基因组中有可能已经整合进了REV的LTR序列。     

Detection and differentiation of re-emerging fowlpox virus (FWPV) strains carrying integrated reticuloendotheliosis virus (FWPV-REV) by real-time PCR

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.     

雀形目野生鸟类REV感染状况的调查及分离株gp90基因序列分析

为了解雀形目野生鸟类感染禽内皮组织增生症病毒（Reticuloendotheliosis virus,REV）的情况,采集了352份样品,应用PCR方法进行了初筛。然后将阳性样品适当处理后接种CEF细胞,利用IFA等方法进一步鉴定。结果分离到2株病毒WB11042和WB11043,分别来自黄喉鹀和燕雀。对2株REV分离株的gp90基因进行扩增、测序和序列分析。结果显示,其gp90序列与REV亚型Ⅲ的代表毒株（CSV、APC-566）亲缘关系最近,高达99%以上;与亚型Ⅱ代表株（SNV）和亚型Ⅰ代表株（HA9901）的亲缘关系相对较远,在95.5%~97.1%之间。遗传进化树分析也表明这2株野生鸟类分离株属于REV亚型Ⅲ。结果表明,雀形目野生鸟类也可以感染REV。     

Molecular characterization of three recombinant isolates of avian leukosis virus obtained from contaminated Marek's disease vaccines

Three natural recombinant avian leukosis viruses (ALV; PDRC-1039, PDRC-3246, and PDRC-3249) expressing a subgroup A gp85 envelope protein and containing long terminal repeats (LTR) of endogenous ALV-E viruses were isolated from contaminated commercial Marek's disease vaccines, cloned, and completely sequenced. Their full genomes were analyzed and compared with representative strains of ALV. The proviral DNA of all three isolates displayed 99.3% identity to each other, suggesting a possible common ancestor, even though the contaminating viruses were obtained from three separate vaccine serials produced by two different vaccine manufacturing companies. The contaminating viruses have a genetic organization typical of replication-competent alpharetroviruses. The proviral genomes of PDRC-1039 and PDRC-3246 are 7497 bp long, and the PDRC-3249 is three base pairs shorter because of a deletion of a threonine residue within the gp85 coding region. The LTR, gag, pol, and the transmembrane (TM) region (gp37) of the env gene of all three viruses displayed high identity to endogenous counterpart sequences (>98%). Only the surface (SU) region (gp85) of the env gene displayed high identity with exogenous ALV-A (98.7%). Locus-specific polymerase chain reaction (PCR) analysis for ALV endogenous sequences (ev loci) in the chicken embryo fibroblasts used to produce the original vaccine vials identified the presence of ev-1, ev-2, ev-3, ev-4, and ev-6 in all three vaccines. Homologous recombination most likely took place to involve the SU region of the env gene because the recombinant viruses only differ in this particular region from the consensus ALV-E. These results suggest that the contaminating ALV isolates probably emerged by recombination of ALV-A with endogenous virus sequences before vaccine preparation.     

Molecular epidemiology of classical swine fever in the Russian Federation

The ability to discriminate between various classical swine fever virus (CSFV) strains and isolates is a prerequisite for following the spread of the virus after an outbreak. To determine the relatedness between Russian CSFV isolates from different geographical regions, three fragments of the viral genome (5' NTR, the variable region of the E2 gene and a fragment of the NS5B gene) were sequenced and used for genetic typing. Thirty-one field isolates were obtained from CSF outbreaks which occurred between 1994 and 1999. In addition, three attenuated strains were included in the study, namely the LK and CS vaccine strains, and the moderately virulent 238H isolate. The vaccine strains have been used in Russia for more than 30 years. Our results showed that all field isolates are in subgroup 1.1 together with Alfort 187 and with the highly virulent strain Shimen. In contrast, the CS and LK vaccine strains belong to subgroup 1.2. While there is no evidence for the reversion of the two vaccine strains to wild type, it is feasible that the highly virulent Shimen strain, which has been used as a challenge strain for many years, contributed to field strain generation. The Russian field isolates from the 1990s can be distinguished from the CSF virus isolates which occurred in the EU Member States in the same decade, as here all outbreaks were caused by CSF viruses belonging to subgroup 2.     

Identification and characterization of fowlpox virus strains using monoclonal antibodies.

The use of 2 monoclonal antibodies (MAbs), P1D9 and P2D4, which recognize different fowlpox virus (FPV) antigens, for the identification and characterization of FPV strains was evaluated. Initially, the MAbs were used in conjunction with a dot blot assay that enabled FPV to be differentiated from the avian herpesvirus, infectious laryngotracheitis virus. Confirmation of the specificity of these MAbs was provided by the demonstration that only FPV antigens were recognized by a combination of both antibodies when used for immunoblotting proteins contained in various avipoxviruses. Later, an antigenic characterization of 11 FPV field isolates, 6 FPV vaccine strains, and 3 pigeonpox virus vaccines was performed by Western blotting with the individual MAbs. Whereas MAb P2D4 consistently recognized a protein with an apparent molecular weight of 60 kD, there was variability in the size of the antigen that was immunoreactive with the other MAb. For example, MAb P1D9 recognized an antigen of apparent molecular weight of 46 kD in all vaccine strains except 2 of FPV origin. In these exceptions, either only a 39-kD or both a 42- and 46-kD protein were immunoreactive. As for the field isolates, a 39-kD antigen was recognized in 8 of them, whereas a 42-kD antigen was detected in the remaining 3. Therefore, the more extensive immunoblotting technique may facilitate FPV strain differentiation, whereas routine diagnosis of fowlpox could be accomplished by using the MAb-based dot blot assay.