一株鸡传染性贫血病毒野毒株致病性及其全基因组序列比较

从山东某商品代肉鸡场表现生长迟缓的14日龄病鸡群分离到一株鸡传染性贫血病毒(CAV)C14株。C14株感染1日龄SPF鸡能抑制对禽流感病毒(AIV)的抗体反应,还能与禽网状内皮增生病病毒(REV)在免疫抑制上起协同作用。用PCR方法分段扩增出C14基因组的三条部分重叠片段,分别克隆于T载体并进行测序,拼接后得到其全基因组序列。测序结果表明,CAV-C14株基因组全长2298bp,含有3个互相重叠的开放阅读框和1个调控区。将C14与国内外已发表的CAV参考株基因组比较,同源性为97.2%~99.2%。序列比较表明CAV非编码区中含有的多个与复制及转录调控相关已知基序的序列都非常保守。CAV的3个编码基因VP1、VP2和VP3均有一定程度变异,以VP1变异性最大,且不同毒株间的3个蛋白质氨基酸序列的变异是互不相关的。     

Characterization of cloned chicken anemia virus DNA that contains all elements for the infectious replication cycle.

Circular double-stranded replication intermediates were identified in low-molecular-weight DNA of cells of the avian leukemia virus-induced lymphoblastoid cell line 1104-X-5 infected with chicken anemia virus (CAV). To characterize the genome of CAV, we cloned linearized CAV DNA into the vector pIC20H. Transfection of the circularized cloned insert into chicken cell lines caused a cytopathogenic effect, which was arrested when a chicken serum with neutralizing antibodies directed against CAV was added. Chickens inoculated at 1 day of age with CAV collected from cell lines transfected with cloned CAV DNA developed clinical signs of CAV. The 2,319-bp cloned CAV DNA contained all the genetic information needed for the complete replication cycle of CAV. The CAV DNA sequence has three partially overlapping major reading frames coding for putative peptides of 51.6, 24.0, and 13.6 kDa. The CAV genome probably contains only one promoter region and only one poly(A) addition signal. Southern blot analysis using oligomers derived from the CAV DNA sequence showed that infected cells contained double- and single-stranded CAV DNAs, whereas purified virus contained only the minus strand. It is the first time that the genome of one of the three known single-stranded circular DNA viruses has been completely analyzed.     

Analysis of chicken anemia virus genome: evidence of intersubtype recombination

Background

Chicken anemia virus (CAV) is the causative agent of chicken infectious anemia. CAV putative intergenotypic recombinants have been reported previously. This fact is based on the previous classification of CAV sequences into three genotypes. However, it is unknown whether intersubtype recombination occurs between the recently reported four CAV genotypes and five subtypes of genome sequences.

Results

Phylogenetic analysis, together with a variety of computational recombination detection algorithms, was used to investigate CAV approximately full genomes. Statistically significant evidence of intersubtype recombination was detected in the parent-like and two putative CAV recombinant sequences. This event was shown to occur between CAV subgroup A1 and A2 sequences in the phylogenetic trees.

Conclusions

We revealed that intersubtype recombination in CAV genome sequences played a role in generating genetic diversity within the natural population of CAV.

     

Investigation of the attenuation exhibited by a molecularly cloned chicken anemia virus isolate by utilizing a chimeric virus approach.

Molecular cloning of the Cux-1 isolate of chicken anemia virus (CAV), which had been passaged 173 times in cell culture, resulted in the isolation of an attenuated strain, designated cloned isolate 10, which reverted to virulence following 10 passages in young chicks (D. Todd, T. J. Connor, V. M. Calvert, J. L. Creelan, B. M. Meehan, and M. S. McNulty, Avian Pathol. 24:171-187, 1995). The attenuated cloned isolate 10 differs from the molecularly cloned pathogenic Cux-1 isolate in that it possesses a 21-nucleotide insertion within the nontranscribed region of the CAV genome and 17 individual nucleotide substitutions dispersed throughout the genome. Comparative analyses with other published CAV sequences indicated that cloned isolate 10 was unique at nine nucleotide positions and at five amino acid positions. The molecular basis of the attenuation exhibited by cloned isolate 10 was investigated by evaluating the pathogenicities of two sets of complementary chimeric viruses. These sets were produced by transfection with chimeric double-stranded replicative-form (RF) DNA equivalents that contained DNA sequences derived from cloned isolate 10 and the pathogenic cloned Cux-1 isolate. The construction of the chimeric RFs exploited the occurrence of unique EcoRI, PstI, and BamHI restriction sites, which allowed their respective circular CAV RFs to be manipulated as three restriction fragments of 0.58, 0.93, and 0.71 kbp. Examination of the levels of anemia and gross pathology in the thymuses and bone marrows of 14 day-old specific-pathogen-free chicks following infection of 1-day-old chicks with the chimeric and cloned parental isolates indicated that nucleotide changes in each of the three genomic regions contributed towards attenuation. The significance of this result to the development and use of live attenuated CAV vaccines is discussed.     

Chicken anemia virus and avian gyrovirus 2 as contaminants in poultry vaccines

This study focuses on the detection of chicken anemia virus (CAV) and avian gyrovirus 2 (AGV2) genomes in commercially available poultry vaccines. A duplex quantitative real-time PCR (dqPCR), capable of identifying genomes of both viruses in a single assay, was employed to determine the viral loads of these agents in commercially available vaccines. Thirty five vaccines from eight manufacturers (32 prepared with live and 3 with inactivated microorganisms) were examined. Genomes of CAV were detected as contaminants in 6/32 live vaccines and in 1/3 inactivated vaccines. The CAV genome loads ranged from 6.4 to 173.4 per 50 ng of vaccine DNA (equivalent to 0.07 to 0.69 genome copies per dose of vaccine). Likewise, AGV2 genomes were detected in 9/32 live vaccines, with viral loads ranging from 93 to 156,187 per 50 ng of vaccine DNA (equivalent to 0.28–9176 genome copies per dose of vaccine). These findings provide evidence for the possibility of contamination of poultry vaccines with CAV and AGV2 and they also emphasize the need of searching for these agents in vaccines in order to ensure the absence of such potential contaminants.     

Evaluation of the analytical sensitivity of a polymerase chain reaction assay for the detection of chicken infectious anemia virus in avian vaccines

Chicken infectious anemia virus (CAV) is a ubiquitous pathogen of chickens causing significant disease in commercial flocks worldwide. During CAV outbreaks, the Center for Veterinary Biologics requires manufacturers of veterinary biologicals to test materials derived from infected flocks for extraneous CAV by polymerase chain reaction (PCR). The analytical sensitivity of a PCR assay for detection of CAV was determined and the applicability of a CAV DNA standard as a positive control for assay validity was evaluated. The analytical sensitivity of the CAV PCR assay was assessed to be 100 copies per reaction for the DNA standard and 1 × 10^1.9 TCID₅₀/reaction for infectious virus. Establishing the analytical sensitivity of this CAV PCR assay and the inclusion of internal and external positive controls for validity provide a basis for determining whether suspect materials are safe for use in the production of veterinary biologics.     

Identification of the first human gyrovirus, a virus related to chicken anemia virus

We have identified in a skin swab sample from a healthy donor a new virus that we have named human gyrovirus (HGyV) because of its similarity to the chicken anemia virus (CAV), the only previously known member of the Gyrovirus genus. In particular, this virus encodes a homolog of the CAV apoptin, a protein that selectively induces apoptosis in cancer cells. By PCR screening, HGyV was found in 5 of 115 other nonlesional skin specimens but in 0 of 92 bronchoalveolar lavages or nasopharyngeal aspirates and in 0 of 92 fecal samples.     

鸡贫血病毒哈尔滨分离株全基因克隆和序列分析

通过PCR方法克隆了从哈尔滨分离的一株鸡贫血病毒(CAV)的全基因,并对其进行了测序,该病毒基因组为环状,全长2298bp,含有三个互相重叠的开放读码框和一个调控区。将克隆的基因与GenBank收录的CAV基因比较,同源性至少为97%,未发现与本次克隆的CAV基因完全一致的分离株。与德国分离株Cuxla、26p4和马来西亚分离株分别有42、42和72个核苷酸不同,同源性分别为98.2%、98.2%和96.9%。与德国分离株Cux1b相比,除在调控区内少一个类似增强子的重复序列外,尚有39处核苷酸不同。它与分离于欧洲的几株CAV的亲源性要比来自亚洲的马来西亚株近。对CAV哈尔滨分离株、26p4、Cux1b、Cux1a和马来西亚株的VP1、VP2和VP3蛋白比较,VP2的保守性最高。     

Quantitative analytical technique applied to histopathology of birds infected experimentally by the virus of chicken anemia virus

This research was conducted on ten glass slides selected from the histopathology evaluation chickens. Five slides of control's chickens healthy and five slides of chickens infected experimentally with chicken anemia virus (CAV slide) between one and twenty-one days post infection (PI), they were analyzed in magnifications of 200x and 400x. Histopathology showed severe bone marrow hypoplasia to complete aplasia, fully depletion of the erythrocytic and granulocytic series, both accompanied by space occupying adipocytic replacement. Foci of erythropoietic hyperplasia with intense mielopoietic activity, some hemocytoblast increased of size, with large nucleus. A quantitative analytical technique by Positive Pixel Count Algorithm was applied. It demonstrated that measures area stained of control slides were higher than CAV slides (Average: 61% vs. 25%, respectively). So, the control slides showed strong positivity, due to the presence of bigger quantity of cells of erythrocytic and granulocytic series. The CAV slides of seven days PI had high positivity (average: 94%), it was explained because the chicken anemia virus takes place severe lesions between ten to seventeen days PI, after 21 days PI the cellular regeneration is observed that is evidenced by means of focuses of erythroblastoid cells hyperplasia. This technique demonstrates in a quantitative way the severe decrease of the cellular components of the bone marrow in presence of the infection for CAV, supporting with numeric data the histology image evaluated by the pathologist. Therefore, it can be used as support to the histopathology of field samples to evaluate the presence of lesions taken place by CAV and this way to improve the quality and efficiency of the veterinary pathology services.     

Epidemiology of chicken anemia virus in Central African Republic and Cameroon

ABSTRACT: BACKGROUND: Although chicken anemia virus (CAV) has been detected on all continents, little is known about this virus in sub-Saharan Africa. This study aimed to detect and characterize CAV for the first time in Central African Republic and in Cameroon. RESULTS: An overall flock seroprevalence of 36.7% was found in Central African Republic during the 2008--2010 period. Virus prevalences were 34.2% (2008), 14.3% (2009) and 10.4% (2010) in Central African Republic and 39% (2007) and 34.9% (2009) in Cameroon. CAV DNA was found in cloacal swabs of 76.9% of seropositive chickens, suggesting that these animals excreted the virus despite antibodies. On the basis of VP1 sequences, most of the strains in Central African Republic and Cameroon belonged to 9 distinct phylogenetic clusters at the nucleotide level and were not intermixed with strains from other continent. Several cases of mixed infections in flocks and individual chickens were identified. CONCLUSIONS: Our results suggest multiple introductions of CAV in each country that later spread and diverged locally. Mixed genotype infections together with the observation of CAV DNA in cloacal samples despite antibodies suggest a suboptimal protection by antibodies or virus persistence.     

A comparison of complete genome sequences of a rabies virus chinese isolate SH06 with the vaccine strains

In this study, we determined the complete nucleotide and deduced amino acid sequence of a primary isolate of rabies virus (SH06) obtained from the brain of a rabid dog. The overall length of the genome was 11 924 nucleotides. Comparison of the genomic sequence showed the homology of SH06 at nucleotide level with full-length genomes of reference vaccine strains ranged from 82.2% with the PV strain to 86.9% with the CTN strain. A full-length genome-based phylogenetic analysis was performed with sequences available from GenBank. Phylogenetic analysis of the complete genome sequences indicated that the SH06 exhibited the highest homology with rabies street virus BD06 and CTN vaccine strain originated from China.     

Complete genome sequence of an avian leukosis virus isolate associated with hemangioma and myeloid leukosis in egg-type and meat-type chickens

Subgroup J avian leukosis virus (ALV-J) was first isolated from meat-type chickens that developed myeloid leukosis (ML). In recent years, field cases of hemangioma (HE) or HE and ML, rather than ML alone, have been reported in commercial layer flocks exposed to ALV-J with a high incidence in China. Here we report the complete genomic sequence of an ALV-J isolate that caused both HE and ML in egg-type and meat-type chickens in China. These findings will provide additional insights into the molecular characteristics in genomes, host range, and pathogenicity of ALV-J.     

<Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> as a live vehicle for oral immunization against chicken anemia virus

The AcmA binding domains of Lactococcus lactis were used to display the VP1 protein of chicken anemia virus (CAV) on Lactobacillus acidophilus. One and two repeats of the cell wall binding domain of acmA gene were amplified from L. lactis MG1363 genome and then inserted into co-expression vector, pBudCE4.1. The VP1 gene of CAV was then fused to the acmA sequences and the VP2 gene was cloned into the second MCS of the same vector before transformation into Escherichia coli. The expressed recombinant proteins were purified using a His-tag affinity column and mixed with a culture of L. acidophilus. Whole cell ELISA and immunofluorescence assay showed the binding of the recombinant VP1 protein on the surface of the bacterial cells. The lactobacilli cells carrying the CAV VP1 protein were used to immunize specific pathogen-free chickens through the oral route. A moderate level of neutralizing antibody to CAV was detected in the serum of the immunized chickens. A VP1-specific proliferative response was observed in splenocytes of the chickens after oral immunization. The vaccinated groups also showed increased levels of Th1 cytokines interleukin (IL)-2, IL-12, and IFN-γ. These observations suggest that L. acidophilus can be used in the delivery of vaccines to chickens.     

Identification of a novel GC-rich 113-nucleotide region to complete the circular, single-stranded DNA genome of TT virus, the first human circovirus

The sequence data (H. Okamoto et al., Hepatol. Res. 10:1-16, 1998) of a newly discovered single-stranded DNA virus, TT virus (TTV), showed that it did not have the terminal structure typical of a parvovirus. Elucidation of the complete genome structure was necessary to understand the nature of TTV. We obtained a 1.0-kb amplified product from serum samples of four TTV carriers by an inverted, nested long PCR targeted for nucleotides (nt) 3025 to 3739 and 1 to 216 of TTV. The sequence of a clone obtained from serum sample TA278 was compared with those registered in GenBank. The complete circular TTV genome contained a novel sequence of 113 nt (nt 3740 to 3852 [=0]) in between the known 3'- and 5'-end arms, forming a 117-nt GC-rich stretch (GC content, 90.6% at nt 3736 to 3852). We found a 36-nt stretch (nt 3816 to 3851) with an 80.6% similarity to chicken anemia virus (CAV) (nt 2237 to 2272 of M55918), a vertebrate circovirus. A putative SP-1 site was located at nt 3834 to 3839, followed by a TATA box at nt 85 to 90, the first initiation codon of a putative VP2 at nt 107 to 109, the termination codon of a putative VP1 at nt 2899 to 2901, and a poly(A) signal at nt 3073 to 3078. The arrangement was similar to that of CAV. Furthermore, several AP-2 and ATF/CREB binding sites and an NF-kappaB site were arranged around the GC-rich region in both TTV and CAV. The data suggested that TTV is circular and similar to CAV in its genomic organization, implying that TTV is the first human circovirus.     

A single chicken anemia virus protein induces apoptosis.

Chicken anemia virus (CAV) causes cytopathogenic effects in chicken thymocytes and cultured transformed mononuclear cells via apoptosis. Early after infection of chicken mononuclear cells, the CAV-encoded protein VP3 exhibits a finely granular distribution within the nucleus. At a later stage after infection, VP3 forms aggregates. At this point, the cell becomes apoptotic and the cellular DNA is fragmented and condensed. By immunogold electron microscopy VP3 was shown to be associated with apoptotic structures. In vitro, expression of VP3 induced apoptosis in chicken lymphoblastoid T cells and myeloid cells, which are susceptible to CAV infection, but not in chicken embryo fibroblasts, which are not susceptible to CAV. Expression of a C-terminally truncated VP3 induced much less pronounced apoptosis in the chicken lymphoblastoid T cells.     

Complete Genome Sequence of a J Subgroup Avian Leukosis Virus Isolated from Local Commercial Broilers

Subgroup J avian leukosis virus (ALV-J) isolate GDKP1202 was isolated from a 50-day-old local yellow commercial broiler in the Guangdong province of China in 2012. Here we report the complete genomic sequence of the GDKP1202 isolate, which caused high mortality, serious growth suppression, thymic atrophy, and liver enlargement in commercial broilers. A novel potential binding site (5′-GGCACCTCC-3′) for c-myb was identified in the GDKP1202 genome. These findings will provide additional insights into the molecular characteristics in the genomes and pathogenicity of ALV-J.     

Chicken anemia virus VP2 is a novel dual specificity protein phosphatase

The function of viral protein 2 (VP2) of the immunosuppressive circovirus chicken anemia virus (CAV) has not yet been established. We show that the CAV VP2 amino acid sequence has some similarity to a number of eukaryotic, receptor, protein-tyrosine phosphatase (PTPase) alpha proteins as well as to a cluster of human TT viruses within the Sanban group. To investigate if CAV VP2 functions as a PTPase, purified glutathione S-transferase (GST)-VP2 fusion protein was assayed for PTPase activity using the generalized peptide substrates ENDpYINASL and DADEpYLIPQQG (where pY represents phosphotyrosine), with free phosphate detected using the malachite green colorimetric assay. CAV GST-VP2 was shown to catalyze dephosphorylation of both substrates. CAV GST-VP2 PTPase activity for the ENDpYINASL substrate had a V(max) of 14,925 units/mg.min and a K(m) of 18.88 microm. Optimal activity was observed between pH 6 and 7, and activity was specifically inhibited by 0.01 mm orthovanadate. We also show that the ORF2 sequence of the CAV-related human virus TT-like minivirus (TLMV) possessed PTPase activity and steady state kinetics equivalent to CAV GST-VP2 when expressed as a GST fusion protein. To establish whether these viral proteins were dual specificity protein phosphatases, the CAV GST-VP2 and TLMV GST-ORF2 fusion proteins were also assayed for serine/threonine phosphatase (S/T PPase) activity using the generalized peptide substrate RRApTVA, with free phosphate detected using the malachite green colorimetric assay. Both CAV GST-VP2 and TLMV GST-ORF2 fusion proteins possessed S/T PPase activity, which was specifically inhibited by 50 mm sodium fluoride. CAV GST-VP2 exhibited S/T PPase activity with a V(max) of 28,600 units/mg.min and a K(m) of 76 microm. Mutagenesis of residue Cys(95) to serine in CAV GST-VP2 abrogated both PTPase and S/T PPase activity, identifying it as the catalytic cysteine within the proposed signature motif. These studies thus show that the circoviruses CAV and TLMV encode dual specificity protein phosphatases (DSP) with an unusual signature motif that may play a role in intracellular signaling during viral replication. This is the first DSP gene to be identified in a small viral genome.     

Identification of a hypervariable region in the long terminal repeat of equine infectious anemia virus.

An avirulent, field-derived isolate of equine infectious anemia virus (EIAV), designated MA-1, was molecularly cloned, and the complete nucleotide sequence was determined for the 3' half of the viral genome. Comparisons between MA-1 and the prototype Wyoming strain of EIAV identified a 66-nucleotide stretch between CAAT (-91) and TATAA (-25) in the U3 region of the long terminal repeat, where sequence divergence was as high as 39.3%. The polymerase chain reaction was used to amplify and clone long terminal repeat sequences from Th-1, the in vivo parental stock of MA-1. Results indicated that the nucleotide sequences of MA-1 and Th-1 clones were less variable than was observed between MA-1 and Wyoming. However, MA-1 and Th-1 markedly differed in the types of enhancer sequences located in the hypervariable region. These results suggest that variation in lentivirus regulatory sequences may be important in EIAV host cell tropism and pathogenesis.