Chicken Anemia Virus Isolates Research Articles

Molecular evolution and pathogenicity of chicken anemia virus isolates in China.

Chicken infectious anemia (CIA), caused by chicken anemia virus (CAV), is an important immunosuppressive disease that seriously threatens the global poultry industry. Here, we isolated and identified 30 new CAV strains from CAV-positive flocks. The VP1 genes of these strains were sequenced and analyzed at the nucleotide and amino acid levels and were found to have very similar nucleotide sequences (>97% identity); however, they showed 93.9-100.0% sequence identity to the VP1 genes of 55 reference strains. Furthermore, alignment of the deduced amino acid sequences revealed some unique mutations. Phylogenetic analysis indicated the division of VP1 amino acid sequences into two groups (A and B) and four subgroups (A1, A2, A3 and A4). Interestingly, 22 of the newly isolated strains and some Asian reference strains belonged to the A1 group, whereas the remaining eight new isolates belonged to the A3 group. To evaluate the pathogenicity of the epidemic CAV strains from China, the representative strains CAV-JL16/8901 and CAV-HeN19/3001 and the reference strain Cux-1 were selected for animal experiments. Chickens infected with the isolates and reference strain all showed thymus atrophy and bone marrow yellowing. The mortality rates for CAV-JL16/8901, CAV-HeN19/3001, and the reference strain was 30%, 20%, and 0%, respectively, indicating that the epidemic strains pose a more serious threat to chickens. We not only analyzed the molecular evolution of the epidemic strains but also showed for the first time that the epidemic strains in China are more pathogenic than reference strain Cux-1. Effective measures should be established to prevent the spread of CIA in China.

Molecular characterization of VP2 and VP3 proteins of Chicken Anemia Virus isolates in Turkey

Chicken anemia virus (CAV) has a circular 2.3 kb DNA genome encoding VP1, VP2, and VP3 proteins. VP1 protein is assembled into viral capsid. VP2 is a nonstructural protein required for the correct assembly of VP1. Also, there is an interaction between VP2 and VP3 protein, known as apoptin. Mostly, VP1 protein of CAV isolates have been investigated in previous studies but the information about VP2 and VP3 proteins is insufficient. The aim of this study is molecular characterization of VP2 and VP3 proteins from 20 CAV isolates previously collected in different regions of Turkey using in silico tools. The open reading frames (ORFs) for VP2 and VP3 were determined to be 651 and 366 base pairs (bp) encoding 216 and 121 amino acids, respectively. Nucleotide substitutions were identified at 21 and 14 positions for VP2 and VP3, respectively. Three isolates had one apiece amino acid substitutions in VP2 sequences. The MW and pI values for VP2 were predicted as 24.08?24.138 kDa and 6.96?8.35, respectively. Moreover, seven isolates had one to three amino acid substitutions for VP3 protein. The MW and pI values for VP3 were calculated as 13.188-13.372 kDa and 9.44-9.95, respectively. The secondary structures were predicted and phylogenetic trees were constructed for VP2 and VP3 proteins. As a conclusion, molecular characterization of VP2 and VP3 proteins from 20 CAV isolates contributes to the current knowledge on these proteins having diverse functions and immunological potentials.

Epidemiology, molecular characterization, and recombination analysis of chicken anemia virus in Guangdong province, China.

Chicken anemia virus (CAV) causes severe anemia and immunosuppression in young chickens and a compromised immune response in older birds, resulting in great economic losses to the poultry industry worldwide. Here, we report the molecular epidemiology and characterization of CAV circulating in poultry in Guangdong province, China. Ninety-one of 277 chickens collected from 2016 to 2017 were CAV positive. Full-genome sequencing revealed the presence of eight separate strains. Phylogenetic analysis based on the genome sequences obtained in this study and related sequences available in the GenBank database showed that all of the CAV isolates exhibit a close relationship to each other and belong to the same genotypic group. Putative recombination events were also detected in the genomes of the newly isolated CAVs. Collectively, our findings underscore the importance of CAV surveillance and provide information that will lead to a better understanding of the evolution of CAV.

Full-Length Genomic Characterization of Chicken Anemia Virus in Turkey

ABSTRACT Chicken infectious anemia (CIA) is an immune-suppressive disease caused by chicken anemia virus (CAV). It is characterized by lymphoid atrophy, aplastic anemia, especially in chicks. In this study, full-length genomic characterization of CAV DNA from the broiler flocks in Turkey and phylogenetic analysis were aimed. In the study, CAV DNA were found positive for 37 (53%) flocks with PCR studies from thymus tissues of each 70 broiler flocks. And 17 purified CAV DNA PCR products from these 37 CAV isolates were full length sequenced with the NGS method (Illumina MiSeq). Also with the phylogenetic analyses, full length PCR products of 17 purified CAV isolates have been determined as 2298bp genome size and 99% similarity with each other. The highest similarity (99%) has been detected with the isolates from China and Taiwan. Furthermore, a 97-98% similarity has been detected with vaccine strains (Cux-1, 26P4 and Del Ros) and also 88-90 % similarity has been detected with GyV4 and GyV3 isolates. As a result, in the study full length genomic characterization of CAV DNA from the 7 regions of Turkey were determined. And also all Turkish CAV isolates and vaccine strains were in group 2 according to the phylogenetic tree were obtained. But these isolates and vaccine strains were not found in the same group with GyV3 and GyV4 strains. Besides, these CAV isolates were showed more similarity to the isolates reported from Taiwan and China than the vaccine strains.

Restriction endonuclease analysis of the genomes of different isolates of chicken anemia virus amplified by polymerase chain reaction

Four DNA fragments (fragments A, B, C and D) covering the whole genome of chicken anemia virus (CAV) were amplified enzymatically by polymerase chain reaction (PCR) using four pairs of oligonucleotide primers. The DNA fragments were amplified from each of nine CAV isolates including eight Malaysian isolates and one European isolate (Cux-1). For all nine CAV isolates, fragment A (1518 bp) was digested with one restriction enzyme, Eco130I (StyI); fragment B (926 bp) with three enzymes, Eco130I (StyI), HpaII and MboI separately; fragment C (675 bp) with also three enzymes, BsuRI (HaeIII), HinfI, and HpaII separately; and the fragment D (552 bp) with one enzyme, EcoRI. Enzyme digested products of different fragments were separated by agarose gel or polyacrylamide gel electrophoresis. Each of the eightenzymatic reactions differentiated at least two isolates except the HpaII digestion of fragment C where no isolate was distinguished. The overall restriction endonuclease (RE) analysis separated four isolates (BL-1, BL- 2, BL-4 and BL-5) in one group and the rest five isolates (SMSC-1, SMSC-2, 3-1, BL-3 and Cux-1) were differentiated from each other and also from the group of four isolates, based on the number of restriction site differences and the fragments generated by different enzymatic digestions. The study revealed that RE analysis could be used to identify and differentiate CAV isolates based on the number of restriction site differences. The study showed that more isolates, even the isolates from the same poultry farm, could be differentiated with proper genomic diversity after RE analysis of more genome fragments compared to that of single genome fragment.SAARC J. Agri., 15(2): 1-18 (2017)

Clinical Association of Chicken Anaemia Virus with Other Infectious Poultry Diseases in North India and Nepal: Its Pathological Studies, Molecular Epidemiology and RFLP Pattern of PCR Amplified Full Length Viral Genome

The aim of the study was to assess the role of chicken anaemia virus (CAV) in precipitating important poul- try diseases in commercial poultry flocks and its molecular surveillance in poultry flocks of North India (Uttarakhand and Uttar Pradesh states) and Nepal (Mahendra Nagar). Study was conducted during September-2012 to April-2013 on 13 randomly selected poultry flocks. Necropsy was performed for making tentative diagnosis of other concomitant diseases and samples were collected and epidemiological data was recorded. Flocks were screened for CAV by VP2 gene based PCR followed by PCR-RFLP analysis of viral genome. Haemato-biochemical parameters of flocks were also studied. Out of 185 tissue samples, PCR screening confirmed the presence of CAV genome in 95 samples, while 50 showed other viruses like infectious bursal disease (IBD), Inclusion Body Hepatitis (IBH) and Marek's Disease (MD). PCR-RFLP analysis of the full genome of different CAV isolates with Hin d III, Pst I, Nhe I and Mbo II re- striction enzymes showed indistinguishable restriction patterns. A significant decline in erythrocytic and leukocytic cell counts, and increase in the activities of degenerating enzymes was observed in CAV positive flocks. In conclusion, CAV was detected at different poultry farms of India and Nepal and its association with precipitation of other poultry diseases through its marked immunosuppressive potential is evident from findings of present study. Thus, the role of CAV in precipitating other infectious diseases in birds along with molecular epidemiology and immunosuppressive effects needs to be studied in detail so that a comprehensive strategy may be formulated for timely adoption of effective prevention and control measures, subsequently alleviating economic losses in poultry rearing countries.

Molecular characterization of chicken anemia virus circulating in chicken flocks in egypt.

Introduction. Although many previous studies reported detection of chicken anemia virus (CAV) in Egypt since 1990, genomic characterization of this circulating CAV has not been published. In the present study, four nucleotide sequences of detected CAV were genetically characterized. Methods. These nucleotide sequences were obtained from commercial chicken flocks in two different locations of Egypt during 2010. The target region for sequencing was 675 bp nucleotide of partial coding region of VP1 protein. The nucleotide and deduced amino acid sequences of the detected CAV were aligned and compared to worldwide CAV isolates including commonly used vaccine strains. Phylogenetic analysis of these sequences was also carried out. Results. Our results showed that all the Egyptian CAV sequences were grouped in one group with viruses from diverse geographic regions. This group is characterized by amino acids profile 75I, 97L, 139Q, and 144Q in VP1. The phylogenetic and amino acid analyses of deduced amino acid indicated that the detected CAV sequences differ from CAV vaccine strains. Conclusion. This is the first report that describes molecular characterization of circulating CAV in Egypt. The study showed that the detected CAV, in Egypt are field viruses and unrelated to vaccine strains.

Phylogenetic analysis and genetic characterization of chicken anemia virus isolates from Cambodia

Three chicken anemia viruses (CAV) were detected by PCR during screening of field samples from village chickens collected in Cambodia in 2011/2012. Nearly full-length VP1 viral structural protein genes (nt 1-1,293) from the 3 CAV were sequenced and characterized. Phylogenetic analysis revealed that all 3 of the Cambodian CAV were clustered with CAV strains belonging to genotype II and were most closely related to CAV strains from Guangdong province, China. On the amino acid level, major substitutions were observed at 12 residues in the VP1 protein (positions 22, 75, 97, 125, 139, 144, 254, 287, 290, 370, 376, and 413) when compared with published reference CAV strains. In motifs associated with virulence, all Cambodian CAV had virulence-associated motifs composed of 75I, 89T, 125I, 139Q, 141Q, 144Q, and 394Q, which are commonly found in highly virulent genotype II viruses and some genotype III viruses. This is the first report of CAV isolated from village chickens in Southeast Asia as well as Cambodia.

Molecular characterization of the chicken anaemia viruses isolated from broiler farms of west Azerbaijan, Iran

Chicken anaemia virus (CAV) is an economically important pathogen of chickens with worldwide distribution. CAV is the causative agent of chicken anaemia disease, causing severe anaemia, lymphoid atrophy and immunosuppression in young birds. In the present study, the genetic variation of circulating CAVs in west Azerbaijan broiler farms was investigated and compared with CAVs from other countries. Extracted viral DNA from livers of chickens positive for CAV (46 out of 100) was used and a fragment of the VP1 gene 1390 base pairs in size was amplified. The purified products were subjected to restriction fragment-length polymorphism (RFLP) using HinfI endonuclease and nucleotide sequencing. Four different RFLP patterns were identified from all examined CAV DNAs. Sequence analysis of the VP1 gene of isolated CAV viruses revealed a high genetic distance (0.5 to 4.7%) among CAV isolates. Phylogenetic analysis showed that CAVs isolated from Iranian poultry farms clustered with CAVs isolated from different parts of the world. It was concluded that the circulating CAVs in broiler farms of west Azerbaijan had a high genomic variation.

Molecular Characterization of Contaminating Infectious Anemia Virus of Chickens in Live Commercial Vaccines Produced in the 1990s

The presence of infectious chicken anemia virus (CAV) was detected in a previous study by nested-PCR as a contaminant in seven commercial vaccines, produced in the 1990s by three different manufacturers, prepared against the most relevant virus etiologies. In order to phylogenetically characterize the genome and compare it to CAV isolates from Brazil and other parts of the world, sequences of approximately 675 bp of the gene encoding the hypervariable region of VP1 protein of three CAV vaccine contaminant strains were studied. The CAV genome in contaminated vaccines showed high similarity (> 98.9%) with the Brazilian BR91/99 and Argentinian ArgA001028 (> 99%) strains. However, the comparison with the Cuxhaven-1 vaccine strain showed a lower identity of between 96.8% and 97.7%, and comparing it with the CAV26P4 vaccine strain showed an identity between 97.2% and 98.2%; both are available in Brazil. Such differences might be relevant for the highly conserved CAV genome. CAV contaminants were positioned in the same genetic group (clusters) with the Brazilian strain BR91/99 and Argentinian strain ArgA001028. Results indicated that the contamination of live vaccines by CAV may have influenced CAV epidemiology in the Brazilian and Argentinian poultry industry.

Complete Genome Sequence Analysis of a Recent Chicken Anemia Virus Isolate and Comparison with a Chicken Anemia Virus Isolate from Human Fecal Samples in China

A new isolate of chicken anemia virus (CAV) was designated GD-1-12. GD-1-12 was isolated from a 12-day-old commercial broiler in Guangdong province, China, in 2012. The GD-1-12 CAV caused high mortality, severe anemia, thymic atrophy, and subcutaneous hemorrhage in commercial broilers. Here, we report the complete genome sequence of GD-1-12 CAV and comparison with the complete genome sequence of another CAV that was isolated from human fecal samples in China (GenBank accession no. JQ690762). The genomes of the two CAV isolates shared high homology, although a deletion was identified by comparison. The findings from this study provide additional insights into the molecular characteristics of the CAV genomes and should advance knowledge for continuous monitoring and, perhaps, preventing the spread of the virus in chickens as well as in humans.

Improving the potency of DNA vaccine against Chicken Anemia Virus (CAV) by fusing VP1 protein of CAV to Marek's Disease Virus (MDV) Type-1 VP22 protein

BackgroundStudies have shown that the VP22 gene of Marek's Disease Virus type-1 (MDV-1) has the property of movement between cells from the original cell of expression into the neighboring cells. The ability to facilitate the spreading of the linked proteins was used to improve the potency of the constructed DNA vaccines against chicken anemia virus (CAV).MethodsThe VP1 and VP2 genes of CAV isolate SMSC-1 were amplified and inserted into eukaryotic co-expression vector, pBudCE4.1 to construct pBudVP2-VP1. We also constructed pBudVP2-VP1/VP22 encoding CAV VP2 and the VP22 of MDV-1 linked to the CAV VP1. In vitro expression of the genes was confirmed by using RT-PCR, Western blot and indirect immunofluorescence. The vaccines were then tested in 2-week-old SPF chickens which were inoculated with the DNA plasmid constructs by the intramuscular route. After in vivo expression studies, immune responses of the immunized chickens were evaluated pre- and post-immunization.ResultsChickens vaccinated with pBudVP2-VP1/VP22 exhibited a significant increase in antibody titers to CAV and also proliferation induction of splenocytes in comparison to the chickens vaccinated with pBudVP2-VP1. Furthermore, the pBudVP2-VP1/VP22-vaccinated group showed higher level of the Th1 cytokines IL-2 and IFN-γ.ConclusionsThis study showed that MDV-1 VP22 gene is capable of enhancing the potency of DNA vaccine against CAV when fused with the CAV VP1 gene.

Genome sequencing analysis of Brazilian chicken anemia virus isolates that lack MSB-1 cell culture tropism

Specific amino acid (aa) substitutions in VP1, VP2 and VP3 genes were reported as a distinctive feature of the American CIA-1 strain, characterized as having a variable rate of growth and tropism for different MSB-1 cell sublines [Renshaw RW, Soiné C, Weinkle T, O'Connell PH, Ohashi K, Watson S, et al. A hypervariable region in VP1 of chicken anemia virus mediates rate of spread and cell tropism in tissue culture. J Virol 1996;70(12):8872-8]. DNA sequencing of 878 nucleotides from twelve Brazilian CAV, eight of which tested for in vitro isolation in three different sources of MDCC-MSB1 cell line and identified as lacking capacity to propagate in any of these cells, were compared to sequence data available for CAV strains propagated or not in cell culture. Alignment of the deduced aa resulted in a lack of singled out amino acid substitutions in the partial genomic sequences of Brazilian isolates that would entirely contrast them to viruses propagated in MSB-1 cells, indicating that the combined VP1, VP2 and VP3 substitutions observed may not entirely account as sole determinants of CAV isolation and propagation in MDCC-MSB-1 cells.

Biological and Molecular Characterization of Chicken Anemia Virus Isolates from Slovenia

The presence of chicken anemia virus (CAV) in Slovenia was confirmed by inoculation of 1-day-old chickens without antibodies against CAV and isolation of the virus on the Marek's disease chicken cell-MSB1 line and by polymerase chain reaction (PCR). Experimental inoculation of 1-day-old chickens resulted in lower hematocrit values, atrophy of the thymus, and atrophy of bone marrow. CAV was confirmed by PCR in the thymus, bone marrow, bursa of Fabricius, liver, spleen, ileocecal tonsils, duodenum, and proventriculus. The nucleotide sequence of the whole viral protein (VP)1 gene was determined by direct sequencing. Alignment of VP1 nucleotide sequences of Slovenian CAV isolates (CAV-69/00, CAV-469/01, and CAV-130/03) showed 99.4% to 99.9% homology. The VP1 nucleotide sequence alignment of Slovenian isolates with 19 other CAV strains demonstrated 94.4% to 99.4% homology. Slovenian isolates shared highest homology with the BD-3 isolate from Bangladesh. Alignment of the deduced VP1 amino acids showed that the Slovenian isolates shared 100% homology and had an amino acid sequence most similar to the BD-3 strain from Bangladesh (99.6%) and were 99.1% similar to the G6 strain from Japan and the L-028 strain from the United States. The Slovenian isolates were least similar (96.6%) to the 82-2 strain from Japan. A phylogeneric analysis on the basis of the alignment of the VP1 amino acids showed that CAV isolates used in the study formed three groups that indicated the possible existence of genetic groups among CAV strains. The CAV isolates were grouped together independent of their geographic origin and pathogenicity.

Biological and molecular characterization of chicken anaemia virus isolates of Indian origin

In the present study, four chicken anaemia virus (CAV) isolates (CAV-A, -B, -E and -P) recovered from different geographical regions of India were characterized. CAV genome of 1766 bp nucleotide region containing the complete coding region of VP2 and VP3 proteins, and partial coding region of VP1 protein were sequenced. The nucleotide and deduced amino acid sequence of the Indian CAV isolates were aligned and compared with CAV isolates of European, Asian, American and Australian origin. Phylogenetic analysis of the Indian CAV isolates were also carried out based on the nucleotide and deduced amino acid sequences. The results indicated that Indian isolates were genetically evolved from different parts of the world. Indian isolate, CAV-A was found closely related to European Cux-1 strain, CAV-B and -P were closely related to Bangladesh BD-3 strain and CAV-E was closely related to Australian 704 strain. The pathogenicity of the four CAV isolates was studied in day-old specific pathogen free (SPF) chicks. Day-old SPF chicks ( n = 50) were divided into five groups comprised of 10 chicks in each group. Group 1 was kept as control and groups 2–5 were infected with each CAV isolate separately. The chicks were infected at a dose rate of 1 ml cell culture fluid (10 4.5 TCID 50/0.1 ml) per bird intramuscularly. The clinical signs, mortality and packed cell volume (PCV) and body weight gain were recorded on 5, 10 and 15 days post-infection. At 15th day, all the birds were sacrificed and various organs, viz., thymus, bone marrow, spleen, liver and bursa were examined for gross and microscopic changes. The pathogenicity study indicated that all the CAVs except CAV-B were able to produce clinical disease and immunosuppression in young chicks whereas the isolate CAV-B produced no clinical disease but only induced immunosuppression, which was revealed by microscopic examination of the lymphoid organs. The study showed valuable information on molecular epidemiological status of CAV isolates prevalent in India for the first time.

Pathogenesis of Chicken Anemia Virus: Comparison of the Oral and the Intramuscular Routes of Infection

The events during the pathogenesis of chicken anemia virus (CAV) infection following intramuscular (IM) and oral inoculation were further elucidated and compared by sequential clinical, pathologic, and morphometric histopathologic evaluations, and by sequential determination of CAV genome concentrations in different organs. Specific-pathogen-free chickens were inoculated by IM or oral routes with the same dose (2 x 10(6) mean tissue culture infective dose [TCID50]) of CAV isolate 03-4876 at 1 day of age. Weights and hematocrits were obtained at 7, 10, 14, 18, 21, 25, and 28 days postinoculation (DPI). Seven birds from each group were necropsied at 7, 10, 14, and 28 DPI, and samples of thymus, Harderian gland, and cecal tonsils (CT) were obtained for histopathologic examination and CAV genome quantification by real-time polymerase chain reaction. Peak CAV genome concentrations were detected in the thymus at 10 and 14 DPI in the IM and orally infected chickens, respectively. High CAV DNA concentrations were maintained throughout the experimental period until 28 DPI, despite specific seroconversion occurring by 14 DPI in the IM-inoculated chickens. CAV was isolated from both orally and IM-infected chickens 28 DPI. Peak CAV genomes in the thymuses of IM and orally infected chickens coincided with peak lymphocyte depletion in these organs. Lymphocyte repopulation of the thymus occurred by 28 DPI in spite of the presence of the virus in the organs of both infected chicken groups. CAV genomes were detected in the CT, but histopathologic changes were not observed. Compared with the IM route of infection, orally infected chickens did not show apparent signs of illness. Clinical parameters, including reduction of weight gains and hematocrits, and gross and histopathologic changes were delayed and less severe in the orally inoculated chickens. This was concurrent with a delay in accumulation of CAV genomes in the thymus of these chickens.

Chicken anemia virus induced apoptosis: underlying molecular mechanisms

In 1990, the chicken anemia virus (CAV) genome was cloned by us and proven to be representative for CAV isolates worldwide. This genome contains unique promoter/enhancer replication elements and genes. Upon infection of its target cells, CAV replicates via a double-stranded (ds) DNA intermediate. From this ds CAV molecule, a single mRNA is transcribed, which encodes for three distinct proteins VP1, VP2, and VP3 or apoptin. Its capsid contains only the VP1 protein. However, for the production of the neutralizing epitope, co-synthesis of VP1 and VP2 are needed. CAV genomes with mutations in the 12 bp insert of the promoter/enhancer region were shown to produce immunogenic functional CAV particles. Mutations in these and other regulatory elements of CAV might also decrease its virus load resulting in a reduced pathogenic effect. CAV causes fatal cytopathogenic effects in e.g. chicken thymocytes via apoptosis. Under in vitro conditions, CAV replicates only in transformed chicken cell lines, which indicates that at least a part of the CAV life-cycle requires transformed-like cellular events. In these transformed cell lines, the synthesis of the apoptin protein alone mimics the CAV-induced apoptosis, whereas the VP2 protein also harbors some apoptotic activity. Extensive studies on apoptin resulted in the characterization of domains essential for its apoptotic activity and nuclear localization, which seems to be related with its ability to induce apoptosis. Therefore, both VP2 and apoptin are of interest in reducing the pathogenicity of CAV infections. A series of biomedical studies on apoptin have been carried out in human cell systems, which are informative about the mechanism of CAV-induced apoptosis in chicken (transformed) cells. Synthesis of apoptin alone induces apoptosis in various human transformed and/or tumorigenic cell lines, but not in normal human diploid cells. A striking difference in the cellular localization of apoptin was observed in human normal diploid cells versus tumor cells. In all tumor cells, apoptin is located mainly in the heterochromatic regions of the nucleus, whereas in normal cells it is present in peri-nuclear structures. Apoptin contains a bipartite nuclear localization signal, and one domain that resemble a nuclear export signal. Elucidation of parts of the apoptin-induced apoptotic pathway revealed unique characteristics: apoptin-induced apoptosis is independent of the tumor suppressor p53. The anti-apoptotic protein Bcl-2 does not inhibit but even accelerates apoptin-induced apoptosis in tumor cells, whereas over expression of Bcl-2 in normal cells has no effect on the apoptin activity. Upstream caspases are not involved, whereas downstream caspase 3 is, but seems not to be essential. A number of novel proteins were shown to interact with apoptin in transformed cells. Future studies of apoptin, VP2 and related cellular proteins in chicken cells will unravel the regulatory aspects of CAV-induced apoptosis.

Molecular basis of the attenuation exhibited by molecularly cloned highly passaged chicken anemia virus isolates.

Chimeric virus experiments indicated that the pathogenicity and monoclonal antibody reactivity differences between two molecularly cloned, highly passaged chicken anemia virus isolates could be attributed to the VP1 amino acid change at residue 89. The introduction of this change into a pathogenic cloned low-passage isolate was not sufficient to cause attenuation.

Isolation, identification and characterization of chicken anaemia virus in Malaysia.

A study was conducted to isolate and identify chicken anaemia virus (CAV) from field samples of clinically infected broiler chickens in Malaysia. A total of 125 samples were collected from chickens aged 2-6 weeks with clinically depressed and retarded growth, of which five samples were found positive to CAV directly by polymerase chain reaction (PCR). Later, five isolates of CAV from the respective five PCR positive samples were isolated in MDCC-MSB1 cells at passage 4 based on cytopathic effects, PCR and indirect immunofluorescent antibody test. The isolates were identified as BL-1, BL-2, BL-3, BL-4 and BL-5. These CAV isolates were found to resist treatment with chloroform and heat at 37 degrees C for 2 h, 56 degrees C for 30 min and 70 degrees C for 5 min. One of the isolates, BL-5 produced significant reduction (p < 0.001) of hematocrit values (9-19%), pale bone marrow, thymus atrophy and haemorrhages in skin/muscle when inoculated into 1-day old SPF chickens. Restriction enzyme digestion of 926 bp genomic fragments of all the isolates including Cux-1 isolate with HindIII exhibited a similar pattern of bands in 2% agarose gel. The present findings confirmed the presence of CAV in Malaysia.

Genetic Characterization of Chicken Anemia Virus from Commercial Broiler Chickens in Alabama

Chicken anemia virus (CAV) isolates show extremely limited genetic variability worldwide. We determined the nucleotide sequence of an 823-nucleotide portion of the 2.3-kb CAV genome found in 10 liver and/or spleen specimens of Alabama 29-to-49-day-old commercial broiler chickens exhibiting lymphocyte depletion of the thymus submitted to the state diagnostic laboratory because of problems unrelated to anemia. We determined the nucleotide sequence directly from DNA isolated from tissues, without isolation of virus in culture. This procedure enabled us to characterize CAV that might not have replicated in culture and avoided the potential for changes during passage. Results confirmed the limited genetic variability of CAV. All sequences were identical in 93% of nucleotide positions. The sequences encoded only two distinct VP1 hypervariable regions, and both had been found previously in other CAV isolates. A novel amino acid, glutamine, was found at VP1 position 22 in half the sequences, replacing the histidine residue encoded by most previously characterized CAV genomes. We were able to distinguish among CAV genomes with different codons at VP1 amino acid 22 and different hypervariable regions by restriction endonuclease analysis of polymerase chain reaction products.