Investigation of the unstable attenuation exhibited by a chicken anaemia virus isolate.

An attenuated chicken anaemia virus (CAV) isolate, cloned isolate 10, which was molecularly cloned from the Cuxhaven-1 CAV after 173 cell-culture passages, was shown previously to recover pathogenicity following 10 passages in young chicks. The consensus nucleotide sequence of the 'revertant' (Rev) virus, present as a tissue homogenate, differed from cloned isolate 10 at a single nucleotide residue (nucleotide 1739) that changed amino acid 287 of the capsid protein from alanine to aspartic acid. Subjecting Rev virus to 10 cell-culture passages reselected viruses with an alanine at this amino acid position. Experimental infections using a molecularly cloned Rev virus isolate demonstrated that the mutation at nucleotide 1739 was not in itself responsible for the recovery of pathogenicity exhibited by the Rev virus. Additional sequence analyses of cloned amplicons provided evidence that the Rev virus population comprised minor, genetically different subpopulations, and provided an indication of CAV's potential for genetic change.     

Antigenicity and pathogenicity characteristics of molecularly cloned chicken anaemia virus isolates obtained after multiple cell culture passages

Summary.  The Cux-1 isolate of chicken anaemia virus (CAV), which had received 310 (P310) cell culture passages, was substantially less pathogenic than virus that had been passaged 13 times (P13). Molecularly cloned virus isolates, selected from the P310 and P13 virus populations using recombinant DNA cloning and transfection procedures, reacted differently with 4 CAV-specific monoclonal antibodies (MAbs), which had been raised to low-passage Cux-1 virus. In contrast to the strong immunofluorescence (IF) reactivities exhibited by all P13 cloned isolates tested, 80% and 57% of the P310 cloned isolates reacted weakly with MAbs 2A9 and 4H4, which are directed against conformational epitopes on the capsid protein, VP1. Sequence analysis of the VP1 coding regions possessed by ten P310 and two P13 cloned isolates showed that 6 amino acid changes within VP1 had been selected by multiple-cell culture passage. One of these at position 89 in VP1 appeared to be crucial for determining reactivity with MAb 2A9. Of nine P310 cloned isolates evaluated, 8 were substantially attenuated compared to the low-passage Cux-1 virus pool. It is concluded that the individual virus variants comprising the P310 virus pool differ with regards to their antigenicity and pathogenicity. Accepted April 18, 1999 Received November 19, 1998     

Pathogenicity,sequence and phylogenetic analysis of <Emphasis Type="Italic">Malaysian Chicken anaemia virus</Emphasis> obtained after low and high passages in MSB-1 cells

Summary. Specific-pathogen-free (SPF) chickens inoculated with low passage Chicken anaemia virus (CAV), SMSC-1 and 3-1 isolates produced lesions suggestive of CAV infection. Repeated passages of the isolates in cell culture until passage 60 (P60) and passage 123 produced viruses that showed a significantly reduced level of pathogenicity in SPF chickens compared to the low passage isolates. Sequence comparison indicated that nucleotide changes in only the coding region of the P60 passage isolates were thought to contribute to virus attenuation. Phylogenetic analysis indicated that SMSC-1 and 3-1 were highly divergent, but their P60 passage derivatives shared significant homology to a Japanese isolate A2.     

Pathogenicity of chicken anaemia virus (isolate 10343) for young and older chickens.

One-day-old chicks, inoculated intramuscularly (i.m.) with the chicken anaemia virus (CAV) isolate 10343, showed depression of body weight gain and anaemia, particularly between days 14 and 21 post-inoculation (p.i.)- The weights of thymus and bursa were substantially reduced compared to controls at days 14 and 21 p.i. The histological lesions detected in thymus, bursa, spleen and liver were similar in frequency at days 14 and 21 p.i. Eosinophilic intranuclear inclusion bodies, lymphocyte depletion, and focal necrosis were detected in the thymus, spleen, bursa and liver of more than 50% of the inoculated chicks at days 14 and 21 p.i. Focal necrosis and vacuolar degeneration in the liver, as well as apoptosis in different organs were more evident at days 14 and 21 p.i. Ten-week-old broiler breeders, inoculated i.m. with isolate 10343 showed pathological changes that were less severe than the changes shown by 1-day-old chicks. No anaemia could be detected in this group. However, severe thymus atrophy, and histological lesions in bursa, spleen, and liver, were also evident at days 14 and 21 p.i. in some of the inoculated birds. Viral detection by immunofluorescence using a monoclonal antibody revealed a wide distribution of the CAV isolate. CAV antigen was detected until day 21 p.i. in thymus, spleen, bursa and liver. According to the severity of the lesions shown by 1-day-old chicks, the length of the period in which CAV antigen could be detected in tissues, and the fact that CAV isolate 10343 was capable of inducing disease in 10-week-old chickens, it seems that this CAV isolate may be particularly virulent.     

Investigation of the transfection capability of cloned tandemly-repeated chicken anaemia virus DNA fragments

Summary Chicken anaemia virus (CAV) is an icosahedral virus, 25 nm in diameter, which, on the basis of its circular single-stranded DNA genome, has recently been classified in the family,Circoviridae. We have investigated whether infectious, monomeric CAV DNA from recombinant plasmids containing tandemly-repeated CAV replicative form (RF) DNAs, following transfection, was generated by homologous recombination or a replicational release mechanism involving rolling circle replication (RCR) of DNA. Experiments designed to locate the virus strand origin of RCR and/or sites of recombination were performed by sequence analyses of hybrid viruses generated after transfection with cloned tandemly-repeated RFs specified by the sequence-distinct Cux-1 and 26P4 isolates. Positive transfection results obtained from 2 recombinant plasmid constructs were shown to have resulted from homologous recombination occurring at different sites within the RF sequence. Three of 5 hybrid viruses analysed were circularised within the same 105bp sequence, that contains four 19bp repeats and with which promoter/enhancer activity has been associated. This region may represent a novel origin or recombination hot-spot within the CAV genome. A distinctive cruciform-loop structure within the non-coding region was shown to contain an S1 nuclease-sensitive site, detected in CAV RF and in recombinant plasmids containing RF inserts.     

Effect of multiple cell culture passages on the biological behaviour of chicken anaemia virus.

The Cux-1 isolate of chicken anaemia virus (CAV) was passaged over 320 times in Marek's disease virus transformed chicken lymphoblastoid (MDCC-MSB1) cells. Comparison of the infectivity titres of virus pools derived from viruses that had received 0 (P0), 49 (P49), 170 (P170) and 320 (P320) passages in our laboratory indicated that the yields of infectious virus increased over 100-fold with passage number from P0 to P170. P320 exhibited unusual cell culture growth characteristics in that, unlike its lower passage counterparts, virus-specific immunofluorescence (IF) and cytopathic effect were detected at very low levels at 2 days post infection, with an additional passage of infected cells into fresh medium being required to produce high levels of infectious virus. Experimental infection of 1-day-old SPF chicks showed that P170 and P320 were substantially attenuated compared to P49 and a pathogenic, low-passage isolate used as control. When assessed by the indirect IF test, infection of 1-day-old chicks with the P49 and P170 isolates elicited similar levels of CAV-specific antibody to those elicited by infection with the pathogenic, low-passage virus and higher than those elicited by infection with the P320 isolate. Experimental infection of 5-week-old chicks indicated that the attenuated P170 and P320 isolates invoked similar CAV-specific antibody levels to those invoked by the pathogenic P49 and control isolates.     

Detection of chicken anaemia virus by DNA hybridization and polymerase chain reaction

A clone containing the complete genome of chicken anaemia virus (CAV) was used in hybridizations with DNA from various field isolates of CAV. CAV DNA from all field isolates was detected in a polymerase chain reaction with oligonucleotides derived from the sequence of the cloned CAV DNA as primers. By way of Southern blot analysis with (32)P-labelled DNA probes derived from cloned CAV DNA, all field isolates were shown to contain DNA molecules of about 2.3 kb, i.e. the size of cloned CAV DNA. In a dot-blot assay it was demonstrated that non-radioactively-labelled cloned CAV DNA hybridized specifically to DNA from field isolates. The cloned CAV DNA is highly similar to the DNA of field isolates, as borne out by restriction-enzyme mapping. We conclude that our cloned CAV genome is representative for CAV in the field. The described PCR and hybridization techniques, may, therefore, be used for research and diagnosis of CAV infections.     

Experimental evaluation of an inactivated vaccine against chicken anaemia virus.

An assessment of the performance of an inactivated chicken anaemia virus (CAV) vaccine following administration to specific-pathogen-free (SPF) breeder hens before the egg production period is reported. Antibodies to CAV in adult hens and maternally-derived antibodies to CAV in the progeny chicks were assayed by ELISA. Progeny of vaccinated chickens showed sufficient levels of maternally-derived antibody to CAV for up to 3 to 4 weeks of age. Following challenge at 1, 10, 20 and 30 days of age of chicks from CAV-vaccinated and non-vaccinated hens with virulent CAV, haematocrit levels and weights of thymus in vaccinates remained within normal ranges. Chicks derived from non-vaccinated hens developed anaemia and thymus atrophy following challenge. These results support the view that vaccination of breeder flocks with an inactivated vaccine to CAV could be an effective means of control of CAV-induced clinical disease.     

Effects of chicken anaemia virus and infectious bursal disease virus-induced immunodeficiency on infectious bronchitis virus replication and genotypic drift

We followed changes in a portion of the S1 gene sequence of the dominant populations of an infectious bronchitis virus (IBV) Arkansas (Ark) vaccine strain during serial passage in chickens infected with the immunosuppressive chicken anaemia virus (CAV) and/or infectious bursal disease virus (IBDV) as well as in immunocompetent chickens. The IBV-Ark vaccine was applied ocularly and tears were collected from infected chickens for subsequent ocular inoculation in later passages. The experiment was performed twice. In both experiments the dominant S1 genotype of the vaccine strain was rapidly and negatively selected in all chicken groups (CAV, IBDV, CAV + IBDV and immunocompetent). Based on the S1 genotype, the same IBV subpopulations previously reported in immunocompetent chickens and named component (C) 1 to C5 emerged both in immunocompetent and immunodeficient chickens. During the first passage different subpopulations emerged, followed by the establishment of one or two predominant populations after further passages. Only when the subpopulation designated C2 became established in either CAV-infected or IBDV-infected chickens was IBV maintained for more than four passages. These results indicate that selection does not cease in immunodeficient chickens and that phenotype C2 may show a distinct adaptation to this environment. Subpopulations C1 or C4 initially became established in immunocompetent birds but became extinct after only a few succeeding passages. A similar result was observed in chickens co-infected with CAV + IBDV. These results suggest that the generation of genetic diversity in IBV is constrained. This finding constitutes further evidence for phenotypic drift occurring mainly as a result of selection.     

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 1,766 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.     

Quantification of chicken anaemia virus by competitive polymerase chain reaction

A quantitative method for chicken anaemia virus (CAV) was developed using competitive polymerase chain reaction (PCR). Competitive template was constructed by deletion of 33 nucleotides from a wildtype DNA clone of CAV. Quantification of CAV DNA molecules by the competitive PCR was rapid and highly reproducible when compared with conventional infectivity titration methods. The ratios of the viral DNA molecules and infectivity titres in MDCC-MSB1 cells varied between 1.3 and 3.55 log10 among several isolates, suggesting the existence of different infection efficiencies to MDCC-MSB1 cells by isolates. The competitive PCR will be useful for studying CAV infection in vivo and/or in vitro.     

Characterisation of a chicken anaemia virus variant population that resists neutralisation with a group-specific monoclonal antibody

Summary.  A variant population of chicken anaemia virus (CAV), termed P310 2A9-resist, that resists neutralisation by the monoclonal antibody (MAb) 2A9, was selected from Cux-1 virus that had been passaged 310 times (P310) in MDCC-MSB1 cells. Substantially higher concentrations of MAb 2A9 were required to neutralise the selected virus compared to those required to neutralise a low-passage (P13) Cux-1 isolate. Virus neutralisation tests showed that serum from chickens infected with the P310 2A9-resist virus neutralised P13 virus and that serum from chickens infected with P13 virus conversely neutralised the P310 2A9-resist virus. MDCC-MSB1 cells infected with the P310 2A9-resist virus produced no staining with low dilutions (1:100) of Mab 2A9 in an indirect immunofluorescence (IF) test, whereas cells infected with P13 virus reacted positively at high MAb dilutions (1:80,000). Experimental infections of 1-day-old SPF chicks showed that the P310 2A9-resist virus was substantially attenuated. Chimaeric viruses constructed using PCR-amplified regions from the P310 2A9-resist and a pathogenic low-passage cloned Cux-1 isolate showed that the reduced MAb reactivity and attenuation exhibited by the P310 2A9-resist virus were mainly associated with a region encoding the N-terminal half of the 50 kDa capsid protein VP1 and C-terminal regions of VP2 and VP3. The nucleotide sequence of the protein-coding region of the P310 2A9-resist virus is reported and the amino acid sequences of the 3 encoded proteins compared with those of other Cux-1 isolates. Received June 19, 2000 Accepted October 24, 2000     

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.     

Replication of chicken anemia virus (CAV) requires apoptin and is complemented by VP3 of human torque teno virus (TTV)

To test requirement for apoptin in the replication of chicken anemia virus (CAV), an apoptin-knockout clone, pCAV/Ap(−), was constructed. DNA replication was completely abolished in cells transfected with replicative form of CAV/Ap(−). A reverse mutant competent in apoptin production regained the full level of DNA replication. DNA replication and virus-like particle (VLP) production of CAV/Ap(−) was fully complemented by supplementation of the wild-type apoptin. The virus yield of a point mutant, CAV/ApT¹⁰⁸I, was 1/40 that of the wild type, even though its DNA replication level was full. The infectious titer of CAV was fully complemented by supplementing apoptin. Progeny virus was free from reverse mutation for T¹⁰⁸I. To localize the domain within apoptin molecule inevitable for CAV replication, apoptin-mutant expressing plasmids, pAp1, pAp2, pAp3, and pAp4, were constructed by deleting amino acids 10-36, 31-59, 59-88 and 80-112, respectively. While Ap1 and Ap2 were preferentially localized in nuclei, Ap3 and Ap4 were mainly present in cytoplasm. Although complementation capacity of Ap3 and Ap4 was 1/10 of the wild type, neither of them completely lost its activity. VP3 of TTV did fully complement the DNA replication and VLP of CAV/Ap(−). These data suggest that apoptin is inevitable not only for DNA replication but also VLP of CAV. The common feature of apoptin and TTV-VP3 presented another evidence for close relatedness of CAV and TTV.     

Identification of a new human DNA virus (TTV-like mini virus, TLMV) intermediately related to TT virus and chicken anemia virus

Summary.  TT virus (TTV) is the only known human virus with single-stranded circular DNA, with a possible but yet unclear relationship to chicken anemia virus (CAV) of the family Circoviridae. Here we report a new human virus resembling TTV and CAV, designated TTV-like mini virus (TLMV). This non-enveloped virus was smaller (>30 nm) but had a similar density (1. 31–1.34 g/ml in CsCl) to TTV, when a TLMV/TTV-coinfected plasma was analyzed. Full-length sequencing revealed that the TLMV genome was a circular DNA comprising 2860 nt (isolate CBD231); significantly shorter than TTV (TA278, 3852 nt) but longer than CAV (CAECUX1, 2319 nt). A strand-specific hybridization assay using oligonucleotide-coated beads suggested TLMV was negative-stranded, like TTV and CAV. In genomic organization, TLMV was similar to both TTV and CAV. The untranslated region of TLMV resembled CAV in that both had direct repeats, whereas the sequence homology was more evident between TLMV and TTV. The predicted ORF1 protein of TLMV was rich in R/W/F residues at its amino terminus; the richness in W was shared by TTV, F by CAV, and R by both. ORF2 proteins of the three viruses had a common motif, WX₇HX₃CXCX₅H. Thus, TLMV is an intermediate between the remotely related TTV and CAV. Since CAV differs much from other circoviruses, it may better be classified together with TTV and TLMV under a new family: we would coin the Paracircoviridae. Accepted November 19, 1999/Received September 13, 1999     

Effect of chemicals on the infectivity of chicken anaemia virus

None of five commercial disinfectants, invert soap, amphoteric soap, orthodichlorobenzene, iodine disinfectant and sodium hypochlorite, was completely effective in destroying the infectivity of chicken anaemia virus (CAV) in liver material at 5% concentration. However, the iodine disinfectant and sodium hypochlorite completely inactivated the virus in tissue culture (TC) material when used at 1% concentration. CAV was resistant to organic solvents such as methyl alcohol, ethyl alcohol, acetone and chloroform. Beta-propiolactone and glutaraldehyde inactivated CAV. Fumigation with formaldehyde for 24 h only partly inactivated both liver and TC materials. It is presumed very hard to disinfect CAV in poultry facilities.     

Avian encephalomyelitis following oral vaccination

Outbreaks of clinical avian encephalomyelitis (AE) which developed after vaccination of 14-week-old birds by the oral route are reported. Mortality during weeks 2 to 5 following vaccination reached 2%. Experimental studies showed that, in contrast to popular opinion, AE vaccine virus given orally can spread to the central nervous system and produce encephalomyelitis (mild). The severity of the vaccine induced lesions was not affected by chicken anaemia virus (CAV) infection 2 weeks before AE vaccination. It is postulated that non-CAV-induced immunosuppression allowed vaccine virus to produce the severe lesions and deaths occurring during the disease outbreaks, and that in one case, Marek's disease virus was the immunosup-pressive agent.