Analysis of chicken anemia virus genome: evidence of intersubtype recombination
Eltahir et al. Virology Journal 2011, 8:512
http://www.virologyj.com/content/8/1/512
RESEARCH
Open Access
Analysis of chicken anemia virus genome:
evidence of intersubtype recombination
Yassir M Eltahir1,3, Kun Qian1, Wenjie Jin1 and Aijian Qin1,2*
Abstract
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.
Background
Chicken anemia virus (CAV) was first reported in 1979 in
specific-pathogen-free (SPF) chickens [1]. CAV belongs to
the Circoviridae and is a non-enveloped, icosahedral virus
with a negative-sense, single-stranded circular DNA. The
viral genome consists of 2.3 kb, with three partially overlapping open reading frames. CAV infection is an economically important clinical and subclinical disease in broiler
chickens, with a worldwide distribution [2].
CAV isolates show extremely limited genetic variability
worldwide [3]. All isolates of CAV are suspected to belong
to a single serotype [4]. Little is known about CAV genome recombination analysis. CAV putative intergenotype
recombinants have been reported to occurs in the virus
gene VP1 and results in a new virus genotype [5].
Recently, after adding more CAV approximately full
genome sequences to GenBank, CAV sequences arising
from different parts of world have been categorized into
four genotype groups (A-D) and five subtypes (A1, A2,
A3, D1 and D2) [6]. Therefore, the necessity of exploring
these CAV genotypes for evidence of recombination as
an important tool for genetic variability has been raised,
* Correspondence:
1
Ministry of Education Key Lab for Avian Preventive Medicine, Yangzhou
University, Yangzhou, 225009, PR China
Full list of author information is available at the end of the article
to establish if any would help in understanding the evolutionary process in the CAV genome.
Here, we report evidence of intersubtype recombination
based on sequence analysis of the entire coding regions
(VP1, VP2 and VP3) of CAV genomes.
Methods
Samples
DNA extraction, PCR screening, amplification of CAV
genome, cloning and sequencing were carried out as previously described, briefly, primers VP1F: 5’AGCCGACCCCGAACCGCAAGAA’3 and VP1R: 5’ TCA GGG
CTG CGT CCC CCA GTA CA’3 were used to amplify
the VP1 region and primers VP2F: 5’ GCG CAC ATA
CCG GTC GGC AGT’3 and VP2R: 5’ GGG GTT CGG
CAG CCT CAC ACT AT’3 were used to amplify the VP2
region [6] for 10 spleen samples collected between April
2010 and December 2010 at Yangzhou University Veterinary Hospital during necropsy. Samples originated from different commercial chicken farms in Anhui (n = 3) and
Jiangsu (n = 7) provinces. For each samples, DNA extraction and PCR was run at least twice. Animal experiments
were conducted in accordance with the guidelines provided by the Chinese Council on Animal Care. All experiments complied with institutional animal care guidelines
© 2011 Eltahir et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Eltahir et al. Virology Journal 2011, 8:512
http://www.virologyj.com/content/8/1/512
Page 2 of 7
JS-China 72
JS-China 85
China|HQ872030
China|HQ872029
JS-China 96
China|HQ872034
China|HQ872031
China|HQ872032
AN-China 95
USA|L14767
India|AY583758 CAV-P
India|AY583756 CAV-B
Bangladesh|AF395114
Malaysia|AY150576 BL-5P90
Malaysia|AF527037
Netherland|D10068
China|AF475908 Harbin
Malaysia|AF390038
UK|U66304
Germ any|M81223|Cuxhaven 1
India|AY583755 CAV-A
Germ any|M55918|CAECUX1
China|HQ872023
USA|AF311892
USA|AF313470 Del Ros
Malyasia |AY040632
Japan|AB031296
J
Malyasia|AF390102
China|AY843527 TJBD33
China|HQ872044
China|HQ872046
China|HQ872045
JS-China 81
AN-China 13
JS-China 80
JS-China 3
China|HQ872041
China|HQ872025
China|AY846844TJBD40
China|HQ872039
China|HQ872040
Japan|AB046590
China|HQ872033
China|HQ872035
China|HQ872038
China|HQ872036
China|HQ872047
Japan|D31965
China|HQ872028
China|HQ872042
China|HQ872043
China|HQ872026
China|HQ872027
USA|AF311900
JS-China 90
AN-China 92
China|HQ872024
China|HQ872037
Japnan|AB027470 TR20
India|AY583757 CAV-E
Austrlaia|U65414
Japan|AB119448
Malaysia|AF285882 SMSC-1
Australia|AF227982
Australia|EF683159
A1
A2
A3
B
D1
D2
C
Figure 1 Phylogenetic analysis of the nucleic acid sequence of the 10 new complete VP1, VP2 and VP3 sequences from Anhui (AN)
and Jiangsu (JS) provinces, China, and the 55 relevant VP1, VP2 and VP3 sequences currently available in GenBank. Values ≥ 70 are
indicated on the branches (as percentages). Sequences from the present study (colored closed symbols) are named as PP-China, where PP is the
area of origin. Sequences from GenBank were given the country name followed by accession number. The four major groups were identified as
A, B, C and D.
Eltahir et al. Virology Journal 2011, 8:512
http://www.virologyj.com/content/8/1/512
and were approved by University of Yangzhou Animal
Care Committee (protocol number 06R015).
Sequence analysis
To look for recombination in CAV sequences, we used 55
published full genomes sequences that are currently available in GenBank, together with 10 sequences characterized
in the present study. Multiple alignments of either CAV
full or subgenomic regions were performed and analyzed
using ClustalW [7]. Unrooted phylogenetic trees were
constructed using the neighbor-joining (NJ) method, and
visualized and edited using MEGA 3.1 software [8]. The
evolutionary distances were estimated using the Kimura
two-parameter method. Bootstrap analyses were performed with 1,000 repeat samples of the data sets. To
reduce redundancy, isolates with previous reports of intergenotypic recombination [5] were excluded from analysis.
Identification of recombination
Recombination breakpoint events in the multiple alignments were detected with the Recombination Detection
Program 3 (RDP3), using the automated suite of algorithms implemented in the RDP3 with default settings.
These included RDP, GENECONV (...truncated)
