Detection and sequence analysis of avian polyomavirus and psittacine beak and feather disease virus from psittacine birds in Taiwan

Avian polyomavirus (APV) and psittacine beak and feather disease virus (PBFDV) are the most common viral diseases of psittacine birds. In Taiwan, however, the existence of these viruses in psittacine birds has not been established. Polymerase chain reaction (PCR) methodology was therefore employed to ascertain whether APV and PBFDV genomes were present in isolates from psittacine birds of Taiwan. A total of 165 psittacine birds belonging to 22 genera were examined between 2002 and 2005. Findings revealed an APV-positive rate of 15.2%, a PBFDV-positive rate of 41.2%, and an APV/PBFDV dual infection rate of 10.3%. After cloning and sequencing, sequences of the PCR products were compared with sequences obtained from GenBank. For APV, the nucleotide identity among VP1 and t/T antigen coding regions ranged from 97.5% to 100% and 97.6% to 100%, respectively. For PBFDV, the nucleotide identity of ORF V1 and ORF C1 sequences ranged from 92.2% to 100% and 83.3% to 100%, respectively. The derived amino acid sequence alignment for PBFDV ORF V1 fragments revealed the conservation of two replication motifs and of the nucleotide binding site motif. In PBFDV, six of 42 deduced positions in the ORF C1 amino acid sequence were considered hypervariable. The established phylogenetic trees based on the four genome fragments examined in this study did not allow the assignment of particular APV or PBFDV nucleotide sequences to distinct avian species.     

Changes in VP2 gene during the attenuation of very virulent infectious bursal disease virus strain Gx isolated in China

Very virulent (vv) infectious bursal disease virus (IBDV) Gx strain with high pathogenicity was attenuated through replication in specific-pathogen-free (SPF) chicken embryos and in chicken embryo fibroblast (CEF) cell cultures. The changes in VP2 nucleotide and the deduced amino acid sequences were obtained during attenuation of vvIBDV in CEF culture. Sequence analysis of selected passages from numbers 0 to 20 in CEFs (designated here Gx to CEF-20) showed that no changes were detectable in the VP2 gene before CEF-7. There were a few changes in the nucleotide sequence of the VP2 gene but no amino acid substitutions at CEF-8. The virus of CEF-9 was an intermediate with some amino acid changes that possibly were related to virulence. CEF-10 virus had become similar to CU-1 strain. The VP2 gene sequence remained the same from CEF-10 to CEF-20. The results of pathogenicity tests showed that the mortalities of Gx, CEF-5, CEF-8, and CEF-9 in 4-wk-old SPF chickens were 64%, 60%, 60%, and 32%, respectively; whereas CEF-10, CEF-15, and CEF-20 were nonpathogenic. Virus neutralization tests with Gx strain showed that the antigenicities are similar from Gx to CEF-20.     

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.     

兔出血症病毒JL株分离鉴定及VP60基因主要抗原表位分析

采用血凝试验、电镜观察、RT－PCR方法分离鉴定了1株JL株兔出血症病毒（RHDV），扩增衣壳蛋白VP60基因，将扩增片段克隆到pMDl8－T载体上，经酶切鉴定后测序。结果显示，VP60基因全长1740bp，编码580个氨基酸；JL株与其他RHDV分离株比较，核苷酸同源性为93．7％－99．2％，氨基酸同源性在97．3％－99．5％，在VP606个区中，A、B、D、F是稳定区，氨基酸变异多发生在衣壳蛋白C、E区，表明毒株具有高度保守性；将JL株与国内外标准株蛋白氨基酸变畀及其亲水性、柔性区、抗原区和表面结构进行比较分析，预测RHDV VP60细胞表位。     

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.     

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.     

A universal polymerase chain reaction for the detection of psittacine beak and feather disease virus.

A universal PCR assay was designed that consistently detected psittacine beak and feather disease virus (BFDV) in psittacine birds affected with psittacine beak and feather disease (PBFD) from different geographic regions across Australia. Primers within open reading frame 1 (ORF1) of the BFDV genome consistently amplified a 717 bp product from blood and/or feathers of 32 birds with PBFD lesions. The PCR did not amplify a product from the feathers or blood from 7 clinically normal psittacine birds. Primers based on regions outside of ORF1 did not consistently produce a PCR product, suggesting there was some genomic variation outside ORF1. The amplified ORF1 PCR products of 10 BFDV isolates, from different psittacine species and from various regions around Australia, were cloned and comparative DNA sequence analysis demonstrated 88-99% of the ORF1 fragments. The derived amino acid sequences of the amplified ORF1 fragments demonstrated similar identity between all 10 isolates. Within ORF1, there was complete conservation of the putative nucleotide binding site and marked conservation of 2 other motifs previously identified as essential components of the replication-associated proteins of other circoviruses and geminiviruses.     

Molecular characterization of a 13-amino acid deletion in VP1 (1D) protein and novel amino acid substitutions in 3D polymerase protein of foot and mouth disease virus subtype A/Iran87

The nucleotide sequence of the VP1 (1D) and partial 3D polymerase (3D^pol) coding regions of the foot and mouth disease virus (FMDV) vaccine strain A/Iran87, a highly passaged isolate (~150 passages), was determined and aligned with previously published FMDV serotype A sequences. Overall analysis of the amino acid substitutions revealed that the partial 3D^pol coding region contained four amino acid alterations. Amino acid sequence comparison of the VP1 coding region of the field isolates revealed deletions in the highly passaged Iranian isolate (A/Iran87). The prominent G-H loop of the FMDV VP1 protein contains the conserved arginine-glycine-aspartic acid (RGD) tripeptide, which is a well-known ligand for a specific cell surface integrin. Despite losing the RGD sequence of the VP1 protein and an Asp₂₆→Glu substitution in a beta sheet located within a small groove of the 3D^pol protein, the virus grew in BHK 21 suspension cell cultures. Since this strain has been used as a vaccine strain, it may be inferred that the RGD deletion has no critical role in virus attachment to the cell during the initiation of infection. It is probable that this FMDV subtype can utilize other pathways for cell attachment.     

Genetic diversity in twenty variants of the avian polyomavirus.

To determine if different pathotypes of the avian polyomavirus (APV) exist and to compare the genomes of APVs originating from different geographic areas, dates, and species of birds, the partial sequences of 18 APVs were determined. New viral sequences were compared with three published APV sequences. Two of the new viruses had identical sequences. Forty point mutations were found at 31 loci. A 27-bp deletion was found in the VP2 and VP3 open reading frames of one virus. A duplication of the putative origin of replication and adjacent enhancer region was previously reported in one APV. Smaller duplications involving the origin in one APV and a second enhancer region in another were discovered. All duplications were in tissue culture-adapted viruses, suggesting they occurred during the isolation process. Excluding duplications and the deletion, maximum variation between viruses was small (11 bp). A maximum parsimony tree was constructed that contained three major branches. The three earliest isolates were on separate branches. The European viruses were confined to branch I, but APVs from the United States were on all three branches. Lovebird, budgerigar, and macaw APVs were also on each of the three branches, suggesting that species-specific pathotypes have not developed. Most nonsynonymous mutations occurred in a small portion of the VP2 and VP3 open reading frames, demonstrating a selection for these mutations. That a glycine at VP2 221 will inhibit virus replication in chicken embryo fibroblasts (CEFs) has been previously reported. In contrast, six of seven of the new APVs isolated in CEFs had a glycine at VP2 221.     

Sequence analysis of the protein-coding regions of foot-and-mouth disease virus O/HK/2001

The nucleotide sequence of the protein-coding region of foot-mouth-disease virus (FMDV) strain O/HK/2001 was determined and compared with the sequences of other FMDVs that were registered in GenBank. The protein-coding region was 6966 nucleotides in length and encoded a protein of 2322 amino acid residues. Comparison of the nucleotide sequence and its deduced amino acid sequence with those of other isolates indicated that O/HK/2001 belonged to the Cathay topotype. A genomic coding region nucleotide sequence phylogenetic tree of several FMDV-O isolates showed that O/HK/2001 was most closely related to FMDV isolates found in Taiwan during 1997, and especially shared significant similarity to HKN/2002, suggesting that the virus causing outbreaks in Hong Kong was genetically most-closely related to that causing an outbreak of type O in Taiwan. Mutations in O/HK/2001 were revealed, including frequent substitutions in the VP1 and L proteins, and deletions involving 10 amino acid residues in the 3A protein. This study was undertaken to assess the regional variation of prevalent FMDV type O viruses and to establish a sequence database for FMDV molecular epidemiological investigation.     

猪圆环病毒2型江西株的全基因组克隆和序列分析

为了解江西地区猪圆环病毒2型（PCV-2）的流行和进化情况,根据GenBank上已发表的PCV-2全基因序列设计1对引物,PCR扩增后得到9条PCV-2全基因序列,并对其全基因序列核苷酸和蛋白序列进行分析,绘制遗传进化树。结果表明,江西地区流行的9株PCV-2中,基因组序列全长分为8株1 767 bp和1株1 768 bp,9株PCV-2的核苷酸同源性为94.7%~99.9%,与GenBank己发表的PCV-2分离株全基因组同源性介于94.3%~99.8%之间,而9株PCV-2的ORF1核苷酸序列同源性为96.9%~100.0%。ORF2和ORF3编码的蛋白氨基酸序列存在部分位点突变。遗传进化树显示为3种基因型：5株PCV-2b、3株PCV-2d、1株PCV-2a。本研究有助于江西地区PCV-2的监测和防制。     

Cloning and Sequence Analysis of Complete Genomes of Porcine Circovirus Type 2 (PCV-2) Jiangxi Strains

In order to understand the epidemiology and evolution of PCV-2 in Jiangxi province, a pair of primers was designed according to the PCV-2 gene sequence published in GenBank. After PCR amplification, we got the whole genome sequence of 9 strains PCV-2 isolates, and the nucleotide and protein sequences were analyzed, the genetic evolutionary tree was constructed. The results showed that the complete genome of 8 out of the 9 strains were 1 767 bp in length and one strain was 1 768 bp. By analyzing the whole genome sequences of the nucleotide,the homology of nucleotide sequences of the 9 strains was 94.7% to 99.9%.Compared with other whole genome sequences of PCV-2 in GenBank, the homology was 94.3% to 99.8%. The homology of nucleotide sequences of the ORF1 of the 9 strain was 96.9% to 100.0%. ORF2 and ORF3 encoding protein amino acid sequence had some locus mutation. Phylogenetic tree analysis showed that the 9 strains could be divided into 3 genotypes,5 strains belonged to PCV-2b, 3 strains belonged to PCV-2d, and 1 strain belonged to PCV-2a. This study was helpful to monitor and control of PCV-2 in Jiangxi province.     

Liver virome of a Little Corella (Cacatua sanguinea) reveals coinfection with a novel parvovirus and two beak and feather disease viruses

Emerging diseases are acknowledged as a growing threat to wildlife, with the continued identification of pathogenic and potentially pathogenic viruses in avian species resulting from ongoing advances in molecular diagnostic techniques. Parvoviruses under the genus Chaphamaparvovirus (subfamily Hamaparvovirinae) are highly divergent. The detection and characterisation of parvoviruses in psittacine birds is limited. This study reports a novel parvovirus, tentatively named psittaciform chaphamaparvovirus 3 (PsChV-3) under the genus Chaphamaparvovirus, identified in an Australian free-ranging little corella (Cacatua sanguinea). The PsChV-3 genome is 4277 bp in length and encompasses four predicted open-reading frames, including two major genes, a nonstructural replicase gene (NS1), and a structural capsid gene (VP1). The NS1 and VP1 genes showed the closest amino acid identities of 78.8% and 69.7%, respectively, with a recently sequenced psittaciform chaphamaparvovirus 2 from Australian Neophema species grass parrots. In addition, the presence of two complete novel beak and feather disease (BFDV) genomes, 1993 and 1868 nt in length, respectively, were detected from the same bird. Both these BFDV genomes contained two bidirectional ORFs encoding the putative Rep and Cap proteins. Phylogenetic analysis showed that the sequenced novel BFDV genomes clustered in a distinct subclade with other BFDVs isolated from Australian cockatoos. This study contributes to the characterisation chaphamaparvoviruses and BFDV in Australian parrots and supports the need for ongoing monitoring and molecular studies into the avian virome in native Australian psittacine bird species.     

Two fiber genes of nearly equal lengths are a common and distinctive feature of Fowl adenovirus C members

The complete genome or the genome region containing the two fiber genes of two reference strains and one field isolate representing both serotypes of Fowl adenovirus C were sequenced. Two fiber genes were revealed in the genomes of all three isolates. Fiber-1 and fiber-2 genes of several Fowl adenovirus C isolates were sequenced as well. Both serotypes 4 and 10 have two fiber genes. The genome region containing the fiber gene was also sequenced for the reference strain of Fowl adenovirus B. Just one fiber gene was revealed in this strain. Predicted amino acid sequences were compared to already published fiber sequences of different adenovirus isolates and one amino acid substitution within fiber-2 was detected in all of the Fowl adenovirus C isolates that were isolated from chickens with hepatitis-hydropericardium syndrome in comparison to apathogenic isolates. Phylogenetic analyses provided insights about the evolution of fiber genes in avian adenoviruses and their genetic relationships.     

2006年至2010年山东省鸡传染性支气管炎病毒分子变异的研究

为研究山东省鸡传染性支气管炎病毒(IBV)的遗传变异规律,本研究2006年～2010年从山东省发病的商品鸡中分离鉴定了17株IBV,并对其S1基因、N基因和M基因分别进行RT-PCR扩增、测序及遗传进化分析.序列分析结果表明:与疫苗株H120相比,17个分离株S1蛋白的变异程度较大,存在广泛的基因突变和氨基酸替代,多数病毒株还存在氨基酸的插入;N蛋白无碱基的缺失和插入,仅存在核苷酸的突变和氨基酸的替代;M蛋白除病毒株CK/CH/SD09/005插入3个碱基外,其它16个分离株仅存在少数的碱基突变和氨基酸替代.S1基因、N基因和M基因的系统进化分析结果表明多数分离株的3个基因在进化上相对平行,与国内分离株LX4同属一个进化分支,同源性较高;分离株SDYT0605的3个基因与疫苗株H120同源性较高,可能是免疫压力下变异的疫苗株;分离株SDTA06111、SDWF0608和CK/CH/SD09/005的S1基因、N基因和M基因分属于不同的进化分支,可能发生了基因重组.本研究结果显示基因突变、插入和不同基因之间的重组是免疫压力下IBV变异的主要方式.     

A single subtype of avian pneumovirus circulates among Minnesota turkey flocks.

The recent emergence of avian pneumovirus (APV) infection among US turkey flocks has resulted in a major economic threat to the turkey industry. In order to elucidate the molecular epidemiology of APV, comparative sequence analysis of the fusion (F) protein gene of APV was performed for 3 cell culture-adapted isolates and 10 APV positive clinical samples recovered from US turkey flocks. Relatively modest levels of nucleotide and amino acid sequence divergence were identified, suggesting the prevalence of a single lineage of APV among US turkey flocks. Additionally, numerous polymorphisms were identified that were only represented in the clinical samples but not in the in vitro propagated isolates of APV. Phylogenetic analyses confirm that the subtype of APV circulating in the upper Midwestern United States is evolutionarily related to, but distinct from, European APV subgroups A and B. Overall, the results of the present investigation suggest that there has been only a single recent introduction of APV into US turkey populations in the upper Midwestern United States.     

Molecular characterizations of avian polyomavirus isolated from budgerigar in China

Budgerigar fledgling disease is an acute viral infectious disease caused by avian polyomavirus (APV). In this study, 34 liver tissue samples of young, dead budgerigar with typical symptoms were collected in 2004. All the samples had positive polymerase chain reaction (PCR) test based on the VP1 specific primers. VP1 genes of these samples were sequenced and had high similarities to each other (99%-100%). A strain (HBYM02) was isolated and sequenced. As shown in the phylogenetic tree, there are two branches. One branch was composed by strains isolated from Passeriformes, and the other was composed only by one strain isolated from Falconiformes. The genome similarities between our isolate and other reported isolates were very high (> 99%), and the evolution distances in the phylogenetic tree were very short (< 0.005), which suggests that APV in China has the same genotype as those in other regions. The results will be useful for the diagnoses of, and vaccine development for, APV.     

禽流感病毒A/Ostrich/Denmark/72420/96(LPAI-H5N2)株HA全基因克隆与序列分析

为进一步分析禽流感病毒(AIV)H5N2分离株血凝素(HA)基因的特性,参照已发表H5亚型禽流感HA基因序列设计了1对引物,采用RT-PCR技术,以禽流感病毒A/Ostrich/Denmark/72420/96(D96)RNA为模板,扩增了HA全基因并进行核苷酸同源性比较,氨基酸编码分析,绘制系统发育进化树。结果表明,扩增片段长1737个核苷酸,包含了完整的HA基因的开放阅读框架,与Genbank已发表的H5N1和H5N2分离株的HA基因序列比较,发现与国内H5N1分离株同源性较低,只有80%左右,而与H5N2各株序列具有很高的同源性,最高达97.5%,印证了AIV基因组8个片段间频繁的重组及AIV高变异性的特点。推导的氨基酸序列分析表明,HA蛋白裂解位点上游丢失了4个连续碱性氨基酸(R-R-R-K),裂解位点处氨基酸序列为E-T-R,仅包含一个碱性氨基酸(R-)残基,符合低致病性毒株的特征,证明为低致病性毒株。其HA推导后氨基酸序列与H5N1AIV的同源性接近90%,以其研究的疫苗,可以有效抵御我国流行的H5亚型AIV病毒的感染,同时因为是弱毒株,以其研制的疫苗具有更好的安全性,也更符合公共卫生学的要求。