Taxonomic updates for the genus Gyrovirus (family Anelloviridae): recognition of several new members and establishment of species demarcation criteria.

The genus Gyrovirus was assigned to the family Anelloviridae in 2017 with only one recognized species, Chicken anemia virus. Over the last decade, many diverse viruses related to chicken anemia virus have been identified but not classified. Here, we provide a framework for the classification of new species in the genus Gyrovirus and communicate the establishment of nine new species. We adopted the 'Genus + freeform epithet' binomial system for the naming of these species.

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.

The diseases suspected of the involvement of chicken anemia virus infection in 11 to 14-weeks old replacement pullets from eastern Japan: a case report.

Three strains of chicken anemia virus (CAV) were detected in 11 to 14-weeks old chickens, showing depression, wasting, and increased mortality, from three farms in eastern Japan. Another strain was detected in 12-weeks old chickens from one farm without clinical signs. Bacterial infections were suggested in three farms with clinical signs and its involvement in the occurrence of the diseases might be suspected. Sequence analysis of the VP1, VP2, and VP3 genes of four CAV strains revealed that the three from farms with clinical signs belonged to genotype A2, whereas that from the apparently-normal farm belonged to A3. This may be a rare case report about the diseases suspected of the involvement of the CAV infection in older birds.

Chicken infectious anaemia virus infections in chickens in northern Vietnam: epidemiological features and genetic characterization of the causative agent.

Since the first report of chicken infectious anaemia virus (CIAV) in Vietnam in 2013, there have not been many studies focused on the detection of CIAV or the molecular characteristics of the virus. This study attempted to investigate the presence of CIAV in northern Vietnam by molecular-based methods. Regarding the spatial distribution of CIAV, the PCR-based results showed that CIAV was detected in 47 out of 64 farms (73.4%) and in all 10 investigated provinces. Of the 119 samples assayed by PCR, 74 (62.2%) tested positive for CIAV DNA. By arranging the samples into different categories, it was found that CIAV was detected at high rates (above 50%) based on all 4 evaluated criteria as follows: production type of chicken, housing system, flock size and age group. Different housing systems were significantly associated with the detection rates of CIAV (P = 0.003). By genetic analyses, all of the Vietnamese CIAVs were found to (i) lack substitutions related to attenuation substitutions, (ii) group separately from vaccine-like CIAVs and (iii) belong to genogroups G2 and G3 of CIAV. Because of the wide distribution of CIAV and because the virus was confirmed not to be vaccine-like viruses, it is suggested that further studies be conducted on the clinical form of chicken infectious anaemia, as well as the immunosuppressive effect of CIAV on chickens in Vietnam.RESEARCH HIGHLIGHTS Wide distribution of chicken infectious anaemia virus (CIAV) in northern Vietnam.Vietnamese CIAVs belong to genogroups G2 and G3 of CIAV.

Chicken anemia virus in northern Vietnam: molecular characterization reveals multiple genotypes and evidence of recombination.

Chicken anemia virus (CAV) has a ubiquitous and worldwide distribution in the chicken production industry. Our group previously reported a high seroprevalence of CAV in chickens from northern Vietnam. In the present study, tissue samples collected from a total of 330 broiler and breeder commercial chickens in eleven provinces of northern Vietnam were tested for CAV infection. All samples were collected from clinically suspected flocks and diseased birds. The CAV genome was detected in 157 out of 330 (47.58%) chicken samples by real-time PCR. The rate of CAV genome detection in young chickens at 2-3 weeks of age (61.43%), which had not been previously reported in Vietnam, was significantly higher than that in older chickens at 4-11 (44.83%) and 12-28 (35.71%) weeks of age. For nine representative CAV strains from broiler chickens, analysis of the entire protein-coding region of the viral genome was conducted. Phylogenetic analysis of the VP1 gene indicated that the CAVs circulating in northern Vietnam were divided into three distinct genotypes: II, III, and V. Only one of the nine Vietnamese CAV strains clustered with a vaccine strain (Del-Ros), whereas the other eight strains did not cluster with any vaccine strains. Among the three genotypes, genotype III was most widely found in northern Vietnam and this included three sub-genotypes (IIIa, IIIb, and IIIc). The Vietnamese CAV strains were closely related to the Chinese, Taiwanese, and USA strains. One strain was defined to be of genotype V, which is a newly reported CAV genotype. Moreover, recombination analysis suggests that this novel genotype V was generated by recombination between genotype II and sub-genotype IIIc.

Chicken anemia virus: A deadly pathogen of poultry.

Chicken infectious anemia (CIA) is an immunosuppressive disease that causes great economic loss in poultry industry globally. This disease is caused by chicken anemia virus (CAV), an icosahedral and single-stranded DNA virus that is transmitted both vertically and horizontally. CAV, which belongs to the genus Gyrovirus has been reported in human, mouse and dog feces. Rapid identification of different strains of gyrovirus with high similarity to CAV has heightened public concern on this virus. Clinical symptoms of this disease such as intramuscular hemorrhage, weight loss, anemia and bone marrow aplasia are prominent in young chickens, while adult chickens experience subclinical symptoms. Biosecurity measures such as good management practice and vaccination have been the most reliable control strategy against this virus. Therefore, this study reviews the current state of CAV under the following subheadings (i) Chicken anemia virus (ii) Pathogenesis of CAV (iii) Serological evaluation of host antibodies to CAV (iv) Association of Marek's disease and infectious bursa disease with CAV infection (v) Genetic diversity and phylogenetics of CAV strains (vi) Current and future vaccine strategy in the control of CAV. In conclusion, improvement on DNA and recombinant vaccines strategy could curtail the economic impact of CAV on poultry birds. Keywords: adjuvant; CAV; chicken; disease.

Characterization of full genome sequences of chicken anemia viruses circulating in Egypt reveals distinct genetic diversity and evidence of recombination.

Chicken anemia virus (CAV) is one of the commercially important diseases of poultry worldwide. In Egypt, CAV has been reported to be a potential threat to the commercial poultry sectors. Hence, this study was aimed at isolation and full genomic analysis of CAVs circulating in chicken populations in different geographical location in Egypt. A total of 42 samples were collected from broiler chicken flocks in 9 governorates in Egypt from 12 to 42 days of age. The mortality rate observed among chickens was ranging from 3% to 22%. Nineteen out of 42 farms were found positive for the CAV genome by polymerase chain reaction (PCR). Full genome sequencing was conducted for 18 positive samples. Genetic analysis revealed a high similarity of >99% in 11 viruses with the vaccine strain Del-Ros; while the other seven samples shared close similarity to CAV field strains isolated from China, Taiwan, and Brazil. The data also indicated Q139 and Q144 amino acids substitutions among the VP1 of Egyptian field strains, which are known to be important in virus replication and spread. Phylogenetic analysis of the sequenced viruses (n = 18) based on either the full gene nucleotide sequence or VP1 coding sequence, suggested the circulation of four distinct genotypes in Egypt designated as group A, B, C and D. Moreover, evidence of recombination was detected among four Egyptian CAVs located within group A. The findings of this study succeeded to elucidate the epidemiological and genetic features of CAVs circulating in Egypt, and underscores the important of CAVs surveillance.

Co-infection of fowl adenovirus with different immunosuppressive viruses in a chicken flock.

In poultry, fowl adenovirus (FAdV) and immunosuppressive virus co-infection is likely to cause decreased egg production, inclusion body hepatitis, and pericardial effusion syndrome. In this study, fowl adenovirus infection was found in parental and descendent generations of chickens. We used quantitative polymerase chain reaction (PCR) and dot blot hybridization to detect the infection of reticuloendotheliosis (REV), avian leukosis virus (ALV), and chicken infectious anemia virus (CIAV) in 480 plasma samples. The test samples were 34.58% FADV-positive, 22.29% REV-positive, 7.5% CAV-positive, and 0.63% ALV-positive. Sequence analysis showed that FADV belonged to serotype 7, which can cause inclusion body hepatitis. The ALV strain was ALV-A, in which the homology of gp85 gene and SDAU09C1 was 97.3%. The positive rate was lower because of the purification of avian leukemia, whereas the phylogenetic tree analysis of REV showed that the highest homology was with IBD-C1605, which was derived from a vaccine isolate. Through pathogen detection in poultry we present, to our knowledge, the first discovery of fowl adenovirus type 7 infection in parental chickens and found that there was co-infection of FAdV and several immunosuppressive viruses, such as the purified ALV and CIAV. This indicates that multiple infection of different viruses is ever-present, and more attention should be given in the diagnosis process.

Molecular characterization of chicken anemia virus outbreaks in Nagpur province, India from 2012 to 2015.

Chicken anemia virus (CAV) is one of the important poultry pathogen. CAV infection can cause immunosuppression, aggravation of co-infections, vaccination failures and mortality. We are reporting the CAV outbreaks from the Nagpur province of India, between the years 2012-2015. The breeds included cockerel and Black Australorp of age varying from 29 to 50 days. The mortality rate observed among poultry was from 20% to 62.5%. Clinical symptoms like anemia, subcutaneous hemorrhages, growth retardation, abnormal feathers and hind limb paralysis suggested CAV infection. Postmortem analysis showed hemorrhages in thigh muscle and atrophy of the thymus and bone marrow. Seven out of 11 samples showed positive amplification of the CAV genome upon PCR. Phylogenetic analysis of all the seven isolates based on VP1 gene nucleotide sequence suggested circulation of genotype A strains in Maharashtra. The study will help us understand the circulating genotype of CAV in India and formulate its diagnosis and vaccination.

Phylogenetic and molecular characterization of chicken anemia virus in southern China from 2011 to 2012.

Chicken anemia virus (CAV) is an important pathogen that causes severe immunosuppression in young chickens. We have characterized 13 CAVs isolated from different commercial farms in southern China between 2011 and 2012. We discovered 92 variable residues compared to 37 other CAV complete genome sequences from other parts of the world listed in GenBank; these residues have not been previously observed. All of the Chinese CAV genomes that were characterized in this study had a glutamine at position 394, a hallmark of highly pathogenic CAVs. We also discovered that intra-group genetic recombination plays a role in generating genetic diversity in natural populations of CAV. The GD-J-12 isolate was a possible recombinant between GD-C-12 and GD-M-12 in the genomic region that encompassed both the coding and non-coding regions.

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.

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.

Molecular epidemiology of chicken anemia virus in commercial farms in China.

BACKGROUND: Chicken anemia virus (CAV) is the causative agent of chicken infectious anemia (CIA). A high prevalence of CAV has been reported in China. However, VP1 sequences of Chinese isolates show no clear genotype clustering or correlation with geographic origin. Therefore, the present study aimed to detect and characterize CAV isolates from China based on sequence and phylogenetic analysis of the VP1, VP2 and VP3 genes. RESULTS: Of 460 spleen samples tested by PCR, 47 (10.22%) were found to be positive for CAV. A total of 25 CAV, approximately full genomes, from different commercial farms were characterized. Phylogenetic analysis of the Chinese CAV sequences together with strains from different countries resulted in four distinct groups (A-D) with significant high bootstrap values. The Chinese viral sequences were located as four different clusters within groups A and D. All the Chinese CAV genomes characterized in this study had glutamine (Q) at amino acid position 394, which indicated that all are highly pathogenic. Mutations associated with attenuation and weaker reactivity with monoclonal antibody 2A9 were absent in the Chinese sequences. CONCLUSIONS: We revealed that CAV prevalence was lower than that reported previously in commercial farms in China. We also showed four distinct sequence groups (A-D), and genetic variability in local CAV sequences that could be divided into four groups based on phylogenetic analysis.

Discovery of a genome of a distant relative of chicken anemia virus reveals a new member of the genus Gyrovirus.

A 2.4-kb phi29 polymerase amplification product from serum of a diseased chicken was cloned and sequenced. The 2383-nucleotide sequence showed about 40% identity to a representative genome of chicken anemia virus (CAV), the only member of the genus Gyrovirus, family Circoviridae. The new genome had an organization similar to that of CAV: a putative 5' untranscribed region of about 400 nt followed by three partially overlapping open reading frames encoding VP1, VP2 and VP3 homologs. The amino acid identities between these homologs and those of CAV were 38.8%, 40.3%, and 32.2%, respectively. Based on these limited similarities, it is proposed that the newly identified virus is a member of a new species in the genus Gyrovirus. For this new species, the name Avian gyrovirus 2 (AGV2) is proposed.

Molecular characterization of chicken infectious anemia viruses detected from breeder and broiler chickens in South Korea.

In South Korea, 32 sequences of chicken infectious anemia virus (CIAV) from various flocks of breeder and commercial chickens were genetically characterized for the first time. Phylogenetic analysis of the viral protein 1 gene, including a hypervariable region of the CIAV genome, indicated that Korean CIAV strains were separated into groups II, IIIa, and IIIb. Strains were commonly identified in great-grandparent and grandparent breeder farms as well as commercial chicken farms. In the field, CIAV strains from breeder farms had no clinical effects, but commercial farm strains were associated with depression, growth retardation, and anemia regardless of the group from which the strain originated. In addition, we identified 7 CIAV genomes that were similar to vaccine strains from vaccinated and unvaccinated breeder flocks. These data suggest that further studies on pathogenicity and vaccine efficacy against the different CIAV group are needed, along with continuous CIAV surveillance and genetic analysis at breeder farms.

Relationship of Torque teno virus to chicken anemia virus.

This chapter examines the correlation between Torque teno virus (TTV) and chicken anemia virus (CAV). Each has a circular single-stranded (ss)DNA genome with every one of its known open reading frames (ORF) on its antigenomic strand. This structure is distinct from those of circoviruses. The genomic sizes of TTV and CAV are different, 3.8 kb and 2.3 kb, respectively. While the spectrum of the TTV genome is enormously diverse, that of the CAV genome is quite narrow. Although a 36-nt stretch near the replication origin of TA278 TTV possesses more than 80% similarity to that of CAV, the sequence of the other genomic regions does not exhibit a significant similarity. Nevertheless, the relative allocation of ORFs on each frame in these viruses mimics each other. Three or more messenger RNA (mRNAs) are generated by transcription in both of them. The structural protein with the replicase domain is coded for by frame 1 in each virus, and a nonstructural protein with a phosphatase domain is coded for by frame 2. A protein on frame 3 in each virus induces apoptosis in transformed cells. Recently, we confirmed that apoptin is necessary for the replication of CAV. TTV has been proposed to constitute a new family, Anelloviridae. Considering these similarities and dissimilarities between CAV and TTV, it seems more reasonable to place CAV, the only member of genus Gyrovirus, into Anelloviridae together with TTV, or into a new independent family.

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/ApT(108)I, 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 T(108)I. 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.

Detection and characterization of chicken anemia virus from commercial broiler breeder chickens.

BACKGROUND: Chicken anemia virus (CAV) is the causative agent of chicken infectious anemia (CIA). Study on the type of CAV isolates present and their genetic diversity, transmission to their progeny and level of protection afforded in the breeder farms is lacking in Malaysia. Hence, the present study was aimed to detect CAV from commercial broiler breeder farms and characterize CAV positive samples based on sequence and phylogenetic analysis of partial VP1 gene. RESULTS: A total of 12 CAV isolates from different commercial broiler breeder farms were isolated and characterized. Detection of CAV positive embryos by the PCR assay in the range of 40 to 100% for different farms indicated high level of occurrence of vertical transmission of viral DNA to the progeny. CAV antigen was detected in the thymus and in the bone marrow but not in spleen, liver, duodenum, ovary and oviduct by indirect immunoperoxidase staining. The 12 CAV isolates were characterized based on partial sequences of VP1 gene. Six isolates (MF1A, MF3C, M3B5, NF4A, P12B and P24A) were found to have maximum homology with previously characterized Malaysian isolate SMSC-1, four isolates (M1B1, NF3A, PYT4 and PPW4) with isolate BL-5 and the remaining two (NF1D and NF2C) have maximum homology both with isolates 3-1 and BL-5. Meanwhile, seven of the isolates with amino acid profile of 75-I, 97-L, 139-Q and 144-Q were clustered together in cluster I together with other isolates from different geographical places. The remaining five isolates with amino acid profile of 75-V, 97-M, 139-K and 144-E were grouped under cluster II. All the CAV isolates demonstrated omega values (Ka/Ks) of less than one (the values ranging from 0.07 to 0.5) suggesting the occurrence of purifying (negative) selection in all the studied isolates. CONCLUSION: The present study showed that CAV is widespread in the studied commercial broiler breeder farms. The result also indicated the occurrence of genetic variability in local CAV isolates that can be divided at least into two groups based on characteristic amino acid substitutions at positions 75, 97, 139 and 144 of the VP1 protein.

Molecular epidemiology of chicken anemia virus (CAV) in southeastern Chinese live birds markets.

Between January 2004 and December 2005, cloacal swabs from essentially healthy chickens and silky chickens from live birds markets in Guangdong and Hunan provinces in southeastern China were screened for chicken anemia virus (CAV) by polymerase chain reaction. Phylogenetic analysis of the major structural protein VP1 sequences showed no clear genotype cluster and no correlation with the geographic origin of CAV strains. Virus evolution at the amino acid level was very slow, which corresponds to a strong negative selection of the VP1 gene in China and worldwide. A high proportion (87%) of birds was CAV positive, suggesting that many farms in the region were infected. Further investigations are necessary to evaluate the economic losses caused by CAV and the cost-benefit of vaccination.

Sequence and phylogenetic analysis of chicken anaemia virus obtained from backyard and commercial chickens in Nigeria.

This work reports the first molecular analysis study of chicken anaemia virus (CAV) in backyard chickens in Africa using molecular cloning and sequence analysis to characterize CAV strains obtained from commercial chickens and Nigerian backyard chickens. Partial VP1 gene sequences were determined for three CAVs from commercial chickens and for six CAV variants present in samples from a backyard chicken. Multiple alignment analysis revealed that the 6% and 4% nucleotide diversity obtained respectively for the commercial and backyard chicken strains translated to only 2% amino acid diversity for each breed. Overall, the amino acid composition of Nigerian CAVs was found to be highly conserved. Since the partial VP1 gene sequence of two backyard chicken cloned CAV strains (NGR/CI-8 and NGR/CI-9) were almost identical and evolutionarily closely related to the commercial chicken strains NGR-1, and NGR-4 and NGR-5, respectively, we concluded that CAV infections had crossed the farm boundary.