Genetic characterization of chicken infectious anaemia viruses isolated in Korea and their pathogenicity in chicks.

Chicken infectious anaemia virus (CIAV) causes severe anemia and immunosuppression through horizontal or vertical transmission in young chickens. Especially, vertical transmission of virus through the egg can lead to significantly economic losses due to the increased mortality in the broiler industry. Here, 28 CIAV complete sequences circulating in Korea were first characterized using the newly designed primers. Phylogenetic analysis based on the complete sequences revealed that CIAV isolates were divided into four groups, IIa (2/28, 7.1%), IIb (9/28, 32.1%), IIIa (8/28, 28.6%) and IIIb (9/28, 32.1%), and exhibited a close relationship to each other. The major groups were IIb, IIIa and IIIb, and no strains were clustered with a vaccine strain available in Korea. Also, for viral titration, we newly developed a quantitative PCR assay that is highly sensitive, reliable and simple. To investigate the pathogenicity of three major genotypes, 18R001(IIb), 08AQ017A(IIIa), and 17AD008(IIIb) isolates were challenged into one-day-old specific-pathogen-free (SPF) chicks. Each CIAV strain caused anaemia, severe growth retardation and immunosuppression in chickens regardless of CIAV genotypes. Notably, a 17AD008 strain showed stable cellular adaptability and higher virus titer in vitro as well as higher pathogenicity in vivo. Taken together, our study provides valuable information to understand molecular characterization, genetic diversity and pathogenicity of CIAV to improve management and control of CIA in poultry farm.

Subclinical circulation of Chicken infectious anaemia virus- A Sero-molecular study.

Chicken infectious anaemia-an important immunosuppressive viral disease of chicken-gained much attention in the recent past. Based on huge mortality and production loss observed in the fast-growing poultry sector, the present study aimed to find out the current status of the chicken infectious anaemia virus (CIAV), among chicken flocks in the Punjab state of India by sero-molecular study. The sera from the blood samples were tested for anti-CIAV antibodies by indirect ELISA and also compared with haematological parameters. DNA from sero-positive samples underwent PCR amplification, sequencing and phylogenetic analysis of the most conserved genomic region (VP3 gene) to detect viraemia in asymptomatic birds. The serological study using indirect ELISA showed a high sero-positivity of 77.27% in chicken flocks. Additionally, the present study also revealed the high molecular evidence (72.54%) of CIAV in apparently healthy birds. Genetic analysis showed that all CIAVs have conserved VP3 genes without any nucleotide substitutions, indicating presence of CIAV and its subclinical circulation among apparently healthy flocks. The wide distribution of CIAV among birds may be the reason for huge mortality and production loss. Further, it is suggested that studies be conducted to find out the co-involvement of CIAV with other immunosuppressive microbial agents and the immunosuppressive effect of CIAV in apparently healthy birds. Also, its role in vaccine failure and outbreaks of various other avian diseases needs to be explored.

First detection and genetic characterization of chicken infectious anemia virus in the Mekong Delta, Vietnam.

Background: Chicken infectious anemia (CIA) caused by the CIA virus (CIAV) is considered one of the most important immunosuppressive diseases affecting chickens and recently poses a great economic burden to the poultry industry worldwide. Aim: This study aims to identify the presence of CIAV in the Mekong Delta (MD), Vietnam, and to determine genotypes of CIAVs that are currently circulating in this area. Methods: Organ samples (spleen, liver, and thymus) of 144 chickens suspected with CIA from 47 poultry farms were collected. A total of 47 pooled samples, each containing 2-4 chickens from each farm, were tested for the presence of CIAV. Results: Twenty out of 47 pooled organ samples (pool of 2-4 chickens per farm) were positive for CIAV using polymerase chain reaction targeting the viral VP1 gene. The VP1 amplicons of eight representative CIAVs were subjected to sequencing and genetic characterization. Phylogenetic analysis based on partial VP1 gene sequence revealed that the CIAVs detected in the MD grouped into different genotypes of II, IIIa, and IIIc together with CIAVs previously detected in the northern Vietnam and other Asian countries. The phylogenetic analysis also confirmed that detected CIAVs genetically differed from vaccine strains. In addition, deduced amino acids of the VP1 identified several critical amino acid substitutions in the VP1 protein that are likely associated with the virulence of CIAV. Conclusion: This is the first report to detect and determine the genetic characterization of the circulating CIAVs in the MD. Therefore, this study provides an important understanding of the evolution of CIAVs and highlights the importance of implementing prompt control measures against CIAVs in the MD and Vietnam.

Nonstructural protein VP2 of chicken anemia virus triggers IFN-ß expression via host cGAS.

Chicken anemia virus (CAV) constitutes an important economic threat for the poultry industry. Advancing the understanding of the pathogenic process of CAV infection, we had previously demonstrated that CAV VP1 has the ability to inhibit expression of IFN-ß via cGAS-STING signalling pathway. Here to go further to reveal this regulatory role of viral phosphatase VP2, we have performed protein-protein interaction assays with cGAS adaptors, as well as IFN-ß induction screenings. Contrary to VP1, VP2 of CAV stimulates the expression of IFN-ß, a regulatory effect more closely associated with cGAS (in the context of the cGAS-STING axis) than with STING, TBK1 or IRF7. The results reported here offer new insights about the molecular mechanisms that varied viral proteins act in a timely manner on the host during CAV infection.

Molecular characterization and pathogenicity study of a highly pathogenic strain of chicken anemia virus that emerged in China.

Chicken infectious anemia (CIA) is caused by chicken anemia virus (CAV). Recently, severe anemia has emerged in layer chickens (8 to 10-week-old) on poultry farms in China. However, the etiological characteristics and pathogenic potential of CAV in chickens at 6 weeks or older are not well understood. In this study, we isolated a CAV strain, termed SD15, from two-month-old chicken with severe anemia and analyzed the genetic evolution relationship. We found that strain SD15 had the highest homology (98.9%) with CAV18 strain. Comparison with 33 reference strains revealed 16 amino acid mutations in strain SD15, two of which were previously unknown (F210S in VP1 and L25S in Vp3). Compared with low pathogenic strains (Cux-1 and C14), highly pathogenic strains (SDLY08 and SD15) had three base mutations in their noncoding region. To further understand its pathogenicity, 10-week-old specific-pathogen-free (SPF) chickens were challenged with the novel strain and SDLY08. No obvious clinical symptoms were observed in the SDLY08 group. However, SD15-infected chickens showed significant growth retardation and immunosuppression. The main manifestations of immunosuppression were the significantly reduced thymus and bursa indices and AIV-H9 vaccine-induced antibody levels (P < 0.05). The lowest number of red blood cells in the SD15 group was just 60% of that in the control group. Taken together, the novel strain SD15 not only showed higher pathogenicity but also exhibited the potential ability to break the age resistance of older chickens to CAV. Our study enhanced the understanding of the epidemiological characteristics of chickens infected with severe anemia and can facilitate the development of improved control strategies of CIA in China.

Pathology and molecular characterization of chicken infectious anemia virus and in silico antigen prediction.

The present study investigated five poultry flocks (size 142-600 birds) suspected of chicken infectious anemia (CIA) from Maharashtra, India. The necropsy of dead birds revealed severe atrophy of the thymus, gelatinization of bone marrow, subcutaneous hemorrhages, growth impairment, and severe anemia. Specific PCR targeting, 1390 bp fragment of the CIAV, VP1 gene was used in this study. Sequence analysis revealed that CIAV sequences of this study were grouped in genotype A. At the nucleotide level identity of 99.6% or more was seen between field sequences. At the amino acid level identity of 100% was seen between field sequences and NGP-1. Also, VP1 protein sequences of this study showed high identity with TJBD40, GD-K-12 strains from China and AB046590 strain from Japan. Further, the protein sequences of field CIAV had 0.7% to 2.5% divergence from VP1 sequences of vaccine strains. Antigenic epitopes of VP1 protein were predicted by SVMTriPtool and the field CIAV presented substitutions in two epitopes. To conclude, present study confirms the circulation of genotype A of CIAV in Maharashtra, India and predicted VP1 proteins of field CIAV revealed changes in two epitopes compared to vaccine strains.

Molecular evolution analysis of three species gyroviruses in China from 2018 to 2019.

Gyrovirus (GyV) is a widespread ssDNA virus with a high population diversity, and several of its species, including the chicken anemia virus (CAV), gyrovirus galga 1 (GyG1), and gyrovirus homsa 1 (GyH1), have been shown to be pathogenic to poultry. The evolution of these viruses, however, is still unclear. Our study analyzed epidemiology and molecular evolution of three species of GyVs (CAV, GyG1, and GyH1) from 2018 to 2019 in China. The survey results indicated that GyV was widespread in China. It is vital to consider the coinfections among the three species of GyV. The phylogenetic analysis showed that CAV was divided into three clades and GyG1 and GyH1 were divided into two clades. Based on the recombination analysis, CAV and GyG1 had similar recombination regions associated with viral replication and transcription. Furthermore, the substitution rates for CAV and GyG1 were approximately 6.09 × 10-4 and 2.784 × 10-4 nucleotides per site per year, respectively. The high substitution rate and recombination were the main factors for the high diversity of GyVs. Unfortunately, GyH1 strains have not been discovered in enough numbers to allow evolutionary analysis. The GyVs had several positively selected sites, possibly related to their potential to escape the host immune response. In summary, our study provides insights into the time of origin, evolution rate, and recombination of GyV for assessing their evolutionary process and genetic diversity.

A multiplex fluorescence-based loop-mediated isothermal amplification assay for identifying chicken parvovirus, chicken infectious anaemia virus, and fowl aviadenovirus serotype 4.

Chicken parvovirus (ChPV), chicken infectious anaemia virus (CIAV) and fowl adenovirus serotype 4 (FAdV-4) are avian viruses that have emerged in recent years and have endangered the global poultry industry, causing great economic loss. In this study, a multiplex fluorescence-based loop-mediated isothermal amplification (mLAMP) assay for detecting ChPV, CIAV and FAdV-4 was developed to simultaneously diagnose single and mixed infections in chickens. Three primer sets and composite probes were designed according to the conserved regions of the NS gene of ChPV, VP1 gene of CIAV and hexon gene of FAdV-4. Each composite probe was labelled with a different fluorophore, which was detached to release the fluorescence signal after amplification. The target viruses were distinguished based on the colour of the mLAMP products. The mLAMP assay was shown to be sensitive, with detection limits of 307 copies of recombinant plasmids containing the ChPV target genes, 749 copies of CIAV and 648 copies of FAdV-4. The assay exhibited good specificity and no cross-reactivity with other symptomatically related avian viruses. When used on field materials, the results of the mLAMP assay were in 100% agreement with those of the previously published PCR assay. The mLAMP assay is rapid, economical, sensitive and specific, and the results of amplification are directly observable by eye. Therefore, the mLAMP assay is a useful tool for the clinical detection of ChPV, CIAV and FAdV-4 and can be applied in rural areas.

Chicken anemia virus VP1 negatively regulates type I interferon via targeting interferon regulatory factor 7 of the DNA-sensing pathway.

The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway plays a vital role in sensing viral DNA in the cytosol, stimulating type I interferon (IFN) production and triggering the innate immune response against DNA virus infection. However, viruses have evolved effective inhibitors to impede this sensing pathway. Chicken anemia virus (CAV), a nonenveloped ssDNA virus, is a ubiquitous pathogen causing great economic losses to the poultry industry globally. CAV infection is reported to downregulate type I IFN induction. However, whether the cGAS-STING signal axis is used by CAV to regulate type I IFN remains unclear. Our results demonstrate that CAV infection significantly elevates the expression of cGAS and STING at the mRNA level, whereas IFN-ß levels are reduced. Furthermore, IFN-ß activation was completely blocked by the structural protein VP1 of CAV in interferon stimulatory DNA (ISD) or STING-stimulated cells. VP1 was further confirmed as an inhibitor by interacting with interferon regulatory factor 7 (IRF7) by binding its C-terminal 143-492 aa region. IRF7 dimerization induced by TANK binding kinase 1 (TBK1) could be inhibited by VP1 in a dose-dependent manner. Together, our study demonstrates that CAV VP1 is an effective inhibitor that interacts with IRF7 and antagonizes cGAS-STING pathway-mediated IFN-ß activation. These findings reveal a new mechanism of immune evasion by CAV.

Molecular characterization of chicken infectious anaemia virus (CIAV) in China during 2020-2021.

Chicken infectious anaemia virus (CIAV) has been identified as the causative agent of chicken infectious anaemia (CIA), causing huge economic losses to the poultry industry globally. In this study, a total of 573 clinical samples were collected from 197 broiler farms in 17 provinces of China during 2020-2021. Among them, 375 samples (375/573, 65.4%) were positive for CIAV by real-time PCR. The positive rate of CIAV detection between different regions of China ranged from 46.67% (North China) to 81.25% (Central China). The nucleotide sequences of the VP1 gene were obtained for 91 CIAV strains, whole genome sequencing was successful for 72 out of 91 strains. Phylogenetic analysis based on the VP1 gene revealed that 91 CIAV strains currently circulating in China belong to three genotypes (II, IIIa and IIIb), and most of the CIAV strains belong to genotype IIIa. Phylogenetic analysis of the whole genome showed that 71 CIAV strains belong to genotype IIIa, and one strain belongs to genotype II. Sequence analysis showed several amino acid substitutions in both the VP1, VP2 and VP3 proteins. Our results enhance the understanding of the molecular characterization of CIAV infection in China.RESEARCH HIGHLIGHTS A molecular systematic survey of CIAV in China during 2020-2021.CIAV genotype IIIa is the predominant genotype in China.

DNA Prime and Recombinant Protein Boost Vaccination Confers Chickens with Enhanced Protection against Chicken Infectious Anemia Virus.

Chicken infectious anemia (CIA) is an immunosuppressive disease caused by chicken infectious anemia virus (CIAV) that poses a great threat to the poultry industry worldwide. At present, vaccination is an important way to prevent and control CIA. Apart from a CIAV-attenuated vaccine used in clinical practice, the research and development of a genetically engineered vaccine has good prospects. However, it is difficult to induce a strong protective effect with a single subunit vaccine or DNA vaccine. Therefore, the goal of this study is to develop and evaluate a DNA prime/protein boost vaccine strategy for defense against CIAV infection and spread. In this study, the recombinant proteins of CIAV VP1 and VP2 were prepared using an Escherichia coli (E. coli) expression system, and the eukaryotic expression plasmid pBud-VP1-VP2 was constructed. Subsequently, the effects of the DNA prime/protein boost strategy on antibody production and cellular immunity response were measured. The results showed that combined vaccination could induce a higher antibody titer than those of a DNA vaccine or subunit vaccine alone. In addition, spleen lymphocyte index (SI) and IL-2, IL-4, and IFN-γ levels were also significant in chickens the received the combined vaccination. To further investigate the protective effect of DNA prime/protein boost vaccination, a CIAV challenge experiment was carried out. The results showed that infection with CIAV reduced the hematocrit value (Hct) and thymus index, while vaccination recovered this reduction, and the combined immunization group was the least affected by CIAV infection. Furthermore, the CIAV viral load in the combined immunization group was the lowest, indicating that the combined immunization could provide a better protective efficacy. In conclusion, the DNA prime and recombinant protein boost vaccination can be used as an important anti-CIAV strategy, which can induce both enhanced cellular and humoral immunity responses in chickens and provide a new avenue for CIAV prevention and control.

Molecular characterization of chicken anemia virus in Guangxi Province, southern China, from 2018 to 2020.

BACKGROUND: Chicken anemia virus (CAV) causes chicken infectious anemia, which results in immunosuppression; the virus has spread widely in chicken flocks in China. OBJECTIVES: The aim of this study was to understand recent CAV genetic evolution in chicken flocks in Guangxi Province, southern China. METHODS: In total, 350 liver samples were collected from eight commercial broiler chicken farms in Guangxi Province in southern China from 2018 to 2020. CAV was detected by conventional PCR, and twenty CAV complete genomes were amplified and used for the phylogenetic analysis and recombination analysis. RESULTS: The overall CAV-positive rate was 17.1%. The genetic analysis revealed that 84 CAVs were distributed in groups A, B, C (subgroups C1-C3) and D. In total, 30 of 47 Chinese CAV sequences from 2005-2020 belong to subgroup C3, including 15 CAVs from this study. There were some specific mutation sites among the intergenotypes in the VP1 protein. The amino acids at position 394Q in the VP1 protein of 20 CAV strains were consistent with the characteristics of a highly pathogenic strain. GX1904B was a putative recombinant. CONCLUSIONS: Subgroup C3 was the dominant genotype in Guangxi Province from 2018-2020. The 20 CAV strains in this study might be virulent according to the amino acid residue analysis. These data help improve our understanding of the epidemiological trends of CAV in southern China.

Design of a Multiepitope Vaccine against Chicken Anemia Virus Disease.

Chicken anemia virus (CAV) causes severe clinical and sub-clinical infection in poultry globally and thus leads to economic losses. The drawbacks of the commercially available vaccines against CAV disease signal the need for a novel, safe, and effective vaccine design. In this study, a multiepitope vaccine (MEV) consisting of T-cell and B-cell epitopes from CAV viral proteins (VP1 and VP2) was computationally constructed with the help of linkers and adjuvant. The 3D model of the MEV construct was refined and validated by different online bioinformatics tools. Molecular docking showed stable interaction of the MEV construct with TLR3, and this was confirmed by Molecular Dynamics Simulation. Codon optimization and in silico cloning of the vaccine in pET-28a (+) vector also showed its potential expression in the E. coli K12 system. The immune simulation also indicated the ability of this vaccine to induce an effective immune response against this virus. Although the vaccine in this study was computationally constructed and still requires further in vivo study to confirm its effectiveness, this study marks a very important step towards designing a potential vaccine against CAV disease.

Long read sequencing revealed proventricular virome of broiler chicken with transmission viral proventriculitis.

BACKGROUND: Transmissible viral proventriculitis (TVP) causes significant economic loss to the poultry industry. However, the exact causative agents are obscure. Here we examine the virome of proventriculus from specified pathogen free (SPF) chickens that reproduced by infection of proventricular homogenate from broiler chicken with TVP using long read sequencing of the Pacific Biosciences RSII platform. The normal SPF chickens were used as control. RESULTS: Our investigation reveals a virome of proventriculitis, including three Gyrovirus genera of the Aneloviridae: Gyrovirus homsa1 (GyH1) (also known as Gyrovirus 3, GyV3) (n = 2662), chicken anemia virus (CAV) (n = 482) and Gyrovirus galga1 (GyG1) (also known as avian Gyrovirus 2, AGV2) (n = 11); a plethora of novel CRESS viral genomes (n = 26) and a novel genomovirus. The 27 novel viruses were divided into three clusters. Phylogenetic analysis showed that the GyH1 strain was more closely related to the strains from chicken (MG366592) than mammalian (human and cat), the GyG1 strain was closely related to the strains from cat in China (MK089245) and from chicken in Brazil (HM590588), and the CAV strain was more closely related to the strains from Germany (AJ297684) and United Kingdom (U66304) than that previously found in China. CONCLUSION: In this study, we revealed that Gyrovirus virome showed high abundance in chickens with TVP, suggesting their potential role in TVP, especially GyH1. This study is expected to contribute to the knowledge of the etiology of TVP.

Cross-species transmission potential of chicken anemia virus and avian gyrovirus 2.

The Gyrovirus genus consists of nonenveloped, icosahedral viruses with small circular single-stranded DNA genomes. Gyroviruses have been detected in diverse hosts, including humans, chickens, rodents, and cats. Two Gyroviruses were detected in canine serum samples using PCR in this study. The results indicated that four serum samples were positive for CAV (0.28%, 2/700) or AGV2 (0.28%, 2/700). Additionally, recombination analysis showed that AGV2 and CAV might have originated from the recombination of viruses similar to those detected in chickens and humans. We detected a total of 14 mutations in CAV VP1 amino acid sequences and identified new mutations at positions 31, 388, 390, 399, and 421 for the first time. The identification of T390C, C912T, T1230C, and T1297C mutations in AGV2 VP1, R93C mutations in AGV2 VP2, and R58C mutations AGV2 VP3 indicated that the differences might be related to a transboundary movement among hosts, which requires further elucidation. To the best of our knowledge, this study is the first report of an AGV2-infected dog in China, suggesting that the cross-species transmission of viruses with circular single-stranded DNA genomes is a public health concern.

A sensitive triple nanoparticle-assisted PCR assay for detection of fowl adenovirus, infectious bursal disease virus and chicken anemia virus.

Fowl adenovirus (FAdV) infections in chickens have resulted in global economic losses in the poultry industry. Infectious bursal disease virus (IBDV) and chicken anemia virus (CAV) infections lead to immunosuppression in chickens, and concomitant co- infection with FAdV usually produces severe and lethal infections. These co-infections are common occurrences on chicken farms and affect large number of chickens. Thus, a rapid, sensitive and specific diagnostic test for these viruses becomes a prerequisite to effective control and isolation measures. We developed a triplex nanoparticle-assisted PCR (nano-PCR) assay that can simultaneously detect these 3 viruses in a single assay tube using PCR primers directed at respective specific genes of each virus. The assay was specific for FAdVs, CAV and IBDV, and it did not amplify Newcastle disease virus, infectious bronchitis virus, egg drop syndrome virus or Marek's disease virus. The minimum detection limit was 27.2 femtogram (fg) for all three viruses and was 1000-fold more sensitive than multiplex PCR using identical primers. Screening of 69 clinical samples from 40 to 50 days old chickens with obvious lesions in liver using the nano-PCR compared with a multiplex PCR yielded identical results. Of the 69 samples, 13 were detected positive including 4 for FAdV, 4 for IBDV and 6 for CAV single virus infections, respectively, as well as 5 for FAdV/CAV, 2 for FAdV/IBDV and 3 for IBDV/CAV co-infections. The triple nano-PCR assay developed in our laboratory is a sensitive, specific and simple method that can be used for detection of FAdV, CAV and IBDV as single or mixed infections.

Molecular and pathological characterization of natural co-infection of poultry farms with the recently emerged Leucocytozoon caulleryi and chicken anemia virus in Egypt.

In the summers of 2018 and 2019, a disease outbreak stroke 25 broiler chicken farms and 3 broiler breeder farms in different Governorates in Egypt. The disease caused a mortality rate ranging from 3.2 to 9%. Postmortem examination showed petechial hemorrhage in the breast and thigh muscles, thymus gland, and peritoneal cavity and extensive hemorrhages in the kidneys. A total of 140 liver, kidney, lung, skeletal muscles, thymus, and spleen samples were collected. Twenty-eight pooled samples were created and examined by PCR and histopathological examination to identify the causative pathogens. All collected samples were PCR-negative to Newcastle disease virus (NDV), avian influenza viruses (H5, H9, and H7), infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), and fowl adenovirus (FadV). Leucocytozoon caulleryi (L. caulleryi) genetic material was identified by PCR in 17 out of the 28 collected samples (61%). Five chicken farms (18%) showed positive PCR results for both L. caulleryi and chicken anemia virus (CAV). Histopathological examination revealed unilocular megaloschizonts in thymus, skeletal muscle, and lung as well as massive hemorrhages in parenchymatous organs. Nucleotide sequences of the identified pathogens were compared with other reference sequences available in the GenBank. The identified L. caulleryi has a close relationship with those previously detected in Asia, indicating potential transmission route of the parasite. The CAV has a close genetic relation with CAVs previously identified in Egypt. Furthermore, a real-time PCR for rapid, specific, and quasiquantitative detection of L. caulleryi was developed with a detection limit of 100 genome copies per reaction.

Whole genome analysis and molecular characterization of chicken infectious anemia virus from an outbreak in a layer flock reveals circulation of genogroup IIIb in South India.

The complete genome (2298 nucleotides) of the economically important and immunosuppressive, chicken infectious anemia virus (CAV), from a disease outbreak in a layer flock is discussed. This is the first report of a complete genome sequence of CAV from India. The phylogenetic analyses grouped this isolate with CAV genogroup IIIb based on both complete genome and capsid protein (VP1) sequences. The analyses further revealed the presence of CAV genogroups II, IIIa and IIIb in India. The VP1 sequence identity ranged between 84.4 to 99.3% with that of the Indian isolates and carried a unique substitution at position 447 (serine instead of threonine). Two novel amino acid substitutions were observed at position 52 of VP1 (serine instead of proline) and at position 26 of VP2 (asparagine instead of serine). Sequence analyses of VP1, VP2 and VP3 suggested that the isolate could be attenuated. Comparison with CAV variants, isolated from mammalian species, showed similarities in the numbers of certain transcription factor binding sites in the non-coding regions. Recombination analysis detected no recombination events in this isolate. Further investigations are needed to understand the implications of the unique features of this isolate on viral virulence.

Genetic characterization of chicken anemia viruses newly isolated from diseased chicks in Japan in 2020.

In this study, a total of nine chicken samples obtained from two broiler flocks in Oita and Tottori prefectures in 2020 were examined for Chicken anemia virus (CAV) infection. The samples were collected from clinically suspected flocks and diseased chickens. The CAV genome was detected in all nine samples tested by real-time PCR. Phylogenetic analyses and sequence comparisons of the full-length VP1 gene sequences indicated that all the Japanese CAV strains obtained in this study formed a similar cluster of genotype III and shared high nucleotide (99.62-100%) identity. The current Japanese CAV strains were closely related to Chinese CAV strains but not related to vaccine strains. One positive selection site of VP1 was detected among the Japanese CAV strains.

Development of TaqMan Real-Time Polymerase Chain Reaction Assay for Detection of Chicken Anemia Virus in Newcastle Disease Vaccines.

Exogenous chicken anemia virus (CAV) has been detected in commercial poultry vaccines in various countries of the world. The presence of unwanted CAV in vaccines not only influences the epidemiology of chicken infectious anemia disease, but may also lead to vaccine failure and confusing results when vaccine responses are monitored. To detect CAV in contaminated vaccines, nucleic acid testing (unlike conventional testing) has a shorter processing time and does not require cell culture or live animals. The aim of the current study was to develop a TaqMan real-time polymerase chain reaction (PCR) assay to detect and quantify CAV in poultry vaccines and investigate CAV contamination in Razi live Newcastle disease vaccines. The TaqMan real-time PCR assay was set up, optimized, and validated in successive experiments. A standard plasmid pUC-VP2 containing viral protein 2 of CAV was constructed and used in the assay to generate a standard curve to quantify CAV genomes. A clear linear correlation was observed between threshold cycle (Ct) values and plasmid copy numbers in the amplification plots of 10-fold serial dilution of the plasmid. Total DNA of three samples of each of four different Razi live Newcastle disease vaccines, namely LaSota, B1, clone.12IR, and thermo-resistant strains, were extracted and subjected to real-time PCR assay. No CAV contamination was detected in the Razi Live Newcastle vaccines. The developed TaqMan real-time PCR assay provides a quick, specific, and sensitive method for use in detecting CAV in quality control vaccine testing and viral load studies.