Chicken Infectious Anemia Virus Research Articles

Synergistic pathogenicity of vertically transmitted chicken infectious anemia virus and avian leukosis virus subgroup J coinfection in chickens

Synergistic pathogenicity of vertically transmitted chicken infectious anemia virus and avian leukosis virus subgroup J coinfection in chickens

Synergy of Subgroup J Avian Leukosis Virus and Chicken Infectious Anemia Virus Enhances the Pathogenicity in Chickens.

Subgroup J avian leukemia virus (ALV-J) and chicken infectious anemia virus (CIAV) are widely acknowledged as significant immunosuppressive pathogens that commonly co-infect chickens, causing substantial economic losses in the poultry industry. However, whether co-infection of ALV-J and CIAV have synergistic pathogenicity remains uncertain. To explore their synergistic pathogenesis, we established a co-infection model of ALV-J and CIAV in HD11 cells and specific-pathogen-free (SPF) chickens. We discovered that ALV-J and CIAV can synergistically promote the secretion of IL-6, IL-10, IFN-α, and IFN-γ and apoptosis in HD11 cells. In vivo, compared to the ALV-J and CIAV mono-infected group, the mortality increased significantly by 27% (20 to 47%) and 14% (33 to 47%) in the co-infected group, respectively. We also discovered that ALV-J and CIAV synergistically inhibited weight gain and exhibited more severe organ damage in co-infected chickens. Furthermore, we found that CIAV can promote the replication of ALV-J in HD11 cells and significantly enhance ALV-J viral load in blood and tissues of co-infected chickens, but ALV-J cannot promote the replication of CIAV. Moreover, by measuring the immune organ indexes and proportions of blood CD3+CD4+ and CD3+CD8+ lymphocytes, more serious instances of immunosuppression were observed in ALV-J and CIAV co-infected chickens than in mono-infected chickens. Taken together, our findings demonstrate that ALV-J and CIAV synergistically enhance pathogenicity and immunosuppression.

Isolation and genomic characterization of chicken infectious anemia virus in Jiangsu province of China during 2020-2022.

As an immunosuppressive disease virus, chicken infectious anemia virus (CIAV) mainly infects chickens, causing aplastic anemia and systemic lymphoid tissue atrophy. In recent years, the prevalence of CIAV in the poultry industry globally has caused huge economic losses. In this study, a total of 223 clinical samples, including anal swabs, tissues, blood, and vaccines, were collected from 19 broiler farms or breeding companies in Jiangsu province, with symptoms of significant anemia and immunosuppression during 2020-2022. Among them, 75 samples (75/223, 33.6%) were positive for CIAV in polymerase chain reaction (PCR) test, and 20 CIAV strains were successfully isolated. The phylogenetic trees of the 20 isolates and 42 CIAV strains deposited in GenBank formed four distinct groups (A-D). And the isolates mainly belonged to Group A but with high genetic diversity. Analysis for VP1 indicated that these isolates possess key characteristics of highly pathogenic strains. Meanwhile, VP2 and VP3 were much conserved with much fewer mutations compare to VP1. The above epidemiological study of CIAV provides novel insights into molecular characterization of CIAV and lays the foundation for developing efficient strategies for control of CIAV in China.

First report of co-infections of Marek's disease virus and chicken infectious anaemia virus in poultry flocks in Nigeria

Marek's disease (MD) and chicken infectious anaemia (CIA) are viral immunosuppressive diseases of poultry caused by the MD virus (MDV) and CIA virus (CIAV) respectively. Despite vaccination against MD, the incidence of the disease in vaccinated poultry flocks in Nigeria persists. However, underlying factors like co-infection with CIAV have not been investigated in the country. This study was designed to investigate possible co-infections of MDV and CIAV in poultry flocks in Nigeria. In 2016, tumorous tissue samples were collected from suspected cases of MD at necropsy in Jos, Plateau State, Nigeria. The samples collected were fixed in formalin for histopathological examination, genomic DNA was extracted from a second part and analysed by polymerase chain reaction (PCR), targeting the meq and VP1 genes of the MDV and CIAV, respectively. The histology results revealed that the cutaneous and proventricular lymphomas were characterized by large numbers of mononuclear cellular infiltrates admixed with heterophils. The PCR results revealed that MDV was detected in 66.7% (16/24), CIAV in 45.8% (11/24), and co-infections of MDV and CIAV were detected in 45.8% (11/24) of the samples analysed. In addition, co-infections of MD and CIA were recorded in 100% (6/6) and 27.7% (5/18) of broilers and layer/pullet’ samples respectively. Phylogenetic analysis of the meq gene sequences revealed that the Nigerian MDV clusters with very virulent MDV from Egypt and Italy. While, CIAV sequences were genotype II and genotype III and clustered with CIAVs from Cameroon and China. This is the first report of co-infections of MD and CIA in Nigeria.

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.

Molecular Characteristics of Chicken Infectious Anemia Virus in Central and Eastern China from 2020 to 2022.

To evaluate the recent evolution of CIAV in China, 43 flocks of chickens from the provinces of Henan, Jiangsu, Hubei, and Anhui were screened via polymerase chain reaction during 2020-2022. Of these, 27 flocks tested positive for CIAV nucleic acids, including 12 which were positive for other immunosuppression viruses. Additionally, 27 CIAV strains were isolated, and their whole genomes were sequenced. The AH2001 and JS2002 strains shared the highest identity at 99.56%, and the HB2102 and HB2101 strains shared the lowest identity at 95.34%. Based on the genome sequences of these strains and reference strains, a phylogenetic tree was constructed and divided into eight main branches. Most of the strains were grouped with the East Asian strains, whereas the HB2101 strain belonged to the Brazil and Argentina cluster. A recombination event was detected in multiple strains, in which AH2002 recombined from KJ728827/China/2014 (from Taiwan Province) and HN2203, and AH2202 recombined from KX811526/China/2017 (from Shandong Province) and HN2203. All the obtained strains had a highly pathogenic Gln amino acid site at position 394 of the VP1. Overall, our findings demonstrate the importance of CIAV monitoring and provide data that aid in understanding the evolution of CIAV.

Detection and Molecular Characterization of VP1 Gene of Chicken Infectious Anaemia Virus in Suspected Layer Birds

Background: Chicken Infectious Anaemia Virus (CIAV) is one of the emerging pathogen of the poultry. The Genus of this virus is Gyrovirus and the family Anelloviridae. Viral DNA contains three open reading frames (ORFs) encoding for three proteins VP1, VP2 and VP3. VP1 protein is major structural protein and is a capsid protein having major role in growth and spread of the virus. Present study was aimed for molecular detection of VP1 gene of the CIA virus in the samples collected from the outbreaks occurred at four different district of Maharashtra. Methods: Total 50 samples comprising of Spleen, bone marrow, thymus and liver, were collected. DNA extracted from those samples was used for PCR amplification targeting, VP1 gene. Selected positive purified PCR products of VP1 were subjected for Nucleotide sequencing and Phylogenetic analysis. Results: Total out of 50 pooled samples, 10 samples were positive for VP1 gene i.e. 20 %. Sequencing of coding region of four CAV positive samples was conducted. Genetic analysis showed three isolates had high similarity with Del-Ros vaccine strainfrom USA ; while one isolate shared close similarity to attenuated cloned isolate 10 of Cux-1 strain. The data also indicated Q139 and Q144 and Q394 amino acids substitutions among the VP1. Phylogenetic analysis of the sequenced viruses based on either the coding nucleotide sequence or VP1 coding sequence, suggested the circulation of III genotypes in Maharashtra.

Isolation, Identification, and Whole Genome Analysis of Chicken Infectious Anemia Virus in an Outbreak of Disease in Adult Layer Hens.

Chicken infectious anemia (CIA) poses a significant threat to the chicken industry in China. Due to its non-specific symptoms, the disease is often overlooked. This study aimed to conduct a comprehensive analysis of the etiology and pathology of CIA in Guangxi Province, China. Three strains of the chicken infectious anemia virus (CIAV) were isolated from liver samples of diseased 20-week-old chickens. The complete genomes of these strains were sequenced, and experiments on specific pathogen-free (SPF) chicks revealed that the GX21121 strain exhibited high virulence. Histopathological examination of the deceased chickens showed liver cell necrosis, fibrous serous exudation, inflammatory cell infiltration, hemorrhage in liver tissues, and congestion in lung and renal tissues. Phylogenetic analysis of the genome revealed that the three strains had a close genetic relationship to the Heilongjiang wild-type strain (GenBank KY486144). The genetic evolution of their VP1 genes indicated that all three CIAV isolates belonged to genotype IIIc. In summary, this study demonstrated the genomic diversity of three CIAV strains in adult layer hens. The isolation and characterization of the GX21121 strain as a highly virulent isolate provide valuable information for further investigations into the etiology, molecular epidemiology, and viral evolution of CIAV.

Effects of garlic, onion, and apple cider vinegar as a herbal mixture on performance and blood traits of broilers inoculated with chicken infectious anemia virus

This study assessed the effects of a herbal mixture (HM) to protect poultry against chicken infectious anemia (CIA) and to modulate the adverse effects of this virus on performance, mortality, blood profile, white blood cells (WBCs) count, liver enzymes, liver histopathology, and intestinal morphology. Therefore, 240 one-day-old male broiler chicks (Ross 308) were divided into four experimental groups, with six replicates and ten chicks per group. The experimental groups consisted of a control group and groups with 2.5%, 5%, and 7.5% HM, all based on corn-soybean meal. All chicks were inoculated with the CIA virus (CIAV) on day 7. The results showed that supplementation of 2.5% of HM to broiler diet increased feed intake (FI) (P < 0.05) and also increased body weight (BW) and weight gain (WG) slightly (P > 0.05). Adding 7.5% HM caused a reversible decrease in FI, BW, and WG and increased FCR. Compared with the control group, mortality rates declined with an additional dose of HM in CIAV-infected chickens. HM supplementation in the diet of CIAV-infected chickens increased hematocrit (HCT), hemoglobin (Hb), and mean corpuscular volume (MCV) and decreased mean corpuscular hemoglobin concentration (MCHC) compared to the control (P < 0.05). Lymphocyte percentage and lymphocyte/heterophile ratio increased in HM-supplemented groups, especially at 2.5% (P < 0.05), and heterophile and granulocyte percentages were reduced (P < 0.05). Liver enzyme alkaline phosphatase (ALP) and liver steatosis declined in the 2.5% HM-treated group compared to the control (P < 0.05). It was concluded that adding 2.5% of the HM to the CIAV-infected broiler’s diet did not negatively affect chicken performance. In addition to its hypolipidemic effects, it could prevent HCT and Hb from decreasing in chicks infected with CIAV and positively affect leukocyte types and liver enzymes. Interestingly, an additional dose of HM in the diet of the CIAV-infected broilers reduced mortality. Therefore, adding 2.5% of HM could prevent the adverse effects of CIA on hematological traits in broiler chicken flocks without adverse effects on performance.

Virome Profiling of Chickens with Hepatomegaly Rupture Syndrome Reveals Coinfection of Multiple Viruses.

Liver diseases seriously challenge the health of chickens raised on scaled farms and cause tremendous economic losses to farm owners. The causative agents for liver diseases are still elusive, even though various pathogens, such as the hepatitis E virus, have been reported. In the winter of 2021, a liver disease was observed on a chicken farm in Dalian, China, which increased chicken mortality by up to 18%. We conducted panvirome profiling of the livers, spleens, kidneys, and recta of 20 diseased chickens. The viromic results revealed coinfection of multiple viruses, including pathogenic ones, in these organs. The viruses were highly identical to those detected in other provinces, and the vaccine and field strains of avian encephalomyelitis virus (AEV) and chicken infectious anemia virus (CIAV) cocirculated on the farm. In particular, the liver showed higher abundance of AEV and multiple fowl adenoviruses than other organs. Furthermore, the liver also contracted avian leukemia virus and CIAV. Experimental animals with infected liver samples developed minor to medium lesions of the liver and showed a virus abundance profile for AEV across internal organs similar to that in the original samples. These results suggest that coinfection with multiple pathogenic viruses influences the occurrence and development of infectious liver disease. The results also highlight that strong farm management standards with strict biosafety measures are needed to minimize the risk of pathogenic virus introduction to the farm.

Isolation, Identification, Sequence Analysis, and Pathogenicity of a CIAV Strain from Aegypius monachus

A cluster of vultures, artificially bred in a zoo in Shandong Province, China, displayed signs of emaciation and some even died, which raised the suspicion of an immunosuppressive pathogen. Upon conducting nucleic acid testing on the clinical samples, it was found that the CIAV was present in the tissue of dead vulture. In this study, samples of dead vulture tissue were used to isolate a chicken infectious anemia virus (CIAV) via the MDCC-MSB1 cell line, which was designated the SDTY2021-TJ strain. The full-length genomic sequence of SDTY2021-TJ was determined and analyzed in detail. The full genomic DNA of SDTY2021-TJ was found to be 2298 bp, with no omissions or additions in the coding region. The homology between the full-length genomic sequence of SDTY2021-TJ and the reference strain ranged from 95.5% (Del-Ros) to 98.7% (YN04). In comparison with the reference strain, the VP1 protein of SDTY2021-TJ contained a number of mutations. To assess the virulence of SDTY2021-TJ, one-day-old SPF chickens were inoculated with both high and low doses of the pathogen. The results demonstrated that SDTY2021-TJ had a considerable pathogenicity to SPF chickens, as the high-dose group caused a 50% mortality rate, and even the low-dose group caused a 30% mortality rate. The chickens infected with the disease exhibited paleness in the cockscomb and stunted growth, as well as a compromised response to NDV and AIV-H9 inactivated vaccine. Furthermore, histological observation revealed an atrophy and degeneration of the thymus. To the best of our knowledge, this is the first instance of CIAV being isolated from artificially bred Aegypius monachus, implying that wild birds may be involved in the transmission and spread of CIAV.

Retrospective Investigation and Genetic Variation Analysis of Chicken Infectious Anemia in Shandong Province, 2020–2022

Chicken infectious anemia (CIA) is a vertical transmission infectious chicken disease caused by the chicken infectious anemia virus (CAV). The disease can induce stunting and immunosuppression in chicks by infecting bone marrow-derived stem cells, causing huge economic losses for the poultry industry. To determine the prevalence of CIA in Shandong Province, China, 854 suspected CIA samples were collected and analyzed in 13 cities in Shandong from 2020 to 2022. The PCR results showed that a total of 115 CAV were isolated. The CAV-positive rates were 17.21% (26/151) in 2020, 12.23% (35/286) in 2021, and 12.94% (54/417) in 2022, with severe mixed infections. Among them, CAV and fowl adenovirus (FAdV) were the most common, accounting for 40.86%. VP1 gene homology analysis showed that isolated strains shared 96.1-100% homology with the previously reported CAV strains. Genetic variation analysis showed that most of the isolated CAV strains were located in genotype A. These results indicate that CIA infection in Shandong chickens in recent years has been prevalent and mixed infections are common, but there were no significant genetic variations. Our results extend the understanding of the prevalence and genetic evolution of CIA in Shandong Province. They will offer new references for further study of the epidemiology and virus variation and the prevention and control of this disease.

Chicken infectious anemia virus (CIAV) VP1 antagonizes type I interferon (IFN-I) production by inhibiting TBK1 phosphorylation

Chicken infectious anemia virus (CIAV) infection induces immunosuppression or subclinical immunosuppression in chickens. CIAV infection has been reported to repress type I interferon (IFN-I) expression, but the underlying mechanisms are not yet understood. Here we reported that VP1, the capsid protein of CIAV, the major immunogenic protein that triggers the production of neutralizing antibodies in chickens, inhibited type I interferon (IFN-I) expression induced by cGAS-STING signaling. We showed that VP1 inhibited TBK1 phosphorylation and down stream signal transduction, leading to the inhibition of IFN-I expression. Subsequently, we demonstrated that VP1 interacted with TBK1. Finally, we clarified that aa 120–150 in VP1 was essential for VP1 to interact with TBK1 and inhibit cGAS-STING signaling. These findings will help us further understand the pathogenesis of CIAV in chickens.

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

ABSTRACT 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.

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

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. 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. 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. 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. 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.

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

ABSTRACT 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.

Research Note: A novel peptide-based ELISA for efficient detection of antibody against chicken infectious anemia virus

Chicken infectious anemia virus (CIAV) is the pathogen of chicken infectious anemia. Currently, due to the lack of effective diagnostics technology and prevention approach, CIAV has spread globally and caused huge economic losses to poultry industry. In this study, a novel peptide-based ELISA (pELISA) for efficient detection of antibody against CIAV was developed. The peptide (25CRLRRRYKFRHRRRQRYRRRAF45) used in pELISA was highly conserved in VP1 protein of different CIAV isolates. The specificity and reproducibility showed that the pELISA only reacted with sera against CIAV, not with sera against other pathogens tested, and the CV of the intra-/inter-assay of the pELISA was 6.8 to 9.22%. Moreover, the comparison assay using 56 clinical samples showed that the positive rate of the pELISA and the commercial ELISA kit (IDEXX) was 85.7 and 80.4%, respectively. The pELISA generated here provides a rapid and efficient serological detection method for diagnosis of CIAV infection and evaluation of the efficacy of CIAV vaccination.

A new rapid and sensitive method for detecting chicken infectious anemia virus.

Since the chicken infectious anemia virus (CIAV) was discovered in 1979, which has been reported as an economically significant and immunosuppressive poultry disease in the world. A novel clinical detection method for the prevention and control of CIAV in the poultry sector is urgently needed. Here, we established a real-time recombinase-aided amplification assay (RAA) for CIAV on-site with a rapid, highly sensitive, strongly specific, low-cost, and simple operational molecular diagnosis detection method. The primers and probe were developed using the CIAV VP2 gene sequence, which has a 117-bp specific band. This assay, which could be carried out at 41°C and completed in 30 min without cross-reactivity with other viruses, had the lowest detection limit of 10 copies of CIAV DNA molecules per reaction. Furthermore, the kappa value of this assay was 0.947, the sensitivity was 93.33%, and the specificity was 100% when compared to the real-time quantitative polymerase chain reaction assay (real-time qPCR). These results indicate that using a real-time RAA assay to detect CIAV on-site could be beneficial. In the future, the real-time RAA test may be a regular assay for the prevention and control of CIAV, as well as help the reduction of economic losses in the poultry business.

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.