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

BackgroundChicken anemia virus (CAV) causes chicken infectious anemia, which results in immunosuppression; the virus has spread widely in chicken flocks in China.ObjectivesThe aim of this study was to understand recent CAV genetic evolution in chicken flocks in Guangxi Province, southern China.MethodsIn 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.ResultsThe 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.ConclusionsSubgroup 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.     

Detection of chicken anemia virus in the gonads and in the progeny of broiler breeder hens with high neutralizing antibody titers

Previous evidence for the presence of chicken anemia virus (CAV) in the gonads of immune specific-pathogen-free chickens raised the question whether this occurs also in commercial breeders. The presence of CAV was investigated by nested PCR in the gonads and spleens of hens from two 55- and 59-week-old, CAV-vaccinated (flocks 2 and 3), and two 48- and 31-week-old non-vaccinated broiler breeder flocks (flocks 1 and 4). In addition, lymphoid tissues of 20-day-old embryos from these hens were also investigated for the presence of CAV. CAV was detected in the gonads and of 5/6 and 11/22 of the vaccinated hens and in some hens also in the spleen alone. Embryos from 7/8 and 5/18 of these hens were positive. In the non-vaccinated flocks, CAV was detected in the gonads of 11/34 and 10/10 hens in flocks 1 and 4, respectively. In addition, 11 birds in flock 1 had positive spleens. CAV DNA was detected in 3/11 and 2/10 of their embryos. CAV-positive gonads and embryos were detected in samples from hens with moderate as well as high VN antibody titers. Vaccinated chickens positive for CAV in the gonads and in their embryos had VN titers ranging from >1:512 to <1:2048. In non-vaccinated chickens, the VN titers of CAV positive chickens ranged from 1:128 to 1:4096. These results demonstrate that CAV genome can remain present in the gonads of hens in commercial broiler breeder flocks even in the presence of high neutralizing antibody titers that have been associated with protection against CAV vertical transmission. It also suggests that transmission to the progeny may occur irrespectively of the level of the humoral immune response in the hens.     

Sequence analysis of the full-length cloned DNA of a chicken anaemia virus (CAV) strain from Bangladesh: evidence for genetic grouping of CAV strains based on the deduced VP1 amino acid sequences

Chicken anaemia virus (CAV) was detected in the bursa of Fabricius of a 4-week-old chicken obtained from an outbreak of acute infectious bursal disease in Bangladesh. Repeated attempts to grow this virus in MDCC-MSB1 cells were not successful. A full-length PCR amplicon of the genome of this strain, designated as BD-3 CAV, was cloned and sequenced. The complete nucleotide sequence and the deduced amino acid sequence were compared with those of 12 other CAV strains. The genetic analysis of the amino acid sequences of VP1 indicated the possible existence of genetic groups among CAV strains, as BD-3 CAV along with four other strains (CIA-1, L-028, Isolate 704 and TR-20) formed a distinct lineage. These strains have four signatory amino acids in VP1, such as 75I/T, 97L, 139Q and 144Q, out of which the latter two are located in a small hydrophilic peak.     

Sequence Analysis of the Full‐Length Cloned DNA of a Chicken Anaemia Virus (CAV) Strain from Bangladesh: Evidence for Genetic Grouping of CAV Strains Based on the Deduced VP1 Amino Acid Sequences

Chicken anaemia virus (CAV) was detected in the bursa of Fabricius of a 4‐week‐old chicken obtained from an outbreak of acute infectious bursal disease in Bangladesh. Repeated attempts to grow this virus in MDCC‐MSB1 cells were not successful. A full‐length PCR amplicon of the genome of this strain, designated as BD‐3 CAV, was cloned and sequenced. The complete nucleotide sequence and the deduced amino acid sequence were compared with those of 12 other CAV strains. The genetic analysis of the amino acid sequences of VP1 indicated the possible existence of genetic groups among CAV strains, as BD‐3 CAV along with four other strains (CIA‐1, L‐028, Isolate 704 and TR‐20) formed a distinct lineage. These strains have four signatory amino acids in VP1, such as ⁷⁵I/T, ⁹⁷L, ¹³⁹Q and ¹⁴⁴Q, out of which the latter two are located in a small hydrophilic peak.     

Correlation of enzyme-linked immunosorbent assay titers with protection against infectious bursal disease virus.

We examined the utility of baculovirus-expressed infectious bursal disease virus (IBDV) proteins to act as antigens in the enzyme-linked immunosorbent assay (ELISA). The three IBDV protein antigens tested included 1) a truncated VP2, 2) whole VP2, and 3) the polyprotein products VP2, VP3, and VP4. Serum samples from 2-wk-old commercially reared broilers were collected and tested in the three ELISAs. Serum samples were obtained from 34 different commercial broiler flocks. An average of 14 serum samples (range = 11-17) were tested for each flock. The ELISA results were compared with the percentage of protection of these birds following challenge with IBDV. Fifty 2-wk-old chicks from each of the 34 broiler flocks were challenged with STC classic virus or Del-E variant virus. At 7 days postchallenge, the bursa from each of the birds was removed and bursa/body weights were recorded. Percentage of protection was determined by the number of birds in each challenge group that had normal relative bursal weights compared with unchallenged controls. No evidence was found of a relationship between ELISA data generated with the polyprotein antigen (VP2, VP3, VP4) and percentage of protection observed in the STC and Del-E challenged birds. A significant relationship was found between ELISA data and percentage of protection to STC and Del-E when the truncated VP2 or whole VP2 antigens were used in the ELISA. The results of this study indicate that predicting the percentage of protection against classic or variant IBDV strains in broilers from vaccinated breeder flocks can be improved when VP2 is used as the only antigen in the ELISA.     

Inclusion body hepatitis as a primary disease in broilers in Saskatchewan, Canada

In recent years inclusion body hepatitis (IBH) has emerged as an economically important disease in Western Canada. Historically, infections with infectious bursal disease virus (IBDV) and chicken anemia virus (CAV) have been known to suppress the immune system of broilers and make them more susceptible to a secondary disease such as IBH. Recently it has been reported that virulent adenoviruses are able to cause IBH as a primary disease in broilers without apparent involvement of IBDV and CAV. The objectives of this study were to examine the possible association of IBH with IBDV and CAV infections in Western Canada and to identify adenoviruses involved in outbreaks. Serum samples from 17 broiler-breeder flocks and their progeny were collected when broilers were hatched and then again from broilers at the time of slaughter, and these samples were tested for IBDV and CAV antibodies by enzyme-linked immunosorbent assay (ELISA). Based on the ELISA titers the antibody response to vaccination against IBDV and CAV was at an expected level in all broiler flocks. Therefore, IBH outbreaks in these flocks were not due to inadequate levels of antibodies against IBDV and CAV. Moreover, there was no correlation found between occurrences of IBH outbreaks in broilers and their IBDV or CAV titers at the time of processing. Viruses that were isolated from livers of birds suffering from IBH could be classified into four different genotypes. Their hexon gene loop 1 sequences showed high percentages of identity to FAdV-7, FAdV-8a, FAdV-8b, and FAdV-11. The results of this study could not demonstrate an association of IBH with IBDV and CAV infections, but they supported the hypothesis that IBH in broilers in Western Canada is a primary disease with no apparent immunosuppressive involvement.     

我国白羽肉用型鸡群中CAV、REV和REOV感染状况的血清学调查

为了解鸡传染性贫血病毒（CAV）、禽网状内皮增生病病毒（REV）和呼肠孤病毒（REOV）在我国白羽肉用型鸡中的感染状态，在2003—2004年，检测了来自5省市8个公司不同年龄鸡群血清样品中3种病毒抗体的存在状况。结果表明，在送检的75个鸡群中，对CAV、REV和REOV呈现抗体阳性的鸡群分别有64个（85．3％）、36个（48％）和74个（96％）。在总共检测的1764份血清样品中，对这3种病毒的平均抗体阳性率分别为51．4％、9．8％和75．1％。在1日龄雏鸡，对CAV和REOV的平均母源抗体阳性率可达100％和81．1％，但对REV只有7．4％。抗体阳性率随年龄变化的动态分析表明，对REV和REOV的母源抗体在出壳后2～3周内消失，而对CAV的母源抗体则可持续3～4周。对CAV和REOV的抗体从5周龄起再次出现，到20周龄时，所有送检鸡群全部阳性，平均阳性率在90％以上。有近一半送检鸡群对REV呈现抗体阳性，抗体阳性率普遍较低，即使在达到开产年龄后，仍还有很高比例鸡为抗体阴性，即对REV仍为易感鸡。研究表明，我国多数鸡群中都同时存在着这3种病毒的感染，但它们在感染的程度和动态等流行病学特点上显著不同，应根据鸡群中抗体的阳性率分别采取不同的措施。     

犊牛腹泻粪便样本中纽布病毒VP1和RNA依赖性RNA聚合酶基因的序列分析

纽布病毒（Nebovirus，NeV）是国内犊牛腹泻的新发病原，其VP1蛋白含有受体结合位点和中和抗原表位，与病毒的感染和免疫密切相关，本研究旨在分析VP1基因1.2型毒株的分子特征。采用RT-PCR方法，对2019年宁夏和河南的犊牛腹泻粪便样本进行NeV检测，扩增阳性样本完整的主要衣壳蛋白（VP1）和RNA依赖性RNA聚合酶（RdRp）。结果显示，宁夏和河南地区NeV检出率分别为11.32%和8.62%。从4个样本中成功获得了1.2型毒株完整VP1和RdRp序列。4个完整VP1与GenBank中73个完整VP1的氨基酸相似性为75.4%~97.8%；与GenBank中仅有的3个1.2型VP1相比，4个毒株在P2区有1个共同的氨基酸突变，在P1区有2个共同的氨基酸突变。与国内基因1.1型、1.3型和1.4型毒株相比，在P2区分别有9、18和14个共同的氨基酸突变，在P1区分别有2个共同的氨基酸突变，在S区分别有1个共同的氨基酸突变。4个完整RdRp均为NB-like基因型，与GenBank中8个完整RdRp的核苷酸相似性为67.2%~94.8%。本文首次在我国检测到VP1基因1.2型毒株，成功获得了4条基因1.2型毒株的完整VP1和RdRp序列，为国内NeV的分子流行病学和遗传进化研究等提供了参考。     

Development and characterization of a potential diagnostic monoclonal antibody against capsid protein VP1 of the chicken anemia virus

Chicken anemia virus (CAV) is an important viral pathogen that causes anemia and severe immunodeficiency syndrome in chickens worldwide. In this study, a potential diagnostic monoclonal antibody against the CAV VP1 protein was developed which can precisely recognize the CAV antigen for diagnostic and virus recovery purposes. The VP1 gene of CAV encoding the N-terminus-deleted VP1 protein, VP1Nd129, was cloned into an Escherichia (E.) coli expression vector. After isopropyl-β-D-thiogalactopyronoside induction, VP1Nd129 protein was shown to be successfully expressed in the E. coli. By performing an enzyme-linked immunoabsorbent assay using two coating antigens, purified VP1Nd129 and CAV-infected liver tissue lysate, E3 monoclonal antibody (mAb) was found to have higher reactivity against VP1 protein than the other positive clones according to the result of limiting dilution method from 64 clones. Using immunohistochemistry, the presence of the VP1-specific mAb, E3, was confirmed using CAV-infected liver and thymus tissues as positive-infected samples. Additionally, CAV particle purification was also performed using an immunoaffinity column containing E3 mAb. The monoclonal E3 mAb developed in this study will not only be very useful for detecting CAV infection and performing histopathology studies of infected chickens, but may also be used to purify CAV particles in the future.     

Application of high-resolution melting curve analysis for typing of fowl adenoviruses in field cases of inclusion body hepatitis

Objective Fowl adenoviruses (FAdVs) cause inclusion body hepatitis (IBH) in chickens. In this study, clinical cases of IBH from Australian broiler flocks were screened for the presence and genotype of FAdVs. Methods Twenty‐six IBH cases from commercial poultry farms were screened. Polymerase chain reaction (PCR) coupled with high‐resolution melt (HRM) curve analysis (PCR/HRM genotyping) was used to determine the presence and genotype of FAdVs. For comparison, field isolates were also assessed by virus microneutralisation and nucleotide sequence analysis of the hexon loop 1 (Hex L1) gene. PCR detection of chicken anaemia virus (CAV) and infectious bursal disease virus (IBDV) was also employed. Results FAdV‐8b and FAdV‐11 were identified in 13 cases each. In one case, FAdV‐1 was also identified. Cross‐neutralisation was observed between the FAdV‐11 field strain and the reference FAdV‐2 and 11 antisera, a result also seen with the type 2 and 11 reference FAdVs. Field strains 1 and 8b were neutralised only by their respective type antisera. The FAdV‐8b field strain was identical to the Australian FAdV vaccine strain (type 8b) in the Hex L1 region. The Hex L1 sequence of the FAdV‐11 field strain had the highest identity to FAdV‐11 (93.2%) and FAdV‐2 (92.7%) reference strains. In the five cases tested for CAV and IBDV, neither virus was detected. The evidence suggested the presence of sufficient antibodies against CAV and IBD in the parent flocks and there was no indication of immunosuppression caused by these viruses. Conclusion These results indicate that PCR/HRM genotyping is a reliable diagnostic method for FAdV identification and is more rapid than virus neutralisation and direct sequence analysis. Furthermore, they suggest that IBH in Australian broiler flocks is a primary disease resulting from two alternative FAdV strains from different species.     

Isolation and preliminary characterization of chicken anemia virus from chickens in Nigeria

Chicken anemia virus (CAV) was isolated for the first time from the Nigerian chicken population. The virus was recovered from necropsied birds from broiler and pullet flocks that suffered disease outbreaks tentatively diagnosed as infectious bursal disease. A sensitive polymerase chain reaction (PCR) assay detected CAV DNA in tissues of necropsied birds. Restriction endonuclease analysis performed with the 733-bp PCR product and the Cfo I enzyme indicated at least two different CAVs were circulating among the Nigerian chicken population. Four isolates were obtained from pooled liver and thymus tissues using the MDCC-MSB1 cell line. These isolates were found to be antigenically closely related to the Cuxhaven-1 (Cux-1) reference strain of CAV when reacted with four monoclonal antibodies prepared against the Cux-1 virus. One of the isolates (isolate A) induced thymus atrophy, bone marrow aplasia, and low hematocrit values when inoculated into 1-day-old specific-pathogen-free chickens. These findings not only demonstrate that CAV is present in Nigeria, but they also likely represent the first cell culture isolation of the virus in Africa.     

鸡贫血病毒衣壳蛋白VP1基因的克隆、原核表达和纯化

根据GenBank中CAV哈尔滨分离株基因序列,设计出针对CAV VP1大片段(608 bp)的引物,利用PCR方法从CAV基因组序列中扩增出VP1基因的大片段(608 bp),按照正确的读码框克隆到原核表达载体pET32a( )上,得到含VP1大片段的pET32a( )重组子,转化大肠埃希菌BL21(DE3)感受态细胞,IPTG诱导重组蛋白表达,经SDS-PAGE和Western blotting检测发现有分子质量约42 ku的融合蛋白表达,与预期分子质量大小一致,通过Ni亲和层析柱纯化出融合蛋白,经Western blotting鉴定,融合蛋白与His单抗能够结合.CAV VP1基因的克隆表达及其融合蛋白的纯化为后续制备多克隆抗体和单克隆抗体提供了良好的抗原来源,也为研究VP1与其他CAV蛋白之间的相互关系奠定了基础.     

商品肉鸡群中MDV、REV、CAV和ARV多重感染的检测

从山东省东营、日照、潍坊、聊城等地区自然发病和临床健康AA商品肉鸡群中分别采集脏器样品,用特异性核酸探针对样品进行马立克氏病病毒（Marek’s disease virus,MDV）、网状内皮组织增生症病毒（Reticuloendotheliosis virus,REV）、鸡传染性贫血病病毒（Chicken anemia virus,CAV）和禽呼肠孤病毒（Avian reoviruses,ARV）检测。结果显示,自然发病AA商品肉鸡群中MDV、REV、CAV和ARV的检出率均较高,分别为69．30％、57．46％、63．60％和67．11％;临床健康AA商品肉鸡群中MDV、REV、CAV的检出率分别为36．96％、43．48％和30．42％,且自然发病和健康鸡群中均存在不同病毒组合的多重感染,感染率分别为85．96％和43．46％。用x2检验进行分析发现,自然发病商品肉鸡群与临床健康商品肉鸡群中MDV、CAV、MDV＋REV、REV＋CAV的检出率和未检出的比例差异极显著（P〈0．01）;REV、MDV＋CAV检出率差异显著（P〈0．05）。对自然发病商品肉鸡的肝脏、脾脏、法氏囊中4种病毒检出率进行X2检验分析发现,MDV在脾脏中检出率显著高于肝脏和法氏囊;REV在法氏囊中检出率显著高于肝脏和脾脏,而CAV和ARV分别在脾脏和肝脏中检出率较高。结果表明,多种免疫抑制性病毒的共感染已普遍存在,是目前AA商品肉鸡易发病且生长缓慢的重要流行病学因素之一。     

Serological evidence of chicken infectious anemia virus in the United States at least since 1959

A retrospective, serological survey was performed to determine an approximate time frame for when chickens were first exposed to chicken anemia virus (CAV) in the southeastern United States. A serum collection covering most of the period between 1959 and 2005 was available for the present study. These sera were obtained from adult chicken flocks that were maintained in experimental chicken farms at Auburn University's Department of Poultry Science. Sera were tested for the presence of CAV-specific antibodies using a commercially available competitive enzyme-linked immunosorbent assay (ELISA) kit. Values <0.6 were considered positive. Fresh sera obtained from hens in 2005 showed 45.5% negative and 54.5% positive for CAV antibodies. The assessment of serum samples covering the time period of 1959 through 1979 resulted in most sera being positive for CAV antibodies. The percentage of positive samples between years varied from 43% to 100%. These serological results support assumptions based on circumstantial evidence that CAV must have been present in the United States long before its first isolation in 1989.     

Biological characteristics of chicken anemia virus regenerated from clinical specimen by PCR

Our previous genetic characterization of chicken anemia virus (CAV) in commercial broiler chickens in Alabama revealed a previously undetected polymorphism: a glutamine codon at VP1 position 22, in 7 of the 14 sequences. The novel glutamine codon was always found in association with a VP1 "hypervariable region" identical to CAV field isolates that replicate poorly in culture. The complete genome of CAV73, representative of the sequences with the novel polymorphism, was generated from cloned polymerase chain reaction (PCR) fragments amplified directly from naturally infected tissues. CAV73 had been detected in 31-day-old broilers submitted for examination for reasons unrelated to anemia. After electroporation of the cloned genomes into MDCC-CU147 lymphoblastoid cells, the regenerated CAV caused the culture to fail within 9 days, and the medium contained 5 X 10(6) TCID50 CAV/ml. Use of MDCC-CU147 cells was essential, as identical electroporation of MDCC-MSB1 cells failed to generate CAV able to destroy the culture within 8 wk. Regenerated CAV73 produced anemia and severe lymphocytic depletion of the thymus when inoculated into susceptible 3-day-old chickens and was reisolated from these chickens. Furthermore, it replicated in low- and high-passage MDCC-MSB1 cells similarly to a low-passage CAV field isolate that contains a different VP 1 "hypervariable region." The regeneration of CAV from PCR products directly from naturally infected carcasses, as performed in this study, provides a tool for the evaluation of distinct genetic polymorphisms that may be detected in specimens where infective virions are no longer available. Our results also provide some insight into the differential susceptibility of cell lines for low-passage CAV field isolates.     

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.     

Development of a DNA vaccine against chicken anemia virus by using a bicistronic vector expressing VP1 and VP2 proteins of CAV

In the present study, we describe the development of a DNA vaccine against chicken anemia virus. The VP1 and VP2 genes of CAV were amplified and cloned into pBudCE4.1 to construct two DNA vaccines, namely, pBudVP1 and pBudVP2-VP1. In vitro and in vivo studies showed that co-expression of VP1 with VP2 are required to induce significant levels of antibody against CAV. Subsequently, the vaccines were tested in 2-week-old SPF chickens. Chickens immunized with the DNA-plasmid pBudVP2-VP1 showed positive neutralizing antibody titer against CAV. Furthermore, VP1-specific proliferation induction of splenocytes and also high serum levels of Th1 cytokines, IL-2 and IFN-γ were detected in the pBudVP2-VP1-vaccinated chickens. These results suggest that the recombinant DNA plasmid co-expressing VP1 and VP2 can be used as a potential DNA vaccine against CAV.