Molecular characterization of Malaysian fowl adenovirus (FAdV) serotype 8b species E and pathogenicity of the virus in specific-pathogen-free chicken

BackgroundInclusion body hepatitis (IBH) is an economically important viral disease primarily affecting broiler and breeder chickens. All 12 serotypes of fowl adenovirus (FAdV) can cause IBH.ObjectivesTo characterize FAdV isolates based on phylogenetic analysis, and to study the pathogenicity of FAdV-8b in specific-pathogen-free (SPF) chickens following virus inoculation via oral and intramuscular (IM) routes.MethodsSuspected organ samples were subjected to virus isolation and polymerase chain reaction (PCR) for FAdV detection. Hexon gene sequencing and phylogenetic analysis were performed on FAdV-positive samples for serotype identification. One FAdV-8b isolate, UPM/FAdV/420/2017, was selected for fiber gene characterization and pathogenicity study and was inoculated in SPF chickens via oral and IM routes.ResultsThe hexon gene phylogenetic analysis revealed that all isolates belonged to FAdV-8b. The fiber gene-based phylogenetic analysis of isolate UPM/FAdV/420/2017 supported the grouping of that isolate into FAdV species E. Pathogenicity study revealed that, chickens infected with UPM/FAdV/420/2017 via the IM route had higher clinical score values, higher percent mortality, higher degree of the liver lesions, higher antibody response (p < 0.05), and higher virus shedding amounts (p < 0.05) than those infected via the oral route. The highest virus copy numbers were detected in liver and gizzard.ConclusionsFAdV-8b is the dominant FAdV serotype in Malaysia, and pathogenicity study of the FAdV-8b isolate UPM/FAdV/420/2017 indicated its ability to induce IBH in young SPF chickens when infected via oral or IM routes.     

The fowl adenovirus (Fadv-11) outbreak in Iranian broiler chicken farms: The first full genome characterization and phylogenetic analysis

Fowl adenoviruses D and E (FAdV-D and E) can cause inclusion body hepatitis (IBH) in commercial chicken flocks. Recently, IBH outbreaks have been increasingly reported in different regions of Iran, particularly in broiler farms. The present study was conducted to perform, for the first time, a complete genome characterization of a FAdV isolate from an IBH outbreak in Iran. Briefly, liver samples were collected from affected broiler flocks and following viral DNA extraction and confirming by PCR technique; one positive sample was selected from an affected flock to conduct a complete genome sequencing. The current FAdV, named "Fowl_Adenovirus_D_isolate_iran/UT-Kiaee_2018", was placed into FAdV-11 serotype (D species). According to the complete genome sequence analysis, UT-Kiaee had high homology with Chinese and Canadian FAdV. The partial sequence of the hexon gene revealed that UT-Kiaee shared 100% identity with previous Iranian FAdVs. The present study was the first to report full genome FAdV in Iran and complete the puzzle of molecular epidemiology of FAdV in Iran through determining the possible origin of Iranian FAdvs, which are the causative agents of recent IBH outbreaks in Iran.     

Application of the polymerase chain reaction to detect fowl adenoviruses.

The possibility of using the polymerase chain reaction (PCR) for the detection of fowl adenoviruses (FAdV) was tested. The optimal reaction parameters were evaluated and defined for purified genomic DNA of type 8 fowl adenovirus (FAdV-8), and then the same conditions were applied for nucleic acid extracted from infected cells. One hundred picograms of purified viral DNA, or 250 FAdV-8-infected cells, were detected by ethidium bromide staining of the PCR products in agarose gels. The sensitivity was increased to 10 pg purified viral DNA, or 25 infected cells, when the PCR products were hybridized with a specific labeled probe. Several field isolates of FAdV and the CELO virus (FAdV serotype 1) could be amplified by the same primers and conditions, but the size of the amplicons was smaller than that for the FAdV-8 PCR product. Other avian viruses and uninfected cell cultures tested negative.     

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.     

Emergence of fowl aviadenovirus C-4 in a backyard chicken flock in California

Fowl aviadenovirus (FAdV) species D and E are associated with inclusion body hepatitis (IBH); species C, serotype 4 (hereafter, FAdV4) is associated with hepatitis–hydropericardium syndrome (HHS) in young chickens. Outbreaks of HHS have led to significant losses in the poultry industry in several countries, predominantly in China. In April 2020, FAdV4 was detected in a remote backyard flock in California. In a mixed flock of chickens of various breeds and ages (6 mo to 2 y old), 7 of 30 were found dead within a week without premonitory signs. One additional bird died after the flock was relocated to fresh pasture, bringing the total mortality to 8 of 30 (27%). Postmortem examination of 3 birds revealed good body condition scores and active laying. One chicken had subtle hemorrhages throughout the liver, and the other 2 had diffusely dark mahogany livers. On histopathology, 2 chickens had hepatic necrosis with hepatocytes containing large, mostly basophilic, intranuclear inclusion bodies, identified by electron microscopy as 82.2-nm diameter adenoviral particles. Virus isolation and genomic sequencing performed on a liver sample revealed strains with 99.9% homology to FAdV4 isolates reported from China. To our knowledge, FAdV4 has not been reported in the United States to date. Furthermore, the chickens affected here were all adults and exhibited a variation of serotype 4 disease in which IBH was present but not hydropericardium.     

2株Ⅰ群禽腺病毒的分离鉴定及致病性分析

为了解山东省禽腺病毒（fowl adenovirus,FAdV）毒株的基因遗传演化情况及致病性,本试验对山东省两家疑似暴发鸡包涵体肝炎和心包积液综合征鸡场采集的病料（肝脏、脾脏）进行PCR鉴定,并将鉴定为FAdV的2份阳性病料提取病毒液,接种鸡肝癌细胞（LMH）进行毒株传代培养和细胞病变（CPE）观察、PCR检测、TCID₅₀测定、鸡胚致病性试验、SPF鸡回归试验、病毒hexon部分基因扩增及序列分析。结果显示,试验成功分离到2株FAdV,2株分离株细胞传第1代即可观察到CPE,PCR均可扩增出大小为500 bp的片段,且2株分离株细胞F5代TCID₅₀分别为10^-7.75/0.1 mL和10^-7.60/0.1 mL,均对7日龄SPF鸡胚有强致病性;第1分离株和第2分离株对21日龄SPF鸡攻毒死亡率分别为80%和15%。hexon基因核苷酸同源性分析结果显示,第1分离株与FAdV-4株同源性最高（99.57%）,第2分离株与FAdV-8b株同源性最高（99.18%）。这2株FAdV分离株均与Ⅰ群禽腺病毒同源,与火鸡出血性肠炎病毒（HEV）所在的血清Ⅱ群及减蛋综合征病毒（EDSV）所在的血清Ⅲ群禽腺病毒位于不同分支。第1分离株基因型为C型,血清型为4型,命名为FAdV-SDC4株;第2分离株基因型为E型,血清型为8b型,命名为FAdV-SDE8b株。综上所述,FAdV-SDC4和FAdV-SDE8b株属于近几年国内流行毒株。本研究结果可为鸡包涵体肝炎、心包积液综合征的防控工作和疫苗株的选择提供科学依据。     

Pathotypic and molecular characterization of a fowl adenovirus associated with inclusion body hepatitis in Saskatchewan chickens

Inclusion body hepatitis (IBH) is one of the major global disease problems, causing significant economic losses to poultry industry of the United States and Canada. The disease is characterized by its sudden onset and high mortalities. Amongst different serotypes of fowl adenoviruses (FAdVs) associated with IBH, serotype 8 of group I FAdV has been isolated from majority of IBH cases. In present studies, we isolated a FAdV from morbid liver of a 17-day-old broiler from a Saskatchewan broiler farm. This newly isolated virus was designated as IBHV(SK). However, based on the sequence analysis of the L1 region of the hexon gene, the IBHV(SK) may be classified as FAdV 8b strain 764. These studies describe for the first time the complete hexon gene sequence of FAdV serotype 8b. Experimental infection of 2-day-old (n = 48) and 2-wk-old (n = 56) chicks caused 83% and 43% mortalities, respectively. Determination of the complete hexon gene sequence of IBHV(SK) with establishment of a disease model in chickens will facilitate the development of type-specific diagnostic reagents and assays for the evaluation of potential experimental vaccines against pathogenic FAdV infections.     

表达FAdV-8b Fiber蛋白重组FAdV-4的构建与鉴定

为了构建能够同时防控鸡肝炎-心包积液综合征(HHS)和包涵体肝炎(IBH)的新型疫苗,本研究利用禽腺病毒4型(FAdV-4)中国流行株CH/HNJZ/2015的反向遗传操作技术,将禽腺病毒8b型(FAdV-8b)中国流行株的全长Fiber基因插入至FAdV-4基因组中1 966 bp自然缺失区,构建感染性克隆并拯救出重组病毒rHNJZ-Fiber/FAdV8b,利用PCR、Western blot和间接免疫荧光试验对重组病毒rHNJZ-Fiber/FAdV8b进行鉴定。结果表明,FAdV-8b的Fiber基因得以准确插入,Fiber蛋白正确并稳定表达,重组病毒rHNJZ-Fiber/FAdV8b具有良好的遗传稳定性,在细胞中具有与亲本毒株相似的高滴度生长特性和复制动态。本研究构建的表达FAdV-8b中国流行株Fiber基因的重组FAdV-4将为同时研发HHS和IBH的有效疫苗提供新的思路。     

Gene Analysis of Penton Protein of 12 Serotypes of Fowl Adenovirus

In order to understand the genetic evolution of Penton protein of fowl adenovirus(FAdV),the specific primers of Penton were designed on the basis of published sequences of different genotypes on GenBank.Then Penton gene of 12 serotypes were amplified by PCR and constructed into pEASY-Blunt Simple Cloning Vector for sequencing.The nucleotide sequences of Penton protein of 12 serotypes were analyzed and compared with the nucleotide sequences of standard strains published on NCBI by ClustalW method to draw a genetic phylogenetic tree.The results showed that the nucleotide sequences of Penton protein of 12 serotypes had more than 99.2% homology with their respective standard strain,among which the homology of FAdV-1,FAdV-2,FAdV-3,FAdV-4,FAdV-6,FAdV-7,FAdV-8a,FAdV-8b,FAdV-10 and FAdV-11 were as high as 100%.This further confirmed the consistency between the laboratory preserved strains and the world standard strains.However,although Penton protein was highly conserved,there were still significant differences among different species.The nucleotide homology between FAdV-A1 and other species was 70.6%-73.3%,while that of FAdV-B5 was 70.6%-77.9%.The nucleotide homology between FAdV-C strains of different serotypes was 99.6%,while homology between FAdV-C4 and other species was 71.2%-73.4%.The nucleotide homology between FAdV-D strains of different serotypes was 95.6%-98.7%,while the highest homology between FAdV-D and other species was 85.3%.The nucleotide homology between FAdV-E strains of different serotypes was 98.2%-99.4%,while the highest homology between FAdV-E and other species was 85.3%.In conclusion,the homology difference between strains of the same species was small,and that of different species was significant.FAdV could be divided into A,B,C,D and E species according to nucleotide sequence of Penton protein.This study successfully cloned 12 serotypes Penton gene of FAdV and performed genetic evolution analysis,which laid the foundation for the diagnosis,monitoring and identification of FAdV in the future.     

鸡腺病毒12个血清型代表毒株Penton蛋白基因分析

为了解鸡腺病毒（fowl adenovirus,FAdV）Penton基因的遗传进化规律,试验根据GenBank中已公布的各基因型序列设计特异性引物,通过PCR技术分别扩增12个血清型毒株的Penton基因,连接至pEASY-Blunt Simple Cloning Vector上进行序列测定,并将12个血清型毒株Penton蛋白的核苷酸序列与NCBI上已公布的标准毒株的核苷酸序列使用ClustalW法进行分析比对,绘制遗传进化树。结果显示,12种血清型Penton蛋白核苷酸序列与各血清型标准毒株同源性均在99.2%以上,其中FAdV-1、FAdV-2、FAdV-3、FAdV-4、FAdV-6、FAdV-7、FAdV-8a、FAdV-8b、FAdV-10、FAdV-11与对应标准株同源性高达100%,这进一步证实了实验室保存毒株与世界相应标准毒株的一致性。Penton蛋白虽然保守性较高,但不同种之间仍存在明显差异。FAdV-A1与其他种毒株同源性为70.6%~73.3%;FAdV-B5与其他种毒株同源性为70.6%~77.9%;FAdV-C同种不同血清型之间核苷酸同源性为99.6%,FAdV-C4与其他种之间同源性71.2%~73.4%;FAdV-D同种不同血清型之间核苷酸同源性为95.6%~98.7%,与其他种之间最高为85.3%;FAdV-E同种不同血清型之间核苷酸同源性为98.2%~99.4%,不同种间最高为85.3%。即相同种毒株之间差异较小,不同种毒株之间差异较大。依据Penton蛋白的核苷酸序列同样可以将FAdV分为A、B、C、D、E 5个种。本研究通过成功克隆FAdV 12种血清型Penton基因,并进行遗传进化分析,为今后对FAdV诊断、监测及毒株鉴定提供了参考。     

广西某健康鸡群Ⅰ群禽腺病毒的hexon蛋白loop 1基因遗传进化分析

为调查广西某规模化鸡场健康鸡群Ⅰ群禽腺病毒(FAdV-Ⅰ)的感染情况,于2017年11月-2018年12月采集不同日龄鸡群咽喉拭子和泄殖腔拭子样品4700份,对样品进行FAdV-Ⅰ检测和hexon蛋白loop 1基因扩增,应用LaserGene7.1和MEGA4.1软件对loop1基因序列进行核苷酸和遗传进化分析。结果显示,FAdV-Ⅰ的阳性检出率为8.72%;40~70日龄和71~99日龄鸡群阳性FAdV-Ⅰ的检出率分别为28.17%和22.91%;测序获得的38条loop 1基因序列分析表明该鸡场健康鸡群感染了5个不同种的FAdV-Ⅰ,其中26.32%的序列为A种(FAdV-A)、2.63%为B种(FAdV-B)、10.53%为C种(FAdV-C)、39.47%为D种(FAdV-D)和21.05%为E种(FAdV-E);遗传进化分析表明该鸡场健康鸡群主要感染的种为FAdV-D,血清2型(FAdV-2)为优势血清型,其次为A种的血清1型(FAdV-1)和E种的血清8a型(FAdV-8a)和8b型(FAdV-8b)。结果表明,40~70日龄健康鸡群的FAdV-Ⅰ的检出率最高,在健康鸡群中感染的FAdV-Ⅰ主要为FAdV-D种中的FAdV-2。     

Serologic monitoring of a broiler breeder flock previously affected by inclusion body hepatitis and testing of the progeny for vertical transmission of fowl adenoviruses

The increasing number of clinical cases of inclusion body hepatitis (IBH) associated with fowl adenoviruses (FAdVs) is a growing concern in different parts of the world, including Canada. After an outbreak of IBH in a 10-d-old pullet broiler breeder flock, we serologically monitored the flock from 8 to 46 wk of age, using the agar gel precipitation test (AGPT) offered by diagnostic laboratories and an FAdV group-specific enzyme-linked immunosorbemt assay (ELISA) developed earlier. In addition, we tested 1-d-old progeny for possible vertical transmission of FAdV when the breeder flock approached the peak of egg production by performing virus isolation and polymerase chain reaction (PCR) procedures on target organs. As in previous studies comparing the 2 tests, ELISA was more sensitive than AGPT. With ELISA, a few birds had weakly positive results at 8 wk of age, and all the birds had strongly positive results from 12 wk of age until the end of the study. This group-specific ELISA is therefore a sensitive and practical way to monitor FAdV antibodies in commercial flocks. None of the 1-d-old chicks tested were positive by PCR, nor was FAdV isolated from the same tissues, indicating an absence of transmission of infectious virus to the progeny. The lack of virus production and transmission could be due to the presence of high antibody titers in the layers.     

Study of vertical transmission of fowl adenoviruses

The vertical transmission of fowl adenoviruses (FAdVs) was studied by polymerase chain reaction (PCR) and virus isolation. Liver, spleen, kidney, and bursa of Fabricius were collected from 60 chicks 1 d old representing progenies hatched to 6 broiler breeder flocks in 6 geographically different premises in Ontario, Canada. The presence of FAdV DNA sequences was detected by PCR with the use of primers specific for the conserved pVI gene of FAdV-9 in 58 (24%) of the 240 samples tested. All samples from 1 flock were negative for FAdV sequences, and only a few samples were positive in 3 flocks, whereas 32% and 72% of the samples from the other 2 flocks were positive. Testing of 1 sample with primers designed to amplify the L1 region of the hexon protein gene and amino acid sequence analysis of the PCR product indicated that the sequences were similar to serotype-8a FAdV sequences. No fowl adenoviruses were isolated in chicken hepatoma cells from any of the 30 samples inoculated. These findings imply that vertical transmission and establishment of latent infection with FAdVs can occur in chickens.     

Outbreaks of hydropericardium syndrome and molecular characterization of Korean fowl adenoviral isolates

Outbreaks ofhydropericardium syndrome (HPS), caused by fowl adenovirus serotype 4 (FAdV-4), have occurred in Korea and caused severe economic loss due to mortality and weight loss. From these outbreaks, several adenoviruses were isolated and identified in samples from broilers, layers, breeders, and native Korean fowl. In pathologic examinations, hydropericardium and multifocal hepatic necrosis, with an intranuclear inclusion body in hepatocytes, were observed. Specific adenovirus particles were also observed in the nucleus of hepatocytes, by electron microscopic examination. Polymerase chain reaction (PCR) analysis of the hexon gene identified all of the isolates as FAdV, serotype 4 and genotype C. To reproduce FAdV-4 field cases, 8- and 52-week-old specific pathogen free (SPF) chicks were infected intramuscularly with the field isolate CBU070244. The mortality rate of infected chicks ranged from 10%-40%, and specific pathologic lesions, such as swollen livers and hydropericardium, were observed. Further studies to determine the prevalence of infection, and analysis of the economic impact to the poultry industry, are needed in the near future.     

4株禽腺病毒hexon基因片段序列分析

目的验证购自中国兽医微生物菌种保藏管理中心（China Veterinary Culture Collection Center,CVCC）的4株禽腺病毒（FAdV-1 AV208、FAdV-4 AV211、FAdV-8 AV215、FAdV-11 AV218）的血清型。方法采用PCR的方法对4株FAdV hexon基因片段进行特异性扩增、克隆与测序,并与GenBank上公布的FAdV各血清型进行亲缘性分析。结果hexon基因片段核苷酸及其编码Hexon蛋白氨基酸亲源性分析结果表明,FAdV-1 AV208为禽腺病毒A血清1型;FAdV-4 AV211为禽腺病毒C中的血清4型;FAdV-8 AV215为禽腺病毒E中的血清8b型;FAdV-11 AV218为禽腺病毒C中的血清10型。结论FAdV-1 AV208、FAdV-4 AV211的血清型与CVCC标注一致;FAdV-8 AV215为FAdV-8b,相比CVCC该研究进一步细化了其血清型;而FAdV-11 AV218为FAdV-10,与CVCC标注的血清型不一致。该研究为今后正确使用CVCC标准毒株FAdV-8 AV215、FAdV-11 AV218提供了参考。     

禽腺病毒血清4型纳米PCR检测试剂盒的研制及应用

为研制一种高效、快捷、灵敏的禽腺病毒血清4型（FAdV-4）检测试剂盒，本研究根据FAdV-4的保守序列设计了一对引物及探针，并在下游引物标记FITC，探针标记Biotin，应用纳米PCR技术结合横向流动试纸条技术（LFD），优化反应及杂交条件后建立了检测FAdV-4的纳米PCR-LFD方法，并组装了FAdV-4纳米PCR检测试剂盒。同时对试剂盒进行了特异性、敏感性、批内批间可重复性、稳定性、保存期评估等试验及临床样品检测。特异性试验表明，此试剂盒能够特异性检测出FAdV-4，而对于禽的其他主要病毒性及细菌性病原如鸡传染性喉气管炎病毒（ILTV）、鸡传染性支气管炎病毒（IBV）、鸡传染性法氏囊病病毒（IBDV）、鸡马立克病病毒（MDV）、鸡减蛋综合征病毒（EDSV）、鸭瘟病毒（DPV）、新城疫病毒（NDV）、禽流感病毒H9亚型（AIV（H9））、禽呼肠孤病毒（ARV）、FAdV标准株（FAdV-1、FAdV-2、FAdV-8a、FAdV-8b和FAdV-11）、鸡大肠杆菌（E.coli）、金黄色葡萄球菌（SA）、鸡白痢沙门氏菌（SP）、鸡毒支原体（MG）的检测结果均为阴性。敏感性试验表明，此试剂盒的敏感性比常规PCR方法敏感10倍，最低核酸拷贝数检出量可以达到56.0拷贝/μL。试剂盒的批内、批间检测结果具有良好的一致性和稳定性。稳定性试验结果说明试剂盒至少可以耐受20次的反复冻融，依然有很好的检测效果。保存期试验证实，试剂盒在4 ℃条件下至少可以保存3个月，在-20 ℃条件下至少可保存12个月。此试剂盒实现了对FAdV-4检测结果的可视化，简化了操作流程，提高了检测效率。对天津及周边地区临床送检的117份样品检测结果显示，FAdV-4阳性率为17.95%（21/117）。本试验研制的试剂盒可用于FAdV-4的临床诊断和分子流行病学调查，对养禽场FAdV-4感染的早期检测与制定综合防控措施提供了重要的技术支撑。     

Ⅰ群禽腺病毒及其疫苗研究进展

1987年以来,禽腺病毒(Fowl adenoviruses,FAdV)在世界范围内感染普遍。2015年,我国山东省首先爆发该病毒引起的疫病,之后各省市地区相继蔓延流行,给养禽业造成了很大的经济损失,特别以心包积液综合征(hydropericardium syndrome,HPS)和包涵体肝炎(inclusion body hepatitis,IBH)造成的危害最大。本文主要将禽腺病毒的病原学、流行病学、临床特征研究及疫苗的研制等进展加以总结,以期为本病的防控提供参考。     

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.     

Isolation and Molecular Characterization of Fowl Adenovirus and Avian Reovirus from Breeder Chickens in Japan in 2019–2021

Fowl adenoviruses (FAdVs) and avian reoviruses (ARVs) are ubiquitous in poultry farms and most of them are not pathogenic, yet often cause damage to chicks. A total of 104 chicken fecal samples were collected from 7 farms of breeder chickens (layers and broilers) in Japan from 2019 to 2021, and yielded 26 FAdV plus 14 ARV isolates. By sequencing, FAdV isolates were classified as FAdV-1, 5 and 8b. ARV isolates were classified as genotype II, IV and V. These results suggest that FAdVs and ARVs are resident in the breeder chicken farms in Japan.     

表达禽腺病毒血清4型Fiber2蛋白的重组耐热新城疫病毒的构建及免疫效果评估

【目的】 研究针对新城疫病毒(Newcastle disease virus, NDV)和禽腺病毒(Fowl adenovirus, FAdV)的耐热基因工程疫苗。【方法】 利用反向遗传学操作技术将NDV耐热株的HN基因替换到LaSota疫苗株上, 再将禽腺病毒血清4型(Fowl adenovirus serotype 4, FAdV-4)的Fiber2基因插入到其基因组上, 构建表达Fiber2蛋白的重组耐热NDV质粒pTS-HN-Fiber2。通过病毒拯救技术拯救重组NDV rTS-HN-Fiber2, 并测定其生物学特性和作为疫苗候选株的免疫原性和攻毒保护性。【结果】 rTS-HN-Fiber2的鸡胚平均致死时间>168 h, 且脑内接种致病指数为0, 属于弱毒的范畴; 在细胞上的生长曲线结果表明, rTS-HN-Fiber2与亲本LaSota株有相似的生长曲线, 但最终的生长滴度略低于LaSota株; rTS-HN-Fiber2在56 ℃处理15 min后, 病毒滴度下降约10³ TCID₅₀/mL, 而LaSota株56 ℃处理5 min几乎无感染性; 间接免疫荧光试验结果表明, rTS-HN-Fiber2能表达Fiber2蛋白。免疫和攻毒试验结果显示, rTS-HN-Fiber2能产生NDV抗体, 且能显著提高雏鸡在FAdV-4强毒下的存活率, 减轻FAdV-4强毒引起的组织病变, 降低组织中的病毒载量。【结论】 本研究成功构建了表达FAdV-4 Fiber2蛋白的重组耐热NDV, 该病毒保持了亲本LaSota株的弱毒生物学特性, 但热稳定性有显著提升; 重组NDV免疫雏鸡可产生针对NDV和FAdV-4强毒的保护, 该重组NDV可作为开发针对FAdV-4和NDV二联基因工程疫苗的候选病毒株。