Two Akabane virus glycoprotein Gc domains induce neutralizing antibodies in mice

Akabane virus (AKAV), belonging to the genus Orthobunyavirus and family Peribunyaviridae, causes reproductive and congenital abnormalities in ruminants. Its envelope glycoprotein Gc is a neutralizing antigen, on which at least five distinct antigenic regions have been identified. We attempted to identify the domains using truncated recombinant AKAV Gc proteins expressed in Escherichia coli and monoclonal antibodies (mAbs) with AKAV-neutralizing activity. Dot blot analysis revealed that amino acid positions 1–97 and 189–397 (Gc_1–97 and Gc_189–397) in the truncated recombinant proteins reacted with the mAbs. Additionally, AKAV was neutralized by sera from mice immunized with these recombinant proteins. The results suggested that the two domains contain neutralizing epitopes and could be potential subunit vaccines against AKAV.     

犬呼肠病毒3型在BHK-21细胞上增殖的研究

采用酶标ＳＰＡ法进行了犬呼肠３ 型病毒在ＢＨＫ２１ 细胞上定位的研究。结果表明：接毒后６ ｈ 即可在胞浆见到絮状阳性反应,２４ ｈ 后呈强阳性着染;Ｈ．Ｅ．染色在胞浆内可见强嗜酸性着染或嗜酸性包涵体,提示可能是病毒感染引起的特征性病变,感染后细胞病变发展较快,６０ ｈ 细胞大部脱落;病毒血凝滴度结果也证实了细胞病变与病毒增殖有关。     

3种禽类呼肠孤病毒血清学相关性及致细胞病变差异分析

本研究旨在研究3种不同疾病型禽类呼肠孤病毒间的抗原性关系及病毒的培养特性。作者通过血清中和试验测定了禽呼肠孤病毒(ARV S1133株)、番鸭呼肠孤病毒(MDRV 9710株)、新型鸭呼肠孤病毒(NDRV NP01株)3种禽类呼肠孤病毒的血清学相关性,统计抗原相关性R值;并应用部分禽胚原代细胞及哺乳动物传代细胞对这3种病毒的培养特性进行了初步研究。结果表明,3种病毒株之间的R值很小,抗原相关性较低;三者具有广泛的细胞亲嗜性,能在多种细胞中增殖,并产生细胞病变,但病毒致细胞病变特征有所差异,ARV和NDRV均以巨融合为主,而MDRV则以细胞圆缩坏死为主。上述结果表明导致禽类不同疾病的ARV、MDRV和NDRV三者之间的抗原相关性较低,病毒的细胞培养特性也不同,细胞病变类型的差别提供了一种初步鉴别禽类呼肠孤病毒的方法。     

Acquired Fanconi syndrome in a dog exposed to jerky treats in Japan

A 6-year-old spayed female Jack Russell Terrier presented with a 1-month history of lethargy, anorexia, vomiting and weight loss. The dog was fed beef and chicken jerky treats daily in addition to a commercial diet. Laboratory tests revealed azotemia, hypokalemia, hyperchloremia, metabolic acidosis and glucosuria with normoglycemia. Urine amino acid analysis showed significant amino acid loss into the urine. Thus, Fanconi syndrome was diagnosed, and based on the case history and extensive diagnostic testing, excessive consumption of jerky treats was strongly suspected as the cause. Glucosuria resolved 7 days after the withdrawal of jerky treats and fluid therapy. Aminoaciduria was substantially, but not completely, improved 3 months after diagnosis. Mild azotemia remained, suggesting chronic renal disease. To the best of our knowledge, this is the first reported case of Fanconi syndrome following the consumption of jerky treats in Japan.     

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.     

Comparison of Immunoperoxidase Plaque Staining and Neutralizing Tests for Canine Distemper Virus

The use of immunoperoxidase plaque staining (IP) in the antibody test for canine distemper virus (CDV) and its diagnostic value were investigated. Antibody titres by the IP test and neutralizing test (NT) against CDV were compared in 114 specific pathogen-free (SPF) dogs, including 84 CDV-vaccinated and 30 unimmunized dogs. All of the unimmunized dogs, which had negative NT titres, also showed negative IP titres, and all of the CDV-vaccinated dogs, which had positive NT titres, showed positive IP titres. Furthermore, the IP titres showed similar changes with time after vaccination to the NT titres in five CDV-vaccinated SPF dogs. These findings indicate that the IP test has similar specificity to the NT.Next, the IP and NT titres were compared in 190 clinically suspected cases of canine distemper (CD), including 107 dogs with neurological signs and 83 dogs without neurological signs, and also in 50 clinically healthy dogs. Among the suspected cases of CD, of 46 dogs with neurological signs and 29 dogs without neurological signs, and which had negative NT titres, 33 (72%) and 19 (66%), respectively, showed positive IP titres. Many suspected cases of CD showed a high IP titre despite a low NT titre. In contrast, among healthy dogs, none of 9 NT-negative dogs showed positive IP titres.The difference in the IP and NT titres (log₁₀ [reciprocal of IP titre] – log₁₀ [reciprocal of NT titre]) was measured to investigate the agreement between IP test and NT. The differences were 1.488±0.867, 1.245±0.833, 0.595±0.351 and 0.594±0.372, respectively, in the suspected cases of CD with neurological signs, the suspected cases of CD without neurological signs, the healthy dogs and the SPF dogs. The differences in the suspected cases of CD were significantly high compared to those for the SPF dogs (p<0.001).Furthermore, in 8 dogs diagnosed as cases of CD from their paired sera, whose initial sera showed a markedly low NT titre compared to the IP titre, 5 dogs without neurological signs developed neurological signs later.These findings suggest that the IP test can detect CD during its early phases and in dogs with low antibody levels, undetectable by NT, and so would be useful for serological diagnosis.     

Antibody Testing Against Canine Coronavirus by Immunoperoxidase Plaque Staining

The application of the immunoperoxidase (IP) plaque staining procedure (IP test) to the diagnosis of canine coronavirus (CCV) infection was investigated. The IP test did not react with sera from either 15 specific pathogen-free (SPF) dogs or 7 SPF dogs immunized with a multivalent vaccine, including canine parvovirus type 2, canine distemper virus, canine adenovirus type 2, and canine parainfluenza virus. To compare the IP test with the neutralizing test (NT), sera from 240 healthy dogs and from 3 experimentally CCV-infected dogs were examined. All 60 sera positive for NT antibody were positive for IP antibody, and all 180 sera negative for NT antibody were negative for IP antibody in the healthy dogs. The IP titres showed similar changes with time after CCV inoculation to those of the NT titres in the experimentally infected dogs. These findings indicate that the IP test specifically detected anti-CCV antibodies. When the IP test and NT were compared in dogs with diarrhoeic signs. 2.1% of 48 sera and 20.3% of 74 sera, which were all negative for NT antibody, were positive for IP antibody in the dogs of under one year of age and at least one year of age, respectively. The difference between the IP and NT titres (log₁₀ [reciprocal of IP titre] – log₁₀ [reciprocal of NT titre]) for the diarrhoeic dogs of under one year of age (2.350±0.931) was significantly larger than that for the healthy dogs (0.982±0.447) (p<0.0001), the NT titre being negative or very low, despite a high IP titre in many diarrhoeic dogs. Hence, the IP test is more able to detect anti-CCV antibodies, especially in dogs showing clinical signs. The IP-positivity rate was significantly higher in the diarrhoeic dogs of under one year of age (48.7%) than in the healthy dogs (25.0%) (² = 19.844, p<0.0001), suggesting that CCV may contribute to diarrhoea in many juvenile dogs.     

The oncolytic effects of reovirus in canine solid tumor cell lines

Oncolytic virotherapy is a new strategy for cancer treatment for humans and dogs. Reovirus has been proven to be a potent oncolytic virus in human medicine. Our laboratory has previously reported that canine mast cell tumor and canine lymphoma were susceptible to reovirus. In this study, canine solid tumor cell lines (mammary gland tumor, osteosarcoma and malignant melanoma) were tested to determine their susceptibility towards reovirus. We demonstrated that reovirus induces more than 50% cell death in three canine mammary gland tumors and one canine malignant melanoma cell line. The reovirus-induced cell death occurred via the activation of caspase 3. Ras activation has been shown to be one of the important mechanisms of reovirus-susceptibility in human cancers. However, Ras activation was not related to the reovirus-susceptibility in canine solid tumor cell lines, which was similar to reports in canine mast cell tumor and canine lymphoma. The results of this study highly suggest that canine mammary gland tumor and canine malignant melanoma are also potential candidates for reovirus therapy in veterinary oncology.     

新型鸭呼肠孤病毒σB蛋白的原核表达及其多克隆抗体制备

为制备新型鸭呼肠孤病毒（new-type duck reovirus,NDRV）XX株σB蛋白的多克隆抗体,试验经RT-PCR扩增NDRV XX株σB基因编码序列,构建原核表达质粒pET-32a（+）-σB,将其转化至大肠杆菌BL21（DE3）感受态细胞后,经IPTG诱导获得His-σB重组蛋白。SDS-PAGE显示成功表达出约55 ku的融合蛋白,主要以包涵体形式存在,其表达时的最佳诱导时间、IPTG诱导浓度分别为3 h和0.25 mmol/L。经Ni²⁺柱亲和层析纯化获得可溶性重组蛋白,将蛋白经Western blotting和蛋白质谱鉴定为高纯度的σB重组蛋白,将纯化后σB重组蛋白按合理免疫程序免疫家兔,获得多抗隆抗体经Western blotting分析显示出特异性的反应。本试验结果为NDRV σB蛋白功能的深入研究及基因工程疫苗的研发奠定了基础。