H3N2亚型犬流感病毒实验感染模型的建立

Establishment of a Canine Experimental Infection Model with a H3N2 Subtype Canine Influenza Virus

【目的】建立H3N2亚型犬流感病毒(canine influenza virus,CIV)实验感染模型,了解犬流感的发病特征,为疫苗效力评价奠定基础。【方法】6—13月龄CIV血凝抑制(haemagglutination inhibition,HI)抗体阴性(HI<1﹕10)比格犬26只,其中3只鼻腔喷雾PBS作为阴性对照,另23只分5组(10、103、105、106、107 EID50/只),分别为3、5、5、5、5只/组,每只犬各鼻腔喷雾H3N2亚型CIV(A/canine/China/Huabei-170607/2017(H3N2),简称HB株)病毒液1mL。观察临床症状、肺脏病变和肺脏组织病理学变化,计算肺实变率,检测病毒和HI抗体效价。【结果】10 EID50剂量感染组,3只犬均未出现明显临床症状,肺脏均无明显眼观病变,肺脏实变率0%,无组织病理学变化,病毒检测均为阴性,HI抗体效价几何平均值(geometric mean titer,GMT)为1﹕15.9;103 EID50剂量感染组,5只犬中有4只喘息、流鼻汁和咳嗽,1只犬未表现出临床症状,2只犬肺脏出现轻微实变,实变率平均为1.4%,4只犬病毒分离阳性,HI抗体效价GMT为1﹕320;105 EID50剂量感染组,5只犬在攻毒后5 d开始出现流鼻汁和咳嗽等临床症状,肺脏均有实变,实变率平均为4.2%,肺泡间隔增宽,病毒分离均为阳性,HI抗体效价GMT为1﹕2940.7;106 EID50剂量感染组,5只犬在攻毒后4 d体温升高、流鼻汁、咳嗽,临床症状出现较105 EID50剂量感染组早1 d,肺脏实变程度增加,实变率平均为17.9%,肺泡间隔增宽,病毒分离均为阳性,HI抗体效价GMT为1﹕2228.7;107 EID50剂量感染组,5只犬在攻毒后3 d表现出体温升高、流鼻汁、咳嗽等严重的临床症状,症状出现较106 EID50剂量感染组早1 d,肺脏严重实变,实变率平均为29.0%,肺泡间隔增宽,病毒分离均为阳性,HI抗体效价GMT为1﹕2940.7;对照犬均未出现明显临床症状,肺脏均无明显眼观病变,无组织病理学变化,病毒检测均为阴性,HI抗体效价均<1﹕10。【结论】106 EID50剂量病毒是能引起犬明显发病的最小病毒接种剂量,建立起H3N2亚型CIV实验感染模型。

【Objective】In this research,an experimental animal infection model of canine influenza virus(CIV,H3N2 subtype)was established to better understand the pathogenesis of canine influenza,so as to lay the foundation for vaccine efficacy evaluation.【Method】26 beagles aged 6-13 months with negative CIV Haemagglutination Inhibition(HI)antibody(HI<1﹕10)were selected,three beagles of which were challenged by 1mL nasal spray of PBS,and 23 beagles of which were challenged by 1mL nasal spray of H3N2 CIV(A/canine/China/Huabei-170607/2017(H3N2),HB strain for short)with 5 groups(10,103,105,106,and 10750%EID50)as 3,5,5,5,and 5 beagles each group,respectively.Clinical symptoms,lung lesions,histopathological changes of lung,calculating the proportion of consolidation mass,HI antibody titer and virus shedding were examined at 14 days after virus challenge together with three control beagles.【Result】3 beagles inoculated with a dose of 10 EID50 H3N2 CIV did not show any clinical symptoms,gross lesions in lungs and histopathological changes,the consolidation rate was 0%,and the virus shedding was not detected.The geometric mean titer(GMT)of HI antibodies was 1﹕15.9.However,4/5 of beagles inoculated with a dose of 103 EID50 H3N2 CIV showed the clinical symptoms and virus shedding,such as puffing,runny nose and cough.2/5 of beagles showed light lung consolidation,whose rate was 1.4%.The GMT of HI antibodies was 1:320.All beagles(5/5)infected with a dose of 105 EID50 H3N2 CIV showed clinical symptoms at day 5 after challenge,such as runny nose and cough,virus shedding,lung consolidation,and widened alveolar septum,and the consolidation rate was 4.2%.The GMT of HI antibodies was 1﹕2940.7.5/5 of beagles infected with a dose of 106 EID50 all showed severe clinical symptoms at day 4 after challenge,such as cough and elevated body temperature,virus shedding,obvious pathological features in the lungs,and the GMT of HI antibodies was 1﹕2228.7.The clinical symptoms appeared earlier 1 day than that in 105 EID50 dose infection group,and the degree of lung consolidation increased.The lung consolidation ratio was 17.9%.5/5 of beagles infected with doses of 107 EID50 all showed severe clinical symptoms at day 3 after challenge which appeared earlier 1 day than that in 106 EID50 dose infection group,virus shedding and widened alveolar septum in the lungs,the GMT of HI antibodies was 1﹕2940.7,and the consolidation rate of lung was 29.0%.The control beagles did not show any clinical symptoms,gross lesions and histopathological changes in lungs,the virus shedding was not detected,and GMT of HI antibodies was<1﹕10.【Conclusion】The dose of 106 EID50 was the minimum virus inoculation dose that could cause obvious pathogenesis in beagles.An experimental animal infection model of CIV subtype H3N2 in beagles was established.