禽流感病毒感染SPF鸭抗体消长规律及相关microRNA表达研究
摘要
MicroRNA (miRNA)是一类长度约22个核苷酸的内源性非编码的小RNA分子，广泛存在于各种生物体内。miRNA通过作用于靶mRNA的3′UTR区，引起靶mRNA的降解或抑制其翻译，从而实现对靶基因表达水平的调控，研究发现，miRNA在细胞发育分化，组织器官形成以及病毒感染等多种生物学过程中均发挥重要作用。
禽流感是由正粘病毒科流感病毒属A型流感病毒引起的以禽类为主的可导致人畜共患的烈性传染性。随着高致病性禽流感病毒感染水禽导致发病死亡的事实出现后，人们越来越意识到禽流感病毒对禽类乃至于对人类的危害。高通量测序技术是近年来发展起来的用于各物种miRNA分析鉴定的强有力工具。鸭miRNA的研究目前还处于空白的阶段，尤其对于感染禽流感病毒后miRNA的变化在水禽中还没有报道，因此本研究在对3月龄SPF鸭感染H5N1亚型禽流感病毒的基础上，采集感染鸭和SPF鸭的脾脏组织，利用高通量测序分析技术，鉴定感染与非感染H5N1亚型禽流感病毒状态下鸭脾脏组织的miRNA并对其进行差异表达分析，同时由于miRNA具有一定的组织特异性，这一特性使得miRNA对生物体的不同生长发育过程及不同的组织器官发挥多种不同的作用。因此本研究进一步对部分差异表达miRNA，利用荧光定量PCR的方法分析其在感染与非感染H5N1亚型禽流感病毒的鸭不同组织中的表达情况。
结果表明，SPF鸭感染H5N1亚型禽流感病毒后的第7天，可以检测到鸭体内病毒的表达，并在第15天达到最大值。利用高通量测序技术，成功获得感染与非感染鸭脾脏组织小RNA文库，总体分析显示，SPF鸭脾脏组织的小RNA种类和数量明显多于感染禽流感病毒鸭的脾脏组织。同时获得了322个差异表达的miRNA，其中105个显著上调，217个显著下调miRNA。另外在新发现的17个小RNA中，有1个显著上调，4个显著下调。随机选取9个差异表达miRNA进行定量分析，检测结果与高通量测序所得结果相一致，证明了高通量测序结果的准确性。不同组织定量分析结果显示，在H5N1亚型禽流感病毒感染状态下，miR-17a*，miR-30c*和miR-10在肌肉中的相对表达量最高，miR-4006f-5p和miR-6在气管中的相对表达量最高，miR-92b-3p在肺脏中的相对表达量最高，miR-4499在脾脏中的相对表达量最高，miR-2808e和miR-4002-5p在心脏中的相对表达量最高。而miR-17a*，miR-92b-3p，miR-30c*，miR-10和miR-6均在肝脏中的相对表达量最低，另外miR-4006f-5p，miR-4499和miR-2808e在肺脏中几乎不表达。在SPF鸭中miR-17a*，miR-30c*和miR-10在肺脏中的相对表达量最高，miR-4006f-5p在肌肉中相对表达量最高，miR-92b-3p和miR-4499在脾脏中的相对表达量最高，miR-6在肾脏中的相对表达量最高，miR-2808e和miR-4002-5p在心脏中的相对表达量最高。而miR-17a*，miR-4006f-5p，miR-10，miR-6和miR-4499均在肝脏中的相对表达量最低。所研究miRNA在病毒感染前后均在不同组织中具有不同的表达量。
本研究对鸭感染与非感染H5N1亚型禽流感病毒状态下的脾脏组织间miRNA组学差异进行了研究，结果表明miRNA在禽流感病毒作用下的水禽中可能具有重要调控作用，该结果为进一步研究miRNA对水禽感染禽流感病毒下的作用机制研究奠定了基础，同时为抗病育种的发展提供新的理论依据。
MicroRNA is a non-coding small RNA with about22nucleotides in lengthendogenous that widely present in a variety of organisms. MiRNA complementarypairing with the target mRNA3′UTR region, specificity base caused the degradationof the target mRNA or inhibiting their translation in order to achieve the level oftarget gene expression regulation. It may paly important role in a variety of biologicalprocesses such as cell development, differentiation, tissues and organa formation,viral infection, et al.
Avian influenza belongs to the orthomyxovirus virus family influenza virus, is apetent infectious poultry, caused by influenza A virus which can lead to human andanimal comorbid. The fact of that the incidence of death with highly pathogenic avianinfluenza virus infection waterfowl appears, human are increasingly aware of hazardsof AIV. High-throughput sequencing technology as a powerful tool for miRNAanalysis identified. Now ducks miRNA research is still in a blank stage, eapeciallyinfected and non-infected AIV duck.
In this study, on the basis of collected3-month-old SPF ducks spleentissueswhich infected and non-infected H5N1subtype avian influenza virus, usinghigh-throughput sequencing analysis techniques, identification and differentiallyexpressed analysis of miRNA in infected and non-infected with the H5N1subtype ofavian influenza virus ducks spleen tissue. The miRNA has a feature of tissue-specific,it makes miRNA play a variety of different role in the process of growth anddevelopment of organisms and different tissues and organs. Further, using quantitativePCR, we analyzed the expression of some differentially expressed miRNAs in ducksvarious tissue under infected and non-infected with the H5N1subtype of avian influenza virus.
The results show that we can detect the avian influenza virus expression in duckvivo after infected avian influenza virus seven days, and reaches a maximum at15days. Deep sequencing of the two libraries by using the high-throughput sequencingtechnology successfully generated. The result shown that the small RNA aresignificant more in types and quantity in SPF duck than the duck that infected H5N1subtype of avian influenza virus.322known differentially expressed miRNAs wereidentified, of which105were significantly up-regulated and217were significantlydown-regulated. In addition,17novel small RNA were identified, including1wassignificantly up-regulated miRNA and4were significantly down-regulated miRNA.The quantitative analysis of nine differentially expressed miRNA that randomlyselected, the result verified the accuracy of the results of the high-throughputsequencing.
The different tissues Quantitative analysis results show that the ducks under theH5N1subtype of avian influenza virus infection, miR-17a*, miR-30c*and miR-10have highest relative expression level in muscle, miR-4006f-5p and miR-6havehighest relative expression level in trachea, miR-92b-3p has highest relativeexpression level in lungs, miR-4499highest relative expression level in spleen,miR-2808e and miR-4002-5p have highest relative expression level in heart.However，miR-17a*, miR-92b-3p, miR-30c*, miR-10and miR-6have lowest relativeexpression lever in liver. In addition, miR-4006f-5p, miR-4499and miR-2808e almosthave no relative expression in lung. In SPF ducks, miR-17a*, miR-30c*, miR-10havehighest relative expression level in lung, miR-4006f-5p has highest relative expressionlevel in muscle, miR-92b-3p and miR-4499have highest relative expression level inspleen, miR-6has highest relative expression level in kidney, miR-2808e andmiR-4002-5p have highest relative expression level in heart. However, miR-17a*,miR-4006f-5p, miR-10, miR-6and miR-4499in liver have lowest relative expressionlevel. In this study, we found that miRNA has a different expression of the same tissue,in SPF duck and infected ducks.
In this study, the miRNA genomics analysis between Duck spleen tissue infectedand non-infected with the H5N1subtype of avian influenza virus were performed, theresults show that miRNA may play an important regulation role in waterfowl in therole of avian influenza virus. This result may lay the foundation for the further studyin miRNA research on the machanism of waterfowl under infected with avianinfluenza virus. At the same time provide a new theoretical basis for the developmentof disease-resistant breeding.
禽流感是由正粘病毒科流感病毒属A型流感病毒引起的以禽类为主的可导致人畜共患的烈性传染性。随着高致病性禽流感病毒感染水禽导致发病死亡的事实出现后，人们越来越意识到禽流感病毒对禽类乃至于对人类的危害。高通量测序技术是近年来发展起来的用于各物种miRNA分析鉴定的强有力工具。鸭miRNA的研究目前还处于空白的阶段，尤其对于感染禽流感病毒后miRNA的变化在水禽中还没有报道，因此本研究在对3月龄SPF鸭感染H5N1亚型禽流感病毒的基础上，采集感染鸭和SPF鸭的脾脏组织，利用高通量测序分析技术，鉴定感染与非感染H5N1亚型禽流感病毒状态下鸭脾脏组织的miRNA并对其进行差异表达分析，同时由于miRNA具有一定的组织特异性，这一特性使得miRNA对生物体的不同生长发育过程及不同的组织器官发挥多种不同的作用。因此本研究进一步对部分差异表达miRNA，利用荧光定量PCR的方法分析其在感染与非感染H5N1亚型禽流感病毒的鸭不同组织中的表达情况。
结果表明，SPF鸭感染H5N1亚型禽流感病毒后的第7天，可以检测到鸭体内病毒的表达，并在第15天达到最大值。利用高通量测序技术，成功获得感染与非感染鸭脾脏组织小RNA文库，总体分析显示，SPF鸭脾脏组织的小RNA种类和数量明显多于感染禽流感病毒鸭的脾脏组织。同时获得了322个差异表达的miRNA，其中105个显著上调，217个显著下调miRNA。另外在新发现的17个小RNA中，有1个显著上调，4个显著下调。随机选取9个差异表达miRNA进行定量分析，检测结果与高通量测序所得结果相一致，证明了高通量测序结果的准确性。不同组织定量分析结果显示，在H5N1亚型禽流感病毒感染状态下，miR-17a*，miR-30c*和miR-10在肌肉中的相对表达量最高，miR-4006f-5p和miR-6在气管中的相对表达量最高，miR-92b-3p在肺脏中的相对表达量最高，miR-4499在脾脏中的相对表达量最高，miR-2808e和miR-4002-5p在心脏中的相对表达量最高。而miR-17a*，miR-92b-3p，miR-30c*，miR-10和miR-6均在肝脏中的相对表达量最低，另外miR-4006f-5p，miR-4499和miR-2808e在肺脏中几乎不表达。在SPF鸭中miR-17a*，miR-30c*和miR-10在肺脏中的相对表达量最高，miR-4006f-5p在肌肉中相对表达量最高，miR-92b-3p和miR-4499在脾脏中的相对表达量最高，miR-6在肾脏中的相对表达量最高，miR-2808e和miR-4002-5p在心脏中的相对表达量最高。而miR-17a*，miR-4006f-5p，miR-10，miR-6和miR-4499均在肝脏中的相对表达量最低。所研究miRNA在病毒感染前后均在不同组织中具有不同的表达量。
本研究对鸭感染与非感染H5N1亚型禽流感病毒状态下的脾脏组织间miRNA组学差异进行了研究，结果表明miRNA在禽流感病毒作用下的水禽中可能具有重要调控作用，该结果为进一步研究miRNA对水禽感染禽流感病毒下的作用机制研究奠定了基础，同时为抗病育种的发展提供新的理论依据。
MicroRNA is a non-coding small RNA with about22nucleotides in lengthendogenous that widely present in a variety of organisms. MiRNA complementarypairing with the target mRNA3′UTR region, specificity base caused the degradationof the target mRNA or inhibiting their translation in order to achieve the level oftarget gene expression regulation. It may paly important role in a variety of biologicalprocesses such as cell development, differentiation, tissues and organa formation,viral infection, et al.
Avian influenza belongs to the orthomyxovirus virus family influenza virus, is apetent infectious poultry, caused by influenza A virus which can lead to human andanimal comorbid. The fact of that the incidence of death with highly pathogenic avianinfluenza virus infection waterfowl appears, human are increasingly aware of hazardsof AIV. High-throughput sequencing technology as a powerful tool for miRNAanalysis identified. Now ducks miRNA research is still in a blank stage, eapeciallyinfected and non-infected AIV duck.
In this study, on the basis of collected3-month-old SPF ducks spleentissueswhich infected and non-infected H5N1subtype avian influenza virus, usinghigh-throughput sequencing analysis techniques, identification and differentiallyexpressed analysis of miRNA in infected and non-infected with the H5N1subtype ofavian influenza virus ducks spleen tissue. The miRNA has a feature of tissue-specific,it makes miRNA play a variety of different role in the process of growth anddevelopment of organisms and different tissues and organs. Further, using quantitativePCR, we analyzed the expression of some differentially expressed miRNAs in ducksvarious tissue under infected and non-infected with the H5N1subtype of avian influenza virus.
The results show that we can detect the avian influenza virus expression in duckvivo after infected avian influenza virus seven days, and reaches a maximum at15days. Deep sequencing of the two libraries by using the high-throughput sequencingtechnology successfully generated. The result shown that the small RNA aresignificant more in types and quantity in SPF duck than the duck that infected H5N1subtype of avian influenza virus.322known differentially expressed miRNAs wereidentified, of which105were significantly up-regulated and217were significantlydown-regulated. In addition,17novel small RNA were identified, including1wassignificantly up-regulated miRNA and4were significantly down-regulated miRNA.The quantitative analysis of nine differentially expressed miRNA that randomlyselected, the result verified the accuracy of the results of the high-throughputsequencing.
The different tissues Quantitative analysis results show that the ducks under theH5N1subtype of avian influenza virus infection, miR-17a*, miR-30c*and miR-10have highest relative expression level in muscle, miR-4006f-5p and miR-6havehighest relative expression level in trachea, miR-92b-3p has highest relativeexpression level in lungs, miR-4499highest relative expression level in spleen,miR-2808e and miR-4002-5p have highest relative expression level in heart.However，miR-17a*, miR-92b-3p, miR-30c*, miR-10and miR-6have lowest relativeexpression lever in liver. In addition, miR-4006f-5p, miR-4499and miR-2808e almosthave no relative expression in lung. In SPF ducks, miR-17a*, miR-30c*, miR-10havehighest relative expression level in lung, miR-4006f-5p has highest relative expressionlevel in muscle, miR-92b-3p and miR-4499have highest relative expression level inspleen, miR-6has highest relative expression level in kidney, miR-2808e andmiR-4002-5p have highest relative expression level in heart. However, miR-17a*,miR-4006f-5p, miR-10, miR-6and miR-4499in liver have lowest relative expressionlevel. In this study, we found that miRNA has a different expression of the same tissue,in SPF duck and infected ducks.
In this study, the miRNA genomics analysis between Duck spleen tissue infectedand non-infected with the H5N1subtype of avian influenza virus were performed, theresults show that miRNA may play an important regulation role in waterfowl in therole of avian influenza virus. This result may lay the foundation for the further studyin miRNA research on the machanism of waterfowl under infected with avianinfluenza virus. At the same time provide a new theoretical basis for the developmentof disease-resistant breeding.
引文
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