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, 最新更新时间 , doi: 10.1007/s12250-021-00355-1
收稿日期: 2020-07-30
录用日期: 2020-12-29
出版日期: 2021-01-28
杆状病毒作为载体被广泛应用于外源基因的表达。苜蓿银纹夜蛾核型多角体病毒（AcMNPV）是最常用的杆状病毒，它编码155个开放阅读框（ORFs）。本研究中，我们通过构建基因敲除型bacmids并进行转染和感染实验，系统地研究了42个AcMNPV基因对子代出芽型病毒粒子（BVs）产生的影响。结果表明，在39个功能未被证实的基因和3个新近报道的基因中，有36个基因是非必需的，它们敲除后，缺失型病毒的一步生长曲线与对照组病毒没有显著差异。ac62、ac82和ac106/107是产生感染性BV所必需的，因为它们的缺失导致BV的感染性完全丧失，而回复型病毒与对照病毒相比没有显著差异。此外， ac13、ac51和ac120对感染性BV的产生重要但非必需，因为基因敲除后缺失型病毒产生的BV水平明显低于对照病毒或回复型病毒。随后我们根据对BV滴度的影响将AcMNPV的155个基因分为三类（非必需、必需或重要）。基于我们的研究结果和目前已有的相关报道，我们提出了一个理论上可能的AcMNPV最小基因组，它只包含必需基因和重要基因。该研究结果有助于我们加深对杆状病毒功能基因组学的理解，为将来杆状病毒表达系统的人工合成与改造打下了基础。Baculoviruses have been widely used as a vector for expressing foreign genes. Among numerous baculoviruses, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the most frequently used and it encodes 155 open reading frames (ORFs). Here, we systematically investigated the impact of 42 genes of AcMNPV on the production of infectious budded viruses (BVs) by constructing gene-knockout bacmids and subsequently conducting transfection and infection assays. The results showed that among the 39 functionally unverified genes and 3 recently reported genes, 36 are dispensable for infectious BV production, as the one-step growth curves of the gene-knockout viruses were not significantly different from those of the parental virus. Three genes (ac62, ac82 and ac106/107) are essential for infectious BV production, as deletions thereof resulted in complete loss of infectivity while the repaired viruses showed no significant difference in comparison to the parental virus. In addition, three genes (ac13, ac51 and ac120) are important but not essential for infectious BV production, as gene-knockout viruses produced significantly lower BV levels than that of the parental virus or repaired viruses. We then grouped the 155 AcMNPV genes into three categories (Dispensable, Essential, or Important for infectious BV production). Based on our results and previous publications, we constructed a schematic diagram of a potential mini-genome of AcMNPV, which contains only essential and important genes. The results shed light on our understanding of functional genomics of baculoviruses and provide fundamental information for future engineering of baculovirus expression system.
, 最新更新时间 , doi: 10.1007/s12250-021-00351-5
收稿日期: 2020-08-03
录用日期: 2020-12-28
出版日期: 2021-01-28
人SAMHD1蛋白主要通过其自身的dNTP水解酶活性阻断慢病毒的逆转录过程，来抑制病毒感染。除了人类以外，猫和牛也能够被慢病毒感染，并且也能表达其各自的SAMHD1蛋白。但是，猫和牛的SAMHD1（fSAM和bSAM）在细胞中是如何分布的以及这种分布情况对于其各自抗病毒功能的影响尚未有人报道。在本研究中，我们发现野生型fSAM和bSAM均主要定位在细胞核中，而核定位信号（11KRPR14）缺失的fSAM和bSAM会重定位到细胞质中。细胞质中的fSAM和bSAM均保留了针对不同慢病毒的抗病毒功能，且细胞质中的fSAM与细胞质中的hSAM类似，比其野生型蛋白具有更高的抗SIV和HIV-2活性。进一步研究发现，细胞质中的hSAM和fSAM相比其野生型蛋白更不易被Vpx降解，而bSAM在细胞核和细胞质中的被降解程度相同，但它们均具有同样的体外dNTP水解酶活性和与Vpx的结合能力，这表明细胞质中的hSAM和fSAM由于具有更好的抗Vpx降解能力而可以抑制更多的SIV和HIV-2。这些结果表明fSAM和bSAM介导的抗病毒作用并不需要其定位在细胞核中，fSAM在多个方面均与hSAM具有更高的相似性，因此，猫可能更适用于作为动物模型来开展对SAMHD1体内生物学功能的研究。本研究将为建立更理想的SAMHD1体内研究动物模型提供数据支持。Human SAMHD1 (hSAM) restricts lentiviruses at the reverse transcription step through its dNTP triphosphohydrolase (dNTPase) activity. Besides humans, several mammalian species such as cats and cows that carry their own lentiviruses also express SAMHD1. However, the intracellular distribution of feline and bovine SAMHD1 (fSAM and bSAM) and its significance in their lentiviral restriction function is not known. Here, we demonstrated that fSAM and bSAM were both predominantly localized to the nucleus and nuclear localization signal (11KRPR14)-deleted fSAM and bSAM relocalized to the cytoplasm. Both cytoplasmic fSAM and bSAM retained the antiviral function against different lentiviruses and cytoplasmic fSAM could restrict Vpx-encoding SIV and HIV-2 more efficiently than its wild-type (WT) protein as cytoplasmic hSAM. Further investigation revealed that cytoplasmic fSAM was resistant to Vpx-induced degradation like cytoplasmic hSAM, while cytoplasmic bSAM was not, but they all demonstrated the same in vitro dNTPase activity and all could interact with Vpx as their WT proteins, indicating that cytoplasmic hSAM and fSAM can suppress more SIV and HIV-2 by being less sensitive to Vpx-mediated degradation. Our results suggested that fSAM- and bSAM-mediated lentiviral restriction does not require their nuclear localization and that fSAM shares more common features with hSAM. These findings may provide insights for the establishment of alternative animal models to study SAMHD1 in vivo.
, 最新更新时间 , doi: 10.1007/s12250-021-00350-6
收稿日期: 2020-07-18
录用日期: 2020-12-17
出版日期: 2021-01-28
非洲猪瘟是由非洲猪瘟病毒引起家猪和野猪致死的疾病。非洲猪瘟给全球养猪业带来巨大威胁。目前，由于非洲猪瘟病毒基因组的复杂性以及生物安全问题，还没有商业疫苗可用。自然分离或基因手段改造得到的减毒活病毒已经证明能够对同源亲本毒株具有完全保护作用。本研究从II型非洲猪瘟病毒 CN/GS/2018毒株删除MGF-110-9L基因得到一株缺失毒株（命名ASFV-Δ9L）。与亲本毒株相比，MGF-110-9L 基因的缺失明显降低了非洲猪瘟病毒在猪巨噬细胞中的体外复制能力。在21天的观察期内，大多数低剂量肌肉接种ASFV-Δ9L的动物临床正常。感染ASFV-Δ9L的动物有3/5表现出低的血毒症和高的病毒特异性抗体反应，表明ASFV-Δ9L对猪具有部分致弱作用。以上的研究将为防控非洲猪瘟提供一定理论依据。African swine fever virus (ASFV) is the etiological agent of African swine fever (ASF), an often lethal disease in domestic and wild pigs. ASF represents a major threat to the swine industry worldwide. Currently, no commercial vaccine is available because of the complexity of ASFV or biosecurity concerns. Live attenuated viruses that are naturally isolated or genetically manipulated have demonstrated reliable protection against homologous ASFV strain challenge. In the present study, a mutant ASFV strain with the deletion of ASFV MGF-110-9L (ASFV-Δ9L) was generated from a highly virulent ASFV CN/GS/2018 parental strain, a genotype II ASFV. Relative to the parental ASFV isolate, deletion of the MGF-110-9L gene significantly decreased the ability of ASFV-Δ9L to replicate in vitro in primary swine macrophage cell cultures. The majority of animals inoculated intramuscularly with a low dose of ASFV-Δ9L (10 HAD50) remained clinically normal during the 21-day observational period. Three of five ASFV-Δ9L-infected animals displayed low viremia titers and low virus shedding and developed a strong virus-specific antibody response, indicating partial attenuation of the ASFV-Δ9L strain in pigs. The findings imply the potential usefulness of the ASFV-Δ9L strain for further development of ASF control measures.
, 最新更新时间 , doi: 10.1007/s12250-021-00352-4
收稿日期: 2020-09-30
录用日期: 2020-12-17
出版日期: 2021-01-28
过氧化还原酶6（PRDX6）是既具有过氧化酶活性，也具有磷脂酶A2（PLA2）活性的抗氧化酶。其广泛参与细胞内多种应答调控反应。但其在病毒感染过程中发挥的功能并不清楚。本研究发现口蹄疫病毒（FMDV）感染的细胞中PRDX6表达量显著下降。过表达PRDX6能够抑制FMDV的复制，而利用干扰RNA下调PRDX6的表达则能促进FMDV的复制，表明了PRDX6的抗病毒功能。为了查明PRDX6的过氧化酶活性和PLA2活性是否参与抗病毒功能，PRDX6过氧化酶活性的特异抑制剂mercaptosuccinate和PLA2活性特异抑制剂MJ33被用于处理FMDV感染的细胞，结果显示MJ33处理FMDV感染的细胞能够显著促进病毒的复制，这表明PRDX6的PLA2活性在发挥抑制FMDV复制过程中发挥重要作用。同时，研究发现过表达PRDX6可以轻度促进I型干扰素通路的应答。而通过过表达各类FMDV蛋白，鉴定出FMDV的3C蛋白酶能够抑制PRDX6的表达，进一步分析表明3C通过其蛋白水解酶活性降解PRDX6。本研究同时研究了PRDX6对另一种小RNA病毒猪塞内卡病毒（SVA）复制的影响，同样证实了PRDX6发挥的抗病毒功能，而且SVA的3C蛋白酶同样通过其蛋白水解酶活性降解PRDX6发挥拮抗作用。总而言之，我们的研究首次发现了PRDX6对猪小RNA病毒的抑制功能，鉴定了猪小RNA病毒3C蛋白水解酶降解PRDX6发挥的拮抗作用。Peroxiredoxin-6 (PRDX6) is an antioxidant enzyme with both the activities of peroxidase and phospholipase A2 (PLA2), which is involved in regulation of many cellular reactions. However, the function of PRDX6 during virus infection remains unknown. In this study, we found that the abundance of PRDX6 protein was dramatically decreased in foot-and-mouth disease virus (FMDV) infected cells. Overexpression of PRDX6 inhibited FMDV replication. In contrast, knockdown of PRDX6 expression promoted FMDV replication, suggesting an antiviral role of PRDX6. To explore whether the activity of peroxidase and PLA2 was associated with PRDX6-mediated antiviral function, a specific inhibitor of PLA2 (MJ33) and a specific inhibitor of peroxidase activity (mercaptosuccinate) were used to treat the cells before FMDV infection. The results showed that incubation of MJ33 but not mercaptosuccinate promoted FMDV replication. Meanwhile, overexpression of PRDX6 slightly enhanced type I interferon signaling. We further determined that the viral 3Cpro was responsible for degradation of PRDX6, and 3Cpro-induced reduction of PRDX6 was independent of the proteasome, lysosome, and caspase pathways. The protease activity of 3Cpro was required for induction of PRDX6 reduction. Besides, PRDX6 suppressed the replication of another porcine picornavirus Senecavirus A (SVA), and the 3Cpro of SVA induced the reduction of PRDX6 through its proteolytic activity as well. Together, our results suggested that PRDX6 plays an important antiviral role during porcine picornavirus infection, and the viral 3Cpro induces the degradation of PRDX6 to overcome PRDX6-mediated antiviral function.
, 最新更新时间 , doi: 10.1007/s12250-021-00353-3
收稿日期: 2020-10-20
录用日期: 2020-12-18
出版日期: 2021-01-28
苜蓿银纹夜蛾核型多角体病毒(Autographa californica multiple nucleopolyhedrovirus, AcMNPV) orf13(ac13)是α杆状病毒的一个保守基因，然而该基因在病毒生活史中的具体功能尚不清楚。本研究发现，ac13是病毒的一个晚期基因，其编码的蛋白含有一段核定位信号，且该蛋白与核纤层蛋白存在共定位现象。ac13基因缺失并不影响病毒基因组的复制，核衣壳的组装和包涵体（occlusion body, OB）的形成，但缺失ac13基因的缺陷型病毒相较于野生型病毒的病毒粒子出芽减少近400倍。ac13基因缺失会显著影响核衣壳从细胞核向细胞质中的释放，但并不影响OB的形成。所以，ac13是病毒出芽过程中核衣壳有效出核的必需基因，而并不是OB形成的必需基因。Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf13 (ac13) is a conserved gene in all sequenced alphabaculoviruses. However, its function in the viral life cycle remains unknown. In this study, we found that ac13 was a late gene and that the encoded protein, bearing a putative nuclear localization signal motif, colocalized with the nuclear lamina. Deletion of ac13 did not affect viral genome replication, nucleocapsid assembly or occlusion body (OB) formation, but reduced virion budding from infected cells by approximately 400-fold compared with the wild-type virus. Deletion of ac13 substantially impaired the egress of nucleocapsids from the nucleus to the cytoplasm, while the OB morphogenesis was unaffected. Taken together, our results indicated that ac13 was required for efficient nuclear egress of nucleocapsids during virion budding, but was dispensable for OB formation.
, 最新更新时间 , doi: 10.1007/s12250-021-00354-2
收稿日期: 2020-09-26
录用日期: 2020-12-28
出版日期: 2021-01-28
人疱疹病毒是双链DNA病毒，分为9种。90%以上的成年人曾感染过一种或多种疱疹病毒。不同疱疹病毒感染的临床症状不同，从轻度或无症状感染到引起致命的疾病，如侵袭性淋巴瘤和肉瘤。及时、准确地检测疱疹病毒感染是对临床管理和治疗至关重要。在这项研究中，我们建立了一个以管家基因作为内参的二维多重qPCR方法，用于单管检测所有九种疱疹病毒。该方法能检测和鉴别不同类型的疱疹病毒，其灵敏度可达到30至300拷贝/ 25μL单管反应，并且和其他20种人类病毒（包括DNA和RNA）均无交叉反应产生。我们使用170个临床样本对新方法的稳定性进行评估，发现新方法与单重qPCR方法具有高度的一致性 (100%)。简单，快速，高灵敏度，特异性和低成本的特点使新方法具有很大的潜力，可广泛用于临床诊断和患者治疗。Human herpesviruses are double-stranded DNA viruses that are classified into nine species. More than 90% of adults are ever infected with one or more herpesviruses. The symptoms of infection with different herpesviruses are diverse ranging from mild or asymptomatic infections to deadly diseases such as aggressive lymphomas and sarcomas. Timely and accurate detection of herpesvirus infection is critical for clinical management and treatment. In this study, we established a single-tube nonuple qPCR assay for detection of all nine herpesviruses using a 2-D multiplex qPCR method with a house-keeping gene as internal control. The novel assay can detect and distinguish different herpesvirus with 30 to 300 copies per 25 µL single-tube reaction, and does not cross-react with 20 other human viruses, including DNA and RNA viruses. The robustness of the novel assay was evaluated using 170 clinical samples. The novel assay showed a high consistency (100%) with the single qPCR assay for HHVs detection. The features of simple, rapid, high sensitivity, specificity, and low cost make this assay a high potential to be widely used in clinical diagnosis and patient treatment.
, 最新更新时间 , doi: 10.1007/s12250-021-00349-z
收稿日期: 2020-08-05
录用日期: 2020-11-16
出版日期: 2021-01-28
狂犬病是由狂犬病病毒（RABV）引起的一种严重威胁全球公共卫生的传染病。目前，除了狂犬病免疫球蛋白，还没有用于狂犬病暴露后治疗的小分子药物。然而，狂犬病免疫球蛋白需要冷链运输，成本比较高。因此，开发能够有效抑制RABV复制的新型药物尤为重要。青蒿琥酯（ART）和双氢青蒿素（DHA）是青蒿素的两种衍生物，目前已经被用于治疗成人和小孩的疟疾，具有良好的安全性。本研究中，我们发现ART和DHA在宿主细胞中能够以较低的浓度（0.1 μmmol/L）抑制RABV复制。这种抑制作用不依赖于病毒毒株和细胞系。提前2小时使用ART和DHA处理RABV并不影响病毒复制，提示ART和DHA不与RABV直接作用，也不影响病毒的吸附和侵入。进一步研究发现，ART和DHA抑制了病毒基因组RNA和基因的转录。使用5 mg/kg的ART或者DHA通过肌肉注射RABV攻毒小鼠，结果显示ART和DHA一定程度地提高了攻毒小鼠存活率。ART和DHA与甘露醇联合使用显著提高了RABV攻毒小鼠存活率。以上结果证实了ART和DHA具有开发成治疗狂犬病新型药物的潜力。Rabies is caused by infection of rabies virus (RABV) and remains a serious threat to the global public health. Except for cold chain and high cost of human rabies immune globulin, no small molecule drugs are currently available for clinical treatment of rabies. So, it is of great importance to identify novel compounds that can effectively inhibit RABV infection. Artesunate (ART) and dihydroartemisinin (DHA), two derivatives of artemisinin, are widely used for treatment of malaria in adults and children, showing high safety. In this study, we found that both ART and DHA were able to inhibit RABV replication in host cells at a low concentration (0.1 μmol/L). The antiviral effects of ART and DHA were independent of viral strains and cell lines. Pre-treatment with ART or DHA for 2 h in vitro did not affect the viral replication in host cells, implying that ART and DHA neither reduced the viability of RABV directly nor inhibited the binding and entrance of the virus to host cells. Further studies revealed that ART and DHA inhibited RABV genomic RNA synthesis and viral gene transcription. Treatment with ART or DHA (5 mg/kg) by intramuscular injection improved, to some extent, the survival rate of RABV-challenged mice. Combination treatment with derivatives of artemisinin and mannitol significantly improved the survival rate of RABV-challenged mice. The results suggest that ART and DHA have a great potential to be explored as new anti-rabies agents for treatment of rabies.
Molecular Characterization of Human Respiratory Adenoviruses Infection in Xining City, China In 2018
, 最新更新时间 , doi: 10.1007/s12250-020-00282-7
收稿日期: 2020-03-18
录用日期: 2020-08-05
出版日期: 2020-09-14
35卷第6期 (2020年12月)
ISSN 1674-0769
EISSN 1995-820X
CN 42-1760/Q
主编: 石正丽
影响因子: 3.242*
*源于2019年JCR
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