The impact of sex and species of cells in herpes simplex virus infection

Herpes simplex virus type I (HSV-1) is a large, enveloped DNA virus which belongs to the Herpesviridae family. HSV-1 is a highly infectious human pathogen that contacts mucosal surfaces to initiate infection and causes a range of diseases from mild cold sores to keratitis and encephalitis. A growing number of studies point to a sex difference in the prevalence and severity of viral infections. However, the role for sex in herpes infection is unclear due to the complexity of factors involving in the manifestation of HSV diseases. Furthermore, many primary infections are asymptomatic, contributing to the difficulty of studying HSV infections in humans. The neurotropic properties of HSV make it harder to reach information from infected humans for investigating the immune mechanisms controlling reactivation of latent infection within sensory ganglia or central nervous system. Therefore, mouse models have been used extensively to understand multiple aspects of HSV pathogenesis. However, the molecular basis underpinning cross-species differences between humans and mice in response to HSV-1 infection is unknown. In this thesis, we asked whether these two factors, sex and species, may influence HSV-1 replication. Much progress towards these goals has been made using reductionist approaches such as in vitro cell cultures and in many situations this is the only way that cell-intrinsic mechanisms can be dissected. We demonstrated that HSV-1 can adapt to specific sex and generate different mutations due to selective pressure derived from different sexes. Next, profiling of male and female transcriptomic programs revealed that the cytosolic sensing pathway is induced to a greater degree in female primary mouse skin cells (female cells), correlating with higher yields of infectious virions in male counterparts (male cells). In addition, female cells distinctively reactivated Xist, a critical component of X-inactivation, to silence the expression of the transcriptional repressor on the X chromosome, which thereby maintained higher innate immune responses and further explains the different growth phenotypes in HSV-1 replication between the two sexes. Collectively, we propose a model in which HSV-1 triggers a sex-specific regulation of antiviral response in the cytosolic sensing signalling via the control of Xist. During investigation of the sex difference, we coincidently found that viperin, an interferon-stimulated gene (ISG), is upregulated in mouse but not in all tested human cells, which is related to previous findings in the field and encouraged us to comprehensively study human-mouse differences in HSV-1 infection. We show that the growth kinetics of HSV-1 differed substantially in human and mouse cells. A computational pipeline was developed to analyse the cross-species RNA sequencing data, revealing over 60% of differentially regulated pathways between these two hosts in cell cultures. Strikingly, mouse cells upregulated more genes in the antiviral pathway driven by ISGs. To identify the key factor that influences the cross-species difference, we identified that Janus kinase 1 (Jak1) is essential for contributing to the human-mouse difference in HSV-1 replication. Lastly, we utilized virus mutants to show that HSV-1 vhs plays an important role to regulate JAK1 expression and therefore activation between human and mouse cells. In summary, this thesis has expanded our understanding on basic differences of cell cultures that sex and species can affect scientific interpretations in virus infection. Furthermore, the results presented in this study provide rich resources to translate data between the two sexes or mouse experiments into the human disease

Ciprofloxacin-resistant Salmonella enterica Typhimurium and Choleraesuis from Pigs to Humans, Taiwan

We evaluated the disk susceptibility data of 671 nontyphoid Salmonella isolates collected from different parts of Taiwan from March 2001 to August 2001 and 1,261 nontyphoid Salmonella isolates from the National Taiwan University Hospital from 1996 to 2001. Overall, ciprofloxacn resistance was found in 2.7% (18/671) of all nontyphoid Salmonella isolates, in 1.4% (5/347) of Salmonella enterica serotype Typhimurium and in 7.5% (8/107) in S. enterica serotype Choleraesuis nationwide. MICs of six newer fluoroquinolones were determined for the following isolates: 37 isolates of ciprofloxacin-resistant (human) S. enterica Typhimurium (N = 26) and Choleraesuis (N = 11), 10 isolates of ciprofloxacin-susceptible (MIC <1 μg/mL) (human) isolates of these two serotypes, and 15 swine isolates from S. enterica Choleraesuis (N = 13) and Typhmurium (N = 2) with reduced susceptibility to ciprofloxacin (MIC >0.12 μg/mL). Sequence analysis of the gryA, gyrB, parC, parE, and acrR genes, ciprofloxacin accumulation; and genotypes generated by pulsed-field gel electrophoresis with three restriction enzymes (SpeI, XbaI, and BlnI) were performed. All 26 S. enterica Typhimurium isolates from humans and pigs belonged to genotype I. For S. enterica Choleraesuis isolates, 91% (10/11) of human isolates and 54% (7/13) of swine isolates belonged to genotype B. These two genotypes isolates from humans all exhibited a high-level of resistance to ciprofloxacin (MIC 16–64 μg/mL). They had two-base substitutions in the gyrA gene at codons 83 (Ser83Phe) and 87 (Asp87Gly or Asp87Asn) and in the parC gene at codon 80 (Ser80Arg, Ser80Ile, or Ser84Lys). Our investigation documented that not only did these two S. enterica isolates have a high prevalence of ciprofloxacin resistance nationwide but also that some closely related ciprofloxacin-resistant strains are disseminated from pigs to humans

The Role of Perfusion Computed Tomography in the Prediction of Cerebral Hyperperfusion Syndrome

Hyperperfusion syndrome (HPS) following carotid angioplasty with stenting (CAS) is associated with significant morbidity and mortality. At present, there are no reliable parameters to predict HPS. The aim of this study was to clarify whether perfusion computed tomography (CT) is a feasible and reliable tool in predicting HPS after CAS.We performed a retrospective case-control study of 54 patients (11 HPS patients and 43 non-HPS) with unilateral severe stenosis of the carotid artery who underwent CAS. We compared the prevalence of vascular risk factors and perfusion CT parameters including regional cerebral blood volume (rCBV), regional cerebral blood flow (rCBF), and time to peak (TTP) within seven days prior to CAS. Demographic information, risk factors for atherosclerosis, and perfusion CT parameters were evaluated by multivariable logistic regression analysis. The rCBV index was calculated as [(ipsilateral rCBV - contralateral rCBV)/contralateral rCBV], and indices of rCBF and TTP were similarly calculated. We found that eleven patients had HPS, including five with intracranial hemorrhages (ICHs) of whom three died. After a comparison with non-HPS control subjects, independent predictors of HPS included the severity of ipsilateral carotid artery stenosis, 3-hour mean systolic blood pressure (3 h SBP) after CAS, pre-stenting rCBV index >0.15 and TTP index >0.22.The combination of severe ipsilateral carotid stenosis, 3 h SBP after CAS, rCBV index and TTP index provides a potential screening tool for predicting HPS in patients with unilateral carotid stenosis receiving CAS. In addition, adequate management of post-stenting blood pressure is the most important treatable factor in preventing HPS in these high risk patients

比較分析羊傳染性化膿性病毒OV20.0蛋白異構體之功能

Orf virus (ORFV) infection causes pustule and ulceration around muzzle of small ruminants. Although it often occurs with low mobility and mortality, orf may be fatal in juvenile hosts. ORFV, belonging to genus parapoxvirus of the family Poxviridae, is an enveloped virus with double-stranded DNA genome. OV20.0 protein is produced from OV20.0L gene, an E3L ortholog, which is conserved in the genome of most members in Poxviridae. Vaccinia E3 (VV E3) has been studied extensively in these days; however sequences of VV E3 shares low identity with those of OV20.0. According to previous publication, OV20.0L could be translated into to two isoforms, full-length OV20.0 and N-terminal truncated one (sh20). Due to limited information of OV20.0 protein, our research focused on the translation mechanism of the isoform, comparative analysis of their cellular distribution and realizing their functions as well as their contribution to pathogenicity in mice model. First, we proved sh20 was translated from the third start codon of OV20.0L gene. In in vitro and in virus infection condition, both ORFV 20.0 and sh20 can hold the ability to bind double-stranded RNA (dsRNA), sequester the substrate of dsRNA-dependent protein kinase (PKR) that in turns inhibits the activity of downstream factor, eukaryotic initiation factor 2 (eIF2α), and influences cytokine releasing in different levels. Moreover, constructing recombinant virus and animal experiments clarify the role ORFV OV20.0 played in vivo. Although full-length OV20.0 and sh20 shared most functions in vitro, the ORFV recombinant virus which only expressed sh20, was attenuated in vivo. This data implied N-terminus of OV20.0 was required to induce intact pathogenicity in live animals.羊傳染性化膿性病毒(Orf virus，ORFV)感染主要發生在反芻動物，造成其口腔潰瘍和膿皰等症狀，發生率和死亡率不高，然而對於年幼的宿主動物ORFV感染具有致死性。ORFV隸屬於痘病毒科的副痘病毒屬，具有封套，含雙股DNA的基因體。OV20.0 蛋白是由OV20.0L基因所轉譯，為E3的同源蛋白； E3基因高度保留於痘病毒科的許多成員，其中以牛痘病毒(vaccinia virus，VACV)的E3蛋白被研究最為透徹，但是牛痘病毒E3與羊傳染性化膿性病毒的OV20.0蛋白的胺基酸序列相似度低 (約為31%)。前人的研究顯示ORFV OV20.0L基因轉譯出全長及一分子量稍小的isoform (sh20蛋白)，有鑒於現今對於ORFV OV20蛋白的功能所知有限，本研究試圖探討這兩個ORFV OV20.0異構體被轉譯的機制、比較兩者的功能、以及其對於病毒複製和致病力的影響。首先確認sh20蛋白為N-端缺陷的OV20.0異構體；是以第三個ATG當起始密碼所轉譯。在in vitro和病毒感染細胞中均可觀察到ORFV OV20.0和sh20蛋白皆具有結合雙股RNA (double-stranded RNA；dsRNA)的能力；dsRNA為dsRNA-dependent protein kinase (PKR)的受質，OV20.0與dsRNA結合推測可與PKR競爭其活化所需的受質。本研究進一步證實ORFV20.0的確抑制PKR的磷酸化，進而抑制PKR下游的訊息傳遞；例如，降低eukaryotic initiation factor 2 (eIF2α)的磷酸化、導致相關細胞素的合成與分泌受到不等程度的影響。雖然這兩個OV20.0異構體在in vitro以及細胞實驗系統中功能性極為相似，但是在小鼠攻毒試驗結果得知僅表現sh20的重組ORFV的致病性遠低於野外ORFV (具有全長OV20.0蛋白)，由此推測 OV20.0異構體於生物體感染時可能扮演不同的角色，而N端為完整OV20.0功能所必須。第一章 序言 1 第二章 文獻探討 2 第一節 羊傳染性化膿性病毒 (orf virus) 2 第二節 OV20.0和其同源蛋白 5 第三節 PKR 7 第三章 材料與方法 9 第一節 各式質體之製備 9 一、ORFV OV20.0 基因的選殖 9 二、原核表現載體之建構 11 三、真核表現載體之建構 12 四、製備重組病毒之載體 14 第二節 重組蛋白之表現 15 一、轉型作用(transformation) 15 二、Isopropyl-β-thiogalactopyranoside (IPTG) 誘導表現 16 三、蛋白質膠體電泳分析 16 四、重組蛋白純化 18 第三節 OV20.0和dsRNA結合能力 19 一、dsRNA pull down assay 20 二、ELISA 20 第四節 蛋白質交互作用 20 一、細胞培養 20 二、轉染(transfection) 21 三、共同免疫沈澱(co-immunoprecipitation，co-IP) 21 四、螢光分析(fluorescence analysis，FA) 21 第五節 重組病毒之建立 22 一、病毒斑挑選與PCR基因型鑑定 22 二、南方墨點法 22 第六節 動物實驗 23 第四章 結果 33 第一節 OV20.0與其同源序列之胺基酸序列比對和其異構體sh20起始位置之推測 33 第二節 OV20.0與其異構體sh20於細胞中之分佈 33 第三節 分析OV20.0蛋白質和dsRNA結合之能力 35 一、大腸桿菌原核系統表現與 Ni-NTA管柱純化各式OV20.0和E3重組融合蛋白 35 二、poly I:C pull down assay分析OV20.0和sh20結合dsRNA之特性 35 三、經設計之ELISA分析OV20.0和sh20與dsRNA結合之能力 36 四、羊傳染性化膿性病毒感染條件下OV20.0結合dsRNA的情形 37 第四節 OV20.0、sh20與PKR之間的交互作用 37 一、利用免疫螢光染色觀察各式OV20.0和PKR於細胞中位置之對應關係 37 二、利用共同免疫沈澱法分析各式OV20.0和PKR結合之情形 38 第五節 OV20.0抑制PKR及其下游eIF2α之磷酸化 39 第六節 OV20.0和sh20抑制抗病毒感染時細胞素的產生 40 第七節 羊傳染性化膿性重組病毒之建立 41 第八節 羊傳染性化膿性病毒感染之動物模式 43 第五章 討論 7

Establishment and characterization of transformed goat primary cells by expression of simian virus 40 large T antigen for orf virus propagations.

Due to the limited host range of orf virus (ORFV), primary cells derived from its natural hosts, such as goats and sheep, are recommended for isolation and propagation of wild type ORFV. This situation limits the option for the study of virus-host interaction during ORFV infection since primary cells only support a few numbers of passages. SV40 T antigen is a viral oncoprotein that can abrogate replicative senescence, leading to an extended life span of cells. In this study, the transformation of two goat primary cells, fibroblast (FB) and testis (GT) cells, were achieved by stably expressing SV40 T antigen using the lentiviral technique. The presence of the gene encoding SV40 T antigen was validated by polymerase chain reaction (PCR) and western blot analyses. As evidenced by immunofluorescent microscopy, the two types of cells expressing SV40 T antigen (namely, FBT and GTT) were purified to homogeneity. Moreover, faster growth kinetics and a lower serum dependency were noticed in FBT and GTT, as compared with their counterpart parental cells. FBT and GTT remain permissive and can form plaque of ORFV, despite with different profiles; generally speaking, with SV40 T expression, ORFV forms plaques with smaller size and distinct margin. Most importantly, the prolonged life span of goat FBT and GTT serves as an ideal cell culture resource for ORFV isolation from the field, studies of ORFV pathogenesis and efficient vaccine development

Establishment and characterization of transformed goat primary cells by expression of simian virus 40 large T antigen for orf virus propagations

Due to the limited host range of orf virus (ORFV), primary cells derived from its natural hosts, such as goats and sheep, are recommended for isolation and propagation of wild type ORFV. This situation limits the option for the study of virus-host interaction during ORFV infection since primary cells only support a few numbers of passages. SV40 T antigen is a viral oncoprotein that can abrogate replicative senescence, leading to an extended life span of cells. In this study, the transformation of two goat primary cells, fibroblast (FB) and testis (GT) cells, were achieved by stably expressing SV40 T antigen using the lentiviral technique. The presence of the gene encoding SV40 T antigen was validated by polymerase chain reaction (PCR) and western blot analyses. As evidenced by immunofluorescent microscopy, the two types of cells expressing SV40 T antigen (namely, FBT and GTT) were purified to homogeneity. Moreover, faster growth kinetics and a lower serum dependency were noticed in FBT and GTT, as compared with their counterpart parental cells. FBT and GTT remain permissive and can form plaque of ORFV, despite with different profiles; generally speaking, with SV40 T expression, ORFV forms plaques with smaller size and distinct margin. Most importantly, the prolonged life span of goat FBT and GTT serves as an ideal cell culture resource for ORFV isolation from the field, studies of ORFV pathogenesis and efficient vaccine development

Increasing antigen presentation on HSV-1-infected cells increases lesion size but does not alter neural infection or latency

CD8+ T cells have a role in the control of acute herpes simplex virus (HSV) infection and may also be important in the maintenance of latency. In this study we have explored the consequences of boosting the efficacy of CD8+ T cells against HSV by increasing the amount of an MHC I-presented epitope on the surface of infected cells. To do this we used HSVs engineered to express an extra copy of the immunodominant CD8+ T cell epitope in C57Bl/6 mice, namely gB498 (SSIEFARL). Despite greater presentation of gB498 on infected cells, CD8+ T cell responses to these viruses in mice were similar to those elicited by a control virus. Further, the expression of extra gB498 did not significantly alter the extent or stability of latency in our mouse model, and virus loads in skin and sensory ganglia of infected mice were not affected. Surprisingly, mice infected with these viruses developed significantly larger skin lesions than those infected with control viruses and notably, this phenotype was dependent on MHC haplotype. Therefore increasing the visibility of HSV-infected cells to CD8+ T cell attack did not impact neural infection or latency, but rather enhanced pathology in the skin

Adenosine Deaminase Acting on RNA 1 Associates with Orf Virus OV20.0 and Enhances Viral Replication

Orf virus (ORFV) infects sheep and goats and is also an important zoonotic pathogen. The viral protein OV20.0 has been shown to suppress innate immunity by targeting the double-stranded RNA (dsRNA)-activated protein kinase (PKR) by multiple mechanisms. These mechanisms include a direct interaction with PKR and binding with two PKR activators, dsRNA and the cellular PKR activator (PACT), which ultimately leads to the inhibition of PKR activation. In the present study, we identified a novel association between OV20.0 and adenosine deaminase acting on RNA 1 (ADAR1). OV20.0 bound directly to the dsRNA binding domains (RBDs) of ADAR1 in the absence of dsRNA. Additionally, OV20.0 preferentially interacted with RBD1 of ADAR1, which was essential for its dsRNA binding ability and for the homodimerization that is critical for intact adenosine-to-inosine (A-to-I)-editing activity. Finally, the association with OV20.0 suppressed the A-to-I-editing ability of ADAR1, while ADAR1 played a proviral role during ORFV infection by inhibiting PKR phosphorylation. These observations revealed a new strategy used by OV20.0 to evade antiviral responses via PKR.IMPORTANCE Viruses evolve specific strategies to counteract host innate immunity. ORFV, an important zoonotic pathogen, encodes OV20.0 to suppress PKR activation via multiple mechanisms, including interactions with PKR and two PKR activators. In this study, we demonstrated that OV20.0 interacts with ADAR1, a cellular enzyme responsible for converting adenosine (A) to inosine (I) in RNA. The RNA binding domains, but not the catalytic domain, of ADAR1 are required for this interaction. The OV20.0-ADAR1 association affects the functions of both proteins; OV20.0 suppressed the A-to-I editing of ADAR1, while ADAR1 elevated OV20.0 expression. The proviral role of ADAR1 is likely due to the inhibition of PKR phosphorylation. As RNA editing by ADAR1 contributes to the stability of the genetic code and the structure of RNA, these observations suggest that in addition to serving as a PKR inhibitor, OV20.0 might modulate ADAR1-dependent gene expression to combat antiviral responses or achieve efficient viral infection

Interaction between NS1 and Cellular MAVS Contributes to NS1 Mitochondria Targeting

Influenza A virus nonstructural protein 1 (NS1) plays an important role in evading host innate immunity. NS1 inhibits interferon (IFN) responses via multiple mechanisms, including sequestering dsRNA and suppressing retinoic acid-inducible gene I (RIG-I) signaling by interacting with RIG-I and tripartite motif-containing protein 25 (TRIM25). In the current study, we demonstrated the mitochondrial localization of NS1 at the early stage of influenza virus infection. Since NS1 does not contain mitochondria-targeting signals, we suspected that there is an association between the NS1 and mitochondrial proteins. This hypothesis was tested by demonstrating the interaction of NS1 with mitochondrial antiviral-signaling protein (MAVS) in a RIG-I-independent manner. Importantly, the association with MAVS facilitated the mitochondrial localization of NS1 and thereby significantly impeded MAVS-mediated Type I IFN production