Trichosanthin affects HSV-1 replication in Hep-2 cells

Trichosanthin (TCS) is a type I ribosome-inactivating protein that inhibits the replication of both human immunodeficiency virus type 1 (HIV-1) and herpes simplex virus type 1 (HSV-1). The mechanism of inhibition is not clear. This investigation explored the effects of TCS on the stages of HSV-1 infection in Hep-2 cells, from attachment to release. We demonstrated that TCS reduced HSV-1 antigen and DNA content and interfered with viral replication as early as 3-15 h after infection. TCS had no effect on HSV-1 attachment, penetration or immediate-early gene expression. However, the expression of early and late genes and virion release were diminished. In summary, this study demonstrates that TCS primarily affects HSV-1 replication in Hep-2 cells during the early to late infection period.     

Immunization with herpes simplex virus 2 (HSV-2) genes plus inactivated HSV-2 is highly protective against acute and recurrent HSV-2 disease

To date, no vaccine that is safe and effective against herpes simplex virus 2 (HSV-2) disease has been licensed. In this study, we evaluated a DNA prime-formalin-inactivated-HSV-2 (FI-HSV2) boost vaccine approach in the guinea pig model of acute and recurrent HSV-2 genital disease. Five groups of guinea pigs were immunized and intravaginally challenged with HSV-2. Two groups were primed with plasmid DNAs encoding the secreted form of glycoprotein D2 (gD2t) together with two genes required for viral replication, either the helicase (UL5) and DNA polymerase (UL30) genes or the single-stranded DNA binding protein (UL29) and primase (UL52) genes. Both DNA-primed groups were boosted with FI-HSV2 formulated with monophosphoryl lipid A (MPL) and alum adjuvants. Two additional groups were primed with the empty backbone plasmid DNA (pVAX). These two groups were boosted with MPL and alum (MPL-alum) together with either formalin-inactivated mock HSV-2 (FI-Mock) or with FI-HSV2. The final group was immunized with gD2t protein in MPL-alum. After challenge, 0/9 animals in the group primed with UL5, UL30, and gD2t DNAs and all 10 animals in the mock-immunized control group (pVAX-FI-Mock) developed primary lesions. All mock controls developed recurrent lesions through day 100 postchallenge. Only 1 guinea pig in the group primed with pVAX DNA and boosted with FI-HSV2 (pVAX-FI-HSV2 group) and 2 guinea pigs in the group primed with UL5, UL30, and gD2t DNAs and boosted with FI-HSV2 (UL5, UL30, gD2t DNA-FI-HSV2 group) developed recurrent lesions. Strikingly, the UL5, UL30, gD2t DNA-FI-HSV2 group showed a 97% reduction in recurrent lesion days compared with the mock controls, had the highest reduction in days with recurrent disease, and contained the lowest mean HSV-2 DNA load in the dorsal root ganglia.     

Characterization of natural killer cell phenotype and function during recurrent human HSV-2 infection

Human natural killer (NK) cell differentiation, characterized by a loss of NKG2A in parallel with the acquisition of NKG2C, KIRs, and CD57 is stimulated by a number of virus infections, including infection with human cytomegalovirus (CMV), hantavirus, chikungunya virus, and HIV-1. Here, we addressed if HSV-2 infection in a similar way drives NK cell differentiation towards an NKG2A(-)NKG2C(+)KIR(+)CD57(+) phenotype. In contrast to infection with CMV, hantavirus, chikungunya virus, and HIV-1, recurrent HSV-2 infection did not yield an accumulation of highly differentiated NK cells in human peripheral blood. This outcome indicates that human HSV-2 infection has no significant imprinting effect on the human NK cell repertoire.     

Herpes Simplex Virus 2 (HSV-2) Infected Cell Proteins Are among the Most Dominant Antigens of a Live-Attenuated HSV-2 Vaccine

Virion glycoproteins such as glycoprotein D (gD) are believed to be the dominant antigens of herpes simplex virus 2 (HSV-2). We have observed that mice immunized with a live HSV-2 ICP0^- mutant virus, HSV-2 0ΔNLS, are 10 to 100 times better protected against genital herpes than mice immunized with a HSV-2 gD subunit vaccine (PLoS ONE 6:e17748). In light of these results, we sought to determine which viral proteins were the dominant antibody-generators (antigens) of the live HSV-2 0ΔNLS vaccine. Western blot analyses indicated the live HSV-2 0ΔNLS vaccine elicited an IgG antibody response against 9 or more viral proteins. Many antibodies were directed against infected-cell proteins of >100 kDa in size, and only 10 ± 5% of antibodies were directed against gD. Immunoprecipitation (IP) of total HSV-2 antigen with 0ΔNLS antiserum pulled down 19 viral proteins. Mass spectrometry suggested 44% of immunoprecipitated viral peptides were derived from two HSV-2 infected cells proteins, RR-1 and ICP8, whereas only 14% of immunoprecipitated peptides were derived from HSV-2’s thirteen glycoproteins. Collectively, the results suggest the immune response to the live HSV-2 0ΔNLS vaccine includes antibodies specific for infected cell proteins, capsid proteins, tegument proteins, and glycoproteins. This increased breadth of antibody-generating proteins may contribute to the live HSV-2 vaccine’s capacity to elicit superior protection against genital herpes relative to a gD subunit vaccine.     

Herpes simplex virus type 1 and 2 (HSV-1,2) DNA detection by PCR during genital herpes

The oligonucleotide primers for herpes simplex virus type 1 and 2 DNA detection are developed. In examined group of patients with genital herpes the virus of type 2 and type 1 was detected in 63% and 26% cases, respectively. The mixed infection of both types is revealed in 11% of the patients.     

Herpes simplex virus 2 (HSV-2) evolves faster in cell culture than HSV-1 by generating greater genetic diversity

Herpes simplex virus type 1 and 2 (HSV-1 and HSV-2, respectively) are prevalent human pathogens of clinical relevance that establish long-life latency in the nervous system. They have been considered, along with the Herpesviridae family, to exhibit a low level of genetic diversity during viral replication. However, the high ability shown by these viruses to rapidly evolve under different selective pressures does not correlates with that presumed genetic stability. High-throughput sequencing has revealed that heterogeneous or plaque-purified populations of both serotypes contain a broad range of genetic diversity, in terms of number and frequency of minor genetic variants, both in vivo and in vitro. This is reminiscent of the quasispecies phenomenon traditionally associated with RNA viruses. Here, by plaque-purification of two selected viral clones of each viral subtype, we reduced the high level of genetic variability found in the original viral stocks, to more genetically homogeneous populations. After having deeply characterized the genetic diversity present in the purified viral clones as a high confidence baseline, we examined the generation of de novo genetic diversity under culture conditions. We found that both serotypes gradually increased the number of de novo minor variants, as well as their frequency, in two different cell types after just five and ten passages. Remarkably, HSV-2 populations displayed a much higher raise of nonconservative de novo minor variants than the HSV-1 counterparts. Most of these minor variants exhibited a very low frequency in the population, increasing their frequency over sequential passages. These new appeared minor variants largely impacted the coding diversity of HSV-2, and we found some genes more prone to harbor higher variability. These data show that herpesviruses generate de novo genetic diversity differentially under equal in vitro culture conditions. This might have contributed to the evolutionary divergence of HSV-1 and HSV-2 adapting to different anatomical niche, boosted by selective pressures found at each epithelial and neuronal tissue.     

Morphological characteristics of fruits of Fagopyrum (Polygonaceae) from China

Macro- and micro-morphological characteristics of fruits in eight species and one variety of the genus Fagopyrum Mill. (Polygonaceae) from China were observed under stereoscope and scanning electron microscope(SEM). Based on the results, the fruits of the species studied are divided into type Ⅰ , Ⅱ and Ⅲ. The fruits of type Ⅰ are triangular-pyramidal; their surface are rugosely reticulate, neither smooth nor shiny. Two species, F. tataricum and F. dibotrys have this fruit type. Those of type Ⅱ are ovoid-triangular-pyramidal; their surface are smooth and shiny, and striately reticulate. Three species, F. esculentum, F. statice and F. lineare, have this fruit type. In type Ⅲ, the fruits are ovoid-triangular-pyramidal; their surface are smooth and shiny, and covered with many warty grains and sparsely finely striate. F. urophyllum, F. gracilipes, F. leptopodum var. leptopodum, and F. leptopodum var. grossii have this fruit type. Judging from the morphological characteristics of fruits, F. dibotrys might be more closely related to F. tataricumthan to F. esculentum.     

Protection Provided by a Herpes Simplex Virus 2 (HSV-2) Glycoprotein C and D Subunit Antigen Vaccine against Genital HSV-2 Infection in HSV-1-Seropositive Guinea Pigs

A prophylactic vaccine for genital herpes disease remains an elusive goal. We report the results of two studies performed collaboratively in different laboratories that assessed immunogenicity and vaccine efficacy in herpes simplex virus 1 (HSV-1)-seropositive guinea pigs immunized and subsequently challenged intravaginally with HSV-2. In study 1, HSV-2 glycoproteins C (gC2) and D (gD2) were produced in baculovirus and administered intramuscularly as monovalent or bivalent vaccines with CpG and alum. In study 2, gD2 was produced in CHO cells and given intramuscularly with monophosphoryl lipid A (MPL) and alum, or gC2 and gD2 were produced in glycoengineered Pichia pastoris and administered intramuscularly as a bivalent vaccine with Iscomatrix and alum to HSV-1-naive or -seropositive guinea pigs. In both studies, immunization boosted neutralizing antibody responses to HSV-1 and HSV-2. In study 1, immunization with gC2, gD2, or both immunogens significantly reduced the frequency of genital lesions, with the bivalent vaccine showing the greatest protection. In study 2, both vaccines were highly protective against genital disease in naive and HSV-1-seropositive animals. Comparisons between gD2 and gC2/gD2 in study 2 must be interpreted cautiously, because different adjuvants, gD2 doses, and antigen production methods were used; however, significant differences invariably favored the bivalent vaccine. Immunization of naive animals with gC2/gD2 significantly reduced the number of days of vaginal shedding of HSV-2 DNA compared with that for mock-immunized animals. Surprisingly, in both studies, immunization of HSV-1-seropositive animals had little effect on recurrent vaginal shedding of HSV-2 DNA, despite significantly reducing genital disease.     

一个水稻内颖退化突变体的形态特征及基因的精确定位

水稻产量和品质受花器官发育的直接影响,因此对水稻颖花发育机理的研究将有助于水稻产量提高和品质的改良。文章利用60Coγ射线辐照亲本8PW33(籼稻背景)获得一个性状能稳定遗传的内颖退化突变体(编号:MU102),并对其农艺性状和花器官进行了观察和分析。结果显示,相对于野生型,该突变体的株高、每穗总粒数及剑叶宽均显著增加,而结实率则显著降低,差异均达显著水平。解剖镜下观察表明,该突变体内颖退化,外颖弯曲呈现镰刀状,其余器官与野生型表型基本一致。扫描电镜观察显示,突变体与野生型叶片维管束的结构组成以及外颖表皮细胞组成、排列均正常,没有明显差异;与野生型相比,突变体内颖表皮细胞排列较为紧密,推测可能是内颖收缩退化导致的。遗传分析显示该突变性状是由隐性单基因控制,并命名为pd2。利用实验室现有的SSR分子标记将PD2基因定位于水稻第9号染色体上,通过进一步扩大群体和开发新的Indel标记,将PD2基因定位在2个Indel标记之间,两者间的物理距离大约是82 kb。在该物理区间内有一个已经克隆的内颖发育基因REP1,经过测序和比对分析,推测REP1与PD2为等位基因。     

中国荞麦属果实形态特征

应用解剖镜和扫描电镜对中国产荞麦属Fagopyrum Mill．8种1变种的果实形态和微形态特征进行了观察。结果表明这些种类的果实可分为3类：(1)果实三棱锥状,表面不光滑,无光泽,具皱纹网状纹饰,此种类型的植物有苦荞麦、金荞麦。(2)果实卵圆三棱锥状,表面光滑,有光泽,具条纹纹饰,此种类型的植物有荞麦、长柄野荞麦、线叶野荞麦。(3)果实卵圆三棱锥状,表面光滑,有光泽,具大量的瘤状颗粒和少数模糊的细条纹纹饰,此种类型的植物有硬枝野荞麦、细柄野荞麦、小野荞麦、疏穗小野荞麦。研究结果认为金荞麦与栽培种苦荞麦关系较近。     

Okazaki fragment processing-independent role for human Dna2 enzyme during DNA replication

Dna2 is an essential helicase/nuclease that is postulated to cleave long DNA flaps that escape FEN1 activity during Okazaki fragment (OF) maturation in yeast. We previously demonstrated that the human Dna2 orthologue (hDna2) localizes to the nucleus and contributes to genomic stability. Here we investigated the role hDna2 plays in DNA replication. We show that Dna2 associates with the replisome protein And-1 in a cell cycle-dependent manner. Depletion of hDna2 resulted in S/G(2) phase-specific DNA damage as evidenced by increased γ-H2AX, replication protein A foci, and Chk1 kinase phosphorylation, a readout for activation of the ATR-mediated S phase checkpoint. In addition, we observed reduced origin firing in hDna2-depleted cells consistent with Chk1 activation. We next examined the impact of hDna2 on OF maturation and replication fork progression in human cells. As expected, FEN1 depletion led to a significant reduction in OF maturation. Strikingly, the reduction in OF maturation had no impact on replication fork progression, indicating that fork movement is not tightly coupled to lagging strand maturation. Analysis of hDna2-depleted cells failed to reveal a defect in OF maturation or replication fork progression. Prior work in yeast demonstrated that ectopic expression of FEN1 rescues Dna2 defects. In contrast, we found that FEN1 expression in hDna2-depleted cells failed to rescue genomic instability. These findings suggest that the genomic instability observed in hDna2-depleted cells does not arise from defective OF maturation and that hDna2 plays a role in DNA replication that is distinct from FEN1 and OF maturation.     

Development of a glycoprotein D-expressing dominant-negative and replication-defective herpes simplex virus 2 (HSV-2) recombinant viral vaccine against HSV-2 infection in mice

Using the T-REx (Invitrogen, California) gene switch technology and a dominant-negative mutant polypeptide of herpes simplex virus 1 (HSV-1)-origin binding protein UL9, we previously constructed a glycoprotein D-expressing replication-defective and dominant-negative HSV-1 recombinant viral vaccine, CJ9-gD, for protection against HSV infection and disease. It was demonstrated that CJ9-gD is avirulent following intracerebral inoculation in mice, cannot establish detectable latent infection following different routes of infection, and offers highly effective protective immunity against primary HSV-1 and HSV-2 infection and disease in mouse and guinea pig models of HSV infections. Given these favorable safety and immunological profiles of CJ9-gD, aiming to maximize levels of HSV-2 glycoprotein D (gD2) expression, we have constructed an ICP0 null mutant-based dominant-negative and replication-defective HSV-2 recombinant, CJ2-gD2, that contains 2 copies of the gD2 gene driven by the tetracycline operator (tetO)-bearing HSV-1 major immediate-early ICP4 promoter. CJ2-gD2 expresses gD2 as efficiently as wild-type HSV-2 infection and can lead to a 150-fold reduction in wild-type HSV-2 viral replication in cells coinfected with CJ2-gD2 and wild-type HSV-2 at the same multiplicity of infection. CJ2-gD2 is avirulent following intracerebral injection and cannot establish a detectable latent infection following subcutaneous (s.c.) immunization. CJ2-gD2 is a more effective vaccine than HSV-1 CJ9-gD and a non-gD2-expressing dominant-negative and replication-defective HSV-2 recombinant in protection against wild-type HSV-2 genital disease. Using recall response, we showed that immunization with CJ2-gD2 elicited strong HSV-2-specific memory CD4(+) and CD8(+) T-cell responses. Collectively, given the demonstrated preclinical immunogenicity and its unique safety profiles, CJ2-gD2 represents a new class of HSV-2 replication-defective recombinant viral vaccines in protection against HSV-2 genital infection and disease.     

A lentiviral vector-based, herpes simplex virus 1 (HSV-1) glycoprotein B vaccine affords cross-protection against HSV-1 and HSV-2 genital infections

Genital herpes is caused by herpes simplex virus 1 (HSV-1) and HSV-2, and its incidence is constantly increasing in the human population. Regardless of the clinical manifestation, HSV-1 and HSV-2 infections are highly transmissible to sexual partners and enhance susceptibility to other sexually transmitted infections. An effective vaccine is not yet available. Here, HSV-1 glycoprotein B (gB1) was delivered by a feline immunodeficiency virus (FIV) vector and tested against HSV-1 and HSV-2 vaginal challenges in C57BL/6 mice. The gB1 vaccine elicited cross-neutralizing antibodies and cell-mediated responses that protected 100 and 75% animals from HSV-1- and HSV-2-associated severe disease, respectively. Two of the eight fully protected vaccinees underwent subclinical HSV-2 infection, as demonstrated by deep immunosuppression and other analyses. Finally, vaccination prevented death in 83% of the animals challenged with a HSV-2 dose that killed 78 and 100% naive and mock-vaccinated controls, respectively. Since this FIV vector can accommodate two or more HSV immunogens, this vaccine has ample potential for improvement and may become a candidate for the development of a truly effective vaccine against genital herpes.     

The human telomeric proteome during telomere replication

Telomere shortening can cause detrimental diseases and contribute to aging. It occurs due to the end replication problem in cells lacking telomerase. Furthermore, recent studies revealed that telomere shortening can be attributed to difficulties of the semi-conservative DNA replication machinery to replicate the bulk of telomeric DNA repeats. To investigate telomere replication in a comprehensive manner, we develop QTIP-iPOND - Quantitative Telomeric chromatin Isolation Protocol followed by isolation of Proteins On Nascent DNA - which enables purification of proteins that associate with telomeres specifically during replication. In addition to the core replisome, we identify a large number of proteins that specifically associate with telomere replication forks. Depletion of several of these proteins induces telomere fragility validating their importance for telomere replication. We also find that at telomere replication forks the single strand telomere binding protein POT1 is depleted, whereas histone H1 is enriched. Our work reveals the dynamic changes of the telomeric proteome during replication, providing a valuable resource of telomere replication proteins. To our knowledge, this is the first study that examines the replisome at a specific region of the genome.     

条赤须盲蝽形态特征与生物学特性

【目的】明确条赤须盲蝽Trigonotylus coelestialium各虫态的形态特征及其发育历期和成虫繁殖力等生物学特性,为条赤须盲蝽的预测预报及科学防治提供理论依据。【方法】在2021年9-10月郑州室内自然变温(22.0~28.1℃)和25℃恒温条件下,以玉米灌浆期籽粒为食料进行饲养,并观察、记录条赤须盲蝽个体各发育阶段的形态特征,测定其各虫态的发育历期、存活率、成虫寿命及雌成虫产卵量。【结果】条赤须盲蝽卵块产于玉米籽粒基部内颖内侧,卵粒长圆筒形,向一侧略弯。从1龄若虫开始触角呈现红色,随龄期增加红色逐渐明显,至5龄若虫时触角第1节出现3条清晰可见的红色纵纹。翅芽从3龄若虫开始明显可见。雌成虫产卵器长瓣状,平放于生殖节中部的沟槽内。室内自然变温下,条赤须盲蝽卵历期为6.27 d,卵孵化率为89.90%;1-5龄若虫历期分别为2.80, 2.33, 2.70, 2.77和3.90 d,若虫总历期为14.50 d,若虫总存活率为85.97%;雌成虫产卵前期为4.43 d,产卵持续期为13.93 d,单雌产卵19.47块,产卵量为82.55粒。25℃恒温下,条赤须盲蝽卵历期为7.73 d,卵孵化率为81.13%;1-5龄若虫历期分别为2.17, 1.90, 1.77, 1.90和2.93 d;若虫总历期为10.67 d,若虫总存活率为7184%;雌成虫产卵前期为4.17 d,产卵持续期为11.27 d,单雌产卵21.17块,产卵量为72.22粒。【结论】条赤须盲蝽的5龄若虫和成虫的触角第1节的形态特征可用于区分其与该属其他昆虫;其翅芽的发育特征可判别若虫龄期;变温能延长其若虫历期和成虫寿命,同时有利于提高雌成虫产卵量和卵孵化率。     

Morphological and functional characteristics of human temporal-bone cell cultures

Morphology and calcium metabolism have been studied on five different cell cultures from human normal adult temporal-bone biopsies obtained during five stapedectomies. Control cell cultures were obtained from normal human skin. Four different cell types were observed in the bone biopsies: 1) osteoblast-like cells; 2) osteoclast-like cells; 3) fibroblast-like cells; 4) intermediate cells. However, morphology by itself is inadequate for clear differentiation of the four cell types. Hormonal stimulation with calcitonin and dibutyryl-cAMP in presence of 45Ca++ showed a clear-cut difference in 45Ca++ uptake between cultured cells deriving from bone and skin. Functional responses to hormonal stimulation are therefore more specific than cell shape and morphology in differentiating fibroblasts from bone cells. Since responses to hormonal stimulation confirm that temporal-bone cell cultures actually contain bone cells, such cultures seem to be a good experimental model for the study of bone morphology and physiology.