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Porcine circovirus type 1 (PCV1), originally isolated as a contaminant of PK-15 cells, is nonpathogenic, whereas porcine circovirus type 2 (PCV2) causes an economically important disease in pigs. To determine the factors affecting virus replication, we constructed chimeric viruses by swapping open reading frame 1 (ORF1) (rep) or the origin of replication (Ori) between PCV1 and PCV2 and compared the replication efficiencies of the chimeric viruses in PK-15 cells. The results showed that the replication factors of PCV1 and PCV2 are fully exchangeable and, most importantly, that both the Ori and rep of PCV1 enhance the virus replication efficiencies of the chimeric viruses with the PCV2 backbone.Porcine circovirus (PCV) is a single-stranded DNA virus in the family Circoviridae (34). Type 1 PCV (PCV1) was discovered in 1974 as a contaminant of porcine kidney cell line PK-15 and is nonpathogenic in pigs (31-33). Type 2 PCV (PCV2) was discovered in piglets with postweaning multisystemic wasting syndrome (PMWS) in the mid-1990s and causes porcine circovirus-associated disease (PCVAD) (1, 9, 10, 25). PCV1 and PCV2 have similar genomic organizations, with two major ambisense open reading frames (ORFs) (16). ORF1 (rep) encodes two viral replication-associated proteins, Rep and Rep′, by differential splicing (4, 6, 21, 22). The Rep and Rep′ proteins bind to specific sequences within the origin of replication (Ori) located in the intergenic region, and both are responsible for viral replication (5, 7, 8, 21, 23, 28, 29). ORF2 (cap) encodes the immunogenic capsid protein (Cap) (26). PCV1 and PCV2 share approximately 80%, 82%, and 62% nucleotide sequence identity in the Ori, rep, and cap, respectively (19).In vitro studies using a reporter gene-based assay system showed that the replication factors of PCV1 and PCV2 are functionally interchangeable (2-6, 22), although this finding has not yet been validated in a live infectious-virus system. We have previously shown that chimeras of PCV in which cap has been exchanged between PCV1 and PCV2 are infectious both in vitro and in vivo (15), and an inactivated vaccine based on the PCV1-PCV2 cap (PCV1-cap2) chimera is used in the vaccination program against PCVAD (13, 15, 18, 27).PCV1 replicates more efficiently than PCV2 in PK-15 cells (14, 15); thus, we hypothesized that the Ori or rep is directly responsible for the differences in replication efficiencies. The objectives of this study were to demonstrate that the Ori and rep are interchangeable between PCV1 and PCV2 in a live-virus system and to determine the effects of swapped heterologous replication factors on virus replication efficiency in vitro. 相似文献
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Human immunodeficiency virus (HIV) types 1 and 2 (HIV-1 and HIV-2) are the etiologic agents of AIDS. Most HIV-1 infected individuals worldwide are women, who acquire HIV infections during sexual contact. Blocking HIV mucosal transmission and local spread in the female lower genital tract is important in preventing infection and ultimately eliminating the pandemic. Microbicides work by destroying the microbes or preventing them from establishing an infection. Thus, a number of different types of microbicides are under investigation, however, the lack of their solubility and bioavailability, and toxicity has been major hurdles. Herein, we report the development of multifunctional chitosan-lipid nanocomplexes that can effectively deliver plasmids encoding siRNA(s) as microbicides without adverse effects and provide significant protection against HIV in both in vitro and in vivo models. Chitosan or chitosan-lipid (chlipid) was complexed with a cocktail of plasmids encoding HIV-1-specific siRNAs (psiRNAs) and evaluated for their efficacy in HEK-293 cells, PBMCs derived from nonhuman primates, 3-dimensional human vaginal ectocervical tissue (3D-VEC) model and also in non-human primate model. Moreover, prophylactic administration of the chlipid to deliver a psiRNA cocktail intravaginally with a cream formulation in a non-human primate model showed substantial reduction of SHIV (simian/human immunodeficiency virus SF162) viral titers. Taken together, these studies demonstrate the potential of chlipid-siRNA nanocomplexes as a potential genetic microbicide against HIV infections. 相似文献
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A comprehensive vaccine for human immunodeficiency virus type 1 (HIV-1) would block HIV-1 acquisition as well as durably control viral replication in breakthrough infections. Recent studies have demonstrated that Env is required for a vaccine to protect against acquisition of simian immunodeficiency virus (SIV) in vaccinated rhesus monkeys, but the antigen requirements for virologic control remain unclear. Here, we investigate whether CD8(+) T lymphocytes from vaccinated rhesus monkeys mediate viral inhibition in vitro and whether these responses predict virologic control following SIV challenge. We observed that CD8(+) lymphocytes from 23 vaccinated rhesus monkeys inhibited replication of SIV in vitro. Moreover, the magnitude of inhibition prior to challenge was inversely correlated with set point SIV plasma viral loads after challenge. In addition, CD8 cell-mediated viral inhibition in vaccinated rhesus monkeys correlated significantly with Gag-specific, but not Pol- or Env-specific, CD4(+) and CD8(+) T lymphocyte responses. These findings demonstrate that in vitro viral inhibition following vaccination largely reflects Gag-specific cellular immune responses and correlates with in vivo virologic control following infection. These data suggest the importance of including Gag in an HIV-1 vaccine in which virologic control is desired. 相似文献
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In poliovirus-infected HeLa-S3 cells, the protease inhibitors tolylsulfonyl-phenylalanyl chloromethyl ketone and iodoacetamide cause an accumulation of large precursor proteins, and they block viral RNA synthesis most probably via these products. Viral RNA polymerase activity can, however, be extracted by detergent containing buffer (Tris/Nonidet P-40, deoxycholate) from the inhibited cells. Only cytoplasmic extracts from infected cells treated with tolylsulfonyl-phenylalanyl chloromethyl ketone or iodoacetamide contain a protein which inhibits the in vitro polymerase reaction. 相似文献
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Alyssa R. Borges Daniel A. Toledo Bruno R. Fermino Jos Carlos de Oliveira Ariel Mariano Silber Maria Carolina Elias Heloisa D'Avila Kzia K. G. Scopel 《The Journal of eukaryotic microbiology》2019,66(3):385-392
Since the observation of the great pleomorphism of fish trypanosomes, in vitro culture has become an important tool to support taxonomic studies investigating the biology of cultured parasites, such as their structure, growth dynamics, and cellular cycle. Relative to their biology, ex vivo and in vitro studies have shown that these parasites, during the multiplication process, duplicate and segregate the kinetoplast before nucleus replication and division. However, the inverse sequence (the nucleus divides before the kinetoplast) has only been documented for a species of marine fish trypanosomes on a single occasion. Now, this previously rare event was observed in Trypanosoma abeli, a freshwater fish trypanosome. Specifically, from 376 cultured parasites in the multiplication process, we determined the sequence of organelle division for 111 forms; 39% exhibited nucleus duplication prior to kinetoplast replication. Thus, our results suggest that nucleus division before the kinetoplast may not represent an accidental or erroneous event occurring in the main pathway of parasite reproduction, but instead could be a species‐specific process of cell biology in trypanosomes, such as previously noticed for Leishmania. This “alternative” pathway for organelle replication is a new field to be explored concerning the biology of marine and freshwater fish trypanosomes. 相似文献
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An unresolved question in herpesvirus biology is why some herpesviruses contain more than one lytic origin of replication (oriLyt). Using murine gammaherpesvirus 68 (MHV-68) as model virus containing two oriLyts, we demonstrate that loss of either of the two oriLyts was well tolerated in some situations but not in others both in vitro and in vivo. This was related to the cell type, the organ or the route of inoculation. Depending on the cell type, different cellular proteins, for example Hexim1 and Rbbp4, were found to be associated with oriLyt DNA. Overexpression or downregulation of these proteins differentially affected the growth of mutants lacking either the left or the right oriLyt. Thus, multiple oriLyts are required to ensure optimal fitness in different cell types and tissues. 相似文献
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The S2 Gene of Equine Infectious Anemia Virus Is Dispensable for Viral Replication In Vitro 总被引:5,自引:1,他引:4
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Equine infectious anemia virus (EIAV) contains the simplest genome among lentiviruses in that it encodes only three putative regulatory genes (S1, S2, S3) in addition to the canonical gag, pol, and env genes, presumably reflecting its limited tropism to cells of monocyte/macrophage lineage. Tat and Rev functions have been assigned to S1 and S3, respectively, but the specific function for the S2 gene has yet to be determined. Thus, the function of S2 in virus replication in vitro was investigated by using an infectious molecular viral clone, EIAVUK. Various EIAVUK mutants lacking S2 were constructed, and their replication kinetics were examined in several equine cell culture systems, including the natural in vivo target equine macrophage cells. The EIAV S2 mutants showed replication kinetics similar to those of the parental virus in all of the tested primary and transformed equine cell cultures, without any detectable reversion of mutant genomes. The EIAVUK mutants also showed replication kinetics similar to those of the parental virus in an equine blood monocyte differentiation-maturation system. These results demonstrate for the first time that the EIAV S2 gene is not essential and does not appear to affect virus infection and replication properties in target cells in vitro. 相似文献
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B. D. Korant 《Journal of virology》1972,10(4):751-759
The use of protease inhibitors causes the accumulation of very large polypeptides (polyprotein) in tissue culture cells infected with either poliovirus or echovirus 12. The effectiveness of the inhibitor varies, depending on the cell line chosen. In infected monkey kidney cells, polyprotein is not cleaved when a chymotrypsin inhibitor is added, but in infected HeLa cells a trypsin inhibitor is most effective. Therefore, at least a part of the proteolytic activity is supplied by the host cell. Extracted viral polyprotein can be cleaved in vitro by trypsin or chymotrypsin. As estimated by migration in sodium dodecyl sulfate gels and antigenicity, chymotrypsin cleavage of the poliovirus polyprotein yields fragments which are similar to the in vivo product. The polyprotein is not in soluble form but is attached to a fast-sedimenting, membrane-bound structure. Proteolytic activities in cell extracts were assayed using polyprotein as substrate, and infected and uninfected extracts produced qualitatively dissimilar cleavages. 相似文献
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Nadine Eckert Florian Wrensch Sabine G?rtner Navaneethan Palanisamy Ulrike Goedecke Nils J?ger Stefan P?hlmann Michael Winkler 《PloS one》2014,9(5)
Reporter genes inserted into viral genomes enable the easy and rapid quantification of virus replication, which is instrumental to efficient in vitro screening of antiviral compounds or in vivo analysis of viral spread and pathogenesis. Based on a published design, we have generated several replication competent influenza A viruses carrying either fluorescent proteins or Gaussia luciferase. Reporter activity could be readily quantified in infected cultures, but the virus encoding Gaussia luciferase was more stable than viruses bearing fluorescent proteins and was therefore analyzed in detail. Quantification of Gaussia luciferase activity in the supernatants of infected culture allowed the convenient and highly sensitive detection of viral spread, and enzymatic activity correlated with the number of infectious particles released from infected cells. Furthermore, the Gaussia luciferase encoding virus allowed the sensitive quantification of the antiviral activity of the neuraminidase inhibitor (NAI) zanamivir and the host cell interferon-inducible transmembrane (IFITM) proteins 1–3, which are known to inhibit influenza virus entry. Finally, the virus was used to demonstrate that influenza A virus infection is sensitive to a modulator of endosomal cholesterol, in keeping with the concept that IFITMs inhibit viral entry by altering cholesterol levels in the endosomal membrane. In sum, we report the characterization of a novel influenza A reporter virus, which allows fast and sensitive detection of viral spread and its inhibition, and we show that influenza A virus entry is sensitive to alterations of endosomal cholesterol levels. 相似文献
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Zhang Leike Liu Jia Cao Ruiyuan Xu Mingyue Wu Yan Shang Weijuan Wang Xi Zhang Huanyu Jiang Xiaming Sun Yuan Hu Hengrui Li Yufeng Zou Gang Zhang Min Zhao Lei Li Wei Guo Xiaojia Zhuang Xiaomei Yang Xing-Lou Shi Zheng-Li Deng Fei Hu Zhihong Xiao Gengfu Wang Manli Zhong Wu 《中国病毒学》2020,35(6):776-784
Virologica Sinica - The recent outbreak of novel coronavirus pneumonia (COVID-19) caused by a new coronavirus has posed a great threat to public health. Identifying safe and effective antivirals is... 相似文献
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Poliovirus RNA-Dependent RNA Polymerase Synthesizes Full-Length Copies of Poliovirion RNA, Cellular mRNA, and Several Plant Virus RNAs In Vitro 总被引:20,自引:16,他引:4
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The poliovirus RNA-dependent RNA polymerase was active on synthetic homopolymeric RNA templates as well as on every natural RNA tested. The polymerase copied polyadenylate. oligouridylate [oligo(U)], polycytidylate . oligoinosinate, and polyinosinate. oligocytidylate templates to about the same extent. The observed activity on polyuridylate. oligoadenylate was about fourfold less. Full-length copies of both poliovirion RNA and a wide variety of other polyadenylated RNAs were synthesized by the polymerase in the presence of oligo(U). Polymerase elongation rates on poliovirion RNA and a heterologous RNA (squash mosaic virus RNA) were about the same. Changes in the Mg(2+) concentration affected the elongation rates on both RNAs to the same extent. With two non-polyadenylated RNAs (tobacco mosaic virus RNA and brome mosaic virus RNA3), the results were different. The purified polymerase synthesized a subgenomic-sized product RNA on brome mosaic virus RNA3 in the presence of oligo(U). This product RNA appeared to initiate on oligo(U) hybridized to an internal oligoadenylate sequence in brome mosaic virus RNA3. No oligo(U)-primed product was synthesized on tobacco mosaic virus RNA. When partially purified polymerase was used in place of the completely purified enzyme, some oligo(U)-independent activity was observed on the brome mosaic virus and tobacco mosaic virus RNAs. The size of the product RNA from these reactions suggested that at least some of the product RNA was full-sized and covalently linked to the template RNA. Thus, the polymerase was found to copy many different types of RNA and to make full-length copies of the RNAs tested. 相似文献
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Tracey R. O’Donovan Simon Rajendran Seamus O’Reilly Gerald C. O’Sullivan Sharon L. McKenna 《PloS one》2015,10(8)
Many epithelial cancers, particularly gastrointestinal tract cancers, remain poor prognosis diseases, due to resistance to cytotoxic therapy and local or metastatic recurrence. We have previously shown that apoptosis incompetent esophageal cancer cells induce autophagy in response to chemotherapeutic agents and this can facilitate their recovery. However, known pharmacological inhibitors of autophagy could not enhance cytotoxicity. In this study, we have examined two well known, clinically approved autophagy inducers, rapamycin and lithium, for their effects on chemosensitivity in apoptosis incompetent cancer cells. Both lithium and rapamycin were shown to induce autophagosomes in esophageal and colorectal cancer cells by western blot analysis of LC3 isoforms, morphology and FACS quantitation of Cyto-ID or mCherry-GFP-LC3. Analysis of autophagic flux indicates inefficient autophagosome processing in lithium treated cells, whereas rapamycin treated cells showed efficient flux. Viability and recovery was assessed by clonogenic assays. When combined with the chemotherapeutic agent 5-fluorouracil, rapamycin was protective. In contrast, lithium showed strong enhancement of non-apoptotic cell death. The combination of lithium with 5-fluorouracil or oxaliplatin was then tested in the syngenic mouse (balb/c) colorectal cancer model—CT26. When either chemotherapeutic agent was combined with lithium a significant reduction in tumor volume was achieved. In addition, survival was dramatically increased in the combination group (p < 0.0001), with > 50% of animals achieving long term cure without re-occurrence (> 1 year tumor free). Thus, combination treatment with lithium can substantially improve the efficacy of chemotherapeutic agents in apoptosis deficient cancer cells. Induction of compromised autophagy may contribute to this cytotoxicity. 相似文献
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Inhibition of Influenza A Virus Replication by Compounds Interfering with the Fusogenic Function of the Viral Hemagglutinin
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Stephen J. Plotch Bryan OHara John Morin Olga Palant James LaRocque Jonathan D. Bloom Stanley A. Lang Jr. Martin J. DiGrandi Mary Bradley Ramaswamy Nilakantan Yakov Gluzman 《Journal of virology》1999,73(1):140-151
Several compounds that specifically inhibited replication of the H1 and H2 subtypes of influenza virus type A were identified by screening a chemical library for antiviral activity. In single-cycle infections, the compounds inhibited virus-specific protein synthesis when added before or immediately after infection but were ineffective when added 30 min later, suggesting that an uncoating step was blocked. Sequencing of hemagglutinin (HA) genes of several independent mutant viruses resistant to the compounds revealed single amino acid changes that clustered in the stem region of the HA trimer in and near the HA2 fusion peptide. One of the compounds, an N-substituted piperidine, could be docked in a pocket in this region by computer-assisted molecular modeling. This compound blocked the fusogenic activity of HA, as evidenced by its inhibition of low-pH-induced cell-cell fusion in infected cell monolayers. An analog which was more effective than the parent compound in inhibiting virus replication was synthesized. It was also more effective in blocking other manifestations of the low-pH-induced conformational change in HA, including virus inactivation, virus-induced hemolysis of erythrocytes, and susceptibility of the HA to proteolytic degradation. Both compounds inhibited viral protein synthesis and replication more effectively in cells infected with a virus mutated in its M2 protein than with wild-type virus. The possible functional relationship between M2 and HA suggested by these results is discussed. 相似文献