AIM: To analyze the association of HCV-RNA with peripheral blood mononuclear cells (PBMC) and to answer the question whether HCV-RNA positivity in PBMC is due to viral replication. METHODS: HCV-RNA was monitored in se...AIM: To analyze the association of HCV-RNA with peripheral blood mononuclear cells (PBMC) and to answer the question whether HCV-RNA positivity in PBMC is due to viral replication. METHODS: HCV-RNA was monitored in serum and PBMC preparations from 15 patients with chronic HCV infection before, during and after an IFN-alpha therapy using a nested RT/PCR technique. In a second approach, PBMC from healthy donors were incubated in HCV positive plasma. RESULTS: In the IFN-alpha responding patients,HCV-RNA disappeared first from total RNA preparations of PBMC and then from serum. In contrast, in relapsing patients, HCV-RNA reappeared first in serum and then in PBMC. A quantitative analysis of the HCV-RNA concentration in serum was performed before and after transition from detectable to non detectable HCV-RNA in PBMC-RNA and vice versa. When HCV-RNA was detectable in PBMC preparations, the HCV concentration in serum was significantly higher than the serum HCV-RNA concentration when HCV-RNA in PBMC was not detectable. Furthermore, at no time during the observation period was HCV specific RNA observed in PBMC, if HCV-RNA in serum was under the detection limit. Incubation of PBMC from healthy donors with several dilutions of HCV positive plasma for two hours showed a concentration dependent PCR positivity for HCV-RNA in reisolated PBMC. CONCLUSION: The detectability of HCV-RNA in total RNA from PBMC seems to depend on the HCV concentration in serum. Contamination or passive adsorption by circulating virus could be the reason for detection of HCV-RNA in PBMC preparations of chronically infected patients.展开更多
This study utilizes the enzyme-substrate complex theory to predict the clinical efficacy of COVID-19 treatments at the biological systems level, using molecular docking stability indicators. Experimental data from the...This study utilizes the enzyme-substrate complex theory to predict the clinical efficacy of COVID-19 treatments at the biological systems level, using molecular docking stability indicators. Experimental data from the Protein Data Bank and molecular structures generated by AlphaFold 3 were used to create macromolecular complex templates. Six templates were developed, including the holo nsp7-nsp8-nsp12 (RNA-dependent RNA polymerase) complex with dsRNA primers (holo-RdRp-RNA). The study evaluated several ligands—Favipiravir-RTP, Remdesivir, Abacavir, Ribavirin, and Oseltamivir—as potential viral RNA polymerase inhibitors. Notably, the first four of these ligands have been clinically employed in the treatment of COVID-19, allowing for comparative analysis. Molecular docking simulations were performed using AutoDock 4, and statistical differences were assessed through t-tests and Mann-Whitney U tests. A review of the literature on COVID-19 treatment outcomes and inhibitors targeting RNA polymerase enzymes was conducted, and the inhibitors were ranked according to their clinical efficacy: Remdesivir > Favipiravir-RTP > Oseltamivir. Docking results obtained from the second and third templates aligned with clinical observations. Furthermore, Abacavir demonstrated a predicted efficacy comparable to Favipiravir-RTP, while Ribavirin exhibited a predicted efficacy similar to that of Remdesivir. This research, focused on inhibitors of SARS-CoV-2 RNA-dependent RNA polymerase, establishes a framework for screening AI-generated drug templates based on clinical outcomes. Additionally, it develops a drug screening platform based on molecular docking binding energy, enabling the evaluation of novel or repurposed drugs and potentially accelerating the drug development process.展开更多
AIM To investigate the existence of hepatitis C virus (HCV) RNA in serum and in peripheral blood mononuclear cells (PBMC) of patients with hepatitis C and its clinical significance. METHODS HCV RNA was detected by ...AIM To investigate the existence of hepatitis C virus (HCV) RNA in serum and in peripheral blood mononuclear cells (PBMC) of patients with hepatitis C and its clinical significance. METHODS HCV RNA was detected by nested polymerase chain reaction (Nested PCR) in serum and in PBMC of 46 patients with acute hepatitis C (AHC) and in 42 with chronic hepatitis C (CHC). RESULTS The positive rate of HCV RNA in PBMC of patients with CHC was markedly higher than that of patients with AHC ( P <0 01). The positive rates of HCV RNA in serum of patients with AHC and CHC and in PBMC of patients with CHC were significantly higher than those of anti HCV positive patients with normal ALT level ( P <0 01). HCV RNA was negative in serum of 2 patients, but could be detected in PBMC. In 12 patients anti HCV was negative while HCV RNA was positive in serum. CONCLUSION ① detection of serum HCV RNA by nested PCR might be helpful in the early diagnosis of anti HCV negative hepatitis C; ② liver damage in patients with hepatitis C might be correlated with HCV viremia; ③ infection of PBMC by HCV might play an important role in the chronic liver damage in patients with HC and the chronicity of its clinical course; ④ PBMC might be considered as a “reservoir” for HCV.展开更多
RNA interference (RNAi) is an evolutionally conserved gene silencing mechanism present in a variety of eukaryotic species. RNAi uses short double-stranded RNA (dsRNA) to trigger degradation or translation repression o...RNA interference (RNAi) is an evolutionally conserved gene silencing mechanism present in a variety of eukaryotic species. RNAi uses short double-stranded RNA (dsRNA) to trigger degradation or translation repression of homologous RNA targets in a sequence-specific manner. This system can be induced effectively in vitro and in vivo by direct application of small interfering RNAs (siRNAs), or by expression of short hairpin RNA (shRNA) with non-viral and viral vectors. To date, RNAi has been extensively used as a novel and effective tool for functional genomic studies, and has displayed great potential in treating human diseases, including human genetic and acquired disorders such as cancer and viral infections. In the present review, we focus on the recent development in the use of RNAi in the prevention and treatment of viral infections. The mechanisms, strategies, hurdles and prospects of employing RNAi in the pharmaceutical industry are also discussed.展开更多
Objective To investigate distinctive features in drug-resistant mutations （DRMs） and interpretations for reverse transcriptase inhibitors （RTIs） between proviral DNA and paired viral RNA in HIV-l-infected patients...Objective To investigate distinctive features in drug-resistant mutations （DRMs） and interpretations for reverse transcriptase inhibitors （RTIs） between proviral DNA and paired viral RNA in HIV-l-infected patients. Methods Forty-three HIV-l-infected individuals receiving first-line antiretroviral therapy were recruited to participate in a multicenter AIDS Cohort Study in Anhui and Henan Provinces in China in 2004. Drug resistance genotyping was performed by bulk sequencing and deep sequencing on the plasma and whole blood of 77 samples, respectively. Drug-resistance interpretation was compared between viral RNA and paired proviral DNA. Results Compared with bulk sequencing, deep sequencing could detect more DRMs and samples with DRMs in both viral RNA and proviral DNA. The mutations M1841 and M2301 were more prevalent in proviral DNA than in viral RNA （Fisher＇s exact test, P〈0.05）. Considering ＇majority resistant variants＇, 15 samples （19.48%） showed differences in drug resistance interpretation between viral RNA and proviral DNA, and 5 of these samples with different DRMs between proviral DNA and paired viral RNA showed a higher level of drug resistance to the first-line drugs. Considering ＇minority resistant variants＇, 22 samples （28.57%） were associated with a higher level of drug resistance to the tested RTIs for proviral DNA when compared with paired viral RNA. Conclusion Compared with viral RNA, the distinctive information of DRMs and drug resistance interpretations for proviral DNA could be obtained by deep sequencing, which could provide more detailed and precise information for drug resistance monitoring and the rational design of optimal antiretroviral therapy regimens.展开更多
Safe and effective gene therapy approaches require targeted tissue-specific transfer of a therapeutic transgene.Besides traditional approaches, such as transcriptional and transductional targeting, micro RNA-dependent...Safe and effective gene therapy approaches require targeted tissue-specific transfer of a therapeutic transgene.Besides traditional approaches, such as transcriptional and transductional targeting, micro RNA-dependent posttranscriptional suppression of transgene expression has been emerging as powerful new technology to increase the specificity of vector-mediated transgene expression. Micro RNAs are small non-coding RNAs and often expressed in a tissue-, lineage-, activation- or differentiation-specific pattern. They typically regulate gene expression by binding to imperfectly complementary sequences in the 3' untranslated region(UTR) of the m RNA. To control exogenous transgene expression, tandem repeats of artificial micro RNA target sites are usually incorporated into the 3' UTR of the transgene expression cassette, leading to subsequent degradation of transgene m RNA in cel s expressing the corresponding micro RNA. This targeting strategy, first shown for lentiviral vectors in antigen presenting cells, has now been used for tissue-specific expression of vector-encoded therapeutic transgenes, to reduce immune response against the transgene, to control virus tropism for oncolytic virotherapy, to increase safety of live attenuated virus vaccines and to identify and select cell subsets for pluripotent stem cell therapies, respectively. This review provides an introduction into the technical mechanism underlying micro RNA-regulation, highlights new developments in this field and gives an overview of applications of micro RNA-regulated viral vectors for cardiac, suicide gene cancer and hematopoietic stem cell therapy, as well as for treatment of neurological and eye diseases.展开更多
Small brown planthopper(SBPH, Laodelphax striatellus Fallén) is an important vector of major crop pathogen rice stripe virus(RSV). Controlling SBPH population is an efficient approach to control RSV. Long non-cod...Small brown planthopper(SBPH, Laodelphax striatellus Fallén) is an important vector of major crop pathogen rice stripe virus(RSV). Controlling SBPH population is an efficient approach to control RSV. Long non-coding RNAs(lnc RNA) have been reported to block virus replication in hosts. However, the function of lnc RNAs in RSV infection and replication is still unknown. Here, we aimed to study regulatory mechanisms of lnc RNA in an immune system during RSV infection. First, lnc RNA genes were predicted from SBPH transcriptomes using a bioinformatics pipeline based on characteristics of lnc RNA. We identified 4 786 lnc RNA genes corresponding to 5 790 transcripts in SBPH from an RNA-Seq dataset of 15 transcriptomes. Differential expression analysis indicated that 3, 11, and 25 lnc RNA genes were highly expressed in gut, salivary gland, and ovary, respectively, of viruliferous SBPH(Student’s t-test, P<0.05). We randomly selected eight lnc RNAs for expression validation using quantitative real-time PCR, confirming the differential expression of these lnc RNAs between viruliferous and non-viruliferous SBPH. In summary, we present evidence that the expression of lnc RNA genes was induced by RSV infection, suggesting that RSV might be involved in the antivirus immune system in SBPH and participate in regulating the RSV replication mechanism. These data provide helpful information for future investigations of the interaction between lncRNA and RSV.展开更多
Negative-sense RNA viruses comprise several zoonotic pathogens that mutate rapidly and frequently emerge in people including Influenza, Ebola, Rabies, Hendra and Nipah viruses. Acute respiratory distress syndrome, enc...Negative-sense RNA viruses comprise several zoonotic pathogens that mutate rapidly and frequently emerge in people including Influenza, Ebola, Rabies, Hendra and Nipah viruses. Acute respiratory distress syndrome, encephalitis and vasculitis are common disease outcomes in people as a result of pathogenic viral infection, and are also associated with high case fatality rates. Viral spread from exposure sites to systemic tissues and organs is mediated by virulence factors, including viral attachment glycoproteins and accessory proteins, and their contribution to infection and disease have been delineated by reverse genetics; a molecular approach that enables researchers to experimentally produce recombinant and reassortant viruses from cloned cD NA. Through reverse genetics we have developed a deeper understanding of virulence factors key to disease causation thereby enabling development of targeted antiviral therapies and well-defined live attenuated vaccines. Despite the value of reverse genetics for virulence factor discovery, classical reverse genetic approaches may not provide sufficient resolution for characterization of heterogeneous viral populations, because current techniques recover clonal virus, representing a consensus sequence. In this review the contribution of reverse genetics to virulence factor characterization is outlined, while the limitation of the technique is discussed withreference to new technologies that may be utilized to improve reverse genetic approaches.展开更多
HIV-1 matrix protein (MA) is a multifunctional structural protein localized on N terminus of Gag precursor p55. MA participates in HIV-1 assembly as membranotropic part of Gag precursor as well as an individual protei...HIV-1 matrix protein (MA) is a multifunctional structural protein localized on N terminus of Gag precursor p55. MA participates in HIV-1 assembly as membranotropic part of Gag precursor as well as an individual protein spliced from Gag early in infection. MA is found in the nuclei of infected cells and in plasma membrane, the site of virus assembly, in association with viral genome RNA. MA mutated variant M4 which contains two changed amino acids in N-terminal regions is also associated with viral RNA, but it is localized in the nuclear and cytoskeleton fractions but not in the plasma membrane suggesting that the mutant is deprived of membranotropic signal and “sticks” in the nuclei an d cytoskeleton, its previous location sites. These data allow suggesting that MA involved into transmission of viral RNA is transported to plasma membrane by cytoskeleton.展开更多
This is the first systematic investigation of viral pathogens in <i>Vitis</i> <i>vinifera</i> from Hangzhou vicinity of China. About 7 viruses and 5 viroids were annotated from four production ...This is the first systematic investigation of viral pathogens in <i>Vitis</i> <i>vinifera</i> from Hangzhou vicinity of China. About 7 viruses and 5 viroids were annotated from four production bases “Dushicun”, “Wangjiayuan”, “Xiajiangcun”, and “Yangducun” covering 15 cultivars through sRNAseq technique. At least 3 viruses<a name="OLE_LINK4"></a>—grapevine leaf roll-associated virus 3 (GLRaV-3), grapevine fleck <span>virus (GFkV) and grapevine geminivirus A (GGVA), and 4 viroids—hop stunt</span> viroid (HSVd), citrus viroid II (CVd-II), grapevine yellow speckle viroid 1 (GYSVd-1) and grapevine yellow speckle viroid 2 (GYSVd-2) infected all four bases. “Yangducun” base showed 11, the most infected pathogens. GYSVd-1 showed the highest accumulation in host of Wangjiayuan base. The main in<span>fected pathogens were verified by reverse-transcription polymerase chain reaction</span> (RT-PCR) technique, the detected rate reached to 85% - 100%. The results provide an important basis for effective and precise detection of viral diseases in the area and for the virus-free cultivation in future.展开更多
基金Supported by a grant of DFG (SFB 402 Teilprojekt C1 (Mihm))by a grant of Hoffmann La Roche (Grenzach-Wyhden, Germany)Part of the data has been presented as poster at the 1999 EASL-meeting in Neaples
文摘AIM: To analyze the association of HCV-RNA with peripheral blood mononuclear cells (PBMC) and to answer the question whether HCV-RNA positivity in PBMC is due to viral replication. METHODS: HCV-RNA was monitored in serum and PBMC preparations from 15 patients with chronic HCV infection before, during and after an IFN-alpha therapy using a nested RT/PCR technique. In a second approach, PBMC from healthy donors were incubated in HCV positive plasma. RESULTS: In the IFN-alpha responding patients,HCV-RNA disappeared first from total RNA preparations of PBMC and then from serum. In contrast, in relapsing patients, HCV-RNA reappeared first in serum and then in PBMC. A quantitative analysis of the HCV-RNA concentration in serum was performed before and after transition from detectable to non detectable HCV-RNA in PBMC-RNA and vice versa. When HCV-RNA was detectable in PBMC preparations, the HCV concentration in serum was significantly higher than the serum HCV-RNA concentration when HCV-RNA in PBMC was not detectable. Furthermore, at no time during the observation period was HCV specific RNA observed in PBMC, if HCV-RNA in serum was under the detection limit. Incubation of PBMC from healthy donors with several dilutions of HCV positive plasma for two hours showed a concentration dependent PCR positivity for HCV-RNA in reisolated PBMC. CONCLUSION: The detectability of HCV-RNA in total RNA from PBMC seems to depend on the HCV concentration in serum. Contamination or passive adsorption by circulating virus could be the reason for detection of HCV-RNA in PBMC preparations of chronically infected patients.
文摘This study utilizes the enzyme-substrate complex theory to predict the clinical efficacy of COVID-19 treatments at the biological systems level, using molecular docking stability indicators. Experimental data from the Protein Data Bank and molecular structures generated by AlphaFold 3 were used to create macromolecular complex templates. Six templates were developed, including the holo nsp7-nsp8-nsp12 (RNA-dependent RNA polymerase) complex with dsRNA primers (holo-RdRp-RNA). The study evaluated several ligands—Favipiravir-RTP, Remdesivir, Abacavir, Ribavirin, and Oseltamivir—as potential viral RNA polymerase inhibitors. Notably, the first four of these ligands have been clinically employed in the treatment of COVID-19, allowing for comparative analysis. Molecular docking simulations were performed using AutoDock 4, and statistical differences were assessed through t-tests and Mann-Whitney U tests. A review of the literature on COVID-19 treatment outcomes and inhibitors targeting RNA polymerase enzymes was conducted, and the inhibitors were ranked according to their clinical efficacy: Remdesivir > Favipiravir-RTP > Oseltamivir. Docking results obtained from the second and third templates aligned with clinical observations. Furthermore, Abacavir demonstrated a predicted efficacy comparable to Favipiravir-RTP, while Ribavirin exhibited a predicted efficacy similar to that of Remdesivir. This research, focused on inhibitors of SARS-CoV-2 RNA-dependent RNA polymerase, establishes a framework for screening AI-generated drug templates based on clinical outcomes. Additionally, it develops a drug screening platform based on molecular docking binding energy, enabling the evaluation of novel or repurposed drugs and potentially accelerating the drug development process.
文摘AIM To investigate the existence of hepatitis C virus (HCV) RNA in serum and in peripheral blood mononuclear cells (PBMC) of patients with hepatitis C and its clinical significance. METHODS HCV RNA was detected by nested polymerase chain reaction (Nested PCR) in serum and in PBMC of 46 patients with acute hepatitis C (AHC) and in 42 with chronic hepatitis C (CHC). RESULTS The positive rate of HCV RNA in PBMC of patients with CHC was markedly higher than that of patients with AHC ( P <0 01). The positive rates of HCV RNA in serum of patients with AHC and CHC and in PBMC of patients with CHC were significantly higher than those of anti HCV positive patients with normal ALT level ( P <0 01). HCV RNA was negative in serum of 2 patients, but could be detected in PBMC. In 12 patients anti HCV was negative while HCV RNA was positive in serum. CONCLUSION ① detection of serum HCV RNA by nested PCR might be helpful in the early diagnosis of anti HCV negative hepatitis C; ② liver damage in patients with hepatitis C might be correlated with HCV viremia; ③ infection of PBMC by HCV might play an important role in the chronic liver damage in patients with HC and the chronicity of its clinical course; ④ PBMC might be considered as a “reservoir” for HCV.
基金RFCID, No 01030152, RGC, CUHK4428/06M, ITF ITS091/03 of Hong Kong Government, and Faculty Direct Fund of the Chinese University of Hong Kong
文摘RNA interference (RNAi) is an evolutionally conserved gene silencing mechanism present in a variety of eukaryotic species. RNAi uses short double-stranded RNA (dsRNA) to trigger degradation or translation repression of homologous RNA targets in a sequence-specific manner. This system can be induced effectively in vitro and in vivo by direct application of small interfering RNAs (siRNAs), or by expression of short hairpin RNA (shRNA) with non-viral and viral vectors. To date, RNAi has been extensively used as a novel and effective tool for functional genomic studies, and has displayed great potential in treating human diseases, including human genetic and acquired disorders such as cancer and viral infections. In the present review, we focus on the recent development in the use of RNAi in the prevention and treatment of viral infections. The mechanisms, strategies, hurdles and prospects of employing RNAi in the pharmaceutical industry are also discussed.
基金supported by grants from the State Key Laboratory of Infectious Disease Prevention and Control(2011SKLID102)the National Nature Science Foundation of China(81172733 and 81561128006)the 12th Five-Year National Science and Technology Major Project(2013ZX10001-006)
文摘Objective To investigate distinctive features in drug-resistant mutations （DRMs） and interpretations for reverse transcriptase inhibitors （RTIs） between proviral DNA and paired viral RNA in HIV-l-infected patients. Methods Forty-three HIV-l-infected individuals receiving first-line antiretroviral therapy were recruited to participate in a multicenter AIDS Cohort Study in Anhui and Henan Provinces in China in 2004. Drug resistance genotyping was performed by bulk sequencing and deep sequencing on the plasma and whole blood of 77 samples, respectively. Drug-resistance interpretation was compared between viral RNA and paired proviral DNA. Results Compared with bulk sequencing, deep sequencing could detect more DRMs and samples with DRMs in both viral RNA and proviral DNA. The mutations M1841 and M2301 were more prevalent in proviral DNA than in viral RNA （Fisher＇s exact test, P〈0.05）. Considering ＇majority resistant variants＇, 15 samples （19.48%） showed differences in drug resistance interpretation between viral RNA and proviral DNA, and 5 of these samples with different DRMs between proviral DNA and paired viral RNA showed a higher level of drug resistance to the first-line drugs. Considering ＇minority resistant variants＇, 22 samples （28.57%） were associated with a higher level of drug resistance to the tested RTIs for proviral DNA when compared with paired viral RNA. Conclusion Compared with viral RNA, the distinctive information of DRMs and drug resistance interpretations for proviral DNA could be obtained by deep sequencing, which could provide more detailed and precise information for drug resistance monitoring and the rational design of optimal antiretroviral therapy regimens.
基金Supported by The Deutsche Forschungsgemeinschaft,Nos.FE785/2-2 and FE785/4-1the Bundesministerium für Bildung und Entwicklung,No.031A331
文摘Safe and effective gene therapy approaches require targeted tissue-specific transfer of a therapeutic transgene.Besides traditional approaches, such as transcriptional and transductional targeting, micro RNA-dependent posttranscriptional suppression of transgene expression has been emerging as powerful new technology to increase the specificity of vector-mediated transgene expression. Micro RNAs are small non-coding RNAs and often expressed in a tissue-, lineage-, activation- or differentiation-specific pattern. They typically regulate gene expression by binding to imperfectly complementary sequences in the 3' untranslated region(UTR) of the m RNA. To control exogenous transgene expression, tandem repeats of artificial micro RNA target sites are usually incorporated into the 3' UTR of the transgene expression cassette, leading to subsequent degradation of transgene m RNA in cel s expressing the corresponding micro RNA. This targeting strategy, first shown for lentiviral vectors in antigen presenting cells, has now been used for tissue-specific expression of vector-encoded therapeutic transgenes, to reduce immune response against the transgene, to control virus tropism for oncolytic virotherapy, to increase safety of live attenuated virus vaccines and to identify and select cell subsets for pluripotent stem cell therapies, respectively. This review provides an introduction into the technical mechanism underlying micro RNA-regulation, highlights new developments in this field and gives an overview of applications of micro RNA-regulated viral vectors for cardiac, suicide gene cancer and hematopoietic stem cell therapy, as well as for treatment of neurological and eye diseases.
基金supported by the National Natural Science Foundation of China (31701785 and 31760514)the Natural Science Foundation of Zhejiang Province, China (LZ18C060001)+1 种基金the China Postdoctoral Science Foundation (2018M632481)the Postdoctoral Research Advanced Project of Zhejiang Province, China (ZJ20180120)
文摘Small brown planthopper(SBPH, Laodelphax striatellus Fallén) is an important vector of major crop pathogen rice stripe virus(RSV). Controlling SBPH population is an efficient approach to control RSV. Long non-coding RNAs(lnc RNA) have been reported to block virus replication in hosts. However, the function of lnc RNAs in RSV infection and replication is still unknown. Here, we aimed to study regulatory mechanisms of lnc RNA in an immune system during RSV infection. First, lnc RNA genes were predicted from SBPH transcriptomes using a bioinformatics pipeline based on characteristics of lnc RNA. We identified 4 786 lnc RNA genes corresponding to 5 790 transcripts in SBPH from an RNA-Seq dataset of 15 transcriptomes. Differential expression analysis indicated that 3, 11, and 25 lnc RNA genes were highly expressed in gut, salivary gland, and ovary, respectively, of viruliferous SBPH(Student’s t-test, P<0.05). We randomly selected eight lnc RNAs for expression validation using quantitative real-time PCR, confirming the differential expression of these lnc RNAs between viruliferous and non-viruliferous SBPH. In summary, we present evidence that the expression of lnc RNA genes was induced by RSV infection, suggesting that RSV might be involved in the antivirus immune system in SBPH and participate in regulating the RSV replication mechanism. These data provide helpful information for future investigations of the interaction between lncRNA and RSV.
文摘Negative-sense RNA viruses comprise several zoonotic pathogens that mutate rapidly and frequently emerge in people including Influenza, Ebola, Rabies, Hendra and Nipah viruses. Acute respiratory distress syndrome, encephalitis and vasculitis are common disease outcomes in people as a result of pathogenic viral infection, and are also associated with high case fatality rates. Viral spread from exposure sites to systemic tissues and organs is mediated by virulence factors, including viral attachment glycoproteins and accessory proteins, and their contribution to infection and disease have been delineated by reverse genetics; a molecular approach that enables researchers to experimentally produce recombinant and reassortant viruses from cloned cD NA. Through reverse genetics we have developed a deeper understanding of virulence factors key to disease causation thereby enabling development of targeted antiviral therapies and well-defined live attenuated vaccines. Despite the value of reverse genetics for virulence factor discovery, classical reverse genetic approaches may not provide sufficient resolution for characterization of heterogeneous viral populations, because current techniques recover clonal virus, representing a consensus sequence. In this review the contribution of reverse genetics to virulence factor characterization is outlined, while the limitation of the technique is discussed withreference to new technologies that may be utilized to improve reverse genetic approaches.
文摘HIV-1 matrix protein (MA) is a multifunctional structural protein localized on N terminus of Gag precursor p55. MA participates in HIV-1 assembly as membranotropic part of Gag precursor as well as an individual protein spliced from Gag early in infection. MA is found in the nuclei of infected cells and in plasma membrane, the site of virus assembly, in association with viral genome RNA. MA mutated variant M4 which contains two changed amino acids in N-terminal regions is also associated with viral RNA, but it is localized in the nuclear and cytoskeleton fractions but not in the plasma membrane suggesting that the mutant is deprived of membranotropic signal and “sticks” in the nuclei an d cytoskeleton, its previous location sites. These data allow suggesting that MA involved into transmission of viral RNA is transported to plasma membrane by cytoskeleton.
文摘This is the first systematic investigation of viral pathogens in <i>Vitis</i> <i>vinifera</i> from Hangzhou vicinity of China. About 7 viruses and 5 viroids were annotated from four production bases “Dushicun”, “Wangjiayuan”, “Xiajiangcun”, and “Yangducun” covering 15 cultivars through sRNAseq technique. At least 3 viruses<a name="OLE_LINK4"></a>—grapevine leaf roll-associated virus 3 (GLRaV-3), grapevine fleck <span>virus (GFkV) and grapevine geminivirus A (GGVA), and 4 viroids—hop stunt</span> viroid (HSVd), citrus viroid II (CVd-II), grapevine yellow speckle viroid 1 (GYSVd-1) and grapevine yellow speckle viroid 2 (GYSVd-2) infected all four bases. “Yangducun” base showed 11, the most infected pathogens. GYSVd-1 showed the highest accumulation in host of Wangjiayuan base. The main in<span>fected pathogens were verified by reverse-transcription polymerase chain reaction</span> (RT-PCR) technique, the detected rate reached to 85% - 100%. The results provide an important basis for effective and precise detection of viral diseases in the area and for the virus-free cultivation in future.
