Background:The injection of rabies immune globulin(RIG)is of the utmost importance in the management of category III exposures to rabies-suspect animals.Because of the high cost and limited availability of existing RI...Background:The injection of rabies immune globulin(RIG)is of the utmost importance in the management of category III exposures to rabies-suspect animals.Because of the high cost and limited availability of existing RIG,one possible replacement for RIG is monoclonal antibodies(MAbs)against the rabies virus(RABV).Consequently,it is necessary to determine the neutralizing activity of the MAbs against rabies viruses,especially street rabies virus.However,the method to detect the neutralizing activity of MAbs against street rabies virus remains undefined.Methods:To establish a method for detecting the neutralizing activity of MAbs against street rabies virus,we constructed a library consisting of 12 strains of street RABV from 11 provinces in China.Using this street RABV library and the Reed-Muench formula,we established a method for detecting the neutralizing titer of the MAbs.The reliability and repeatability of the method were evaluated by repeatedly measuring the neutralizing activity of a MAb and a post vaccination serum.Results:A total of 12 strains of street RABV were chosen for inclusion in the street RABV library,which covered six Chinese lineages(China I-China VI)and grew to high titers in N2A cells(>105 FFD50/ml).On the basis of the library,we constructed the method to detect the neutralizing activity of the MAbs.The results of repeatedly measuring the MAbs and positive serum showed excellent reliability and repeatability of the method established in this study.Conclusions:This study established a street RABV library reflecting the epidemiological features of Chinese rabies viruses,which provides a platform for detecting the neutralizing activity of MAbs against rabies viruses circulating in China.展开更多
Many studies suggest that severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)can infect various animals and transmit among animals,and even to humans,posing a threat to humans and animals.There is an urgent ne...Many studies suggest that severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)can infect various animals and transmit among animals,and even to humans,posing a threat to humans and animals.There is an urgent need to develop inexpensive and efficient animal vaccines to prevent and control coronavirus disease 2019(COVID-19)in animals.Rabies virus(RABV)is another important zoonotic pathogen that infects almost all warmblooded animals and poses a great public health threat.The present study constructed two recombinant chimeric viruses expressing the S1 and RBD proteins of the SARS-CoV-2 Wuhan01 strain based on a reverse genetic system of the RABV SRV9 strain and evaluated their immunogenicity in mice,cats and dogs.The results showed that both inactivated recombinant viruses induced durable neutralizing antibodies against SARS-CoV-2 and RABV and a strong cellular immune response in mice.Notably,inactivated SRV-nCoV-RBD induced earlier antibody production than SRV-nCoV-S1,which was maintained at high levels for longer periods.Inactivated SRV-nCoV-RBD induced neutralizing antibodies against both SARS-CoV-2 and RABV in cats and dogs,with a relatively broadspectrum cross-neutralization capability against the SARS-CoV-2 pseudoviruses including Alpha,Beta,Gamma,Delta,and Omicron,showing potential to be used as a safe bivalent vaccine candidate against COVID-19 and rabies in animals.展开更多
Although inactivated vaccines against rabies have the advantage of high safety,effective protection against rabies virus(RABV)infection often requires multiple,high-dose immunization.Incorporating a molecular adjuvant...Although inactivated vaccines against rabies have the advantage of high safety,effective protection against rabies virus(RABV)infection often requires multiple,high-dose immunization.Incorporating a molecular adjuvant into the viral particles has been found to be a useful strategy to promote the immune effectiveness of inactivated vaccines.In this study,we constructed a recombinant virus,rCVS11-LTB,which chimerically expresses a molecular adjuvant heat-labile enterotoxin B subunit(LTB)protein on the surface of the RABV particles.Immunogenicity in vivo was found to be promoted by rCVS11-LTB through the activation of dendritic cells(DCs).Our results demonstrated that inactivated rCVS11-LTB was able to induce higher levels of virusneutralizing antibodies(VNAs)in both mice and dogs than the parent virus rCVS11,to enhance the cellular immune response and T cell immune memory in mice,and was also able to provide 100%protection in mice from lethal doses of rabies virus,indicating its potential as a safe and effective inactivated rabies vaccine candidate.展开更多
Rabies virus(RABV) is a highly neurotropic virus that follows clathrin-mediated endocytosis and p H-dependent pathway for trafficking and invasion into endothelial cells. Early(Rab5, EEA1) and late(Rab7, LAMP1) endoso...Rabies virus(RABV) is a highly neurotropic virus that follows clathrin-mediated endocytosis and p H-dependent pathway for trafficking and invasion into endothelial cells. Early(Rab5, EEA1) and late(Rab7, LAMP1) endosomal proteins play critical roles in endosomal sorting, maturity and targeting various molecular cargoes, but their precise functions in the early stage of RABV neuronal infection remain elusive. In this study, the relationship between enigmatic entry of RABV with these endosomal proteins into neuronal and SH-SY5 Y cells was investigated.Immunofluorescence, TCID_(50) titers, electron microscopy and western blotting were carried out to determine the molecular interaction of the nucleoprotein(N) of RABV with early or late endosomal proteins in these cell lines. The expression of N was also determined by down-regulating Rab5 and Rab7 in both cell lines through RNA interference. The results were indicative that N proficiently colocalized with Rab5/EEA1 and Rab7/LAMP1 in both cell lines at 24 and 48 h post-infection, while N titers significantly decreased in early infection of RABV. Down-regulation of Rab5 and Rab7 did not inhibit N expression, but it prevented productive infection via blocking the normal trafficking of RABV in a low pH environment. Ultrathin sections of cells studied by electron microscope also verified the close association of RABV with Rab5 and Rab7 in neurons. From the data it was concluded that primary entry of RABV strongly correlates with the kinetics of Rab-proteins present on early and late vesicles, which provides helpful clues to explain the early events of RABV in nerve cells.展开更多
Rabies is caused by infection of rabies virus(RABV)and remains a serious threat to the global public health.Except for the requirement for cold chain and high cost of human rabies immune globulin,no small molecule dru...Rabies is caused by infection of rabies virus(RABV)and remains a serious threat to the global public health.Except for the requirement 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(0.1μmol/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.展开更多
基金This work was supported by the National Program on Key Research Project of China(No.2016YFD0500400)the National Natural Science Foundation of China(No.31500152)the China Mega-Project for Infectious Disease(No.2017ZX10104001–004-001).
文摘Background:The injection of rabies immune globulin(RIG)is of the utmost importance in the management of category III exposures to rabies-suspect animals.Because of the high cost and limited availability of existing RIG,one possible replacement for RIG is monoclonal antibodies(MAbs)against the rabies virus(RABV).Consequently,it is necessary to determine the neutralizing activity of the MAbs against rabies viruses,especially street rabies virus.However,the method to detect the neutralizing activity of MAbs against street rabies virus remains undefined.Methods:To establish a method for detecting the neutralizing activity of MAbs against street rabies virus,we constructed a library consisting of 12 strains of street RABV from 11 provinces in China.Using this street RABV library and the Reed-Muench formula,we established a method for detecting the neutralizing titer of the MAbs.The reliability and repeatability of the method were evaluated by repeatedly measuring the neutralizing activity of a MAb and a post vaccination serum.Results:A total of 12 strains of street RABV were chosen for inclusion in the street RABV library,which covered six Chinese lineages(China I-China VI)and grew to high titers in N2A cells(>105 FFD50/ml).On the basis of the library,we constructed the method to detect the neutralizing activity of the MAbs.The results of repeatedly measuring the MAbs and positive serum showed excellent reliability and repeatability of the method established in this study.Conclusions:This study established a street RABV library reflecting the epidemiological features of Chinese rabies viruses,which provides a platform for detecting the neutralizing activity of MAbs against rabies viruses circulating in China.
基金funded by the National Key Research and Development Program of China(grant No.2021YFC2600202)the National Natural Science Foundation of China(grant numbers 31872487).
文摘Many studies suggest that severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)can infect various animals and transmit among animals,and even to humans,posing a threat to humans and animals.There is an urgent need to develop inexpensive and efficient animal vaccines to prevent and control coronavirus disease 2019(COVID-19)in animals.Rabies virus(RABV)is another important zoonotic pathogen that infects almost all warmblooded animals and poses a great public health threat.The present study constructed two recombinant chimeric viruses expressing the S1 and RBD proteins of the SARS-CoV-2 Wuhan01 strain based on a reverse genetic system of the RABV SRV9 strain and evaluated their immunogenicity in mice,cats and dogs.The results showed that both inactivated recombinant viruses induced durable neutralizing antibodies against SARS-CoV-2 and RABV and a strong cellular immune response in mice.Notably,inactivated SRV-nCoV-RBD induced earlier antibody production than SRV-nCoV-S1,which was maintained at high levels for longer periods.Inactivated SRV-nCoV-RBD induced neutralizing antibodies against both SARS-CoV-2 and RABV in cats and dogs,with a relatively broadspectrum cross-neutralization capability against the SARS-CoV-2 pseudoviruses including Alpha,Beta,Gamma,Delta,and Omicron,showing potential to be used as a safe bivalent vaccine candidate against COVID-19 and rabies in animals.
基金the National Key Research and Development Program of China(No.2022YFD1800100).
文摘Although inactivated vaccines against rabies have the advantage of high safety,effective protection against rabies virus(RABV)infection often requires multiple,high-dose immunization.Incorporating a molecular adjuvant into the viral particles has been found to be a useful strategy to promote the immune effectiveness of inactivated vaccines.In this study,we constructed a recombinant virus,rCVS11-LTB,which chimerically expresses a molecular adjuvant heat-labile enterotoxin B subunit(LTB)protein on the surface of the RABV particles.Immunogenicity in vivo was found to be promoted by rCVS11-LTB through the activation of dendritic cells(DCs).Our results demonstrated that inactivated rCVS11-LTB was able to induce higher levels of virusneutralizing antibodies(VNAs)in both mice and dogs than the parent virus rCVS11,to enhance the cellular immune response and T cell immune memory in mice,and was also able to provide 100%protection in mice from lethal doses of rabies virus,indicating its potential as a safe and effective inactivated rabies vaccine candidate.
基金supported by the National Key Research and Development Program of China(Grant No.216YFD0500402)Natural Science Foundation of China(Grants No.31272579 and 31472208)
文摘Rabies virus(RABV) is a highly neurotropic virus that follows clathrin-mediated endocytosis and p H-dependent pathway for trafficking and invasion into endothelial cells. Early(Rab5, EEA1) and late(Rab7, LAMP1) endosomal proteins play critical roles in endosomal sorting, maturity and targeting various molecular cargoes, but their precise functions in the early stage of RABV neuronal infection remain elusive. In this study, the relationship between enigmatic entry of RABV with these endosomal proteins into neuronal and SH-SY5 Y cells was investigated.Immunofluorescence, TCID_(50) titers, electron microscopy and western blotting were carried out to determine the molecular interaction of the nucleoprotein(N) of RABV with early or late endosomal proteins in these cell lines. The expression of N was also determined by down-regulating Rab5 and Rab7 in both cell lines through RNA interference. The results were indicative that N proficiently colocalized with Rab5/EEA1 and Rab7/LAMP1 in both cell lines at 24 and 48 h post-infection, while N titers significantly decreased in early infection of RABV. Down-regulation of Rab5 and Rab7 did not inhibit N expression, but it prevented productive infection via blocking the normal trafficking of RABV in a low pH environment. Ultrathin sections of cells studied by electron microscope also verified the close association of RABV with Rab5 and Rab7 in neurons. From the data it was concluded that primary entry of RABV strongly correlates with the kinetics of Rab-proteins present on early and late vesicles, which provides helpful clues to explain the early events of RABV in nerve cells.
基金We thank Dr.Xianzhu Xia(Academy of Military Medical Sciences)for providing the CVS-11 virus.We thank Dr.Zhenfang Fu(Huazhong Agricultural University)for providing the CVS-24 virus.This work was supported by the National Key Research and Development Program of China(2016YFD0500400)National Natural Science Foundation of China(31772742)Natural Science Foundation of Guangdong(2015A03031103).
文摘Rabies is caused by infection of rabies virus(RABV)and remains a serious threat to the global public health.Except for the requirement 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(0.1μmol/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.
