Porcine reproductive and respiratory syndrome virus(PRRSV)is characterized by its genetic variation and limited cross protection among heterologous strains.Even though several viral structural proteins have been regar...Porcine reproductive and respiratory syndrome virus(PRRSV)is characterized by its genetic variation and limited cross protection among heterologous strains.Even though several viral structural proteins have been regarded as inducers of neutralizing antibodies(NAs)against PRRSV,the mechanism underlying limited cross-neutralization among heterologous strains is still controversial.In the present study,examinations of NA cross reaction between a highly pathogenic PRRSV(HP-PRRSV)strain,JXwn06,and a low pathogenic PRRSV(LP-PRRSV)strain,HB-1/3.9,were conducted with viral neutralization assays in MARC-145 cells.None of the JXwn06-hyperimmuned pigs’sera could neutralize HB-1/3.9 in vitro and vice versa.To address the genetic variation between these two viruses that are associated with limited crossneutralization,chimeric viruses with coding regions swapped between these two strains were constructed.Viral neutralization assays indicated that variations in nonstructural protein 2(nsp2)and structural proteins together contribute to weak cross-neutralization activity between JXwn06 and HB-1/3.9.Furthermore,we substituted the nsp2-,glycoprotein2(GP2)-,GP3-,and GP4-coding regions together,or nsp2-,GP5-,and membrane(M)protein-coding regions simultaneously between these two viruses to construct chimeric viruses to test cross-neutralization reactivity with hyperimmunized sera induced by their parental viruses.The results indicated that the swapped nsp2 and GP5-M viruses increased the neutralization reactivity with the donor strain antisera in MARC-145 cells.Taken together,these results show that variations in nsp2 and GP5-M correlate with the limited neutralization reactivity between the heterologous strains HP-PRRSV JXwn06 and LP-PRRSV HB-1/3.9.展开更多
Although African swine fever(ASF) has been prevalent for more than a century, it remains the number one swine disease that seriously endangers the global pig industry, and there is no effective means of prevention and...Although African swine fever(ASF) has been prevalent for more than a century, it remains the number one swine disease that seriously endangers the global pig industry, and there is no effective means of prevention and treatment(Wang et al. 2023). Due to its enormous economic and social impact, it is listed as a notifiable animal disease by the World Organization for Animal Health(Costard et al. 2013). Although ASF has been present in Sub-Saharan Africa since its first discovery in Kenya.展开更多
Porcine Contagious Pleuropneumonia(PCP)is a respiratory infectious disease of pigs caused by Actinobacillus pleuropneumoniae.The disease has been prevalent in pig farms since it was first identified in 1957(Pattison e...Porcine Contagious Pleuropneumonia(PCP)is a respiratory infectious disease of pigs caused by Actinobacillus pleuropneumoniae.The disease has been prevalent in pig farms since it was first identified in 1957(Pattison et al.1957).展开更多
Porcine reproductive and respiratory syndrome virus (PRRSV) is a member within the family Arteriviridae of the order Nidovirales. Replication of this positive-stranded RNA virus within the host cell involves expressio...Porcine reproductive and respiratory syndrome virus (PRRSV) is a member within the family Arteriviridae of the order Nidovirales. Replication of this positive-stranded RNA virus within the host cell involves expression of viral replicase proteins encoded by two ORFs, namely ORF1 a and ORF1 b. In particular, translation of ORF1 b depends on a-1-ribosomal frameshift strategy. Thus, nonstructural protein 9 (nsp9), the first protein within ORF1 b that specifies the function of the viral RNA-dependent RNA polymerase, is expressed as the C-terminal extension of nsp8, a small nsp that is encoded by ORF1 a. However, it has remained unclear whether the mature form of nsp9 in virus-infected cells still retains nsp8,addressing which is clearly critical to understand the biological function of nsp9. By taking advantage of specific antibodies to both nsp8 and nsp9, we report the following findings. (1) In infected cells, PRRSV nsp9 was identified as a major product with a size between 72 and 95 k Da (72–95 KDa form), which exhibited the similar mobility on the gel to the in vitro expressed nsp8–9 ORF1 b, but not the ORF1 b-coded portion (nsp9 ORF1 b). (2) The antibodies to nsp8, but not to nsp7 or nsp10, could detect a major product that had the similar mobility to the 72–95 KDa form of nsp9. Moreover, nsp9 could be co-immunoprecipitated by antibodies to nsp8, and vice versa. (3) Neither nsp4 nor nsp2 PLP2 was able to cleave nsp8–nsp9 in vitro. Together, our studies provide experimental evidence to suggest that nsp8 is an N-terminal extension of nsp9.Our findings here paves way for further charactering the biological function of PRRSV nsp9.展开更多
Porcine reproductive and respiratory syndrome virus(PRRSV)is an economically important pathogen for the global pork industry.Although modified live virus(MLV)vaccines are commonly used for PRRSV prevention and control...Porcine reproductive and respiratory syndrome virus(PRRSV)is an economically important pathogen for the global pork industry.Although modified live virus(MLV)vaccines are commonly used for PRRSV prevention and control,they still carry a risk of infecting the host and replicating in target cells,thereby increasing the likehood of virus recombination and reversion to virulence.In this study,we inserted the target sequence of miR-142 into the nsp2 hypervariable region of PRRSV to inhibit viral replication in its host cells of pigs,with the aim of achieving virus attenuation.The chimeric virus RvJX-miR-142t was successfully rescued and retained its growth characteristics in MARC-145 cells.Furthermore,it did not replicate in MARC-145 cells transfected with miRNA-142 mimic.We also observed limited replication ability of RvJX-miR-142t in pulmonary alveolar macrophages,which are the main cell types that PRRSV infects.Our animal inoculation study showed that pigs infected with RvJX-miR-142t displayed less severe clinical symptoms,lower viremia titers,lighter lung lesions,and significantly lower mortality rates during the first 7 days post-inoculation,in comparison to pigs infected with the backbone virus RvJXwn.We detected a partially deletion of the miR-142 target sequence in the RvJX-miR-142t genome at 14 dpi.It is highly possible that the reversion of viral virulence observed in the later timepoints of our animal experiment was caused by that.Our study provided a new strategy for attenuating PRRSV and confirmed its effectiveness.However,further studies are necessary to increase the stability of this virus under host selection pressure.展开更多
The genome segment for replicase protein nsp2 represents the fastest evolving region of porcine reproductive and respiratory syndrome virus(PRRSV),and our previous studies have shown that the PRRSV nsp2 genetic variat...The genome segment for replicase protein nsp2 represents the fastest evolving region of porcine reproductive and respiratory syndrome virus(PRRSV),and our previous studies have shown that the PRRSV nsp2 genetic variation contributes to poor cross-neutralization.By using in vitro antibody absorption assay,here we show that the papainlike protease 2(PLP2)domain of nsp2 is a target of neutralizing antibodies.This was further verified by cross-neutralization assay with a series of inter-lineage chimeric mutants between the Chinese highly pathogenic PRRSV(HP-PRRSV)strain JXwn06 and the low virulent NADC30-like strain CHsx1401(lineage 1).The role of nsp2 in protective immunity was subsequently tested in a one-month SPF piglet model by immunizing the piglets with CHsx1401 or its derivatives carrying JXwn06 structural protein region(SP)alone(CHsx1401-SPJX)or in combination with PLP2 region(CHsx1401-SPplp2JX),or the whole nsp2 region(CHsx1401-SPnsp2JX),followed by challenge with JXwn06 at 42 days post immunization,a time point when the viremia was undetectable.All chimera groups were protected from the challenge by JXwn06,whereas the group CHsx1401 failed to provide beneficial protection.Interestingly,the group CHsx1401-SPnsp2JX,but not CHsx1401-SPplp2JX,showed the lowest lung microscopic lesions and viral tissue load.Significantly,the vaccine virus CHsx1401-SPnsp2JX was undetectable in the examined tissues,and so was for the challenge virus except for one piglet,highlighting an important role of HP-PRRSV nsp2 in promoting viral clearance.The findings provide insight into the mechanisms underlying the protective immunity against PRRSV and have important implications in PRRSV vaccine development.展开更多
基金supported by the Major Program of National Natural Science Foundation of China (31490603, 31572549)the National Key Technology R & D Program of China (2015BAD12B01-2)
文摘Porcine reproductive and respiratory syndrome virus(PRRSV)is characterized by its genetic variation and limited cross protection among heterologous strains.Even though several viral structural proteins have been regarded as inducers of neutralizing antibodies(NAs)against PRRSV,the mechanism underlying limited cross-neutralization among heterologous strains is still controversial.In the present study,examinations of NA cross reaction between a highly pathogenic PRRSV(HP-PRRSV)strain,JXwn06,and a low pathogenic PRRSV(LP-PRRSV)strain,HB-1/3.9,were conducted with viral neutralization assays in MARC-145 cells.None of the JXwn06-hyperimmuned pigs’sera could neutralize HB-1/3.9 in vitro and vice versa.To address the genetic variation between these two viruses that are associated with limited crossneutralization,chimeric viruses with coding regions swapped between these two strains were constructed.Viral neutralization assays indicated that variations in nonstructural protein 2(nsp2)and structural proteins together contribute to weak cross-neutralization activity between JXwn06 and HB-1/3.9.Furthermore,we substituted the nsp2-,glycoprotein2(GP2)-,GP3-,and GP4-coding regions together,or nsp2-,GP5-,and membrane(M)protein-coding regions simultaneously between these two viruses to construct chimeric viruses to test cross-neutralization reactivity with hyperimmunized sera induced by their parental viruses.The results indicated that the swapped nsp2 and GP5-M viruses increased the neutralization reactivity with the donor strain antisera in MARC-145 cells.Taken together,these results show that variations in nsp2 and GP5-M correlate with the limited neutralization reactivity between the heterologous strains HP-PRRSV JXwn06 and LP-PRRSV HB-1/3.9.
基金supported by the National Key Research and Development Program of China (2021YFD1800100)the earmarked fund for China Agriculture Research System (CARS-35)。
文摘Although African swine fever(ASF) has been prevalent for more than a century, it remains the number one swine disease that seriously endangers the global pig industry, and there is no effective means of prevention and treatment(Wang et al. 2023). Due to its enormous economic and social impact, it is listed as a notifiable animal disease by the World Organization for Animal Health(Costard et al. 2013). Although ASF has been present in Sub-Saharan Africa since its first discovery in Kenya.
基金supported by the University-Industry Col aborative Education Program,China(220904860093831)。
文摘Porcine Contagious Pleuropneumonia(PCP)is a respiratory infectious disease of pigs caused by Actinobacillus pleuropneumoniae.The disease has been prevalent in pig farms since it was first identified in 1957(Pattison et al.1957).
基金supported by the National Key Basic Research Plan Grant from the Chinese Ministry of Science and Technology(2014CB542700)the China National Thousand Youth Talents program(1051-21986001)the earmarked fund for China Agriculture Research System(CARS-35)from the Chinese Ministry of Agriculture
文摘Porcine reproductive and respiratory syndrome virus (PRRSV) is a member within the family Arteriviridae of the order Nidovirales. Replication of this positive-stranded RNA virus within the host cell involves expression of viral replicase proteins encoded by two ORFs, namely ORF1 a and ORF1 b. In particular, translation of ORF1 b depends on a-1-ribosomal frameshift strategy. Thus, nonstructural protein 9 (nsp9), the first protein within ORF1 b that specifies the function of the viral RNA-dependent RNA polymerase, is expressed as the C-terminal extension of nsp8, a small nsp that is encoded by ORF1 a. However, it has remained unclear whether the mature form of nsp9 in virus-infected cells still retains nsp8,addressing which is clearly critical to understand the biological function of nsp9. By taking advantage of specific antibodies to both nsp8 and nsp9, we report the following findings. (1) In infected cells, PRRSV nsp9 was identified as a major product with a size between 72 and 95 k Da (72–95 KDa form), which exhibited the similar mobility on the gel to the in vitro expressed nsp8–9 ORF1 b, but not the ORF1 b-coded portion (nsp9 ORF1 b). (2) The antibodies to nsp8, but not to nsp7 or nsp10, could detect a major product that had the similar mobility to the 72–95 KDa form of nsp9. Moreover, nsp9 could be co-immunoprecipitated by antibodies to nsp8, and vice versa. (3) Neither nsp4 nor nsp2 PLP2 was able to cleave nsp8–nsp9 in vitro. Together, our studies provide experimental evidence to suggest that nsp8 is an N-terminal extension of nsp9.Our findings here paves way for further charactering the biological function of PRRSV nsp9.
基金funded by the National Natural Science Foundation of China(31490603 and 31772759)the earmarked fund for CARS(CARS-35).
文摘Porcine reproductive and respiratory syndrome virus(PRRSV)is an economically important pathogen for the global pork industry.Although modified live virus(MLV)vaccines are commonly used for PRRSV prevention and control,they still carry a risk of infecting the host and replicating in target cells,thereby increasing the likehood of virus recombination and reversion to virulence.In this study,we inserted the target sequence of miR-142 into the nsp2 hypervariable region of PRRSV to inhibit viral replication in its host cells of pigs,with the aim of achieving virus attenuation.The chimeric virus RvJX-miR-142t was successfully rescued and retained its growth characteristics in MARC-145 cells.Furthermore,it did not replicate in MARC-145 cells transfected with miRNA-142 mimic.We also observed limited replication ability of RvJX-miR-142t in pulmonary alveolar macrophages,which are the main cell types that PRRSV infects.Our animal inoculation study showed that pigs infected with RvJX-miR-142t displayed less severe clinical symptoms,lower viremia titers,lighter lung lesions,and significantly lower mortality rates during the first 7 days post-inoculation,in comparison to pigs infected with the backbone virus RvJXwn.We detected a partially deletion of the miR-142 target sequence in the RvJX-miR-142t genome at 14 dpi.It is highly possible that the reversion of viral virulence observed in the later timepoints of our animal experiment was caused by that.Our study provided a new strategy for attenuating PRRSV and confirmed its effectiveness.However,further studies are necessary to increase the stability of this virus under host selection pressure.
基金supported by the National Natural Science Foundation of China(32025035)China Agriculture Research System of MOF and MARA(CARS-35).
文摘The genome segment for replicase protein nsp2 represents the fastest evolving region of porcine reproductive and respiratory syndrome virus(PRRSV),and our previous studies have shown that the PRRSV nsp2 genetic variation contributes to poor cross-neutralization.By using in vitro antibody absorption assay,here we show that the papainlike protease 2(PLP2)domain of nsp2 is a target of neutralizing antibodies.This was further verified by cross-neutralization assay with a series of inter-lineage chimeric mutants between the Chinese highly pathogenic PRRSV(HP-PRRSV)strain JXwn06 and the low virulent NADC30-like strain CHsx1401(lineage 1).The role of nsp2 in protective immunity was subsequently tested in a one-month SPF piglet model by immunizing the piglets with CHsx1401 or its derivatives carrying JXwn06 structural protein region(SP)alone(CHsx1401-SPJX)or in combination with PLP2 region(CHsx1401-SPplp2JX),or the whole nsp2 region(CHsx1401-SPnsp2JX),followed by challenge with JXwn06 at 42 days post immunization,a time point when the viremia was undetectable.All chimera groups were protected from the challenge by JXwn06,whereas the group CHsx1401 failed to provide beneficial protection.Interestingly,the group CHsx1401-SPnsp2JX,but not CHsx1401-SPplp2JX,showed the lowest lung microscopic lesions and viral tissue load.Significantly,the vaccine virus CHsx1401-SPnsp2JX was undetectable in the examined tissues,and so was for the challenge virus except for one piglet,highlighting an important role of HP-PRRSV nsp2 in promoting viral clearance.The findings provide insight into the mechanisms underlying the protective immunity against PRRSV and have important implications in PRRSV vaccine development.
