| Atypical porcine reproductive and respiratory syndrome (PRRS) caused by Chinese highly pathogenic PRRS virus (HP-PRRSV), has resulted in huge economic losses to the swine industry since its outbreak in2006. The disease is characterized by high body temperature (41℃-42℃), high morbidity and mortality of the affected pigs. Although the genomic marker, the30-amino-acid deletion in its Nsp2-coding region has been previously verified to have no relation with its increased pathogenicity, the genomic region (s) associated with the fatal virulence of HP-PRRSV remains unknown. According to our previous study, the ORF1b had a close relation to the pathogenicity of HP-PRRSV. In the present study, in order to further find out the fatal virulence-determining gene (s) within the ORF1b region, we generated a series of full-length infectious cDNA clones with swapped nonstructural protein (Nsp)9-, Nsp10-, Nsp11-and Nsp12-coding region separately, or Nsp9-and Nsp10-coding region together, or Nsp9-, Nsp10-and Nsp11-coding region simultaneously between the highly pathogenic RvJXwn and low pathogenic RvHB-1/3.9by using rever genetic manipulation, and then the in vitro and in vivo replication ability and pathogenicity for piglets of the rescued chimeric viruses were systematically analyzed and compared with their backbone viruses.For constructing the chimeric full-length cDNA clones, the Nsp9-, Nsp10-, Nsp11-and Nsp12-coding region separately, or Nsp9-and Nsp10-coding region together, or Nsp9-, Nsp10-and Nspll-coding region simultaneously of pWSK-HB-1/3.9and pWSK-JXwn were amplified, then a series of new fragment C or D of pWSK-JXwn and new fragment D or E of pWSK-HB-1/3.9were constructed by fusion PCR. After digested by the restriction enzymes and linked, the chimeric full-length cDNA clones with pWSK-JXwn backbone, containing Nsp-coding region from pWSK-HB-1/3.9, were individually designated as pWSK-JHn9, pWSK-JHn10, pWSK-JHnll, pWSK-JHn12, pWSK-JHn9n10and pWSK-JHn9n10n11, and the chimeric clones with RvHB-1/3.9backbone were designated as pWSK-HJn9, pWSK-HJn10, pWSK-HJnlly pWSK-HJnl2, pWSK-HJn9n10and pWSK-HJn9n10n11, respectively. Each full-length cDNA clone plasmid was separately linearized, transcribed, capped and transfected into BHK-21cells. The recovery of chimeric viruses was comfirmed by IFA and the correctness of exchanging regions were verified by sequencing. These rescued viruses were individually designated as RvJHn9, RvJHn10, RvJHn11, RvJHn12, RvJHn9n10, RvJHn9n1On11, RvHJn9, RvHJn10, RvHJn11, RvHJn12, RvHJn9n10and RvHJn9n10n11.To characterize and compare the growth properities between these chimeras and their parental viruses RvJXwn and RvHB-1/3.9, the growth kinetics were analyzed by infection of MARC-145cells or primary PAMs with the respective virus. In MARC-145cells, RvJHn9showed significantly lower titers than RvJXwn at all time points, with significant differences from60h to96h pi (P<0.05; P<0.01); Moreover both RvJHn9n10and RvJHn9n10n11displayed obviously a lower replication efficiency than RvJXwn, with significant differences at many time points (P<0.05; P<0.01). In primary PAMs, RvJHn9and RvJHnlO had lower virus titers than RvJXwn at majority of time points without significant differences; furthermore, compared with RvJXwn, the replication level of RvJHn9n10was lower, and its peak titer delayed a24-h to reach. Similarly, the virus titers of RvJHn9n10n11were obviously lower than those of RvJXwn at some time points, but without significant difference. On the other hand, in MARC-145cells, the replication efficiency of all the four rescued viruses with the substituted single Nsp-coding region were significantly lower than the parental virus RvHB-1/3.9at many time points (P<0.05; P<0.01; P<0.001); while the virus titers of RvHJn9nlO were slightly lower than those of RvHB-1/3.9, with significant differences at12h pi (P<0.05), and the virus yield of RvHJn9n10n11were slightly higher than RvHB-1/3.9at some time points, and moreover, the peak titers of both RvHJn9nlO and RvHJn9n10n11were higher than that of RvHB-1/3.9, with no significant difference. In primary PAMs, RvHJn9and RvHJn10showed higher virus titers than RvHB-1/3.9, with no significant difference, and moreover, both RvHJn9n10and RvHJn9n10n11had obviously higher replication efficiency than RvHB-1/3.9, with significant differences from12h to60h pi (P<0.01;P<0.001), and moreover their peak titers were10times higher than that of RvHB-1/3.9. Taken together, these results showed that the substitution of Nsp9-or/and Nsp10-coding region could obviously affect the virus replication efficiency in vitro, in particular, exchanging both of them together, indicating that Nsp9-and Nsp10-coding region together are related to the replication ability of HP-PRRSV in vitro.We further investigated the pathogenicities of the chimeric viruses and compared with their parental viruses. Fifty-five six-week-old healthy SPF landrace piglets were randomly allotted to eleven groups (n=5). Each piglet in each infection group was intranasally inoculated with2ml of each virus containing2x105TCID50. The results showed that piglets in RvJHn10-infected group exhibited severe clinical symptoms and high fever, and all of them died, similar to RvJXwn-infected group; the rising rate of body temperature and disease progress of RvJHn9-infected group was slower, and three of them died; while all piglets of RvJHn9n10-and RvJHn9n10n11-infected group survived, they just exhibited transient temperature elevation and mild respiratory symptoms, with a significantly lower clinical sign score than RvJXwn-, RvJHn9-or RvJHn10-infected group (P<0.01; P<0.001). Meanwhile, the ADG of RvJHn9n10-and RvJHn9n1On11-infected group showed a significantly higher ADG than other three infected groups (P<0.05; P<0.01; P<0.001). Their in vivo replication efficiency were both significantly lower than those of RvJXwn-infected group (P<0.001); also their seroconversion for antibodies to theN protein of PRRSV was delayed and the antibody level was statistically lower (P<0.01; P<0.001), compared with RvJXwn-infected group. Moreover, the lung lesion and PRRSV-positive signal of the piglets inoculated with RvJHn9n10or RvJHn9n10n11were significantly lower than that of RvJXwn-infected group. On the other hand, no obvious clinical signs were observed in RvHJn10-or RvHB-1/3.9-infected group, and no piglet died in these two groups; one piglet died in RvHJn9group; however, three piglets in RvHJn9n10and two piglets in RvHJn9n10n11group died, respectively, and their body temperature elevated quickly to41℃, accompanied by severe clinical signs, with significantly higher clinical scores than RvHB-1/3.9-or RvHJn10-infected group on day9to18pi (P<0.05; P<0.01; P<0.001). Meanwhile, RvHJn9n10-or RvHJn9n10n11-infected group showed a remarkably lower ADG compared with that of RvHB-1/3.9-or RvJHnlO-infected group during the experiment, with significant differences on day7and14pi (P<0.01;P<0.001). The virus loads of RvHJn9n10-or RvHJn9n10n11-infected piglets were obviously higher than their parental virus RvHB-1/3.9, with significant differences on day3,5and7pi (P<0.05; P<0.01). Their seroconversion for antibodies to the N protein of PRRSV occurred earlier and the antibody level was significantly higher than that of RvHB-1/3.9group (on day14,21pi)(P<0.01). RvHJn9n10-or RvHJn9n10n11-infected group displayed more sever lung lesions and PRRSV positive signals in dead piglets, similer to RvJXwn-infected group, and other surival piglets in these two groups showed severe and extensive lung lesions. The histopathological and immunohistochemical scores in both RvHJn9n1On11-infected group were significantly higher than those of RvHB-1/3.9-infected group (P<0.05). Taken together, the above results indicated that the replacement of Nsp9-and Nsp10-coding regions together of RvJXwn by RvHB-1/3.9could greatly attenuate the replication efficiency and virulence of RvJXwn, and in contrast, the substitution of the corresponding regions of RvHB-1/3.9by RvJXwn could remarkably increase the virus titers in vivo and virulence of the chimeric virus. Therefore, our sufficient findings suggest that Nsp9and Nsp10together contribute to the increased pathogenicity and fatal virulence of Chinese HP-PRRSV.In summary, our results indicate that Nsp9-and Nsp10-coding regions together not only contribute to the in vitro and in vivo replication efficiency of Chinese HP-PRRSV, but also contribute to the fatal virulence of Chinese HP-PRRSV. Meanwhile, our findings also suggest that the replication efficiency is one of the key factors related to the virulence of Chinese HP-PRRSV, providing a novel insight for understanding the pathogenesis of this virus. |
PDF Full Text Request