Bioluminescence imaging BLI is a powerful alternative, enabling rapid measurements of viral load and tissue distribution 11 — Spatial and temporal progression of infection can be quantified, and viral replication and dissemination in the animals can be identified The traditional approaches for viral pathogenicity studies require the killing of animals at diverse time points for the determination of viral titers in excised organs and tissues, whereas BLI does not In addition, BLI can identify unexpected sites or patterns of viral infection that could be missed if organs are not collected or if entire organs are not analyzed for viral titers This approach has been exploited in multiple viruses, including dengue virus, herpes simplex virus type 1, Sindbis virus, influenza virus, and Sendai virus 12 , 14 , 15 , 17 , These results demonstrated that the recombinant PRV was not attenuated both in vitro and in vivo.
Furthermore, we reported the visualization of PRV infection in mice. These data suggest that imaging of the recombinant PRV can be used to rapidly assess the replication and pathogenicity characteristics of emerging PRVs; these will provide a reference for control PR caused by PRV variants.
The resulting L arm, R arm, and exogenous gene were amplified; — ng of DNA template was used per reaction for overlap PCR; the long DNA segment was cloned into blunt T-vector and sequenced to make sure to get the target sequence.
The first generation recombinant viruses were collected at 2—3 days after transfection. A total of five generations of plaque purification were performed to obtain the purified recombinant viruses. After the sample returned to room temperate, the cellular debris was removed by centrifugation, and the TCID 50 of supernatant was detected on PK cells.
Average values and standard deviations of the three independent experiments were calculated. The PCR product was analyzed using 1. The supernatants were removed, and cells were washed once with PBS before cell culture lysis buffer Promega was added.
Two mice experiments were made in this work: one for the pathogen detection and another one for the live imaging test. The mice were randomly allocated into seven groups, five mice in one box. Mice of groups 1, 2, and 3 were injected intramuscularly i.
Groups 4, 5, and 6 were injected i. Group 7 was the mock-inoculations medium only in parallel. Mice were scored daily for symptoms of PRV infection using the following three-point system adapted from the protocol previously described All the mice in the infected and control groups were anesthetized by CO 2 before euthanasia, using the broken-neck method at 7 dpi.
Real-time PCR qPCR was used to quantitatively analyze the viral loading in the brain, heart, liver, spleen, lung, kidney, spinal cord, and trigeminal ganglion using the reported method of Meng The samples were fixed in buffered formalin and embedded in paraffin wax. Tissue sections were prepared and stained with hematoxylin and eosin assay for histopathological examinations.
All the mice in the infected and control groups were anesthetized by CO 2 before euthanasia, using the broken-neck method after imaging the experiment. All data in composite images utilized the same scale. The comparison between groups was performed by a Student's t -test with a two-tailed analysis.
As reported, the noncoding interval sequence between U S 9 open reading frame and U S 2 open reading frame in the U S regions is long enough to tolerate large exogenous genes We obtained the purified virus through five rounds of plaque purification.
These results indicated there were gB and gI genes in the genome backbone of the recombinant PRV, which was the same as its parental strain. Also, the reporter genes could exist in the backbone simultaneously and stably, which were detected in the following passages. Middle panel and right panel, identification of the NLuc and DsRed reporter genes in different passages in infected PK cells, respectively.
G Correlation curve between titers of virus and luciferase activities using Pearson's correlation coefficient. NLuc luciferase activities were measured at 12 hpi. The result demonstrated that the luminescent flux could represent the titer of the recombinant PRV in the infected cell. Clinical signs, including pruritus, anxiety, rolling, and scratching of the injection site, were recorded every day throughout the experiment.
The mice infected with 10 4 TCID 50 viruses groups 1 and 4 began showing clinical signs around 72 hpi. The mice infected with 10 3 TCID 50 viruses groups 2 and 5 showed similar symptoms but delayed.
The mice infected with 10 2 TCID 50 doses of viruses groups 3 and 6 showed barely clinical symptoms. These results demonstrated that the clinical symptoms of the recombinant PRV did not change after the insertion of the reporter genes.
Extended scoring systems for clinical signs. B Survival rates of mice infected indicated a dose of viruses. C Viral DNA copies in different tissue. All the mock-infected and PRV-infected mice were euthanized and subjected to dissection at a moribund stage or 7 dpi.
Next, we determined whether the fatal clinical outcome and distinct pathology of the mice that were infected could be attributed to viral replication in specific tissues. As shown in Figure 2C , the virus could be detected in the brain, kidney, spinal cord, and trigeminal ganglion of peracute death mice infected with 10 4 or 10 3 TCID 50 PRVs groups groups 1, 2, 4, and 5.
No virus was detected in the 10 2 TCID 50 groups groups 3 and 6 , as well as the control group group 7. The underlying mechanisms, however, remain unclear. Pseudorabies virus infection of its natural host, the pig, provides a suitable infection model to study this more profoundly.
We show here that the severe neurological disease observed in 2-week-old pigs does not appear to be related to a hampered innate immune response but is more likely to reflect the immature development state of the trigeminal ganglia TG and central nervous system CNS neurons, resulting in an inefficient suppression of viral replication.
The survival of mice was recorded. The average neutralizing antibody titer of three measurements was calculated according to Reed-Muench method. Then the collected mouse serum was diluted with PBS at the ratio of , and the positive serum control murine polyclonal antibody of S protein and negative serum control were set at the same time.
The data were analyzed using Kaluza 2. Primers were designed using Oligo 6. Extract 1. Extract the total RNA of feces collected in 2. YH performed the experiments. YH and JZ drafted the manuscript. YZ substantively revised this manuscript.
All authors read and approved the final manuscript. The funders had no role in the design of the collection, analysis, and interpretation of data. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Yao Huang and Zhiwen Xu contributed equally to this work and should be considered co-first authors. National Center for Biotechnology Information , U. BMC Vet Res. Published online Jan 4. Author information Article notes Copyright and License information Disclaimer. Ling Zhu, Email: moc. Corresponding author. Received Aug 29; Accepted Dec The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material.
If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. Associated Data Supplementary Materials Additional file 1. Abstract Background Porcine deltacoronavirus PDCoV is a new pathogenic porcine intestinal coronavirus, which has appeared in many countries since Supplementary Information The online version contains supplementary material available at Open in a separate window.
Spleen lymphocyte proliferation test In order to evaluate the proliferation of T lymphocytes in the spleen of experimental mice, mouse splenocytes were isolated at 14 dpi and 28 dpi respectively, and the proliferation response of specific lymphocytes was measured. Detoxification of mice Although there is no literature report that PDCoV can infect mice, we still administered PDCoV disease material to mice to detect the detoxification of mice.
Discussion The emerging coronavirus could cause widespread transmission among humans and animals, posing a major threat to human health and causing significant economic losses to the global livestock and trade industries. Table 1 Sequences of oligonucleotides used for PCR in this study.
Supplementary Information Additional file 1. Acknowledgments We thank Prof. Xu for his insightful comments on the design of the study. Availability of data and materials The raw data is available from the corresponding author upon reasonable request.
References 1. Characteristics of the life cycle of porcine Deltacoronavirus PDCoV in vitro: replication kinetics, cellular ultrastructure and Virion morphology, and evidence of inducing autophagy. Discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus.
J Virol. Detection and genetic characterization of deltacoronavirus in pigs, Ohio, USA, Emerg Infect Dis. Rapid detection, complete genome sequencing, and phylogenetic analysis of porcine deltacoronavirus. Biosecurity to prevent feral swine exposure is important to protect swine. In general, the virus infects the central nervous system CNS and the respiratory tract. Clinical signs observed depend on the age at time of infection.
Young swine are very susceptible and can develop severe CNS symptoms. Mortality can reach percent in piglets. Clinical signs in weaned pigs, depends on age. Few weaned pigs develop a fatal CNS disease but more often will develop a respiratory disease. Grower finishing swine typically develop only a respiratory disease component.
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