Breaking Latent Infection: How ORF37/38-Deletion Mutants Offer New Hope against EHV-1 Neuropathogenicity
<p>Replication properties in vitro and pathogenicity in hamsters of ∆ORF38 and ∆ORF37/38. (<b>A</b>) Herpes-characteristic CPEs (syncytium formation, rounded cells) were observed after infecting RK-13 cells with ∆ORF38, ∆ORF37/38, ∆ORF38-eGFP, and ∆ORF37/38-eGFP strains at an MOI of 0.01 for 16 h, (fluorescence microscope, emission filter bandpass, 505–530 nm); the white arrow points to the syncytium. (<b>B</b>) The mean plaque size of each virus is shown as a percentage relative to the WT plaque size. (<b>C</b>) One-step growth curve. (<b>D</b>) Body-weight loss of hamsters within 14 days post-infection (n = 6). Green asterisks indicate a significant difference (∆ORF38-infected group compared to the control group) ** <span class="html-italic">p</span> < 0.01. (<b>E</b>) Clinical sign scoring. The dashed line represents a clinical sign score of 3; scores above 3 indicate the presence of typical clinical signs. (<b>F</b>) Survival curve. (<b>G</b>) Pathological scoring of lung and brain tissue. The dashed line represents a pathological score of 3; scores above 3 indicate the presence of pathological damage. (<b>H</b>) Hematoxylin and eosin (HE) staining was utilized to detect pathological lesions. Blue markings indicate alveolar inflammatory exudates, red markings indicate hemorrhage, yellow markings indicate infiltration of inflammatory cells, green markings indicate vacuolated neurons, and black markings indicate activation of microglia. (<b>I</b>) Viral load in different tissues of infected hamsters. Statistical differences were analyzed by one-way ANOVA. * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001, ns <span class="html-italic">p</span> > 0.05.</p> "> Figure 2
<p>Evaluation of reactivation of ∆ORF38 and ∆ORF37/38. (<b>A</b>) Schematic diagram of infection, DEX stimulation, and sample collection in hamsters. (<b>B</b>) Body weight. Blank asterisks indicate a significant difference (WT and ∆ORF38 compared to the control group) ** <span class="html-italic">p</span> < 0.01. (<b>C</b>) Clinical sign scoring. (<b>D</b>) Viral DNA load analysis in lung, brain, and lymph nodes at day 0, day 5, and day 10. (<b>E</b>) Pathological scoring and (<b>F</b>) histopathological lesions in the brain tissues of the hamsters at day 10. Yellow markings indicate perivascular cuffing of macrophage–lymphocyte cells (nonsuppurative encephalitis), green markings indicate necrosis of neurons, and black markings indicate activation of microglia. Statistical differences were analyzed by one-way ANOVA. * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001, ns <span class="html-italic">p</span> > 0.05. The dashed line represents a score of 3.</p> "> Figure 3
<p>Immunogenicity and protection efficacy of intranasal inoculation with ∆ORF37/38. (<b>A</b>) Schematic diagram of vaccination, challenge, and sample collection in hamsters. (<b>B</b>) EHV-1 gG specific serum antibody and (<b>C</b>) neutralizing antibody titers. Splenocytes from ∆ORF37/38-immunized groups exhibited significantly higher (<b>D</b>) stimulation index and (<b>E</b>) cytokine levels (IFN-γ and IL-10) when comparing with ∆ORF38-immunized group. (<b>F</b>) Body-weight loss was recorded until 14 dpc. Green asterisks indicate a significant difference (∆ORF38-immunized group compared to the ∆ORF37/38-immunized group) ** <span class="html-italic">p</span> < 0.01. (<b>G</b>) Clinical sign scoring. (<b>H</b>) Viral load analysis in lung, brain, and lymph nodes at 14 dpc. (<b>I</b>) Lesion scoring and (<b>J</b>) histopathological lesions. Blue markings indicate alveolar inflammatory exudates, red markings indicate hemorrhage, yellow markings indicate infiltration of inflammatory cells, green markings indicate necrosis of neurons, and black markings indicate activation of microglia. Statistical differences were determined by one-way ANOVA analysis with Bonferroni’s multiple comparison test. * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001, ns <span class="html-italic">p</span> > 0.05. The dashed line represents a score of 3.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Virus, Cell Lines, Primers, and Plasmids
2.2. Construction of EHV-1 ∆ORF38 and ∆ORF37/38 Mutants
2.3. Viral Growth Kinetics
2.4. Evaluation of Neuropathogenicity
2.5. Evaluation of the Latency of Reactivation
2.6. Evaluation of Immune Efficacy
2.7. Serological Test
2.8. Cell-Mediated Immune Responses
3. Results
3.1. The ∆ORF37/38-Gene-Deletion Virus Does Not Induce Neurological Signs in Hamsters during Acute Infection
3.2. Inability of the ∆ORF37/38 Virus to Cause an Increase in Clinical Signs or Viral DNA Loads with Dexamethasone Stimulation
3.3. Intranasal Vaccination with the ∆ORF37/38 Mutant Effectively Protects Hamsters from Lethal Infections
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Hu, Y.; Zhang, S.-Y.; Sun, W.-C.; Feng, Y.-R.; Gong, H.-R.; Ran, D.-L.; Zhang, B.-Z.; Liu, J.-H. Breaking Latent Infection: How ORF37/38-Deletion Mutants Offer New Hope against EHV-1 Neuropathogenicity. Viruses 2024, 16, 1472. https://doi.org/10.3390/v16091472
Hu Y, Zhang S-Y, Sun W-C, Feng Y-R, Gong H-R, Ran D-L, Zhang B-Z, Liu J-H. Breaking Latent Infection: How ORF37/38-Deletion Mutants Offer New Hope against EHV-1 Neuropathogenicity. Viruses. 2024; 16(9):1472. https://doi.org/10.3390/v16091472
Chicago/Turabian StyleHu, Yue, Si-Yu Zhang, Wen-Cheng Sun, Ya-Ru Feng, Hua-Rui Gong, Duo-Liang Ran, Bao-Zhong Zhang, and Jian-Hua Liu. 2024. "Breaking Latent Infection: How ORF37/38-Deletion Mutants Offer New Hope against EHV-1 Neuropathogenicity" Viruses 16, no. 9: 1472. https://doi.org/10.3390/v16091472