Study of latent infection and reactivation of pseudorabies wild type virus and temperature-sensitive mutant in BALB/c mice and Vero cell culture models

• Main Authors: Ching-Chien Lee, 李青蒨
• Other Authors: Shih-Hui Chen
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/84735191974998021282

碩士 === 國立成功大學 === 生物學系碩博士班 === 91 === 　　Pseudorabies Virus (PRV), a member of the alphaherpesviridae, can cause latent infection in trigeminal ganglia (TG) of infected adult swine. When host’s immunity was suppressed, reactivation may occur and virus was transmitted to uninfected animals causing economic losses in the husbandry industry. Current PRV vaccines used still cannot offer full protection and the PRV prevalence rate in Taiwan swine farms still reaches 96%. We have previously screened a selected temperature- sensitive PRV mutant, ts5-7, which could offer 80% protection against challenge in BALB/c mice. In this research, BALB/c mice and Vero cell culture models were adopted to test ts5-7 mutant’s ability in causing latent infection and subsequent viral reactivation. PRV wild type P7 strain was used as positive control. 　　In animal model, the BALB/c mice were passively immunized by intraperitoneal inoculation with 1:25 diluted anti-PRV P7 mice antiserum. After 30 minutes, the pre-immunized mice were infected by intranasal inoculation with 20 μl of 103 TCID50 (i.e. 0.67 LD50) of P7 and 103.59 TCID50 (i.e. 1LD50) of ts5-7 mutant, respectively. After 90 days, some of the surviving mice were irradiated by ultraviolet light (UV, 366 nm) and intravenously inoculated with dexamethasone for inducing viral reactivation. The remaining mice were scarified for viral DNA detection. The results showed the viral glycoprotein E (gE) gene could be detected by PCR (gE1 and gE2 primers) in TG of P7-infected mice, but not in ts5-7 infected group. However, the supposed latency- associated LLT mRNA could be detected neither by RT-PCR using LLT-1 and LLT-3 primers, nor by nested-PCR using CM3.1 and LLT3.1 primers. Negative results were obtained by explant co-culture. On the 8th day after induction, viral gE was also detected in P7-infected mice but not in ts5-7 infected group. Moreover, the explant co-cultures with lung and TG tissues were successful in virus isolation in P7-infected mice (20% and 10% positive rates, respectively). In contrast, negative results were obtained for ts5-7 infected group. Furthermore, sera from 2 of 10 P7-infected mice showed antibody rising against PRV on the 4th and the 8th days after induction. Sera from 1 of 10 ts5-7 infected mice also showed antibody rising. No clinical signs were noted during latency or after reactivation in all mice. 　　In cell culture model, cells were pre-fed with cytosine arabinoside for 5 hours, then infected with P7 or ts5-7. The infected Vero cells were subsequently cultivated for 11 days. On the 7th day of culture, survived cells were irradiated with ultraviolet (wavelength 254 nm) for inducing viral reactivation. The viral gE gene for survived cells on the 4th and 7th days can be detected for both viral infections. However, LLT mRNA and active viral replication could not be found. On the 11th day, the highest frequency of virus reactivation was detected with UV irradiation for 60 seconds. Frequencies were 21.8% and 9.8% for P7 and ts5-7 infection, respectively. All survived cells also showed the presence of gE gene. 　　These findings indicate that ts5-7 latent-infection and reactivation can be detected in Vero cell model, but the frequency of reactivation is lower than P7 infection. BALB/c mice model for this experiment was failed for ts5-7 mutant, however.