Costantini V, Lewis P, Alsop J, Templeton C, Saif L J
Food Animal Health Research Program, Department of Veterinary Preventive Medicine, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, 44691, USA.
Arch Virol. 2004 May;149(5):957-74. doi: 10.1007/s00705-003-0245-z. Epub 2003 Nov 26.
Porcine respiratory coronavirus (PRCV), a spike (S) gene deletion mutant of Transmissible gastroenteritis virus (TGEV), causes mild or subclinical respiratory infections in pigs. The shedding of PRCV/TGEV was studied at different days post-arrival in fecal and nasal swabs from PRCV/TGEV seronegative sentinel pigs introduced into a PRCV seropositive herd with questionable TGEV serology and diarrhea. Nasal shedding of PRCV was detected in 57% and 63% of samples by nested-RT-PCR and cell culture immunofluorescence (CCIF), respectively. However fecal shedding of PRCV was detected in 37% of the samples by nested-RT-PCR and 19% by CCIF. Four respiratory and 5 fecal PRCV strains were isolated in swine testicle cells including nasal/fecal PRCV pairs (isolated at the same time) from 3 pigs. Comparison of nasal/fecal PRCV pairs from individual pigs revealed different deletions in the spike (S) gene (648 or 681 nt) in 2 pairs and a consistent change in nt 790/791 (aa T to V) for all pairs. In preliminary studies, inoculation of gnotobiotic pigs with each plaque-purified pair of the nasal and fecal PRCV isolates, revealed no clinical disease but different tropisms. The nasal isolate was shed both nasally and in feces, but the fecal isolate was shed only marginally in feces, and not nasally. Our results show that nested-RT-PCR was as sensitive as CCIF for PRCV detection in nasal swabs, but was more sensitive than CCIF for PRCV detection in fecal samples; alternatively PRCV shed in feces was more labile with loss of infectivity. The S-gene sequence differences found between the fecal and respiratory PRCV isolates may influence their tissue tropism. These new PRCV isolates should be useful to understand the molecular basis of coronavirus tropism and evolution in infected swine.
猪呼吸道冠状病毒(PRCV)是传染性胃肠炎病毒(TGEV)的一种刺突(S)基因缺失突变体,可引起猪的轻度或亚临床呼吸道感染。对引入到PRCV血清阳性、TGEV血清学情况存疑且有腹泻症状猪群的PRCV/TGEV血清阴性哨兵猪,在到达后的不同天数采集粪便和鼻拭子,研究PRCV/TGEV的排毒情况。通过巢式RT-PCR和细胞培养免疫荧光法(CCIF)分别在57%和63%的样本中检测到PRCV的鼻腔排毒。然而,通过巢式RT-PCR在37%的样本中检测到PRCV的粪便排毒,通过CCIF检测到的比例为19%。在猪睾丸细胞中分离出4株呼吸道PRCV毒株和5株粪便PRCV毒株,包括来自3头猪的鼻/粪PRCV配对毒株(同时分离)。对来自个体猪的鼻/粪PRCV配对毒株进行比较发现,2对毒株的刺突(S)基因存在不同的缺失(648或681个核苷酸),所有配对毒株的核苷酸790/791均发生一致变化(氨基酸T变为V)。在初步研究中,用每对噬斑纯化的鼻和粪PRCV分离株接种无菌猪,未发现临床疾病,但存在不同的嗜性。鼻腔分离株在鼻腔和粪便中均有排毒,但粪便分离株仅在粪便中有少量排毒,在鼻腔中无排毒。我们的结果表明,巢式RT-PCR在检测鼻拭子中的PRCV时与CCIF一样敏感,但在检测粪便样本中的PRCV时比CCIF更敏感;或者说,粪便中排出的PRCV更不稳定,传染性丧失。粪便和呼吸道PRCV分离株之间发现的S基因序列差异可能影响它们的组织嗜性。这些新的PRCV分离株将有助于理解冠状病毒在感染猪体内嗜性和进化的分子基础。