Laboratory of Virology, Department of Translational Physiology, Infectiology and Public Health, Faculty of Veterinary Medicine, Ghent Universitygrid.5342.0, Merelbeke, Belgium.
Laboratory of Veterinary Pathology, Department of Pathobiology, Pharmacology and Zoological Medicine, Faculty of Veterinary Medicine, Ghent Universitygrid.5342.0, Merelbeke, Belgium.
J Virol. 2022 Jun 8;96(11):e0051922. doi: 10.1128/jvi.00519-22. Epub 2022 May 12.
Virus strains in the live attenuated influenza vaccine (LAIV) for swine in the United States that was on the market until 2020 encode a truncated nonstructural protein 1 of 126 amino acids (NS1del126). Their attenuation is believed to be due to an impaired ability to counteract the type I interferon (IFN)-mediated antiviral host response. However, this mechanism has been documented only for H3N2 strain A/swine/Texas/4199-2/98 NS1del126 (lvTX98), and several cases of clinical respiratory disease in the field were associated with the LAIV strains. We therefore further examined the pathobiology, including type I IFN induction, of lvTX98 in pigs and compared it with IFN induction in pig kidney-15 (PK-15) cells. lvTX98 induced up to 3-fold-higher type I IFN titers than wild-type TX98 (wtTX98) after inoculation of PK-15 cells at a high multiplicity of infection, while virus replication kinetics were similar. Mean nasal lvTX98 excretion by intranasally inoculated pigs was on average 50 times lower than that for wtTX98 but still reached titers of up to 4.3 log 50% tissue culture infective doses/mL. After intratracheal inoculation, mean lvTX98 titers in the lower respiratory tract were significantly reduced at 18 to 48 h postinoculation (hpi) but similar to wtTX98 titers at 72 hpi. lvTX98 caused milder clinical signs than wtTX98 but induced comparable levels of microscopic and macroscopic lung lesions, peak neutrophil infiltration, and peak type I IFN. Thus, lvTX98 was partly attenuated in pigs, but this could not be associated with higher type I IFN levels. Swine influenza A viruses (swIAVs) with a truncated NS1del126 protein were strongly attenuated in previous laboratory-based safety studies and therefore approved for use as LAIVs for swine in the United States. In the field, however, the LAIV strains were detected in diagnostic samples and could regain a wild-type NS1 via reassortment with endemic swIAVs. This suggests a significant degree of LAIV replication and urges further investigation of the level and mechanism of attenuation of these LAIV strains . Here, we show that H3N2 LAIV strain lvTX98 is only partly attenuated in pigs and is excreted at significant titers after intranasal vaccination. Attenuation and restricted replication of lvTX98 seemed to be associated with the loss of NS1 functions other than type I IFN antagonism. Our findings can help to explain the occurrence of clinical respiratory disease and reassortment events associated with NS1del126-based LAIV strains in the field.
美国市场上直至 2020 年的猪用减毒活流感疫苗（LAIV）中的病毒株编码截短的 126 个氨基酸的非结构蛋白 1（NS1del126）。它们的减毒作用被认为是由于其对抗 I 型干扰素（IFN）介导的抗病毒宿主反应的能力受损所致。然而，这种机制仅在 H3N2 株 A/swine/Texas/4199-2/98 NS1del126（lvTX98）中得到证实，并且田间的几种临床呼吸道疾病与 LAIV 株有关。因此，我们进一步研究了 lvTX98 在猪中的病理生物学，包括 I 型 IFN 的诱导，并将其与猪肾-15（PK-15）细胞中的 IFN 诱导进行了比较。在高感染复数下接种 PK-15 细胞后，lvTX98 诱导的 I 型 IFN 滴度比野生型 TX98（wtTX98）高 3 倍，而病毒复制动力学相似。经鼻接种的猪平均鼻腔 lvTX98 排出量比 wtTX98 低 50 倍，但仍达到高达 4.3 log50%组织培养感染剂量/mL 的滴度。经气管内接种后，lvTX98 在感染后 18 至 48 小时（hpi）的下呼吸道中的滴度明显降低，但在 72 hpi 时与 wtTX98 的滴度相似。lvTX98 引起的临床症状比 wtTX98 轻，但诱导的显微镜和大体肺病变、峰值中性粒细胞浸润和峰值 I 型 IFN 水平相当。因此，lvTX98 在猪中部分减毒，但这与更高的 I 型 IFN 水平无关。具有截短的 NS1del126 蛋白的猪流感 A 病毒（swIAVs）在以前的基于实验室的安全性研究中被强烈减毒，因此被批准作为美国猪用 LAIV。然而，在野外，LAIV 株在诊断样本中被检测到，并可通过与地方性 swIAVs 重组而恢复野生型 NS1。这表明 LAIV 有显著的复制，因此需要进一步研究这些 LAIV 株的减毒程度和机制。在这里，我们表明 H3N2 LAIV 株 lvTX98 在猪中仅部分减毒，经鼻腔接种后可排出高滴度的疫苗。lvTX98 的衰减和有限复制似乎与 NS1 的除 I 型 IFN 拮抗以外的功能丧失有关。我们的研究结果可以帮助解释与基于 NS1del126 的 LAIV 株相关的临床呼吸道疾病和重组事件在野外的发生。
