Simian varicella virus (SVV), the primate counterpart of varicella-zoster virus, causes varicella (chickenpox), establishes latency in ganglia, and reactivates to produce zoster. We previously demonstrated that a recombinant SVV expressing enhanced green fluorescent protein (rSVV.eGFP) is slightly attenuated both in culture and in infected monkeys. Here, we generated two additional recombinant SVVs to visualize infected cells in vitro and in vivo. One harbors eGFP fused to the N terminus of open reading frame 9 (ORF9) (rSVV.eGFP-2a-ORF9), and another harbors eGFP fused to the C terminus of ORF66 (rSVV.eGFP-ORF66). Both recombinant viruses efficiently expressed eGFP in cultured cells. Both recombinant SVV infections in culture were comparable to that of wild-type SVV (SVV.wt). Unlike SVV.wt, eGFP-tagged SVV did not replicate in rhesus cells in culture. Intratracheal (i.t.) or i.t. plus intravenous (i.v.) inoculation of rhesus macaques with these new eGFP-tagged viruses resulted in low viremia without varicella rash, although SVV DNA was abundant in bronchoalveolar lavage (BAL) fluid at 10 days postinoculation (dpi). SVV DNA was also found in trigeminal ganglia of one monkey inoculated with rSVV.eGFP-ORF66. Intriguingly, a humoral response to both SVV and eGFP was observed. In addition, monkeys inoculated with the eGFP-expressing viruses were protected from superinfection with SVV.wt, suggesting that the monkeys had mounted an efficient immune response. Together, our results show that eGFP expression could be responsible for their reduced pathogenesis.

IMPORTANCE SVV infection in nonhuman primates has served as an extremely useful animal model to study varicella-zoster virus (VZV) pathogenesis. eGFP-tagged viruses are a great tool to investigate their pathogenesis. We constructed and tested two new recombinant SVVs with eGFP inserted into two different locations in the SVV genome. Both recombinant SVVs showed robust replication in culture but reduced viremia compared to that with SVV.wt during primary infection in rhesus macaques. Our results indicate that conclusions on eGFP-tagged viruses based on in vitro results should be handled with care, since eGFP expression could result in attenuation of the virus.

水痘带状疱疹病毒的灵长类动物猿猴水痘病毒（SVV）引起水痘（水痘），在神经节中形成潜伏期，并重新激活以产生带状疱疹。我们先前证明，在培养物中和感染的猴子中，表达增强的绿色荧光蛋白（rSVV.eGFP）的重组SVV都略有减弱。在这里，我们生成了两个额外的重组SVV，以在体外和体内可视化受感染的细胞。一个带有与开放阅读框9（ORF9）的N末端融合的eGFP（rSVV.eGFP-2a-ORF9），另一个带有与ORF66的C末端融合的eGFP（rSVV.eGFP-ORF66）。两种重组病毒均在培养的细胞中有效表达eGFP。培养中的两种重组SVV感染都与野生型SVV（SVV.wt）相当。与SVV.wt不同，eGFP标签的SVV在培养的恒河猴细胞中不复制。气管内（it）或静脉内（iv）用这些新的带有eGFP标记的病毒接种猕猴可导致低病毒血症，无水痘疹，尽管在接种后10天（dpi），支气管肺泡灌洗液（BAL）中的SVV DNA含量很高。在一只接种了rSVV.eGFP-ORF66的猴子的三叉神经节中也发现了SVV DNA。有趣的是，观察到了对SVV和eGFP的体液反应。此外，接种了表达eGFP的病毒的猴子受到保护，不会被SVV.wt过度感染，这表明猴子已经建立了有效的免疫反应。在一起，我们的结果表明，eGFP的表达可能是其减少发病机理的原因。

重要事项非人灵长类动物中的SVV感染已成为研究水痘带状疱疹病毒（VZV）发病机理的极为有用的动物模型。带有eGFP标签的病毒是研究其发病机理的好工具。我们构建并测试了两个新的重组SVV，其中的eGFP插入了SVV基因组中的两个不同位置。与恒河猴猕猴初次感染期间的SVV.wt相比，两种重组SVV在培养物中均显示出强大的复制能力，但病毒血症降低。我们的结果表明，应谨慎处理基于eGFP标记的病毒的体外结论，因为eGFP的表达可能导致病毒的减毒。