Gendler Paul, Barbachano-Guerrero Arturo, Chappell Samuel D, Ratnayake Oshani C, Pinto Samantha M, Perera Rushika, Sawyer Sara L
Department of Molecular, Cellular, and Developmental Biology, University of Colorado Boulder, Boulder, Colorado, USA.
BioFrontiers Institute, University of Colorado Boulder, Boulder, Colorado, USA.
J Virol. 2025 Jul 22;99(7):e0045025. doi: 10.1128/jvi.00450-25. Epub 2025 Jun 4.
There are an unknown number of sylvatic ("sylvan" = "of the forest") dengue viruses currently being sustained by nonhuman primates and mosquitoes in the forests of Africa and Asia. Humans are regularly infected with these viruses, occasionally resulting in small epidemics. One barrier to understanding sylvatic dengue virus biology is the scarcity of stocks available for study. While the full-length genome sequences of 28 sylvatic viruses exist on GenBank, accessible infectious stocks exist for only a handful of these. One way to overcome this obstacle is to rescue infectious virus stocks using reverse genetics. However, reverse genetic engineering of dengue viruses is notoriously difficult. Here, we optimize a reverse genetics method specifically for the rescue of sylvatic dengue virus stocks from sequence data. The key to our approach was the observation that mosquito cells, rather than mammalian cells, must be used to launch sylvatic dengue virus replication from assembled genomes. We demonstrate the success of this technique by rescuing seven sylvatic dengue viruses. With this unique collection, we then characterized the relative fitness of each virus strain on human, monkey, and mosquito cells. While mosquito cells are universally permissive for the growth of sylvatic dengue viruses, some sylvatic dengue virus strains showed significantly better replication in human and monkey cells than others. These sylvatic dengue virus strains may have a greater potential for human adaptation.
Given the enormous burden of the four human dengue viruses, which emerged from the sylvatic dengue virus reservoir, it is important that we consider the possibility of a new dengue virus emerging into the human population. Nonhuman primate species in Asia and Africa are suspected to be the natural reservoir hosts for sylvatic dengue viruses. Occasionally, these sylvatic dengue viruses infect humans, although there are few stocks of these viruses available for study in the lab. Here, we optimize a reverse genetics technique for sylvatic dengue viruses, and we rescue stocks of seven strains. With this method, theoretically, any sylvatic dengue virus sequence deposited on GenBank can be transformed into a high-titer infectious virus stock.
目前，在非洲和亚洲的森林中，有数量不明的野生型（“sylvan”意为“森林的”）登革病毒由非人灵长类动物和蚊子传播。人类经常感染这些病毒，偶尔会引发小规模疫情。了解野生型登革病毒生物学特性的一个障碍是可供研究的病毒株稀缺。虽然GenBank上存在28种野生型病毒的全长基因组序列，但只有少数几种有可获取的感染性病毒株。克服这一障碍的一种方法是利用反向遗传学拯救感染性病毒株。然而，登革病毒的反向基因工程 notoriously difficult。在这里，我们专门优化了一种反向遗传学方法，用于从序列数据中拯救野生型登革病毒株。我们方法的关键在于观察到，必须使用蚊子细胞而非哺乳动物细胞，才能从组装好的基因组启动野生型登革病毒的复制。我们通过拯救七种野生型登革病毒证明了该技术的成功。有了这个独特的病毒株集合，我们随后对每种病毒株在人、猴和蚊子细胞上的相对适应性进行了表征。虽然蚊子细胞对野生型登革病毒的生长普遍具有易感性，但一些野生型登革病毒株在人细胞和猴细胞中的复制明显优于其他病毒株。这些野生型登革病毒株可能具有更大的适应人类的潜力。
鉴于源自野生型登革病毒库的四种人类登革病毒造成的巨大负担，我们必须考虑新的登革病毒出现并传播给人类的可能性。亚洲和非洲的非人灵长类物种被怀疑是野生型登革病毒的天然宿主。这些野生型登革病毒偶尔会感染人类，尽管在实验室中可供研究的此类病毒株很少。在这里，我们优化了一种针对野生型登革病毒的反向遗传学技术，并拯救了七种病毒株。通过这种方法，理论上，任何存于GenBank上的野生型登革病毒序列都可以转化为高滴度的感染性病毒株。
