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Shi, P. Y.

Publications and source records attributed to Shi, P. Y..

2 recordsLinked to original sources
bioRxiv2021

Ecology and transmission of a dengue virus serotype 4 identified in wild Aedes aegypti in Florida

1Dengue virus is the most prevalent mosquito-borne virus, causing approximately 390 million infections and 25,000 deaths per year. Aedes aegypti, the primary mosquito vector of dengue virus, is well established throughout the state of Florida, USA. Autochthonous transmission of dengue virus to humans in Florida has been increasing since 2009, alongside consistent importation of dengue cases. However, most cases of first infection with dengue are asymptomatic and the virus can be maintained in mosquito populations, complicating surveillance and leading to an underestimation of disease risk. Metagenomic sequencing of Aedes aegypti mosquitoes in Manatee County, Florida revealed the presence of dengue virus serotype 4 (DENV-4) genomes in mosquitoes from multiple trapping sites over 2 years, in the absence of a human DENV-4 index case and even though a locally acquired case of DENV-4 has never been reported in Florida. This finding suggested that: i) DENV-4 may circulate amongst humans undetected, ii) the virus was being maintained in the mosquito population, or iii) the detected complete genome sequence may not represent a viable virus. This study demonstrates that an infectious clone generated from the Manatee County DENV-4 (DENV-4M) sequence is capable of infecting mammalian and insect tissue culture systems, as well as adult female Aedes aegypti mosquitoes when fed in a blood meal. However, the virus is subject to a dose dependent infection barrier in mosquitoes, and has a kinetic delay compared to a phylogenetically related wild-type (WT) control virus from a symptomatic child, DENV-4H (strain Homo sapiens/Haiti-0075/2015, GenBank accession MK514144.1). DENV-4M disseminates from the midgut to the ovary and saliva at 14 days post-infection. Viral RNA was also detectable in the adult female offspring of DENV-4M infected mosquitoes. These results demonstrate that the virus is capable of infecting vector mosquitoes, is transmissible by bite, and is vertically transmitted, indicating a mechanism for maintenance in the environment without human-mosquito transmission. These findings suggest undetected human-mosquito transmission and/or long-term maintenance of the virus in the mosquito population is occurring in Florida, and underscore the importance of proactive surveillance for viruses in mosquitoes. Graphical AbstractIn order to better assess the public health risk posed by a detection of DENV-4 RNA in Manatee County, FL Aedes aegypti, we produced an infectious clone using the sequence from the wild-caught mosquitoes and characterized it via laboratory infections of mosquitoes and mosquito tissues.

microbiology↗
bioRxiv2020

Furin Cleavage Site Is Key to SARS-CoV-2 Pathogenesis

SARS-CoV-2 has resulted in a global pandemic and shutdown economies around the world. Sequence analysis indicates that the novel coronavirus (CoV) has an insertion of a furin cleavage site (PRRAR) in its spike protein. Absent in other group 2B CoVs, the insertion may be a key factor in the replication and virulence of SARS-CoV-2. To explore this question, we generated a SARS-CoV-2 mutant lacking the furin cleavage site ({Delta}PRRA) in the spike protein. This mutant virus replicated with faster kinetics and improved fitness in Vero E6 cells. The mutant virus also had reduced spike protein processing as compared to wild-type SARS-CoV-2. In contrast, the {Delta}PRRA had reduced replication in Calu3 cells, a human respiratory cell line, and had attenuated disease in a hamster pathogenesis model. Despite the reduced disease, the {Delta}PRRA mutant offered robust protection from SARS-CoV-2 rechallenge. Importantly, plaque reduction neutralization tests (PRNT50) with COVID-19 patient sera and monoclonal antibodies against the receptor-binding domain found a shift, with the mutant virus resulting in consistently reduced PRNT50 titers. Together, these results demonstrate a critical role for the furin cleavage site insertion in SARS-CoV-2 replication and pathogenesis. In addition, these findings illustrate the importance of this insertion in evaluating neutralization and other downstream SARS-CoV-2 assays. ImportanceAs COVID-19 has impacted the world, understanding how SARS-CoV-2 replicates and causes virulence offers potential pathways to disrupt its disease. By removing the furin cleavage site, we demonstrate the importance of this insertion to SARS-CoV-2 replication and pathogenesis. In addition, the findings with Vero cells indicate the likelihood of cell culture adaptations in virus stocks that can influence reagent generation and interpretation of a wide range of data including neutralization and drug efficacy. Overall, our work highlights the importance of this key motif in SARS-CoV-2 infection and pathogenesis. Article SummaryA deletion of the furin cleavage site in SARS-CoV-2 amplifies replication in Vero cells, but attenuates replication in respiratory cells and pathogenesis in vivo. Loss of the furin site also reduces susceptibility to neutralization in vitro.

microbiology↗