Flaviviruses and other arthropod-transmitted viruses represent a widespread global health problem, with limited treatment options currently available. Thus, a better understanding of the cellular requirements for their infection is needed. Both DENV and ZIKV rely on expansion of the endoplasmic reticulum (ER) and the induction of autophagy to establish productive infections. However, little is known regarding the interplay between the requirements for autophagy initiation during infection and the mechanisms used by these viruses to avoid clearance through the autophagic pathway. Our study highlights the importance of the host factor BPIFB3 in regulating flavivirus replication and further confirms that the RETREG1-dependent reticulophagy pathway is antiviral to both DENV and ZIKV.

Autophagy is a degradative cellular pathway that targets cytoplasmic contents and organelles for turnover by the lysosome. Various autophagy pathways play key roles in the clearance of viral infections, and many families of viruses have developed unique methods for avoiding degradation. Some positive stranded RNA viruses, such as enteroviruses and flaviviruses, usurp the autophagic pathway to promote their own replication. We previously identified the endoplasmic reticulum-localized protein BPIFB3 as an important negative regulator of non-canonical autophagy that uniquely impacts the replication of enteroviruses and flaviviruses. Here, we find that many components of the canonical autophagy machinery are not required for BPIFB3 depletion induced autophagy and identify the host factors that facilitate its role in the replication of enteroviruses and flaviviruses. Using proximity-dependent biotinylation (BioID) followed by mass spectrometry, we identify ARFGAP1 and TMED9 as two cellular components that interact with BPIFB3 to regulate autophagy and viral replication. Importantly, our data demonstrate that non-canonical autophagy in mammalian cells can be controlled outside of the traditional pathway regulators and define the role of two proteins in BPIFB3 depletion mediated non-canonical autophagy.

Enteroviruses manipulate host membranes to form replication organelles, which concentrate viral and host factors to allow for efficient replication. However, this process has not been well-studied in living cells throughout the course of infection. To define the dynamic process of enterovirus membrane remodeling of major secretory pathway organelles, we have developed plasmid-based reporter systems that utilize viral protease-dependent release of a nuclear-localized fluorescent protein from the endoplasmic reticulum (ER) membrane during infection, while retaining organelle-specific fluorescent protein markers such as the ER and Golgi. This system thus allows for the monitoring of organelle-specific changes induced by infection in real-time. Using long-term time-lapse imaging of living cells infected with coxsackievirus B3 (CVB), we detected reporter translocation to the nucleus beginning ~4 h post-infection, which correlated with a loss of Golgi integrity and a collapse of the peripheral ER. Lastly, we applied our system to study the effects of a calcium channel inhibitor, 2APB, on virus-induced manipulation of host membranes. We found that 2APB treatment had no effect on the kinetics of infection or the percentage of infected cells. However, we observed aberrant ER structures in CVB-infected cells treated with 2APB and a significant decrease in viral-dependent cell lysis, which corresponded with a decrease in extracellular virus titers. Thus, our system provides a tractable platform to monitor the effects of inhibitors, gene silencing, and/or gene editing on viral manipulation of host membranes, which can help determine the mechanism of action for antivirals.

Aim: Vaginitis and other vaginal discharge syndromes lead to high healthcare utilization. Molecular tests like syndromic multiplex real-time (RT) polymerase chain reaction (PCR)-based tests are highly sensitive and specific at diagnosing the infectious causes of vaginitis. This study compared the healthcare resource utilization (HCRU) and direct all-cause healthcare costs among patients with vaginitis in the US receiving next-day syndromic multiplex RT-PCR tests with those receiving other PCR tests or no diagnostic test of interest. Patients & methods: This retrospective study utilized claims data from IQVIA PharMetrics ^® Plus database to identify adult patients with a diagnosis for vaginitis (first claim = index) from January 2021 to April 2023, with 6 months of continuous enrollment prior to (baseline) and after index (follow-up). Pairwise comparisons were conducted between RT-PCR and 1:1 propensity matched Other PCR and No Test subcohorts for all-cause HCRU and costs during follow-up. Results: Each of the RT-PCR, Other PCR and No Test subcohorts included 1946 matched patients. Mean(SD) follow-up total cost was significantly lower for the RT-PCR than the No Test subcohort ($5607 [$15,122] vs $6680 [$20,751], p = 0.0023). Mean(SD) overall outpatient and other medical service costs were lower for RT-PCR versus Other PCR (outpatient: $2964 [$9666] vs $3174 [$7113], p = 0.0110; other medical: $1961 [$9244] vs $2099 [$6475], p = 0.0002) and No Test subcohorts (outpatient: $2964 [$9666] vs $4067 [$12,341], p < 0.0001; other medical: $1961 [$9244] vs $2973 [$11,685]; p < 0.0001). A lower proportion had any outpatient service HCRU in RT-PCR versus Other PCR subcohort (92.6% vs 94.2%, p = 0.0349). A lower proportion had any other medical service claim in RT-PCR versus Other PCR (78.3% vs 83.2%, p < 0.0001) and No Test subcohorts (78.3% vs 83.0%, p = 0.0001). Physician office, emergency room (ER), prescription use and costs were similar between the subcohorts. Conclusion: The use of syndromic multiplex RT-PCR diagnostics with next day test results in patients with vaginitis was associated with lower outpatient costs and total healthcare costs than those in the no test cohort over 6 months. These findings indicate that use of syndromic multiplex RT-PCR diagnostics may contribute to improved patient management compared with clinical diagnosis alone.

Purpose: Advancements in pathogen identification by diagnostic testing may improve patient outcomes. This study evaluated healthcare utilization and costs following diagnostic testing for acute oropharyngeal and respiratory tract infections (RTIs). Patients and Methods: Healthcare utilization and costs were evaluated in patients with acute oropharyngeal infections (n=1,172,693), and RTIs (n=4,005,228) who received a syndromic panel-based PCR test with next-day results (HealthTrackRx, Denton, TX), or no test in the IQVIA PharMetrics^® Plus adjudicated claims database. Results: Statistically significant differences were observed between patients who received the PCR test compared to those who received no test. The PCR test cohort had lower total healthcare costs (mean = $5,601±$29,170, median = $807) versus the no test cohort (mean = $7,460±$40,817, median = $1,163) (p = 0.0014) over 6 months, and fewer outpatient visits, other medical service visits, emergency room visits, and inpatient stays (p< 0.0001). Similarly, those who received the PCR test for oropharyngeal infection trended towards lower total healthcare costs (mean = $4,393±$13,524, median=$844) than those who received no test (mean = $5,503±$34,141, median = $956) (p=0.0525) and had fewer outpatient and other medical services (p< 0.0001). Conclusion: Next-day molecular testing for respiratory and oropharyngeal infection lowers healthcare utilization and costs, suggesting improved patient care through reduced need for healthcare resources.

The flavivirus genus, which includes dengue virus (DENV) and Zika virus (ZIKV), are significant human pathogens and the prevalence of infected vectors continues to geographically expand. Both DENV and ZIKV rely on expansion of the endoplasmic reticulum (ER) and the induction of autophagy to establish a productive viral infection. However, little is known regarding the interplay between the requirements for autophagy initiation during infection and the mechanisms used by these viruses to avoid clearance through the autophagic pathway. We recently showed that DENV and ZIKV inhibit reticulophagy (specific degradation of the ER through autophagy) by cleaving reticulophagy regulator 1 (RETREG1), an autophagy receptor responsible for targeted ER sheet degradation. These data suggest that DENV and ZIKV require specific autophagic pathways for their replication, while other autophagic pathways are antiviral. We previously identified BPI Fold Containing Family B Member 3 (BPIFB3) as a regulator of autophagy that negatively controls enterovirus replication. Here, we show that in contrast to enteroviruses, BPIFB3 functions as a positive regulator of DENV and ZIKV infection and that its RNAi-mediated silencing drastically inhibits the formation of viral replication organelles. We show that BPIFB3 depletion enhances ER fragmentation, while its overexpression protects against autophagy-induced ER degradation, demonstrating that BPIFB3 serves as a specific regulator of ER turnover. We further show that the antiviral effects of BPIFB3 depletion on flavivirus infection are reversed in RETREG1-depleted cells, and that BPIFB3 associates with RETREG1 within the ER, suggesting that BPIFB3 regulates a RETREG1-specific reticulophagy pathway. Collectively, these studies identify BPIFB3 as a regulator of the reticulophagy pathway and define the requirements for a novel host regulator of flavivirus infection. Author Summary: Flaviviruses and other arthropod transmitted viruses represent a widespread global health problem with limited treatment options currently available. Thus, greater knowledge of the host factors required for replication and transmission is needed to provide a better understanding of the cellular requirements for infection. Here, we show that the endoplasmic reticulum (ER) localized protein, BPIFB3 is required to facilitate flavivirus infection. Depletion of BPIFB3 in cells inhibits dengue virus and Zika virus infection prior to replication of the viral genome. Mechanistically, we show that BPIFB3 inhibits ER degradation in an autophagy-specific manner and that loss of BPIFB3 decreases the availability of ER membranes needed for flavivirus replication. We further show that BPIFB3 specifically regulates the RETREG1 pathway, but not other pathways of ER turnover. Together, our data define a previously uncharacterized method of regulating ER degradation and show that BPIFB3 is an essential host factor for a productive flavivirus infection.