Chronic shedding of a SARS-CoV-2 Alpha variant in wastewater.

BACKGROUND: Central Michigan University (CMU) participated in a state-wide SARS-CoV-2 wastewater monitoring program since 2021. Wastewater samples were collected from on-campus sites and nine off-campus wastewater treatment plants servicing small metropolitan and rural communities. SARS-CoV-2 genome copies were quantified using droplet digital PCR and results were reported to the health department. RESULTS: One rural, off-campus site consistently produced higher concentrations of SARS-CoV-2 genome copies. Samples from this site were sequenced and contained predominately a derivative of Alpha variant lineage B.1.1.7, detected from fall 2021 through summer 2023. Mutational analysis of reconstructed genes revealed divergence from the Alpha variant lineage sequence over time, including numerous mutations  in the Spike RBD and NTD. CONCLUSIONS: We discuss the possibility that a chronic SARS-CoV-2 infection accumulated adaptive mutations that promoted long-term infection. This study reveals that small wastewater treatment plants can enhance resolution of rare events and facilitate reconstruction of viral genomes due to the relative lack of contaminating sequences.

Adaptation of an NLP system to a new healthcare environment to identify social determinants of health.

Social determinants of health (SDoH) are increasingly important factors for population health, healthcare outcomes, and care delivery. However, many of these factors are not reliably captured within structured electronic health record (EHR) data. In this work, we evaluated and adapted a previously published NLP tool to include additional social risk factors for deployment at Vanderbilt University Medical Center in an Acute Myocardial Infarction cohort. We developed a transformation of the SDoH outputs of the tool into the OMOP common data model (CDM) for re-use across many potential use cases, yielding performance measures across 8 SDoH classes of precision 0.83 recall 0.74 and F-measure of 0.78.

Real-time tracking of stem cell viability, proliferation, and differentiation with autonomous bioluminescence imaging.

BACKGROUND: Luminescent reporter proteins are vital tools for visualizing cells and cellular activity. Among the current toolbox of bioluminescent systems, only bacterial luciferase has genetically defined luciferase and luciferin synthesis pathways that are functional at the mammalian cell temperature optimum of 37 °C and have the potential for in vivo applications. However, this system is not functional in all cell types, including stem cells, where the ability to monitor continuously and in real-time cellular processes such as differentiation and proliferation would be particularly advantageous. RESULTS: We report that artificial subdivision of the bacterial luciferin and luciferase pathway subcomponents enables continuous or inducible bioluminescence in pluripotent and mesenchymal stem cells when the luciferin pathway is overexpressed with a 20-30:1 ratio. Ratio-based expression is demonstrated to have minimal effects on phenotype or differentiation while enabling autonomous bioluminescence without requiring external excitation. We used this method to assay the proliferation, viability, and toxicology responses of iPSCs and showed that these assays are comparable in their performance to established colorimetric assays. Furthermore, we used the continuous luminescence to track stem cell progeny post-differentiation. Finally, we show that tissue-specific promoters can be used to report cell fate with this system. CONCLUSIONS: Our findings expand the utility of bacterial luciferase and provide a new tool for stem cell research by providing a method to easily enable continuous, non-invasive bioluminescent monitoring in pluripotent cells.

Identification of coronavirus sequences in carp cDNA from Wuhan, China.

Severe acute respiratory syndrome (SARS)-like coronavirus sequences were identified in two separate complementary DNA (cDNA) pools. The first pool was from a Carassius auratus (crusian carp) cell line and the second was from Ctenopharyngodon idella (grass carp) head kidney tissue. BLAST analysis suggests that these sequences belong to SARS-like coronaviruses, and that they are not evolutionarily conserved in other species. Investigation of the submitting laboratories revealed that two laboratories from the Institute of Hydrobiology at the Chinese Academy of Sciences in Wuhan, China performed the research and submitted the cDNA libraries to GenBank. This institution is very close in proximity to the Wuhan South China Seafood Wholesale Market where SARS-CoV-2 first amplified in the human population. It is possible that these sequences are an artifact of the bioinformatics pipeline that was used. It is also possible that SARS-like coronaviruses are a common environmental pathogen in the region that may be in aquatic habitats.

Complex Ventral Hernia Repair in the Class III Morbidly Obese Patient.

INTRODUCTION: Patients undergoing complex ventral hernia repair (VHR) often present with significant medical comorbidities, the most prevalent of which is obesity. Although recent advancements in abdominal wall reconstruction techniques have provided the general hernia patient population with markedly improved recurrence and postoperative complication rates, many patients have been precluded from these procedures owing to excessive body mass index (BMI). In this study, we investigate the viability of complex ventral hernia repair with epigastric artery perforator sparing skin incisions, component separation, and wide-spanning retrorectus mesh reinforcement for patients with BMI of greater than or equal to 40 kg/m(2) (class III obesity). METHODS: A single surgeon retrospective review of our prospectively maintained database was performed. We restricted this data to class III morbidly obese patients undergoing open VHR with component muscle separation and wide-spanning mesh reinforcement. RESULTS: Between 2010 and 2017, 131 patients met the inclusion criteria for our study. The mean patient BMI was 46.7 kg/m(2). Operative wounds were categorized according to the National Healthcare Safety Network Wound Class Definitions. There was no statistically significant association between wound class and postoperative complication rates. After our implementation of epigastric artery perforator sparing skin incisions in 2013, significantly less wound breakdown was observed (26.3%) as opposed to before (49.0%) (P < 0.01). Furthermore, significantly less cases required return to the operating room after this technique was implemented (31.3%) as compared with before (60.8%) (P < 0.001). Postoperatively, 28 patients developed an infection requiring antibiotic treatment (21.4%), and the overall hernia recurrence rate was 5.3%. Three patients expired. CONCLUSIONS: Complex VHR with abdominal wall reconstruction may be a viable option for class III morbidly obese patients. Preliminary data suggest that implementation of epigastric artery perforator sparing skin incisions may reduce the risk of postoperative wound complications, and we have demonstrated hernia recurrence and wound complications comparable with those seen in the general population.

Zika virus induces astrocyte differentiation in neural stem cells.

Zika virus (ZIKV) is a rapidly emerging flavivirus that has been associated with a number of congenital neurological manifestations. Here, we show that ZIKV replicated efficiently in mouse neural stem cells (mNSCs). ZIKV infection caused a cytopathic effect without affecting cell viability, yet led to a significant decrease in the number of proteins secreted into mNSC supernatants. A gene expression array of neural stem cell progenitor and differentiation markers suggested that infection reduced the number of neuronal and oligodendrocyte progenitors while increasing the number of astrocyte progenitors. Infection in astrocytes increased transcription of key genes involved in the antiviral response. These data provide molecular and cellular evidence that ZIKV significantly alters neural development in the vertebrate host and that astrocyte differentiation may be a protective response that limits neuropathogenesis.

A novel mosquito ubiquitin targets viral envelope protein for degradation and reduces virion production during dengue virus infection.

BACKGROUND: Dengue virus (DENV) is a mosquito-borne flavivirus that causes significant human disease and mortality in the tropics and subtropics. By examining the effects of virus infection on gene expression, and interactions between virus and vector, new targets for prevention of infection and novel treatments may be identified in mosquitoes. We previously performed a microarray analysis of the Aedes aegypti transcriptome during infection with DENV and found that mosquito ubiquitin protein Ub3881 (AAEL003881) was specifically and highly down-regulated. Ubiquitin proteins have multiple functions in insects, including marking proteins for proteasomal degradation, regulating apoptosis and mediating innate immune signaling. METHODS: We used qRT-PCR to quantify gene expression and infection, and RNAi to reduce Ub3881 expression. Mosquitoes were infected with DENV through blood feeding. We transfected DENV protein expression constructs to examine the effect of Ub3881 on protein degradation. We used site-directed mutagenesis and transfection to determine what amino acids are involved in Ub3881-mediated protein degradation. Immunofluorescence, Co-immunoprecipitation and Western blotting were used to examine protein interactions and co-localization. RESULTS: The overexpression of Ub3881, but not related ubiquitin proteins, decreased DENV infection in mosquito cells and live Ae. aegypti. The Ub3881 protein was demonstrated to be involved in DENV envelope protein degradation and reduce the number of infectious virions released. CONCLUSIONS: We conclude that Ub3881 has several antiviral functions in the mosquito, including specific viral protein degradation. GENERAL SIGNIFICANCE: Our data highlights Ub3881 as a target for future DENV prevention strategies in the mosquito transmission vector.

Dengue Virus Infection of Aedes aegypti Requires a Putative Cysteine Rich Venom Protein.

Dengue virus (DENV) is a mosquito-borne flavivirus that causes serious human disease and mortality worldwide. There is no specific antiviral therapy or vaccine for DENV infection. Alterations in gene expression during DENV infection of the mosquito and the impact of these changes on virus infection are important events to investigate in hopes of creating new treatments and vaccines. We previously identified 203 genes that were ≥5-fold differentially upregulated during flavivirus infection of the mosquito. Here, we examined the impact of silencing 100 of the most highly upregulated gene targets on DENV infection in its mosquito vector. We identified 20 genes that reduced DENV infection by at least 60% when silenced. We focused on one gene, a putative cysteine rich venom protein (SeqID AAEL000379; CRVP379), whose silencing significantly reduced DENV infection in Aedes aegypti cells. Here, we examine the requirement for CRVP379 during DENV infection of the mosquito and investigate the mechanisms surrounding this phenomenon. We also show that blocking CRVP379 protein with either RNAi or specific antisera inhibits DENV infection in Aedes aegypti. This work identifies a novel mosquito gene target for controlling DENV infection in mosquitoes that may also be used to develop broad preventative and therapeutic measures for multiple flaviviruses.

Co-Cultured Continuously Bioluminescent Human Cells as Bioreporters for the Detection of Prodrug Therapeutic Impact Pre- and Post-Metabolism.

Modern drug discovery workflows require assay systems capable of replicating the complex interactions of multiple tissue types, but that can still function under high throughput conditions. In this work, we evaluate the use of substrate-free autobioluminescence in human cell lines to support the performance of these assays with reduced economical and logistical restrictions relative to substrate-requiring bioluminescent reporter systems. The use of autobioluminescence was found to support assay functionality similar to existing luciferase reporter targets. The autobioluminescent assay format was observed to correlate strongly with general metabolic activity markers such as ATP content and the presence of reactive oxygen species, but not with secondary markers such as glutathione depletion. At the transcriptional level, autobioluminescent dynamics were most closely associated with expression of the CYP1A1 phase I detoxification pathway. These results suggest constitutively autobioluminescent cells can function as general metabolic activity bioreporters, while pairing expression of the autobioluminescent phenotype to detoxification pathway specific promoters could create more specific sensor systems.

Differential contributions of infralimbic prefrontal cortex and nucleus accumbens during reward-based learning and extinction.

Using environmental cues for the prediction of future events is essential for survival. Such cue-outcome associations are thought to depend on mesolimbic circuitry involving the nucleus accumbens (NAc) and prefrontal cortex (PFC). Several studies have identified roles for both NAc and PFC in the expression of stable goal-directed behaviors, but much remains unknown about their roles during learning of such behaviors. To further address this question, we used in vivo oxygen amperometry, a proxy for blood oxygen level-dependent (BOLD) signal measurement in human functional magnetic resonance imaging, in rats performing a cued lever-pressing task requiring discrimination between a rewarded and nonrewarded cue. Simultaneous oxygen recordings were obtained from infralimbic PFC (IFC) and NAc throughout both acquisition and extinction of this task. Activation of NAc was specifically observed following rewarded cue onset during the entire acquisition phase and also during the first days of extinction. In contrast, IFC activated only during the earliest periods of acquisition and extinction, more specifically to the nonrewarded cue. Thus, in vivo oxygen amperometry permits a novel, stable form of longitudinal analysis of brain activity in behaving animals, allowing dissociation of the roles of different brain regions over time during learning of reward-driven instrumental action. The present results offer a unique temporal perspective on how NAc may promote actions directed toward anticipated positive outcome throughout learning, while IFC might suppress actions that no longer result in reward, but only during critical periods of learning.

Mosquito saliva serine protease enhances dissemination of dengue virus into the mammalian host.

Dengue virus (DENV), a flavivirus of global importance, is transmitted to humans by mosquitoes. In this study, we developed in vitro and in vivo models of saliva-mediated enhancement of DENV infectivity. Serine protease activity in Aedes aegypti saliva augmented virus infectivity in vitro by proteolyzing extracellular matrix proteins, thereby increasing viral attachment to heparan sulfate proteoglycans and inducing cell migration. A serine protease inhibitor reduced saliva-mediated enhancement of DENV in vitro and in vivo, marked by a 100-fold reduction in DENV load in murine lymph nodes. A saliva-mediated infectivity enhancement screen of fractionated salivary gland extracts identified serine protease CLIPA3 as a putative cofactor, and short interfering RNA knockdown of CLIPA3 in mosquitoes demonstrated its role in influencing DENV infectivity. Molecules in mosquito saliva that facilitate viral infectivity in the vertebrate host provide novel targets that may aid in the prevention of disease.

Susceptibility of high-risk human papillomavirus type 16 to clinical disinfectants.

OBJECTIVES: Little to nothing is known about human papillomavirus (HPV) susceptibility to disinfection. HPV is estimated to be among the most common sexually transmitted diseases in humans. HPV is also the causative agent of cervical cancers and other anogenital cancers and is responsible for a significant portion of oropharyngeal cancers. While sexual transmission is well documented, vertical and non-sexual transmission may also be important. METHODS: Using recombinant HPV16 particles (quasivirions) and authentic HPV16 grown in three-dimensional organotypic human epithelial culture, we tested the susceptibility of high-risk HPV to clinical disinfectants. Infectious viral particles were incubated with 11 common clinical disinfectants, appropriate neutralizers were added to inactivate the disinfectant and solutions were filter centrifuged. Changes in the infectivity titres of the disinfectant-treated virus were measured compared with untreated virus. RESULTS: HPV16 is a highly resistant virus; more so than other non-enveloped viruses previously tested. The HPV16 quasivirions showed similar resistance to native virions, except for being susceptible to isopropanol, the triple phenolic and the lower concentration peracetic acid-silver (PAA-silver)-based disinfectant. Authentic virus and quasivirus were resistant to glutaraldehyde and ortho-phthalaldehyde and susceptible to hypochlorite and the higher concentration PAA-silver-based disinfectant. CONCLUSIONS: We present the first disinfectant susceptibility data on HPV16 native virions, which show that commonly used clinical disinfectants, including those used as sterilants in medical and dental healthcare facilities, have no effect on HPV16 infectivity. Policy changes concerning disinfectant use are needed. The unusually high resistance of HPV16 to disinfection supports other data suggesting the possibility of fomite or non-sexual transmission of HPV16.

West Nile Virus: biology, transmission, and human infection.

West Nile Virus was introduced into the Western Hemisphere during the late summer of 1999 and has been causing significant and sometimes severe human diseases since that time. This article briefly touches upon the biology of the virus and provides a comprehensive review regarding recent discoveries about virus transmission, virus acquisition, and human infection and disease.

Hemodynamic responses in amygdala and hippocampus distinguish between aversive and neutral cues during Pavlovian fear conditioning in behaving rats.

Lesion and electrophysiological studies in rodents have identified the amygdala and hippocampus (HPC) as key structures for Pavlovian fear conditioning, but human functional neuroimaging studies have not consistently found activation of these structures. This could be because hemodynamic responses cannot detect the sparse neuronal activity proposed to underlie conditioned fear. Alternatively, differences in experimental design or fear levels could account for the discrepant findings between rodents and humans. To help distinguish between these alternatives, we used tissue oxygen amperometry to record hemodynamic responses from the basolateral amygdala (BLA), dorsal HPC (dHPC) and ventral HPC (vHPC) in freely-moving rats during the acquisition and extinction of conditioned fear. To enable specific comparison with human studies we used a discriminative paradigm, with one auditory cue [conditioned stimulus (CS)+] that was always followed by footshock, and another auditory cue (CS-) that was never followed by footshock. BLA tissue oxygen signals were significantly higher during CS+ than CS- trials during training and early extinction. In contrast, they were lower during CS+ than CS- trials by the end of extinction. dHPC and vHPC tissue oxygen signals were significantly lower during CS+ than CS- trials throughout extinction. Thus, hemodynamic signals in the amygdala and HPC can detect the different patterns of neuronal activity evoked by threatening vs. neutral stimuli during fear conditioning. Discrepant neuroimaging findings may be due to differences in experimental design and/or fear levels evoked in participants. Our methodology offers a way to improve translation between rodent models and human neuroimaging.

Targeting Aedes aegypti Metabolism with Next-Generation Insecticides.

Aedes aegypti is the primary vector of dengue virus (DENV), zika virus (ZIKV), and other emerging infectious diseases of concern. A key disease mitigation strategy is vector control, which relies heavily on the use of insecticides. The development of insecticide resistance poses a major threat to public health worldwide. Unfortunately, there is a limited number of chemical compounds available for vector control, and these chemicals can have off-target effects that harm invertebrate and vertebrate species. Fundamental basic science research is needed to identify novel molecular targets that can be exploited for vector control. Next-generation insecticides will have unique mechanisms of action that can be used in combination to limit selection of insecticide resistance. Further, molecular targets will be species-specific and limit off-target effects. Studies have shown that mosquitoes rely on key nutrients during multiple life cycle stages. Targeting metabolic pathways is a promising direction that can deprive mosquitoes of nutrition and interfere with development. Metabolic pathways are also important for the virus life cycle. Here, we review studies that reveal the importance of dietary and stored nutrients during mosquito development and infection and suggest strategies to identify next-generation insecticides with a focus on trehalase inhibitors.

SARS-CoV-2 wastewater monitoring in rural and small metropolitan communities in Central Michigan.

Central Michigan University (CMU) participated in a state-wide SARS-CoV-2 wastewater monitoring program throughout the 2021-2022 academic year. Wastewater samples were collected weekly from ten on-campus sites and nine off-campus wastewater treatment plants servicing small metropolitan and rural communities. SARS-CoV-2 genome copies were quantified using droplet digital PCR. Case data reported by Central Michigan District Health Department and CMU were collected and compared with wastewater data. During the delta wave, wastewater detection and on-campus case reports increased rapidly with the start of the academic semester and peaked quickly, compared with a more gradual and prolonged increase in detection and case reports off-campus. During the omicron wave, transmission dynamics were similar on-campus and off-campus. Normalization of on-campus and off-campus wastewater data with pepper mild mottle virus gene expression suggested lower SARS-CoV-2 shedding per person in on-campus compared to off-campus samples during the delta wave, but no difference in virus shedding during the omicron wave. We discuss the possibility that a higher on-campus vaccination rate may have reduced virus shedding per person during the delta wave, but that this effect was lost with the omicron variant. This study suggests that wastewater monitoring is effective in rural and small metropolitan communities when used in conjunction with case reports to understand regional transmission dynamics and the impact of public health policies at a public university on virus shedding in the community.

Changes in reward-related signals in the rat nucleus accumbens measured by in vivo oxygen amperometry are consistent with fMRI BOLD responses in man.

Real-time in vivo oxygen amperometry, a technique that allows measurement of regional brain tissue oxygen (O(2)) has been previously shown to bear relationship to the BOLD signal measured with functional magnetic resonance imaging (fMRI) protocols. In the present study, O(2) amperometry was applied to the study of reward processing in the rat nucleus accumbens to validate the technique with a behavioural process known to cause robust signals in human neuroimaging studies. After acquisition of a cued-lever pressing task a robust increase in O(2) tissue levels was observed in the nucleus accumbens specifically following a correct lever press to the rewarded cue. This O(2) signal was modulated by cue reversal but not lever reversal, by differences in reward magnitudes and by the motivational state of the animal consistent with previous reports of the role of the nucleus accumbens in both the anticipation and representation of reward value. Moreover, this modulation by reward value was related more to the expected incentive value rather than the hedonic value of reward, also consistent with previous reports of accumbens coding of "wanting" of reward. Altogether, these results show striking similarities to those obtained in human fMRI studies suggesting the use of oxygen amperometry as a valid surrogate for fMRI in animals performing cognitive tasks, and a powerful approach to bridge between different techniques of measurement of brain function.

Chronic depletion of vertebrate lipids in Aedes aegypti cells dysregulates lipid metabolism and inhibits innate immunity without altering dengue infectivity.

Aedes aegypti is the primary vector of dengue virus (DENV) and other arboviruses. Previous literature suggests that vertebrate and invertebrate lipids and the nutritional status of mosquitoes modify virus infection. Here, we developed a vertebrate lipid-depleted Ae. aegypti cell line to investigate if chronic depletion of vertebrate lipids normally present in a blood meal and insect cell culture medium would impact cell growth and virus infection. Chronic depletion of vertebrate lipids reduced cell size and proliferation, although cells retained equivalent total intracellular lipids per cell by reducing lipolysis and modifying gene expression related to sugar and lipid metabolism. Downregulation of innate immunity genes was also observed. We hypothesized that chronic depletion of vertebrate lipids would impact virus infection; however, the same amount of DENV was produced per cell. This study reveals how Ae. aegypti cells adapt in the absence of vertebrate lipids, and how DENV can replicate equally well in cells that contain predominately vertebrate or invertebrate lipids.

Adeno-associated virus type 2 infection activates caspase dependent and independent apoptosis in multiple breast cancer lines but not in normal mammary epithelial cells.

BACKGROUND: In normal cells proliferation and apoptosis are tightly regulated, whereas in tumor cells the balance is shifted in favor of increased proliferation and reduced apoptosis. Anticancer agents mediate tumor cell death via targeting multiple pathways of programmed cell death. We have reported that the non-pathogenic, tumor suppressive Adeno-Associated Virus Type 2 (AAV2) induces apoptosis in Human Papillomavirus (HPV) positive cervical cancer cells, but not in normal keratinocytes. In the current study, we examined the potential of AAV2 to inhibit proliferation of MCF-7 and MDA-MB-468 (both weakly invasive), as well as MDA-MB-231 (highly invasive) human breast cancer derived cell lines. As controls, we used normal human mammary epithelial cells (nHMECs) isolated from tissue biopsies of patients undergoing breast reduction surgery. RESULTS: AAV2 infected MCF-7 line underwent caspase-independent, and MDA-MB-468 and MDA-MB-231 cell lines underwent caspase-dependent apoptosis. Death of MDA-MB-468 cells was marked by caspase-9 activation, whereas death of MDA-MB-231 cells was marked by activation of both caspase-8 and caspase-9, and resembled a mixture of apoptotic and necrotic cell death. Cellular demise was correlated with the ability of AAV2 to productively infect and differentially express AAV2 non-structural proteins: Rep78, Rep68 and Rep40, dependent on the cell line. Cell death in the MCF-7 and MDA-MB-231 lines coincided with increased S phase entry, whereas the MDA-MB-468 cells increasingly entered into G2. AAV2 infection led to decreased cell viability which correlated with increased expression of proliferation markers c-Myc and Ki-67. In contrast, nHMECs that were infected with AAV2 failed to establish productive infection or undergo apoptosis. CONCLUSION: AAV2 regulated enrichment of cell cycle check-point functions in G1/S, S and G2 phases could create a favorable environment for Rep protein expression. Inherent Rep associated endonuclease activity and AAV2 genomic hair-pin ends have the potential to induce a cellular DNA damage response, which could act in tandem with c-Myc regulated/sensitized apoptosis induction. In contrast, failure of AAV2 to productively infect nHMECs could be clinically advantageous. Identifying the molecular mechanisms of AAV2 targeted cell cycle regulation of death inducing signals could be harnessed for developing novel therapeutics for weakly invasive as well as aggressive breast cancer types.

Downregulation of Cdc2/CDK1 kinase activity induces the synthesis of noninfectious human papillomavirus type 31b virions in organotypic tissues exposed to benzo[a]pyrene.

Epidemiological studies suggest that human papillomavirus (HPV)-infected women who smoke face an increased risk for developing cervical cancer. We have previously reported that exposure of HPV-positive organotypic cultures to benzo[a]pyrene (BaP), a major carcinogen in cigarette smoke, resulted in enhanced viral titers. Since BaP is known to deregulate multiple pathways of cellular proliferation, enhanced virion synthesis could result from carcinogen/host cell interaction. Here, we report that BaP-mediated upregulation of virus synthesis is correlated to an altered balance between cell cycle-specific cyclin-dependent kinase (CDK) activity profile compared with controls. Specifically, BaP treatment increased accumulation of hyperphosphorylated retinoblastoma protein (pRb) which coincided with increased cdc2/CDK1 kinase activity, but which further conflicted with the simultaneous upregulation of CDK inhibitors p16(INK4) and p27(KIP1), which normally mediate pRb hypophosphorylation. In contrast, p21(WAF1) and p53 levels remained unchanged. Under these conditions, CDK6 and CDK2 kinase activities were decreased, whereas CDK4 kinase activity remained unchanged. The addition of purvalanol A, a specific inhibitor of CDK1 kinase, to BaP-treated cultures, resulted in the production of noninfectious HPV type 31b (HPV31b) particles. In contrast, infectivity of control virus was unaffected by purvalanol A treatment. BaP targeting of CDK1 occurred independently of HPV status, since BaP treatment also increased CDK1 activity in tissues derived from primary keratinocytes. Our data indicate that HPV31b virions synthesized in the presence of BaP were dependent on BaP-mediated alteration in CDK1 kinase activity for maintaining their infectivity.