Biology subjects

Patel, D. R.

Publications and source records attributed to Patel, D. R..

7 recordsLinked to original sources

bioRxiv2026

A c-di-AMP-controlled glutamine synthesis pathway promotes persistence of Staphylococcus aureus thymidine-dependent small colony variants in the lung

Children with cystic fibrosis (CF) commonly harbor Staphylococcus aureus thymidine-dependent small-colony variants (TD-SCVs), which are associated with reduced lung function and increased respiratory exacerbations. How TD-SCVs survive in the thymidine-limited CF lung is unknown. Here, we show that TD-SCVs exhibit impaired glutamine uptake and depend on c-di-AMP-regulated de novo glutamine synthesis for survival in the murine lung. We found that transcription of the glutamine synthetase gene glnA is cooperatively repressed by the transcriptional regulator GlnR, the c-di-AMP-binding protein PstA, and GlnA itself. Glutamine starvation elevates c-di-AMP levels, relieving repression by this ternary complex and promoting glutamine synthesis. Reducing c-di-AMP levels causes a profound growth defect in TD-SCVs under low-thymidine conditions, which is rescued by glnA overexpression. Moreover, pharmacological inhibition of GlnA markedly impairs TD-SCV growth in murine lung. These findings elucidate the molecular mechanism underlying S. aureus TD-SCV survival during infection and identify glutamine synthesis as a promising therapeutic target for treating infections caused by antifolate-resistant bacteria.

microbiology↗

bioRxiv2025

Quantifying viral pandemic potential from experimental transmission studies

In an effort to avert future pandemics, surveillance studies aimed at identifying zoonotic viruses at high risk of spilling over into humans act to monitor the viral chatter at the animal-human interface. These studies are hampered, however, by the diversity of zoonotic viruses and the limited tools available to assess pandemic risk. Methods currently in use include the characterization of candidate viruses using in vitro laboratory assays and experimental transmission studies in animal models. However, transmission experiments yield relatively low-resolution outputs that are not immediately translatable to projections of viral dynamics at the level of a host population. To address this gap, we present an analytical framework to extend the use of measurements from experimental transmission studies to generate more quantitative risk assessments. Specifically, we use within-host viral titer data from index and contact animals to estimate parameters relevant to transmission, including an estimate of transmissibility. We then extended this model to estimate epidemiological parameters, such as the basic reproductive number and generation interval. To illustrate these approaches, we present them in the context of two influenza A virus (IAV) ferret transmission experiments: one using influenza A/California/07/2009 (Cal/2009) and one using influenza A/Hong Kong/1/1968 (Hong Kong/1968). Despite estimating broadly similar transmissibilities for Cal/2009 and Hong Kong/1968, we conclude that Cal/2009 has a higher pandemic potential. This difference in pandemic potential seems to be primarily driven by its more robust within-host replication. Our results critically depend on several assumptions, namely that the within-host viral dynamics in humans and those in the model animal used (here, ferrets) share quantitative similarities and that viral transmissibility between model animals reflects viral transmissibility between humans. The methods we present to assess relative pandemic risk across viral isolates can be used to improve quantitative risk assessment of other emerging viruses of pandemic concern. Author summaryPandemic viruses often originate in animal reservoirs. Surveillance studies at the animal-human interface try to identify which of the many viruses circulating in animals could cause a pandemic in humans. Experimental transmission studies in animal models are one such method used to characterize potential pandemic viruses. However, only simple outcomes usually get reported from these experiments, such as the fraction of animals that become infected in such an experiment. Here, we develop a framework to extract more information from these experiments. Specifically, we use data on the virus population within infected ferrets to extrapolate information about transmission. We then simulate many transmission events to estimate epidemiological parameters commonly used in public health to quantify pandemic potential. Our methods therefore allow us to use commonly gathered data from experimental transmission studies to estimate epidemic dynamics in humans. This set of methods can be used to compare the relative pandemic risk of animal viruses before they emerge in humans.

microbiology↗

bioRxiv2025

Defining the transmissible dose 50%, the donor inoculation dose that results in airborne transmission to 50% of contacts, for two pandemic influenza viruses in ferrets

Ferrets are widely used to model airborne transmission of influenza viruses in humans. Airborne transmission is evaluated by infecting donor ferrets with a high virus dose (106 infectious units) and monitoring transmission to contact animals sharing the same airspace. However, humans can be infected with a broad range of influenza virus doses. Therefore, we evaluated the relationship between virus inoculation dose and transmission for two pandemic influenza viruses in ferrets. Donor ferrets were inoculated with 100 to 106 tissue culture infectious dose 50 (TCID50) of the 2009 pandemic H1N1 or 1968 H3N2 pandemic virus, and were then paired with respiratory contacts. Using the proportion of donors that became infected across virus doses, we calculated the infectious dose 50 (ID50). Subsequently, by comparing the proportion of respiratory contacts that became infected, we calculated the transmissible dose 50% (TD50): the donor inoculation dose that resulted in transmission to 50% of contacts. For the 2009 H1N1 virus, the ID50 and TD50 were equivalent at <1 TCID50. However, for the 1968 H3N2 virus, the ID50 and TD50 were <4 and 103.49 TCID50, respectively. The increased TD50 for the H3N2 virus was associated with reductions in peak viral titers and viral shedding in donors over decreasing virus inoculation doses. Collectively, these studies define a new measure of transmission that permits comparisons of transmissibility over a log scale. Using this metric, we show the 1968 pandemic H3N2 virus has reduced transmissibility in ferrets relative to the 2009 pandemic H1N1 virus. ImportanceFerrets are the gold standard animal model used to assess the transmissibility of influenza viruses. Airborne transmission is evaluated by infecting donor ferrets with a high virus dose and monitoring transmission to contact animals sharing the same airspace. However, the relationship between inoculation dose and transmission has not been evaluated in ferrets. Therefore, we performed studies evaluating airborne transmission of the 2009 pandemic H1N1 and 1968 pandemic H3N2 viruses over log scale reductions in donor inoculation doses. Using the results of these studies, we define a new measure of transmission, the transmissible dose 50%, which is the donor inoculation dose at which a virus is transmitted to 50% of contacts. Importantly, this metric permits the evaluation of transmissibility over a 10-100 fold scale, and using this metric, we demonstrate the 1968 pandemic H3N2 virus has reduced transmissibility compared to the 2009 pandemic H1N1 virus in ferrets.

microbiology↗

bioRxiv2022

Intranasal virus-particle mimicking vaccine enhances SARS-CoV-2 clearance in the Syrian hamster model

Multiple vaccines have been developed and licensed for SARS-CoV-2. While these vaccines reduce disease severity, they do not prevent infection, and SARS-CoV-2 continues to spread and evolve. To prevent infection and limit transmission, vaccines must be developed that induce immunity in the respiratory tract. Therefore, we performed proof-of-principle vaccination studies with an intranasal nanoparticle vaccine against SARS-CoV-2. The vaccine candidate consisted of the self-assembling 60-subunit I3-01 protein scaffold covalently decorated with the SARS-CoV-2 receptor binding domain (RBD) using the SpyCatcher-SpyTag system. We verified the intended antigen display features by reconstructing the I3-01 scaffold to 3.4A using cryo-EM, and then demonstrated that the scaffold was highly saturated when grafted with RBD. Using this RBD-grafted SpyCage scaffold (RBD+SpyCage), we performed two unadjuvanted intranasal vaccination studies in the "gold-standard" preclinical Syrian hamster model. Hamsters received two vaccinations 28 days apart, and were then challenged 28 days post-boost with SARS-CoV-2. The initial study focused on assessing the immunogenicity of RBD+SpyCage, which indicated that vaccination of hamsters induced a non-neutralizing antibody response that enhanced viral clearance but did not prevent infection. In an expanded study, we demonstrated that covalent bonding of RBD to the scaffold was required to induce an antibody response. Consistent with the initial study, animals vaccinated with RBD+SpyCage more rapidly cleared SARS-CoV-2 from both the upper and lower respiratory tract. These findings demonstrate the intranasal SpyCage vaccine platform can induce protection against SARS-CoV-2 and, with additional modifications to improve immunogenicity, is a versatile platform for the development of intranasal vaccines targeting respiratory pathogens. IMPORTANCEDespite the availability of efficacious COVID vaccines that reduce disease severity, SARS-CoV-2 continues to spread. To limit SARS-CoV-2 transmission, the next generation of vaccines must induce immunity in the mucosa of the upper respiratory tract. Therefore, we performed proof-of-principle, unadjuvanted intranasal vaccination studies with a recombinant protein nanoparticle scaffold, SpyCage, decorated with the receptor-binding domain (RBD) of the S protein (SpyCage+RBD). We show that SpyCage+RBD was immunogenic and enhanced SARS-CoV-2 clearance from the nose and lungs of Syrian hamsters. Moreover, covalent grafting of the RBD to the scaffold was required to induce an immune response when given via the intranasal route. These proof-of-concept findings indicate that with further enhancements to immunogenicity (e.g., adjuvant incorporation, antigen optimization), the SpyCage scaffold has potential as a versatile, intranasal vaccine platform for respiratory pathogens.

immunology↗

bioRxiv2022

Vitamin D and the ability to produce 1,25(OH)2D are critical for protection from viral infection of the lungs.

Vitamin D supplementation has been linked to improved outcomes from respiratory virus infection, and the COVID19 pandemic has renewed interest in understanding the potential role of vitamin D in protecting the lung from viral infections. Therefore, we evaluated the role of Vitamin D using animal models of pandemic H1N1 influenza and SARS-CoV-2 infection. In mice, dietary induced vitamin D deficiency resulted in lung inflammation that was present prior to infection. Vitamin D sufficient (D+) and deficient (D-) wildtype (WT) and D+ and D-Cyp27B1 (Cyp) knockout (KO, cannot produce 1,25(OH)2D) mice were infected with pandemic H1N1. D- WT, D+ Cyp KO, and D- Cyp KO mice all exhibited significantly reduced survival compared to D+ WT mice. Importantly, survival was not the result of reduced viral replication as influenza M gene expression in the lungs was similar for all animals. Based on these findings, additional experiments were performed using the mouse and hamster models of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. In these studies, high dose vitamin D supplementation reduced lung inflammation in mice but not hamsters. A trend to faster weight recovery was observed in 1,25(OH)2D treated mice that survived SARS-CoV-2 infection. There was no effect of vitamin D on SARS-CoV-2 N gene expression in the lung of either mice or hamsters. Therefore, vitamin D deficiency enhanced disease severity, while vitamin D sufficient/supplementation reduced inflammation following infections with H1N1 influenza and SARS-CoV-2.

immunology↗

bioRxiv2022

Robustness of the ferret model for influenza risk assessment studies: a cross-laboratory exercise

Ferrets represent the preferred animal model for assessing the transmission potential of newly emerged zoonotic influenza viruses. However, heterogeneity among established experimental protocols and facilities across different laboratories may lead to variable results, complicating interpretation of transmission experimental data. Between 2018-2020, a global exercise was conducted by 11 participating laboratories to assess the range of variation in ferret transmission experiments using two common stock H1N1 influenza viruses that possess different transmission characteristics in ferrets. Inoculation route, dose, and volume were standardized, and all participating laboratories followed the same experimental conditions for respiratory droplet transmission, including a strict 1:1 donor:contact ratio. Additional host and environmental parameters likely to affect influenza transmission kinetics were monitored throughout. Overall transmission outcomes for both viruses across 11 laboratories were concordant, suggesting the robustness of the ferret model for zoonotic influenza risk assessment. To attain high confidence in identifying zoonotic influenza viruses with moderate-to-high or low transmissibility, our analyses support that as few as three but as many as five laboratories, respectively, would need to independently perform viral transmission experiments with concordant results. This exercise facilitates the development of a more homogenous protocol for ferret transmission experiments that are employed for the purposes of risk assessment.

microbiology↗

bioRxiv2020

Transmission and protection against re-infection in the ferret model with the SARS-CoV-2 USA-WA1/2020 reference isolate

SARS-CoV-2 has initiated a global pandemic and vaccines are being rapidly developed. Using the reference strain SARS-CoV-2 USA-WA1/2020, we evaluated modes of transmission and the ability of prior infection or vaccine-induced immunity to protect against infection in ferrets. Ferrets were semi-permissive to infection with the USA-WA1/2020 isolate. When transmission was assessed via the detection of vRNA at multiple timepoints, direct contact transmission was efficient to 3/3 and 3/4 contact animals in two respective studies, while respiratory transmission was poor to only 1/4 contact animals. To assess the durability of immunity, ferrets were re-challenged 28 or 56 days post-primary infection. Following viral challenge, no infectious virus was recovered in nasal wash samples. In addition, levels of vRNA in the nasal wash were several orders of magnitude lower than during primary infection, and vRNA was rapidly cleared. To determine if intramuscular vaccination protected ferrets against infection, ferrets were vaccinated using a prime-boost strategy with the S-protein receptor-binding domain formulated with an oil-in-water adjuvant. Upon viral challenge, none of the mock or vaccinated animals were protected against infection, and there were no significant differences in vRNA or infectious virus titers in the nasal wash. Combined these studies demonstrate that in ferrets direct contact is the predominant mode of transmission of the SARS-CoV-2 USA-WA1/2020 isolate and immunity to SARS-CoV-2 is maintained for at least 56 days. Our studies also indicate protection of the upper respiratory tract against SARS-CoV-2 will require vaccine strategies that mimic natural infection or induce site-specific immunity. ImportanceThe SARS-CoV-2 USA-WA1/2020 strain is a CDC reference strain used by multiple research laboratories. Here, we show the predominant mode of transmission of this isolate in ferrets is by direct contact. We further demonstrate ferrets are protected against re-infection for at least 56 days even when levels of neutralizing antibodies are low or undetectable. Last, we show that when ferrets were vaccinated by the intramuscular route to induce antibodies against SARS-CoV-2, ferrets remain susceptible to infection of the upper respiratory tract. Collectively, these studies suggest protection of the upper respiratory tract will require vaccine approaches that mimic natural infection.

microbiology↗