Aerosol Infection Research Articles

Pulmonary Mycobacterium tuberculosisinfection alters the hepatic immunometabolic landscape and compromises insulin responsiveness early after infection

Abstract Immunity against Mycobacterium tuberculosis(Mtb) is associated with both regional and systemic inflammation as well as with metabolic disruption in tuberculosis (TB) patients. The metabolic and immunological responses are intertwined but the specific impact of localized disease on systemic metabolism is not well defined. We investigated the impact of low dose aerosol infection of the lung with Mtbon the systemic metabolic regulatory organ – the liver. Using an unbiased approach we defined the mRNA and protein dysregulation occurring in the early stage of infection. We found that infection leads to immune pathway activation and reduced metabolic activity in liver. Comparison of the gene expression patterns between of the lung, liver and blood revealed organ specific metabolic gene dysregulation in the context of non-organ specific activation of interferon response pathways. We have also found that infection leads to suppression of carbohydrate and lipid metabolism, without alteration of fasting or random blood glucose levels. Infected mice show reduced sensitivity to exogenous insulin but the glucose tolerance remains intact indicating insulin resistance. Analysis of metabolomics data from public repository reveals a metabolic signature of insulin resistance correlates with TB progression. Taken together, our finding shows that aerosol Mtbinfection influences hepatic metabolism very soon after infection and may contribute to early metabolic disease. Defining the early interplay between localised infection, inflammation and metabolic regulation is key to understanding the impact of latent and subclinical TB on the large proportion of the World’s population who do not exhibit TB symptoms but who are infected with Mtb. Supported by Grants from Royal Society Wolfson Merit Award (AMC) Newton Fellowship (MKD), Wellcome Trust Institutional Support Fund Fellowship (MKD), MRC award MR/P011136/1 (AMC).

State of the Lung Microbiota in С57bl/6 Mice in the Experimental Tuberculosis Model

The objective: to study the changes in the lung microbiota in inbred C57BL/6 mice after aerogenic infection with M. tuberculosis in an experimental tuberculosis model.Subjects and Methods. This study was carried out on 20 female mice of inbred line C57BL/6 weighing 20-22 grams which were infected in a Glas-Col aerosol chamber (USA) with the culture of M. tuberculosis of virulent strain H37Rv at the dose of 400 CFU/lung. Morphological and microbiological assessment of the lungs state was performed before (day 0) (n=5) and 7 (n=5), 30 (n=5) and 60 (n=5) days after the infection. The results obtained were subjected to statistical processing using ANOVA test and Student t-test.Results. 7, 30, and 60 days after aerosol infection with M. tuberculosis against the background of successive morphological and microbiological changes typical of the experimental tuberculosis model, we established an imbalance of bacterial population in the lung microbiota. Before infection with M. tuberculosis, a scanty biotope was recorded with a predominance of lactobacilli –Lactobacillus murinus, Lactobacillus apodeme. 7, 30 and 60 days after infection with M. tuberculosis, consistent changes were recorded, such as increase in the number and diversity of the bacterial population. The most indicative markers of the recorded imbalance were: Streptococcus thoraltensis, Streptococcus acidominiminus, Arthrobacter crystallopoietes, Staphylococcus hominis, Micrococcus luteus.Conclusion. Tuberculosis infection is a significant factor affecting the state of the lung microbiota. With increased duration of the infection with M. tuberculosis, imbalance of the bacterial flora is formed in the lungs of C57BL/6 mice, accompanied by characteristic tissue inflammation and growing mycobacterial load.

International guidelines for controlling aerosol infections in dental services: which countries were prepared?

International guidelines for controlling aerosol infections in dental services: which countries were prepared?

A novel vaccine formulation candidate based on lipooligosaccharides and pertussis toxin against Bordetella pertussis.

Pertussis is a severe human respiratory tract infectious disease caused by Bordetella pertussis that primarily affects infants and young children. However, the acellular pertussis vaccine currently administered can induce antibody and Th2 immune responses but fails to prevent the nasal colonization and transmission of B. pertussis, causing a resurgence of pertussis, so improved pertussis vaccines are urgently needed. In this study, we created a two-component pertussis vaccine candidate containing a conjugate prepared from oligosaccharides and pertussis toxin. After demonstrating the ability of the vaccine to induce a mixed Th1/Th2/Th17 profile in a mouse model, the strong in vitro bactericidal activity and IgG response of the vaccine were further demonstrated. In addition, the vaccine candidate further induced efficient prophylactic effects against B. pertussis in a mouse aerosol infection model. In summary, the vaccine candidate in this paper induces antibodies with bactericidal activity to provide high protection, shorten the duration of bacterial existence, and further reduce disease outbreaks. Therefore, the vaccine has the potential to be the next generation of pertussis vaccines.

Quantitatively Visualizing Airborne Disease Transmission Risks of Different Exhalation Activities through CO2 Imaging.

Aerosol transmission has played a leading role in COVID-19 pandemic. However, there is still a poor understanding about how it is transmitted. This work was designed to study the exhaled breath flow dynamics and transmission risks under different exhaling modes. Using an infrared photography device, exhaled flow characteristics of different breathing activities, such as deep breathing, dry coughing, and laughing, together with the roles of mouth and nose were characterized by imaging CO2 flow morphologies. Both mouth and nose played an important role in the disease transmission though in the downward direction for the nose. In contrast to the trajectory commonly modeled, the exhaled airflows appeared with turbulent entrainments and obvious irregular movements, particularly the exhalations involving mouth were directed horizontal and had a higher propagation capacity and transmission risk. While the cumulative risk was high for deep breathing, those transient ones from dry coughing, yawning, and laughing were also shown to be significant. Various protective measures including masks, canteen table shields, and wearable devices were visually demonstrated to be effective for altering the exhaled flow directions. This work is useful to understanding the risk of aerosol infection and guiding the formulation of its prevention and control strategies. Experimental data also provide important information for refining model boundary conditions.

Assessing Indoor Air Quality and Ventilation to Limit Aerosol Dispersion—Literature Review

The COVID-19 pandemic highlighted the importance of indoor air quality (IAQ) and ventilation, which researchers have been warning about for years. During the pandemic, researchers studied several indicators using different approaches to assess IAQ and diverse ventilation systems in indoor spaces. To provide an overview of these indicators and approaches in the case of airborne transmission through aerosols, we conducted a literature review, which covered studies both from before and during the COVID-19 pandemic. We searched online databases for six concepts: aerosol dispersion, ventilation, air quality, schools or offices, indicators, and assessment approaches. The indicators found in the literature can be divided into three categories: dose-, building-, and occupant-related indicators. These indicators can be measured in real physical spaces, in a controlled laboratory, or modeled and analyzed using numerical approaches. Rather than organizing this paper according to these approaches, the assessment methods used are grouped according to the following themes they cover: aerosol dispersion, ventilation, infection risk, design parameters, and human behavior. The first finding of the review is that dose-related indicators are the predominant indicators used in the selected studies, whereas building- and occupant-related indicators are only used in specific studies. Moreover, for a better understanding of airborne transmission, there is a need for a more holistic definition of IAQ indicators. The second finding is that although different design assessment tools and setups are presented in the literature, an optimization tool for a room’s design parameters seems to be missing. Finally, to efficiently limit aerosol dispersion in indoor spaces, better coordination between different fields is needed.

Severe fever with thrombocytopenia syndrome virus aerosol infection in C57/BL6 mice

Although secondary cases have become infected with the SFTSV after being in the same space without direct contact with the index case, it has not been experimentally determined if the SFTSV can be transmitted through aerosols. Here, this study aimed to verify if the SFTSV could be transmitted by aerosols. Firstly, we demonstrated that the SFTSV can infect BEAS-2B cells, and SFTSV genomes can be isolate from mild patient's sputum, which provided a foundation for the existence of SFTSV aerosol transmission. Then, we evaluated total antibody production in serum and viral load in tissue of mice infected with SFTSV by aerosols. The results showed that the presence of antibodies is related to the dose of virus infection and the SFTSV preferentially replicates in the lungs of mice following an aerosol exposure. Our study will help update the prevention and treatment guidelines for SFTSV and prevent the spread of the SFTSV in hospitals.

Differential Requirement for IRGM Proteins during Tuberculosis Infection in Mice.

Mycobacterium tuberculosis (Mtb) is a bacterium that exclusively resides in human hosts and remains a dominant cause of morbidity and mortality among infectious diseases worldwide. Host protection against Mtb infection is dependent on the function of immunity-related GTPase clade M (IRGM) proteins. Polymorphisms in human IRGM associate with altered susceptibility to mycobacterial disease, and human IRGM promotes the delivery of Mtb into degradative autolysosomes. Among the three murine IRGM orthologs, Irgm1 has been singled out as essential for host protection during Mtb infections in cultured macrophages and in vivo. However, whether the paralogous murine Irgm genes, Irgm2 and Irgm3, play roles in host defense against Mtb or exhibit functional relationships with Irgm1 during Mtb infection remains undetermined. Here, we report that Irgm1-/- mice are indeed acutely susceptible to aerosol infection with Mtb, yet the additional deletion of the paralogous Irgm3 gene restores protective immunity to Mtb infections in Irgm1-deficient animals. Mice lacking all three Irgm genes (panIrgm-/-) are characterized by shifted lung cytokine profiles at 5 and 24 weeks postinfection, but control disease until the very late stages of the infection, when panIrgm-/- mice display increased mortality compared to wild-type mice. Collectively, our data demonstrate that disruptions in the balance between Irgm isoforms is more detrimental to the Mtb-infected host than total loss of Irgm-mediated host defense, a concept that also needs to be considered in the context of human Mtb susceptibility linked to IRGM polymorphisms.

Ventilation Methods against Indoor Aerosol Infection of COVID-19 in Japan

The importance of effective ventilation as one of the measures against COVID-19 is widely recognized worldwide. In Japan, at the early stage of the pandemic, in March 2020, an official announcement was made about basic ventilation measures against COVID-19. WHO also used the term “long-range aerosol or long-range airborne transmission” for the first time in December 2021. Based on the aerosol infection control measures before 2021 by the Japanese government, we conducted experiments on methods related to partition placement as an element of effective ventilation methods. In July 2022, the governmental subcommittee on Novel Coronavirus Disease Control provided an emergent proposal about effective ventilation methods to prevent two types of aerosol infection; infection by large aerosol on the air current and infection by small floating aerosol diffusion in a room. They also showed the way of setting droplet prevention partitions, which do not block off ventilation based on this investigation’s results.

In Vitro and In Vivo Phenotypes of Venezuelan, Eastern and Western Equine Encephalitis Viruses Derived from cDNA Clones of Human Isolates.

The Department of Defense recently began an effort to improve and standardize virus challenge materials and efficacy determination strategies for testing therapeutics and vaccines. This includes stabilization of virus genome sequences in cDNA form where appropriate, use of human-derived virus isolates, and noninvasive strategies for determination of challenge virus replication. Eventually, it is desired that these approaches will satisfy the FDA "Animal Rule" for licensure, which substitutes animal efficacy data when human data are unlikely to be available. To this end, we created and examined the virulence phenotype of cDNA clones of prototypic human infection-derived strains of the alphaviruses, Venezuelan (VEEV INH9813), eastern (EEEV V105) and western (WEEV Fleming) equine encephalitis viruses, and created fluorescent and luminescent reporter expression vectors for evaluation of replication characteristics in vitro and in vivo. Sequences of minimally passaged isolates of each virus were used to synthesize full-length cDNA clones along with a T7 transcription promoter-based bacterial propagation vector. Viruses generated from the cDNA clones were compared with other "wild type" strains derived from cDNA clones and GenBank sequences to identify and eliminate putative tissue culture artifacts accumulated in the cell passaged biological stocks. This was followed by examination of aerosol and subcutaneous infection and disease in mouse models. A mutation that increased heparan sulfate binding was identified in the VEEV INH9813 biological isolate sequence and eliminated from the cDNA clone. Viruses derived from the new human isolate cDNA clones showed similar mouse virulence to existing clone-derived viruses after aerosol or subcutaneous inoculation.

Ventilation improvement and evaluation of its effectiveness in a Japanese manufacturing factory

A coronavirus disease 2019 (COVID-19) cluster emerged in a manufacturing factory in early August 2021. In November 2021, we conducted a ventilation survey using the tracer gas method. Firstly, we reproduce the situation at the time of cluster emergence and examined whether the ventilation in the office was in a condition that increased the risk of aerosol transmission. Secondly, we verified the effectiveness of the factory’s own countermeasure implemented immediately after the August cluster outbreak. Furthermore, we verified the effectiveness of several additional improvement measures on the factory’s own countermeasures already installed in August. Under the conditions of the cluster emergence, the air changes per hour (ACH) value was 0.73 ACH on average. The ACH value was less than 2 ACH recommended by the Ministry of Health, Labour, and Welfare, suggesting an increased risk of aerosol transmission. The factory’s own countermeasures taken immediately in August were found to be effective, as the ACH value increased to 3.41 ACH on average. Moreover, it was confirmed that additional improvement measures on the factory’s own countermeasures increased the ACH value to 8.33 ACH on average. In order to prevent the re-emergence of COVID-19 clusters due to aerosol infection in the office, it was found that while continuing the factory’s own countermeasure, additional improvement measures should also be added depending on the number of workers in the room. In a company, it is important that workers themselves continue to take infection control measures autonomously, and confirming the effectiveness of the measures will help maintain workers’ motivation. We believe it is helpful that external researchers in multiple fields and internal personnel in charge of the health and safety department and occupational health work together to confirm the effectiveness of conducted measures, such as in this case.

Targeted dose delivery of Mycobacterium tuberculosis in mice using silicon antifoaming agent via aerosol exposure system.

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that forms aggregates (clumps) on solid agar plates and in liquid media. Detergents such as Tween 80/Tyloxapol are considered the gold standard to disrupt clump formation in Mtb cultures. The presence of detergent, however, may generate foam and hinder Mtb aerosolization thus requiring addition of an antifoam agent for optimal Mtb aerosol-based procedures. Aerosol inhalation can be technically challenging, in particular to achieve a reproducible inhaled target dose. In this study, the impact of an antifoam, the silicon antifoaming agent (SAF), on Mtb aerosolization and whole-body mouse aerosol infection was investigated. A comparative study using SAF in a liquid suspension containing Mycobacterium bovis BCG (M. bovis BCG) or Mtb H37Rv did not cause any adverse effect on bacterial viability. Incorporation of SAF during mycobacteria inhalation procedures revealed that aerosolized mycobacterial strains were maintained under controlled environmental conditions such as humidity, temperature, pressure, and airflow inside the aerosol chamber. In addition, environmental factors and spray factors were not affected by the presence of SAF in mycobacterial cultures during aerosolization. Spray factor was significantly less during aerosol procedures with a low-input dose of mycobacteria in comparison to high-dose, as predicted. The mycobacterial load recovered in the biosampler (AGI) was ~2–3 logs lower than nebulizer or input bacterial load. A consistent Mtb bacillary load determined in mouse lungs indicates that SAF does not affect mycobacteria aerosolization during the aerosol generation process. These data confirmed that 1) SAF prevents formation of excessive foam during aerosolization, 2) SAF had no negative impact on mycobacterial viability within aerosol droplets, 3) Mtb droplets within aerosol-generated particles are well within the range required for reaching and depositing deep into lung tissue, and 4) SAF had no negative impact on achieving a target dose in mice exposed to Mtb aerosol.

Development of the Photocatalytic Bioaerosol Elimination System Specialized in the Control of Aerosol Infection such as COVID-19

Development of the Photocatalytic Bioaerosol Elimination System Specialized in the Control of Aerosol Infection such as COVID-19

Development and evaluation of a fluidic facemask for airborne transmission mitigation

Recently, a fluidic facemask concept was proposed to mitigate the transmission of virus-laden aerosol and droplet infections, such as SARS-CoV-2 (COVID-19). This paper describes an experimental investigation of the first practical fluidic facemask prototype, or “Air-Screen”. It employs a small, high-aspect-ratio, crossflow fan mounted on the visor of a filter-covered cap to produce a rectangular air jet, or screen, in front of the wearer’s face. The entire assembly weighs less than 200 g. Qualitative flow visualization experiments using a mannequin clearly illustrated the Air-Screen’s ability to effectively block airborne droplets (∼100 µm) from the wearer’s face. Quantitative experiments to simulate droplets produced during sneezing or a wet cough (∼102 µm) were propelled (via a transmitter) at an average velocity of 50 m/s at 1 m from the mannequin or a target. The Air-Screen blocked 62% of all droplets with a diameter of less than 150 µm. With an Air-Screen active on the transmitter, 99% of all droplets were blocked. When both mannequin and transmitter Air-Screens were active, 99.8% of all droplets were blocked. A mathematical model, based on a weakly-advected jet in a crossflow, was employed to gain greater insight into the experimental results. This investigation highlighted the remarkable blocking effect of the Air-Screen and serves as a basis for a more detailed and comprehensive experimental evaluation.

The Influence of Plastic Barriers on Aerosol Infection Risk during Airport Security Checks

Plastic barriers physically separate queuing passengers in airport security check areas as a measure against aerosol transmission. However, this may create “canyons” that interfere with the existing ventilation design: potentially inhibiting airflow, concentrating exhaled viruses, and exacerbating aerosol transmission risk. Accordingly, this study investigated the transmission implications of installing plastic barriers in a security check area with computational fluid dynamics (CFD). Two air distribution schemes were modeled: one with linear air supply diffusers aligned vertically to (Case 1) and another with diffusers parallel with (Case 2) the orientation of partitions. The drift-flux model was used to calculate the spread of viral bioaerosols with 5 µm in diameter; then the Wells–Riley equation was applied to assess aerosol transmission risk for SARS-CoV-2. According to simulation results, in Case 1, installing plastic barriers resulted in relatively small changes in volume with a high infection risk of 1% or greater in the breathing zone within the first 25 min. However, in Case 2, using plastic barriers resulted in the continuous increase in this volume within the first 25 min while this volume was near zero if without plastic barriers. In conclusion, installing plastic barriers needs careful consideration because they do not reduce the risk of airborne SARS-CoV-2 transmission and might even exacerbate it without localized ventilation and air cleaning.

Nebulization as a more efficient method than atomizer for experimental reproduction of avian colibacillosis in young chickens

ABSTRACT Infection and immunity studies involving genetically modified organisms (GMOs), such as gene knockout bacterial mutants require stringent physical containment to prevent the accidental spread of these organisms into the environment. Experimental respiratory tract infection models often require the animals, for example birds, to be transported several times between a negative pressure housing isolator and a bespoke aerosol exposure chamber under positive pressure. While the exposure chamber is sealed and fitted with HEPA filters, the repeated movements of infected animals and opening of the chamber can still pose a serious risk of breaching containment of the organism in the experimental facility. In the current study, the ability of two aerosol infection protocols that expose birds to avian pathogenic E. coli (APEC) aerosols directly within the housing isolator was evaluated. Young chicks were exposed to APEC E956 within the negative pressure housing isolators using either a nebulizer or an atomizer. Birds exposed twice (days 1 and 4) to aerosols of APEC E956 produced by the nebulizer developed a rapidly progressing disease mimicking field cases of avian colibacillosis. However, birds exposed to aerosols of APEC E956 produced by an atomizer, did not develop colibacillosis even after 3 exposures to APEC E956 on days 1, 4 and 7. Consequently, the current study reports the nebulizer was more efficacious in producing avian colibacillosis under stricter bacterial containment settings.

Research Advances for Virus-vectored Tuberculosis Vaccines and Latest Findings on Tuberculosis Vaccine Development.

Tuberculosis (TB), caused by respiratory infection with Mycobacterium tuberculosis, remains a major global health threat. The only licensed TB vaccine, the one-hundred-year-old Bacille Calmette-Guérin has variable efficacy and often provides poor protection against adult pulmonary TB, the transmissible form of the disease. Thus, the lack of an optimal TB vaccine is one of the key barriers to TB control. Recently, the development of highly efficacious COVID-19 vaccines within one year accelerated the vaccine development process in human use, with the notable example of mRNA vaccines and adenovirus-vectored vaccines, and increased the public acceptance of the concept of the controlled human challenge model. In the TB vaccine field, recent progress also facilitated the deployment of an effective TB vaccine. In this review, we provide an update on the current virus-vectored TB vaccine pipeline and summarize the latest findings that might facilitate TB vaccine development. In detail, on the one hand, we provide a systematic literature review of the virus-vectored TB vaccines are in clinical trials, and other promising candidate vaccines at an earlier stage of development are being evaluated in preclinical animal models. These research sharply increase the likelihood of finding a more effective TB vaccine in the near future. On the other hand, we provide an update on the latest tools and concept that facilitating TB vaccine research development. We propose that a pre-requisite for successful development may be a better understanding of both the lung-resident memory T cell-mediated mucosal immunity and the trained immunity of phagocytic cells. Such knowledge could reveal novel targets and result in the innovative vaccine designs that may be needed for a quantum leap forward in vaccine efficacy. We also summarized the research on controlled human infection and ultra-low-dose aerosol infection murine models, which may provide more realistic assessments of vaccine utility at earlier stages. In addition, we believe that the success in the ongoing efforts to identify correlates of protection would be a game-changer for streamlining the triage of multiple next-generation TB vaccine candidates. Thus, with more advanced knowledge of TB vaccine research, we remain hopeful that a more effective TB vaccine will eventually be developed in the near future.

Impact of ventilation and avoidance measures on SARS-CoV-2 risk of infection in public indoor environments

BackgroundThe literature includes many studies which individually assess the efficacy of protective measures against the spread of the SARS-CoV-2 virus. This study considers the high infection risk in public buildings and models the quality of the indoor environment, related safety measures, and their efficacy in preventing the spread of the SARS-CoV-2 virus. MethodsSimulations are created that consider protective factors such as hand hygiene, face covering and engagement with Covid-19 vaccination programs in reducing the risk of infection in a university foyer. Furthermore, a computational fluid dynamics model is developed to simulate and analyse the university foyer under three ventilation regimes. The probability of transmission was measured across different scenarios. FindingsEstimates suggest that the Delta variant requires the air change rate to be increased >1000 times compared to the original strain, which is practically not feasible. Consequently, appropriate hygiene practices, such as wearing masks, are essential to reducing secondary infections. A comparison of different protective factors in simulations found the overall burden of infections resulting from indoor contact depends on (i) face mask adherence, (ii) quality of the ventilation system, and (iii) other hygiene practices. InterpretationRelying on ventilation, whether natural, mechanical, or mixed, is not sufficient alone to mitigate the risk of aerosol infections. This is due to the internal configuration of the indoor space in terms of (i) size and number of windows, their location and opening frequency, as well as the position of the air extraction and supply inlets, which often induce hotspots with stagnating air, (ii) the excessive required air change rate. Hence, strict reliance on proper hygiene practices, namely adherence to face coverings and hand sanitising, are essential. Consequently, face mask adherence should be emphasized and promoted by policymakers for public health applications. Similar research may need to be conducted using a similar approach on the Omicron (B.1.1.529) variant.

MAVS mediates a protective immune response in the brain to Rift Valley fever virus.

Rift Valley fever virus (RVFV) is a highly pathogenic mosquito-borne virus capable of causing hepatitis, encephalitis, blindness, hemorrhagic syndrome, and death in humans and livestock. Upon aerosol infection with RVFV, the brain is a major site of viral replication and tissue damage, yet pathogenesis in this organ has been understudied. Here, we investigated the immune response in the brain of RVFV infected mice. In response to infection, microglia initiated robust transcriptional upregulation of antiviral immune genes, as well as increased levels of activation markers and cytokine secretion that is dependent on mitochondrial antiviral-signaling protein (MAVS) and independent of toll-like receptors 3 and 7. In vivo, Mavs-/- mice displayed enhanced susceptibility to RVFV as determined by increased brain viral burden and higher mortality. Single-cell RNA sequence analysis identified defects in type I interferon and interferon responsive gene expression within microglia in Mavs-/- mice, as well as dysregulated lymphocyte infiltration. The results of this study provide a crucial step towards understanding the precise molecular mechanisms by which RVFV infection is controlled in the brain and will help inform the development of vaccines and antiviral therapies that are effective in preventing encephalitis.

Tradeoffs between ventilation, air mixing, and passenger density for the airborne transmission risk in airport transportation vehicles

Airport transportation vehicles, such as buses, aerotrains, and shuttles, provide important passenger transfer services in airports. This study quantitatively investigated COVID-19 aerosol infection risk and identified acceptable operational conditions, such as passenger occupancy rates and duration of rides, given the performance of vehicle ventilation. The greatest risk to the largest number of passengers is from an index case whose exhaled breath would take the longest time to exit the vehicle. The study identified such a case based on ventilation patterns, and it tracked the spread of viral aerosols (5 μm) by using the Wells-Riley equation to predict aerosol infection risk distribution. These distributions allowed a definition of an imperfect mixing degree (δ) as the ratio of actual risk and the calculated risk under a perfect mixing condition, and further derived regression equations to predict δ in the far-field (FF) and near-field (NF) of each passenger. These results revealed an order of magnitude higher aerosol infection risk in NF than in FF. For example, with a ventilation rate of 58 ACH (air changes per hour) and a 45% occupancy rate, unmasked passengers should stay up to 15 min in the bus and 35 min in the shuttle to limit infection risk in NF within 10%. These also indicate that masking is an important and effective risk reduction measure in transportation vehicles, especially important in NF. Overall, the analysis of imperfect air mixing allows direct comparison of risks in different transportation vehicles and a structured approach to development of policy recommendations.