Browsing by Subject "Influenza"
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Item Analysis Of Regulatory T Cells With An Interferon Gene Signature During Influenza Infection(2023) Ernest, JordanRegulatory T cells are antigen-specific immune suppressors that can be found in circulation or residing in tissues or secondary lymphoid organs. In an effort to characterize Tregs that arise in response to influenza and are found in the lung, the Farrar lab recently performed single-cell RNA sequencing on murine Tregs isolated at various time points throughout infection from the lung. We hypothesized that different subsets of lung Tregs induced by influenza would play unique roles in either suppressing immunity or in resolving inflammation. As part of this thesis, I have carried out an independent analysis of this dataset investigating both the diversity and kinetics of Tregs over the course of infection. Utilizing a series of mouse models to selectively deplete either all Tregs or specific Treg subsets at various times, my data suggests that depletion of Tregs starting at day 6 post infection may lead to the expansion of influenza-specific IgA+ antibody secreting cells in the lungs. I attempted to determine the contribution of Type 1 interferon stimulated Tregs to the influenza immune response. However, my results ultimately demonstrated that the existing Mx1-Cre transgenic mouse line exhibits variegated expression of Cre-recombinase, and thus cannot be used to accurately mark and deplete all ISG-Tregs. These results give insight into the trafficking of specific Treg subsets during flu infection, and highlight technical hurdles to be overcome if we wish to continue to study Type 1 interferon stimulated Tregs.Item Avian influenza in suphanburi province, Thailand: assessment of transmission dynamics and interventions in the local poultry sector.(2012-06) Beaudoin, Amanda LeighThis dissertation provides a review of avian influenza, with specific regard to highly pathogenic avian influenza (HPAI) H5N1, a description of the Thai poultry system and the country's experiences with influenza outbreaks, and discussions of both poultry and human exposure to and infection with HPAI H5N1. In addition, four research manuscripts provide insight into the relationship between influenza A, including HPAI H5N1, and poultry and human health in rural Suphanburi Province, Thailand. A major goal of this work was to learn more about the management of free-grazing duck (FGD) flocks in Thailand and their role in avian influenza virus maintenance and transmission. FGD have been associated with highly pathogenic avian influenza (HPAI) H5N1 outbreaks and may be a viral reservoir. In July-August 2010, the influenza exposure of Thai FGD and risk factors thereof were assessed. Sera from over 6000 FGDs were analyzed to detect antibodies to influenza A nucleoprotein (NP) and hemagglutinin H5 protein. Eighty-five percent were seropositive for influenza A. Of the NP-seropositive sera tested with H5 assays 39% were H5 ELISA-positive and 4% suspect. Twelve per cent of H5 ELISA-positive or suspect ducks had H5 titers ≥1:20. Risk factors for influenza A seropositivity include older age, poultry contact, flock visitors and older purchase age. Flocks had H5 virus exposure as recently as March 2010, but the last HPAI H5N1 outbreak in Thailand was in 2008, highlighting a need for FGD surveillance. This dissertation also includes an investigation of the seroprevalence of and risk factors for antibodies to HPAI H5N1 in poultry owners. Seroprevalence was 6.3%, and single persons and those working with farmed chickens were at increased risk of seropositivity. Poultry owners reported limited use of personal protective equipment during all activities and inconsistent hand washing practices after carrying poultry and gathering eggs. Lastly, this dissertation includes the description and results of an agent-based model (ABM) of the local Thai poultry sector which was built to simulate contacts among FGD flocks and persons that own poultry and conduct poultry-related activities. Using this model, opportunities for the transmission and control of HPAI H5N1 in this setting were identified.Item Computational analysis and visualization of the evolution of influenza virus(2014-08) Lam, Ham ChingInfluenza viruses can infect a large variety of birds and mammals including humans, pigs, domestic poultry, marine mammals, cats, dogs, horses, and wild carnivores \cite{Webster2002}. Surveillance for influenza viruses circulating in humans has been gradually increased and expanded to many areas around the world. These surveillance programs have produced large amount of influenza genomic data which facilitates the study of the virus by computational methods that are efficient and cost saving.The main focus of this dissertation research is the development of visualization methods to understand the evolution of influenza viruses circulating in humans and other mammals. The methods developed have been applied to different human influenza A subtypes, swine influenza viruses, and avian influenza viruses. The methods are based on unsupervised dimensional reduction techniques which can be applied to each individual genome segments or to the complete genome sequence of the virus. These methods are a departure from the traditional phylogenetic tree construction paradigm because very large number of high dimensional input sequences can be processed and results are viewed directly in a two or three dimensional Euclidean space.We reproduced the evolutionary trajectory of the seasonal human influenza A/H3N2 virus since its introduction to humans in 1968 on a 2D PCA space. The observed pathway led us to hypothesize that vaccination serves as a primary evolutionary pressure on this virus. We provided visual, simulation results, and statistical results to support this. The North American swine influenza H3N2 viruses were also studied using the developed visualization methods. The diversity of this virus is changing since the 2009 H1N1 pandemic outbreak. Five main clusters were observed from the visualization results. The mutations at two positive selected sites on the HA gene were identified as the potential driver for clusters segregation of this virus after the pandemic.A visualization method was developed to visually detect reassortant influenza virus. A reassortant influenza virus is difficult to detect because it consists of genome segments from different parental origin. As two different strains of influenza coinfect a single cell, the capability to exchange genome segments between these two strains can lead to progeny carrying different parental segments within its genome. In order to detect such progeny, a PCA projection based visualization method that is able to examine the full genome sequence of a reference and test strains simultaneously was developed in order to detect any reassorted segments within a full genome. Besides the development of visualization methods, we have also developed a compact Markov Chain model to estimate the probability of viruses with high genetic similarity found after a very large time gap. This model is a two components model where we combined a Markov Chain with a Poisson model. The Markov model uses Hamming distance as the evolution process of the virus and a computed mutation rate as the input to the Poisson model, combined together, we simulated the evolution process of the influenza virus under the neutral evolution process. The computational results from this model led us to conclude that the existence of reservoirs preserving viruses for decades cannot be completely eliminated.In short, our primary goal has been to develop visualization based approaches to understand the evolution of the influenza viruses from different hosts. The results we have so far suggested that the power of visualization paves the way to gain deeper understanding and insight of the evolution of the virus as we utilize the rapidly growing amount of the genomic data of the virus.Item Control and characterization of influenza A viruses in swine(2011-07) Detmer, Susan ElisabethBetween 1958 and 2005, there were 37 human cases of zoonotic swine-origin influenza A virus (IAV) infection reported (Myers et al 2007; Van Reeth 2007). A majority of these infections were with classical swine H1N1 viruses and these 37 cases did not include the Fort Dix cases in 1976 that resulted in 1 death and up to 230 soldiers infected (Myers et al. 2007; Van Reeth 2007). However, a recent report of pig to human transmission was at an Ohio County Fair in 2007 (Vincent et al. 2009b). The sequence analysis of the HA gene segment of the IAVs isolated from the humans and pigs in this case revealed that it was a strain that was currently circulating in the U.S. pig population. The internal genes of this isolate were determined to be of the triple-reassortant swine influenza lineage, including a conserved avian PB2 gene sequence (Vincent et al. 2009b). On June 11, 2009 the first influenza pandemic in 41 years was declared by the World Health Organization. This virus was like no virus previously seen in the human population with gene segments from both Eurasian and North American swine viruses. The 2009 pandemic H1N1 virus was called a "quadruple-reassortant" virus because it is composed of neuraminidase (NA) and matrix (M) gene segments from Eurasian swine influenza viruses combined with triple-reassortant proteins of North American swine influenza viruses (human-origin polymerase B1 (PB1), avian-origin polymerase B2 (PB2) and polymerase A (PA), and classical swine-origin hemagglutinin (HA), nucleoprotein (NP) and non-structural (NS) (Garten et al. 2009; Smith et al. 2009). The evolutionary analysis of the 2009 pandemic H1N1 shows that the generation of this strain was not likely a recent event. In fact, in order to facilitate human-to-human spread, it probably adapted to the human host through secondary reassortments in humans (Ding et al. 2009). However, the original source of this virus has not yet been determined. The emergence of the 2009 pandemic H1N1 virus and scattered reports of human infections with swine-origin isolates underscores the importance of fully understanding the genetic, antigenic, and pathogenic characteristics of influenza A viruses so that we may limit the introduction of novel IAVs to the swine population and monitor for newly emerging and evolving viruses. In order to improve our understanding of IAVs in swine, the goal of this dissertation is to address the ability of genetic characterization to predict variations in virus phenotype, such as viral binding and antigenicity. Understanding the genetic, antigenic and pathogenic features of viruses is important to prevent introduction of human and avian viruses into swine herds and the potential spillback of those viruses to the human population, as wells as preventing the sustained transmission of IAVs within an endemically infected herd. The control of influenza viruses in pig populations continues to be dynamic and complex, and is reliant on a number of factors. Two of these factors include appropriate selection and application of (1) diagnostic tests and (2) vaccines. Routine surveillance for influenza viruses at the farm level, either syndromic or active surveillance, is often accomplished using real time RT-PCR tests on nasal swabs from live pigs and lung tissue samples from post-mortem examinations. Easily collected sample methods, such as oral fluids, could provide additional viruses for characterization of IAVs in swine. Oral fluids have been used extensively for diagnostic tests in human medicine and are now being applied in swine herds for detecting pathogens and antibodies against the pathogens (Prickett et al. 2010). As part of this dissertation, porcine oral fluids were validated as a viable sample collection method for routine RT-PCR and virus isolation tests (chapter 2). Another important control measure for influenza viruses in pigs continues to be vaccines. In order to assure continued efficacy of vaccines against currently circulating strains of virus, vaccine challenge trials are performed. In this dissertation, the efficacy of a commercial vaccine was examined against challenge with a contemporary field isolate (chapter 3). To address the genetic and phenotypic characterization of influenza A viruses from swine, two sets of viruses were selected from the influenza database at the University of Minnesota, Veterinary Diagnostic Laboratory and sequenced. The first set was isolated from a group of endemically infected farms treated by the same veterinarian from 2005 to 2009. The selected viruses were either used to produce autogenous vaccines or they were the epizootic viruses found during outbreaks in the vaccinated herds (chapter 4). The second set of viruses were isolated from nursery pigs in one endemically infected multi-site swine production system (farm M) from 2007 to 2009 and either contained a distinct two amino acid insertion or were presumptive ancestral viruses without the insertion (chapter 5). The viruses from farm M were further characterized along with representative viruses that have been previously studied in vivo using a new technique called virus histochemistry to examine the patterns of virus attachment in the respiratory tract (chapter 6). For the purposes of this dissertation IAVs were classified as virulent increased virulence have some of the following clinical/case presentations: (a) morbidity approaching 90% and mortality approaching 10 percent, (b) sudden, unexpected deaths occurring early in the disease outbreak, (c) gross lesions that are not typical of swine influenza including profuse hemorrhage and/or edema, and (d) sufficient health and production records along with laboratory results that indicate that a highly virulent influenza virus is involved. The characteristics of highly virulent influenza viruses, such as A/swine/KS/77778/2007 H1N1 and A/swine/OH/511445/2007 H1N1, have been previously described in the literature (Ma et al. 2010; Vincent et al. 2009b). This classification was not related to the criteria for classification of avian viruses as having high or low pathogenicity.Item Epidemiology of influenza A viruses of swine: surveilance, airborne detection and dissemination(2012-08) Corzo, Cesar AgustinChapter 2: Swine Influenza Active Surveillance in the United States Influenza A virus (IAV) in swine continues to be an important swine respiratory agent along with being a source of concern to public health authorities. While veterinary diagnostic laboratories are a valuable source of information with regards to the identification and genetic characterization of newly emerged virus through passive surveillance, there is still a need for additional surveillance programs that can aid in detecting new viruses in a timely manner. An active surveillance program was performed in 32 pig farms throughout the Midwestern United States between June 2009 and December 2011. Thirty nasal swabs were collected from growing pigs on a monthly basis and tested for IAV by RRT-PCR. During sample collection, data on sample collection date, pig age, pig group respiratory signs, clinical status and vaccination history were recorded. A total of 16,170 nasal swabs from 540 groups of growing pigs were collected from which 746 (4.6 %) nasal swabs and 117 (21.7 %) groups tested positive for IAV, respectively. Throughout the study, IAV was consistently detected in at least one farm except in two months. From the positive groups of pigs, H1N1, H1N2, H3N2, 2009 pandemic H1N1were detected in 18%, 16%, 7.6% and 14.5% of the groups, respectively. In seven groups, H1N2 or H3N2 reassortants containing genes from 2009 pandemic H1N1 were found. There were eight groups in which an H1N2 and the 2009 pandemic H1N1 were identified simultaneously. Groups of pigs were more likely to test positive for IAV during the spring and summer seasons compared to the fall. Age and group respiratory clinical signs were not predictors of group IAV status. This active IAV surveillance program provided quality data and increased the understanding of the current situation of circulating viruses in the U.S. pig population. Further studies in swine should be conducted to increase our knowledge regarding the characteristics of IAV. Chapter 3: Swine influenza virus risk factors in growing pigs Influenza A virus (IAV) is an important cause of respiratory disease in swine. Understanding the epidemiology of the disease is in its early stages and is needed to develop effective control and prevention strategies. A study was conducted to assess the relationship between the presence of IAV in growing pig farms and farm level risk factors. Twenty-six pig farms participated in the study from which 30 nasal swabs from growing pigs were collected on a monthly basis for 12 or 24 consecutive months between 2009 and 2011. Nasal swabs were tested for IAV by RRT-PCR. Weather stations were located at every participating farm for monitoring temperature, relative humidity, light intensity, wind speed and wind gusts. Farm level data was obtained through a questionnaire to assess the relationship between the presence of IAV and farm level characteristics. At the individual level, 4.6% of the nasal swabs from growing pigs tested positive for IAV. Of the monthly groups of pigs from which nasal swabs were collected, 20.8% had at least one positive nasal swab. Positive nasal swabs originated from 23 of the 26 participating farms. Farm type, pig flow and gilt source were associated with the presence of IAV. Environmental temperature and wind speed were associated with the presence of IAV. Overall, this study provides insights into the ecology of IAV which can aid in the development of control and prevention strategies. Chapter 4: Prevalence and risk factors for H1N1 and H3N2 influenza A virus infections in Minnesota turkey premisesInfluenza virus infections can cause respiratory and systemic disease of variable severity and also result in economic losses for the turkey industry. Several subtypes of influenza can infect turkeys causing diverse clinical signs. Influenza subtypes of swine origin have been diagnosed in turkey premises. However, it is not known how common these infections are nor the likely routes of transmission. We conducted a cross-sectional study to estimate the seroprevalence of influenza viruses in turkeys and examine factors associated with infection on Minnesota turkey premises. Results for influenza diagnostic tests and turkey and pig premises location data were obtained from the Minnesota Poultry Testing Laboratory (MPTL) and the Minnesota Board of Animal Health (MBAH) respectively from January 2007 to September 2008. Diagnostic data from 356 premises were obtained, of which 17 premises tested positive for antibodies to influenza A virus by agar gel immunodiffusion (AGID) assay and were confirmed as either H1N1 or H3N2 influenza viruses by hemagglutination and neuraminidase inhibition assays. Influenza infection status was associated with proximity to pig premises and flock size. The latter had a sparing effect on influenza status. This study suggests that H1N1 and H3N2 influenza virus infections of turkey premises in Minnesota are an uncommon event. The route of influenza virus transmission could not be determined, however, the findings suggest that airborne transmission should be considered in future studies. Chapter 5: Characterization of the temporal dynamics of airborne influenza A virus detection in acutely infected pigs Influenza A viruses infect many species including avians, mammals and humans. Aerosol transmission is one route that enables the virus to infect populations. This study explored the relationship between number of infected pigs and the probability of detecting influenza virus RNA in bioaerosols through the course of an acute infection. Bioaerosols were collected using a cyclonic collector in two groups of seven week-old pigs that were experimentally infected upon exposure with a contact infected pig (seeder pig). After contact exposure, individual pig nasal swab samples were collected daily and air samples were collected three times per day for eight days. All samples were tested for influenza by RRT-PCR targeting the influenza virus matrix gene. All pigs' nasal swabs became influenza virus RRT-PCR positive upon exposure to the infected seeder pig. Airborne influenza was detected in 58% (25/43) of the air samples collected. Temporal dynamics of influenza virus detection in air samples were in close agreement with the nasal shedding pattern in the infected pigs. First detection of positive bioaerosols occurred 2 days post contact (DPC). Positive bioaerosols were consistently detected between 3 and 6 DPC, a time when most pigs were also shedding virus in nasal secretions. Overall, the odds of detecting a positive air sample increased 2.2 times with every additional nasal swab positive pig in the group. In summary, there was a strong relationship between the number of pigs shedding influenza virus in nasal secretions and the detection of bioaerosols during the course of an acute infection in non-immune population. Chapter 6: Detection of airborne influenza A virus in experimentally infected pigs with maternally derived antibodies.This study assessed whether recently weaned piglets with maternally derived antibodies were able to generate infectious influenza aerosols. Three groups of piglets were assembled based on the vaccination status of the dam. Sows were either non vaccinated (CTRL) or vaccinated with the same (VAC-HOM) strain or a different (VAC-HET) strain than the one used for challenge. Piglets acquired the maternally derived antibodies by directly suckling colostrum from their respective dams. At weaning, pigs were challenged with influenza virus by direct contact with an infected pig (seeder pig) and clinical signs were evaluated. Air samples, collected using a liquid cyclonic air collector, and individual nasal swabs were collected daily for 10 days from each group and tested by matrix real-time reverse transcriptase polymerase chain reaction (RRT-PCR) assay. Virus isolation and titration were attempted for air samples on Madin-Darby canine kidney (MDCK) cells. All individual pigs from both VAC-HET and CTRL groups tested positive during the study but only one pig in the VAC-HOM group was positive by nasal swab RRT-PCR. Influenza virus could not be detected or isolated from air samples from the VAC-HOM group. Influenza A virus was isolated from 3.2% and 6.4% air samples from both the VAC-HET and CTRL groups, respectively. Positive RRT-PCR air samples were only detected in VAC-HET and CTRL groups on day 7 post-exposure. Overall, this study provides evidence that recently weaned pigs with maternally derived immunity without obvious clinical signs of influenza infection can generate influenza infectious aerosols which is relevant to the transmission and the ecology of influenza virus in pigs. Chapter 7: Detection of airborne swine influenza A virus in air samples collected inside, outside and downwind from swine barns Airborne transmission of influenza A virus (IAV) in swine is speculated to be an important route of virus dissemination, but data are scarce. This study attempted to detect airborne IAV by virus isolation and RRT-PCR in air samples under field conditions. This was accomplished by collecting air samples from four acutely infected pig farms and locating air samplers inside the barns, at the external exhaust fans and downwind from the farms and at distances up to 2.1 km. Weather data was also collected to explore the relationship between detection of IAV and temperature, relative humidity and sunlight intensity. IAV was detected in air samples collected in all the farms included in the study. On average, 96% and 85% of the air samples collected inside and at the exhaust fans from positive farms tested positive through RRT-PCR, respectively. Isolation of IAV was possible from air samples collected inside the barn at two of the farms and in one farm from the exhausted air. Influenza virus RNA was detected in air samples collected between 1.5 and 2.1 Km away from the farms. The odds of detecting IAV decreased with distance from the farm and greater levels of sunlight intensity. The results from this study prove evidence of the risk of aerosol transmission in pigs under field conditions and perhaps to other species as well.Item Influenza In Pigs Prior To Weaning: Sampling Strategies, Transmission Pathways And Approaches To Reduce Prevalence(2020-01) Garrido Mantilla, Jorge EduardoInfluenza in pigs prior to weaning: sampling strategies, transmission pathways and approaches to reduce prevalence General abstract Influenza is an important disease of swine and it represents a threat to public health because it is caused by influenza A virus (IAV), a zoonotic virus with pandemic potential. One of the objectives that producers and veterinarians have is to control influenza in breed-to-wean (BTW) farms by weaning IAV negative pigs. Pigs prior to weaning play an important role in influenza epidemiology because they can maintain endemic infections in BTW farms and they can disseminate IAV to other farms and regions at weaning. Unfortunately, there is limited information regarding the transmission pathways that lead to piglet infections, nor it is known what effect specific pig-rearing practices and farm management procedures may have on these pathways. To properly detect, isolate and characterize IAV genetically and antigenically, it is necessary to have sampling approaches that accurately define disease status yet are cost-effective to conduct. However, IAV detection and isolation can be challenging in endemic situations. Endemicity and transmission of IAV in pig populations can be affected by certain management practices that are necessary in production and do not allow IAV control. Even though, control of influenza is difficult, it is possible. Vaccination is one of the most common strategies to control influenza transmission and sow vaccination can help to reduce IAV prevalence in piglets. However, the diversity of IAV found in farms requires the use of vaccines that antigenically match the wild-type virus circulating in the pigs in order to provide good cross-protection against the field strains. In an effort to increase vaccine efficacy, custom-made vaccines that include viral strains identified in farms are used to help to control influenza in BTW farms. However, despite the widespread use of custom-made vaccines, there is limited data on the long term effectiveness of using custom-made vaccines in farms. Finally, in this thesis, I aimed to address some of the questions that are central to the transmission and control of IAV in BTW farms and reduce the prevalence of IAV in piglets at weaning. Specifically, I aimed to: 1) identify the best sampling strategy to detect and isolate IAV in weaned and growing pigs, 2) determine the role of nurse sows in the transmission and perpetuation of IAV in pigs prior to weaning, and 3) assess the impact of custom-made vaccines in reducing IAV prevalence in an integrated swine production system over time. The results obtained during my studies determined that IAV detection rates are higher when group and environmental sampling strategies are used compared to samples collected from individual pigs. Nevertheless, individual samples may still be needed to obtain a viral isolate or perform genetic sequencing and virus characterization. As part of my work, I developed the udder skin wipe technique to detect IAV from lactating sows and isolate IAV from litters prior to weaning. Furthermore, I identified that management practices such as the use of nurse sows can transmit IAV between litters thereby perpetuating IAV infection in pigs prior to weaning. Finally, our multi-year evaluations of custom-vaccine usage on BTW farms demonstrated that on-going surveillance and characterization of IAV isolates facilitate vaccine updates with custom-made epidemiologically-relevant strains. In addition to selecting epidemiologically-relevant strains, the strain selection criteria should also include the identification of strains with similar HA antigenic properties, e.g. those with an overall HA protein identity of 95% or more and having identical or nearly identical amino acid motifs. Once selected, these strains can be included in the updated vaccines used to immunize sows and reduce IAV prevalence in their pigs at weaning. The findings on my thesis contribute to the understanding of IAV transmission in pigs before weaning and point to specific strategies to improve surveillance and disease control. Nevertheless, more studies are necessary to elucidate strategies to limit IAV infections and transmission in BTW farms.Item Measurement and filtration of virus aerosols(2014-06) Zuo, ZhiliThe potential involvement of virus aerosols (i.e., airborne virus-carrying particles) in the transmission of human respiratory diseases has led to increased public concern. This dissertation focuses on 1) measurement of laboratory generated virus aerosols as a function of particle size, virus type, and composition of nebulizer suspensions (Chapter 2 and 3) and 2) performance evaluation of filtering facepiece respirators against virus aerosols (Chapter 4 and 5) with the long term goal to better understand and better control the airborne transmission of viral diseases.Item Systems Design and Synthetic Construction of Influenza Virus for Flu Vaccine Application(2021-11) Phan, ThuInfluenza A virus (IAV) is the leading cause of annual flu epidemics, which inflicts about 250,000-500,000 deaths worldwide. The morbidity and mortality rate are much higher when a novel strain of IAV arises, resulting in flu pandemics. Vaccination has been the best prevention strategy for influenza. However, flu viruses constantly evolve and escape the established immunity, thus annual flu vaccination is required. Most current flu vaccine manufacturing platforms use multi-plasmid transfection to rescue seasonal seed viruses, the seed viruses are then used to infect either embryonic chicken eggs or cultured cells to produce viruses. Both production methods have high degrees of variability and produce viruses with a high content of non-infectious particles that reduce vaccine effectiveness. To address the need for more reliable and scalable processes, we applied systems biology and synthetic biology approaches to understand the kinetics of virus replication and to engineer cell lines that can control viral gene expression dynamics. First, we established a new data analysis pipeline using RNA sequencing to study segment-specific kinetics of all IAV RNA molecules. Using the pipeline, InVERT, to study the kinetics of IAV infection, revealed different phases of virus infection, and groups of genes whose kinetics are similar. This was the first-time IAV replication kinetics of all segments is reported. Building on that success, we then developed the second pipeline named InVERT II, which can further differentiate mRNA transcripts made by the viral replication enzyme RdRP from mRNA transcripts synthesized by host cells' RNA Polymerase II, to study the kinetics of virus rescue by transfection. With the understanding gained from the kinetics of virus infection and replication, we engineered the human cell line HEK 293T to express inducible components of IAV that not only have inducible replicative activity but also can package virus particles. This is the first proof of principle to show that mammalian cells can be engineered to produce complex negative-sense RNA viruses. Our integrative approach using both systems biology and synthetic biology has enabled the creation of a platform that could be further optimized for reliable, robust, and scalable flu vaccine manufacturing processes.