Outbreak Response to Circulating Vaccine-Derived Poliovirus in Three Northern Regions of Ghana, 2019.

Background: Circulating Vaccine-Derived Poliovirus Type 2 (cVDPV2) was isolated in sewage and later in stool samples from children with acute flaccid paralysis (AFP) in northern Ghana. Method: A multidisciplinary and multisectoral team investigated this outbreak and reported on epidemiological and laboratory investigations. Sewage/wastewater samples were collected from the environment, while stool samples were collected from AFP/contact children under 5 years of age. The samples were processed for virus isolation, and positive isolates were sequenced. We also conducted a descriptive investigation involving a review of records, active case search, and Monovalent Oral Polio Vaccine 2 campaigns. Additionally, we interviewed caregivers about the vaccination status of their children, as well as their knowledge on polio prevention. Water quality, sanitation, hygiene practices, and health-seeking behaviours were also assessed. Results: A total of 18 cVDPV2 were confirmed in the three regions of Ghana during the outbreak in 2019-2020. All strains were genetically linked to a Nigerian cVDPV2 strain NIE-KWS-KSB-18-006HC29 that circulated in 2018. Evaluation of the surveillance system shows that officers have good knowledge of AFP and know how to collect samples, package them, and ship them to the laboratory. Few communities had access to potable water. Open defecation was common, and the water supply, sanitation, and hygiene practices of the communities were poor. Conclusion: The cVDPV2 outbreak represents the first time cVDPV2 has circulated in the country since Ghana embarked on the polio eradication program in 1996. However, with quality mOPV2 mop-up campaigns, a nationwide IPV catch-up campaign coupled with enhanced surveillance measures, transmission was interrupted.

Sequence-matching adapter trimmers generate consistent quality and assembly metrics for Illumina sequencing of RNA viruses.

Trimming adapters and low-quality bases from next-generation sequencing (NGS) data is crucial for optimal analysis. We evaluated six trimming programs, implementing five different algorithms, for their effectiveness in trimming adapters and improving quality, contig assembly, and single-nucleotide polymorphism (SNP) quality and concordance for poliovirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and norovirus paired data sequenced on Illumina iSeq and MiSeq platforms. Trimmomatic and BBDuk effectively removed adapters from all datasets, unlike FastP, AdapterRemoval, SeqPurge, and Skewer. All trimmers improved read quality (Q ≥ 30, 87.8 - 96.1%) compared to raw reads (83.6 - 93.2%). Trimmers implementing traditional sequence-matching (Trimmomatic and AdapterRemoval) and overlapping algorithm (FastP) retained the highest-quality reads. While all trimmers improved the maximum contig length and genome coverage for iSeq and MiSeq viral assemblies, BBDuk-trimmed reads assembled the shortest contigs. SNP concordance was consistently high (> 97.7 - 100%) across trimmers. However, BBDuk-trimmed reads had the lowest quality SNPs. Overall, the two adapter trimmers that utilized the traditional sequence-matching algorithm performed consistently across the viral datasets analyzed. Our findings guide software selection and inform future versatile trimmer development for viral genome analysis.

Reversion of neurovirulent mutations, recombination and high intra-host diversity in vaccine-derived poliovirus excreted by patients with primary immune deficiency.

Patients with Primary immunodeficiency (PIDs) may be infected by Polioviruses (PVs), especially when vaccinated with live Oral Polio Vaccine before diagnosis. They may establish long-term shedding of divergent strains and may act as reservoirs of PV transmission. This study delved into the effect of the genetic evolution of complete PV genomes, from MHC class II-deficient patients, on the excretion duration and clinical outcomes. Stool samples from three PID patients underwent analysis for PV detection through inoculation on cell culture and real-time PCR, followed by VP1 partial sequencing and full genome sequencing using the Illumina technology. Our findings revealed a low number of mutations for one patient who cleared the virus, while two exhibited a high intra-host diversity favoring the establishment of severe outcomes. Neurovirulence-reverse mutations were detected in two patients, possibly leading to paralysis development. Furthermore, a recombination event, between type 3 Vaccine-Derived Poliovirus and Sabin-like1 (VDPV3/SL1), occurred in one patient. Our findings have suggested an association between intra-host diversity, recombination, prolonged excretion of the virus, and emergence of highly pathogenic strains. Further studies on intra-host diversity are crucial for a better understanding of the virus evolution as well as for the success of the Global Polio Eradication Initiative.

Environmental surveillance of a circulating vaccine-derived poliovirus type 2 outbreak in Israel between 2022 and 2023: a genomic epidemiology study.

BACKGROUND: Similarly to wild poliovirus, vaccine-derived poliovirus (VDPV) strains can cause acute flaccid paralysis, posing a considerable challenge to public health and the eradication of poliovirus. VDPV outbreaks, particularly VDPV type 2 (VDPV2), are increasing worldwide, including in high-income countries with high vaccine coverage. We aimed to conduct a comprehensive analysis of the molecular epidemiology of a widespread VDPV2 outbreak in Israel in 2022-23 using conventional polio identification techniques and whole-genome sequencing. METHODS: In this genomic epidemiology study, we monitored and identified poliovirus type 2 (PV2) through the surveillance of stool samples from individuals with acute flaccid paralysis and related contacts, as well as environmental surveillance of sewage samples. Environmental surveillance involved 15 routine surveillance sites and an additional 30 sites dedicated to monitoring this outbreak, covering approximately 70% of Israel's population between April 1, 2022, and June 30, 2023. Additionally, we performed phylogenetic and mutation analyses using whole-genome, next-generation sequencing of PV2 isolates to identify recombination events, characterise VDPV2 lineages according to the capsid region, and establish the geographical distribution and linkage of PV2 isolates. FINDINGS: We detected 256 genetically linked samples from environmental surveillance, as well as one case of acute flaccid paralysis and four positive contacts associated with the Sabin type 2 oral vaccine strain. Most affected locations showed a high-density population of Jewish Ultra-Orthodox communities. Through high-resolution genomic characterisation and phylogenetic analysis of 202 representative sequences with complete capsid coverage, including isolates from both environmental surveillance and the case of acute flaccid paralysis, a conclusive linkage was established among all detections, confirming them to be part of a single VDPV2 outbreak. This strategy enabled the characterisation of three distinct lineages and established connections between different locations in Israel, including linking the case of acute flaccid paralysis and nearby environmental surveillance detections from the northern region with detections in the geographically distant central region. INTERPRETATION: This study highlights the role of environmental surveillance in the early detection and monitoring of poliovirus circulation, enabling a prompt public health response involving enhanced surveillance and a catch-up campaign with inactivated polio vaccine. Whole-genome sequencing offered valuable insights into the origins of the outbreak, linkage across detections, and the geographical distribution of the virus, with higher resolution than would have been possible with the standard analysis of the VP1 gene alone. FUNDING: None.

Progress Toward Poliomyelitis Eradication - Pakistan, January 2023-June 2024.

Since its launch in 1988, the Global Polio Eradication Initiative has made substantial progress toward the eradication of wild poliovirus (WPV), including eradicating two of the three serotypes, and reducing the countries with ongoing endemic transmission of WPV type 1 (WPV1) to just Afghanistan and Pakistan. Both countries are considered a single epidemiologic block. Despite the occurrence of only a single confirmed WPV1 case during the first half of 2023, Pakistan experienced widespread circulation of WPV1 over the subsequent 12 months, specifically in the historical reservoirs of the cities of Karachi, Peshawar, and Quetta. As of June 30, 2024, eight WPV1 cases had been reported in Pakistan in 2024, compared with six reported during all of 2023. These cases, along with more than 300 WPV1-positive environmental surveillance (sewage) samples reported during 2023-2024, indicate that Pakistan is not on track to interrupt WPV1 transmission. The country's complex sociopolitical and security environment continues to pose formidable challenges to poliovirus elimination. To interrupt WPV1 transmission, sustained political commitment to polio eradication, including increased accountability at all levels, would be vital for the polio program. Efforts to systematically track and vaccinate children who are continually missed during polio vaccination activities should be enhanced by better addressing operational issues and the underlying reasons for community resistance to vaccination and vaccine hesitancy.

In vitro reconstitution reveals membrane clustering and RNA recruitment by the enteroviral AAA+ ATPase 2C.

Enteroviruses are a vast genus of positive-sense RNA viruses that cause diseases ranging from common cold to poliomyelitis and viral myocarditis. They encode a membrane-bound AAA+ ATPase, 2C, that has been suggested to serve several roles in virus replication, e.g. as an RNA helicase and capsid assembly factor. Here, we report the reconstitution of full-length, poliovirus 2C's association with membranes. We show that the N-terminal membrane-binding domain of 2C contains a conserved glycine, which is suggested by structure predictions to divide the domain into two amphipathic helix regions, which we name AH1 and AH2. AH2 is the main mediator of 2C oligomerization, and is necessary and sufficient for its membrane binding. AH1 is the main mediator of a novel function of 2C: clustering of membranes. Cryo-electron tomography reveal that several 2C copies mediate this function by localizing to vesicle-vesicle interfaces. 2C-mediated clustering is partially outcompeted by RNA, suggesting a way by which 2C can switch from an early role in coalescing replication organelles and lipid droplets, to a later role where 2C assists RNA replication and particle assembly. 2C is sufficient to recruit RNA to membranes, with a preference for double-stranded RNA (the replicating form of the viral genome). Finally, the in vitro reconstitution revealed that full-length, membrane-bound 2C has ATPase activity and ATP-independent, single-strand ribonuclease activity, but no detectable helicase activity. Together, this study suggests novel roles for 2C in membrane clustering, RNA membrane recruitment and cleavage, and calls into question a role of 2C as an RNA helicase. The reconstitution of functional, 2C-decorated vesicles provides a platform for further biochemical studies into this protein and its roles in enterovirus replication.

Assessing the mucosal intestinal and systemic humoral immunity of sequential schedules of inactivated poliovirus vaccine and bivalent oral poliovirus vaccine for essential immunization in Bangladesh: An open-label, randomized controlled trial.

In 2012, the Strategic Advisory Group of Experts on Immunization (SAGE) recommended introduction of at least one inactivated poliovirus vaccine (IPV) dose in essential immunization programs. We evaluated systemic humoral and intestinal mucosal immunity of a sequential IPV-bivalent oral poliovirus vaccine (bOPV) schedule compared with a co-administration IPV + bOPV schedule in an open-label, randomized, controlled, non-inferiority, inequality trial in Dhaka, Bangladesh. Healthy infants aged 6 weeks were randomized to either: (A) IPV and bOPV at 6 and bOPV at 10 and 14 weeks (IPV + bOPV-bOPV-bOPV); or (B) IPV at 6 and bOPV at 10 and 14 weeks (IPV-bOPV-bOPV). Of 456 participants enrolled and randomly assigned during May-August 2015, 428 (94%) were included in the modified intention-to-treat analysis (arm A: 211, arm B: 217). Humoral immune responses did not differ at 18 weeks between study arms: type 1 (98% versus 96%; p = 0.42), type 2 (37% versus 39%; p = 0.77), and type 3 (97% versus 93%; p = 0.07). Virus shedding one week after the bOPV challenge dose in arm B was non-inferior to arm A (type 1 difference = -3% [90% confidence interval: -6 - 0.4%]; type 3 difference: -3% [-6 to -0.2%]). Twenty-six adverse events including seven serious adverse events were reported among 25 participants including one death; none were attributed to study vaccines. An IPV-bOPV-bOPV sequential schedule induced comparable systemic humoral immunity to all poliovirus types and types 1 and 3 intestinal mucosal immunity as an IPV + bOPV-bOPV-bOPV co-administration schedule.

The respiratory route of transmission of virulent polioviruses.

AIMS: The route of transmission of wild and circulating vaccine-derived polioviruses remains controversial, between respiratory and faecal-oral, and we aim to identify the most plausible one to settle the controversy. METHODS: We explored available epidemiological clues and evidence in support of either route in order to arrive at an evidence-based conclusion. RESULTS: Historically the original concept was respiratory transmission based on epidemiological features of age distribution, which was later revised to faecal-oral as the rationale for popularising the live attenuated oral polio vaccine in preference to the inactivated poliovirus vaccine. Through epidemiological logic, we find no evidence for the faecal-oral route from available studies and observations, but all available information supports the respiratory route. CONCLUSIONS: The route is respiratory, not faecal-oral. The global polio eradication initiative assumed it was faecal-oral - and its gargantuan efforts based on this assumption have failed in two ways: eradication remains pending and circulating vaccine-derived polioviruses have seeded widely. With clarity on the route of transmission the choice of vaccine is also clear - it can only be the inactivated poliovirus vaccine.

An automated assay platform for the evaluation of antiviral compounds against polioviruses.

High-throughput screening requires assays that have flexibility to test large numbers of specimens while being accurate to ensure reproducibility across all specimens and variables tested. Previously, we used a low-throughput, cell-based assay to identify compounds with antiviral activity against polioviruses. In this report, we report the development and implementation of a high-throughput automation platform for the identification of compounds with antiviral activity against polioviruses. The platform uses off-the-shelf automated equipment combined with a modified assay, with minimal changes to existing laboratory space. We evaluated automation systems from Hudson Robotics Inc., Agilent Technologies, and a microplate reader from PerkinElmer during the platform design. Optimization for high throughput was focused on bulk reagent additions, serial dilutions, microplate washing and measuring results from the tens-to-hundreds of microplates. We evaluated the automated cell-based assay for selectivity, sensitivity, accuracy, precision, and reproducibility. This platform can be applied to screen novel antivirals against polioviruses and non-polio enteroviruses.

Monitoring the VDPV2 outbreak in Egypt during 2020-2021 highlights the crucial role of environmental surveillance and boosting immunization in combating Poliovirus.

BACKGROUND: Poliovirus is a highly infectious enterovirus (EV) that primarily affects children and can lead to lifelong paralysis or even death. Vaccine-derived polioviruses (VDPVs) are a great threat since they are derived from the attenuated virus in the Oral Poliovirus Vaccine (OPV) and can mutate to a more virulent form. The purpose of this study was to identify VDPV serotype 2 through the year 2020-2021 via surveillance of sewage samples collected from different localities and governorates in Egypt and stool specimens from Acute Flaccid Paralysis (AFP) cases. Both were collected through the national poliovirus surveillance system and according to the guidelines recommended by the WHO. METHODS: A total of 1266 sewage samples and 3241 stool samples from January 2020 to December 2021 were investigated in the lab according to World Health Organization (WHO) protocol for the presence of Polioviruses by cell culture, molecular identification of positive isolates on L20B cell line was carried out using real-time polymerase chain reactions (RT-PCR). Any positive isolates for Poliovirus type 2 and isolates suspected of Vaccine Derived Poliovirus Type 1 and type 3 screened by (VDPV1) or Vaccine Poliovirus Type 3 (VDPV3) assay in RT-PCR were referred for VP1 genetic sequencing. RESULTS: The outbreak was caused by circulating VDPV2 (cVDPV2) strains started in January 2021. By the end of February 2021, a total of 11 cVDPV2s were detected in sewage samples from six governorates confirming the outbreak situation. One additional cVDPV2 was detected later in the sewage sample from Qena (June 2021). The first and only re-emergence of VDPV2 in stool samples during the outbreak was in contact with Luxor in June 2021. By November 2021, a total of 80 VDPVs were detected. The Egyptian Ministry of Health and Population (MOHP), in collaboration with the WHO, responded quickly by launching two massive vaccination campaigns targeting children under the age of five. Additionally, surveillance systems were strengthened to detect new cases and prevent further spread of the virus. CONCLUSION: The continued threat of poliovirus and VDPVs requires ongoing efforts to prevent their emergence and spread. Strategies such as improving immunization coverage, using genetically stable vaccines, and establishing surveillance systems are critical to achieving global eradication of poliovirus and efficient monitoring of VDPVs outbreaks.

Safety and 6-month immune persistence of inactivated poliovirus vaccine (Sabin strains) simultaneously administrated with other vaccines for primary and booster immunization in Jiangxi Province, China.

OBJECTIVES: This study aims to evaluate the safety of a new inactivated poliomyelitis vaccine (Sabin strains) (sIPV) for large-scale use in primary and booster immunizations, whether simultaneously administered with other vaccines or not and to explore the persistence of all vaccines at approximately six months after vaccination. METHOD: A total of 3200 infants were recruited into this study, including 2000 infants aged 2-3 months randomly assigned (1:1) into the "sIPV basic" or the "sIPV+DTaP" group for primary immunization of sIPV. Another 1200 children aged 18 months old and above were randomly assigned (2:2:1:1) into the "sIPV booster," "sIPV+HepA-I," "sIPV+MMR", or "sIPV+HepA-L" group for booster immunization of sIPV. Adverse events within 30 days of each vaccination dose in all participants were self-reported by guardians using a WeChat mini-program. Approximately 200 blood samples were collected at 5-7 months after the final vaccination to test for antibodies against poliovirus and other viruses. RESULTS: 3198 participants in total were included in the safety study, including 1999 infants aged 2-3 months old and 1199 children aged 18-26 months old. For primary immunization, the incidence of adverse reactions in the "sIPV basic" and the "sIPV+DTaP" group were 3.19 and 6.21% (P = 0.001), respectively. For booster immunization, the incidences of adverse reaction for the "sIPV booster" group were 2.25%, while the incidence for the "sIPV +others" group in total was 2.50% (P = 0.788). Most adverse reactions were mild. Fever was the most common symptom in all groups. No vaccine-related serious adverse events (SAEs) were observed in this study. The seropositivity rates of antibodies in the "sIPV basic" and the "sIPV+DTaP" group were 92.31 and 100% against type 1 poliovirus (P = 0.031); 96.15% and 98.57% against type 2 poliovirus (P = 0.575); 98.08% and 91.43% against type 3 poliovirus (P = 0.237), respectively. Regarding booster vaccination with sIPV, whether co-administered with other vaccines or not, the seropositivity rates of antibodies against the three types of polioviruses were all 100%. Seropositivity rates of antibodies against hepatitis A, measles, mumps, and rubella were all no <77%, except for pertussis, which was <30%. CONCLUSION: sIPV demonstrated good safety and immune persistence for primary and booster vaccinations, whether administered singly or simultaneously. Antibodies against hepatitis A, measles, mumps and rubella were not disrupted by the co-vaccination. However, the seropositivity rates and geometric mean concentrations (GMCs) of antibodies against pertussis indicate the necessity for a booster dose.

Estimation of the poliovirus type 2 immunity gap in South Africa.

In the context of polio eradication efforts, accurate assessment of vaccination programme effectiveness is essential to public health planning and decision making. Such assessments are often based on zero-dose children, estimated using the number of children who did not receive the first dose of the Diphtheria-Tetanus-Pertussis containing vaccine as a proxy. Our study introduces a novel approach to directly estimate the number of children susceptible to poliovirus type 2 (PV2) and uses this approach to provide district-level estimates for South Africa of susceptible children born between 2017 and 2022. We used district-level data on annual doses of inactivated poliovirus vaccine (IPV) administered, live births, and population sizes, from 2017 through 2022. We imputed missing vaccination data, implemented flexible assumptions regarding dose distribution in the eligible population, and used estimated efficacy values for one, two, three, and four doses of IPV, to compute the number of susceptible and immune children by birth year. We validated our approach by comparing an intermediary output with zero-dose children (ZDC) estimated using data reported by WHO/UNICEF Estimates of National Immunization Coverage (WUENIC). Our results indicate high heterogeneity in susceptibility to PV2 across South Africa's 52 districts as of the end of 2022. In children under 5 years, PV2 susceptibility ranged from approximately 30 % in districts including Xhariep (31.9 %), Ekurhuleni (30.1 %), and Central Karoo (29.8 %), to less than 4 % in Sarah Baartman (1.9 %), Buffalo City (2.1 %), and eThekwini (3.2 %). Our susceptibility estimates were consistently higher than ZDC over the timeframe. We estimated that ZDC decreased nationally from 155,168 (152,737-158,523) in 2017 to 108,593 in 2021, and increased to 127,102 in 2022, a trend consistent with ZDC derived from data reported by WUENIC. While our approach provides a more comprehensive profile of PV2 susceptibility, our susceptibility and ZDC estimates generally agree in the ranking of districts according to risk.

Poliovirus capsid protein VP3 can penetrate vascular endothelial cells.

The poliovirus (PV) enters the central nervous system (CNS) via the bloodstream, suggesting the existence of a mechanism to cross the blood-brain barrier. Here, we report that PV capsid proteins (VP1 and VP3) can penetrate cells, with VP3 being more invasive. Two independent parts of VP3 are responsible for this function. Both peptides can penetrate human umbilical cord vascular endothelial cells, and one peptide of VP3 could also penetrate peripheral blood mononuclear cells. In an in vitro blood-brain barrier model using rat-derived astrocytes, pericytes, and endothelial cells, both peptides were observed to traverse from the blood side to the brain side at 6 h after administration. These results provide insights into the molecular mechanisms underlying PV invasion into the CNS.

Molecular epidemiology of enteroviruses from Guatemalan wastewater isolated from human lung fibroblasts.

The Global Specialized Polio Laboratory at CDC supports the Global Poliovirus Laboratory Network with environmental surveillance (ES) to detect the presence of vaccine strain polioviruses, vaccine-derived polioviruses, and wild polioviruses in high-risk countries. Environmental sampling provides valuable supplementary information, particularly in areas with gaps in surveillance of acute flaccid paralysis (AFP) mainly in children less than 15 years. In collaboration with Guatemala's National Health Laboratory (Laboratorio Nacional de Salud Guatemala), monthly sewage collections allowed screening enterovirus (EV) presence without incurring additional costs for sample collection, transport, or concentration. Murine recombinant fibroblast L-cells (L20B) and human rhabdomyosarcoma (RD) cells are used for the isolation of polioviruses following a standard detection algorithm. Though non-polio-Enteroviruses (NPEV) can be isolated, the algorithm is optimized for the detection of polioviruses. To explore if other EV's are present in sewage not found through standard methods, five additional cell lines were piloted in a small-scale experiment, and next-generation sequencing (NGS) was used for the identification of any EV types. Human lung fibroblast cells (HLF) were selected based on their ability to isolate EV-A genus. Sewage concentrates collected between 2020-2021 were isolated in HLF cells and any cytopathic effect positive isolates used for NGS. A large variety of EVs, including echoviruses 1, 3, 6, 7, 11, 13, 18, 19, 25, 29; coxsackievirus A13, B2, and B5, EV-C99, EVB, and polioviruses (Sabin 1 and 3) were identified through genomic typing in NGS. When the EV genotypes were compared by phylogenetic analysis, it showed many EV's were genomically like viruses previously isolated from ES collected in Haiti. Enterovirus occurrence did not follow a seasonality, but more diverse EV types were found in ES collection sites with lower populations. Using the additional cell line in the existing poliovirus ES algorithm may add value by providing data about EV circulation, without additional sample collection or processing. Next-generation sequencing closed gaps in knowledge providing molecular epidemiological information on multiple EV types and full genome sequences of EVs present in wastewater in Guatemala.

Nanoparticles with a Lipid Core Can Enhance the Infection of Epithelial Cells with an Enterovirus.

INTRODUCTION: The effect of maltodextrin-based nanoparticles with an anionic phospholipid core (lipid-based nanoparticles [NPLs]) on the infection of a human tumoral cell line with poliovirus (PV) has been studied. METHODS: NPLs were synthesized and associated with the PV type 1 Sabin strain, and the formulations were characterized. PV and PV/NPL formulations were inoculated to HEp-2 cells. RESULTS: The surface charge and the diameter of PV/NPL formulation suggest that viral particles were adsorbed onto NPLs. When HEp-2 cells were inoculated with 1 tissue culture 50% infectious dose/mL PV associated with NPLs, the cytopathic effect appeared obvious; the levels of the infectious titer of culture supernatants and the proportion of VP1-positive cells were higher. The level of intracellular viral RNA extracted from HEp-2 cells inoculated with PV/NPL formulation was higher as well. CONCLUSION: These results show that NPLs can enhance the infection with a virus and suggest that they might be used in virotherapy to increase the virus-mediated lysis of tumor cells.

Optimum study of fractional polio model with exponential decay kernel.

This study introduces a fractional order model to investigate the dynamics of polio disease spread, focusing on its significance, unique results, and conclusions. We emphasize the importance of understanding polio transmission dynamics and propose a novel approach using a fractional order model with an exponential decay kernel. Through rigorous analysis, including existence and stability assessment applying the Caputo Fabrizio fractional operator, we derive key insights into the disease dynamics. Our findings reveal distinct disease-free equilibrium (DFE) and endemic equilibrium (EE) points, shedding light on the disease's stability. Furthermore, graphical representations and numerical simulations demonstrate the behavior of the disease under various parameter values, enhancing our understanding of polio transmission dynamics. In conclusion, this study offers valuable insights into the spread of polio and contributes to the broader understanding of infectious disease dynamics.