NIAID/SMB Workshop on Multiscale Modeling of Infectious and Immune-Mediated Diseases.

On July 19th, 2023, the National Institute of Allergy and Infectious Diseases co-organized a workshop with the Society of Mathematical Biology, with the authors of this paper as the organizing committee. The workshop, "Bridging multiscale modeling and practical clinical applications in infectious diseases" sought to create an environment for mathematical modelers, statisticians, and infectious disease researchers and clinicians to exchange ideas and perspectives.

The Landscape of NIAID-funded Observational COVID-19 Cohort Studies.

The spread of SARS-CoV-2 since late 2019 represented an unprecedented public health emergency, which included a need to fully understand COVID-19 disease across all ages and populations. In response, the US National Institute of Allergy and Infectious Diseases (NIAID) rapidly funded epidemiology studies that monitored COVID-19. However, the diversity and breadth of the populations studied in NIAID-funded COVID-19 observational cohorts were not easy to extrapolate because of siloed approaches to collect and report data within NIAID. Here, we describe the effort to develop a harmonized cohort study reporting tool that includes common epidemiological data elements as well as NIAID priorities. We report its implementation to analyze metadata from 58 COVID-19 cohort studies funded February 2020 to June 2021, visualize key metadata including geographic distribution, study duration, participant demographics, sample types collected, and scientific priorities addressed. A bibliographic analysis highlights the scientific publications and citations across these funded studies and demonstrates their enormous impact on the COVID-19 field. These analyses highlight how common data elements and reporting tools can assist funding agencies to capture the landscape and potential gaps during public health responses and how they can assist in decision making.

Developing a standardized but extendable framework to increase the findability of infectious disease datasets.

Biomedical datasets are increasing in size, stored in many repositories, and face challenges in FAIRness (findability, accessibility, interoperability, reusability). As a Consortium of infectious disease researchers from 15 Centers, we aim to adopt open science practices to promote transparency, encourage reproducibility, and accelerate research advances through data reuse. To improve FAIRness of our datasets and computational tools, we evaluated metadata standards across established biomedical data repositories. The vast majority do not adhere to a single standard, such as Schema.org, which is widely-adopted by generalist repositories. Consequently, datasets in these repositories are not findable in aggregation projects like Google Dataset Search. We alleviated this gap by creating a reusable metadata schema based on Schema.org and catalogued nearly 400 datasets and computational tools we collected. The approach is easily reusable to create schemas interoperable with community standards, but customized to a particular context. Our approach enabled data discovery, increased the reusability of datasets from a large research consortium, and accelerated research. Lastly, we discuss ongoing challenges with FAIRness beyond discoverability.

Optimization of qRT-PCR assay for zika virus detection in human serum and urine.

Zika Virus (ZIKV) is a mosquito-borne flavivirus that the World Health Organization (WHO) declared a global concern due to the severity of infection. This study focuses on determining the level of detection of ZIKV RNA in human serum and urine. Known amounts of Zika virus were added to uninfected human serum and urine samples. Different reverse transcriptases were compared to select the optimal enzyme for this application. Zika RNA in these samples was then quantified with qRT-PCR to determine the lower limit of detection in these fluids and to construct a standard curve. Student's t-test of paired samples was used in order to identify statistical differences. The SuperScript III enzyme was able to produce more ZIKV cDNA when compared to PrimeScript. Zika virus RNA was found to be detectable at lower levels (2.5 PFU/mL) in urine than in serum (250 PFU/mL) when using SuperScript III. This study demonstrates how the selection of both the human clinical specimen, and the reverse transcriptase enzyme involved in the molecular detection of ZIKV by quantitative real-time polymerase chain reaction (qRT-PCR), play an important role in enabling improved detection of the virus.

Contemporary Circulating Enterovirus D68 Strains Have Acquired the Capacity for Viral Entry and Replication in Human Neuronal Cells.

Enterovirus D68 (EV-D68) has historically been associated with respiratory illnesses. However, in the summers of 2014 and 2016, EV-D68 outbreaks coincided with a spike in polio-like acute flaccid myelitis/paralysis (AFM/AFP) cases. This raised concerns that EV-D68 could be the causative agent of AFM during these recent outbreaks. To assess the potential neurotropism of EV-D68, we utilized the neuroblastoma-derived neuronal cell line SH-SY5Y as a cell culture model to determine if differential infection is observed for different EV-D68 strains. In contrast to HeLa and A549 cells, which support viral infection of all EV-D68 strains tested, SH-SY5Y cells only supported infection by a subset of contemporary EV-D68 strains, including isolates from the 2014 outbreak. Viral replication and infectivity in SH-SY5Y were assessed using multiple assays: virus production, cytopathic effects, cellular ATP release, and VP1 capsid protein production. Similar differential neurotropism was also observed in differentiated SH-SY5Y cells, primary human neuron cultures, and a mouse paralysis model. Using the SH-SY5Y cell culture model, we determined that barriers to viral binding and entry were at least partly responsible for the differential infectivity phenotype. Transfection of genomic RNA into SH-SY5Y generated virions for all EV-D68 isolates, but only a single round of replication was observed from strains that could not directly infect SH-SY5Y. In addition to supporting virus replication and other functional studies, this cell culture model may help identify the signatures of virulence to confirm epidemiological associations between EV-D68 strains and AFM and allow for the rapid identification and characterization of emerging neurotropic strains.IMPORTANCE Since the EV-D68 outbreak during the summer of 2014, evidence of a causal link to a type of limb paralysis (AFM) has been mounting. In this article, we describe a neuronal cell culture model (SH-SY5Y cells) in which a subset of contemporary 2014 outbreak strains of EV-D68 show infectivity in neuronal cells, or neurotropism. We confirmed the difference in neurotropism in vitro using primary human neuron cell cultures and in vivo with a mouse paralysis model. Using the SH-SY5Y cell model, we determined that a barrier to viral entry is at least partly responsible for neurotropism. SH-SY5Y cells may be useful in determining if specific EV-D68 genetic determinants are associated with neuropathogenesis, and replication in this cell line could be used as rapid screening tool for identification of neurotropic EV-D68 strains. This may assist with better understanding of pathogenesis and epidemiology and with the development of potential therapies.

Whole genome sequencing, variant analysis, phylogenetics, and deep sequencing of Zika virus strains.

The recent emergence of Zika virus (ZIKV) has been concentrated in the Caribbean, Southeastern United States, and South- and Central America; resulting in travel-based cases being reported around the globe. As multi-disciplinary collaborations are combatting the ZIKV outbreak, the need to validate the sequence of existing strains has become apparent. Here, we report high-quality sequence data for multiple ZIKV strains made publicly available through the National Institutes of Health- (NIH) funded biorepository, BEI Resources (www.beiresources.org). Next-generation sequencing, 3' rapid amplification of cDNA ends (RACE), and viral genome annotation pipelines generated GenBank sequence records for 16 BEI Resources strains. Minor variants, consensus mutations, and consensus insertions/deletions were identified within the viral stocks using next-generation sequencing (NGS) and consensus changes were confirmed with Sanger sequencing. Bioinformatics analyses of the sequencing results confirm that the virus stocks available to the scientific research community through BEI Resources adequately represent the viral population diversity of ZIKV.

Conservation of Structure and Immune Antagonist Functions of Filoviral VP35 Homologs Present in Microbat Genomes.

Non-retroviral integrated RNA viral sequences (NIRVs) potentially encoding ∼280 amino acid homologs to filovirus VP35 proteins are present across the Myotis genus of bats. These are estimated to have been maintained for ∼18 million years, indicating their co-option. To address the reasons for co-option, 16 Myotis VP35s were characterized in comparison to VP35s from the extant filoviruses Ebola virus and Marburg virus, in which VP35s play critical roles in immune evasion and RNA synthesis. The Myotis VP35s demonstrated a conserved suppression of innate immune signaling, albeit with reduced potency, in either human or Myotis cells. Their attenuation reflects a lack of dsRNA binding that in the filoviral VP35s correlates with potent suppression of interferon responses. Despite divergent function, evolution has preserved in Myotis the structure of the filoviral VP35s, indicating that this structure is critical for co-opted function, possibly as a regulator of innate immune signaling.

Differing epidemiological dynamics of Chikungunya virus in the Americas during the 2014-2015 epidemic.

Chikungunya virus (CHIKV) has been detected sporadically since the 1950s and includes three distinct co-circulating genotypes. In late 2013, the Asian genotype of CHIKV was responsible for the Caribbean outbreak (CO) that rapidly became an epidemic throughout the Americas. There is a limited understanding of the molecular evolution of CHIKV in the Americas during this epidemic. We sequenced 185 complete CHIKV genomes collected mainly from Nicaragua in Central America and Florida in the United States during the 2014-2015 Caribbean/Americas epidemic. Our comprehensive phylogenetic analyses estimated the epidemic history of the Asian genotype and the recent Caribbean outbreak (CO) clade, revealed considerable genetic diversity within the CO clade, and described different epidemiological dynamics of CHIKV in the Americas. Specifically, we identified multiple introductions in both Nicaragua and Florida, with rapid local spread of viruses in Nicaragua but limited autochthonous transmission in Florida in the US. Our phylogenetic analysis also showed phylogeographic clustering of the CO clade. In addition, we identified the significant amino acid substitutions that were observed across the entire Asian genotype during its evolution and examined amino acid changes that were specific to the CO clade. Deep sequencing analysis identified specific minor variants present in clinical specimens below-consensus levels. Finally, we investigated the association between viral phylogeny and geographic/clinical metadata in Nicaragua. To date, this study represents the largest single collection of CHIKV complete genomes during the Caribbean/Americas epidemic and significantly expands our understanding of the emergence and evolution of CHIKV CO clade in the Americas.

Sequences of Zika Virus Genomes from a Pediatric Cohort in Nicaragua.

We report here the whole-genome sequence of 11 Zika virus (ZIKV) samples from six pediatric patients in Nicaragua. Serum samples were collected, and ZIKV was isolated in tissue culture. Both serum and virus isolates were sequenced. The consensus ZIKV genomes are greater than 99% identical to each other.

A draft genome sequence for the Ixodes scapularis cell line, ISE6.

Background: The tick cell line ISE6, derived from Ixodes scapularis, is commonly used for amplification and detection of arboviruses in environmental or clinical samples. Methods: To assist with sequence-based assays, we sequenced the ISE6 genome with single-molecule, long-read technology. Results: The draft assembly appears near complete based on gene content analysis, though it appears to lack some instances of repeats in this highly repetitive genome. The assembly appears to have separated the haplotypes at many loci. DNA short read pairs, used for validation only, mapped to the cell line assembly at a higher rate than they mapped to the Ixodes scapularis reference genome sequence. Conclusions: The assembly could be useful for filtering host genome sequence from sequence data obtained from cells infected with pathogens.

Genetic stability of foot-and-mouth disease virus during long-term infections in natural hosts.

Foot-and-mouth disease (FMD) is a severe infection caused by a picornavirus that affects livestock and wildlife. Persistence in ruminants is a well-documented feature of Foot-and-mouth disease virus (FMDV) pathogenesis and a major concern for disease control. Persistently infected animals harbor virus for extended periods, providing a unique opportunity to study within-host virus evolution. This study investigated the genetic dynamics of FMDV during persistent infections of naturally infected Asian buffalo. Using next-generation sequencing (NGS) we obtained 21 near complete FMDV genome sequences from 12 sub-clinically infected buffalo over a period of one year. Four animals yielded only one virus isolate and one yielded two isolates of different serotype suggesting a serial infection. Seven persistently infected animals yielded more than one virus of the same serotype showing a long-term intra-host viral genetic divergence at the consensus level of less than 2.5%. Quasi-species analysis showed few nucleotide variants and non-synonymous substitutions of progeny virus despite intra-host persistence of up to 152 days. Phylogenetic analyses of serotype Asia-1 VP1 sequences clustered all viruses from persistent animals with Group VII viruses circulating in Pakistan in 2011, but distinct from those circulating on 2008-2009. Furthermore, signature amino acid (aa) substitutions were found in the antigenically relevant VP1 of persistent viruses compared with viruses from 2008-2009. Intra-host purifying selective pressure was observed, with few codons in structural proteins undergoing positive selection. However, FMD persistent viruses did not show a clear pattern of antigenic selection. Our findings provide insight into the evolutionary dynamics of FMDV populations within naturally occurring subclinical and persistent infections that may have implications to vaccination strategies in the region.

Ross River virus envelope glycans contribute to disease through activation of the host complement system.

Mannose binding lectin (MBL) generally plays a protective role during viral infection, yet MBL-mediated complement activation promotes Ross River virus (RRV)-induced inflammatory tissue destruction, contributing to arthritis and myositis. As MBL binds to carbohydrates, we hypothesized that N-linked glycans on the RRV envelope glycoproteins act as ligands for MBL. Using a panel of RRV mutants lacking the envelope N-linked glycans, we found that MBL deposition onto infected cells was dependent on the E2 glycans. Moreover, the glycan-deficient viruses exhibited reduced disease and tissue damage in a mouse model of RRV-induced myositis compared to wild-type RRV, despite similar viral load and inflammatory infiltrates within the skeletal muscle. Instead, the reduced disease induced by glycan-deficient viruses was linked to decreased MBL deposition and complement activation within inflamed tissues. These results demonstrate that the viral N-linked glycans promote MBL deposition and complement activation onto RRV-infected cells, contributing to the development of RRV-induced myositis.

Analysis of the Aedes albopictus C6/36 genome provides insight into cell line utility for viral propagation.

Background: The 50-year-old Aedes albopictus C6/36 cell line is a resource for the detection, amplification, and analysis of mosquito-borne viruses including Zika, dengue, and chikungunya. The cell line is derived from an unknown number of larvae from an unspecified strain of Aedes albopictus mosquitoes. Toward improved utility of the cell line for research in virus transmission, we present an annotated assembly of the C6/36 genome. Results: The C6/36 genome assembly has the largest contig N50 (3.3 Mbp) of any mosquito assembly, presents the sequences of both haplotypes for most of the diploid genome, reveals independent null mutations in both alleles of the Dicer locus, and indicates a male-specific genome. Gene annotation was computed with publicly available mosquito transcript sequences. Gene expression data from cell line RNA sequence identified enrichment of growth-related pathways and conspicuous deficiency in aquaporins and inward rectifier K+ channels. As a test of utility, RNA sequence data from Zika-infected cells were mapped to the C6/36 genome and transcriptome assemblies. Host subtraction reduced the data set by 89%, enabling faster characterization of nonhost reads. Conclusions: The C6/36 genome sequence and annotation should enable additional uses of the cell line to study arbovirus vector interactions and interventions aimed at restricting the spread of human disease.

Whole-Genome Sequences of Zika Virus FLR Strains after Passage in Vero or C6/36 Cells.

We report 26 complete genomes of Zika virus (ZIKV) isolated after passaging the Zika virus strain FLR in mosquito (C6/36) and mammalian (Vero) cell lines. The consensus ZIKV genomes we recovered show greater than 99% nucleotide identify with each other and with the FLR strain used as input.

A host subtraction database for virus discovery in human cell line sequencing data.

The human cell lines HepG2, HuH-7, and Jurkat are commonly used for amplification of the RNA viruses present in environmental samples. To assist with assays by RNAseq, we sequenced these cell lines and developed a subtraction database that contains sequences expected in sequence data from uninfected cells. RNAseq data from cell lines infected with Sendai virus were analyzed to test host subtraction. The process of mapping RNAseq reads to our subtraction database vastly reduced the number non-viral reads in the dataset to allow for efficient secondary analyses.

IFN-λ4 Attenuates Antiviral Responses by Enhancing Negative Regulation of IFN Signaling.

Type III IFNs are important mediators of antiviral immunity. IFN-λ4 is a unique type III IFN because it is produced only in individuals who carry a dG allele of a genetic variant rs368234815-dG/TT. Counterintuitively, those individuals who can produce IFN-λ4, an antiviral cytokine, are also less likely to clear hepatitis C virus infection. In this study, we searched for unique functional properties of IFN-λ4 that might explain its negative effect on hepatitis C virus clearance. We used fresh primary human hepatocytes (PHHs) treated with recombinant type III IFNs or infected with Sendai virus to model acute viral infection and subsequently validated our findings in HepG2 cell line models. Endogenous IFN-λ4 protein was detectable only in Sendai virus-infected PHHs from individuals with the dG allele, where it was poorly secreted but highly functional, even at concentrations < 50 pg/ml. IFN-λ4 acted faster than other type III IFNs in inducing antiviral genes, as well as negative regulators of the IFN response, such as USP18 and SOCS1 Transient treatment of PHHs with IFN-λ4, but not IFN-λ3, caused a strong and sustained induction of SOCS1 and refractoriness to further stimulation with IFN-λ3. Our results suggest unique functional properties of IFN-λ4 that can be important in viral clearance and other clinical conditions.

Identification of Dezidougou Virus in a DAK AR 41524 Zika Virus Stock.

We report here the complete genome of a Dezidougou virus (DEZV) isolated from a passaged culture of the Zika virus strain DAK AR 41524. The consensus DEZV sequence we recovered shows 99% nucleotide similarity using BLASTN to a previously reported DEZV (accession no. JQ675604.1). The current sequence has additional repeat regions as well as a deleted repeat region, which we confirmed by Sanger sequencing, that were not present in the originally published sequence, JQ675604.1.

Initial genome sequencing of the sugarcane CP 96-1252 complex hybrid.

The CP 96-1252 cultivar of sugarcane is a complex hybrid of commercial importance. DNA was extracted from lab-grown leaf tissue and sequenced. The raw Illumina DNA sequencing results provide 101 Gbp of genome sequence reads. The dataset is available from https://www.ncbi.nlm.nih.gov/bioproject/PRJNA345486/.

The Ebola virus VP35 protein binds viral immunostimulatory and host RNAs identified through deep sequencing.

Ebola virus and Marburg virus are members of the Filovirdae family and causative agents of hemorrhagic fever with high fatality rates in humans. Filovirus virulence is partially attributed to the VP35 protein, a well-characterized inhibitor of the RIG-I-like receptor pathway that triggers the antiviral interferon (IFN) response. Prior work demonstrates the ability of VP35 to block potent RIG-I activators, such as Sendai virus (SeV), and this IFN-antagonist activity is directly correlated with its ability to bind RNA. Several structural studies demonstrate that VP35 binds short synthetic dsRNAs; yet, there are no data that identify viral immunostimulatory RNAs (isRNA) or host RNAs bound to VP35 in cells. Utilizing a SeV infection model, we demonstrate that both viral isRNA and host RNAs are bound to Ebola and Marburg VP35s in cells. By deep sequencing the purified VP35-bound RNA, we identified the SeV copy-back defective interfering (DI) RNA, previously identified as a robust RIG-I activator, as the isRNA bound by multiple filovirus VP35 proteins, including the VP35 protein from the West African outbreak strain (Makona EBOV). Moreover, RNAs isolated from a VP35 RNA-binding mutant were not immunostimulatory and did not include the SeV DI RNA. Strikingly, an analysis of host RNAs bound by wild-type, but not mutant, VP35 revealed that select host RNAs are preferentially bound by VP35 in cell culture. Taken together, these data support a model in which VP35 sequesters isRNA in virus-infected cells to avert RIG-I like receptor (RLR) activation.

Zika Virus Antagonizes Type I Interferon Responses during Infection of Human Dendritic Cells.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that is causally linked to severe neonatal birth defects, including microcephaly, and is associated with Guillain-Barre syndrome in adults. Dendritic cells (DCs) are an important cell type during infection by multiple mosquito-borne flaviviruses, including dengue virus, West Nile virus, Japanese encephalitis virus, and yellow fever virus. Despite this, the interplay between ZIKV and DCs remains poorly defined. Here, we found human DCs supported productive infection by a contemporary Puerto Rican isolate with considerable variability in viral replication, but not viral binding, between DCs from different donors. Historic isolates from Africa and Asia also infected DCs with distinct viral replication kinetics between strains. African lineage viruses displayed more rapid replication kinetics and infection magnitude as compared to Asian lineage viruses, and uniquely induced cell death. Infection of DCs with both contemporary and historic ZIKV isolates led to minimal up-regulation of T cell co-stimulatory and MHC molecules, along with limited secretion of inflammatory cytokines. Inhibition of type I interferon (IFN) protein translation was observed during ZIKV infection, despite strong induction at the RNA transcript level and up-regulation of other host antiviral proteins. Treatment of human DCs with RIG-I agonist potently restricted ZIKV replication, while type I IFN had only modest effects. Mechanistically, we found all strains of ZIKV antagonized type I IFN-mediated phosphorylation of STAT1 and STAT2. Combined, our findings show that ZIKV subverts DC immunogenicity during infection, in part through evasion of type I IFN responses, but that the RLR signaling pathway is still capable of inducing an antiviral state, and therefore may serve as an antiviral therapeutic target.