Defining new therapeutics using a more immunocompetent mouse model of antibody-enhanced dengue virus infection

With over 3.5 billion people at risk and approximately 390 million human infections per year, dengue virus (DENV) disease strains health care resources worldwide. Previously, we and others established models for DENV pathogenesis in mice that completely lack subunits of the receptors (Ifnar and Ifngr) for type I and type II interferon (IFN) signaling; however, the utility of these models is limited by the pleotropic effect of these cytokines on innate and adaptive immune system development and function. Here, we demonstrate that the specific deletion of Ifnar expression on subsets of murine myeloid cells (LysM Cre(+) Ifnar(flox/flox) [denoted as Ifnar(f/f) herein]) resulted in enhanced DENV replication in vivo. The administration of subneutralizing amounts of cross-reactive anti-DENV monoclonal antibodies to LysM Cre(+) Ifnar(f/f) mice prior to infection with DENV serotype 2 or 3 resulted in antibody-dependent enhancement (ADE) of infection with many of the characteristics associated with severe DENV disease in humans, including plasma leakage, hypercytokinemia, liver injury, hemoconcentration, and thrombocytopenia. Notably, the pathogenesis of severe DENV-2 or DENV-3 infection in LysM Cre(+) Ifnar(f/f) mice was blocked by pre- or postexposure administration of a bispecific dual-affinity retargeting molecule (DART) or an optimized RIG-I receptor agonist that stimulates innate immune responses. Our findings establish a more immunocompetent animal model of ADE of infection with multiple DENV serotypes in which disease is inhibited by treatment with broad-spectrum antibody derivatives or innate immune stimulatory agents

Genetic mechanisms of critical illness in COVID-19.

Host-mediated lung inflammation is present1, and drives mortality2, in the critical illness caused by coronavirus disease 2019 (COVID-19). Host genetic variants associated with critical illness may identify mechanistic targets for therapeutic development3. Here we report the results of the GenOMICC (Genetics Of Mortality In Critical Care) genome-wide association study in 2,244 critically ill patients with COVID-19 from 208 UK intensive care units. We have identified and replicated the following new genome-wide significant associations: on chromosome 12q24.13 (rs10735079, P = 1.65 × 10-8) in a gene cluster that encodes antiviral restriction enzyme activators (OAS1, OAS2 and OAS3); on chromosome 19p13.2 (rs74956615, P = 2.3 × 10-8) near the gene that encodes tyrosine kinase 2 (TYK2); on chromosome 19p13.3 (rs2109069, P = 3.98 ×  10-12) within the gene that encodes dipeptidyl peptidase 9 (DPP9); and on chromosome 21q22.1 (rs2236757, P = 4.99 × 10-8) in the interferon receptor gene IFNAR2. We identified potential targets for repurposing of licensed medications: using Mendelian randomization, we found evidence that low expression of IFNAR2, or high expression of TYK2, are associated with life-threatening disease; and transcriptome-wide association in lung tissue revealed that high expression of the monocyte-macrophage chemotactic receptor CCR2 is associated with severe COVID-19. Our results identify robust genetic signals relating to key host antiviral defence mechanisms and mediators of inflammatory organ damage in COVID-19. Both mechanisms may be amenable to targeted treatment with existing drugs. However, large-scale randomized clinical trials will be essential before any change to clinical practice

Inter- and intra-lineage genetic diversity of wild-type Zika viruses reveals both common and distinctive nucleotide variants and clusters of genomic diversity

ABSTRACTZika virus (ZIKV) strains belong to the East African, West African, and Asian/American phylogenetic lineages. RNA viruses, like ZIKV, exist as populations of genetically-related sequences whose heterogeneity may impact viral fitness, evolution, and virulence. Genetic diversity of representative ZIKVs from each lineage was examined using next generation sequencing (NGS) paired with downstream entropy and single nucleotide variant (SNV) analysis. Comparisons showed that inter-lineage diversity was statistically supported, while intra-lineage diversity varied. Intra-lineage diversity was significant for East but not West Africa strains. Furthermore, intra-lineage diversity for the Asian/American lineage was not supported for human serum isolates; however, a placenta isolate differed significantly. Relative entropy values were higher in the pre-membrane/membrane (prM/M) gene of several ZIKV strains. Additionally, the East African lineage contained a greater number of synonymous SNVs, while a greater number of non-synonymous SNVs were identified for American strains. Further, inter-lineage SNVs were dispersed throughout the genome, whereas intra-lineage non-synonymous SNVs for Asian/American strains clustered within prM/M and NS1 gene. This comprehensive analysis of ZIKV genetic diversity provides a repository of SNV positions across lineages. We posit that increased non-synonymous SNV populations and increased relative genetic diversity of the prM/M and NS1 proteins provides more evidence for their role in ZIKV virulence and fitness

Using next generation sequencing to study the genetic diversity of candidate live attenuated zika vaccines

This research was funded in part by a Gillson-Longenbaugh Foundation grant to A.D.T.B. P.-Y.S. was supported in part by NIH grants AI142759, AI145617, AI127744, AI136126, and UL1TR001439, and awards from the Kleberg Foundation, John S. Dunn Foundation, Amon G. Carter Foundation, and Gillson-Longenbaugh Foundation.University of Texas Medical Branch. Department of Microbiology and Immunology. Galveston, TX, USA / Walter Reed Army Institute of Research. Viral Disease Branch. Silver Spring, MD, USA.University of Texas Medical Branch. Department of Biochemistry and Molecular Biology. Galveston, TX, USA / Chinese Academy of Sciences. Wuhan Institute of Virology. Wuhan, China.University of Texas Medical Branch. Department of Biochemistry and Molecular Biology. Galveston, TX, USA / Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.University of Texas Medical Branch. Department of Biochemistry and Molecular Biology. Galveston, TX, USA.University of Texas Medical Branch. Institute for Human Infections and Immunity. Sealy Institute for Vaccine Sciences. Department of Pathology. Galveston, TX, USA.University of Texas Medical Branch. Institute for Human Infections and Immunity. Sealy Institute for Vaccine Sciences. Department of Pathology. Galveston, TX, USA.University of Texas Medical Branch. Department of Biochemistry and Molecular Biology. Galveston, TX, USA.Abstract: Zika virus (ZIKV) is a mosquito-transmitted positive-sense RNA virus in the family Flaviviridae. Candidate live-attenuated vaccine (LAV) viruses with engineered deletions in the 3’ untranslated region (UTR) provide immunity and protection in animal models of ZIKV infection, and phenotypic studies show that LAVs retain protective abilities following in vitro passage. The present study investigated the genetic diversity of wild-type (WT) parent ZIKV and its candidate LAVs using next generation sequencing analysis of five sequential in vitro passages. The results show that genomic entropy of WT ZIKV steadily increases during in vitro passage, whereas that of LAVs also increased by passage number five but was variable throughout passaging. Additionally, clusters of single nucleotide variants (SNVs) were found to be present in the pre-membrane/membrane (prM), envelope (E), nonstructural protein NS1 (NS1), and other nonstructural protein genes, depending on the specific deletion, whereas in the parent WT ZIKV, they are more abundant in prM and NS1. Ultimately, both the parental WT and LAV derivatives increase in genetic diversity, with evidence of adaptation following passage

DENV-4 703–4 produces sustained viremia and disseminated infection in multiple organs.

<p>Six-week-old AG129 were inoculated with 7.3 log₁₀ pfu DENV-4 703–4. On days 1 (n = 8), 2 (n = 8), and 3 (n = 7) post-infection, animals were sacrificed and serum and organs harvested. The organs were weighed, homogenized and virus load in all samples determined by culture. The results shown for (A) serum, (B) liver, (C) spleen, (D) large intestine and (E) brain are combined from two independent experiments. Each symbol represents an individual sample titer. Serum titers are expressed as log₁₀ pfu/ml and organ titers as log₁₀ pfu/g of tissue. Horizontal lines represent the mean daily titer.; *P<0.05 by ANOVA with Tukey’s multiple comparison test.</p

DENV-4 703–4 infection produces leukopenia and thrombocytopenia.

<p>Six-to-eight week old AG129 (N = 8) were inoculated with 7.3 log₁₀ pfu DENV-4 703–4 or served as uninfected controls (N = 10). Blood was collected on days 1, 2 and 3 post-infection and samples were analyzed using a HEMAVET950TS. Bars represent the mean daily value from one experiment performed with groups of 2–4 mice. Asterisks above the 703-4-infected bars are significant (Student’s t test) compared to control values.</p

DENV-4 703–4 infection is lethal in AG129 mice.

<p>(A) Kaplan-Meier survival curves of 6 week old mice inoculated with 7.3 log₁₀ pfu (■; n = 8) or 6.3 log10 pfu (●; n = 8) and 18 week old mice inoculated with 7.3 log₁₀ pfu (▲n = 8) DENV-4 703–4 by i.p. injection. Mice were monitored daily for 4 weeks. Morbidity among the 6 week old (B) and 18 week old (C) animals infected with 7.3 log₁₀ pfu, as measured by weight loss. Values are mean (±SD) percent weight of initial weight.</p