Kalani Ratnasiri

All Publications

• Single-cell RNA-seq methods to interrogate virus-host interactions. Seminars in immunopathology Ratnasiri, K., Wilk, A. J., Lee, M. J., Khatri, P., Blish, C. A. 2022

  Abstract

  The twenty-first century has seen the emergence of many epidemic and pandemic viruses, with the most recent being the SARS-CoV-2-driven COVID-19 pandemic. As obligate intracellular parasites, viruses rely on host cells to replicate and produce progeny, resulting in complex virus and host dynamics during an infection. Single-cell RNA sequencing (scRNA-seq), by enabling broad and simultaneous profiling of both host and virus transcripts, represents a powerful technology to unravel the delicate balance between host and virus. In this review, we summarize technological and methodological advances in scRNA-seq and their applications to antiviral immunity. We highlight key scRNA-seq applications that have enabled the understanding of viral genomic and host response heterogeneity, differential responses of infected versus bystander cells, and intercellular communication networks. We expect further development of scRNA-seq technologies and analytical methods, combined with measurements of additional multi-omic modalities and increased availability of publicly accessible scRNA-seq datasets, to enable a better understanding of viral pathogenesis and enhance the development of antiviral therapeutics strategies.

  View details for DOI 10.1007/s00281-022-00972-2

  View details for PubMedID 36414692

• SARS-CoV-2 infection drives an inflammatory response in human adipose tissue through infection of adipocytes and macrophages. Science translational medicine Martínez-Colón, G. J., Ratnasiri, K., Chen, H., Jiang, S., Zanley, E., Rustagi, A., Verma, R., Chen, H., Andrews, J. R., Mertz, K. D., Tzankov, A., Azagury, D., Boyd, J., Nolan, G. P., Schürch, C. M., Matter, M. S., Blish, C. A., McLaughlin, T. L. 2022: eabm9151

  Abstract

  Obesity, characterized by chronic low-grade inflammation of the adipose tissue, is associated with adverse coronavirus disease 2019 (COVID-19) outcomes, yet the underlying mechanism is unknown. To explore whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of adipose tissue contributes to pathogenesis, we evaluated COVID-19 autopsy cases and deeply profiled the response of adipose tissue to SARS-CoV-2 infection in vitro. In COVID-19 autopsy cases, we identified SARS-CoV-2 RNA in adipocytes with an associated inflammatory infiltrate. We identified two distinct cellular targets of infection: adipocytes and a subset of inflammatory adipose tissue-resident macrophages. Mature adipocytes were permissive to SARS-CoV-2 infection; although macrophages were abortively infected, SARS-CoV-2 initiated inflammatory responses within both the infected macrophages and bystander preadipocytes. These data suggest that SARS-CoV-2 infection of adipose tissue could contribute to COVID-19 severity through replication of virus within adipocytes and through induction of local and systemic inflammation driven by infection of adipose tissue-resident macrophages.

  View details for DOI 10.1126/scitranslmed.abm9151

  View details for PubMedID 36137009

• Single mosquito metatranscriptomics identifies vectors, emerging pathogens and reservoirs in one assay. eLife Batson, J., Dudas, G., Haas-Stapleton, E., Kistler, A. L., Li, L. M., Logan, P., Ratnasiri, K., Retallack, H. 2021; 10

  Abstract

  Mosquitoes are major infectious disease-carrying vectors. Assessment of current and future risks associated with the mosquito population requires knowledge of the full repertoire of pathogens they carry, including novel viruses, as well as their blood meal sources. Unbiased metatranscriptomic sequencing of individual mosquitoes offers a straightforward, rapid and quantitative means to acquire this information. Here, we profile 148 diverse wild-caught mosquitoes collected in California and detect sequences from eukaryotes, prokaryotes, 24 known and 46 novel viral species. Importantly, sequencing individuals greatly enhanced the value of the biological information obtained. It allowed us to a) speciate host mosquito, b) compute the prevalence of each microbe and recognize a high frequency of viral co-infections, c) associate animal pathogens with specific blood meal sources, and d) apply simple co-occurrence methods to recover previously undetected components of highly prevalent segmented viruses. In the context of emerging diseases, where knowledge about vectors, pathogens, and reservoirs is lacking, the approaches described here can provide actionable information for public health surveillance and intervention decisions.

  View details for DOI 10.7554/eLife.68353

  View details for PubMedID 33904402

• Dengue virus NS1 cytokine-independent vascular leak is dependent on endothelial glycocalyx components PLOS PATHOGENS Glasner, D. R., Ratnasiri, K., Puerta-Guardo, H., Espinosa, D. A., Beatty, P., Harris, E. 2017; 13 (11): e1006673

  Abstract

  Dengue virus (DENV) is the most prevalent, medically important mosquito-borne virus. Disease ranges from uncomplicated dengue to life-threatening disease, characterized by endothelial dysfunction and vascular leakage. Previously, we demonstrated that DENV nonstructural protein 1 (NS1) induces endothelial hyperpermeability in a systemic mouse model and human pulmonary endothelial cells, where NS1 disrupts the endothelial glycocalyx-like layer. NS1 also triggers release of inflammatory cytokines from PBMCs via TLR4. Here, we examined the relative contributions of inflammatory mediators and endothelial cell-intrinsic pathways. In vivo, we demonstrated that DENV NS1 but not the closely-related West Nile virus NS1 triggers localized vascular leak in the dorsal dermis of wild-type C57BL/6 mice. In vitro, we showed that human dermal endothelial cells exposed to DENV NS1 do not produce inflammatory cytokines (TNF-α, IL-6, IL-8) and that blocking these cytokines does not affect DENV NS1-induced endothelial hyperpermeability. Further, we demonstrated that DENV NS1 induces vascular leak in TLR4- or TNF-α receptor-deficient mice at similar levels to wild-type animals. Finally, we blocked DENV NS1-induced vascular leak in vivo using inhibitors targeting molecules involved in glycocalyx disruption. Taken together, these data indicate that DENV NS1-induced endothelial cell-intrinsic vascular leak is independent of inflammatory cytokines but dependent on endothelial glycocalyx components.

  View details for DOI 10.1371/journal.ppat.1006673

  View details for Web of Science ID 000416888500010

  View details for PubMedID 29121099

  View details for PubMedCentralID PMC5679539

• Intermittent preventive treatment for malaria in pregnancy and infant growth: a mediation analysis of a randomised trial. EBioMedicine Tong, Y., Ratnasiri, K., Hanif, S., Nguyen, A. T., Roh, M. E., Dorsey, G., Kakuru, A., Jagannathan, P., Benjamin-Chung, J. 2024; 109: 105397

  Abstract

  Intermittent preventive treatment for malaria in pregnancy (IPTp) can improve birth outcomes, but whether it confers benefits to postnatal growth is unclear. We investigated the effect of IPTp on infant growth in Uganda and its pathways of effects using causal mediation analyses.We analysed data from 633 infants born to mothers enrolled in a randomised trial of monthly IPTp with dihydroartemisinin-piperaquine (DP) vs. sulfadoxine-pyrimethamine (SP) (NCT02793622). Weight and length were measured from 0 to 12 months of age. Using generalised linear models, we estimated effects of DP vs. SP on gravidity-stratified mean length-for-age (LAZ) and weight-for-length Z-scores (WLZ). We investigated mediation by placental malaria, gestational weight change, maternal anaemia, maternal inflammation-related proteins, preterm birth, birth length, and birth weight. Mediation models adjusted for infant sex, gravidity, gestational age at enrolment, maternal age, maternal parasitaemia at enrolment, education, and wealth.SP increased mean LAZ by 0.18-0.28 Z from birth through age 4 months compared to DP, while DP increased mean WLZ by 0.11-0.28 Z from 2 to 8 months compared to SP among infants of multigravidae; at these ages, confidence intervals for mean differences excluded 0. We did not observe differences among primigravida. Mediators of SP included birth weight, birth length, maternal stem cell factor, and DNER. Mediators of DP included placental malaria and birth length, maternal IL-18, CDCP1, and CD6 at delivery.In high malaria transmission settings, this exploratory study suggests different IPTp regimens may influence infant growth among multigravidae, potentially through distinct pathways, in the exclusive breastfeeding period, when few other interventions are available.Stanford Center for Innovation in Global Health, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bill & Melinda Gates Foundation.

  View details for DOI 10.1016/j.ebiom.2024.105397

  View details for PubMedID 39418986

• Systems immunology of transcriptional responses to viral infection identifies conserved antiviral pathways across macaques and humans. Cell reports Ratnasiri, K., Zheng, H., Toh, J., Yao, Z., Duran, V., Donato, M., Roederer, M., Kamath, M., Todd, J. M., Gagne, M., Foulds, K. E., Francica, J. R., Corbett, K. S., Douek, D. C., Seder, R. A., Einav, S., Blish, C. A., Khatri, P. 2024; 43 (2): 113706

  Abstract

  Viral pandemics and epidemics pose a significant global threat. While macaque models of viral disease are routinely used, it remains unclear how conserved antiviral responses are between macaques and humans. Therefore, we conducted a cross-species analysis of transcriptomic data from over 6,088 blood samples from macaques and humans infected with one of 31 viruses. Our findings demonstrate that irrespective of primate or viral species, there are conserved antiviral responses that are consistent across infection phase (acute, chronic, or latent) and viral genome type (DNA or RNA viruses). Leveraging longitudinal data from experimental challenges, we identify virus-specific response kinetics such as host responses to Coronaviridae and Orthomyxoviridae infections peaking 1-3 days earlier than responses to Filoviridae and Arenaviridae viral infections. Our results underscore macaque studies as a powerful tool for understanding viral pathogenesis and immune responses that translate to humans, with implications for viral therapeutic development and pandemic preparedness.

  View details for DOI 10.1016/j.celrep.2024.113706

  View details for PubMedID 38294906

• Upper airway gene expression shows a more robust adaptive immune response to SARS-CoV-2 in children NATURE COMMUNICATIONS Mick, E., Tsitsiklis, A., Spottiswoode, N., Caldera, S., Serpa, P., Detweiler, A. M., Neff, N., Pisco, A., Li, L. M., Retallack, H., Ratnasiri, K., Williamson, K. M., Soesanto, V., Simoes, E. F., Smith, C., Abuogi, L., Kistler, A., Wagner, B. D., DeRisi, J. L., Ambroggio, L., Mourani, P. M., Langelier, C. R. 2022; 13 (1): 3937

  Abstract

  Unlike other respiratory viruses, SARS-CoV-2 disproportionately causes severe disease in older adults whereas disease burden in children is lower. To investigate whether differences in the upper airway immune response may contribute to this disparity, we compare nasopharyngeal gene expression in 83 children (<19-years-old; 38 with SARS-CoV-2, 11 with other respiratory viruses, 34 with no virus) and 154 older adults (>40-years-old; 45 with SARS-CoV-2, 28 with other respiratory viruses, 81 with no virus). Expression of interferon-stimulated genes is robustly activated in both children and adults with SARS-CoV-2 infection compared to the respective non-viral groups, with only subtle distinctions. Children, however, demonstrate markedly greater upregulation of pathways related to B cell and T cell activation and proinflammatory cytokine signaling, including response to TNF and production of IFNγ, IL-2 and IL-4. Cell type deconvolution confirms greater recruitment of B cells, and to a lesser degree macrophages, to the upper airway of children. Only children exhibit a decrease in proportions of ciliated cells, among the primary targets of SARS-CoV-2, upon infection. These findings demonstrate that children elicit a more robust innate and especially adaptive immune response to SARS-CoV-2 in the upper airway that likely contributes to their protection from severe disease in the lower airway.

  View details for DOI 10.1038/s41467-022-31600-0

  View details for Web of Science ID 000825867200012

  View details for PubMedID 35803954

  View details for PubMedCentralID PMC9263813

• Quantitative reverse transcription PCR assay to detect a genetic marker of pyrethroid resistance in Culex mosquitoes. PloS one Hager, K. M., Gaona, E., Kistler, A., Ratnasiri, K., Retallack, H., Barretto, M., Wheeler, S. S., Hoover, C. M., Haas-Stapleton, E. J. 2022; 17 (8): e0252498

  Abstract

  Pyrethroid insecticides are widely used to control mosquitoes that transmit pathogens such as West Nile virus (WNV) to people. Single nucleotide polymorphisms (SNP) in the knockdown resistance locus (kdr) of the voltage gated sodium channel (Vgsc) gene in Culex mosquitoes are associated with knockdown resistance to pyrethroids. RNAseq was used to sequence the coding region of Vgsc for Culex tarsalis Coquillett and Culex erythrothorax Dyar, two WNV vectors. The cDNA sequences were used to develop a quantitative reverse transcriptase PCR assay that detects the L1014F kdr mutation in the Vgsc. Because this locus is conserved, the assay was used successfully in six Culex spp. The resulting Culex RTkdr assay was validated using quantitative PCR and sequencing of PCR products. The accuracy of the Culex RTkdr assay was 99%. The L1014F kdr mutation associated with pyrethroid resistance was more common among Cx. pipiens than other Culex spp. and was more prevalent in mosquitoes collected near farmland. The Culex RTkdr assay takes advantage of the RNA that vector control agencies routinely isolate to assess arbovirus prevalence in mosquitoes. We anticipate that public health and vector control agencies may employ the Culex RTkdr assay to define the geographic distribution of the L1014F kdr mutation in Culex species and improve the monitoring of insecticide resistance that will ultimately contribute to effective control of Culex mosquitoes.

  View details for DOI 10.1371/journal.pone.0252498

  View details for PubMedID 35939507

• Upper airway gene expression reveals suppressed immune responses to SARS-CoV-2 compared with other respiratory viruses NATURE COMMUNICATIONS Mick, E., Kamm, J., Pisco, A., Ratnasiri, K., Babik, J. M., Castaneda, G., DeRisi, J. L., Detweiler, A. M., Hao, S. L., Kangelaris, K. N., Kumar, G., Li, L. M., Mann, S. A., Neff, N., Prasad, P. A., Serpa, P., Shah, S. J., Spottiswoode, N., Tan, M., Calfee, C. S., Christenson, S. A., Kistler, A., Langelier, C. 2020; 11 (1): 5854

  Abstract

  SARS-CoV-2 infection is characterized by peak viral load in the upper airway prior to or at the time of symptom onset, an unusual feature that has enabled widespread transmission of the virus and precipitated a global pandemic. How SARS-CoV-2 is able to achieve high titer in the absence of symptoms remains unclear. Here, we examine the upper airway host transcriptional response in patients with COVID-19 (n = 93), other viral (n = 41) or non-viral (n = 100) acute respiratory illnesses (ARIs). Compared with other viral ARIs, COVID-19 is characterized by a pronounced interferon response but attenuated activation of other innate immune pathways, including toll-like receptor, interleukin and chemokine signaling. The IL-1 and NLRP3 inflammasome pathways are markedly less responsive to SARS-CoV-2, commensurate with a signature of diminished neutrophil and macrophage recruitment. This pattern resembles previously described distinctions between symptomatic and asymptomatic viral infections and may partly explain the propensity for pre-symptomatic transmission in COVID-19. We further use machine learning to build 27-, 10- and 3-gene classifiers that differentiate COVID-19 from other ARIs with AUROCs of 0.981, 0.954 and 0.885, respectively. Classifier performance is stable across a wide range of viral load, suggesting utility in mitigating false positive or false negative results of direct SARS-CoV-2 tests.

  View details for DOI 10.1038/s41467-020-19587-y

  View details for Web of Science ID 000594731600005

  View details for PubMedID 33203890

  View details for PubMedCentralID PMC7673985

• Clinical features, diagnostics, and outcomes of patients presenting with acute respiratory illness: A retrospective cohort study of patients with and without COVID-19. EClinicalMedicine Shah, S. J., Barish, P. N., Prasad, P. A., Kistler, A. n., Neff, N. n., Kamm, J. n., Li, L. M., Chiu, C. Y., Babik, J. M., Fang, M. C., Kangelaris, K. N., Langelier, C. n., Abe-Jones, Y. n., Alipanah, N. n., Alvarez, F. N., Botvinnik, O. B., Castaneda, G. n., Dadasovich, R. M., Davis, J. n., Deng, X. n., DeRisi, J. L., Detweiler, A. M., Federman, S. n., Haliburton, J. n., Hao, S. n., Kerkhoff, A. D., Kumar, G. R., Malcolm, K. B., Mann, S. A., Martinez, S. n., Mary, R. K., Mick, E. n., Mwakibete, L. n., Najafi, N. n., Peluso, M. J., Phelps, M. n., Pisco, A. O., Ratnasiri, K. n., Rubio, L. A., Sellas, A. n., Sherwood, K. D., Sheu, J. n., Spottiswoode, N. n., Tan, M. n., Yu, G. n. 2020: 100518

  Abstract

  Most data on the clinical presentation, diagnostics, and outcomes of patients with COVID-19 have been presented as case series without comparison to patients with other acute respiratory illnesses.We examined emergency department patients between February 3 and March 31, 2020 with an acute respiratory illness who were tested for SARS-CoV-2. We determined COVID-19 status by PCR and metagenomic next generation sequencing (mNGS). We compared clinical presentation, diagnostics, treatment, and outcomes.Among 316 patients, 33 tested positive for SARS-CoV-2; 31 without COVID-19 tested positive for another respiratory virus. Among patients with additional viral testing (27/33), no SARS-CoV-2 co-infections were identified. Compared to those who tested negative, patients with COVID-19 reported longer symptoms duration (median 7d vs. 3d, p < 0.001). Patients with COVID-19 were more often hospitalized (79% vs. 56%, p = 0.014). When hospitalized, patients with COVID-19 had longer hospitalizations (median 10.7d vs. 4.7d, p < 0.001) and more often developed ARDS (23% vs. 3%, p < 0.001). Most comorbidities, medications, symptoms, vital signs, laboratories, treatments, and outcomes did not differ by COVID-19 status.While we found differences in clinical features of COVID-19 compared to other acute respiratory illnesses, there was significant overlap in presentation and comorbidities. Patients with COVID-19 were more likely to be admitted to the hospital, have longer hospitalizations and develop ARDS, and were unlikely to have co-existent viral infections.National Center for Advancing Translational Sciences, National Heart Lung Blood Institute, National Institute of Allergy and Infectious Diseases, Chan Zuckerberg Biohub, Chan Zuckerberg Initiative.

  View details for DOI 10.1016/j.eclinm.2020.100518

  View details for PubMedID 32864588

  View details for PubMedCentralID PMC7447618

• Flavivirus NS1 Triggers Tissue-Specific Vascular Endothelial Dysfunction Reflecting Disease Tropism CELL REPORTS Puerta-Guardo, H., Glasner, D. R., Espinosa, D. A., Biering, S. B., Patana, M., Ratnasiri, K., Wang, C., Beatty, P., Harris, E. 2019; 26 (6): 1598-+

  Abstract

  Flaviviruses cause systemic or neurotropic-encephalitic pathology in humans. The flavivirus nonstructural protein 1 (NS1) is a secreted glycoprotein involved in viral replication, immune evasion, and vascular leakage during dengue virus infection. However, the contribution of secreted NS1 from related flaviviruses to viral pathogenesis remains unknown. Here, we demonstrate that NS1 from dengue, Zika, West Nile, Japanese encephalitis, and yellow fever viruses selectively binds to and alters permeability of human endothelial cells from lung, dermis, umbilical vein, brain, and liver in vitro and causes tissue-specific vascular leakage in mice, reflecting the pathophysiology of each flavivirus. Mechanistically, each flavivirus NS1 leads to differential disruption of endothelial glycocalyx components, resulting in endothelial hyperpermeability. Our findings reveal the capacity of a secreted viral protein to modulate endothelial barrier function in a tissue-specific manner both in vitro and in vivo, potentially influencing virus dissemination and pathogenesis and providing targets for antiviral therapies and vaccine development.

  View details for DOI 10.1016/j.celrep.2019.01.036

  View details for Web of Science ID 000457709200019

  View details for PubMedID 30726741