Label-free and amplification-free viral RNA quantification from primate biofluids using a trapping-assisted optofluidic nanopore platform.

Viral outbreaks can cause widespread disruption, creating the need for diagnostic tools that provide high performance and sample versatility at the point of use with moderate complexity. Current gold standards such as PCR and rapid antigen tests fall short in one or more of these aspects. Here, we report a label-free and amplification-free nanopore sensor platform that overcomes these challenges via direct detection and quantification of viral RNA in clinical samples from a variety of biological fluids. The assay uses an optofluidic chip that combines optical waveguides with a fluidic channel and integrates a solid-state nanopore for sensing of individual biomolecules upon translocation through the pore. High specificity and low limit of detection are ensured by capturing RNA targets on microbeads and collecting them by optical trapping at the nanopore location where targets are released and rapidly detected. We use this device for longitudinal studies of the viral load progression for Zika and Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infections in marmoset and baboon animal models, respectively. The up to million-fold trapping-based target concentration enhancement enables amplification-free RNA quantification across the clinically relevant concentration range down to the assay limit of RT-qPCR as well as cases in which PCR failed. The assay operates across all relevant biofluids, including semen, urine, and whole blood for Zika and nasopharyngeal and throat swab, rectal swab, and bronchoalveolar lavage for SARS-CoV-2. The versatility, performance, simplicity, and potential for full microfluidic integration of the amplification-free nanopore assay points toward a unique approach to molecular diagnostics for nucleic acids, proteins, and other targets.

Protective efficacy of a Zika purified inactivated virus vaccine candidate during pregnancy in marmosets.

Zika virus (ZIKV) infection during pregnancy poses significant threats to maternal and fetal health, leading to intrauterine fetal demise and severe developmental malformations that constitute congenital Zika syndrome (CZS). As such, the development of a safe and effective ZIKV vaccine is a critical public health priority. However, the safety and efficacy of such a vaccine during pregnancy remain uncertain. Historically, the conduct of clinical trials in pregnant women has been challenging. Therefore, clinically relevant animal pregnancy models are in high demand for testing vaccine efficacy. We previously reported that a marmoset pregnancy model of ZIKV infection consistently demonstrated vertical transmission from mother to fetus during pregnancy. Using this marmoset model, we also showed that vertical transmission could be prevented by pre-pregnancy vaccination with Zika purified inactivated virus (ZPIV) vaccine. Here, we further examined the efficacy of ZPIV vaccination during pregnancy. Vaccination during pregnancy elicited virus neutralizing antibody responses that were comparable to those elicited by pre-pregnancy vaccination. Vaccination also reduced placental pathology, viral burden and vertical transmission of ZIKV during pregnancy, without causing adverse effects. These results provide key insights into the safety and efficacy of ZPIV vaccination during pregnancy and demonstrate positive effects of vaccination on the reduction of ZIKV infection, an important advance in preparedness for future ZIKV outbreaks.

Impact of prior dengue virus infection on Zika virus infection during pregnancy in marmosets.

Zika virus (ZIKV) infection during pregnancy causes severe developmental defects in newborns, termed congenital Zika syndrome (CZS). Factors contributing to a surge in ZIKV-associated CZS are poorly understood. One possibility is that ZIKV may exploit the antibody-dependent enhancement of infection mechanism, mediated by cross-reactive antibodies from prior dengue virus (DENV) infection, which may exacerbate ZIKV infection during pregnancy. In this study, we investigated the impact of prior DENV infection or no DENV infection on ZIKV pathogenesis during pregnancy in a total of four female common marmosets with five or six fetuses per group. The results showed that negative-sense viral RNA copies increased in the placental and fetal tissues of DENV-immune dams but not in DENV-naïve dams. In addition, viral proteins were prevalent in endothelial cells, macrophages, and neonatal Fc receptor-expressing cells in the placental trabeculae and in neuronal cells in the brains of fetuses from DENV-immune dams. DENV-immune marmosets maintained high titers of cross-reactive ZIKV-binding antibodies that were poorly neutralizing, raising the possibility that these antibodies might be involved in the exacerbation of ZIKV infection. These findings need to be verified in a larger study, and the mechanism involved in the exacerbation of ZIKV infection in DENV-immune marmosets needs further investigation. However, the results suggest a potential negative impact of preexisting DENV immunity on subsequent ZIKV infection during pregnancy in vivo.

Transcriptional induction of TGF-ß1 and endothelial-to-mesenchymal transition cell markers in human umbilical vein endothelial cells by Ebola virus infection.

BACKGROUND: Ebola virus (EBOV) causes a serious hemorrhagic disease in humans, with a mortality rate of up to 80%. Despite significant achievements in the past decades elucidating the pathogenesis of EBOV, there is still much to be elucidated about the cell type-specific host response and their functional roles during infection. OBJECTIVE: This study aimed to gain insight into cell type-specific host responses to EBOV infection. METHODS: Real-time RT-qPCR analysis was used to identify host transcriptional changes in epithelial Caco-2 cells and endothelial HUVECs by EBOV infection. RESULTS: EBOV efficiently infected to both Caco-2 cells and HUVECs, depending on the time of infection. However, changes in the transcriptional levels of several host cellular genes following viral infection showed significant differences between Caco-2 cells and HUVECs. EBOV infection increases the transcription of TGF-ß1, a key factor in epithelium-to-mesenchyme transition (EMT), only in HUVECs, but not in Caco-2 cells. This upregulation in turn induces the transcription of other EMT signaling molecules such as snail, slug and MMP9, ultimately leading to endothelial-to-mesenchymal transition (EndMT). Furthermore, this EndMT process appears to be associated with increased transcription of stem-cell markers such as Klf4, Sox2 and Oct4. However, most of these transcriptional changes due to EBOV infection did not occur in Caco-2 cells, suggesting that EMT or EndMT by EBOV infection is cell type-specific. CONCLUSION: We propose that EBOV infection induces the expression of TGF-ß1-mediated signals in endothelial HUVECs, resulting in EndMT. This could provide broader information to elucidate the pathogenesis of Ebola virus disease.

Molecular Approaches for the Validation of the Baboon as a Nonhuman Primate Model for the Study of Zika Virus Infection.

Nonhuman primates (NHP) are particularly important for modeling infections with viruses that do not naturally replicate in rodent cells. Zika virus (ZIKV) has been responsible for sporadic epidemics, but in 2015 a disseminated outbreak of ZIKV resulted in the World Health Organization declaring it a global health emergency. Since the advent of this last epidemic, several NHP species, including the baboon, have been utilized for modeling and understanding the complications of ZIKV infection in humans; several health issues related to the outcome of infection have not been resolved yet and require further investigation. This study was designed to validate, in baboons, the molecular signatures that have previously been identified in ZIKV-infected humans and macaque models. We performed a comprehensive molecular analysis of baboons during acute ZIKV infection, including flow cytometry, cytokine, immunological, and transcriptomic analyses. We show here that, similar to most human cases, ZIKV infection of male baboons tends to be subclinical, but is associated with a rapid and transient antiviral interferon-based response signature that induces a detectable humoral and cell-mediated immune response. This immunity against the virus protects animals from challenge with a divergent ZIKV strain, as evidenced by undetectable viremia but clear anamnestic responses. These results provide additional support for the use of baboons as an alternative animal model to macaques and validate omic techniques that could help identify the molecular basis of complications associated with ZIKV infections in humans.

Efficacy of an inactivated Zika vaccine against virus infection during pregnancy in mice and marmosets.

Zika virus (ZIKV) is a mosquito-borne arbovirus that can cause severe congenital birth defects. The utmost goal of ZIKV vaccines is to prevent both maternal-fetal infection and congenital Zika syndrome. A Zika purified inactivated virus (ZPIV) was previously shown to be protective in non-pregnant mice and rhesus macaques. In this study, we further examined the efficacy of ZPIV against ZIKV infection during pregnancy in immunocompetent C57BL6 mice and common marmoset monkeys (Callithrix jacchus). We showed that, in C57BL/6 mice, ZPIV significantly reduced ZIKV-induced fetal malformations. Protection of fetuses was positively correlated with virus-neutralizing antibody levels. In marmosets, the vaccine prevented vertical transmission of ZIKV and elicited neutralizing antibodies that remained above a previously determined threshold of protection for up to 18 months. These proof-of-concept studies demonstrate ZPIV's protective efficacy is both potent and durable and has the potential to prevent the harmful consequence of ZIKV infection during pregnancy.

Early post-infection treatment of SARS-CoV-2 infected macaques with human convalescent plasma with high neutralizing activity reduces lung inflammation.

Early in the SARS-CoV-2 pandemic, there was a high level of optimism based on observational studies and small controlled trials that treating hospitalized patients with convalescent plasma from COVID-19 survivors (CCP) would be an important immunotherapy. However, as more data from controlled trials became available, the results became disappointing, with at best moderate evidence of efficacy when CCP with high titers of neutralizing antibodies was used early in infection. To better understand the potential therapeutic efficacy of CCP, and to further validate SARS-CoV-2 infection of macaques as a reliable animal model for testing such strategies, we inoculated 12 adult rhesus macaques with SARS-CoV-2 by intratracheal and intranasal routes. One day later, 8 animals were infused with pooled human CCP with a high titer of neutralizing antibodies (RVPN NT 50 value of 3,003), while 4 control animals received normal human plasma. Animals were monitored for 7 days. Animals treated with CCP had detectable levels of antiviral antibodies after infusion. In comparison to the control animals, they had similar levels of virus replication in the upper and lower respiratory tract, but had significantly reduced interstitial pneumonia, as measured by comprehensive lung histology. By highlighting strengths and weaknesses, data of this study can help to further optimize nonhuman primate models to provide proof-of-concept of intervention strategies, and guide the future use of convalescent plasma against SARS-CoV-2 and potentially other newly emerging respiratory viruses. AUTHOR SUMMARY: The results of treating SARS-CoV-2 infected hospitalized patients with COVID-19 convalescent plasma (CCP), collected from survivors of natural infection, have been disappointing. The available data from various studies indicate at best moderate clinical benefits only when CCP with high titer of neutralizing antibodies was infused early in infection. The macaque model of SARS-CoV-2 infection can be useful to gain further insights in the value of CCP therapy. In this study, animals were infected with SARS-CoV-2 and the next day, were infused with pooled human convalescent plasma, selected to have a very high titer of neutralizing antibodies. While administration of CCP did not result in a detectable reduction in virus replication in the respiratory tract, it significantly reduced lung inflammation. These data, combined with the results of monoclonal antibody studies, emphasize the need to use products with high titers of neutralizing antibodies, and guide the future development of CCP-based therapies.

Early treatment with a combination of two potent neutralizing antibodies improves clinical outcomes and reduces virus replication and lung inflammation in SARS-CoV-2 infected macaques.

There is an urgent need for effective therapeutic interventions against SARS-CoV-2, including new variants that continue to arise. Neutralizing monoclonal antibodies have shown promise in clinical studies. We investigated the therapeutic efficacy of a combination of two potent monoclonal antibodies, C135-LS and C144-LS that carry half-life extension mutations, in the rhesus macaque model of COVID-19. Twelve young adult macaques (three groups of four animals) were inoculated intranasally and intra-tracheally with a high dose of SARS-CoV-2 and 24 hours later, treated intravenously with a high (40 mg/kg) or low (12 mg/kg) dose of the C135-LS and C144-LS antibody combination, or a control monoclonal antibody. Animals were monitored for 7 days. Compared to the control animals, animals treated with either dose of the anti-SARS-CoV-2 antibodies showed similarly improved clinical scores, lower levels of virus replication in upper and lower respiratory tract, and significantly reduced interstitial pneumonia, as measured by comprehensive lung histology. In conclusion, this study provides proof-of-concept in support of further clinical development of these monoclonal antibodies against COVID-19 during early infection.

Inhibition of IL-6 in the LCWE Mouse Model of Kawasaki Disease Inhibits Acute Phase Reactant Serum Amyloid A but Fails to Attenuate Vasculitis.

Objective: Kawasaki disease (KD) is the most common cause of acquired pediatric heart disease in the developed world. 10% of KD patients are resistant to front-line therapy, and no interventions exist to address secondary complications such as myocardial fibrosis. We sought to identify proteins and pathways associated with disease and anti-IL-1 treatment in a mouse model of KD. Methods: Vasculitis was induced via Lactobacillus casei cell wall extract (LCWE) injection in 5-week-old male mice. Groups of mice were injected with LCWE alone, LCWE and IL-1 receptor antagonist anakinra, or saline for controls. Upper heart tissue was assessed by quantitative mass spectrometry analysis. Expression and activation of STAT3 was assessed by immunohistochemistry, immunofluorescence and Western blot, and IL-6 expression by RNA-seq and ELISA. A STAT3 small molecular inhibitor and anti-IL-6R antibody were used to evaluate the role of STAT3 and IL-6 in disease development. Results: STAT3 was highly expressed and phosphorylated in cardiac tissue of LCWE-injected mice, and reduced following anakinra treatment. Il6 and Stat3 gene expression was enhanced in abdominal aorta of LCWE-injected mice and reduced with Anakinra treatment. IL-6 serum levels were enhanced in LCWE-injected mice and normalized by anakinra. However, neither inhibition of STAT3 nor blockade of IL-6 altered disease development. Conclusion: Proteomic analysis of cardiac tissues demonstrates differential protein expression between KD-like, control and anakinra treated cardiac tissue. STAT3 and IL-6 were highly upregulated with LCWE and normalized by anakinra treatment. However, both STAT3 and IL-6 were dispensable for disease development indicating they may be bystanders of inflammation.

Optofluidic Amplification-free Multiplex Detection of Viral Hemorrhagic Fevers.

Infectious disease outbreaks such as Ebola and other Viral Hemorrhagic Fevers (VHF) require low-complexity, specific, and differentiated diagnostics as illustrated by the recent outbreak in the Democratic Republic of Congo. Here, we describe amplification-free spectrally multiplex detection of four different VHF total RNA samples using multi-spot excitation on a multimode interference waveguide platform along with combinatorial fluorescence labeling of target nucleic acids. In these experiments, we observed an average of 8-fold greater fluorescence signal amplitudes for the Ebola total RNA sample compared to three other total RNA samples: Lake Victoria Marburg Virus, Ravn Marburg Virus, and Crimean-Congo Hemorrhagic Fever. We have attributed this amplitude amplification to an increased amount of RNA during synthesis of soluble glycoprotein in infection. This hypothesis is confirmed by single molecule detection of the total RNA sample after heat-activated release from the carrier microbeads. From these experiments, we observed at least a 5.3x higher RNA mass loading on the Ebola carrier microbeads compared to the Lake Victoria Marburg carrier microbeads, which is consistent with the known production of soluble glycoprotein during infection.

Placental Autophagy and Viral Replication Co-localize in Human and Non-human Primate Placentae Following Zika Virus Infection: Implications for Therapeutic Interventions.

Background: Multiple studies have shown both induction and inhibition of autophagy during Zika virus (ZIKV) infection. While some have proposed mechanisms by which autophagic dysregulation might facilitate ZIKV vertical transmission, there is a lack of in situ data in human and non-human primate models. This is an especially pertinent question as autophagy-inhibitors, such as hydroxychloroquine, have been proposed as potential therapeutic agents aimed at preventing vertical transmission of ZIKV and other RNA viruses. Objectives: Given the paucity of pre-clinical data in support of either autophagic enhancement or inhibition of placental ZIKV viral infection, we sought to assess cellular, spatial, and temporal associations between placental ZIKV infection and measures of autophagy in human primary cell culture and congenital infection cases, as well as an experimental non-human primate (marmoset, Callithrix jacchus) model. Study Design: Primary trophoblast cells were isolated from human placentae (n = 10) and infected in vitro with ZIKV. Autophagy-associated gene expression (ULK-1, BECN1, ATG5, ATG7, ATG12, ATG16L1, MAP1LC3A, MAP1LC3B, p62/SQSTM1) was then determined by TaqMan qPCR to determine fold-change with ZIKV-infection. In in vivo validation experiments, autophagy genes LC3B and p62/SQSTM1 were probed using in situ hybridization (ISH) in the placentae of human Congenital Zika Syndrome (CZS) cases (n = 3) and ZIKV-infected marmoset placenta (n = 1) and fetal tissue (n = 1). Infected and uninfected villi were compared for mean density and co-localization of autophagic protein markers. Results: Studies of primary cultured human trophoblasts revealed decreased expression of autophagy genes ATG5 and p62/SQSTM1 in ZIKV-infected trophoblasts [ATG5 fold change (±SD) 0.734-fold (±0.722), p = 0.036; p62/SQSTM1 0.661-fold (±0.666), p = 0.029]. Histologic examination by ISH and immunohistochemistry confirmed spatial association of autophagy and ZIKV infection in human congenital infection cases, as well as marmoset placental and fetal tissue samples. When quantified by densitometric data, autophagic protein LC3B, and p62/SQSTM1 expression in marmoset placenta were significantly decreased in in situ ZIKV-infected villi compared to less-infected areas [LC3B mean 0.951 (95% CI, 0.930-0.971), p = 0.018; p62/SQSTM1 mean 0.863 (95% CI, 0.810-0.916), p = 0.024]. Conclusion: In the current study, we observed that in the non-transformed human and non-human primate placenta, disruption (specifically down-regulation) of autophagy accompanies later ZIKV replication in vitro, in vivo, and in situ. The findings collectively suggest that dysregulated autophagy spatially and temporally accompanies placental ZIKV replication, providing the first in situ evidence in relevant primate pre-clinical and clinical models for the importance of timing of human therapeutic strategies aimed at agonizing/antagonizing autophagy. These studies have likely further implications for other congenitally transmitted viruses, particularly the RNA viruses, given the ubiquitous nature of autophagic disruption and dysregulation in host responses to viral infection during pregnancy.

Lethality of SARS-CoV-2 infection in K18 human angiotensin-converting enzyme 2 transgenic mice.

Vaccine and antiviral development against SARS-CoV-2 infection or COVID-19 disease would benefit from validated small animal models. Here, we show that transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) by the human cytokeratin 18 promoter (K18 hACE2) represent a susceptible rodent model. K18 hACE2 transgenic mice succumbed to SARS-CoV-2 infection by day 6, with virus detected in lung airway epithelium and brain. K18 ACE2 transgenic mice produced a modest TH1/2/17 cytokine storm in the lung and spleen that peaked by day 2, and an extended chemokine storm that was detected in both lungs and brain. This chemokine storm was also detected in the brain at day 6. K18 hACE2 transgenic mice are, therefore, highly susceptible to SARS-CoV-2 infection and represent a suitable animal model for the study of viral pathogenesis, and for identification and characterization of vaccines (prophylactic) and antivirals (therapeutics) for SARS-CoV-2 infection and associated severe COVID-19 disease.

Intradermal Immunization of EBOV VLPs in Guinea Pigs Induces Broader Antibody Responses Against GP Than Intramuscular Injection.

Ebolavirus (EBOV) infection in humans causes severe hemorrhagic fevers with high mortality rates that range from 30 to 80% as shown in different outbreaks. Thus the development of safe and efficacious EBOV vaccines remains an important goal for biomedical research. We have shown in early studies that immunization with insect cell-produced EBOV virus-like particles (VLPs) is able to induce protect vaccinated mice against lethal EBOV challenge. In the present study, we investigated immune responses induced by Ebola VLPs via two different routes, intramuscular and intradermal immunizations, in guinea pigs. Analyses of antibody responses revealed that similar levels of total IgG antibodies against the EBOV glycoprotein (GP) were induced by the two different immunization methods. However, further characterization showed that the EBOV GP-specific antibodies induced by intramuscular immunization were mainly of the IgG2 subtype whereas both IgG1 and IgG2 antibodies against EBOV GP were induced by intradermal immunization. In contrast, antibody responses against the EBOV matrix protein VP40 induced by intramuscular or intradermal immunizations exhibited similar IgG1 and IgG2 profiles. More interestingly, we found that the sites that the IgG1 antibodies induced by intradermal immunizations bind to in GP are different from those that bind to the IgG2 antibodies induced by intramuscular immunization. Further analyses revealed that sera from all vaccinated guinea pigs exhibited neutralizing activity against Ebola GP-mediated HIV pseudovirion infection at high levels. Moreover, all EBOV VLP-vaccinated guinea pigs survived the challenge by a high dose (1000 pfu) of guinea pig-adapted EBOV, while all control guinea pigs immunized with irrelevant VLPs succumbed to the challenge. The induction of both IgG1 and IgG2 antibody responses that recognized broader sites in GP by intradermal immunization of EBOV VLPs indicates that this approach may represent a more advantageous route of vaccination against virus infection.

Author Correction: Metagenomic sequencing with spiked primer enrichment for viral diagnostics and genomic surveillance.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Metagenomic sequencing with spiked primer enrichment for viral diagnostics and genomic surveillance.

Metagenomic next-generation sequencing (mNGS), the shotgun sequencing of RNA and DNA from clinical samples, has proved useful for broad-spectrum pathogen detection and the genomic surveillance of viral outbreaks. An additional target enrichment step is generally needed for high-sensitivity pathogen identification in low-titre infections, yet available methods using PCR or capture probes can be limited by high cost, narrow scope of detection, lengthy protocols and/or cross-contamination. Here, we developed metagenomic sequencing with spiked primer enrichment (MSSPE), a method for enriching targeted RNA viral sequences while simultaneously retaining metagenomic sensitivity for other pathogens. We evaluated MSSPE for 14 different viruses, yielding a median tenfold enrichment and mean 47% (±16%) increase in the breadth of genome coverage over mNGS alone. Virus detection using MSSPE arboviral or haemorrhagic fever viral panels was comparable in sensitivity to specific PCR, demonstrating 95% accuracy for the detection of Zika, Ebola, dengue, chikungunya and yellow fever viruses in plasma samples from infected patients. Notably, sequences from re-emerging and/or co-infecting viruses that have not been specifically targeted a priori, including Powassan and Usutu, were successfully enriched using MSSPE. MSSPE is simple, low cost, fast and deployable on either benchtop or portable nanopore sequencers, making this method directly applicable for diagnostic laboratory and field use.

Directional entry and release of Zika virus from polarized epithelial cells.

BACKGROUND: Both vector borne and sexual transmission of Zika virus (ZIKV) involve infection of epithelial cells in the initial stages of infection. Epithelial cells are unique in their ability to form polarized monolayers and their barrier function. Cell polarity induces an asymmetry in the epithelial monolayer, which is maintained by tight junctions and specialized sorting machinery. This differential localization can have a potential impact of virus infection. Asymmetrical distribution of a viral receptor can restrict virus entry to a particular membrane while polarized sorting can lead to a directional release of virions. The present study examined the impact of cell polarity on ZIKV infection and release. METHODS: A polarized Caco-2 cell model we described previously was used to assess ZIKV infection. Transepithelial resistance (TEER) was used to assess epithelial cell polarity, and virus infection was measured by immunofluorescence microscopy and qRT-PCR. Cell permeability was measured using a fluorescein leakage assay. Statistical significance was calculated using one-way ANOVA and significance was set at p < 0.05. RESULTS: Using the Caco-2 cell model for polarized epithelial cells, we report that Zika virus preferentially infects polarized cells from the apical route and is released vectorially through the basolateral route. Our data also indicates that release occurs without disruption of cell permeability. CONCLUSIONS: Our results show that ZIKV has directional infection and egress in a polarized cell system. This mechanism of directional infection may be one of the mechanisms that enables the cross the epithelial barrier effectively without a disruption in cell monolayer integrity. Elucidation of entry and release characteristics of Zika virus in polarized epithelial cells can lead to better understanding of virus dissemination in the host, and can help in developing effective therapeutic interventions.

Rapid and Fully Microfluidic Ebola Virus Detection with CRISPR-Cas13a.

Highly infectious illness caused by pathogens is endemic especially in developing nations where there is limited laboratory infrastructure and trained personnel. Rapid point-of-care (POC) serological assays with minimal sample manipulation and low cost are desired in clinical practice. In this study, we report an automated POC system for Ebola RNA detection with RNA-guided RNA endonuclease Cas13a, utilizing its collateral RNA degradation after its activation. After automated microfluidic mixing and hybridization, nonspecific cleavage products of Cas13a are immediately measured by a custom integrated fluorometer which is small in size and convenient for in-field diagnosis. Within 5 min, a detection limit of 20 pfu/mL (5.45 × 107 copies/mL) of purified Ebola RNA is achieved. This isothermal and fully solution-based diagnostic method is rapid, amplification-free, simple, and sensitive, thus establishing a key technology toward a useful POC diagnostic platform.