Smartphone multiplex microcapillary diagnostics using Cygnus: Development and evaluation of rapid serotype-specific NS1 detection with dengue patient samples.

Laboratory diagnosis of dengue virus (DENV) infection including DENV serotyping requires skilled labor and well-equipped settings. DENV NS1 lateral flow rapid test (LFT) provides simplicity but lacks ability to identify serotype. A simple, economical, point-of-care device for serotyping is still needed. We present a gravity driven, smartphone compatible, microfluidic device using microcapillary film (MCF) to perform multiplex serotype-specific immunoassay detection of dengue virus NS1. A novel device-termed Cygnus-with a stackable design allows analysis of 1 to 12 samples in parallel in 40 minutes. A sandwich enzyme immunoassay was developed to specifically detect NS1 of all four DENV serotypes in one 60-µl plasma sample. This test aims to bridge the gap between rapid LFT and laboratory microplate ELISAs in terms of sensitivity, usability, accessibility and speed. The Cygnus NS1 assay was evaluated with retrospective undiluted plasma samples from 205 DENV infected patients alongside 50 febrile illness negative controls. Against the gold standard RT-PCR, clinical sensitivity for Cygnus was 82% in overall (with 78, 78, 80 and 76% for DENV1-4, respectively), comparable to an in-house serotyping NS1 microplate ELISA (82% vs 83%) but superior to commercial NS1-LFT (82% vs 74%). Specificity of the Cygnus device was 86%, lower than that of NS1-microplate ELISA and NS1-LFT (100% and 98%, respectively). For Cygnus positive samples, identification of DENV serotypes DENV2-4 matched those by RT-PCR by 100%, but for DENV1 capillaries false positives were seen, suggesting an improved DENV1 capture antibody is needed to increase specificity. Overall performance of Cygnus showed substantial agreement to NS1-microplate ELISA (κ = 0.68, 95%CI 0.58-0.77) and NS1-LFT (κ = 0.71, 95%CI 0.63-0.80). Although further refinement for DENV-1 NS1 detection is needed, the advantages of multiplexing and rapid processing time, this Cygnus device could deliver point-of-care NS1 antigen testing including serotyping for timely DENV diagnosis for epidemic surveillance and outbreak prediction.

Gravity-Driven Microfluidic Siphons: Fluidic Characterization and Application to Quantitative Immunoassays.

A range of biosensing techniques including immunoassays are routinely used for quantitation of analytes in biological samples and available in a range of formats, from centralized lab testing (e.g., microplate enzyme-linked immunosorbent assay (ELISA)) to automated point-of-care (POC) and lateral flow immunochromatographic tests. High analytical performance is intrinsically linked to the use of a sequence of reagent and washing steps, yet this is extremely challenging to deliver at the POC without a high level of fluidic control involving, e.g., automation, fluidic pumping, or manual fluid handling/pipetting. Here we introduce a microfluidic siphon concept that conceptualizes a multistep ″dipstick″ for quantitative, enzymatically amplified immunoassays using a strip of microporous or microbored material. We demonstrated that gravity-driven siphon flow can be realized in single-bore glass capillaries, a multibored microcapillary film, and a glass fiber porous membrane. In contrast to other POC devices proposed to date, the operation of the siphon is only dependent on the hydrostatic liquid pressure (gravity) and not capillary forces, and the unique stepwise approach to the delivery of the sample and immunoassay reagents results in zero dead volume in the device, no reagent overlap or carryover, and full start/stop fluid control. We demonstrated applications of a 10-bore microfluidic siphon as a portable ELISA system without compromised quantitative capabilities in two global diagnostic applications: (1) a four-plex sandwich ELISA for rapid smartphone dengue serotype identification by serotype-specific dengue virus NS1 antigen detection, relevant for acute dengue fever diagnosis, and (2) quantitation of anti-SARS-CoV-2 IgG and IgM titers in spiked serum samples. Diagnostic siphons provide the opportunity for high-performance immunoassay testing outside sophisticated laboratories, meeting the rapidly changing global clinical and public health needs.

Aberrant glycosylation of anti-SARS-CoV-2 spike IgG is a prothrombotic stimulus for platelets.

A subset of patients with coronavirus disease 2019 (COVID-19) become critically ill, suffering from severe respiratory problems and also increased rates of thrombosis. The causes of thrombosis in severely ill patients with COVID-19 are still emerging, but the coincidence of critical illness with the timing of the onset of adaptive immunity could implicate an excessive immune response. We hypothesized that platelets might be susceptible to activation by anti-severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) antibodies and might contribute to thrombosis. We found that immune complexes containing recombinant SARS-CoV-2 spike protein and anti-spike immunoglobulin G enhanced platelet-mediated thrombosis on von Willebrand factor in vitro, but only when the glycosylation state of the Fc domain was modified to correspond with the aberrant glycosylation previously identified in patients with severe COVID-19. Furthermore, we found that activation was dependent on FcγRIIA, and we provide in vitro evidence that this pathogenic platelet activation can be counteracted by the therapeutic small molecules R406 (fostamatinib) and ibrutinib, which inhibit tyrosine kinases Syk and Btk, respectively, or by the P2Y12 antagonist cangrelor.

Affordable mobile microfluidic diagnostics: minimum requirements for smartphones and digital imaging for colorimetric and fluorometric anti-dengue and anti-SARS-CoV-2 antibody detection.

Background: Miniaturised bioassays permit diagnostic testing near the patient, and the results can be recorded digitally using inexpensive cameras including smartphone and mobile phone cameras. Although digital cameras are now inexpensive and portable, the minimum performance required for microfluidic diagnostic bioassays has not been defined. We present a systematic comparison of a wide range of different digital cameras for capturing and measuring results of microfluidic bioassays and describe a framework to specify performance requirements to quantify immunoassays. Methods: A set of 200 µm diameter microchannels was filled with a range of concentrations of dyes used in colorimetric and fluorometric enzyme immunoassays. These were imaged in parallel using cameras of varying cost and performance ranging from <£30 to >£500. Results: Higher resolution imaging allowed larger numbers of microdevices to be resolved and analysed in a single image. In contrast, low quality cameras were still able to quantify results but for fewer samples. In some cases, an additional macro lens was added to focus closely. If image resolution was sufficient to identify individual microfluidic channels as separate lines, all cameras were able to quantify a similar range of concentrations of both colorimetric and fluorometric dyes. However, the mid-range cameras performed better, with the lowest cost cameras only allowing one or two samples to be quantified per image. Consistent with these findings, we demonstrate that quantitation (to determine endpoint titre) of antibodies against dengue and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses is possible using a wide range of digital imaging devices including the mid-range smartphone iPhone 6S and a budget Android smartphone costing <£50. Conclusions: In conclusion, while more expensive and higher quality cameras allow larger numbers of devices to be simultaneously imaged, even the lowest resolution and cheapest cameras were sufficient to record and quantify immunoassay results.

Bluetongue Virus Nonstructural Protein 3 Orchestrates Virus Maturation and Drives Non-Lytic Egress via Two Polybasic Motifs.

Bluetongue virus (BTV) is an arthropod-borne virus that infects domestic and wild ruminants. The virion is a non-enveloped double-layered particle with an outer capsid that encloses a core containing the segmented double-stranded RNA genome. Although BTV is canonically released by cell lysis, it also exits non-lytically. In infected cells, the BTV nonstructural glycoprotein 3 (NS3) is found to be associated with host membranes and traffics from the endoplasmic reticulum through the Golgi apparatus to the plasma membrane. This suggests a role for NS3 in BTV particle maturation and non-lytic egress. However, the mechanism by which NS3 coordinates these events has not yet been elucidated. Here, we identified two polybasic motifs (PMB1/PMB2), consistent with the membrane binding. Using site-directed mutagenesis, confocal and electron microscopy, and flow cytometry, we demonstrated that PBM1 and PBM2 mutant viruses retained NS3 either in the Golgi apparatus or in the endoplasmic reticulum, suggesting a distinct role for each motif. Mutation of PBM2 motif decreased NS3 export to the cell surface and virus production. However, both mutant viruses produced predominantly inner core particles that remained close to their site of assembly. Together, our data demonstrates that correct trafficking of the NS3 protein is required for virus maturation and release.

Nonhomologous recombination between defective poliovirus and coxsackievirus genomes suggests a new model of genetic plasticity for picornaviruses.

Most of the circulating vaccine-derived polioviruses (cVDPVs) implicated in poliomyelitis outbreaks in Madagascar have been shown to be recombinants between the type 2 poliovirus (PV) strain of the oral polio vaccine (Sabin 2) and another species C human enterovirus (HEV-C), such as type 17 coxsackie A virus (CA17) in particular. We studied intertypic genetic exchanges between PV and non-PV HEV-C by developing a recombination model, making it possible to rescue defective type 2 PV RNA genomes with a short deletion at the 3' end by the cotransfection of cells with defective or infectious CA17 RNAs. We isolated over 200 different PV/CA17 recombinants, using murine cells expressing the human PV receptor (PVR) and selecting viruses with PV capsids. We found some homologous (H) recombinants and, mostly, nonhomologous (NH) recombinants presenting duplications of parental sequences preferentially located in the regions encoding proteins 2A, 2B, and 3A. Short duplications appeared to be stable, whereas longer duplications were excised during passaging in cultured cells or after multiplication in PVR-transgenic mice, generating H recombinants with diverse sites of recombination. This suggests that NH recombination events may be a transient, intermediate step in the generation and selection of the fittest H recombinants. In addition to the classical copy-choice mechanism of recombination thought to generate mostly H recombinants, there may also be a modular mechanism of recombination, involving NH recombinant precursors, shaping the genomes of recombinant enteroviruses and other picornaviruses. Importance: The multiplication of circulating vaccine-derived polioviruses (cVDPVs) in poorly immunized human populations can render these viruses pathogenic, causing poliomyelitis outbreaks. Most cVDPVs are intertypic recombinants between a poliovirus (PV) strain and another human enterovirus, such as type 17 coxsackie A viruses (CA17). For further studies of the genetic exchanges between PV and CA17, we have developed a model of recombination, making it possible to rescue defective PV RNA genomes with a short deletion by cotransfecting cells with the defective PV genome and CA17 genomic RNA. Numerous recombinants were found, including homologous PV/CA17 recombinants, but mostly nonhomologous recombinants presenting duplications of parental sequences preferentially located in particular regions. Long duplications were excised by passages in cultured cells or in mice, generating diverse homologous recombinants. Recombination leading to nonhomologous recombinants, which evolve into homologous recombinants, may therefore be seen as a model of genetic plasticity in enteroviruses and, possibly, in other RNA viruses.

The Golgi protein ACBD3, an interactor for poliovirus protein 3A, modulates poliovirus replication.

We have shown that the circulating vaccine-derived polioviruses responsible for poliomyelitis outbreaks in Madagascar have recombinant genomes composed of sequences encoding capsid proteins derived from poliovaccine Sabin, mostly type 2 (PVS2), and sequences encoding nonstructural proteins derived from other human enteroviruses. Interestingly, almost all of these recombinant genomes encode a nonstructural 3A protein related to that of field coxsackievirus A17 (CV-A17) strains. Here, we investigated the repercussions of this exchange, by assessing the role of the 3A proteins of PVS2 and CV-A17 and their putative cellular partners in viral replication. We found that the Golgi protein acyl-coenzyme A binding domain-containing 3 (ACBD3), recently identified as an interactor for the 3A proteins of several picornaviruses, interacts with the 3A proteins of PVS2 and CV-A17 at viral RNA replication sites, in human neuroblastoma cells infected with either PVS2 or a PVS2 recombinant encoding a 3A protein from CV-A17 [PVS2-3A(CV-A17)]. The small interfering RNA-mediated downregulation of ACBD3 significantly increased the growth of both viruses, suggesting that ACBD3 slowed viral replication. This was confirmed with replicons. Furthermore, PVS2-3A(CV-A17) was more resistant to the replication-inhibiting effect of ACBD3 than the PVS2 strain, and the amino acid in position 12 of 3A was involved in modulating the sensitivity of viral replication to ACBD3. Overall, our results indicate that exchanges of nonstructural proteins can modify the relationships between enterovirus recombinants and cellular interactors and may thus be one of the factors favoring their emergence.

Common and diverse features of cocirculating type 2 and 3 recombinant vaccine-derived polioviruses isolated from patients with poliomyelitis and healthy children.

BACKGROUND: Five cases of poliomyelitis due to type 2 or 3 recombinant vaccine-derived polioviruses (VDPVs) were reported in the Toliara province of Madagascar in 2005. METHODS: We sequenced the genome of the VDPVs isolated from the patients and from 12 healthy children and characterized phenotypic aspects, including pathogenicity, in mice transgenic for the poliovirus receptor. RESULTS: We identified 6 highly complex mosaic recombinant lineages composed of sequences derived from different vaccine polioviruses and other species C human enteroviruses (HEV-Cs). Most had some recombinant genome features in common and contained nucleotide sequences closely related to certain cocirculating coxsackie A virus isolates. However, they differed in terms of their recombinant characteristics or nucleotide substitutions and phenotypic features. All VDPVs were neurovirulent in mice. CONCLUSIONS: This study confirms the genetic relationship between type 2 and 3 VDPVs, indicating that both types can be involved in a single outbreak of disease. Our results highlight the various ways in which a vaccine-derived poliovirus may become pathogenic in complex viral ecosystems, through frequent recombination events and mutations. Intertypic recombination between cocirculating HEV-Cs (including polioviruses) appears to be a common mechanism of genetic plasticity underlying transverse genetic variability.

Enhanced gene silencing in cells cured of persistent virus infection by RNA interference.

We compared HEp-2-derived cells cured of persistent poliovirus infection by RNA interference (RNAi) with parental cells, to investigate possible changes in the efficiency of RNAi. Lower levels of poliovirus replication were observed in cured cells, possibly facilitating virus silencing by antiviral small interfering RNAs (siRNAs). However, green fluorescent protein (GFP) produced from a measles virus vector and also GFP and luciferase produced from plasmids that do not replicate in human cells were more effectively silenced by specific siRNAs in cured than in control cells. Thus, cells displaying enhanced silencing were selected during curing by RNAi. Our results strongly suggest that the RNAi machinery of cured cells is more efficient than that of parental cells.

Recombination between polioviruses and co-circulating Coxsackie A viruses: role in the emergence of pathogenic vaccine-derived polioviruses.

Ten outbreaks of poliomyelitis caused by pathogenic circulating vaccine-derived polioviruses (cVDPVs) have recently been reported in different regions of the world. Two of these outbreaks occurred in Madagascar. Most cVDPVs were recombinants of mutated poliovaccine strains and other unidentified enteroviruses of species C. We previously reported that a type 2 cVDPV isolated during an outbreak in Madagascar was co-circulating with coxsackieviruses A17 (CA17) and that sequences in the 3' half of the cVDPV and CA17 genomes were related. The goal of this study was to investigate whether these CA17 isolates can act as recombination partners of poliovirus and subsequently to evaluate the major effects of recombination events on the phenotype of the recombinants. We first cloned the infectious cDNA of a Madagascar CA17 isolate. We then generated recombinant constructs combining the genetic material of this CA17 isolate with that of the type 2 vaccine strain and that of the type 2 cVDPV. Our results showed that poliovirus/CA17 recombinants are viable. The recombinant in which the 3' half of the vaccine strain genome had been replaced by that of the CA17 genome yielded larger plaques and was less temperature sensitive than its parental strains. The virus in which the 3' portion of the cVDPV genome was replaced by the 3' half of the CA17 genome was almost as neurovirulent as the cVDPV in transgenic mice expressing the poliovirus cellular receptor gene. The co-circulation in children and genetic recombination of viruses, differing in their pathogenicity for humans and in certain other biological properties such as receptor usage, can lead to the generation of pathogenic recombinants, thus constituting an interesting model of viral evolution and emergence.