Gold Nanourchins Improve Virus Targeting and Plasmonic Coupling for Virus Diagnosis on a Smartphone Platform.

Point-of-care detection of pathogens is critical to monitor and combat viral infections. The plasmonic coupling assay (PCA) is a homogeneous assay and allows rapid, one-step, and colorimetric detection of intact viruses. However, PCA lacks sufficient sensitivity, necessitating further mechanistic studies to improve the detection performance of PCA. Here, we demonstrate that gold nanourchins (AuNUs) provide significantly improved colorimetric detection of viruses in PCA. Using respiratory syncytial virus (RSV) as a target, we demonstrate that the AuNU-based PCA achieves a detection limit of 1400 PFU/mL, or 17 genome equivalent copies/µL. Mechanistic studies suggest that the improved detection sensitivity arises from the higher virus-binding capability and stronger plasmonic coupling at long distances (∼10 nm) by AuNU probes. Furthermore, we demonstrate the virus detection with a portable smartphone-based spectrometer using RSV-spiked nasal swab clinical samples. Our study uncovers important mechanisms for the sensitive detection of intact viruses in PCA and provides a potential toolkit at the point of care.

Digital plasmonic nanobubble detection for rapid and ultrasensitive virus diagnostics.

Rapid and sensitive diagnostics of infectious diseases is an urgent and unmet need as evidenced by the COVID-19 pandemic. Here, we report a strategy, based on DIgitAl plasMONic nanobubble Detection (DIAMOND), to address this need. Plasmonic nanobubbles are transient vapor bubbles generated by laser heating of plasmonic nanoparticles (NPs) and allow single-NP detection. Using gold NPs as labels and an optofluidic setup, we demonstrate that DIAMOND achieves compartment-free digital counting and works on homogeneous immunoassays without separation and amplification steps. DIAMOND allows specific detection of respiratory syncytial virus spiked in nasal swab samples and achieves a detection limit of ~100 PFU/mL (equivalent to 1 RNA copy/µL), which is competitive with digital isothermal amplification for virus detection. Therefore, DIAMOND has the advantages including one-step and single-NP detection, direct sensing of intact viruses at room temperature, and no complex liquid handling, and is a platform technology for rapid and ultrasensitive diagnostics.

mTOR kinase is a therapeutic target for respiratory syncytial virus and coronaviruses.

Therapeutic interventions targeting viral infections remain a significant challenge for both the medical and scientific communities. While specific antiviral agents have shown success as therapeutics, viral resistance inevitably develops, making many of these approaches ineffective. This inescapable obstacle warrants alternative approaches, such as the targeting of host cellular factors. Respiratory syncytial virus (RSV), the major respiratory pathogen of infants and children worldwide, causes respiratory tract infection ranging from mild upper respiratory tract symptoms to severe life-threatening lower respiratory tract disease. Despite the fact that the molecular biology of the virus, which was originally discovered in 1956, is well described, there is no vaccine or effective antiviral treatment against RSV infection. Here, we demonstrate that targeting host factors, specifically, mTOR signaling, reduces RSV protein production and generation of infectious progeny virus. Further, we show that this approach can be generalizable as inhibition of mTOR kinases reduces coronavirus gene expression, mRNA transcription and protein production. Overall, defining virus replication-dependent host functions may be an effective means to combat viral infections, particularly in the absence of antiviral drugs.

Results of COVID-19 Surveillance in a Large United States Pediatric Healthcare System over One Year.

BACKGROUND: The lack of SARS-CoV-2 antigen surveillance testing in the pediatric population has inhibited accurate infection and hospitalization prevalence estimates. We aim to report the estimated prevalence of and risk factors for COVID-19 infection, hospitalization, and intensive care unit (ICU) admission across the three United States (US) waves in one of the largest pediatric healthcare systems in the nation. METHODS: Retrospective electronic health record (EHR) review of all COVID-19 surveillance data among children aged 0-19 years seeking healthcare at one pediatric healthcare system that serves predominantly Medicaid-dependent families from 1 March 2020 to 31 March 2021. COVID-19 infection status (Y/N), hospital admission (Y/N), and ICU admission (Y/N) are the main outcomes. RESULTS: Of 22,377 children aged ≤ 19 years tested for SARS-CoV-2 infection from March 2020-March 2021, 3126 were positive (14.0%), and out of those positive, 53.7% were hospitalized and 2.9% were admitted to the ICU. Compared to Wave 1 (1 March 2020-31 May 2020), the risk of a positive test increased from 16% (RR 1.16, 95% CI, 1.07-1.26) in Wave 2 (1 June 2020-31 October 2020) to 33% (RR 1.33, 95% CI, 1.23-1.44) in Wave 3 (1 November 2020-31 March 2021). Similarly, compared to Wave 1, the risk for hospitalization increased 86% (RR 1.86, 95% CI, 1.86-2.06) in Wave 2 and 89% in Wave 3 (RR 1.89, 95% CI, 1.70-2.08), and the risk for ICU admission increased from 10% in Wave 2 (RR 1.10, 95% CI, 0.39-3.01) to 310% in Wave 3 (RR 3.10, 95% CI, 1.21-7.80). Children with asthma, depressive disorders, type 1 or 2 diabetes, and anemia were more likely to be hospitalized while children with diabetes, obesity, cardiac malformations, and hypertension were more likely to be admitted to the ICU versus children without these conditions. CONCLUSIONS: Children were cumulatively impacted by the COVID-19 pandemic through the three US waves with more than a third hospitalized in Wave 3. Children with underlying health conditions were particularly at risk for severe illness and should be monitored for any long-term impacts.

Single-Particle Counting Based on Digital Plasmonic Nanobubble Detection for Rapid and Ultrasensitive Diagnostics.

Rapid and sensitive diagnostics of infectious diseases is an urgent and unmet need as evidenced by the COVID-19 pandemic. Here we report a novel strategy, based on DIgitAl plasMONic nanobubble Detection (DIAMOND), to address these gaps. Plasmonic nanobubbles are transient vapor bubbles generated by laser heating of plasmonic nanoparticles and allow single-particle detection. Using gold nanoparticles labels and an optofluidic setup, we demonstrate that DIAMOND achieves a compartment-free digital counting and works on homogeneous assays without separation and amplification steps. When applied to the respiratory syncytial virus diagnostics, DIAMOND is 150 times more sensitive than commercial lateral flow assays and completes measurements within 2 minutes. Our method opens new possibilities to develop single-particle digital detection methods and facilitate rapid and ultrasensitive diagnostics. ONE SENTENCE SUMMARY: Single-particle digital plasmonic nanobubble detection allows rapid and ultrasensitive detection of viruses in a one-step homogeneous assay.

Tuning the Gold Nanoparticle Colorimetric Assay by Nanoparticle Size, Concentration, and Size Combinations for Oligonucleotide Detection.

Gold nanoparticle (GNP)-based aggregation assay is simple, fast, and employs a colorimetric detection method. Although previous studies have reported using GNP-based colorimetric assay to detect biological and chemical targets, a mechanistic and quantitative understanding of the assay and effects of GNP parameters on the assay performance is lacking. In this work, we investigated this important aspect of the GNP aggregation assay including effects of GNP concentration and size on the assay performance to detect malarial DNA. Our findings lead us to propose three major competing factors that determine the final assay performance including the nanoparticle aggregation rate, plasmonic coupling strength, and background signal. First, increasing nanoparticle size reduces the Brownian motion and thus aggregation rate, but significantly increases plasmonic coupling strength. We found that larger GNP leads to stronger signal and improved limit of detection (LOD), suggesting a dominating effect of plasmonic coupling strength. Second, higher nanoparticle concentration increases the probability of nanoparticle interactions and thus aggregation rate, but also increases the background extinction signal. We observed that higher GNP concentration leads to stronger signal at high target concentrations due to higher aggregation rate. However, the fact the optimal LOD was found at intermediate GNP concentrations suggests a balance of two competing mechanisms between aggregation rate and signal/background ratio. In summary, our work provides new guidelines to design GNP aggregation-based POC devices to meet the signal and sensitivity needs for infectious disease diagnosis and other applications.

Distinct patterns of innate immune activation by clinical isolates of respiratory syncytial virus.

Respiratory syncytial virus (RSV) is a major respiratory pathogen of infants and young children. Multiple strains of both subgroup A and B viruses circulate during each seasonal epidemic. Genetic heterogeneity among RSV genomes, in large part due to the error prone RNA-dependent, RNA polymerase, could mediate variations in pathogenicity. We evaluated clinical strains of RSV for their ability to induce the innate immune response. Subgroup B viruses were used to infect human pulmonary epithelial cells (A549) and primary monocyte-derived human macrophages (MDM) from a variety of donors. Secretions of IL-6 and CCL5 (RANTES) from infected cells were measured following infection. Host and viral transcriptome expression were assessed using RNA-SEQ technology and the genomic sequences of several clinical isolates were determined. There were dramatic differences in the induction of IL-6 and CCL5 in both A549 cells and MDM infected with a variety of clinical isolates of RSV. Transcriptome analyses revealed that the pattern of innate immune activation in MDM was virus-specific and host-specific. Specifically, viruses that induced high levels of secreted IL-6 and CCL5 tended to induce cellular innate immune pathways whereas viruses that induced relatively low level of IL-6 or CCL5 did not induce or suppressed innate immune gene expression. Activation of the host innate immune response mapped to variations in the RSV G gene and the M2-1 gene. Viral transcriptome data indicated that there was a gradient of transcription across the RSV genome though in some strains, RSV G was the expressed in the highest amounts at late times post-infection. Clinical strains of RSV differ in cytokine/chemokine induction and in induction and suppression of host genes expression suggesting that these viruses may have inherent differences in virulence potential. Identification of the genetic elements responsible for these differences may lead to novel approaches to antiviral agents and vaccines.

Induction of IL-6 and CCL5 (RANTES) in human respiratory epithelial (A549) cells by clinical isolates of respiratory syncytial virus is strain specific.

BACKGROUND: Respiratory syncytial virus (RSV) is the major respiratory pathogen of infants and young children. During each seasonal epidemic, multiple strains of both subgroup A and B viruses circulate in the community. Like other RNA viruses, RSV genome replication is prone to errors that results in a heterogeneous population of viral strains some of which may possess differences in virulence. We sought to determine whether clinical isolates of RSV differ in their capacity to induce inflammatory cytokines IL-6 and CCL5 (previously known as RANTES [regulated upon activation, normal T-cell expressed and secreted protein]), which are known to be induced in vitro and in vivo in response to RSV, during infection of A549 cells. RESULTS: Screening of subgroup A and B isolates revealed heterogeneity among strains to induce IL-6 and CCL5. We chose two subgroup B strains, New Haven (NH)1067 and NH1125, for further analysis because of their marked differences in cytokine inducing properties and because subgroup B strains, in general, are less genetically heterogeneous as compared to subgroup A strains. At 12 and 24 hours post infection RSV strains, NH1067 and NH1125 differed in their capacity to induce IL-6 by an order of magnitude or more. The concentrations of IL-6 and CCL5 were dependent on the dose of infectious virus and the concentration of these cytokines induced by NH1125 was greater than that of those induced by NH1067 when the multiplicity of infection of NH1067 used was as much as 10-fold higher than that of NH1125. The induction of IL-6 was dependent on viable virus as infection with UV-inactivated virus did not induce IL-6. The difference in IL-6 induction most likely could not be explained by differences in viral replication kinetics. The intracellular level of RSV RNA, as determined by quantitative RT-PCR, was indistinguishable between the 2 strains though the titer of progeny virus produced by NH1125 was greater than that produced by NH1067 at 16, 24 and 36 hours but essentially equal at 48 and 72 hours. Full genome sequencing of the 2 strains revealed 193 polymorphisms and 4 insertions in NH1067 when compared to NH1125 (2 single base insertions in non-coding regions and 2 duplications of 3 and 60 bases in the RSV G gene). Of the polymorphisms, 147 occurred in coding regions and only 30 resulted in amino acid changes in 7 of the RSV genes. CONCLUSIONS: These data suggest that RSV strains may not be homogeneous with regard to pathogenesis or virulence. Identification of the genetic polymorphisms associated with variations in cytokine induction may lead to insights into RSV disease and to the development of effective antiviral agents and vaccines.

Seroepidemiology of WU polyomavirus among children exposed perinatally to HIV-1.

WU polyomavirus (WUPyV), a new member of the genus wukipolyomavirus in the family Polyomaviridae, has been detected in serum and tissues of individuals infected with HIV. However, the epidemiology of WUPyV among children exposed perinatally to HIV-1 is unknown. To investigate the epidemiology of WUPyV in children exposed to and infected perinatally with HIV, serum samples from 150 children exposed to HIV and 114 children infected with HIV were screened for IgG antibodies to WUPyV. A subset was screened for IgM antibodies to WUPyV. Both antibody detection assays were performed using a recombinant WUPyV VP1-based ELISA. The overall seroprevalence of WUPyV IgG was 76.3% in children infected with HIV and 62% in children exposed perinatally to HIV. In the group of children infected with HIV, the prevalence of WUPyV IgG antibody reached its peak in 2-3 year olds (90.9%). In children 0-5 months of age, IgG seroprevalence was lower in those children exposed to HIV compared to children infected with HIV (43.1% vs.75%, P = 0.047). However, the seroprevalence of WUPyV IgM antibody was higher in children exposed to HIV compared to infants infected with HIV (27.4% vs. 8.3%, P = 0.044). WUPyV infection is acquired in early childhood in the majority of children born to mothers infected with HIV. The implication of this infection and the specific clinical syndrome that it produces, if any, remain to be defined.

WU polyomavirus in patients infected with HIV or hepatitis C virus, Connecticut, USA, 2007.

WU polyomavirus (WUPyV) was detected in 10 (8.3%) of 121 HIV-positive plasma specimens, 0 (0%) of 120 HIV-negative serum specimens, and 2 (2.5%) of 79 hepatitis C virus (HCV)-positive serum specimens. KI polyomavirus was not detected in HIV-positive plasma or HCV-positive serum specimens. HIV-infected persons may be susceptible to systemic WUPyV infection.

Rhinoviruses are a major cause of wheezing and hospitalization in children less than 2 years of age.

BACKGROUND: Human rhinoviruses (HRV) are now considered major respiratory pathogens. We sought to determine whether HRV are a cause of wheezing and/or hospitalization in children <2 years old. METHODS: A polymerase chain reaction assay was used to screen for HRV infection in 4 categories of children <2 years old: (1) with symptoms of respiratory tract disease without wheezing; (2) with wheezing with or without other symptoms; (3) who were asymptomatic and; (4) who had a respiratory specimen submitted to a diagnostic laboratory. All specimens were collected between January and December 2004. Phylogenetic analyses were performed on most HRV isolates. RESULTS: Twenty-eight (17%) of 165 children with symptoms of respiratory infection without wheezing; 21 (26.3%) of 80 children with wheezing; 3 (3%) of 93 asymptomatic children; and 47 (23.3%) of 202 children with specimens submitted to the diagnostic laboratory tested positive for HRV. The difference between the rates of infection in the asymptomatic group and in each of the 3 other categories was statistically significant (P

Role of human polyomaviruses in respiratory tract disease in young children.

KI virus was detected in respiratory secretions of 8/367 (2.2%) symptomatic and 0/96 asymptomatic children (p = 0.215). WU virus was detected in 26/367 (7.1%) symptomatic and 6/96 (6.3%) asymptomatic children (p = 1.00). These human polyomaviruses may not independently cause respiratory tract disease in young children.

Seroepidemiology of human bocavirus defined using recombinant virus-like particles.

BACKGROUND: Human bocavirus (HBoV) is a newly identified human parvovirus for which seroepidemiology and antigenic properties remain undefined. METHODS: The HBoV VP2 gene, expressed from a baculovirus vector, produced virus-like particles (VLPs), which were used to raise rabbit anti-HBoV antisera and to develop an enzyme-linked immunosorbent assay (ELISA). The VLP-based ELISA was used to screen for HBoV-specific immunoglobulin G antibodies in a convenience sample of 270 serum specimens, mostly from children, obtained at Yale-New Haven Hospital; 208 specimens were also screened for erythrovirus B19-specific antibodies by a B19 VLP-based ELISA. RESULTS: Immunofluorescence and ELISA showed that human parvoviruses HBoV and B19 are antigenically distinct. By the HBoV VLP-based ELISA, 91.8% and 63.6% of serum specimens from infants in the first and second months of life, respectively, were found to be seropositive, as were 45.4% from 3-month-old infants and 25.0% from 4-month-old infants. The percentages of HBoV-seropositive children increased to 40.7%-60.0% for children 5-47 months of age and to >85% for individuals >or=48 months old. However, the overall percentage of B19-seropositive individuals was <40.5% for all age groups screened. CONCLUSIONS: HBoV infection is common during childhood, but a minority of children and young adults screened have evidence of B19 infection.

Seroepidemiology of group I human coronaviruses in children.

BACKGROUND: Recently, several new human coronaviruses have been identified. OBJECTIVES: To define the seroepidemiology of group I human coronaviruses. STUDY DESIGN: A recombinant protein enzyme linked immunosorbent assay (ELISA) based on portions of the nucleocapsid protein of group I human coronaviruses was developed and was used to screen serum from 243 children and young adults. RESULTS: For HCoV-229E, the percentages of seropositive individuals were 57.1% for infants <2 months old; 38.9% for infants 2-3 months old; 4.7% for infants 4-5 months old; 42.9-50.0% for infants 6-12 months old; 34.8-62.5% for individuals 1-20 years old. For HCoV-NL63, the percentages of seropositive individuals were 45.2% for infants <2 months old; 11.1% for infants 2-3 months old; 4.7% for infants 4-5 months old; 28.6-40.0% for infants 6-12 months old; 25.0-70.3% for individuals 1-20 years old. CONCLUSIONS: Infection with these viruses is common in childhood though the prevalence of these viruses may vary from year to year.

Newly discovered respiratory viruses: significance and implications.

With the recent advances in molecular biology and the ability to amplify viral genomes in a non-sequence-dependent manner, several previously unidentified human respiratory viruses have been discovered. There are accumulating data that some of these new pathogens are responsible for a substantial proportion of respiratory tract diseases, particularly in children. This review will focus on several of these newly identified pathogens for which there are clinical data implicating a role of these viruses in respiratory tract disease specifically, human metapneumovirus, human coronaviruses NL63 and HKU1, and the human bocavirus. Antivirals and effective vaccines for these new agents may decrease the burden of respiratory tract diseases.

Human bocavirus infection in young children in the United States: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus.

BACKGROUND: Human bocavirus (HBoV) is a newly identified human parvovirus that was originally identified in the respiratory secretions of children with respiratory tract disease. To further investigate the epidemiological profile and clinical characteristics of HBoV infection, we screened infants and children <2 years of age (hereafter referred to as "children") for HBoV. METHODS: Children for whom respiratory specimens submitted to a diagnostic laboratory tested negative for respiratory syncytial virus, parainfluenza viruses (types 1-3), influenza A and B viruses, and adenovirus, as well as asymptomatic children, underwent screening for HBoV by use of polymerase chain reaction (PCR). Respiratory specimens were obtained from the children from 1 January 2004 through 31 December 2004. RESULTS: Twenty-two (5.2%) of the 425 children who had a respiratory specimen submitted to the diagnostic laboratory and 0 of the 96 asymptomatic children were found to be positive for HBoV by PCR (P=.02). Fever, rhinorrhea, cough, and wheezing were observed in > or =50% of the HBoV-positive children. Of the 17 children who had chest radiography performed, 12 (70.6%) had abnormal findings. HBoV appeared to have a seasonal distribution. Nucleotide polymorphisms were detected in the viral capsid protein (VP) 1/VP2 genes. Two distinct HBoV genotypes circulated during the study period. CONCLUSIONS: HBoV is circulating in the United States and is associated with both upper and lower respiratory tract disease in infants and young children.

Novel mutations in the respiratory syncytial virus G gene identified in viral isolates from a girl with severe combined immune deficiency treated with intravenous immune globulin.

BACKGROUND: Respiratory syncytial virus (RSV) can cause prolonged infections in individuals with compromised immunity. OBJECTIVES: To investigate whether RSV evolves during prolonged infection in an immunocompromised host. STUDY DESIGN: We sequenced the envalope glycoprotein genes of RSV obtained at three time points during a 59-day period from a 4-year-old female with severe combined immune deficiency (SCID) treated with intravenous immunoglobulin (IVIG). RESULTS: Sporadic silent mutations were found in the SH, G and F genes among three RSV samples collected at days 0, 19 and 59. Premature stop codons at amino acid positions 257 and 278 were present in the RSV G glycoprotein gene sequenced from each time point. None of the 48 RSV G sequences available on GenBank or any of 50 genetically diverse clinical isolates of RSV contained these mutations. These premature stop codon mutations occurred at the same positions of the G glycoprotein gene as those described in in vitro monoclonal-antibody resistant mutants reported elsewhere. CONCLUSION: Our patient was most likely infected with a single RSV strain that did not mutate during the study period. This strain contains unique mutations that may have previously evolved in this individual who had prolonged infection and was treated with monthly IVIG.

Epidemiology of human metapneumovirus.

Since the discovery of human metapneumovirus (hMPV) in 2001, the virus has been identified worldwide. hMPV is a common respiratory pathogen, particularly in infants and young children. The virus is associated with both upper and lower respiratory tract infections and may be a trigger for asthma. At least two major genotypes of hMPV circulate during community outbreaks. Whether these genotypes represent distinct serotypes remains controversial. The major challenges faced by the medical and scientific communities are the understanding of the pathogenesis of hMPV disease and the development of a safe and effective vaccine to protect against infection and disease caused by this newly recognized respiratory virus.