Characterization of Human Adenoviruses of Medical Importance: Isolation of Infectious Virus from Clinical Specimens and Molecular Typing.

Human adenoviruses (HAdVs) constitute a group of ubiquitous viruses that currently comprises 51 well-defined serotypes and more than 113 genotypes classified into seven species, HAdV-A through HAdV-G. The members of these species differ considerably in their genomic characteristics and also in their tropism and pathogenicity. Virus isolation in cell culture remains critical for the preservation and comprehensive characterization of viruses of biomedical interest but has been almost completely abandoned by diagnostic laboratories. Currently, the most frequently used approach for the detection of HAdV in clinical specimens is real-time qPCR targeting a region of the hexon gene, conserved among all genotypes described to the present. In the absence of typing, the detection of an HAdV in association with disease provides limited information. Molecular typing is therefore highly desirable and required in the epidemiological investigation of HAdV-associated disease. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Virus isolation from plasma and whole blood Alternate Protocol 1: Virus isolation from stool Alternate Protocol 2: Virus isolation from respiratory specimens and urine Alternate Protocol 3: Virus isolation from tissue specimens Support Protocol: Inoculation of shell vials Basic Protocol 2: Extraction of highly pure viral genomic DNA from infected cells Basic Protocol 3: Molecular detection of human adenovirus by real-time PCR Basic Protocol 4: Molecular typing for basic identification of species and hexon type Basic Protocol 5: Typing human adenoviruses by next-generation whole-genome sequencing and analysis.

A non-enzymatic test for SARS-CoV-2 RNA using DNA nanoswitches.

The emergence of a highly contagious novel coronavirus in 2019 led to an unprecedented need for large scale diagnostic testing. The associated challenges including reagent shortages, cost, deployment delays, and turnaround time have all highlighted the need for an alternative suite of low-cost tests. Here, we demonstrate a diagnostic test for SARS-CoV-2 RNA that provides direct detection of viral RNA and eliminates the need for costly enzymes. We employ DNA nanoswitches that respond to segments of the viral RNA by a change in shape that is readable by gel electrophoresis. A new multi-targeting approach samples 120 different viral regions to improve the limit of detection and provide robust detection of viral variants. We apply our approach to a cohort of clinical samples, positively identifying a subset of samples with high viral loads. Since our method directly detects multiple regions of viral RNA without amplification, it eliminates the risk of amplicon contamination and renders the method less susceptible to false positives. This new tool can benefit the COVID-19 pandemic and future emerging outbreaks, providing a third option between amplification-based RNA detection and protein antigen detection. Ultimately, we believe this tool can be adapted both for low-resource onsite testing as well as for monitoring viral loads in recovering patients.

Detection of SARS-CoV-2 Variants by Multiple Diagnostic Assays Second RESUBMISSION JCV-D-21-00675R2.

INTRODUCTION: The emergence of SARS-CoV-2 Variants of Concern (VOC) and Variants Being Monitored (VBM) have presented additional clinical and public health concerns regarding potential virus transmissibility, disease severity, and immune evasion. It is imperative that diagnostic assays can detect all such variants, and since commercial oligo sequences are commonly not available, empirical testing may be necessary to confirm this. To confirm the sensitivity of the SARS-CoV-2 assays used at the Wadsworth Center for the detection of VOC and VBM, relevant specimens were selected from the specimen archive and tested in the various platforms. MATERIALS AND METHODS: Patient respiratory specimens submitted from clinal laboratories across the state were selected; three samples per variant were chosen to account for inter assay and variant reproducibility. The four molecular diagnostic platforms for SARS-CoV-2 currently in use at our facility were examined. RESULTS: A total of 64 specimens were tested, representing 2 VOC, 8 VBM and 4 other variants circulating in New York State. For certain samples, original Ct values provided by sample submitters were much higher, or lower, than those obtained from this study. The investigation of submitter testing platforms, with consideration of the assay's viral targets, confirmed the differences in Ct were not variant specific. CONCLUSIONS: It was demonstrated that the diagnostic methods investigated in this study detected all the variants tested. Because of the continual evolution of the virus, it is vital to monitor new variants as they emerge for the ability of molecular diagnostic methods to detect them with acceptable sensitivity.

Delta-Omicron recombinant escapes therapeutic antibody neutralization.

Background: The emergence of recombinant viruses is a threat to public health. Recombination of viral variants may combine variant-specific features that together catalyze viral escape from treatment or immunity. The selective advantages of recombinant SARS-CoV-2 isolates over their parental lineages remain unknown. Methods: Multi-method amplicon and metagenomic sequencing of a clinical swab and the in vitro grown virus allowed for high-confidence detection of a novel recombinant variant. Mutational, phylogeographic, and structural analyses determined features of the recombinant genome and spike protein. Neutralization assays using infectious as well as pseudotyped viruses and point mutants thereof defined the recombinant's sensitivity to a panel of monoclonal antibodies and sera from vaccinated and/or convalescent individuals. Results: A novel Delta-Omicron SARS-CoV-2 recombinant was identified in an unvaccinated, immunosuppressed kidney transplant recipient treated with monoclonal antibody Sotrovimab. The recombination breakpoint is located in the spike N-terminal domain, adjacent to the Sotrovimab quaternary binding site, and results in a 5'-Delta AY.45 and a 3'-Omicron BA.1 mosaic spike protein. Delta and BA.1 are sensitive to Sotrovimab neutralization, whereas the Delta-Omicron recombinant is highly resistant to Sotrovimab, both with and without the RBD resistance mutation E340D. Conclusions: Recombination between circulating SARS-CoV-2 variants can functionally contribute to immune escape. It is critical to validate phenotypes of mosaic viruses and monitor immunosuppressed COVID-19 patients treated with monoclonal antibodies for the selection of recombinant and immune escape variants. (Funded by NYU, the National Institutes of Health, and others).

Early introductions and community transmission of SARS-CoV-2 variant B.1.1.7 in the United States.

The emergence and spread of SARS-CoV-2 lineage B.1.1.7, first detected in the United Kingdom, has become a global public health concern because of its increased transmissibility. Over 2500 COVID-19 cases associated with this variant have been detected in the US since December 2020, but the extent of establishment is relatively unknown. Using travel, genomic, and diagnostic data, we highlight the primary ports of entry for B.1.1.7 in the US and locations of possible underreporting of B.1.1.7 cases. Furthermore, we found evidence for many independent B.1.1.7 establishments starting in early December 2020, followed by interstate spread by the end of the month. Finally, we project that B.1.1.7 will be the dominant lineage in many states by mid to late March. Thus, genomic surveillance for B.1.1.7 and other variants urgently needs to be enhanced to better inform the public health response.

Monitoring Coronavirus Disease 2019 (COVID-19) Through Trends in Influenza-like Illness, Laboratory-confirmed Influenza, and COVID-19-New York State, Excluding New York City, 1 January 2020-12 April 2020.

Innovative monitoring approaches are needed to track the coronavirus disease 2019 (COVID-19) epidemic and potentially assess the impact of community mitigation interventions. We present temporal data on influenza-like illness, influenza diagnosis, and COVID-19 cases for all 4 regions of New York State through the first 6 weeks of the outbreak.

Multicenter evaluation of the NeuMoDx™ SARS-CoV-2 Test.

The SARS-CoV-2 virus has caused millions of confirmed COVID-19 cases worldwide and hundreds of thousands of deaths in less than 6 months. Mitigation measures including social distancing were implemented to control disease spread, however, thousands of new cases continue to be diagnosed daily. To resume some suspended social activities, early diagnosis and contact tracing are essential. To meet this required diagnostic and screening capacity, high throughput diagnostic assays are needed. The NeuMoDx™ SARS-CoV-2 assay, performed on a NeuMoDx molecular system, is a rapid, fully automated, qualitative real-time RT-PCR diagnostic test with throughput of up to 288 tests in an 8 -h shift. The assay received emergency use authorization from the FDA and is used in some large testing centers in the US. This paper describes the analytical and clinical performance of the assay at three centers: Johns Hopkins Hospital, St. Jude Children's Research Hospital, and the Wadsworth Center.

Evaluation of the COVID19 ID NOW EUA assay.

BACKGROUND: The SARS-CoV-2 pandemic caused a major surge in needed diagnostic capacity. In response, many EUA assays have become available for clinical laboratories, and more recently, the point of care device, Abbott ID NOW. OBJECTIVES: To determine the analytical performance of the ID NOW assay for detecting SARS-CoV-2. STUDY DESIGN: Residual NP samples collected in viral transport media were tested by the ID NOW platform in two independent laboratories. Results were compared to either the CDC or New York EUA assays, which served as reference methods. RESULTS: Overall agreement of ID NOW was 78.7%. Sensitivity was 71.7% and specificity was 100%. Notably, all false-negative results correlated to those samples that were weakly positive. CONCLUSIONS: ID NOW performs well for strong and moderately positive samples but has reduced sensitivity for weakly positive samples. This sensitivity, among other concerns, should be taken into consideration when using this test for patients with a low suspicion for COVID-19 disease.

Genetic characterization of mumps viruses associated with the resurgence of mumps in the United States: 2015-2017.

Despite high coverage with measles, mumps, and rubella vaccine in the United States, outbreaks of mumps occur in close contact settings such as schools, colleges, and camps. Starting in late 2015, outbreaks were reported from several universities, and by the end of 2017, greater than 13,800 cases had been reported nation-wide. In 2013, the CDC and the Association of Public Health Laboratories contracted four Vaccine Preventable Diseases Reference Centers (VPD-RCs) to perform real-time reverse transcription PCR (RT-qPCR) to detect mumps RNA in clinical samples and to determine the genotype. Twelve genotypes of mumps virus are currently recognized by the World Health Organization, and the standard protocol for genotyping requires sequencing the entire gene coding for the small hydrophobic (SH) protein. Phylogenetic analysis of the 1862 mumps samples genotyped from 2015 through 2017 showed that the overall diversity of genotypes detected was low. Only 0.8 % of the sequences were identified as genotypes C, H, J, or K, and 0.5 % were identified as vaccine strains in genotypes A or N, while most sequences (98.7 %) were genotype G. The majority of the genotype G sequences could be included into one of two large groups with identical SH sequences. Within genotype G, a small number of phylogenetically significant outlier sequences were associated with epidemiologically distinct chains of transmission. These results demonstrate that molecular and epidemiologic data can be used to track transmission pathways of mumps virus; however, the limited diversity of the SH sequences may be insufficient for resolving transmission in all outbreaks.

Use of the immunoglobulin G avidity assay to differentiate between recent Zika and past dengue virus infections.

Zika (ZIKV) and dengue (DENV) virus infections elicit a robust but cross-reactive antibody response against the viral envelope protein, while antibody responses against non-structural proteins (NS) are more virus specific. Building on this premise, we have previously developed a flavivirus multiplex microsphere immunoassay (MIA) for the serologic diagnosis of ZIKV and DENV infections. This assay significantly improved diagnostic accuracy; however, MIA could not differentiate more recent from past infections, which still represents a major diagnostic challenge. Therefore, an immunoglobulin G (IgG) based avidity assay was developed and its diagnostic performance evaluated. Specimens from New York State residents were submitted to the Wadsworth Center New York State Department of Health (NYSDOH) for routine clinical testing by Zika IgM ELISA and plaque reduction neutralization test (PRNT). Using our previously developed flavivirus MIA as a platform, we developed an IgG avidity assay to discriminate recent ZIKV from past DENV infections. Zika IgM positive specimens had an average Zika IgG avidity index of 14.8% (95% CI: 11.0-18.4%), while Zika IgM negative but flavivirus MIA and PRNT positive samples had an average Zika IgG avidity index of 34.9% (95% CI: 31.1-38.7%). Specimens positive for dengue antibodies by flavivirus MIA and PRNT had an average dengue IgG avidity index of 68.7% (95% CI: 62.7-75.0%). The IgG avidity assay accurately distinguished recent ZIKV from past DENV infections in patients who traveled to dengue endemic regions. This assay could be very useful in patients with high risk of Zika complications such as pregnant women and monitoring immune responses in vaccine trials.

Arm Paralysis After Routine Childhood Vaccinations: Application of Advanced Molecular Methods to the Causality Assessment of an Adverse Event After Immunization.

Post-licensure surveillance for adverse events following immunizations (AEFI) can identify rare complications of vaccinations and rigorous vaccine adverse event causality assessments can help to identify possible causal relationships. We report the development of arm paralysis after varicella vaccination in a 1-year-old child. Paralysis was initially presumed to be due to vOka because of the temporal relationship between vaccination and onset of arm weakness; however, molecular studies identified wild-type varicella zoster virus VZV (WT-VZV) in the CSF, leading the authors to conclude that WT-VZV was the probable cause. This case illustrates the complexity of assessing AEFI causality, and the importance of careful and complete evaluations when determining the most likely cause of an AEFI.

Detection and Genomic Characterization of Enterovirus D68 in Respiratory Samples Isolated in the United States in 2016.

The genomic sequences of three 2016 enterovirus D68 (EV-D68) strains were obtained from respiratory samples of patients from Florida, Texas, and New York. These EV-D68 sequences share highest nucleotide identities with strains that circulated in North America, Europe, and Asia in 2014-2015.

Next generation sequencing for whole genome analysis and surveillance of influenza A viruses.

BACKGROUND: The Wadsworth Center, New York State Department of Health (NYSDOH), conducts routine diagnosis and surveillance of influenza viruses. Whole genome sequencing (WGS) with next generation sequencing (NGS) was initiated to provide more rapid, detailed, thorough, and accurate analysis. OBJECTIVES: To optimize and implement a method for routine WGS of influenza A viruses. To use WGS to monitor influenza A viruses for reassortment, mutations associated with antiviral resistance and antigenicity changes, as well as those potentially affecting virulence and tropism. STUDY DESIGN: Multiple extraction and amplification methods were investigated and optimized for the production of template to be used for NGS. Additionally, software options were considered for data analysis. Initial WGS influenza projects have included the comparison of mixed population sequence data obtained with NGS, Sanger dideoxy sequencing, and pyrosequencing, the comparison of sequences obtained from paired primary/cultured samples, the analysis of sequence changes over several influenza seasons, and phylogenetic analysis. RESULTS: Procedures were optimized for extraction and amplification such that WGS could be successfully performed on both cultured isolates and primary specimens. Data is presented on 15 A/H1pdm09 and 44 A/H3N2 samples. Analysis of influenza A viruses identified and confirmed variant and mixed populations affecting antigenicity and antiviral susceptibility in both primary specimens and cultured isolates. CONCLUSIONS: An influenza A whole genome PCR method has been optimized for the reliable production of template for NGS. The WGS method has been successfully implemented for enhanced comprehensive surveillance and the generation of detailed clinical data on drug resistance and virulence. Data obtained with this method will also aid in future vaccine selection.

Clinical and molecular epidemiology of human rhinovirus infections in patients with hematologic malignancy.

BACKGROUND: Human rhinoviruses (HRVs) are common causes of upper respiratory tract infection (URTI) in hematologic malignancy (HM) patients. Predictors of lower respiratory tract infection (LRTI) including the impact of HRV species and types are poorly understood. OBJECTIVES: This study aims to describe the clinical and molecular epidemiology of HRV infections among HM patients. STUDY DESIGN: From April 2012-March 2013, HRV-positive respiratory specimens from symptomatic HM patients were molecularly characterized by analysis of partial viral protein 1 (VP1) or VP4 gene sequence. HRV LRTI risk-factors and outcomes were analyzed using multivariable logistic regression. RESULTS: One hundred and ten HM patients presented with HRV URTI (n=78) and HRV LRTI (n=32). Hypoalbuminemia (OR 3.0; 95% CI, 1.0-9.2; p=0.05) was independently associated with LRTI, but other clinical and laboratory markers of host immunity did not differ between patients with URTI versus LRTI. Detection of bacterial co-pathogens was common in LRTI cases (25%). Among 92 typeable respiratory specimens, there were 58 (64%) HRV-As, 12 (13%) HRV-Bs, and 21 (23%) HRV-Cs, and one Enterovirus 68. LRTI rates among HRV-A (29%), HRV-B (17%), and HRV-C (29%) were similar. HRV-A infections occurred year-round while HRV-B and HRV-C infections clustered in the late fall and winter. CONCLUSIONS: HRVs are associated with LRTI in HM patients. Illness severity is not attributable to specific HRV species or types. The frequent detection of bacterial co-pathogens in HRV LRTIs further substantiates the hypothesis that HRVs predispose to bacterial superinfection of the lower airways, similar to that of other community-acquired respiratory viruses.

Diagnosis of influenza virus.

The laboratory diagnosis of influenza uses a wide range of techniques including rapid immunoassays, immunofluorescence techniques, virus culture methods, and increasingly sophisticated molecular assays. The potential utility of each of these methods has changed over the years, most dramatically perhaps with the emergence of the pandemic H1N1 2009 influenza virus. While rapid immunoassays had previously been widely used in clinics and emergency departments, their poor detection sensitivity for the 2009 subtype brought their application into question. Concerns were also raised about the detection sensitivities of antibody reagents used in immunofluorescence methods, and the safety of virus culture was initially questioned with regard to the newly emerged subtype. Early molecular detection techniques had been labor intensive, and required separate facilities in order to prevent contamination. Those techniques have largely been supplanted by more modern methods, most notably real-time reverse transcription PCR assays, which are currently the method of choice in many laboratories for the detection and subtyping of influenza viruses. Suspension and low-density array assays are also increasingly used, in an effort to detect larger numbers of viruses in a single assay, and microarrays have proven valuable for outbreak analysis and pathogen discovery. Each laboratory must assess the optimal methods for its situation and the best application of each technique, taking into account numerous factors including its budget, equipment, staff expertise, the patient population that it serves, the needs of its submitting clinicians, and its surveillance and public health responsibilities.

Detection of coxsackievirus A10 in multiple tissues of a fatal infant sepsis case.

Non-polio enteroviruses are a common cause of childhood infections varying in symptomatology and severity. While infections with many of the enterovirus serotypes can be severe and even fatal, coxsackievirus A10 (CVA10) has most commonly been associated with more mild disease. Here we present the detection of CVA10 in multiple organ tissues in the investigation of an infant death.

Integrated Amplification Microarrays for Infectious Disease Diagnostics.

This overview describes microarray-based tests that combine solution-phase amplification chemistry and microarray hybridization within a single microfluidic chamber. The integrated biochemical approach improves microarray workflow for diagnostic applications by reducing the number of steps and minimizing the potential for sample or amplicon cross-contamination. Examples described herein illustrate a basic, integrated approach for DNA and RNA genomes, and a simple consumable architecture for incorporating wash steps while retaining an entirely closed system. It is anticipated that integrated microarray biochemistry will provide an opportunity to significantly reduce the complexity and cost of microarray consumables, equipment, and workflow, which in turn will enable a broader spectrum of users to exploit the intrinsic multiplexing power of microarrays for infectious disease diagnostics.

Molecular epidemiology and brief history of emerging adenovirus 14-associated respiratory disease in the United States.

BACKGROUND: First isolated in the Netherlands in 1955 during an outbreak of acute respiratory disease (ARD) among military recruits, human adenovirus 14 (HAdV-14) has historically been considered rare. With no precedent of circulation in North America, HAdV-14 has been isolated from military and civilian cases of ARD of variable severity since 2003 in the United States. METHODS: Ninety-nine isolates from military and civilian cases from different geographic locations and circulation periods were characterized by restriction enzyme analysis of viral DNA and select gene sequencing. RESULTS: All examined viruses were found to be identical and to belong to a new genome type designated "HAdV-14p1" (formerly known as "14a"). Comparative alignments of E1A, hexon, and fiber gene sequences with other subspecies B2 HAdVs suggest that HAdV-14p1, like the closely related HAdV-11a, arose from recombination among similar HAdV-11 and HAdV-14 ancestral strains. A deletion of 2 amino acids in the knob region of the fiber protein is the only identified unique characteristic of HAdV-14p1. CONCLUSION: The current geographic distribution of HAdV-14p1 involves at least 15 states in the Unites States. The role of the fiber mutations in the recent emergence of HAdV-14p1 ARD in North America warrants further study.

Diagnosis of human metapneumovirus infection in immunosuppressed lung transplant recipients and children evaluated for pertussis.

Human metapneumovirus (hMPV) is a recently discovered paramyxovirus that is known to cause respiratory tract infections in children and immunocompromised individuals. Given the difficulties of identifying hMPV by conventional culture, molecular techniques could improve the detection of this virus in clinical specimens. In this study, we developed a real-time reverse transcription-PCR (RT-PCR) assay designed to detect the four genetic lineages of hMPV. This assay and a commercial real-time nucleic acid sequence-based amplification (NASBA) assay (bioMérieux, Durham, NC) were used to determine the prevalence of hMPV in 114 immunosuppressed asymptomatic and symptomatic lung transplant recipients and 232 pediatric patients who were being evaluated for pertussis. hMPV was detected in 4.3% of the immunosuppressed lung transplant recipients and in 9.9% of children evaluated for pertussis. Both RT-PCR and NASBA assays were efficient in detection of hMPV infection in respiratory specimens. Even though hMPV was detected in a small number of the lung transplant recipients, it was still the most prevalent etiologic agent detected in patients with respiratory symptoms. In both of these diverse patient populations, hMPV infection was the most frequent viral respiratory tract infection identified. Given our findings, infection with hMPV infection should be determined as part of the differential diagnosis of respiratory illnesses.

Stochastic processes are key determinants of short-term evolution in influenza a virus.

Understanding the evolutionary dynamics of influenza A virus is central to its surveillance and control. While immune-driven antigenic drift is a key determinant of viral evolution across epidemic seasons, the evolutionary processes shaping influenza virus diversity within seasons are less clear. Here we show with a phylogenetic analysis of 413 complete genomes of human H3N2 influenza A viruses collected between 1997 and 2005 from New York State, United States, that genetic diversity is both abundant and largely generated through the seasonal importation of multiple divergent clades of the same subtype. These clades cocirculated within New York State, allowing frequent reassortment and generating genome-wide diversity. However, relatively low levels of positive selection and genetic diversity were observed at amino acid sites considered important in antigenic drift. These results indicate that adaptive evolution occurs only sporadically in influenza A virus; rather, the stochastic processes of viral migration and clade reassortment play a vital role in shaping short-term evolutionary dynamics. Thus, predicting future patterns of influenza virus evolution for vaccine strain selection is inherently complex and requires intensive surveillance, whole-genome sequencing, and phenotypic analysis.