Virology under the microscope—a call for rational discourse

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns – conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we – a broad group of working virologists – seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology

Identification of variants in primary and recurrent glioblastoma using a cancer-specific gene panel and whole exome sequencing.

Glioblastoma (GBM) is an aggressive, malignant brain tumor typically resulting in death of the patient within one year following diagnosis; and those who survive beyond this point usually present with tumor recurrence within two years (5-year survival is 5%). The genetic heterogeneity of GBM has made the molecular characterization of these tumors an area of great interest and has led to identification of molecular subtypes in GBM. The availability of sequencing platforms that are both fast and economical can further the adoption of tumor sequencing in the clinical environment, potentially leading to identification of clinically actionable genetic targets. In this pilot study, comprised of triplet samples of normal blood, primary tumor, and recurrent tumor samples from three patients; we compared the ability of Illumina whole exome sequencing (ExomeSeq) and the Ion AmpliSeq Comprehensive Cancer Panel (CCP) to identify somatic variants in patient-paired primary and recurrent tumor samples. Thirteen genes were found to harbor variants, the majority of which were exclusive to the ExomeSeq data. Surprisingly, only two variants were identified by both platforms and they were located within the PTCH1 and NF1 genes. Although preliminary in nature, this work highlights major differences in variant identification in data generated from the two platforms. Additional studies with larger samples sizes are needed to further explore the differences between these technologies and to enhance our understanding of the clinical utility of panel based platforms in genomic profiling of brain tumors

Patient demographics and clinical information.

<p>All patients received total resection, followed by temozolomide and external beam radiation of 6,000 Gray in 30 fractions.</p><p>^aW/NH, white not Hispanic or Latino; W/H, white Hispanic or Latino.</p><p>^bSample purity determined by histological review.</p><p>Patient demographics and clinical information.</p

Read alignment view of <i>PTEN</i> (A) and <i>TP53</i> (B) genes zoomed out to exon level.

<p>The AmpliSeq data did not produce any reads covering the variant base in <i>PTEN</i>; however, a single read not shown in the graphic did cover the variant base in <i>TP53</i> in the recurrent tumor. Data from primary tumors are shown. (A) View displayed spans chr10: 89,653,700–89,653,900 of <i>PTEN</i>; variant is T to G mutation located at base 89,653,783. (B) View of <i>TP53</i> displays region of chr17: 7,578,400–7,578,600; variant is G to A mutation located at base 7,578,475. The location of the variant base is indicated by the vertical line crossing the reads.</p

Chikungunya virus' high genomic plasticity enables rapid adaptation to restrictive A549 cells

Chikungunya virus (CHIKV) is an emerging arthropod-borne virus that has spread globally during the last two decades. The virus is mainly transmitted by Aedes aegypti and Aedes albopictus mosquitos and is thus capable of replicating in both human and mosquito cells. CHIKV has a broad tropism in vivo, capable of replicating in various tissues and cell types but largely excluding blood cells. This was reflected in vitro by a broad array of adherent cell lines supporting CHIKV infection. One marked exception to this general rule is the resistance of the lung cancer-derived A549 cell line to CHIKV infection. We verified that A549 cells were restrictive to infection by multiple alphaviruses while being completely permissive to flavivirus infection. The adaptive growth of a primary CHIKV strain through multiple passages allowed the emergence of a CHIKV strain that productively infected A549 cells while causing overt cytopathic effects and without a fitness cost for replication in otherwise CHIKV-susceptible cells. Whole genome sequencing of polyclonal and monoclonal preparations of the adapted virus showed that a limited number of mutations consistently emerged in both structural (2 mutations in E2) and non-structural proteins (1 mutation in nsP1 and 1 mutation in nsP2). The introduction of the adaptive mutations, individually or in combinations, into a wild-type molecular clone of CHIKV allowed us to determine the relative contributions of the mutations to the new phenotype. We found that the mutations in the E2 envelope protein and non-structural proteins contributed significantly to the acquired phenotype. The nsP mutations were introduced in a split-genome trans-replicase assay to monitor their effect on viral genome replication efficiency. Interestingly, neither mutation supported increased viral genomic replication in either Vero or A549 cells

Overview of patient samples and sequencing methods.

<p>Sequencing for the AmpliSeq CCP samples was done using the Ion 318 Chip and whole exome sequencing was preformed using the Genome Analyzer II or HiSeq 2000, both from Illumina.</p

Summary of mutated AmpliSeq CCP genes.

<p>Genes containing variants and the associated sequencing platforms are shown. Most variants were associated with ExomeSeq data and there were a total of 409 genes on the AmpliSeq CCP.</p

Variants identified in Ion AmpliSeq Comprehensive Cancer Panel Genes.

<p>Summary of all variants identified in this study, the majority of which were found in primary tumors. R, recurrent; Chr, chromosome; NS, nonsynonymous; P, primary; SNV, single nucleotide variant.</p><p>Variants identified in Ion AmpliSeq Comprehensive Cancer Panel Genes.</p

Persistent Replication of Human Immunodeficiency Virus Type 1 despite Treatment of Pulmonary Tuberculosis in Dually Infected Subjects

Tuberculosis (TB) is the most common life-threatening infection in human immunodeficiency virus (HIV)-infected persons and frequently occurs before the onset of severe immunodeficiency. Development of TB is associated with increased HIV type 1 (HIV-1) viral load, a fall in CD4 lymphocyte counts, and increased mortality. The aim of this study was to examine how treatment of pulmonary TB affected HIV-1 activity in HIV-1/TB-coinfected subjects with CD4 cell counts of >100 cells/μl. HIV-1/TB-coinfected subjects were recruited in Kampala, Uganda, and were monitored over time. Based upon a significant (0.5 log(10) copies/ml) decrease in viral load by the end of treatment, two patient groups could be distinguished. Responders (n = 17) had more rapid resolution of anemia and pulmonary lesions on chest radiography during TB treatment. This group had a significant increase in viral load to levels not different from those at baseline 6 months after completion of TB treatment. HIV-1 viral load in nonresponders (n = 10) with TB treatment increased and at the 6 month follow-up was significantly higher than that at the time of diagnosis of TB. Compared to baseline levels, serum markers of macrophage activation including soluble CD14 decreased significantly by the end of TB treatment in responders but not in nonresponders. These data further define the impact of pulmonary TB on HIV-1 disease. HIV-1 replication during dual HIV-1/TB infection is not amenable to virologic control by treatment of TB alone. Concurrent institution of highly active antiretroviral treatment needs to be evaluated in patients dually infected with pulmonary TB and HIV-1