Join us for the next Journal of Virology Seminar Series where Jason Botten, Ph.D. and Rebekah Honce, Ph.D. will discuss the ecology and natural history of orthohantavirus infection in reservoir rodents, with an emphasis on the North American Sin Nombre virus, which can cause hantavirus cardiopulmonary syndrome (HCPS) in humans. Orthohantaviruses are enveloped, negative-strand RNA viruses that are maintained in nature in rodents where they are thought to establish life-long, asymptomatic infections. This webinar will outline approaches to study the infection process in reservoir animals, including the development of near-natural infection models featuring wild rodents and matched infectious virus isolates obtained from those same wild populations. To illustrate how this can be done, the presenters will revisit the nontraditional approach used to establish the first experimental model of Sin Nombre virus infection in its reservoir, the deer mouse and discuss key findings from that model. The presentation will close with a summary of knowledge regarding the natural history of infection of other orthohantaviruses as well as mammarenaviruses in their respective reservoir hosts and a discussion of key gaps in knowledge and opportunities for the field. Key Learning Objectives: Understand what rodent-borne orthohantaviruses and mammarenaviruses are. Understand the infection process of Sin Nombre virus in its reservoir, the deer mouse. Understand the approaches available to study rodent-borne virus infection in natural reservoir rodents, including their strengths and limitations. Learn the key gaps in knowledge regarding our understanding of infection in reservoir animals and opportunities available to address these key questions.

Zoonotic viruses represent a significant threat to human health, underscoring the critical need to understand their biology within natural reservoir hosts. Traditional laboratory infection models are often insufficient for this purpose, necessitating a shift towards more ecologically realistic approaches. This study advocates for the use of experimental infection models featuring authentic virus-host pairings, complemented by observational trap-and-release field studies. Such systems are shown to more effectively capture the intricate host-virus interactions that drive viral persistence and transmission within reservoirs, thereby providing crucial insights into spillover mechanisms to humans. The presented primary studies, integrated with findings from a mini-review, offer guidance on optimal strategies for investigating infection processes in natural hosts. A key unresolved question in the field pertains to the restricted geographic distribution of certain rodent-borne viruses, exemplified by Lassa virus, which primarily emerges in West Africa despite its reservoir host being broadly distributed across the African continent. Addressing such questions through advanced experimental models is paramount. Ultimately, a deeper understanding of these intimate virus-host interactions, particularly the identification of critical “choke points” in the viral life cycle, is essential for developing broad-spectrum antivirals and effective vaccines, moving beyond pathogen-specific treatments.

ABSTRACT Rodent-borne viruses pose threats to global health because of their capacity for zoonoses with the potential to cause outbreaks of high morbidity and mortality. Gaining a deeper understanding of how these viruses persist in their reservoir hosts and spillover into human populations requires an integrated approach blending both laboratory- and field-based research. Here, we review our current understanding of rodent-borne virus dynamics—primarily mammarenaviruses and orthohantaviruses—within their natural rodent reservoirs. We highlight key insights gained from experimental infection models seeking to replicate authentic host-pathogen interactions under controlled conditions. We argue that expanding and refining experimental models, particularly those that simulate natural reservoir conditions, is crucial to identifying the host and viral factors facilitating viral persistence in nature, and transmission to human populations. We propose a framework for future research that prioritizes hypothesis-driven research in model systems that are both natural and translatable to bridge these knowledge gaps.

Rodent-borne hantaviruses pose a continual public health threat to humans through zoonotic transmission, with case fatality rates of up to 50% in some cases. Human infections can lead to hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome depending on the viral species. Despite the morbidity and mortality associated with this family of viruses, no anti-viral therapeutics or vaccines are available to treat and prevent hantavirus disease. The relative shortage of commercially available reagents to study hantavirus infections in vitro and in vivo likely contributes to the challenges in developing viral countermeasures. This report describes the generation of a panel of mouse monoclonal antibodies that collectively recognize the four viral proteins of Seoul virus (SEOV, Orthohantavirus seoulense), an Old World (OW) hantavirus with worldwide distribution, and the causative agent of HFRS. We have validated the specificity and versatility of these antibodies against a subset of OW and New World hantaviruses in assays relying on antigen recognition in denatured or native conformations. We present several antibodies that specifically recognize the SEOV nucleoprotein and polymerase protein in Western blotting and immunostaining assays. We also identified three novel antibodies directed against the glycoprotein complex that are capable of binding to the N-terminal glycoprotein of all hantaviruses tested. These antibodies are freely available to all hantavirus researchers to add to the small, but growing, collection of reliable and available reagents to be used to study hantavirus biology, identify novel antiviral compounds, and measure viral prevalence in the laboratory and the field.IMPORTANCEPathogenic hantaviruses cause severe hemorrhagic disease and pose a significant public health threat worldwide. Insufficient research into the biology of these viruses has slowed the development of effective direct-acting antivirals and vaccines. Here, we describe the generation and validation of novel, specific monoclonal antibodies for the detection of Seoul virus proteins in vitro. These reagents can be used to fill in critical gaps in knowledge regarding hantavirus entry, protein expression, and particle generation.

Since the SARS-CoV-2 Omicron virus has gained dominance worldwide, its continual evolution with unpredictable mutations and patterns has revoked all authorized immunotherapeutics. Rapid viral evolution has also necessitated several rounds of vaccine updates in order to provide adequate immune protection. It remains imperative to understand how Omicron evolves into different subvariants and causes immune escape as this could help reevaluate the current intervention strategies mostly implemented in the clinics as emergency measures to counter the pandemic and, importantly, develop new solutions. Here, we provide a review focusing on the major events of Omicron viral evolution, including the features of spike mutation that lead to immune evasion against monoclonal antibody (mAb) therapy and vaccination, and suggest alternative durable options such as the ACE2-based experimental therapies superior to mAbs to address this unprecedented evolution of Omicron virus. In addition, this type of unique ACE2-based virus-trapping molecules can counter all zoonotic SARS coronaviruses, either from unknown animal hosts or from established wild-life reservoirs of SARS-CoV-2, and even seasonal alpha coronavirus NL63 that depends on human ACE2 for infection.

The capacity of SARS-CoV-2 to evolve poses challenges to conventional prevention and treatment options such as vaccination and monoclonal antibodies, as they rely on viral receptor binding domain (RBD) sequences from previous strains. Additionally, animal CoVs, especially those of the SARS family, are now appreciated as a constant pandemic threat. We present here a new antiviral approach featuring inhalation delivery of a recombinant viral trap composed of ten copies of angiotensin-converting enzyme 2 (ACE2) fused to the IgM Fc. This ACE2 decamer viral trap is designed to inhibit SARS-CoV-2 entry function, regardless of viral RBD sequence variations as shown by its high neutralization potency against all known SARS-CoV-2 variants, including Omicron BQ.1, BQ.1.1, XBB.1 and XBB.1.5. In addition, it demonstrates potency against SARS-CoV-1, human NL63, as well as bat and pangolin CoVs. The multivalent trap is effective in both prophylactic and therapeutic settings since a single intranasal dosing confers protection in human ACE2 transgenic mice against viral challenges. Lastly, this molecule is stable at ambient temperature for more than twelve weeks and can sustain physical stress from aerosolization. These results demonstrate the potential of a decameric ACE2 viral trap as an inhalation solution for ACE2-dependent coronaviruses of current and future pandemic concerns.

Reverse transcription–quantitative polymerase chain reaction (RT-qPCR) is used worldwide to test and trace the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). “Extraction-less” or “direct” real time–reverse transcription polymerase chain reaction (RT-PCR) is a transparent and accessible qualitative method for SARS-CoV-2 detection from nasopharyngeal or oral pharyngeal samples with the potential to generate actionable data more quickly, at a lower cost, and with fewer experimental resources than full RT-qPCR. This study engaged 10 global testing sites, including laboratories currently experiencing testing limitations due to reagent or equipment shortages, in an international interlaboratory ring trial. Participating laboratories were provided a common protocol, common reagents, aliquots of identical pooled clinical samples, and purified nucleic acids and used their existing in-house equipment. We observed 100% concordance across laboratories in the correct identification of all positive and negative samples, with highly similar cycle threshold values. The test also performed well when applied to locally collected patient nasopharyngeal samples, provided the viral transport media did not contain charcoal or guanidine, both of which appeared to potently inhibit the RT-PCR reaction. Our results suggest that direct RT-PCR assay methods can be clearly translated across sites utilizing readily available equipment and expertise and are thus a feasible option for more efficient COVID-19 coronavirus disease testing as demanded by the continuing pandemic.

ABSTRACTAmplicon-based sequencing methods have been central in characterizing the diversity, transmission and evolution of SARS-CoV-2, but need to be rigorously assessed for clinical utility. Here, we validated the Swift Biosciences’ SARS-CoV-2 Swift Normalase Amplicon Panels using remnant clinical specimens. High quality genomes meeting our established library and sequence quality criteria were recovered from positive specimens with a 95% limit of detection of ≥ 40.08 SARS-CoV-2 copies/PCR reaction. Breadth of genome recovery was evaluated across a range of Ct values (11.3 – 36.7, median 21.6). Out of 428 positive samples, 406 (94.9%) generated genomes with < 10% Ns, with a mean genome coverage of 13,545X ± SD 8,382X. No genomes were recovered from PCR-negative specimens (n = 30), or from specimens positive for non-SARS-CoV-2 respiratory viruses (n = 20). Compared to whole-genome shotgun metagenomic sequencing (n = 14) or Sanger sequencing for the spike gene (n = 11), pairwise identity between consensus sequences was 100% in all cases, with highly concordant allele frequencies (R2= 0.99) between Swift and shotgun libraries. When samples from different clades were mixed at varying ratios, expected variants were detected even in 1:99 mixtures. When deployed as a clinical test, 268 tests were performed in the first 23 weeks with a median turnaround time of 11 days, ordered primarily for outbreak investigations and infection control.

Flaviviruses are enveloped, positive-strand RNA viruses that cause millions of infections in the human population annually. Although Zika virus (ZIKV) had been detected in humans as early as the 1950s, its reemergence in South America in 2015 resulted in a global health crisis. While flaviviruses encode 10 proteins that can be post-translationally modified by host enzymes, little is known regarding post-translational modifications (PTMs) of the flavivirus proteome. We used mass spectrometry to comprehensively identify host-driven PTMs on the ZIKV proteome. This approach allowed us to identify 43 PTMs across 8 ZIKV proteins, including several that are highly conserved within the Flavivirus genus. Notably, we found two phosphosites on the ZIKV envelope protein that are functionally important for viral propagation and appear to regulate viral budding. Additionally, we discovered 115 host kinases that interacted with ZIKV proteins and determined that Bosutinib, an FDA-approved tyrosine kinase inhibitor that targets ZIKV interacting host kinases, impairs ZIKV growth. Thus, we have defined a high-resolution map of host-driven PTMs on ZIKV proteins as well as cellular interacting kinases, uncovered a novel mechanism of host driven-regulation of ZIKV budding, and identified an FDA-approved inhibitor of ZIKV growth.

The pandemic caused by severe acute respiratory distress syndrome coronavirus 2 (SARS-CoV-2) prompted rapid development of research tools, including plasmid systems carrying cDNA copies of various portions of the viral genome, providing tractable tools for the study of the virus [1, 2].….

ABSTRACTWith the COVID-19 pandemic caused by SARS-CoV-2 now in its second year, there remains an urgent need for diagnostic testing that can identify infected individuals, particularly those who harbor infectious virus. Various RT-PCR strategies have been proposed to identify specific viral RNA species that may predict the presence of infectious virus, including detection of transcriptional intermediates (e.g. subgenomic RNA [sgRNA]) and replicative intermediates (e.g. negative-strand RNA species). Using a novel primer/probe set for detection of subgenomic (sg)E transcripts, we successfully identified 100% of specimens containing culturable SARS-CoV-2 from a set of 126 clinical samples (total sgE CT values ranging from 12.3-37.5). This assay showed superior performance compared to a previously published sgRNA assay and to a negative-strand RNA assay, both of which failed to detect target RNA in a subset of samples from which we isolated live virus. In addition, total levels of viral RNA (genome, negative-strand, and sgE) detected with the WHO/Charité primer-probe set correlated closely with levels of infectious virus. Specifically, infectious virus was not detected in samples with a CT above 31.0. Clinical samples with higher levels of viral RNA also displayed cytopathic effect (CPE) more quickly than those with lower levels of viral RNA. Finally, we found that the infectivity of SARS-CoV-2 samples is significantly dependent on the cell type used for viral isolation, as Vero E6 cells expressing TMRPSS2 extended the analytical sensitivity of isolation by more than 3 CT compared to parental Vero E6 cells and resulted in faster isolation. Our work shows that using a total viral RNA Ct cut-off of >31 or specifically testing for sgRNA can serve as an effective rule-out test for viral infectivity.

There is an urgent need for antiviral agents that treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We screened a library of 1900 clinically safe drugs against OC43, a human beta coronavirus that causes the common cold, and evaluated the top hits against SARS-CoV-2. Twenty drugs significantly inhibited replication of both viruses in cultured human cells. Eight of these drugs inhibited the activity of the SARS-CoV-2 main protease, 3CLpro, with the most potent being masitinib, an orally bioavailable tyrosine kinase inhibitor. X-ray crystallography and biochemistry show that masitinib acts as a competitive inhibitor of 3CLpro. Mice infected with SARS-CoV-2 and then treated with masitinib showed >200-fold reduction in viral titers in the lungs and nose, as well as reduced lung inflammation. Masitinib was also effective in vitro against all tested variants of concern (B.1.1.7, B.1.351, and P.1).

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus responsible for the current COVID-19 pandemic and has now infected more than 200 million people with more than 4 million deaths globally. Recent data suggest that symptoms and general malaise may continue long after the infection has ended in recovered patients, suggesting that SARS-CoV-2 infection has profound consequences in the host cells. Here we report that SARS-CoV-2 infection can trigger a DNA damage response (DDR) in African green monkey kidney cells (Vero E6). We observed a transcriptional upregulation of the Ataxia telangiectasia and Rad3 related protein (ATR) in infected cells. In addition, we observed enhanced phosphorylation of CHK1, a downstream effector of the ATR DNA damage response, as well as H2AX. Strikingly, SARS-CoV-2 infection lowered the expression of TRF2 shelterin-protein complex, and reduced telomere lengths in infected Vero E6 cells. Thus, our observations suggest SARS-CoV-2 may have pathological consequences to host cells beyond evoking an immunopathogenic immune response.

The spillover of pathogenic hantaviruses from rodent reservoirs into the human population poses a continued threat to human health. Here, we show that a recombinant form of the Hantaan virus (HTNV) surface-displayed glycoprotein, Gn, elicits a neutralizing antibody response in rabbits.

The tetravalent live attenuated dengue vaccine candidate TV003 induces neutralizing antibodies against all four dengue virus serotypes (DENV1-4), and protects against experimental DENV2 challenge in humans. Protection occurred with or without a secondary post-challenge rise in DENV2-specific neutralizing antibodies. Here we combined high resolution temporal sampling and analysis of antigen-specific responses after vaccination and challenge to determine the cellular underpinnings of the B cell response in relation to vaccine viremia and serum antibodies. TV003 vaccine-related viremia was associated with an acute plasmablast response that correlated with the development of serum neutralizing antibodies. At six-months following immunization, subjects had developed DENV2-specific memory B cells including serotype-specific and multi-valent responders. DENV2 challenge of vaccinees did not induce a post-challenge plasmablast response, though stronger and earlier post-vaccine plasmablast responses were associated with sterile humoral protection from DENV2 challenge (i.e. a lack of DENV2 antibody boosting). Our findings demonstrate that TV003 vaccine triggers a durable B cell response containing plasmablasts and memory B cells which functionally link early vaccine viremia and the serum antibody responses.

Dengue virus (DENV) is a mosquito-borne flavivirus that causes serious human disease. The current lack of an effective vaccine to simultaneously protect against the four serotypes of DENV in seronegative individuals is a major unmet medical need. Further, the immunological basis for protective immunity in the setting of DENV infection or vaccination is not fully understood. Our team has developed a live attenuated tetravalent dengue virus vaccine that provides complete protection in a human model of dengue virus challenge. The goal of this study was to define, in the context of protective human vaccination, the quality of vaccine-induced DENV-specific CD8(+) and CD4(+) T cells and the temporal dynamics associated with their formation and maintenance. Multifunctional, DENV-specific CD8(+) and CD4(+) T cells developed 8-14 days after vaccination and were maintained for at least 6 months. Virus-specific CD8 T+ cells were a mixture of effector memory T cells (T-EM) and effector memory T cells re-expressing CD45RA (T-EMRA), with T-EM cells predominating until day 21 post-vaccination and T-EMRA cells thereafter. The majority of virus-specific CD4(+) T cells were T-EM with a small fraction being T-EMRA. The frequency of virus-specific CD8(+) and CD4(+) T cells were further skewed to the T-EMRA phenotype following either a second dose of the tetravalent vaccine or challenge with a single serotype of DENV. Collectively, our study has defined the phenotypic profile of antiviral CD8(+) and CD4(+) T cells associated with protective immunity to DENV infection and the kinetics of their formation and maintenance.

Viral defective interfering particles (DIPs) were intensely studied several decades ago but research waned leaving open many critical questions. New technologies and other advances led to a resurgence in DIP studies for negative-strand RNA viruses. While DIPs have long been recognized, their exact contribution to the outcome of acute or persistent viral infections has remained elusive. Recent studies have identified defective viral genomes (DVGs) in human infections, including respiratory syncytial virus and influenza, and growing evidence indicates that DVGs influence disease severity and may contribute to viral persistence. Further, several studies have advanced our understanding of key viral and host factors that regulate DIP formation and activity. Here we review these discoveries and highlight key questions moving forward.

Current demand for SARS-CoV-2 testing is straining material resource and labor capacity around the globe. As a result, the public health and clinical community are hindered in their ability to monitor and contain the spread of COVID-19. Despite broad consensus that more testing is needed, pragmatic guidance toward realizing this objective has been limited. This paper addresses this limitation by proposing a novel and geographically agnostic framework (the 4Ps framework) to guide multidisciplinary, scalable, resource-efficient, and achievable efforts toward enhanced testing capacity. The 4Ps (Prioritize, Propagate, Partition, and Provide) are described in terms of specific opportunities to enhance the volume, diversity, characterization, and implementation of SARS-CoV-2 testing to benefit public health. Coordinated deployment of the strategic and tactical recommendations described in this framework has the potential to rapidly expand available testing capacity, improve public health decision-making in response to the COVID-19 pandemic, and/or to be applied in future emergent disease outbreaks.

SARS-CoV-2 is the newly emerged virus responsible for the global COVID-19 pandemic. There is an incomplete understanding of the host humoral immune response to SARS-CoV-2 during acute infection. Host factors such as age and sex as well the kinetics and functionality of antibody responses are important factors to consider as vaccine development proceeds. The receptor-binding domain of the CoV spike (RBD-S) protein is important in host cell recognition and infection and antibodies targeting this domain are often neutralizing. In a cross-sectional study of anti-RBD-S antibodies in COVID-19 patients we found equivalent levels in male and female patients and no age-related deficiencies even out to 93 years of age. The anti-RBD-S response was evident as little as 6 days after onset of symptoms and for at least 5 weeks after symptom onset. Anti-RBD-S IgG, IgM, and IgA responses were simultaneously induced within 10 days after onset, but isotype-specific kinetics differed such that anti-RBD-S IgG was most sustained over a 5-week period. The kinetics and magnitude of neutralizing antibody formation strongly correlated with that seen for anti-RBD-S antibodies. Our results suggest age- and sex- related disparities in COVID-19 fatalities are not explained by anti-RBD-S responses. The multi-isotype anti-RBD-S response induced by live virus infection could serve as a potential marker by which to monitor vaccine-induced responses.