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We investigated the impact of antiviral treatment on the emergence of SARS-CoV-2 resistance during persistent infections in immunocompromised patients (n = 15). All patients received remdesivir and some also received nirmatrelvir-ritonavir (n = 3) or therapeutic monoclonal antibodies (n = 4). Sequence analysis showed that nine patients carried viruses with mutations in the nsp12 (RNA dependent RNA polymerase), while four had viruses with nsp5 (3C protease) mutations. Infectious SARS-CoV-2 with a double mutation in nsp5 (T169I) and nsp12 (V792I) was recovered from respiratory secretions 77 days after initial COVID-19 diagnosis from a patient sequentially treated with nirmatrelvir-ritonavir and remdesivir. In vitro characterization confirmed its decreased sensitivity to remdesivir and nirmatrelvir, which was overcome by combined antiviral treatment. Studies in golden Syrian hamsters demonstrated efficient transmission to contact animals. This study documents the isolation of SARS-CoV-2 carrying resistance mutations to both nirmatrelvir and remdesivir from a patient and demonstrates its transmissibility in vivo., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Split-virion-inactivated influenza vaccines are formulated based on viral hemagglutinin content. These vaccines also contain the viral neuraminidase (NA) protein, but NA content is not standardized and varies between manufacturers. In clinical studies and animal models, antibodies directed toward NA reduced disease severity and viral load; however, the impact of vaccine-induced NA immunity on airborne transmission of influenza A viruses is not well characterized. Therefore, we evaluated if vaccination against NA could disrupt chains of airborne transmission for the 2009 pandemic H1N1 virus in ferrets. Immunologically naïve donor ferrets were infected with the 2009 pandemic H1N1 virus and then paired in transmission cages with mock- or NA-vaccinated respiratory contacts. The mock- and NA-vaccinated animals were then monitored daily for infection, and once infected, these animals were paired with a naive secondary respiratory contact. In these studies, all mock- and NA-vaccinated animals became infected; however, NA-vaccinated animals shed significantly less virus for fewer days relative to mock-vaccinated animals. For the secondary contacts, 6/6 and 5/6 animals became infected after exposure to mock- and NA-vaccinated animals, respectively. To determine if vaccine-induced immune pressure selected for escape variants, we sequenced viruses recovered from ferrets. No mutations in NA became enriched during transmission. These findings indicate that despite reducing viral load, vaccine-induced NA immunity does not prevent infection during continuous airborne exposure and subsequent onward airborne transmission of the 2009 pandemic H1N1 virus., Importance: In humans and animal models, immunity against neuraminidase (NA) reduces disease severity and viral replication during influenza infection. However, we have a limited understanding of the impact of NA immunity on viral transmission. Using chains of airborne transmission in ferrets as a strategy to simulate a more natural route of infection, we assessed if vaccine-induced NA immunity could disrupt transmission of the 2009 pandemic H1N1 virus. The 2009 pandemic H1N1 virus transmitted efficiently through chains of transmission in the presence of NA immunity, but NA-vaccinated animals shed significantly less virus and had accelerated viral clearance. To determine if immune pressure led to the generation of escape variants, viruses in ferret nasal wash samples were sequenced, and no mutations in NA were identified. These findings demonstrate that vaccine-induced NA immunity is not sufficient to prevent infection via airborne exposure and onward airborne transmission of the 2009 pandemic H1N1 virus.

SARS-CoV-2 infection induces the generation of virus-specific CD4 + and CD8 + effector and memory T cells. However, the contribution of T cells in controlling SARS-CoV-2 during infection is not well understood. Following infection of C57BL/6 mice, SARS-CoV-2-specific CD4 + and CD8 + T cells are recruited to the respiratory tract, and a vast proportion secrete the cytotoxic molecule granzyme B. Using depleting antibodies, we found that T cells within the lungs play a minimal role in viral control, and viral clearance occurs in the absence of both CD4 + and CD8 + T cells through 28 days postinfection. In the nasal compartment, depletion of both CD4 + and CD8 + T cells, but not individually, results in persistent, culturable virus replicating in the nasal epithelial layer through 28 days postinfection. Viral sequencing analysis revealed adapted mutations across the SARS-CoV-2 genome, including a large deletion in ORF6. Overall, our findings highlight the importance of T cells in controlling virus replication within the respiratory tract during SARS-CoV-2 infection.

High error rates of viral RNA-dependent RNA polymerases lead to diverse intra-host viral populations during infection. Errors made during replication that are not strongly deleterious to the virus can lead to the generation of minority variants. However, accurate detection of minority variants in viral sequence data is complicated by errors introduced during sample preparation and data analysis. We used synthetic RNA controls and simulated data to test seven variant-calling tools across a range of allele frequencies and simulated coverages. We show that choice of variant caller and use of replicate sequencing have the most significant impact on single-nucleotide variant (SNV) discovery and demonstrate how both allele frequency and coverage thresholds impact both false discovery and false-negative rates. When replicates are not available, using a combination of multiple callers with more stringent cutoffs is recommended. We use these parameters to find minority variants in sequencing data from SARS-CoV-2 clinical specimens and provide guidance for studies of intra-host viral diversity using either single replicate data or data from technical replicates. Our study provides a framework for rigorous assessment of technical factors that impact SNV identification in viral samples and establishes heuristics that will inform and improve future studies of intra-host variation, viral diversity, and viral evolution. IMPORTANCE When viruses replicate inside a host cell, the virus replication machinery makes mistakes. Over time, these mistakes create mutations that result in a diverse population of viruses inside the host. Mutations that are neither lethal to the virus nor strongly beneficial can lead to minority variants that are minor members of the virus population. However, preparing samples for sequencing can also introduce errors that resemble minority variants, resulting in the inclusion of false-positive data if not filtered correctly. In this study, we aimed to determine the best methods for identification and quantification of these minority variants by testing the performance of seven commonly used variant-calling tools. We used simulated and synthetic data to test their performance against a true set of variants and then used these studies to inform variant identification in data from SARS-CoV-2 clinical specimens. Together, analyses of our data provide extensive guidance for future studies of viral diversity and evolution., Competing Interests: The authors declare no conflict of interest.

Airborne transmission in ferrets is a key component of pandemic risk assessment. However, some emerging avian influenza viruses transmit between ferrets but do not spread in humans. Therefore, we evaluated sequential rounds of airborne transmission as an approach to enhance the predictive accuracy of the ferret model. We reasoned that infection of ferrets via the respiratory route and onward transmission would more closely model transmission in humans. We hypothesized that pandemic and seasonal viruses would transmit efficiently over two rounds of transmission, while emerging avian viruses would fail to transmit in a second round. The 2009 pandemic H1N1 (pdm09) and seasonal H3N2 viruses were compared to avian-origin H7N9 and H3N8 viruses. Depending on the virus strain, transmission efficiency varied from 50 to 100% during the first round of transmission; the efficiency for each virus did not change during the second round, and viral replication kinetics in both rounds of transmission were similar. Both the H1N1pdm09 and H7N9 viruses acquired specific mutations during sequential transmission, while the H3N2 and H3N8 viruses did not; however, a global analysis of host-adaptive mutations revealed that minimal changes were associated with transmission of H1N1 and H3N2 viruses, while a greater number of changes occurred in the avian H3N8 and H7N9 viruses. Thus, influenza viruses that transmit in ferrets maintain their transmission efficiency through serial rounds of transmission. This answers the question of whether ferrets can propagate viruses through more than one round of airborne transmission and emphasizes that transmission in ferrets is necessary but not sufficient to infer transmissibility in humans. IMPORTANCE Airborne transmission in ferrets is used to gauge the pandemic potential of emerging influenza viruses; however, some emerging influenza viruses that transmit between ferrets do not spread between humans. Therefore, we evaluated sequential rounds of airborne transmission in ferrets as a strategy to enhance the predictive accuracy of the ferret model. Human influenza viruses transmitted efficiently (>83%) over two rounds of airborne transmission, demonstrating that, like humans, ferrets infected by the respiratory route can propagate the infection onward through the air. However, emerging avian influenza viruses with associated host-adaptive mutations also transmitted through sequential transmission. Thus, airborne transmission in ferrets is necessary but not sufficient to infer transmissibility in humans, and sequential transmission did not enhance pandemic risk assessment.

Alphaviruses and flaviviruses have class II fusion glycoproteins that are essential for virion assembly and infectivity. Importantly, the tip of domain II is structurally conserved between the alphavirus and flavivirus fusion proteins, yet whether these structural similarities between virus families translate to functional similarities is unclear. Using in vivo evolution of Zika virus (ZIKV), we identified several novel emerging variants, including an envelope glycoprotein variant in β-strand c (V114M) of domain II. We have previously shown that the analogous β-strand c and the ij loop, located in the tip of domain II of the alphavirus E1 glycoprotein, are important for infectivity. This led us to hypothesize that flavivirus E β-strand c also contributes to flavivirus infection. We generated this ZIKV glycoprotein variant and found that while it had little impact on infection in mosquitoes, it reduced replication in human cells and mice and increased virus sensitivity to ammonium chloride, as seen for alphaviruses. In light of these results and given our alphavirus ij loop studies, we mutated a conserved alanine at the tip of the flavivirus ij loop to valine to test its effect on ZIKV infectivity. Interestingly, this mutation inhibited infectious virion production of ZIKV and yellow fever virus, but not West Nile virus. Together, these studies show that shared domains of the alphavirus and flavivirus class II fusion glycoproteins harbor structurally analogous residues that are functionally important and contribute to virus infection in vivo. IMPORTANCE Arboviruses are a significant global public health threat, yet there are no antivirals targeting these viruses. This problem is in part due to our lack of knowledge of the molecular mechanisms involved in the arbovirus life cycle. In particular, virus entry and assembly are essential processes in the virus life cycle and steps that can be targeted for the development of antiviral therapies. Therefore, understanding common, fundamental mechanisms used by different arboviruses for entry and assembly is essential. In this study, we show that flavivirus and alphavirus residues located in structurally conserved and analogous regions of the class II fusion proteins contribute to common mechanisms of entry, dissemination, and infectious-virion production. These studies highlight how class II fusion proteins function and provide novel targets for development of antivirals.

The fundamental basis of how arboviruses evolve in nature and what regulates the adaptive process remain unclear. To address this problem, we established a Zika virus (ZIKV) vector-borne transmission system in immunocompromised mice to study the evolutionary characteristics of ZIKV infection. Using this system, we defined factors that influence the evolutionary landscape of ZIKV infection and show that transmission route and specific organ microenvironments impact viral diversity and defective viral genome production. In addition, we identified in mice the emergence of ZIKV mutants previously seen in natural infections, including variants present in currently circulating Asian and American strains, as well as mutations unique to the mouse infections. With these studies, we have established an insect-to-mouse transmission model to study ZIKV evolution in vivo . We also defined how organ microenvironments and infection route impact the ZIKV evolutionary landscape, providing a deeper understanding of the factors that regulate arbovirus evolution and emergence., (© The Author(s) 2020. Published by Oxford University Press.)

The error-prone replication and life cycle of influenza virus generate a diverse set of genetic variants. Transmission between hosts strictly limits both the number of virus particles and the genetic diversity of virus variants that reach a new host and establish an infection. This sharp reduction in the virus population at transmission--the transmission bottleneck--is significant to the evolution of influenza virus and to its epidemic and pandemic potential. This review describes transmission bottlenecks and their effect on the diversity and evolution of influenza virus. It also reviews the methods for calculating and predicting bottleneck sizes and highlights the host and viral determinants of influenza transmissibility., (Copyright © 2020 Cold Spring Harbor Laboratory Press; all rights reserved.)