Viral and host factors associated with SARS-CoV-2 disease severity in Georgia, USA.

While SARS-CoV-2 vaccines have shown strong efficacy, their suboptimal uptake combined with the continued emergence of new viral variants raises concerns about the ongoing and future public health impact of COVID-19. We investigated viral and host factors, including vaccination status, that were associated with SARS-CoV-2 disease severity in a setting with low vaccination rates. We analyzed clinical and demographic data from 1,957 individuals in the state of Georgia, USA, coupled with viral genome sequencing from 1,185 samples. We found no difference in disease severity between individuals infected with Delta and Omicron variants among the participants in this study, after controlling for other factors, and we found no specific mutations associated with disease severity. Compared to those who were unvaccinated, vaccinated individuals experienced less severe SARS-CoV-2 disease, and the effect was similar for both variants. Vaccination within 270 days before infection was associated with decreased odds of moderate and severe outcomes, with the strongest association observed at 91-270 days post-vaccination. Older age and underlying health conditions, especially immunosuppression and renal disease, were associated with increased disease severity. Overall, this study provides insights into the impact of vaccination status, variants/mutations, and clinical factors on disease severity in SARS-CoV-2 infection when vaccination rates are low. Understanding these associations will help refine and reinforce messaging around the crucial importance of vaccination in mitigating the severity of SARS-CoV-2 disease.

Eastern Equine Encephalitis Virus Diversity in Massachusetts Patients, 1938-2020.

Eastern equine encephalitis virus (EEEV) is a relatively little-studied alphavirus that can cause devastating viral encephalitis, potentially leading to severe neurological sequelae or death. Although case numbers have historically been low, outbreaks have been increasing in frequency and scale since the 2000 s. It is critical to investigate EEEV evolutionary patterns, especially within human hosts, to understand patterns of emergence, host adaptation, and within-host evolution. To this end, we obtained formalin-fixed paraffin-embedded tissue blocks from discrete brain regions from five contemporary (2004-2020) patients from Massachusetts, confirmed the presence of EEEV RNA by in situ hybridization (ISH) staining, and sequenced viral genomes. We additionally sequenced RNA from scrapings of historical slides made from brain sections of a patient in the first documented EEE outbreak in humans in 1938. ISH staining revealed the presence of RNA in all contemporary samples, and quantification loosely correlated with the proportion of EEEV reads in samples. Consensus EEEV sequences were generated for all six patients, including the sample from 1938; phylogenetic analysis using additional publicly available sequences revealed clustering of each study sample with like sequences from a similar region, whereas an intrahost comparison of consensus sequences between discrete brain regions revealed minimal changes. Intrahost single nucleotide variant (iSNV) analysis of four samples from two patients revealed the presence of tightly compartmentalized, mostly nonsynonymous iSNVs. This study contributes critical primary human EEEV sequences, including a historic sequence as well as novel intrahost evolution findings, contributing substantially to our understanding of the natural history of EEEV infection in humans.

Severe Acute Respiratory Syndrome Coronavirus 2 Evolution and Escape From Combination Monoclonal Antibody Treatment in a Person With HIV.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) escape from combination monoclonal antibody treatment is rarely reported. We describe an immunocompromised individual with human immunodeficiency virus and persistent SARS-CoV-2 infection in whom substantial SARS-CoV-2 evolution occurred, including the emergence of 2 mutations associated with escape from the monoclonal antibody cocktail received.

Phylodynamics of deer tick virus in North America.

The burden of ticks and the pathogens they carry is increasing worldwide. Powassan virus (POWV; Flaviviridae: Flavivirus), the only known North American tick-borne flavivirus, is of particular concern due to rising cases and the severe morbidity of POWV encephalitis. Here, we use a multifaceted approach to evaluate the emergence of the II POWV lineage, known as deer tick virus (DTV), in parts of North America where human cases occur. We detected DTV-positive ticks from eight of twenty locations in the Northeast USA with an average infection rate of 1.4 per cent. High-depth, whole-genome sequencing of eighty-four POWV and DTV samples allowed us to assess geographic and temporal phylodynamics. We observed both stable infection in the Northeast USA and patterns of geographic dispersal within and between regions. A Bayesian skyline analysis demonstrated DTV population expansion over the last 50 years. This is concordant with the documented expansion of Ixodes scapularis tick populations and suggests an increasing risk of human exposure as the vector spreads. Finally, we isolated sixteen novel viruses in cell culture and demonstrated limited genetic change after passage, a valuable resource for future studies investigating this emerging virus.

Chikungunya Virus Infects the Heart and Induces Heart-Specific Transcriptional Changes in an Immunodeficient Mouse Model of Infection.

Chikungunya virus (CHIKV) is a mosquito-transmitted pathogen in family Togaviridae, genus Alphavirus. Although CHIKV is well known for its ability to cause debilitating rheumatoid-like arthritis, it has been also been observed to cause cardiovascular symptoms such as arrhythmias. Here, using samples from a previous study, we sequenced RNA from serum, kidney, skeletal muscle, and cardiac muscle from CHIKV- and mock-infected IFN-αR-/- mice using two sequencing techniques to investigate heart-specific changes in virus mutational profiles and host gene expression. Mutation rates were similar across muscle tissues although heart tissue carried heart-specific CHIKV minority variants, one of which had a coding change in the nsP3 gene and another in the 3'UTR. Importantly, heart-specific transcriptional changes included differential expression of genes critical for ion transport and muscle contraction. These results demonstrate that CHIKV replicates in the hearts of immunodeficient mice and induce heart-specific mutations and host responses with implications for cardiac pathologies.

Mouse-adapted SARS-CoV-2 protects animals from lethal SARS-CoV challenge.

The emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has resulted in a pandemic causing significant damage to public health and the economy. Efforts to understand the mechanisms of Coronavirus Disease 2019 (COVID-19) have been hampered by the lack of robust mouse models. To overcome this barrier, we used a reverse genetic system to generate a mouse-adapted strain of SARS-CoV-2. Incorporating key mutations found in SARS-CoV-2 variants, this model recapitulates critical elements of human infection including viral replication in the lung, immune cell infiltration, and significant in vivo disease. Importantly, mouse adaptation of SARS-CoV-2 does not impair replication in human airway cells and maintains antigenicity similar to human SARS-CoV-2 strains. Coupled with the incorporation of mutations found in variants of concern, CMA3p20 offers several advantages over other mouse-adapted SARS-CoV-2 strains. Using this model, we demonstrate that SARS-CoV-2-infected mice are protected from lethal challenge with the original Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), suggesting immunity from heterologous Coronavirus (CoV) strains. Together, the results highlight the use of this mouse model for further study of SARS-CoV-2 infection and disease.

Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants.

High-throughput genomics of SARS-CoV-2 is essential to characterize virus evolution and to identify adaptations that affect pathogenicity or transmission. While single-nucleotide variations (SNVs) are commonly considered as driving virus adaption, RNA recombination events that delete or insert nucleic acid sequences are also critical. Whole genome targeting sequencing of SARS-CoV-2 is typically achieved using pairs of primers to generate cDNA amplicons suitable for next-generation sequencing (NGS). However, paired-primer approaches impose constraints on where primers can be designed, how many amplicons are synthesized and requires multiple PCR reactions with non-overlapping primer pools. This imparts sensitivity to underlying SNVs and fails to resolve RNA recombination junctions that are not flanked by primer pairs. To address these limitations, we have designed an approach called 'Tiled-ClickSeq', which uses hundreds of tiled-primers spaced evenly along the virus genome in a single reverse-transcription reaction. The other end of the cDNA amplicon is generated by azido-nucleotides that stochastically terminate cDNA synthesis, removing the need for a paired-primer. A sequencing adaptor containing a Unique Molecular Identifier (UMI) is appended to the cDNA fragment using click-chemistry and a PCR reaction generates a final NGS library. Tiled-ClickSeq provides complete genome coverage, including the 5'UTR, at high depth and specificity to the virus on both Illumina and Nanopore NGS platforms. Here, we analyze multiple SARS-CoV-2 isolates and clinical samples to simultaneously characterize minority variants, sub-genomic mRNAs (sgmRNAs), structural variants (SVs) and D-RNAs. Tiled-ClickSeq therefore provides a convenient and robust platform for SARS-CoV-2 genomics that captures the full range of RNA species in a single, simple assay.

Isolation of a novel insect-specific flavivirus with immunomodulatory effects in vertebrate systems.

We describe the isolation and characterization of a novel insect-specific flavivirus (ISFV), tentatively named Aripo virus (ARPV), that was isolated from Psorophora albipes mosquitoes collected in Trinidad. The ARPV genome was determined and phylogenetic analyses showed that it is a dual host associated ISFV, and clusters with the main mosquito-borne flaviviruses. ARPV antigen was significantly cross-reactive with Japanese encephalitis virus serogroup antisera, with significant cross-reactivity to Ilheus and West Nile virus (WNV). Results suggest that ARPV replication is limited to mosquitoes, as it did not replicate in the sandfly, culicoides or vertebrate cell lines tested. We also demonstrated that ARPV is endocytosed into vertebrate cells and is highly immunomodulatory, producing a robust innate immune response despite its inability to replicate in vertebrate systems. We show that prior infection or coinfection with ARPV limits WNV-induced disease in mouse models, likely the result of a robust ARPV-induced type I interferon response.

Mouse Adapted SARS-CoV-2 protects animals from lethal SARS-CoV challenge.

The emergence of SARS-CoV-2 has resulted in a worldwide pandemic causing significant damage to public health and the economy. Efforts to understand the mechanisms of COVID-19 disease have been hampered by the lack of robust mouse models. To overcome this barrier, we utilized a reverse genetic system to generate a mouse-adapted strain of SARS-CoV-2. Incorporating key mutations found in SARSCoV-2 variants, this model recapitulates critical elements of human infection including viral replication in the lung, immune cell infiltration, and significant in vivo disease. Importantly, mouse-adaptation of SARS-CoV-2 does not impair replication in human airway cells and maintains antigenicity similar to human SARS-CoV-2 strains. Utilizing this model, we demonstrate that SARS-CoV-2 infected mice are protected from lethal challenge with the original SARS-CoV, suggesting immunity from heterologous CoV strains. Together, the results highlight the utility of this mouse model for further study of SARS-CoV-2 infection and disease.

Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants.

High-throughput genomics of SARS-CoV-2 is essential to characterize virus evolution and to identify adaptations that affect pathogenicity or transmission. While single-nucleotide variations (SNVs) are commonly considered as driving virus adaption, RNA recombination events that delete or insert nucleic acid sequences are also critical. Whole genome targeting sequencing of SARS-CoV-2 is typically achieved using pairs of primers to generate cDNA amplicons suitable for Next-Generation Sequencing (NGS). However, paired-primer approaches impose constraints on where primers can be designed, how many amplicons are synthesized and requires multiple PCR reactions with non-overlapping primer pools. This imparts sensitivity to underlying SNVs and fails to resolve RNA recombination junctions that are not flanked by primer pairs. To address these limitations, we have designed an approach called 'Tiled-ClickSeq', which uses hundreds of tiled-primers spaced evenly along the virus genome in a single reverse-transcription reaction. The other end of the cDNA amplicon is generated by azido-nucleotides that stochastically terminate cDNA synthesis, removing the need for a paired-primer. A sequencing adaptor containing a Unique Molecular Identifier (UMI) is appended to the cDNA fragment using click-chemistry and a PCR reaction generates a final NGS library. Tiled-ClickSeq provides complete genome coverage, including the 5'UTR, at high depth and specificity to the virus on both Illumina and Nanopore NGS platforms. Here, we analyze multiple SARS-CoV-2 isolates and clinical samples to simultaneously characterize minority variants, sub-genomic mRNAs (sgmRNAs), structural variants (SVs) and D-RNAs. Tiled-ClickSeq therefore provides a convenient and robust platform for SARS-CoV-2 genomics that captures the full range of RNA species in a single, simple assay.

Effects of Chikungunya virus immunity on Mayaro virus disease and epidemic potential.

Mayaro virus (MAYV) causes an acute febrile illness similar to that produced by chikungunya virus (CHIKV), an evolutionary relative in the Semliki Forest virus complex of alphaviruses. MAYV emergence is typically sporadic, but recent isolations and outbreaks indicate that the virus remains a public health concern. Given the close phylogenetic and antigenic relationship between CHIKV and MAYV, and widespread distribution of CHIKV, we hypothesized that prior CHIKV immunity may affect MAYV pathogenesis and/or influence its emergence potential. We pre-exposed immunocompetent C57BL/6 and immunocompromised A129 or IFNAR mice to wild-type CHIKV, two CHIKV vaccines, or a live-attenuated MAYV vaccine, and challenged with MAYV. We observed strong cross-protection against MAYV for mice pre-exposed to wild-type CHIKV, and moderately but significantly reduced cross-protection from CHIKV-vaccinated animals. Immunity to other alphavirus or flavivirus controls provided no protection against MAYV disease or viremia. Mechanistic studies suggested that neutralizing antibodies alone can mediate this protection, with T-cells having no significant effect on diminishing disease. Finally, human sera obtained from naturally acquired CHIKV infection cross-neutralized MAYV at high titers in vitro. Altogether, our data suggest that CHIKV infection can confer cross-protective effects against MAYV, and the resultant reduction in viremia may limit the emergence potential of MAYV.

Low-fidelity Venezuelan equine encephalitis virus polymerase mutants to improve live-attenuated vaccine safety and efficacy.

During RNA virus replication, there is the potential to incorporate mutations that affect virulence or pathogenesis. For live-attenuated vaccines, this has implications for stability, as replication may result in mutations that either restore the wild-type phenotype via reversion or compensate for the attenuating mutations by increasing virulence (pseudoreversion). Recent studies have demonstrated that altering the mutation rate of an RNA virus is an effective attenuation tool. To validate the safety of low-fidelity mutations to increase vaccine attenuation, several mutations in the RNA-dependent RNA-polymerase (RdRp) were tested in the live-attenuated Venezuelan equine encephalitis virus vaccine strain, TC-83. Next generation sequencing after passage in the presence of mutagens revealed a mutant containing three mutations in the RdRp, TC-83 3x, to have decreased replication fidelity, while a second mutant, TC-83 4x displayed no change in fidelity, but shared many phenotypic characteristics with TC-83 3x. Both mutants exhibited increased, albeit inconsistent attenuation in an infant mouse model, as well as increased immunogenicity and complete protection against lethal challenge of an adult murine model compared with the parent TC-83. During serial passaging in a highly permissive model, the mutants increased in virulence but remained less virulent than the parent TC-83. These results suggest that the incorporation of low-fidelity mutations into the RdRp of live-attenuated vaccines for RNA viruses can confer increased immunogenicity whilst showing some evidence of increased attenuation. However, while in theory such constructs may result in more effective vaccines, the instability of the vaccine phenotype decreases the likelihood of this being an effective vaccine strategy.

Chikungunya Virus Strains Show Lineage-Specific Variations in Virulence and Cross-Protective Ability in Murine and Nonhuman Primate Models.

Chikungunya virus (CHIKV) is a reemerging arbovirus capable of causing explosive outbreaks of febrile illness, polyarthritis, and polyarthralgia, inflicting severe morbidity on affected populations. CHIKV can be genetically classified into 3 major lineages: West African (WA); East, Central, and South African (ECSA); Indian Ocean (IOL); and Asian. Additionally, the Indian Ocean (IOL) sublineage emerged within the ECSA clade and the Asian/American sublineage emerged within the Asian clade. While differences in epidemiological and pathological characteristics among outbreaks involving different CHIKV lineages and sublineages have been suggested, few targeted investigations comparing lineage virulence levels have been reported. We compared the virulence levels of CHIKV isolates representing all major lineages and sublineages in the type I interferon receptor-knockout A129 mouse model and found lineage-specific differences in virulence. We also evaluated the cross-protective efficacy of the IOL-derived, live-attenuated vaccine strain CHIKV/IRESv1 against the Asian/American CHIKV isolate YO123223 in both murine and nonhuman primate models, as well as the WA strain SH2830 in a murine model. The CHIKV/IRES vaccine provided protection both in mice and in nonhuman primate cohorts against Caribbean strain challenge and protected mice against WA challenge. Taken together, our data suggest that Asian/American CHIKV strains are less virulent than those in the Asian, ECSA, and WA lineages and that despite differences in virulence, IOL-based vaccine strains offer robust cross-protection against strains from other lineages. Further research is needed to elucidate the genetic basis for variation in CHIKV virulence in the A129 mouse model and to corroborate this variation with human pathogenicity.IMPORTANCE Chikungunya virus (CHIKV) is a reemerging human pathogen capable of causing debilitating and disfiguring polyarthritis, which can last for months to years after initial fever has resolved. There are four major genetic lineages of CHIKV, as well as two recently emerged sublineages, none of which have been evaluated for differences in virulence. Moreover, the ability of chikungunya vaccines to cross-protect against heterologous CHIKV lineages has not been explored. Therefore, we sought to compare the virulence levels among CHIKV lineages, as well as to evaluate the cross-protective efficacy of the CHIKV/IRESv1 vaccine candidate, in two different models of CHIKV infection. Our results suggest that, although significant differences in virulence were observed among CHIKV lineages, the CHIKV/IRESv1 vaccine elicits cross-lineage protective immunity. These findings provide valuable information for predicting the severity of CHIKV-associated morbidity in future outbreaks, as well as vaccine development considerations.

Host oxidative folding pathways offer novel anti-chikungunya virus drug targets with broad spectrum potential.

Alphaviruses require conserved cysteine residues for proper folding and assembly of the E1 and E2 envelope glycoproteins, and likely depend on host protein disulfide isomerase-family enzymes (PDI) to aid in facilitating disulfide bond formation and isomerization in these proteins. Here, we show that in human HEK293 cells, commercially available inhibitors of PDI or modulators thereof (thioredoxin reductase, TRX-R; endoplasmic reticulum oxidoreductin-1, ERO-1) inhibit the replication of CHIKV chikungunya virus (CHIKV) in vitro in a dose-dependent manner. Further, the TRX-R inhibitor auranofin inhibited Venezuelan equine encephalitis virus and the flavivirus Zika virus replication in vitro, while PDI inhibitor 16F16 reduced replication but demonstrated notable toxicity. 16F16 significantly altered the viral genome: plaque-forming unit (PFU) ratio of CHIKV in vitro without affecting relative intracellular viral RNA quantities and inhibited CHIKV E1-induced cell-cell fusion, suggesting that PDI inhibitors alter progeny virion infectivity through altered envelope function. Auranofin also increased the extracellular genome:PFU ratio but decreased the amount of intracellular CHIKV RNA, suggesting an alternative mechanism of action. Finally, auranofin reduced footpad swelling and viremia in the C57BL/6 murine model of CHIKV infection. Our results suggest that targeting oxidative folding pathways represents a potential new anti-alphavirus therapeutic strategy.

Molecular Virologic and Clinical Characteristics of a Chikungunya Fever Outbreak in La Romana, Dominican Republic, 2014.

Since emerging in Saint Martin in 2013, chikungunya virus (CHIKV), an alphavirus transmitted by the Aedes aegypti mosquito, has infected approximately two million individuals in the Americas, with over 500,000 reported cases in the Dominican Republic (DR). CHIKV-infected patients typically present with a febrile syndrome including polyarthritis/polyarthralgia, and a macropapular rash, similar to those infected with dengue and Zika viruses, and malaria. Nevertheless, many Dominican cases are unconfirmed due to the unavailability and high cost of laboratory testing and the absence of specific treatment for CHIKV infection. To obtain a more accurate representation of chikungunya fever (CHIKF) clinical signs and symptoms, and confirm the viral lineage responsible for the DR CHIKV outbreak, we tested 194 serum samples for CHIKV RNA and IgM antibodies from patients seen in a hospital in La Romana, DR using quantitative RT-PCR and IgM capture ELISA, and performed retrospective chart reviews. RNA and antibodies were detected in 49% and 24.7% of participants, respectively. Sequencing revealed that the CHIKV strain responsible for the La Romana outbreak belonged to the Asian/American lineage and grouped phylogenetically with recent Mexican and Trinidadian isolates. Our study shows that, while CHIKV-infected individuals were infrequently diagnosed with CHIKF, uninfected patients were never falsely diagnosed with CHIKF. Participants testing positive for CHIKV RNA were more likely to present with arthralgia, although it was reported in just 20.0% of CHIKF+ individuals. High percentages of respiratory (19.6%) signs and symptoms, especially among children, were noted, though it was not possible to determine whether individuals infected with CHIKV were co-infected with other pathogens. These results suggest that CHIKV may have been underdiagnosed during this outbreak, and that CHIKF should be included in differential diagnoses of diverse undifferentiated febrile syndromes in the Americas.

Characterization of a Novel Murine Model to Study Zika Virus.

The mosquito-borne Zika virus (ZIKV) is responsible for an explosive ongoing outbreak of febrile illness across the Americas. ZIKV was previously thought to cause only a mild, flu-like illness, but during the current outbreak, an association with Guillain-Barré syndrome and microcephaly in neonates has been detected. A previous study showed that ZIKV requires murine adaptation to generate reproducible murine disease. In our study, a low-passage Cambodian isolate caused disease and mortality in mice lacking the interferon (IFN) alpha receptor (A129 mice) in an age-dependent manner, but not in similarly aged immunocompetent mice. In A129 mice, viremia peaked at ∼10(7) plaque-forming units/mL by day 2 postinfection (PI) and reached high titers in the spleen by day 1. ZIKV was detected in the brain on day 3 PI and caused signs of neurologic disease, including tremors, by day 6. Robust replication was also noted in the testis. In this model, all mice infected at the youngest age (3 weeks) succumbed to illness by day 7 PI. Older mice (11 weeks) showed signs of illness, viremia, and weight loss but recovered starting on day 8. In addition, AG129 mice, which lack both type I and II IFN responses, supported similar infection kinetics to A129 mice, but with exaggerated disease signs. This characterization of an Asian lineage ZIKV strain in a murine model, and one of the few studies reporting a model of Zika disease and demonstrating age-dependent morbidity and mortality, could provide a platform for testing the efficacy of antivirals and vaccines.

Chikungunya Virus as Cause of Febrile Illness Outbreak, Chiapas, Mexico, 2014.

Since chikungunya virus (CHIKV) was introduced into the Americas in 2013, its geographic distribution has rapidly expanded. Of 119 serum samples collected in 2014 from febrile patients in southern Mexico, 79% were positive for CHIKV or IgM against CHIKV. Sequencing results confirmed CHIKV strains closely related to Caribbean isolates.

First Report of Aedes aegypti Transmission of Chikungunya Virus in the Americas.

During a chikungunya fever outbreak in late 2014 in Chiapas, Mexico, entomovirological surveillance was performed to incriminate the vector(s). In neighborhoods, 75 households with suspected cases were sampled for mosquitoes, of which 80% (60) harbored Aedes aegypti and 2.7% (2) Aedes albopictus. A total of 1,170 Ae. aegypti and three Ae. albopictus was collected and 81 pools were generated. Although none of the Ae. albopictus pools were chikungunya virus (CHIKV)-positive, 18 Ae. aegypti pools (22.8%) contained CHIKV, yielding an infection rate of 32.3/1,000 mosquitoes. A lack of herd immunity in conjunction with high mosquito populations, poor vector control services in this region, and targeted collections in locations of human cases may explain the high infection rate in this vector. Consistent with predictions from experimental studies, Ae. aegypti appears to be the principal vector of CHIKV in southern Mexico, while the role of Ae. albopictus remains unknown.