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1.
Microbiol Spectr ; : e0100321, 2021 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-34756092

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in 2019 and has become a major global pathogen in an astonishingly short period of time. The emergence of SARS-CoV-2 has been notable due to its impacts on residents in long-term care facilities (LTCFs). LTCF residents tend to possess several risk factors for severe outcomes of SARS-CoV-2 infection, including advanced age and the presence of comorbidities. Indeed, residents of LTCFs represent approximately 40% of SARS-CoV-2 deaths in the United States. Few studies have focused on the prevalence and transmission dynamics of SARS-CoV-2 among LTCF staff during the early months of the pandemic, prior to mandated surveillance testing. To assess the prevalence and incidence of SARS-CoV-2 among LTCF staff, characterize the extent of asymptomatic infections, and investigate the genomic epidemiology of the virus within these settings, we sampled staff for 8 to 11 weeks at six LTCFs with nasopharyngeal swabs from March through June of 2020. We determined the presence and levels of viral RNA and infectious virus and sequenced 54 nearly complete genomes. Our data revealed that over 50% of infections were asymptomatic/mildly symptomatic and that there was a strongly significant relationship between viral RNA (vRNA) and infectious virus, prolonged infections, and persistent vRNA (4+ weeks) in a subset of individuals, and declining incidence over time. Our data suggest that asymptomatic SARS-CoV-2-infected LTCF staff contributed to virus persistence and transmission within the workplace during the early pandemic period. Genetic epidemiology data generated from samples collected during this period support that SARS-CoV-2 was commonly spread between staff within an LTCF and that multiple-introduction events were less common. IMPORTANCE Our work comprises unique data on the characteristics of SARS-CoV-2 dynamics among staff working at LTCFs in the early months of the SARS-CoV-2 pandemic prior to mandated staff surveillance testing. During this time period, LTCF residents were largely sheltering-in-place. Given that staff were able to leave and return daily and could therefore be a continued source of imported or exported infection, we performed weekly SARS-CoV-2 PCR on nasal swab samples collected from this population. There are limited data from the early months of the pandemic comprising longitudinal surveillance of staff at LTCFs. Our data reveal the surprisingly high level of asymptomatic/presymptomatic infections within this cohort during the early months of the pandemic and show genetic epidemiological analyses that add novel insights into both the origin and transmission of SARS-CoV-2 within LTCFs.

2.
PLoS Pathog ; 17(11): e1009433, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34752502

RESUMO

Arthropod-borne viruses (arboviruses) require replication across a wide range of temperatures to perpetuate. While vertebrate hosts tend to maintain temperatures of approximately 37°C-40°C, arthropods are subject to ambient temperatures which can have a daily fluctuation of > 10°C. Temperatures impact vector competence, extrinsic incubation period, and mosquito survival unimodally, with optimal conditions occurring at some intermediate temperature. In addition, the mean and range of daily temperature fluctuations influence arbovirus perpetuation and vector competence. The impact of temperature on arbovirus genetic diversity during systemic mosquito infection, however, is poorly understood. Therefore, we determined how constant extrinsic incubation temperatures of 25°C, 28°C, 32°C, and 35°C control Zika virus (ZIKV) vector competence and population dynamics within Aedes aegypti and Aedes albopictus mosquitoes. We also examined fluctuating temperatures which better mimic field conditions in the tropics. We found that vector competence varied in a unimodal manner for constant temperatures peaking between 28°C and 32°C for both Aedes species. Transmission peaked at 10 days post-infection for Aedes aegypti and 14 days for Aedes albopictus. Conversely, fluctuating temperature decreased vector competence. Using RNA-seq to characterize ZIKV population structure, we identified that temperature alters the selective environment in unexpected ways. During mosquito infection, constant temperatures more often elicited positive selection whereas fluctuating temperatures led to strong purifying selection in both Aedes species. These findings demonstrate that temperature has multiple impacts on ZIKV biology, including major effects on the selective environment within mosquitoes.

3.
BMC Infect Dis ; 21(1): 677, 2021 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-34256735

RESUMO

BACKGROUND: SARS-CoV-2 has swept across the globe, causing millions of deaths worldwide. Though most survive, many experience symptoms of COVID-19 for months after acute infection. Successful prevention and treatment of acute COVID-19 infection and its associated sequelae is dependent on in-depth knowledge of viral pathology across the spectrum of patient phenotypes and physiologic responses. Longitudinal biobanking provides a valuable resource of clinically integrated, easily accessed, and quality-controlled samples for researchers to study differential multi-organ system responses to SARS-CoV-2 infection, post-acute sequelae of COVID-19 (PASC), and vaccination. METHODS: Adults with a history of a positive SARS-CoV-2 nasopharyngeal PCR are actively recruited from the community or hospital settings to enroll in the Northern Colorado SARS-CoV-2 Biorepository (NoCo-COBIO). Blood, saliva, stool, nasopharyngeal specimens, and extensive clinical and demographic data are collected at 4 time points over 6 months. Patients are assessed for PASC during longitudinal follow-up by physician led symptom questionnaires and physical exams. This clinical trial registration is NCT04603677 . RESULTS: We have enrolled and collected samples from 119 adults since July 2020, with 66% follow-up rate. Forty-nine percent of participants assessed with a symptom surveillance questionnaire (N = 37 of 75) had PASC at any time during follow-up (up to 8 months post infection). Ninety-three percent of hospitalized participants developed PASC, while 23% of those not requiring hospitalization developed PASC. At 90-174 days post SARS-CoV-2 diagnosis, 67% of all participants had persistent symptoms (N = 37 of 55), and 85% percent of participants who required hospitalization during initial infection (N = 20) still had symptoms. The most common symptoms reported after 15 days of infection were fatigue, loss of smell, loss of taste, exercise intolerance, and cognitive dysfunction. CONCLUSIONS: Patients who were hospitalized for COVID-19 were significantly more likely to have PASC than those not requiring hospitalization, however 23% of patients who were not hospitalized also developed PASC. This patient-matched, multi-matrix, longitudinal biorepository from COVID-19 survivors with and without PASC will allow for current and future research to better understand the pathophysiology of disease and to identify targeted interventions to reduce risk for PASC. Registered 27 October 2020 - Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT04603677 .


Assuntos
Bancos de Espécimes Biológicos , Teste para COVID-19/métodos , COVID-19/complicações , SARS-CoV-2/genética , Sobreviventes , Adulto , Idoso , COVID-19/sangue , COVID-19/epidemiologia , COVID-19/patologia , COVID-19/virologia , Colorado/epidemiologia , Progressão da Doença , Feminino , Seguimentos , Hospitalização , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Manejo de Espécimes , Adulto Jovem
4.
Microbiol Spectr ; 9(1): e0022421, 2021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34287058

RESUMO

SARS-CoV-2 has had a disproportionate impact on nonhospital health care settings, such as long-term-care facilities (LTCFs). The communal nature of these facilities, paired with the high-risk profile of residents, has resulted in thousands of infections and deaths and a high case fatality rate. To detect presymptomatic infections and identify infected workers, we performed weekly surveillance testing of staff at two LTCFs, which revealed a large outbreak at one of the sites. We collected serum from staff members throughout the study and evaluated it for binding and neutralization to measure seroprevalence, seroconversion, and type and functionality of antibodies. At the site with very few incident infections, we detected that over 40% of the staff had preexisting SARS-CoV-2 neutralizing antibodies, suggesting prior exposure. At the outbreak site, we saw rapid seroconversion following infection. Neutralizing antibody levels were stable for many weeks following infection, suggesting a durable, long-lived response. Receptor-binding domain antibodies and neutralizing antibodies were strongly correlated. The site with high seroprevalence among staff had two unique introductions of SARS-CoV-2 into the facility through seronegative infected staff during the period of study, but these did not result in workplace spread or outbreaks. Together, our results suggest that a high seroprevalence rate among staff can contribute to immunity within a workplace and protect against subsequent infection and spread within a facility. IMPORTANCE Long-term care facilities (LTCFs) have been disproportionately impacted by COVID-19 due to their communal nature and high-risk profile of residents. LTCF staff have the ability to introduce SARS-CoV-2 into the facility, where it can spread, causing outbreaks. We tested staff weekly at two LTCFs and collected blood throughout the study to measure SARS-CoV-2 antibodies. One site had a large outbreak and infected individuals rapidly generated antibodies after infection. At the other site, almost half the staff already had antibodies, suggesting prior infection. The majority of these antibodies bind to the receptor-binding domain of the SARS-CoV-2 spike protein and are potently neutralizing and stable for many months. The non-outbreak site had two unique introductions of SARS-CoV-2 into the facility, but these did not result in workplace spread or outbreaks. Our results reveal that high seroprevalence among staff can contribute to immunity and protect against subsequent infection and spread within a facility.


Assuntos
Formação de Anticorpos , COVID-19/epidemiologia , COVID-19/imunologia , Surtos de Doenças , Assistência de Longa Duração , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Infecções Assintomáticas/epidemiologia , Sítios de Ligação de Anticorpos , Teste para COVID-19 , Humanos , Vigilância Imunológica , RNA Viral , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Sensibilidade e Especificidade , Estudos Soroepidemiológicos , Glicoproteína da Espícula de Coronavírus/imunologia
5.
Parasit Vectors ; 14(1): 261, 2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34006306

RESUMO

BACKGROUND: Zika virus (ZIKV) is an arthropod-borne virus (arbovirus) with an urban transmission cycle that primarily involves humans and Aedes aegypti. Evidence suggests that the evolution of some arboviruses is constrained by their dependency on alternating between disparate (vertebrate and invertebrate) hosts. The goals of this study are to compare the genetic changes that occur in ZIKV after serial passaging in mosquito or vertebrate cell lines or alternate passaging in both cell types and to compare the replication, dissemination, and transmission efficiencies of the cell culture-derived viruses in Ae. aegypti. METHODS: An isolate of ZIKV originally acquired from a febrile patient in Yucatan, Mexico, was serially passaged six times in African green monkey kidney (Vero) cells or Aedes albopictus (C6/36) cells or both cell types by alternating passage. A colony of Ae. aegypti from Yucatan was established, and mosquitoes were challenged with the cell-adapted viruses. Midguts, Malpighian tubules, ovaries, salivary glands, wings/legs and saliva were collected at various times after challenge and tested for evidence of virus infection. RESULTS: Genome sequencing revealed the presence of two non-synonymous substitutions in the premembrane and NS1 regions of the mosquito cell-adapted virus and two non-synonymous substitutions in the capsid and NS2A regions of both the vertebrate cell-adapted and alternate-passaged viruses. Additional genetic changes were identified by intrahost variant frequency analysis. Virus maintained by continuous C6/36 cell passage was significantly more infectious in Ae. aegypti than viruses maintained by alternating passage and consecutive Vero cell passage. CONCLUSIONS: Mosquito cell-adapted ZIKV displayed greater in vivo fitness in Ae. aegypti compared to the other viruses, indicating that obligate cycling between disparate hosts carries a fitness cost. These data increase our understanding of the factors that drive ZIKV adaptation and evolution and underscore the important need to consider the in vivo passage histories of flaviviruses to be evaluated in vector competence studies.


Assuntos
Aedes/virologia , Mosquitos Vetores/virologia , Inoculações Seriadas/métodos , Infecção por Zika virus/transmissão , Zika virus/genética , Zika virus/fisiologia , Animais , Linhagem Celular , Chlorocebus aethiops , Vetores de Doenças , Aptidão Genética , Insetos/citologia , Glândulas Salivares/virologia , Células Vero , Carga Viral
6.
PLoS Pathog ; 17(5): e1009585, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34010360

RESUMO

Coronavirus disease-19 (COVID-19) emerged in late 2019 in China and rapidly became pandemic. As with other coronaviruses, a preponderance of evidence suggests the virus originated in horseshoe bats (Rhinolophus spp.) and may have infected an intermediate host prior to spillover into humans. A significant concern is that SARS-CoV-2 could become established in secondary reservoir hosts outside of Asia. To assess this potential, we challenged deer mice (Peromyscus maniculatus) with SARS-CoV-2 and found robust virus replication in the upper respiratory tract, lungs and intestines, with detectable viral RNA for up to 21 days in oral swabs and 6 days in lungs. Virus entry into the brain also occurred, likely via gustatory-olfactory-trigeminal pathway with eventual compromise to the blood-brain barrier. Despite this, no conspicuous signs of disease were observed, and no deer mice succumbed to infection. Expression of several innate immune response genes were elevated in the lungs, including IFNα, IFNß, Cxcl10, Oas2, Tbk1 and Pycard. Elevated CD4 and CD8ß expression in the lungs was concomitant with Tbx21, IFNγ and IL-21 expression, suggesting a type I inflammatory immune response. Contact transmission occurred from infected to naive deer mice through two passages, showing sustained natural transmission and localization into the olfactory bulb, recapitulating human neuropathology. In the second deer mouse passage, an insertion of 4 amino acids occurred to fixation in the N-terminal domain of the spike protein that is predicted to form a solvent-accessible loop. Subsequent examination of the source virus from BEI Resources determined the mutation was present at very low levels, demonstrating potent purifying selection for the insert during in vivo passage. Collectively, this work has determined that deer mice are a suitable animal model for the study of SARS-CoV-2 respiratory disease and neuropathogenesis, and that they have the potential to serve as secondary reservoir hosts in North America.


Assuntos
COVID-19/fisiopatologia , COVID-19/transmissão , Peromyscus/virologia , Doenças dos Roedores/transmissão , Animais , Encéfalo/patologia , Encéfalo/virologia , COVID-19/patologia , Modelos Animais de Doenças , Reservatórios de Doenças , Suscetibilidade a Doenças , Feminino , Masculino , Doenças dos Roedores/patologia , Doenças dos Roedores/virologia , Glicoproteína da Espícula de Coronavírus/genética , Replicação Viral
7.
J Virol ; 2021 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-33536175

RESUMO

Zika virus (ZIKV) has the unusual capacity to circumvent natural alternating mosquito-human transmission and be directly transmitted human-to-human via sexual and vertical routes. The impact of direct transmission on ZIKV evolution and adaptation to vertebrate hosts is unknown. Here we show that molecularly barcoded ZIKV rapidly adapted to a mammalian host during direct transmission chains in mice, coincident with the emergence of an amino acid substitution previously shown to enhance virulence. In contrast, little to no adaptation of ZIKV to mice was observed following chains of direct transmission in mosquitoes or alternating host transmission. Detailed genetic analyses revealed that ZIKV evolution in mice was generally more convergent and subjected to more relaxed purifying selection than in mosquitoes or alternate passages. These findings suggest that prevention of direct human transmission chains may be paramount to resist gains in ZIKV virulence.Importance We used experimental evolution to model chains of direct and indirect Zika virus (ZIKV) transmission by serially passaging a synthetic swarm of molecularly barcoded ZIKV within and between mosquitoes and mice. We observed that direct mouse transmission chains facilitated a rapid increase in ZIKV replication and enhanced virulence in mice. These findings demonstrate that ZIKV is capable of rapid adaptation to a vertebrate host and indicate that direct human-to-human transmission could pose a greater threat to public health than currently realized.

8.
Cryobiology ; 99: 1-10, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33556359

RESUMO

Mosquito-borne diseases are responsible for millions of human deaths every year, posing a massive burden on global public health. Mosquitoes transmit a variety of bacteria, parasites and viruses. Mosquito control efforts such as insecticide spraying can reduce mosquito populations, but they must be sustained in order to have long term impacts, can result in the evolution of insecticide resistance, are costly, and can have adverse human and environmental effects. Technological advances have allowed genetic manipulation of mosquitoes, including generation of those that are still susceptible to insecticides, which has greatly increased the number of mosquito strains and lines available to the scientific research community. This generates an associated challenge, because rearing and maintaining unique mosquito lines requires time, money and facilities, and long-term maintenance can lead to adaptation to specific laboratory conditions, resulting in mosquito lines that are distinct from their wild-type counterparts. Additionally, continuous rearing of transgenic lines can lead to loss of genetic markers, genes and/or phenotypes. Cryopreservation of valuable mosquito lines could help circumvent these limitations and allow researchers to reduce the cost of rearing multiple lines simultaneously, maintain low passage number transgenic mosquitoes, and bank lines not currently being used. Additionally, mosquito cryopreservation could allow researchers to access the same mosquito lines, limiting the impact of unique laboratory or field conditions. Successful cryopreservation of mosquitoes would expand the field of mosquito research and could ultimately lead to advances that would reduce the burden of mosquito-borne diseases, possibly through rear-and-release strategies to overcome mosquito insecticide resistance. Cryopreservation techniques have been developed for some insect groups, including but not limited to fruit flies, silkworms and other moth species, and honeybees. Recent advances within the cryopreservation field, along with success with other insects suggest that cryopreservation of mosquitoes may be a feasible method for preserving valuable scientific and public health resources. In this review, we will provide an overview of basic mosquito biology, the current state of and advances within insect cryopreservation, and a proposed approach toward cryopreservation of Anopheles stephensi mosquitoes.


Assuntos
Anopheles , Mosquitos Vetores , Animais , Abelhas , Criopreservação/métodos , Humanos , Resistência a Inseticidas/genética , Controle de Mosquitos , Mosquitos Vetores/genética
9.
Cell Host Microbe ; 29(1): 3-5, 2021 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-33444554

RESUMO

Troubled by an unstable world beset by new and emerging viruses? Virus evolution is here to help. Through detailed studies of poliovirus vaccine reversion to virulence, Valesano and colleagues remind us that some things in life can, indeed, be counted on.


Assuntos
Poliomielite , Poliovirus , Humanos , Mutação , Poliomielite/prevenção & controle , Poliovirus/genética , Vacina Antipólio Oral , Vacinação
10.
J Virol ; 95(6)2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33328311

RESUMO

Zika virus (ZIKV; Flaviviridae, Flavivirus) is an arthropod-borne infection that can result in severe outcomes, particularly in fetuses infected in utero It has been assumed that infection by ZIKV, as well as other viruses, is largely initiated by individual virus particles binding to and entering a cell. However, recent studies have demonstrated that multiple virus particles are frequently delivered to a cell simultaneously and that this collective particle delivery enhances infection. ZIKV is maintained in nature between Aedes aegypti mosquitos and vertebrate hosts, including humans. Human infection is initiated through the injection of a relatively small initial inoculum comprised of a genetically complex virus population. Since most mutations decrease virus fitness, collective particle transmission could benefit ZIKV and other arthropod-borne diseases by facilitating the maintenance of genetic complexity and adaptability during infection or through other mechanisms. Therefore, we utilized a barcoded ZIKV to quantify the number of virus genomes that initiate a plaque. We found that individual plaques contain a mean of 10 infecting viral genomes (range, 1 to 212). Few plaques contained more than two dominant genomes. To determine whether multigenome infectious units consist of collectively transmitting virions, infectious units of ZIKV were then separated mechanically by centrifugation, and heavier fractions were found to contain more genomes per plaque-forming unit, with larger diameters. Finally, larger/heavier infectious units reformed after removal. These data suggest that ZIKV populations consist of a variety of infectious unit sizes, likely mostly made up of aggregates, and only rarely begin with a single virus genome.IMPORTANCE The arthropod-borne Zika virus (ZIKV) infects humans and can cause severe neurological sequelae, particularly in fetuses infected in utero How this virus has been able to spread across vast geological ranges and evolve in new host populations is not yet understood. This research demonstrates a novel mechanism of ZIKV transmission through multigenome aggregates, providing insight into ZIKV evolution, immunologic evasion, and better future therapeutic design. This study shows that ZIKV plaques result from collections of genomes rather than individual genomes, increasing the potential for interactions between ZIKV genotypes.


Assuntos
Genoma Viral/genética , Polimorfismo Genético , Infecção por Zika virus/virologia , Zika virus/genética , Aedes/virologia , Animais , Linhagem Celular , Variações do Número de Cópias de DNA , Tamanho do Genoma , Genótipo , Humanos , Mosquitos Vetores/virologia , Temperatura , Vírion/metabolismo , Replicação Viral , Zika virus/crescimento & desenvolvimento , Infecção por Zika virus/transmissão
11.
bioRxiv ; 2020 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-32793912

RESUMO

Coronavirus disease-19 (COVID-19) emerged in November, 2019 in China and rapidly became pandemic. As with other coronaviruses, a preponderance of evidence suggests the virus originated in horseshoe bats (Rhinolophus spp.) and likely underwent a recombination event in an intermediate host prior to entry into human populations. A significant concern is that SARS-CoV-2 could become established in secondary reservoir hosts outside of Asia. To assess this potential, we challenged deer mice (Peromyscus maniculatus) with SARS-CoV-2 and found robust virus replication in the upper respiratory tract, lungs and intestines, with detectable viral RNA for up to 21 days in oral swabs and 14 days in lungs. Virus entry into the brain also occurred, likely via gustatory-olfactory-trigeminal pathway with eventual compromise to the blood brain barrier. Despite this, no conspicuous signs of disease were observed and no deer mice succumbed to infection. Expression of several innate immune response genes were elevated in the lungs, notably IFNα, Cxcl10, Oas2, Tbk1 and Pycard. Elevated CD4 and CD8ß expression in the lungs was concomitant with Tbx21, IFNγ and IL-21 expression, suggesting a type I inflammatory immune response. Contact transmission occurred from infected to naive deer mice through two passages, showing sustained natural transmission. In the second deer mouse passage, an insertion of 4 amino acids occurred to fixation in the N-terminal domain of the spike protein that is predicted to form a solvent-accessible loop. Subsequent examination of the source virus from BEI Resources indicated the mutation was present at very low levels, demonstrating potent purifying selection for the insert during in vivo passage. Collectively, this work has determined that deer mice are a suitable animal model for the study of SARS-CoV-2 pathogenesis, and that they have the potential to serve as secondary reservoir hosts that could lead to periodic outbreaks of COVID-19 in North America.

12.
J Virol ; 94(21)2020 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-32847848

RESUMO

Zika virus (ZIKV) can establish infection in immune privileged sites such as the testes, eye, and placenta. Whether ZIKV infection of white blood cells is required for dissemination of the virus to immune privileged sites has not been definitively shown. To assess whether initial ZIKV replication in myeloid cell populations is critical for dissemination during acute infection, recombinant ZIKVs were generated that could not replicate in these specific cells. ZIKV was cell restricted by insertion of a complementary sequence to a myeloid-specific microRNA in the 3' untranslated region. Following inoculation of a highly sensitive immunodeficient mouse model, crucial immune parameters, such as quantification of leukocyte cell subsets, cytokine and chemokine secretion, and viremia, were assessed. Decreased neutrophil numbers in the spleen were observed during acute infection with myeloid-restricted ZIKV that precluded the generation of viremia and viral dissemination to peripheral organs. Mice inoculated with a nontarget microRNA control ZIKV demonstrated increased expression of key cytokines and chemokines critical for neutrophil and monocyte recruitment and increased neutrophil influx in the spleen. In addition, ZIKV-infected Ly6Chi monocytes were identified in vivo in the spleen. Mice inoculated with myeloid-restricted ZIKV had a decrease in Ly6Chi ZIKV RNA-positive monocytes and a lack of inflammatory cytokine production compared to mice inoculated with control ZIKV.IMPORTANCE Myeloid cells, including monocytes, play a crucial role in immune responses to pathogens. Monocytes have also been implicated as "Trojan horses" during viral infections, carrying infectious virus particles to immune privileged sites and/or to sites protected by physical blood-tissue barriers, such as the blood-testis barrier and the blood-brain barrier. In this study, we found that myeloid cells are crucial to Zika virus (ZIKV) pathogenesis. By engineering ZIKV clones to encode myeloid-specific microRNA target sequences, viral replication was inhibited in myeloid cells by harnessing the RNA interference pathway. Severely immunodeficient mice inoculated with myeloid-restricted ZIKV did not demonstrate clinical signs of disease and survived infection. Furthermore, viral dissemination to peripheral organs was not observed in these mice. Lastly, we identified Ly6Cmid/hi murine monocytes as the major myeloid cell population that disseminates ZIKV.


Assuntos
Linhagem da Célula/imunologia , Hospedeiro Imunocomprometido , Células Mieloides/imunologia , Viremia/imunologia , Replicação Viral/imunologia , Infecção por Zika virus/imunologia , Zika virus/imunologia , Animais , Antígenos Ly/genética , Antígenos Ly/imunologia , Encéfalo/imunologia , Encéfalo/patologia , Encéfalo/virologia , Linhagem da Célula/genética , Citocinas/genética , Citocinas/imunologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , MicroRNAs/genética , MicroRNAs/imunologia , Monócitos/imunologia , Monócitos/patologia , Monócitos/virologia , Células Mieloides/classificação , Células Mieloides/patologia , Células Mieloides/virologia , Neutrófilos/imunologia , Neutrófilos/patologia , Neutrófilos/virologia , RNA Viral/genética , RNA Viral/imunologia , Transdução de Sinais , Baço/imunologia , Baço/patologia , Baço/virologia , Testículo/imunologia , Testículo/patologia , Testículo/virologia , Viremia/genética , Viremia/patologia , Viremia/virologia , Zika virus/patogenicidade , Infecção por Zika virus/genética , Infecção por Zika virus/patologia , Infecção por Zika virus/virologia
13.
J Virol ; 94(19)2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32699093

RESUMO

The unwinding of double-stranded RNA intermediates is critical for the replication and packaging of flavivirus RNA genomes. This unwinding activity is achieved by the ATP-dependent nonstructural protein 3 (NS3) helicase. In previous studies, we investigated the mechanism of energy transduction between the ATP and RNA binding pockets using molecular dynamics simulations and enzymatic characterization. Our data corroborated the hypothesis that motif V is a communication hub for this energy transduction. More specifically, mutations T407A and S411A in motif V exhibit a hyperactive helicase phenotype, leading to the regulation of translocation and unwinding during replication. However, the effect of these mutations on viral infection in cell culture and in vivo is not well understood. Here, we investigated the role of motif V in viral replication using West Nile virus (Kunjin subtype) T407A and S411A mutants (T407A and S411A Kunjin, respectively) in cell culture and in vivo We were able to recover S411A Kunjin but unable to recover T407A Kunjin. Our results indicated that S411A Kunjin decreased viral infection and increased cytopathogenicity in cell culture compared to wild-type (WT) Kunjin. Similarly, decreased infection rates in surviving S411A Kunjin-infected Culex quinquefasciatus mosquitoes were observed, but S411A Kunjin infection resulted in increased mortality compared to WT Kunjin infection. Additionally, S411A Kunjin infection increased viral dissemination and saliva positivity rates in surviving mosquitoes compared to WT Kunjin infection. These data suggest that S411A Kunjin increases viral pathogenesis in mosquitoes. Overall, these data indicate that NS3 motif V may play a role in the pathogenesis, dissemination, and transmission efficiency of Kunjin virus.IMPORTANCE Kunjin and West Nile viruses belong to the arthropod-borne flaviviruses, which can result in severe symptoms, including encephalitis, meningitis, and death. Flaviviruses have expanded into new populations and emerged as novel pathogens repeatedly in recent years, demonstrating that they remain a global threat. Currently, there are no approved antiviral therapeutics against either Kunjin or West Nile viruses. Thus, there is a pressing need for understanding the pathogenesis of these viruses in humans. In this study, we investigated the role of the Kunjin virus helicase on infection in cell culture and in vivo This work provides new insight into how flaviviruses control pathogenesis and mosquito transmission through the nonstructural protein 3 helicase.


Assuntos
Culicidae/virologia , RNA Helicases/genética , Serina Endopeptidases/genética , Proteínas não Estruturais Virais/genética , Febre do Nilo Ocidental/mortalidade , Febre do Nilo Ocidental/veterinária , Vírus do Nilo Ocidental/enzimologia , Vírus do Nilo Ocidental/genética , Animais , Linhagem Celular , Chlorocebus aethiops , Culex/virologia , Feminino , Flavivirus/genética , Células HEK293 , Humanos , Modelos Moleculares , Mutação , Domínios e Motivos de Interação entre Proteínas , Células Vero , Replicação Viral , Febre do Nilo Ocidental/transmissão , Vírus do Nilo Ocidental/patogenicidade
14.
medRxiv ; 2020 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-32577700

RESUMO

Background: SARS-CoV-2 emerged in 2019 and has become a major global pathogen. Its emergence is notable due to its impacts on individuals residing within long term care facilities (LTCFs) such as rehabilitation centers and nursing homes. LTCF residents tend to possess several risk factors for more severe SARS-CoV-2 outcomes, including advanced age and multiple comorbidities. Indeed, residents of LTCFs represent approximately 40% of SARS-CoV-2 deaths in the United States. Methods: To assess the prevalence and incidence of SARS-CoV-2 among LTCF workers, determine the extent of asymptomatic SARS-CoV-2 infection, and provide information on the genomic epidemiology of the virus within these unique care settings, we collected nasopharyngeal swabs from workers for 8-11 weeks at six Colorado LTCFs, determined the presence and level of viral RNA and infectious virus within these samples, and sequenced 54 nearly complete genomes. Findings: Our data reveal a strikingly high degree of asymptomatic/mildly symptomatic infection, a strong correlation between viral RNA and infectious virus, prolonged infections and persistent RNA in a subset of individuals, and declining incidence over time. Interpretation: Our data suggest that asymptomatic SARS-CoV-2 infected individuals contribute to virus persistence and transmission within the workplace, due to high levels of virus. Genetic epidemiology revealed that SARS-CoV-2 likely spreads between staff within an LTCF. Funding: Colorado State University Colleges of Health and Human Sciences, Veterinary Medicine and Biomedical Sciences, Natural Sciences, and Walter Scott, Jr. College of Engineering, the Columbine Health Systems Center for Healthy Aging, and the National Institute of Allergy and Infectious Diseases.

15.
Am J Trop Med Hyg ; 103(2): 869-875, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32431284

RESUMO

Chikungunya virus (CHIKV) and Zika virus (ZIKV) are arthropod-borne viruses transmitted mainly by Aedes aegypti mosquitoes. These viruses have become endemic in large parts of North, Central, and South America. Arboviruses persistently infect mosquitoes throughout their life span and become infectious (i.e., expectorate infectious virus in saliva) after a period of time called the extrinsic incubation period (EIP). The duration of this infectiousness, however, is not well characterized. This is an important shortcoming because many epidemiological models assume that mosquitoes continue to be infectious for the duration of their life span. To define the duration of infectiousness for CHIKV and ZIKV, mosquitoes were infected orally with these viruses. Every 2 days, legs/wings, midguts, salivary glands, and saliva were collected from 30 to 60 mosquitoes and viral load measured. In CHIKV-infected mosquitoes, infectious virus in saliva peaked early (2-4 dpi), and then decreased rapidly and was rarely observed after 10 dpi. Viral RNA in infected tissues also decreased after the initial peak (4-8 dpi) but did so much less drastically. In ZIKV-infected mosquitoes, the infectious virus in saliva peaked at 12-14 dpi and dropped off only slightly after 14 dpi. In infected tissues, viral RNA increased early during infection, and then plateaued after 6-10 days. Our findings suggest that significant variation exists in the duration of the infectious period for arboviruses that is in part influenced by virus clearance from expectorated saliva.


Assuntos
Aedes/virologia , Vírus Chikungunya/fisiologia , Intestinos/virologia , Saliva/virologia , Glândulas Salivares/virologia , Replicação Viral/fisiologia , Zika virus/fisiologia , Animais , Febre de Chikungunya/transmissão , Extremidades/virologia , Período de Incubação de Doenças Infecciosas , Mosquitos Vetores/virologia , Asas de Animais/virologia , Infecção por Zika virus/transmissão
16.
Pathogens ; 9(1)2020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31935992

RESUMO

West Nile virus (WNV) continues to be a major cause of human arboviral neuroinvasive disease. Susceptible non-human vertebrates are particularly diverse, ranging from commonly affected birds and horses to less commonly affected species such as alligators. This review summarizes the pathology caused by West Nile virus during natural infections of humans and non-human animals. While the most well-known findings in human infection involve the central nervous system, WNV can also cause significant lesions in the heart, kidneys and eyes. Time has also revealed chronic neurologic sequelae related to prior human WNV infection. Similarly, neurologic disease is a prominent manifestation of WNV infection in most non-human non-host animals. However, in some avian species, which serve as the vertebrate host for WNV maintenance in nature, severe systemic disease can occur, with neurologic, cardiac, intestinal and renal injury leading to death. The pathology seen in experimental animal models of West Nile virus infection and knowledge gains on viral pathogenesis derived from these animal models are also briefly discussed. A gap in the current literature exists regarding the relationship between the neurotropic nature of WNV in vertebrates, virus propagation and transmission in nature. This and other knowledge gaps, and future directions for research into WNV pathology, are addressed.

17.
Elife ; 82019 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-31596239

RESUMO

The magnitude and duration of vertebrate viremia is a critical determinant of arbovirus transmission, geographic spread, and disease severity. We find that multiple alphaviruses, including chikungunya (CHIKV), Ross River (RRV), and o'nyong 'nyong (ONNV) viruses, are cleared from the circulation of mice by liver Kupffer cells, impeding viral dissemination. Clearance from the circulation was independent of natural antibodies or complement factor C3, and instead relied on scavenger receptor SR-A6 (MARCO). Remarkably, lysine to arginine substitutions at distinct residues within the E2 glycoproteins of CHIKV and ONNV (E2 K200R) as well as RRV (E2 K251R) allowed for escape from clearance and enhanced viremia and dissemination. Mutational analysis revealed that viral clearance from the circulation is strictly dependent on the presence of lysine at these positions. These findings reveal a previously unrecognized innate immune pathway that controls alphavirus viremia and dissemination in vertebrate hosts, ultimately influencing disease severity and likely transmission efficiency.


Assuntos
Infecções por Alphavirus/imunologia , Vírus Chikungunya/imunologia , Macrófagos do Fígado/imunologia , Vírus O'nyong-nyong/imunologia , Receptores Imunológicos/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Animais , Modelos Animais de Doenças , Lisina/genética , Lisina/metabolismo , Camundongos , Mutação de Sentido Incorreto
18.
Nat Microbiol ; 4(12): 2298-2309, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31527796

RESUMO

Major histocompatibility complex class II (MHC-II) molecules of multiple species function as cell-entry receptors for the haemagglutinin-like H18 protein of the bat H18N11 influenza A virus, enabling tropism of the viruses in a potentially broad range of vertebrates. However, the function of the neuraminidase-like N11 protein is unknown because it is dispensable for viral infection or the release of H18-pseudotyped viruses. Here, we show that infection of mammalian cells with wild-type H18N11 leads to the emergence of mutant viruses that lack the N11 ectodomain and acquired mutations in H18. An infectious clone of one such mutant virus, designated rP11, appeared to be genetically stable in mice and replicated to higher titres in mice and cell culture compared with wild-type H18N11. In ferrets, rP11 antigen and RNA were detected at low levels in various tissues, including the tonsils, whereas the wild-type virus was not. In Neotropical Jamaican fruit bats, wild-type H18N11 was found in intestinal Peyer's patches and was shed to high concentrations in rectal samples, resulting in viral transmission to naive contact bats. Notably, rP11 also replicated efficiently in bats; however, only restored full-length N11 viruses were transmissible. Our findings suggest that wild-type H18N11 replicates poorly in mice and ferrets and that N11 is a determinant for viral transmission in bats.


Assuntos
Quirópteros/virologia , Vírus da Influenza A/fisiologia , Infecções por Orthomyxoviridae/transmissão , Infecções por Orthomyxoviridae/veterinária , Infecções por Orthomyxoviridae/virologia , Orthomyxoviridae/fisiologia , Animais , Linhagem Celular , Furões/virologia , Células HEK293 , Especificidade de Hospedeiro , Humanos , Vírus da Influenza A/patogenicidade , Mamíferos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Neuraminidase/química , Neuraminidase/genética , Orthomyxoviridae/genética , Orthomyxoviridae/patogenicidade , Receptor de Interferon alfa e beta/genética , Receptores de Interferon/genética , Replicação Viral
19.
J Med Entomol ; 56(6): 1467-1474, 2019 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-31549720

RESUMO

West Nile virus (WNV) was first identified in North America almost 20 yr ago. In that time, WNV has crossed the continent and established enzootic transmission cycles, resulting in intermittent outbreaks of human disease that have largely been linked with climatic variables and waning avian seroprevalence. During the transcontinental dissemination of WNV, the original genotype has been displaced by two principal extant genotypes which contain an envelope mutation that has been associated with enhanced vector competence by Culex pipiens L. (Diptera: Culicidae) and Culex tarsalis Coquillett vectors. Analyses of retrospective avian host competence data generated using the founding NY99 genotype strain have demonstrated a steady reduction in viremias of house sparrows over time. Reciprocally, the current genotype strains WN02 and SW03 have demonstrated an inverse correlation between house sparrow viremia magnitude and the time since isolation. These data collectively indicate that WNV has evolved for increased avian viremia while house sparrows have evolved resistance to the virus such that the relative host competence has remained constant. Intrahost analyses of WNV evolution demonstrate that selection pressures are avian species-specific and purifying selection is greater in individual birds compared with individual mosquitoes, suggesting that the avian adaptive and/or innate immune response may impose a selection pressure on WNV. Phylogenomic, experimental evolutionary systems, and models that link viral evolution with climate, host, and vector competence studies will be needed to identify the relative effect of different selective and stochastic mechanisms on viral phenotypes and the capacity of newly evolved WNV genotypes for transmission in continuously changing landscapes.


Assuntos
Doenças das Aves/epidemiologia , Aves , Culicidae/virologia , Genoma Viral , Interações Hospedeiro-Patógeno , Febre do Nilo Ocidental/veterinária , Vírus do Nilo Ocidental/fisiologia , Animais , Doenças das Aves/virologia , Meio Ambiente , América do Norte , Febre do Nilo Ocidental/epidemiologia , Febre do Nilo Ocidental/virologia , Vírus do Nilo Ocidental/crescimento & desenvolvimento
20.
Viruses ; 11(7)2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31336898

RESUMO

Arthropod-borne viruses (arboviruses) of vertebrates including dengue, zika, chikungunya, Rift Valley fever, and blue tongue viruses cause extensive morbidity and mortality in humans, agricultural animals, and wildlife across the globe. As obligate intercellular pathogens, arboviruses must be well adapted to the cellular and molecular environment of both their arthropod (invertebrate) and vertebrate hosts, which are vastly different due to hundreds of millions of years of separate evolution. Here we discuss the comparative pressures on arbovirus RNA genomes as a result of a dual host life cycle, focusing on pressures that do not alter amino acids. We summarize what is currently known about arboviral genetic composition, such as dinucleotide and codon usage, and how cyclical infection of vertebrate and invertebrate hosts results in different genetic profiles compared with single-host viruses. To serve as a comparison, we compile what is known about arthropod tRNA, dinucleotide, and codon usages and compare this with vertebrates. Additionally, we discuss the potential roles of genetic robustness in arboviral evolution and how it may vary from other viruses. Overall, both arthropod and vertebrate hosts influence the resulting genetic composition of arboviruses, but a great deal remains to be investigated.


Assuntos
Arbovírus/genética , Arbovírus/fisiologia , Uso do Códon , Interações Hospedeiro-Patógeno , Nucleotídeos , Animais , Infecções por Arbovirus/virologia , Artrópodes/virologia , Evolução Molecular , Genoma Viral , Humanos , Estágios do Ciclo de Vida , RNA Viral/genética , Replicação Viral
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