Last data update: Jun 03, 2024. (Total: 46935 publications since 2009)
Records 1-27 (of 27 Records) |
Query Trace: Duggal NK [original query] |
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Differential pathogenesis of Usutu virus isolates in mice (preprint)
Kuchinsky SC , Hawks SA , Mossel EC , Coutermarsh-Ott S , Duggal NK . bioRxiv 2020 2020.08.31.275149 Usutu virus (USUV; Flavivirus), a close phylogenetic and ecological relative of West Nile virus, is a zoonotic virus that can cause neuroinvasive disease in humans. USUV is maintained in an enzootic cycle between Culex mosquitoes and birds. Since the first isolation in 1959 in South Africa, USUV has spread throughout Africa and Europe. Reported human cases have increased over the last few decades, primarily in Europe, with symptoms ranging from mild febrile illness to severe neurological effects. In this study, we investigated whether USUV has become more pathogenic during emergence in Europe. Interferon α/β receptor knockout (Ifnar1-/-) mice were inoculated with recent USUV isolates from Africa and Europe, as well as the historic 1959 South African strain. The three tested African strains and one European strain from Spain caused 100% mortality in inoculated mice, with similar survival times and histopathology in tissues. Unexpectedly, a European strain from the Netherlands caused only 12% mortality and significantly less histopathology in tissues from mice compared to mice inoculated with the other strains. Viremia was highest in mice inoculated with the recent African strains and lowest in mice inoculated with the Netherlands strain. Based on phylogenetics, the USUV isolates from Spain and the Netherlands were derived from separate introductions into Europe, suggesting that disease outcomes may differ for USUV strains circulating in Europe. These results also suggest that while more human USUV disease cases have been reported in Europe recently, circulating African USUV strains are still a potential major health concern.Author Summary Usutu virus (USUV) is an emerging mosquito-borne virus that causes severe neuroinvasive disease in humans. USUV was first detected in Africa in 1959, and cases of human disease have increased in recent years. Most USUV disease cases are now reported in Europe, where the virus currently circulating. One possibility for the increase in case numbers is that USUV strains have become more pathogenic over time during its spread from Africa into Europe. We compared the pathogenesis of five USUV isolates from Africa and Europe in a mouse model. Three isolates from Africa and one isolate from Europe caused 100% mortality in mice. Surprisingly, one isolate from Netherlands caused only 12% motality. Significantly less histopathology was observed in tissues from mice inoculated with the Netherlands strain compared to mice inoculated with the other four strains. Our results suggest that, even though more human USUV disease cases have been reported in Europe recently, African USUV strains are also highly pathogenic.Competing Interest StatementThe authors have declared no competing interest. |
Prolonged shedding of Zika virus in human semen is associated with male reproductive tract inflammation.
Vogt MB , McDonald EM , Delorey M , Mead PS , Hook SA , Hinckley AF , Werre SR , Brault AC , Duggal NK . J Infect Dis 2022 226 (7) 1140-1150 Zika virus (ZIKV) is a mosquito-borne flavivirus that causes congenital defects. Sexual transmission of ZIKV was confirmed in a recent epidemic; however, mechanisms behind ZIKV infection and persistence in the male reproductive tract are unknown. Previously, we found that ∼33% of men with symptomatic ZIKV infections shed ZIKV RNA in semen, and some men shed ZIKV RNA for >3 months. Here, we evaluated the semen of 49 ZIKV-infected men to identify immune factors correlating with long-term ZIKV shedding in semen and ZIKV-infected cell types in semen. We found prolonged ZIKV RNA shedding in semen was associated with male reproductive tract inflammation, indicated by higher leukocyte counts and inflammatory cytokine concentrations in semen of long-term versus short-term shedders. Additionally, we found ZIKV RNA in seminal leukocytes and epithelial cells. This study of human semen from ZIKV-infected men provides critical insights into impacts of ZIKV on male reproductive tract health. |
West nile virus vaccination protects against usutu virus disease in mice
Salgado R , Hawks SA , Frere F , Vázquez A , Huang CYH , Duggal NK . Viruses 2021 13 (12) West Nile virus (WNV) and Usutu virus (USUV) are mosquito‐borne flaviviruses that can cause neuroinvasive disease in humans. WNV and USUV circulate in both Africa and Europe and are closely related. Due to antigenic similarity, WNV‐specific antibodies and USUV‐specific antibodies have the potential to bind heterologous viruses; however, it is unclear whether this interaction may offer protection against infection. To investigate how prior WNV exposure would influ-ence USUV infection, we used an attenuated WNV vaccine that contains the surface proteins of WNV in the backbone of a dengue virus 2 vaccine strain and protects against WNV disease. We hypothesized that vaccination with this attenuated WNV vaccine would protect against USUV in-fection. Neutralizing responses against WNV and USUV were measured in vitro using sera following vaccination. Sera from vaccinated CD‐1 and Ifnar1−/− mice cross‐neutralized with WNV and USUV. All mice were then subsequently challenged with an African or European USUV strain. In CD‐1 mice, there was no difference in USUV titers between vaccinated and mock‐vaccinated mice. However, in the Ifnar1−/− model, vaccinated mice had significantly higher survival rates and significantly lower USUV viremia compared to mock‐vaccinated mice. Our results indicate that exposure to an attenuated form of WNV protects against severe USUV disease in mice and elicits a neutralizing response to both WNV and USUV. Future studies will investigate the immune mechanisms responsible for the protection against USUV infection induced by WNV vaccination, providing critical insight that will be essential for USUV and WNV vaccine development. © 2021 by the authors. Licensee MDPI, Basel, Switzerland. |
Differential pathogenesis of Usutu virus isolates in mice
Kuchinsky SC , Hawks SA , Mossel EC , Coutermarsh-Ott S , Duggal NK . PLoS Negl Trop Dis 2020 14 (10) e0008765 Usutu virus (USUV; Flavivirus), a close phylogenetic and ecological relative of West Nile virus, is a zoonotic virus that can cause neuroinvasive disease in humans. USUV is maintained in an enzootic cycle between Culex mosquitoes and birds. Since the first isolation in 1959 in South Africa, USUV has spread throughout Africa and Europe. Reported human cases have increased over the last few decades, primarily in Europe, with symptoms ranging from mild febrile illness to severe neurological effects. In this study, we investigated whether USUV has become more pathogenic during emergence in Europe. Interferon α/β receptor knockout (Ifnar1-/-) mice were inoculated with recent USUV isolates from Africa and Europe, as well as the historic 1959 South African strain. The three tested African strains and one European strain from Spain caused 100% mortality in inoculated mice, with similar survival times and histopathology in tissues. Unexpectedly, a European strain from the Netherlands caused only 12% mortality and significantly less histopathology in tissues from mice compared to mice inoculated with the other strains. Viremia was highest in mice inoculated with the recent African strains and lowest in mice inoculated with the Netherlands strain. Based on phylogenetics, the USUV isolates from Spain and the Netherlands were derived from separate introductions into Europe, suggesting that disease outcomes may differ for USUV strains circulating in Europe. These results also suggest that while more human USUV disease cases have been reported in Europe recently, circulating African USUV strains are still a potential major health concern. |
On the Fly: Interactions Between Birds, Mosquitoes, and Environment That Have Molded West Nile Virus Genomic Structure Over Two Decades.
Duggal NK , Langwig KE , Ebel GD , Brault AC . J Med Entomol 2019 56 (6) 1467-1474 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. |
Duration of seminal Zika viral RNA shedding in immunocompetent mice inoculated with Asian and African genotype viruses.
McDonald EM , Duggal NK , Delorey MJ , Oksanish J , Ritter JM , Brault AC . Virology 2019 535 1-10 Prior to the emergence of Asian genotype Zika virus (ZIKV) in the Western hemisphere, sexual transmission in humans was documented. Sexual transmission by African genotype ZIKVs has not been assessed in laboratory animal models, due to rapid and high mortality rates of immunodeficient mice following inoculation. To overcome these limitations, immunocompetent C57Bl/6 mice were used to longitudinally assess Asian and African genotype ZIKV sexual transmission potential. Furthermore, to determine if enhanced pathogenesis of African genotype ZIKVs is due to structural determinants, PRVABC59 prM/E was replaced with African MR766 prM/E (chimeric ZIKV). The African genotype and chimeric ZIKV elicited greater pathogenic effects in the male reproductive tract and generated higher viremias. Yet, the duration, magnitude and efficiency of seminal shedding of infectious virus and viral RNA were similar between chimeric-, African and Asian genotype ZIKV-inoculated mice. These data show that increased male reproductive tract pathology does not increase sexual transmission potential. |
Mutations present in a low-passage Zika virus isolate result in attenuated pathogenesis in mice
Duggal NK , McDonald EM , Weger-Lucarelli J , Hawks SA , Ritter JM , Romo H , Ebel GD , Brault AC . Virology 2019 530 19-26 Zika virus (ZIKV) infection can result in neurological disorders including Congenital Zika Syndrome in infants exposed to the virus in utero. Pregnant women can be infected by mosquito bite as well as by sexual transmission from infected men. Herein, the variants of ZIKV within the male reproductive tract and ejaculates were assessed in inoculated mice. We identified two non-synonymous variants at positions E-V330L and NS1-W98G. These variants were also present in the passage three PRVABC59 isolate and infectious clone relative to the patient serum PRVABC59 sequence. In subsequent studies, ZIKV E-330L was less pathogenic in mice than ZIKV E-330V as evident by increased average survival times. In Vero cells, ZIKV E-330L/NS1-98G outcompeted ZIKV E-330V/NS1-98W within 3 passages. These results suggest that the E-330L/NS1-98G variants are attenuating in mice and were enriched during cell culture passaging. Cell culture propagation of ZIKV could significantly affect animal model development and vaccine efficacy studies. |
Heartland virus epidemiology, vector association, and disease potential
Brault AC , Savage HM , Duggal NK , Eisen RJ , Staples JE . Viruses 2018 10 (9) First identified in two Missouri farmers exhibiting low white-blood-cell and platelet counts in 2009, Heartland virus (HRTV) is genetically closely related to severe fever with thrombocytopenia syndrome virus (SFTSV), a tick-borne phlebovirus producing similar symptoms in China, Korea, and Japan. Field isolations of HRTV from several life stages of unfed, host-seeking Amblyomma americanum, the lone star tick, implicated it as a putative vector capable of transstadial transmission. Laboratory vector competence assessments confirmed transstadial transmission of HRTV, demonstrated vertical infection, and showed co-feeding infection between A. americanum. A vertical infection rate of 33% from adult females to larvae in the laboratory was observed, while only one of 386 pools of molted nymphs (1930) reared from co-feeding larvae was positive for HRTV (maximum-likelihood estimate of infection rate = 0.52/1000). Over 35 human HRTV cases, all within the distribution range of A. americanum, have been documented. Serological testing of wildlife in areas near the index human cases, as well as in widely separated regions of the eastern United States where A. americanum occur, indicated many potential hosts such as raccoons and white-tailed deer. Attempts, however, to experimentally infect mice, rabbits, hamsters, chickens, raccoons, goats, and deer failed to produce detectable viremia. Immune-compromised mice and hamsters are the only susceptible models. Vertical infection augmented by co-feeding transmission could play a role in maintaining the virus in nature. A more complete assessment of the natural transmission cycle of HRTV coupled with serosurveys and enhanced HRTV disease surveillance are needed to better understand transmission dynamics and human health risks. |
Infection of epididymal epithelial cells and leukocytes drives seminal shedding of Zika virus in a mouse model
McDonald EM , Duggal NK , Ritter JM , Brault AC . PLoS Negl Trop Dis 2018 12 (8) e0006691 While primarily a mosquito-borne virus, Zika virus (ZIKV; genus Flavivirus in the Flaviviridae family) is capable of being sexually transmitted. Thirty to fifty percent of men with confirmed ZIKV infection shed ZIKV RNA in their semen, and prolonged viral RNA shedding in semen can occur for more than 6 months. The cellular reservoir of ZIKV in semen is unknown, although spermatozoa have been shown to contain ZIKV RNA and antigen. Yet, spermatozoa are not a requisite for sexual transmission, as at least one case of ZIKV sexual transmission involved a vasectomized man. To determine the cellular reservoirs of ZIKV in semen, an established animal model of sexual transmission was used. The majority of virus detected in the seminal fluid of infected mice during the peak timing of sexual transmission was from the supernatant fraction, suggesting cell-free ZIKV may be largely responsible for sexual transmission. However, some ZIKV RNA was cell-associated. In the testes and epididymides of infected mice, intracellular staining of ZIKV RNA was more pronounced in spermatogenic precursors (spermatocytes and spermatogonia) than in spermatids. Visualization of intracellular negative strand ZIKV RNA demonstrated ZIKV replication intermediates in leukocytes, immature spermatids and epididymal epithelial cells in the male urogenital tract. Epididymal epithelial cells were the principal source of negative-strand ZIKV RNA during the peak timing of sexual transmission potential, indicating these cells may be the predominant source of infectious cell-free ZIKV in seminal fluid. These data promote a more complete understanding of sexual transmission of ZIKV and will inform further model development for future studies on persistent ZIKV RNA shedding. |
Zika Virus Shedding in Semen of Symptomatic Infected Men.
Mead PS , Duggal NK , Hook SA , Delorey M , Fischer M , Olzenak McGuire D , Becksted H , Max RJ , Anishchenko M , Schwartz AM , Tzeng WP , Nelson CA , McDonald EM , Brooks JT , Brault AC , Hinckley AF . N Engl J Med 2018 378 (15) 1377-1385 BACKGROUND: Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that has been linked to adverse birth outcomes. Previous reports have shown that person-to-person transmission can occur by means of sexual contact. METHODS: We conducted a prospective study involving men with symptomatic ZIKV infection to determine the frequency and duration of ZIKV shedding in semen and urine and to identify risk factors for prolonged shedding in these fluids. Specimens were obtained twice per month for 6 months after illness onset and were tested by real-time reverse-transcriptase-polymerase-chain-reaction (RT-PCR) assay for ZIKV RNA and by Vero cell culture and plaque assay for infectious ZIKV. RESULTS: A total of 1327 semen samples from 184 men and 1038 urine samples from 183 men were obtained 14 to 304 days after illness onset. ZIKV RNA was detected in the urine of 7 men (4%) and in the semen of 60 (33%), including in semen samples from 22 of 36 men (61%) who were tested within 30 days after illness onset. ZIKV RNA shedding in semen decreased substantially during the 3 months after illness onset but continued for 281 days in 1 man (1%). Factors that were independently associated with prolonged RNA shedding included older age, less frequent ejaculation, and the presence of certain symptoms at the time of initial illness. Infectious ZIKV was isolated from 3 of 78 semen samples with detectable ZIKV RNA, all obtained within 30 days after illness onset and all with at least 7.0 log10 ZIKV RNA copies per milliliter of semen. CONCLUSIONS: ZIKV RNA was commonly present in the semen of men with symptomatic ZIKV infection and persisted in some men for more than 6 months. In contrast, shedding of infectious ZIKV appeared to be much less common and was limited to the first few weeks after illness onset. (Funded by the Centers for Disease Control and Prevention.). |
Sexual transmission of Zika virus enhances in utero transmission in a mouse model
Duggal NK , McDonald EM , Ritter JM , Brault AC . Sci Rep 2018 8 (1) 4510 Zika virus (ZIKV) is an emerging mosquito-borne virus that can cause ZIKV congenital syndrome when a pregnant woman is infected. Sexual transmission has also been described for ZIKV, though the relationship between sexual transmission and vertical transmission has not been investigated. Here, viral dissemination to the female reproductive tract and fetuses was assessed in immunodeficient (AG129) female mice that were exposed to ZIKV by subcutaneous (s.c.) inoculation, intravaginal (ivag.) inoculation, or sexual transmission from infected male AG129 mice. Pregnant females had significantly increased ZIKV dissemination to the female reproductive tract compared to non-pregnant females when exposed by s.c. or ivag. inoculation. Sexual transmission resulted in significantly greater morbidity and mortality in females and higher ZIKV titers in the female reproductive tract than s.c. or ivag. inoculation. Ovaries from females infected sexually contained ZIKV RNA within the ovarian follicles. Furthermore, ZIKV titers were significantly higher in fetuses from dams exposed sexually compared to fetuses from dams exposed by s.c. or ivag. inoculation. These results demonstrate that sexual transmission enhances dissemination of ZIKV to the female reproductive tract and developing fetuses in a mouse model. |
Increases in the competitive fitness of West Nile virus isolates after introduction into California
Worwa G , Hutton AA , Frey M , Duggal NK , Brault AC , Reisen WK . Virology 2017 514 170-181 To investigate the phenotypic evolution of West Nile virus (WNV) in California, we competed sixteen isolates made during 2007-08 against COAV997-5nt, a genetically marked clone from the founding 2003 California isolate COAV997-2003. Using in vivo fitness competitions in House Finches (HOFI) and Culex tarsalis mosquitoes, we found that the majority of WNV WN02 and SW03 genotype isolates exhibited elevated replicative fitness in both hosts compared to COAV997-5nt. Increased replicative capacity in HOFIs was not associated with increased mortality, indicating that these isolates had not gained avian virulence. One WN02 isolate from Coachella Valley, a region geographically close to the isolation of COAV997, showed neutral fitness in HOFIs and reduced fitness in Cx. tarsalis. Two isolates from Kern County and Sacramento/Yolo County out-competed COAV997-nt in HOFIs, but were transmitted less efficiently by Cx. tarsalis. Competition demonstrated neutral or increased fitness that appeared independent of both WN02 and SW03 genotypes. |
A Zika vaccine targeting NS1 protein protects immunocompetent adult mice in a lethal challenge model
Brault AC , Domi A , McDonald EM , Talmi-Frank D , McCurley N , Basu R , Robinson HL , Hellerstein M , Duggal NK , Bowen RA , Guirakhoo F . Sci Rep 2017 7 (1) 14769 Zika virus (ZIKV) is a mosquito-borne flavivirus that has rapidly extended its geographic range around the world. Its association with abnormal fetal brain development, sexual transmission, and lack of a preventive vaccine have constituted a global health concern. Designing a safe and effective vaccine requires significant caution due to overlapping geographical distribution of ZIKV with dengue virus (DENV) and other flaviviruses, possibly resulting in more severe disease manifestations in flavivirus immune vaccinees such as Antibody-Dependent Enhancement (ADE, a phenomenon involved in pathogenesis of DENV, and a risk associated with ZIKV vaccines using the envelope proteins as immunogens). Here, we describe the development of an alternative vaccine strategy encompassing the expression of ZIKV non-structural-1 (NS1) protein from a clinically proven safe, Modified Vaccinia Ankara (MVA) vector, thus averting the potential risk of ADE associated with structural protein-based ZIKV vaccines. A single intramuscular immunization of immunocompetent mice with the MVA-ZIKV-NS1 vaccine candidate provided robust humoral and cellular responses, and afforded 100% protection against a lethal intracerebral dose of ZIKV (strain MR766). This is the first report of (i) a ZIKV vaccine based on the NS1 protein and (ii) single dose protection against ZIKV using an immunocompetent lethal mouse challenge model. |
Pathogenesis and sexual transmission of Spondweni and Zika viruses
McDonald EM , Duggal NK , Brault AC . PLoS Negl Trop Dis 2017 11 (10) e0005990 The Spondweni serogroup of viruses (Flaviviridae, Flavivirus) is comprised of Spondweni virus (SPONV) and Zika virus (ZIKV), which are mosquito-borne viruses capable of eliciting human disease. Numerous cases of ZIKV sexual transmission in humans have been documented following the emergence of the Asian genotype in the Americas. The African ZIKV genotype virus was previously implicated in the first reported case of ZIKV sexual transmission. Reports of SPONV infection in humans have been associated with non-specific febrile illness, but no association with sexual transmission has been reported. In order to assess the relative efficiency of sexual transmission of different ZIKV strains and the potential capacity of SPONV to be sexually transmitted, viral loads in the male reproductive tract and in seminal fluids were assessed in interferon alpha/beta and -gamma receptor deficient (AG129) mice. Male mice were inoculated subcutaneously with Asian genotype ZIKV strains PRVABC59 (Puerto Rico, 2015), FSS13025 (Cambodia, 2010), or P6-740 (Malaysia, 1966); African genotype ZIKV strain DakAr41524 (Senegal, 1984); or SPONV strain SAAr94 (South Africa, 1955). Infectious virus was detected in 60-72% of ejaculates collected from AG129 mice inoculated with ZIKV strains. In contrast, only 4% of ejaculates from SPONV-inoculated AG129 males were found to contain infectious virus, despite viral titers in the testes that were comparable to those of ZIKV-inoculated mice. Based on these results, future studies should be undertaken to assess the role of viral genetic determinants and host tropism that dictate the differential sexual transmission potential of ZIKV and SPONV. |
Differential neurovirulence of African and Asian genotype Zika virus isolates in outbred immunocompetent mice
Duggal NK , Ritter JM , McDonald EM , Romo H , Guirakhoo F , Davis BS , Chang GJ , Brault AC . Am J Trop Med Hyg 2017 97 (5) 1410-1417 Although first isolated almost 70 years ago, Zika virus (ZIKV; Flavivirus, Flaviviridae) has only recently been associated with significant outbreaks of disease in humans. Several severe ZIKV disease manifestations have also been recently documented, including fetal malformations, such as microcephaly, and Guillain-Barre syndrome in adults. Although principally transmitted by mosquitoes, sexual transmission of ZIKV has been documented. Recent publications of several interferon receptor knockout mouse models have demonstrated ZIKV-induced disease. Herein, outbred immunocompetent CD-1/ICR adult mice were assessed for susceptibility to disease by intracranial (i.c.) and intraperitoneal (i.p.) inoculation with the Ugandan prototype strain (MR766; African genotype), a low-passage Senegalese strain (DakAr41524; African genotype) and a recent ZIKV strain isolated from a traveler infected in Puerto Rico (PRVABC59; Asian genotype). Morbidity was not observed in mice inoculated by the i.p. route with either MR766 or PRVABC59 for doses up to 6 log10 PFU. In contrast, CD-1/ICR mice inoculated i.c. with the MR766 ZIKV strain exhibited an 80-100% mortality rate that was age independent. The DakAr41524 strain delivered by the i.c route caused 30% mortality, and the Puerto Rican ZIKV strain failed to elicit mortality but did induce a serum neutralizing immune response in 60% of mice. These data provide a potential animal model for assessing neurovirulence determinants of different ZIKV strains as well as a potential immunocompetent challenge model for assessing protective efficacy of vaccine candidates. |
Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone.
Weger-Lucarelli J , Duggal NK , Brault AC , Geiss BJ , Ebel GD . J Vis Exp 2017 (124) Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively. |
Rapid and specific detection of Asian- and African-lineage Zika viruses.
Chotiwan N , Brewster CD , Magalhaes T , Weger-Lucarelli J , Duggal NK , Ruckert C , Nguyen C , Garcia Luna SM , Fauver JR , Andre B , Gray M , Black WCth , Kading RC , Ebel GD , Kuan G , Balmaseda A , Jaenisch T , Marques ETA , Brault AC , Harris E , Foy BD , Quackenbush SL , Perera R , Rovnak J . Sci Transl Med 2017 9 (388) Understanding the dynamics of Zika virus transmission and formulating rational strategies for its control require precise diagnostic tools that are also appropriate for resource-poor environments. We have developed a rapid and sensitive loop-mediated isothermal amplification (LAMP) assay that distinguishes Zika viruses of Asian and African lineages. The assay does not detect chikungunya virus or flaviviruses such as dengue, yellow fever, or West Nile viruses. The assay conditions allowed direct detection of Zika virus RNA in cultured infected cells; in mosquitoes; in virus-spiked samples of human blood, plasma, saliva, urine, and semen; and in infected patient serum, plasma, and semen samples without the need for RNA isolation or reverse transcription. The assay offers rapid, specific, sensitive, and inexpensive detection of the Asian-lineage Zika virus strain that is currently circulating in the Western hemisphere, and can also detect the African-lineage Zika virus strain using separate, specific primers. |
Transmission incompetence of Culex quinquefasciatus and Culex pipiens pipiens from North America for Zika virus
Kenney JL , Romo H , Duggal NK , Tzeng WP , Burkhalter KL , Brault AC , Savage HM . Am J Trop Med Hyg 2017 96 (5) 1235-1240 AbstractIn late 2014, Zika virus (ZIKV; Flaviviridae, Flavivirus) emerged as a significant arboviral disease threat in the Western hemisphere. Aedes aegypti and Aedes albopictus have been considered the principal vectors of ZIKV in the New World due to viral isolation frequency and vector competence assessments. Limited reports of Culex transmission potential have highlighted the need for additional vector competence assessments of North American Culex species. Accordingly, North American Culex pipiens and Culex quinquefasciatus were orally exposed and intrathoracically inoculated with the African prototype ZIKV strain and currently circulating Asian lineage ZIKV strains to assess infection, dissemination, and transmission potential. Results indicated that these two North American Culex mosquito species were highly refractory to oral infection with no dissemination or transmission observed with any ZIKV strains assessed. Furthermore, both Culex mosquito species intrathoracically inoculated with either Asian or African lineage ZIKVs failed to expectorate virus in saliva. These in vivo results were further supported by the observation that multiple mosquito cell lines of Culex species origin demonstrated significant growth restriction of ZIKV strains compared with Aedes-derived cell lines. In summation, no evidence for the potential of Cx. pipiens or Cx. quinquefasciatus to serve as a competent vector for ZIKV transmission in North America was observed. |
Frequent Zika virus sexual transmission and prolonged viral RNA shedding in an immunodeficient mouse model
Duggal NK , Ritter JM , Pestorius SE , Zaki SR , Davis BS , Chang GJ , Bowen RA , Brault AC . Cell Rep 2017 18 (7) 1751-1760 Circulation of Zika virus (ZIKV) was first identified in the Western hemisphere in late 2014. Primarily transmitted through mosquito bite, ZIKV can also be transmitted through sex and from mother to fetus, and maternal ZIKV infection has been associated with fetal malformations. We assessed immunodeficient AG129 mice for their capacity to shed ZIKV in semen and to infect female mice via sexual transmission. Infectious virus was detected in semen between 7 and 21 days post-inoculation, and ZIKV RNA was detected in semen through 58 days post-inoculation. During mating, 73% of infected males transmitted ZIKV to uninfected females, and 50% of females became infected, with evidence of fetal infection in resulting pregnancies. Semen from vasectomized mice contained significantly lower levels of infectious virus, though sexual transmission still occurred. This model provides a platform for studying the kinetics of ZIKV sexual transmission and prolonged RNA shedding also observed in human semen. |
Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone.
Weger-Lucarelli J , Duggal NK , Bullard-Feibelman K , Veselinovic M , Romo H , Nguyen C , Ruckert C , Brault AC , Bowen RA , Stenglein M , Geiss BJ , Ebel GD . J Virol 2016 91 (1) Zika virus (ZIKV; Family Flaviviridae, genus Flavivirus) is a rapidly expanding global pathogen that has been associated with severe clinical manifestations, including devastating neurological disease in infants. There are currently no molecular clones of a New World ZIKV available, hindering progress toward understanding determinants of transmission and pathogenesis. Here we report the development and characterization of a novel ZIKV reverse genetics system based on a 2015 isolate from Puerto Rico (PRVABC59). We generated a two-plasmid infectious clone system from which infectious virus was rescued that replicates in human and mosquito cells with growth kinetics representative of wild-type ZIKV. Infectious clone-derived virus initiated comparable infection and transmission rates in Aedes aegypti mosquitoes compared to the primary isolate and displayed similar pathogenesis in AG129 mice. This infectious clone system provides a valuable resource to the research community to explore ZIKV molecular biology, vaccine development, antiviral development, diagnostics, vector competence, and disease pathogenesis. IMPORTANCE: ZIKV is a rapidly spreading mosquito-borne pathogen that has been linked to Guillain-Barre syndrome in adults and congenital microcephaly in developing fetuses and infants. ZIKV can also be sexually transmitted. The viral molecular determinants of any of these phenotypes are not well understood. There is no reverse genetics system available for the current epidemic virus that will allow researchers to study ZIKV immunity, develop novel vaccines, or develop antiviral drugs. Here we provide a novel infectious clone system generated from a recent ZIKV isolated from a patient infected in Puerto Rico. This infectious clone produces virus with similar in vitro and in vivo characteristics to the primary isolate, providing a critical tool to study ZIKV infection and disease. |
MicroRNA reduction of neuronal West Nile virus replication attenuates and affords a protective immune response in mice
Brostoff T , Pesavento PA , Barker CM , Kenney JL , Dietrich EA , Duggal NK , Bosco-Lauth AM , Brault AC . Vaccine 2016 34 (44) 5366-5375 West Nile virus (WNV) is an important agent of human encephalitis that has quickly become endemic across much of the United States since its identification in North America in 1999. While the majority ( approximately 75%) of infections are subclinical, neurologic disease can occur in a subset of cases, with outcomes including permanent neurologic damage and death. Currently, there are no WNV vaccines approved for use in humans. This study introduces a novel vaccine platform for WNV to reduce viral replication in the central nervous system while maintaining peripheral replication to elicit strong neutralizing antibody titers. Vaccine candidates were engineered to incorporate microRNA (miRNA) target sequences for a cognate miRNA expressed only in neurons, allowing the host miRNAs to target viral transcription through endogenous RNA silencing. To maintain stability, these targets were incorporated in multiple locations within the 3'-untranslated region, flanking sequences essential for viral replication without affecting the viral open reading frame. All candidates replicated comparably to wild type WNV in vitro within cells that did not express the cognate miRNA. Insertional control viruses were also capable of neuroinvasion and neurovirulence in vivo in CD-1 mice. Vaccine viruses were safe at all doses tested and did not demonstrate mutations associated with a reversion to virulence when serially passaged in mice. All vaccine constructs were protective from lethal challenge in mice, producing 93-100% protection at the highest dose tested. Overall, this is a safe and effective attenuation strategy with broad potential application for vaccine development. |
Potential for co-infection of a mosquito-specific flavivirus, Nhumirim virus, to block West Nile virus transmission in mosquitoes
Goenaga S , Kenney JL , Duggal NK , Delorey M , Ebel GD , Zhang B , Levis SC , Enria DA , Brault AC . Viruses 2015 7 (11) 5801-12 Nhumirim virus (NHUV) is an insect-specific virus that phylogenetically affiliates with dual-host mosquito-borne flaviviruses. Previous in vitro co-infection experiments demonstrated prior or concurrent infection of Aedes albopictus C6/36 mosquito cells with NHUV resulted in a 10,000-fold reduction in viral production of West Nile virus (WNV). This interference between WNV and NHUV was observed herein in an additional Ae. albopictus mosquito cell line, C7-10. A WNV 2K peptide (V9M) mutant capable of superinfection with a pre-established WNV infection demonstrated a comparable level of interference from NHUV as the parental WNV strain in C6/36 and C7-10 cells. Culex quinquefasciatus and Culex pipiens mosquitoes intrathoracically inoculated with NHUVandWNV, or solely withWNVas a control, were allowed to extrinsically incubate the viruses up to nine and 14 days, respectively, and transmissibility and replication of WNV was determined. The proportion of Cx. quinquefasciatus mosquitoes capable of transmitting WNV was significantly lower for the WNV/NHUV group than the WNV control at seven and nine days post inoculation (dpi), while no differences were observed in the Cx. pipiens inoculation group. By dpi nine, a 40% reduction in transmissibility in mosquitoes from the dual inoculation group was observed compared to the WNV-only control. These data indicate the potential that infection of some Culex spp. vectors with NHUV could serve as a barrier for efficient transmissibility of flaviviruses associated with human disease. |
Genotype-specific variation in West Nile virus dispersal in California.
Duggal NK , Reisen WK , Fang Y , Newman RM , Yang X , Ebel GD , Brault AC . Virology 2015 485 79-85 West Nile virus (WNV) is an arbovirus that was first reported in North America in New York in 1999 and, by 2003, had spread more than 4000km to California. However, variation in viral genetics associated with spread is not well understood. Herein, we report sequences for more than 100 WNV isolates made from mosquito pools that were collected from 2003 to 2011 as part of routine surveillance by the California Mosquito-borne Virus Surveillance System. We performed phylogeographic analyses and demonstrated that 5 independent introductions of WNV (1 WN02 genotype strain and 4 SW03 genotype strains) occurred in California. The SW03 genotype of WNV was constrained to the southwestern U.S. and had a more rapid rate of spread. In addition, geographic constraint of WNV strains within a single region for up to 6 years suggest viral maintenance has been driven by resident, rather than migratory, birds and overwintering in mosquitoes. |
Experimental evolution of an RNA virus in wild birds: evidence for host-dependent impacts on population structure and competitive fitness.
Grubaugh ND , Smith DR , Brackney DE , Bosco-Lauth AM , Fauver JR , Campbell CL , Felix TA , Romo H , Duggal NK , Dietrich EA , Eike T , Beane JE , Bowen RA , Black WC , Brault AC , Ebel GD . PLoS Pathog 2015 11 (5) e1004874 Within hosts, RNA viruses form populations that are genetically and phenotypically complex. Heterogeneity in RNA virus genomes arises due to error-prone replication and is reduced by stochastic and selective mechanisms that are incompletely understood. Defining how natural selection shapes RNA virus populations is critical because it can inform treatment paradigms and enhance control efforts. We allowed West Nile virus (WNV) to replicate in wild-caught American crows, house sparrows and American robins to assess how natural selection shapes RNA virus populations in ecologically relevant hosts that differ in susceptibility to virus-induced mortality. After five sequential passages in each bird species, we examined the phenotype and population diversity of WNV through fitness competition assays and next generation sequencing. We demonstrate that fitness gains occur in a species-specific manner, with the greatest replicative fitness gains in robin-passaged WNV and the least in WNV passaged in crows. Sequencing data revealed that intrahost WNV populations were strongly influenced by purifying selection and the overall complexity of the viral populations was similar among passaged hosts. However, the selective pressures that control WNV populations seem to be bird species-dependent. Specifically, crow-passaged WNV populations contained the most unique mutations (~1.7x more than sparrows, ~3.4x more than robins) and defective genomes (~1.4x greater than sparrows, ~2.7x greater than robins), but the lowest average mutation frequency (about equal to sparrows, ~2.6x lower than robins). Therefore, our data suggest that WNV replication in the most disease-susceptible bird species is positively associated with virus mutational tolerance, likely via complementation, and negatively associated with the strength of selection. These differences in genetic composition most likely have distinct phenotypic consequences for the virus populations. Taken together, these results reveal important insights into how different hosts may contribute to the emergence of RNA viruses. |
Evidence for co-evolution of West Nile Virus and house sparrows in North America
Duggal NK , Bosco-Lauth A , Bowen RA , Wheeler SS , Reisen WK , Felix TA , Mann BR , Romo H , Swetnam DM , Barrett AD , Brault AC . PLoS Negl Trop Dis 2014 8 (10) e3262 West Nile virus (WNV) has been maintained in North America in enzootic cycles between mosquitoes and birds since it was first described in North America in 1999. House sparrows (HOSPs; Passer domesticus) are a highly competent host for WNV that have contributed to the rapid spread of WNV across the U.S.; however, their competence has been evaluated primarily using an early WNV strain (NY99) that is no longer circulating. Herein, we report that the competence of wild HOSPs for the NY99 strain has decreased significantly over time, suggesting that HOSPs may have developed resistance to this early WNV strain. Moreover, recently isolated WNV strains generate higher peak viremias and mortality in contemporary HOSPs compared to NY99. These data indicate that opposing selective pressures in both the virus and avian host have resulted in a net increase in the level of host competence of North American HOSPs for currently circulating WNV strains. |
Host competence and helicase activity differences exhibited by West Nile viral variants expressing NS3-249 amino acid polymorphisms
Langevin SA , Bowen RA , Reisen WK , Andrade CC , Ramey WN , Maharaj PD , Anishchenko M , Kenney JL , Duggal NK , Romo H , Bera AK , Sanders TA , Bosco-Lauth A , Smith JL , Kuhn R , Brault AC . PLoS One 2014 9 (6) e100802 A single helicase amino acid substitution, NS3-T249P, has been shown to increase viremia magnitude/mortality in American crows (AMCRs) following West Nile virus (WNV) infection. Lineage/intra-lineage geographic variants exhibit consistent amino acid polymorphisms at this locus; however, the majority of WNV isolates associated with recent outbreaks reported worldwide have a proline at the NS3-249 residue. In order to evaluate the impact of NS3-249 variants on avian and mammalian virulence, multiple amino acid substitutions were engineered into a WNV infectious cDNA (NY99; NS3-249P) and the resulting viruses inoculated into AMCRs, house sparrows (HOSPs) and mice. Differential viremia profiles were observed between mutant viruses in the two bird species; however, the NS3-249P virus produced the highest mean peak viral loads in both avian models. In contrast, this avian modulating virulence determinant had no effect on LD50 or the neurovirulence phenotype in the murine model. Recombinant helicase proteins demonstrated variable helicase and ATPase activities; however, differences did not correlate with avian or murine viremia phenotypes. These in vitro and in vivo data indicate that avian-specific phenotypes are modulated by critical viral-host protein interactions involving the NS3-249 residue that directly influence transmission efficiency and therefore the magnitude of WNV epizootics in nature. |
Sequence analyses of 2012 West Nile virus isolates from Texas fail to associate viral genetic factors with outbreak magnitude.
Duggal NK , D'Anton M , Xiang J , Seiferth R , Day J , Nasci R , Brault AC . Am J Trop Med Hyg 2013 89 (2) 205-210 In 2012, Texas experienced the largest outbreak of human West Nile encephalitis (WNE) since the introduction of West Nile virus (WNV) in 2002. Despite the large number of WNV infections, data indicated the rate of reported WNE among human cases was no higher than in previous years. To determine whether the increase in WNV human cases could have been caused by viral genetic changes, the complete genomes of 17 isolates made from mosquito pools in Dallas and Montgomery Counties in 2012 were sequenced. The 2012 Texas isolates were found to be composed of two distinct clades, both circulating in Dallas and Montgomery Counties despite a 5-fold higher disease incidence in the former. Although minor genetic differences existed between Dallas and Montgomery WNV populations, there was weak support for population subdivision or adaptive changes. On the basis of these data, alternative explanations for increased WNV disease incidence in 2012 are proposed. |
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