Newly Recognized Spotted Fever Group Rickettsia as Cause of Severe Rocky Mountain Spotted Fever-Like Illness, Northern California, USA.

The incidence of spotted fever group (SFG) rickettsioses in the United States has tripled since 2010. Rocky Mountain spotted fever, the most severe SFG rickettsiosis, is caused by Rickettsia rickettsii. The lack of species-specific confirmatory testing obfuscates the relative contribution of R. rickettsii and other SFG Rickettsia to this increase. We report a newly recognized rickettsial pathogen, Rickettsia sp. CA6269, as the cause of severe Rocky Mountain spotted fever-like illness in 2 case-patients residing in northern California. Multilocus sequence typing supported the recognition of this pathogen as a novel Rickettsia genotype most closely related to R. rickettsii. Cross-reactivity observed for an established molecular diagnostic test indicated that Rickettsia sp. CA6269 might be misidentified as R. rickettsii. We developed a Rickettsia sp. CA6269-specific real-time PCR to help resolve this diagnostic challenge and better characterize the spectrum of clinical disease and ecologic epidemiology of this pathogen.

Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone.

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 that lack significant attenuation, 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 infection and transmission rates in Aedes aegypti mosquitoes comparable to those of 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-Barré 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 in vitro and in vivo characteristics similar to those of the primary isolate, providing a critical tool to study ZIKV infection and disease.

Experimental evolution of an RNA virus in wild birds: evidence for host-dependent impacts on population structure and competitive fitness.

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 (~1.7× more than sparrows, ~3.4× more than robins) and defective genomes (~1.4× greater than sparrows, ~2.7× greater than robins), but the lowest average mutation frequency (about equal to sparrows, ~2.6× 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.

Susceptibility of Carrion Crows to Experimental Infection with Lineage 1 and 2 West Nile Viruses.

West Nile virus (WNV) outbreaks in North America have been characterized by substantial die-offs of American crows (Corvus brachyrhynchos). In contrast, a low incidence of bird deaths has been observed during WNV epidemic activity in Europe. To examine the susceptibility of the western European counterpart of American crows, we inoculated carrion crows (Corvus corone) with WNV strains isolated in Greece (Gr-10), Italy (FIN and Ita09), and Hungary (578/10) and with the highly virulent North American genotype strain (NY99). We also inoculated American crows with a selection of these strains to examine the strains' virulence in a highly susceptible bird species. Infection with all strains, except WNV FIN, resulted in high rates of death and high-level viremia in both bird species and virus dissemination to several organs. These results suggest that carrion crows are highly susceptible to WNV and may potentially be useful as part of dead bird surveillance for early warning of WNV activity in Europe.

Identification of a motif in BMRP required for interaction with Bcl-2 by site-directed mutagenesis studies.

Bcl-2 is an anti-apoptotic protein that inhibits apoptosis elicited by multiple stimuli in a large variety of cell types. BMRP (also known as MRPL41) was identified as a Bcl-2 binding protein and shown to promote apoptosis. Previous studies indicated that the amino-terminal two-thirds of BMRP contain the domain(s) required for its interaction with Bcl-2, and that this region of the protein is responsible for the majority of the apoptosis-inducing activity of BMRP. We have performed site-directed mutagenesis analyses to further characterize the BMRP/Bcl-2 interaction and the pro-apoptotic activity of BMRP. The results obtained indicate that the 13-17 amino acid region of BMRP is necessary for its binding to Bcl-2. Further mutagenesis of this motif shows that amino acid residue aspartic acid (D) 16 of BMRP is essential for the BMRP/Bcl-2 interaction. Functional analyses conducted in mammalian cells with BMRP site-directed mutants BMRP(13Ala17) and BMRP(D16A) indicate that these mutants induce apoptosis through a caspase-mediated pathway, and that they kill cells slightly more potently than wild-type BMRP. Bcl-2 is still able to counteract BMRP(D16A)-induced cell death significantly, but not as completely as when tested against wild-type BMRP. These results suggest that the apoptosis-inducing ability of wild-type BMRP is blocked by Bcl-2 through several mechanisms.

Geographic distribution of hantaviruses associated with neotomine and sigmodontine rodents, Mexico.

To increase our knowledge of the geographic distribution of hantaviruses associated with neotomine or sigmodontine rodents in Mexico, we tested 876 cricetid rodents captured in 18 Mexican states (representing at least 44 species in the subfamily Neotominae and 10 species in the subfamily Sigmodontinae) for anti-hantavirus IgG. We found antibodies against hantavirus in 35 (4.0%) rodents. Nucleotide sequence data from 5 antibody-positive rodents indicated that Sin Nombre virus (the major cause of hantavirus pulmonary syndrome [HPS] in the United States) is enzootic in the Mexican states of Nuevo León, San Luis Potosí, Tamaulipas, and Veracruz. However, HPS has not been reported from these states, which suggests that in northeastern Mexico, HPS has been confused with other rapidly progressive, life-threatening respiratory diseases. Analyses of nucleotide sequence data from 19 other antibody-positive rodents indicated that El Moro Canyon virus and Limestone Canyon virus are geographically widely distributed in Mexico.

Deletion mutational analysis of BMRP, a pro-apoptotic protein that binds to Bcl-2.

Bcl-2 is an anti-apoptotic member of the Bcl-2 family of proteins that protects cells from apoptosis induced by a large variety of stimuli. The protein BMRP (MRPL41) was identified as a Bcl-2 binding partner and shown to have pro-apoptotic activity. We have performed deletion mutational analyses to identify the domain(s) of Bcl-2 and BMRP that are involved in the Bcl-2/BMRP interaction, and the region(s) of BMRP that mediate its pro-apoptotic activity. The results of these studies indicate that both the BH4 domain of Bcl-2 and its central region encompassing its BH1, BH2, and BH3 domains are required for its interaction with BMRP. The loop region and the transmembrane domain of Bcl-2 were found to be dispensable for this interaction. The Bcl-2 deletion mutants that do not interact with BMRP were previously shown to be functionally inactive. Deletion analyses of the BMRP protein delimited the region of BMRP needed for its interaction with Bcl-2 to the amino-terminal two-thirds of the protein (amino acid residues 1-92). Further deletions at either end of the BMRP(1-92) truncated protein resulted in lack of binding to Bcl-2. Functional studies performed with BMRP deletion mutants suggest that the cell death-inducing domains of the protein reside mainly within its amino-terminal two-thirds. The region of BMRP required for the interaction with Bcl-2 is very relevant for the cell death-inducing activity of the protein, suggesting that one possible mechanism by which BMRP induces cell death is by binding to and blocking the anti-apoptotic activity of Bcl-2.

Complex Oncological Decision-Making Utilizing Fast-and-Frugal Trees in a Community Setting-Role of Academic and Hybrid Modeling.

Non-small cell lung cancer is a devastating disease and with the advent of targeted therapies and molecular testing, the decision-making process has become complex. While established guidelines and pathways offer some guidance, they are difficult to utilize in a busy community practice and are not always implemented in the community. The rationale of the study was to identify a cohort of patients with lung adenocarcinoma at a City of Hope community site (n = 11) and utilize their case studies to develop a decision-making framework utilizing fast-and-frugal tree (FFT) heuristics. Most patients had stage IV (N = 9, 81.8%) disease at the time of the first consultation. The most common symptoms at initial presentation were cough (N = 5, 45.5%), shortness of breath (N = 3, 27.2%), and weight loss (N = 3, 27.2%). The Eastern Cooperative Oncology Group (ECOG) performance status ranged from 0-1 in all patients in this study. Distribution of molecular drivers among the patients were as follows: EGFR (N = 5, 45.5%), KRAS (N = 2, 18.2%), ALK (N = 2, 18.2%), MET (N = 2, 18.2%), and RET (N = 1, 9.1%). Seven initial FFTs were developed for the various case scenarios, but ultimately the decisions were condensed into one FFT, a molecular stage IV FFT, that arrived at accurate decisions without sacrificing initial information. While these FFT decision trees may seem arbitrary to an experienced oncologist at an academic site, the simplicity of their utility is essential for community practice where patients often do not get molecular testing and are not assigned proper therapy.

Mutations present in a low-passage Zika virus isolate result in attenuated pathogenesis in mice.

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.

Restriction of Zika virus infection and transmission in Aedes aegypti mediated by an insect-specific flavivirus.

Previous studies demonstrated an insect-specific flavivirus, Nhumirim virus (NHUV), can suppress growth of West Nile virus (WNV) and decrease transmission rates in NHUV/WNV co-inoculated Culex quinquefasciatus. To assess whether NHUV might interfere with transmission of other medically important flaviviruses, the ability of NHUV to suppress viral growth of Zika virus (ZIKV) and dengue-2 virus (DENV-2) was assessed in Aedes albopictus cells. Significant reductions in ZIKV (100,000-fold) and DENV-2 (10,000-fold) were observed in either cells concurrently inoculated with NHUV or pre-inoculated with NHUV. In contrast, only a transient 10-fold titer reduction was observed with an alphavirus, chikungunya virus. Additionally, restricted in vitro mosquito growth of ZIKV was associated with lowered levels of intracellular ZIKV RNA in NHUV co-inoculated cultures. To assess whether NHUV could modulate vector competence for ZIKV, NHUV-inoculated Aedes aegypti were orally exposed to ZIKV. NHUV-inoculated mosquitoes demonstrated significantly lower ZIKV infection rates (18%) compared to NHUV unexposed mosquitoes (51%) (p < 0.002). Similarly, lower ZIKV transmission rates were observed for NHUV/ZIKV dually intrathoracically inoculated mosquitoes (41%) compared to ZIKV only inoculated mosquitoes (78%) (p < 0.0001), suggesting that NHUV can interfere with both midgut infection and salivary gland infection of ZIKV in Ae. aegypti. These results indicate NHUV could be utilized to model superinfection exclusion mechanism(s) and to study the potential for the mosquito virome to impact transmission of medically important flaviviruses.

Comparative Vector Competence of North American Culex pipiens and Culex quinquefasciatus for African and European Lineage 2 West Nile Viruses.

West Nile virus (WNV) is a mosquito-borne flavivirus that is phylogenetically separated into distinct lineages. Lineage 1 (L1) and lineage 2 (L2) encompass all WNV isolates associated with human and veterinary disease cases. Although L1 WNV is globally distributed, including North America, L2 WNV only recently emerged out of sub-Saharan Africa into Europe and Russia. The spread of L2 WNV throughout and beyond Europe depends, in part, on availability of competent vectors. The vector competence of mosquitoes within the Culex genus for WNV is well established for L1 WNV but less extensively studied for L2 WNV. Assessing the vector competence of North American Culex mosquitoes for L2 WNV will be critical for predicting the potential for L2 WNV emergence in North America. We address the vector competence of North American Culex pipiens and Culex quinquefasciatus for L2 WNV. Both mosquito species were highly competent for each of the L2 WNV strains assessed, but variation in infection, dissemination, and transmission was observed. An L2 WNV strain (NS10) isolated during the Greek outbreak in 2010 exhibited a reduced capacity to infect Cx. pipiens compared with other L2 WNV strains. In addition, a South African L2 WNV strain (SA89) displayed a significantly shorter extrinsic incubation period in Cx. quinquefasciatus compared with other L2 WNV strains. These results demonstrate that North American Culex mosquito species are competent vectors of African and European L2 WNV and that emergence of L2 WNV is unlikely to be hindered by poor competence of North American vectors.

Generation of a Lineage II Powassan Virus (Deer Tick Virus) cDNA Clone: Assessment of Flaviviral Genetic Determinants of Tick and Mosquito Vector Competence.

The Flavivirus genus comprises a diverse group of viruses that utilize a wide range of vertebrate hosts and arthropod vectors. The genus includes viruses that are transmitted solely by mosquitoes or vertebrate hosts as well as viruses that alternate transmission between mosquitoes or ticks and vertebrates. Nevertheless, the viral genetic determinants that dictate these unique flaviviral host and vector specificities have been poorly characterized. In this report, a cDNA clone of a flavivirus that is transmitted between ticks and vertebrates (Powassan lineage II, deer tick virus [DTV]) was generated and chimeric viruses between the mosquito/vertebrate flavivirus, West Nile virus (WNV), were constructed. These chimeric viruses expressed the prM and E genes of either WNV or DTV in the heterologous nonstructural (NS) backbone. Recombinant chimeric viruses rescued from cDNAs were characterized for their capacity to grow in vertebrate and arthropod (mosquito and tick) cells as well as for in vivo vector competence in mosquitoes and ticks. Results demonstrated that the NS elements were insufficient to impart the complete mosquito or tick growth phenotypes of parental viruses; however, these NS genetic elements did contribute to a 100- and 100,000-fold increase in viral growth in vitro in tick and mosquito cells, respectively. Mosquito competence was observed only with parental WNV, while infection and transmission potential by ticks were observed with both DTV and WNV-prME/DTV chimeric viruses. These data indicate that NS genetic elements play a significant, but not exclusive, role for vector usage of mosquito- and tick-borne flaviviruses.

Differential Neurovirulence of African and Asian Genotype Zika Virus Isolates in Outbred Immunocompetent Mice.

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-Barré 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.

Transmission Incompetence of Culex quinquefasciatus and Culex pipiens pipiens from North America for Zika Virus.

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.

Evidence for co-evolution of West Nile Virus and house sparrows in North America.

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.

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.

zRICH, a protein induced during optic nerve regeneration in zebrafish, promotes neuritogenesis and interacts with tubulin.

Mammals do not regenerate axons in their central nervous system (CNS) spontaneously. This phenomenon is the cause of numerous medical conditions after damage to nerve fibers in the CNS of humans. The study of the mechanisms of nerve regeneration in other vertebrate animals able to spontaneously regenerate axons in their CNS is essential for understanding nerve regeneration from a scientific point of view, and for developing therapeutic approaches to enhance nerve regeneration in the CNS of humans. RICH proteins are a novel group of proteins implicated in nerve regeneration in the CNS of teleost fish, yet their mechanisms of action are not well understood. A number of mutant versions of the zebrafish RICH (zRICH) protein were generated and characterized at biochemical and cellular levels in our laboratory. With the aim of understanding the effects of RICH proteins in neuronal axon outgrowth, stable transfectants derived from the neuronal model PC12 cell line expressing zRICH Wild-Type or mutant versions of zRICH were studied. Results from differentiation experiments suggest that RICH proteins enhance neuronal plasticity by facilitating neurite branching. Biochemical co-purification results have demonstrated that zRICH binds to the cytoskeletal protein tubulin. The central domain of the protein is sufficient for tubulin binding, but a mutant version of the protein lacking the terminal domains, which cannot bind to the plasma membrane, was not able to enhance neurite branching. RICH proteins may facilitate axon regeneration by regulating the axonal cytoskeleton and facilitating the formation of new neurite branches.