The genomic origin of Zana of Abkhazia.

Enigmatic phenomena have sparked the imagination of people around the globe into creating folkloric creatures. One prime example is Zana of Abkhazia (South Caucasus), a well-documented 19th century female who was captured living wild in the forest. Zana's appearance was sufficiently unusual, that she was referred to by locals as an Almasty-the analog of Bigfoot in the Caucasus. Although the exact location of Zana's burial site was unknown, the grave of her son, Khwit, was identified in 1971. The genomes of Khwit and the alleged Zana skeleton were sequenced to an average depth of ca. 3× using ancient DNA techniques. The identical mtDNA and parent-offspring relationship between the two indicated that the unknown woman was indeed Zana. Population genomic analyses demonstrated that Zana's immediate genetic ancestry can likely be traced to present-day East-African populations. We speculate that Zana might have had a genetic disorder such as congenital generalized hypertrichosis which could partially explain her strange behavior, lack of speech, and long body hair. Our findings elucidate Zana's unfortunate story and provide a clear example of how prejudices of the time led to notions of cryptic hominids that are still held and transmitted by some today.

Expression of flavivirus capsids enhance the cellular environment for viral replication by activating Akt-signalling pathways.

Flaviviruses depend on multiple host pathways during their life cycles and have evolved strategies to avoid the innate immune response. Previously, we showed that the West Nile virus capsid protein plays a role in this process by blocking apoptosis. In this study, we examined how expression of capsid proteins from several flaviviruses affects apoptosis and other host processes that impact virus replication. All of the tested capsid proteins protected cells from Fas-dependent apoptosis through a mechanism that requires activated Akt. Capsid expression upregulated other Akt-dependent cellular processes including expression of glucose transporter 1 and mitochondrial metabolism. Protein phosphatase 1, which is known to inactivate Akt, was identified as a DENV capsid interacting protein. This suggests that DENV capsid expression activates Akt by sequestering phosphatases that downregulate phospho-Akt. Capsid-dependent upregulation of Akt would enhance downstream signalling pathways that affect cell survival and metabolism, thus providing a favourable environment for virus replication.

Development of a human live attenuated West Nile infectious DNA vaccine: Identification of a minimal mutation set conferring the attenuation level acceptable for a human vaccine.

For the development of a human West Nile (WN) infectious DNA (iDNA) vaccine, we created highly attenuated chimeric virus W1806 with the serological identity of highly virulent WN-NY99. Earlier, we attempted to utilize mutations found in the E protein of the SA14-14-2 vaccine to bring safety of W1806 to the level acceptable for human use (Yamshchikov et al., 2016). Here, we analyzed effects of the SA14-14-2 changes on growth properties and neurovirulence of W1806. A set including the E138K, K279M, K439R and G447D changes was identified as the perspective subset for satisfying the target safety profile without compromising immunogenicity of the vaccine candidate. The genetic stability of the attenuated phenotype was found to be unsatisfactory being dependent on a subset of attenuating changes incorporated in W1806. Elucidation of underlying mechanisms influencing selection of pathways for restoration of the envelope protein functionality will facilitate resolution of the emerged genetic stability issue.

Development of a human live attenuated West Nile infectious DNA vaccine: Suitability of attenuating mutations found in SA14-14-2 for WN vaccine design.

Direct attenuation of West Nile (WN) virus strain NY99 for the purpose of vaccine development is not feasible due to its high virulence and pathogenicity. Instead, we created highly attenuated chimeric virus W1806 with the serological identity of NY99. To further attenuate W1806, we investigated effects of mutations found in Japanese encephalitis virus vaccine SA14-14-2. WN viruses carrying all attenuating mutations lost infectivity in mammalian, but not in mosquito cells. No single reversion restored infectivity in mammalian cells, although increased infectivity in mosquito cells was observed. To identify a subset of mutations suitable for further attenuation of W1806, we analyzed effects of E138K and K279M changes on virulence, growth properties, and immunogenicity of derivatized W956, from which chimeric W1806 inherited its biological properties and attenuation profile. Despite strong dominant attenuating effect, introduction of only two mutations was not sufficient for attenuating W1806 to the safety level acceptable for human use.

Development of a human live attenuated West Nile infectious DNA vaccine: conceptual design of the vaccine candidate.

West Nile virus has become an important epidemiological problem attracting significant attention of health authorities, mass media, and the public. Although there are promising advancements toward addressing the vaccine need, the perspectives of the commercial availability of the vaccine remain uncertain. To a large extent this is due to lack of a sustained interest for further commercial development of the vaccines already undergoing the preclinical and clinical development, and a predicted insignificant cost effectiveness of mass vaccination. There is a need for a safe, efficacious and cost effective vaccine, which can improve the feasibility of a targeted vaccination program. In the present report, we summarize the background, the rationale, and the choice of the development pathway that we selected for the design of a live attenuated human West Nile vaccine in a novel infectious DNA format.

Course of seasonal influenza A/Brisbane/59/07 H1N1 infection in the ferret.

Every year, influenza viruses infect approximately 5-20% of the population in the United States leading to over 200,000 hospitalizations and 36,000 deaths from flu-related complications. In this study, we characterized the immune and pathological progression of a seasonal strain of H1N1 influenza virus, A/Brisbane/59/2007 in a ferret model. The immune response of the animals showed a dose-dependent increase with increased virus challenge, as indicated by the presence of virus specific IgG, IgM, and neutralizing antibodies. Animals infected with higher doses of virus also experienced increasing severity of clinical symptoms and fever at 2 days post-infection (DPI). Interestingly, weight loss was more pronounced in animals infected with lower doses of virus compared to those infected with a higher dose; these results were consistent with viral titers of swabs collected from the nares, but not the throat. Analyzed specimens included nasal and throat swabs from 1, 3, 5, and 7 DPI as well as tissue samples from caudal lung and nasal turbinates. Viral titers of the swab samples in all groups were higher on 1 and 3 DPI and returned to baseline levels by 7 DPI. Analysis of nasal turbinates indicated presence of virus at 3 DPI in all infected groups, whereas virus was only detected in the lungs of animals in the two highest dose groups. Histological analysis of the lungs showed a range of pathology, such as chronic inflammation and bronchial epithelial hypertrophy. The results provided here offer important endpoints for preclinical testing of the efficacy of new antiviral compounds and experimental vaccines.

Genetically delivered antibody protects against West Nile virus.

Gene-based delivery of recombinant antibody genes is a promising therapeutic strategy offering numerous advantages including sustained antibody levels, better safety profile and lower production cost. Here we describe generation of a recombinant antibody Fc-9E2 comprising a fusion protein between human Fc of IgG1 and a single-chain Fv derived from a hybridoma 9E2 secreting a mAb neutralizing West Nile virus (WNV). Fc-9E2 was shown to retain parental mAb's specificity and WNV-neutralizing capacity. Adenovirus-mediated in vivo delivery of the antibody gene resulted in sustained Fc-9E2 serum levels leading to abrogation of lethal WNV infection in an animal model.

Immunogenicity of West Nile virus infectious DNA and its noninfectious derivatives.

The exceptionally high virulence of the West Nile NY99 strain makes its suitability in the development of a live WN vaccine uncertain. The aim of this study is to investigate the immunogenicity of noninfectious virus derivatives carrying pseudolethal mutations, which preclude virion formation without affecting preceding steps of the viral infectious cycle. When administered using DNA immunization, such constructs initiate an infectious cycle but cannot lead to a viremia. While the magnitude of the immune response to a noninfectious replication-competent construct was lower than that of virus or infectious DNA, its overall quality and the protective effect were similar. In contrast, a nonreplicating construct of similar length induced only a marginally detectable immune response in the dose range used. Thus, replication-competent noninfectious constructs derived from infectious DNA may offer an advantageous combination of the safety of noninfectious formulations with the quality of the immune response characteristic of infectious vaccines.

Biological properties of chimeric West Nile viruses.

Recently, we have described a lineage 2 attenuated WN virus suitable for the development of a live WN vaccine. To design vaccine candidates with an improved immunogenicity, we assembled an infectious clone of the NY99 strain and created several chimeric constructs with reciprocal exchanges of structural protein genes between attenuated W956 and virulent NY99 and investigated their biological properties. Our data indicated that, while the growth rates of NY99 and chimeric viruses in tissue culture are determined primarily by properties of the structural proteins, determinants responsible for a highly cytopathic phenotype of NY99 or lack thereof for W956 are located within the nonstructural protein region of the WN genome. The high virulence of NY99 and the attenuated phenotype of W956 were found to be associated with determinants in the nonstructural region. Chimeric viruses carrying the NY99 structural proteins were attenuated in neuroinvasiveness and demonstrated an immunogenicity superior to W956.

The suitability of yellow fever and Japanese encephalitis vaccines for immunization against West Nile virus.

Seven volunteers involved in flavivirus studies have been immunized with commercial Japanese encephalitis and yellow fever vaccines JE-VAX and YF-VAX. Strong homologous and cross-reactive with West Nile virus (WNV) antibody responses with titers 1:1600 to 1:51200 were found in all donors. All donors developed high levels of yellow fever virus (YFV) and Japanese encephalitis virus (JEV) neutralizing antibodies with titers 1:50 to 1:1600 and 1:20 to 1:640, respectively, and WNV neutralizing antibodies with titers 1:10 to 1:80. In contrast, predominantly YF-specific cell-mediated immunity was detected in all immunized donors. Responses to YFV were long lasting, but the anti-JEV humoral immunity was found to decrease with time. Cross-reactive anti-WNV responses were following the same trend dropping below detectable level at 4 years post-immunization and sharply coming back after booster immunization with the JE vaccine. Thus, immunization with the commercial flavivirus JE vaccine may be beneficial for individuals at high risk of exposure to WNV, such as personnel involved in WN research.

Error-prone replication of West Nile virus caused by ribavirin.

Ribavirin has been reported to cause error-prone replication and viral extinction in RNA viruses. The antiviral activity of ribavirin against West Nile virus (WNV) was evaluated in various cell lines to select a model in which mutagenic effects could be studied. The antiviral activity was greatest in HeLa cells as compared to CV-1, L929, Vero, or MA-104 cells. WNV was also passaged sequentially in cell monolayers treated with ribavirin to determine whether cumulative mutations could lead to viral extinction in these cell lines. The virus was abrogated in HeLa cells after 4 passages, while high viral titers persisted after many passages in other cells. A molecular clone of WNV was propagated in HeLa cells treated with 15 microg/mL ribavirin, and sequencing of viral genome segments revealed significant increases in transition mutations, demonstrating that ribavirin induced error-prone replication. The relative infectivity of viral RNA synthesized in the presence of ribavirin was shown to be reduced compared with untreated controls. These data support the hypothesis that error catastrophe is one of the modes of action for ribavirin against WNV.

An attenuated West Nile prototype virus is highly immunogenic and protects against the deadly NY99 strain: a candidate for live WN vaccine development.

In a short time, West Nile virus has developed into a nationwide health and veterinary problem. The high virulence of the circulating virus and related lineage 1 WN strains hinders development of an attenuated live vaccine. We describe an attenuated WN isolate, WN1415, which is a molecularly cloned descendant of the WN prototype B956 strain. The parent virus belongs to lineage 2, members of which have not been associated with epidemic or epizootic outbreaks. A set of non-conservative mutations, mostly in non-structural protein genes, distinguishes the WN1415 isolate from the parent B956 prototype strain. Immunization with WN1415 (55-550,000 pfu) established a potent immunity, which protected the majority of mice against lethal challenge with WN NY99. The attenuated nature of the isolate and its excellent growth characteristics combined with the availability of a highly stable infectious clone make the isolate an attractive candidate for live WN vaccine development.

Differential contribution of IL-1Ra isoforms to allele-specific IL-1Ra mRNA accumulation.

The interleukin-1 (IL-1) receptor antagonist (IL-1Ra) gene produces two isoforms of IL-1Ra, intracellular (icIL-1Ra) and secreted (sIL-1Ra). Distinct promoter regions control synthesis of each isoform. Five alleles of this gene, defined by sIL-1Ra intron 2 polymorphism, have been described. Although differences in IL-1Ra protein production have been demonstrated in various tissues and cells obtained from individuals carrying allele 1 vs. allele 2, the underlying mechanisms of this discrepancy remain poorly understood. We hypothesize that one mechanism contributing to differences in protein levels may be allele-specific accumulation of icIL-1Ra or sIL-1Ra mRNA. Quantification of allele-specific differences in mRNA accumulation in colonic biopsies and peripheral blood mononuclear cells (PBMC) of heterozygous individuals shows that the amount of allele 1-specific icIL-1Ra mRNA averaged four times higher relative to allele 2. In transfection assays, gene expression directed by the allele 1-specific icIL-1Ra promoter fragment was found to be greater than that directed by the allele 2 promoter, suggesting that icIL-1Ra promoter activity contributes to the disparity in the allele-specific icIL-1Ra mRNA accumulation. Our data show that differences in the transcriptional regulation of icIL-1Ra alleles 1 and 2 may be involved in the production of icIL-1Ra protein. Disregulated icIL-1Ra production may play a role in chronic inflammatory diseases where the balance between IL-1 and IL-1Ra has been implicated as a key pathogenic mechanism.

A possible role of IL-1ra 3'-untranslated region in modulation of protein production.

Human interleukin 1 (IL-1) receptor antagonist (hIL-1ra), an anti-inflammatory cytokine and naturally occurring antagonist of IL-1, may be regulated at both the transcriptional and post-transcriptional levels. The aim of this study was to evaluate post-transcriptional regulation of hIL-1ra with specific focus on the 3'-untranslated region (3'-UTR) of hIL-1ra mRNA using the luciferase reporter gene system. Constructs were created containing the luciferase reporter gene followed by the hIL-1ra 3'-UTR or its modified variants. In monocyte/macrophage cell lines RAW264.7 and U937, the presence of the hIL-1ra 3'-UTR resulted in a 5.7-fold (n=6, P<0.001) and a 3.9-fold (n=7, P<0.001) decrease in transient reporter gene expression, respectively, with only a less than 2-fold mean difference in steady-state mRNA levels in the former case. In a cell-free translation system, the presence of the middle segment of the 3'-UTR caused a 5.2-fold (n=5, P<0.001) decrease in the amount of luciferase synthesized. In contrast to synthetic 3'-UTR and that derived from bovine growth hormone RNA, the presence of hIL-1ra 3'-UTR resulted in accumulation of unprocessed transcripts in transfected cells. We conclude that hIL-1ra synthesis may be regulated at the post-transcriptional level through mechanisms involving the 3'-UTR of IL-1ra transcripts.