Generalized Modules for Membrane Antigens (GMMA), an outer membrane vesicle-based vaccine platform, for efficient viral antigen delivery.

Vaccine platforms enable fast development, testing, and manufacture of more affordable vaccines. Here, we evaluated Generalized Modules for Membrane Antigens (GMMA), outer membrane vesicles (OMVs) generated by genetically modified Gram-negative bacteria, as a vaccine platform for viral pathogens. Influenza A virus hemagglutinin (HA), either physically mixed with GMMA (HA+STmGMMA mix), or covalently linked to GMMA surface (HA-STmGMMA conjugate), significantly increased antigen-specific humoral and cellular responses, with HA-STmGMMA conjugate inducing further enhancement than HA+STmGMMA mix. HA-STmGMMA conjugate protected mice from lethal challenge. The versatility for this platform was confirmed by conjugation of rabies glycoprotein (RABVG) onto GMMA through the same method. RABVG+STmGMMA mix and RABVG-STmGMMA conjugate exhibited similar humoral and cellular response patterns and protection efficacy as the HA formulations, indicating relatively consistent responses for different vaccines based on the GMMA platform. Comparing to soluble protein, GMMA was more efficiently taken up in vivo and exhibited a B-cell preferential uptake in the draining lymph nodes (LNs). Together, GMMA enhances immunity against viral antigens, and the platform works well with different antigens while retaining similar immunomodulatory patterns. The findings of our study imply the great potential of GMMA-based vaccine platform also against viral infectious diseases.

SARS-CoV-2 infection produces chronic pulmonary epithelial and immune cell dysfunction with fibrosis in mice.

A subset of individuals who recover from coronavirus disease 2019 (COVID-19) develop post-acute sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (PASC), but the mechanistic basis of PASC-associated lung abnormalities suffers from a lack of longitudinal tissue samples. The mouse-adapted SARS-CoV-2 strain MA10 produces an acute respiratory distress syndrome in mice similar to humans. To investigate PASC pathogenesis, studies of MA10-infected mice were extended from acute to clinical recovery phases. At 15 to 120 days after virus clearance, pulmonary histologic findings included subpleural lesions composed of collagen, proliferative fibroblasts, and chronic inflammation, including tertiary lymphoid structures. Longitudinal spatial transcriptional profiling identified global reparative and fibrotic pathways dysregulated in diseased regions, similar to human COVID-19. Populations of alveolar intermediate cells, coupled with focal up-regulation of profibrotic markers, were identified in persistently diseased regions. Early intervention with antiviral EIDD-2801 reduced chronic disease, and early antifibrotic agent (nintedanib) intervention modified early disease severity. This murine model provides opportunities to identify pathways associated with persistent SARS-CoV-2 pulmonary disease and test countermeasures to ameliorate PASC.

Prevalence and Mechanisms of Mucus Accumulation in COVID-19 Lung Disease.

Rationale: The incidence and sites of mucus accumulation and molecular regulation of mucin gene expression in coronavirus (COVID-19) lung disease have not been reported. Objectives: To characterize the incidence of mucus accumulation and the mechanisms mediating mucin hypersecretion in COVID-19 lung disease. Methods: Airway mucus and mucins were evaluated in COVID-19 autopsy lungs by Alcian blue and periodic acid-Schiff staining, immunohistochemical staining, RNA in situ hybridization, and spatial transcriptional profiling. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected human bronchial epithelial (HBE) cultures were used to investigate mechanisms of SARS-CoV-2-induced mucin expression and synthesis and test candidate countermeasures. Measurements and Main Results: MUC5B and variably MUC5AC RNA concentrations were increased throughout all airway regions of COVID-19 autopsy lungs, notably in the subacute/chronic disease phase after SARS-CoV-2 clearance. In the distal lung, MUC5B-dominated mucus plugging was observed in 90% of subjects with COVID-19 in both morphologically identified bronchioles and microcysts, and MUC5B accumulated in damaged alveolar spaces. SARS-CoV-2-infected HBE cultures exhibited peak titers 3 days after inoculation, whereas induction of MUC5B/MUC5AC peaked 7-14 days after inoculation. SARS-CoV-2 infection of HBE cultures induced expression of epidermal growth factor receptor (EGFR) ligands and inflammatory cytokines (e.g., IL-1α/ß) associated with mucin gene regulation. Inhibiting EGFR/IL-1R pathways or administration of dexamethasone reduced SARS-CoV-2-induced mucin expression. Conclusions: SARS-CoV-2 infection is associated with a high prevalence of distal airspace mucus accumulation and increased MUC5B expression in COVID-19 autopsy lungs. HBE culture studies identified roles for EGFR and IL-1R signaling in mucin gene regulation after SARS-CoV-2 infection. These data suggest that time-sensitive mucolytic agents, specific pathway inhibitors, or corticosteroid administration may be therapeutic for COVID-19 lung disease.

Protease-anti-protease compartmentalization in SARS-CoV-2 ARDS: Therapeutic implications.

BACKGROUND: Interleukin-6 (IL-6) is elevated in SARS-CoV-2 infection. IL-6 regulates acute-phase proteins, such as alpha-1 antitrypsin (AAT), a key lung anti-protease. We investigated the protease-anti-protease balance in the circulation and pulmonary compartments in SARS-CoV-2 acute respiratory distress syndrome (ARDS) compared to non-SARS-CoV-2 ARDS (nsARDS) and the effects of tocilizumab (IL-6 receptor antagonist) on anti-protease defence in SARS-CoV-2 infection. METHODS: Levels and activity of AAT and neutrophil elastase (NE) were measured in plasma, airway tissue and tracheal secretions (TA) of people with SARS-CoV-2 ARDS or nsARDS. AAT and IL-6 levels were evaluated in people with moderate SARS-CoV-2 infection who received standard of care +/- tocilizumab. FINDINGS: AAT plasma levels doubled in SARS-CoV-2 ARDS. In lung parenchyma AAT levels were increased, as was the percentage of neutrophils involved in NET formation. A protease-anti-protease imbalance was detected in TA with active NE and no active AAT. The airway anti-protease, secretory leukoprotease inhibitor was decreased in SARS-CoV-2-infected lungs and cleaved in TA. In nsARDS, plasma AAT levels were elevated but TA samples had less AAT cleavage, with no detectable active NE in most samples. Induction of AAT in ARDS occurred mainly through IL-6. Tocilizumab down-regulated AAT during SARS-CoV-2 infection. INTERPRETATION: There is a protease-anti-protease imbalance in the airways of SARS-CoV-2-ARDS patients. This imbalance is a target for anti-protease therapy. FUNDING: NIH Serological Sciences Network, National Heart, Lung, and Blood Institute and National Institute of Diabetes and Digestive and Kidney Diseases.

A model of persistent post SARS-CoV-2 induced lung disease for target identification and testing of therapeutic strategies.

COVID-19 survivors develop post-acute sequelae of SARS-CoV-2 (PASC), but the mechanistic basis of PASC-associated lung abnormalities suffers from a lack of longitudinal samples. Mouse-adapted SARS-CoV-2 MA10 produces an acute respiratory distress syndrome (ARDS) in mice similar to humans. To investigate PASC pathogenesis, studies of MA10-infected mice were extended from acute disease through clinical recovery. At 15-120 days post-virus clearance, histologic evaluation identified subpleural lesions containing collagen, proliferative fibroblasts, and chronic inflammation with tertiary lymphoid structures. Longitudinal spatial transcriptional profiling identified global reparative and fibrotic pathways dysregulated in diseased regions, similar to human COVID-19. Populations of alveolar intermediate cells, coupled with focal upregulation of pro-fibrotic markers, were identified in persistently diseased regions. Early intervention with antiviral EIDD-2801 reduced chronic disease, and early anti-fibrotic agent (nintedanib) intervention modified early disease severity. This murine model provides opportunities to identify pathways associated with persistent SARS-CoV-2 pulmonary disease and test countermeasures to ameliorate PASC.

Rapid idiosyncratic mechanisms of clinical resistance to KRAS G12C inhibition.

BACKGROUNDThe KRAS proto-oncogene is among the most frequently mutated genes in cancer, yet for 40 years it remained an elusive therapeutic target. Recently, allosteric inhibitors that covalently bind to KRAS G12C mutations have been approved for use in lung adenocarcinomas. Although responses are observed, they are often short-lived, thus making in-depth characterization of the mechanisms of resistance of paramount importance.METHODSHere, we present a rapid-autopsy case of a patient who had a KRASG12C-mutant lung adenocarcinoma who initially responded to a KRAS G12C inhibitor but then rapidly developed resistance. Using deep-RNA and whole-exome sequencing comparing pretreatment, posttreatment, and matched normal tissues, we uncover numerous mechanisms of resistance to direct KRAS inhibition.RESULTSIn addition to decreased KRAS G12C-mutant allele frequency in refractory tumors, we also found reactivation of the MAPK pathway despite no new mutations in KRAS or its downstream mediators. Tumor cell-intrinsic and non-cell autonomous mechanisms included increased complement activation, coagulation, and tumor angiogenesis, and several lines of evidence of immunologic evasion.CONCLUSIONTogether, our findings reveal numerous mechanisms of resistance to current KRAS G12C inhibitors through enrichment of clonal populations, KRAS-independent downstream signaling, and diverse remodeling of the tumor microenvironment.FUNDINGRichard and Fran Duley, Jimmy and Kay Mann, the NIH, and the North Carolina Biotechnology Center.

Fatal Pediatric COVID-19 Case With Seizures and Fulminant Cerebral Edema.

The novel coronavirus, SARS-CoV-2, can present with a wide range of neurological manifestations, in both adult and pediatric populations. We describe here the case of a previously healthy 8-year-old girl who presented with seizures, encephalopathy, and rapidly progressive, diffuse, and ultimately fatal cerebral edema in the setting of acute COVID-19 infection. CSF analysis, microbiological testing, and neuropathology yielded no evidence of infection or acute inflammation within the central nervous system. Acute fulminant cerebral edema (AFCE) is an often fatal pediatric clinical entity consisting of fever, encephalopathy, and new-onset seizures followed by rapid, diffuse, and medically-refractory cerebral edema. AFCE occurs as a rare complication of a variety of common pediatric infections and a CNS pathogen is identified in only a minority of cases, suggesting a para-infectious mechanism of edema. This report suggests that COVID-19 infection can precipitate AFCE, and highlights the need for high suspicion and early recognition thereof.

Decline in circulating viral and human tumor markers after resection of head and neck carcinoma.

BACKGROUND: DNA sequencing panels can simultaneously quantify human and viral tumor markers in blood. We explored changes in levels of plasma tumor markers following surgical resection of head and neck carcinoma. METHODS: In preresection and postresection plasmas, targeted DNA sequencing quantified variants in 28 human cancer genes and levels of oncogenic pathogens (human papillomavirus [HPV], Epstein-Barr virus [EBV], Helicobacter pylori) from 21 patients with head and neck squamous cell carcinoma. RESULTS: Preresection, 11 of 21 patients (52%) had detectable tumor markers in plasma, most commonly TP53 mutation or HPV genome. Several days postresection, levels fell to undetectable in 8 of 10 evaluable patients, while two high-stage patients retained circulating tumor markers. CONCLUSIONS: Modern sequencing technology can simultaneously quantify human gene variants and oncogenic viral genomes in plasma. Falling levels of cancer-specific markers upon resection can help identify viral and human markers to track at subsequent timepoints as a means to evaluate efficacy of interventions.

Transmissible spongiform encephalopathy in goats: is PrP rapid test sensitivity affected by genotype?

Transmissible spongiform encephalopathy (TSE) surveillance in goats relies on tests initially approved for cattle, subsequently assessed for sheep, and approval extrapolated for use in "small ruminants." The current EU-approved immunodetection tests employ antibodies against various epitopes of the prion protein PrPSc, which is encoded by the host PRNP gene. The caprine PRNP gene is polymorphic, mostly at codons different from the ovine PRNP. The EU goat population is much more heterogeneous than the sheep population, with more PRNP-related polymorphisms, and with marked breed-related differences. The ability of the current tests to detect disease-specific PrPSc generated against these different genetic backgrounds is currently assumed, rather than proven. We examined whether common polymorphisms within the goat PRNP gene might have any adverse effect on the relative performance of EU-approved rapid tests. The sample panel comprised goats from the UK, Cyprus, France, and Italy, with either experimental or naturally acquired scrapie at both the preclinical and/or unknown and clinical stages of disease. Test sensitivity was significantly lower and more variable when compared using samples from animals that were preclinical or of unknown status. However, all of the rapid tests included in our study were able to correctly identify all samples from animals in the clinical stages of disease, apart from samples from animals polymorphic for serine or aspartic acid at codon 146, in which the performance of the Bio-Rad tests was profoundly affected. Our data show that some polymorphisms may adversely affect one test and not another, as well as underline the dangers of extrapolating from other species.

Evidence for infection but not transmission of Zika virus by Aedes albopictus (Diptera: Culicidae) from Spain.

BACKGROUND: A number of mosquito-borne viruses such as dengue virus (DENV), Usutu virus (USUV), West Nile virus (WNV) are autochthonously transmitted in Europe and six invasive mosquito species have been detected in this temperate region. This has increased the risk for the emergence of further mosquito-borne diseases. However, there is a paucity of information on whether European populations of invasive mosquito species are competent to transmit arboviruses. In this study, the susceptibility of Aedes albopictus originating from Spain and a laboratory-adapted colony of Aedes aegypti, was assessed for infection with, and transmission of Zika virus (ZIKV). Vertical transmission in both species was also assessed. METHODS: Aedes albopictus colonised from eggs collected in Spain and an existing colony of Ae. aegypti were fed infectious blood meals containing ZIKV (Polynesian strain) at 1.6 × 107 PFU/ml. Blood-fed mosquitoes were separated and maintained at 20 °C or 25 °C. Legs, saliva and bodies were sampled from specimens at 7, 14 and 21 days post-infection (dpi) in order to determine infection, dissemination and transmission rates. All samples were analysed by real-time RT-PCR using primers targeting the ZIKV NS1 gene. RESULTS: At 14 dpi and 21 dpi, ZIKV RNA was detected in the bodies of both species at both temperatures. However, live virus only was detected in the saliva of Ae. aegypti at 25 °C with a transmission rate of 44%. No evidence for virus expectoration was obtained for Ae. albopictus under any condition. Notably, ZIKV RNA was not detectable in the saliva of Ae. aegypti at 20 °C after 21 days. No vertical transmission of ZIKV was detected in this study. CONCLUSIONS: Experimental infection of Ae. albopictus colonized from Spain with ZIKV did not result in expectoration of virus in saliva in contrast to results for Ae. aegypti. No evidence of vertical transmission of virus was observed in this study. This suggests that this strain of Ae. albopictus is not competent for ZIKV transmission under the conditions tested.

Incomplete inactivation of atypical scrapie following recommended autoclave decontamination procedures.

Prions are highly resistant to the decontamination procedures normally used to inactivate conventional pathogens. This is a challenging problem not only in the medical and veterinary fields for minimizing the risk of transmission from potentially infective sources but also for ensuring the safe disposal or subsequent use of animal by-products. Specific pressure autoclaving protocols were developed for this purpose, but different strains of prions have been reported to have differing resistance patterns to established prion decontamination procedures, and as additional TSE strains are identified it is necessary to determine the effectiveness of such procedures. In this study we assessed the efficacy of sterilization using the EU recommended autoclave procedure for prions (133°C, 3 Bar for 20 min) on the atypical or Nor98 (AS/Nor98) scrapie strain of sheep and goats. Using a highly sensitive murine mouse model (tg338) that overexpresses ovine PrPC , we determined that this method of decontamination reduced the infectivity titre by 1010 . Infectivity was nonetheless still detected after applying the recommended autoclaving protocol. This shows that AS/Nor98 can survive the designated legislative decontamination conditions, albeit with a significant decrease in titre. The infectivity of a classical scrapie isolate subjected to the same decontamination conditions was reduced by 106 suggesting that the AS/Nor98 isolate is less sensitive to decontamination than the classical scrapie source.

DNA barcoding of British mosquitoes (Diptera, Culicidae) to support species identification, discovery of cryptic genetic diversity and monitoring invasive species.

Correct mosquito species identification is essential for mosquito and disease control programs. However, this is complicated by the difficulties in morphologically identifying some mosquito species. In this study, variation of a partial sequence of the cytochrome c oxidase unit I (COI) gene was used for the molecular identification of British mosquito species and to facilitate the discovery of cryptic diversity, and monitoring invasive species. Three DNA extraction methods were compared to obtain DNA barcodes from adult specimens. In total, we analyzed 42 species belonging to the genera Aedes Meigen, 1818 (21 species), Anopheles Meigen, 1818 (7 species), Coquillettidia Theobald, 1904 (1 species), Culex Linnaeus, 1758 (6 species), Culiseta Felt, 1904 (7 species), and Orthopodomyia Theobald, 1904 (1 species). Intraspecific genetic divergence ranged from 0% to 5.4%, while higher interspecific divergences were identified between Aedesgeminus Peus, 1971/Culisetalitorea (Shute, 1928) (24.6%) and Ae.geminus/An.plumbeus Stephens, 1828 (22.5%). Taxonomic discrepancy was shown between An.daciae Linton, Nicolescu & Harbach, 2004 and An.messeae Falleroni, 1828 indicating the poor resolution of the COI DNA barcoding region in separating these taxa. Other species such as Ae.cantans (Meigen, 1818)/Ae.annulipes (Meigen, 1830) showed similar discrepancies indicating some limitation of this genetic marker to identify certain mosquito species. The combination of morphology and DNA barcoding is an effective approach for the identification of British mosquitoes, for invasive mosquitoes posing a threat to the UK, and for the detection of hidden diversity within species groups.

Re-evaluating the effect of Favipiravir treatment on rabies virus infection.

There is no antiviral treatment available once clinical disease following rabies virus infection has initiated. Considered a neglected tropical disease, >60,000 human rabies deaths are estimated each year despite the availability of pre- and post-exposure prophylaxis for pre-immunisation or administration following a potential exposure before the onset of clinical disease. Such post-exposure treatments include administration of rabies immunoglobulin (RIG) and vaccination at a distant site to prime a humoral immune response. However, current therapeutic options are limited. Regardless there is a need for molecules that target virus infection following the onset of clinical disease where the outcome of infection is invariably fatal. Numerous molecules have been assessed as potential antivirals against rabies virus (RABV) but with little promise. Favipiravir (T-705) is a broad-spectrum RNA polymerase inhibitor, which has been shown to have antiviral activity against a range of RNA viruses including some against RABV. In the present study, the utility of T-705 has been reassessed in vitro as well as in vivo in a murine model using intraperitoneal administration to investigate any immune protective effect of the molecule. In vitro T-705 effectively reduces RABV replication. However, in vivo, following assessment of various applications of the molecule in both pre- and post-exposure scenarios, the effect was limited. T-705 treatment delayed the onset of clinical signs when virus was delivered intramuscularly at a higher dose (106.8 TCID50/ml) and reduced the number of mice that developed clinical signs when virus was delivered at a lower dose (105.8 TCID50/ml) during the observation period. The day at which treatment commenced did not appear to have a statistically significant effect on the results in either experiment. The use of T-705 as a single biological entity may be limited, however, further work is required to assess the synergistic effect of T-705 as a component of a multi-drug therapy for treating human rabies infections.

Molecular characterisation of atypical BSE prions by mass spectrometry and changes following transmission to sheep and transgenic mouse models.

The prion hypothesis proposes a causal relationship between the misfolded prion protein (PrPSc) molecular entity and the disease transmissible spongiform encephalopathy (TSE). Variations in the conformation of PrPSc are associated with different forms of TSE and different risks to animal and human health. Since the discovery of atypical forms of bovine spongiform encephalopathy (BSE) in 2003, scientists have progressed the molecular characterisation of the associated PrPSc in order to better understand these risks, both in cattle as the natural host and following experimental transmission to other species. Here we report the development of a mass spectrometry based assay for molecular characterisation of bovine proteinase K (PK) treated PrPSc (PrPres) by quantitative identification of its N-terminal amino acid profiles (N-TAAPs) and tryptic peptides. We have applied the assay to classical, H-type and L-type BSE prions purified from cattle, transgenic (Tg) mice expressing the bovine (Tg110 and Tg1896) or ovine (TgEM16) prion protein gene, and sheep brain. We determined that, for classical BSE in cattle, the G96 N-terminal cleavage site dominated, while the range of cleavage sites was wider following transmission to Tg mice and sheep. For L-BSE in cattle and Tg bovinised mice, a C-terminal shift was identified in the N-TAAP distribution compared to classical BSE, consistent with observations by Western blot (WB). For L-BSE transmitted to sheep, both N-TAAP and tryptic peptide profiles were found to be changed compared to cattle, but less so following transmission to Tg ovinised mice. Relative abundances of aglycosyl peptides were found to be significantly different between the atypical BSE forms in cattle as well as in other hosts. The enhanced resolution provided by molecular analysis of PrPres using mass spectrometry has improved insight into the molecular changes following transmission of atypical BSE to other species.

Assessment of vector competence of UK mosquitoes for Usutu virus of African origin.

BACKGROUND: Usutu virus (USUV) is an emerging zoonotic virus originally from sub-Saharan Africa. It has been introduced into Europe on multiple occasions, causing substantial mortality within the Eurasian blackbird (Turdus merula) population. It is transmitted by the mosquito species Culex pipiens in Europe and Africa. Vector competence studies indicate that European strains of USUV are readily transmitted by indigenous Cx. pipiens. However, there is limited information on the ability of an African strain to infect European mosquitoes. METHODS: We evaluated the ability of African strain SAAR-1776 to infect two lines of Cx. pipiens colonised within the United Kingdom (UK). Mosquitoes were fed blood meals containing this virus and maintained at 25 °C for up to 21 days. Individual mosquitoes were tested for the presence of virus in the body, legs and an expectorate saliva sample. Changes to the consensus of the virus genome were monitored in samples derived from infected mosquitoes using amplicon based next generation sequencing. RESULTS: Infection, dissemination and the presence of virus in saliva in one mosquito line was observed, but no evidence for dissemination in the second mosquito line. This suggests a strong barrier to infection in UK Cx. pipiens for this strain of USUV. When comparing the genome of input virus within the blood meal with USUV recovered from an infected mosquito, we observed limited changes in the consensus genome sequence. CONCLUSIONS: The evaluation of vector competence of UK populations of Cx. pipiens for Usutu virus suggests a limited susceptibility to infection with USUV strain SAAR-1776 of African origin. However, within a single mosquito there was complete dissemination and expectoration of USUV, indicating that infection, and potentially transmission, is possible. Sequence changes were observed that may represent early adaption to the mosquito host and could reflect the early events of USUV establishment in European mosquito populations.

Isolation, antigenicity and immunogenicity of Lleida bat lyssavirus.

The lyssaviruses are an important group of viruses that cause a fatal encephalitis termed rabies. The prototypic lyssavirus, rabies virus, is predicted to cause more than 60 000 human fatalities annually. The burden of disease for the other lyssaviruses is undefined. The original reports for the recently described highly divergent Lleida bat lyssavirus were based on the detection of virus sequence alone. The successful isolation of live Lleida bat lyssavirus from the carcass of the original bat and in vitro characterization of this novel lyssavirus are described here. In addition, the ability of a human rabies vaccine to confer protective immunity following challenge with this divergent lyssavirus was assessed. Two different doses of Lleida bat lyssavirus were used to challenge vaccinated or naïve mice: a high dose of 100 focus-forming units (f.f.u.) 30 µl-1 and a 100-fold dilution of this dose, 1 f.f.u. 30 µl-1. Although all naïve control mice succumbed to the 100 f.f.u. 30 µl-1 challenge, 42 % (n=5/12) of those infected intracerebrally with 1 f.f.u. 30 µl-1 survived the challenge. In the high-challenge-dose group, 42 % of the vaccinated mice survived the challenge (n=5/12), whilst at the lower challenge dose, 33 % (n=4/12) survived to the end of the experiment. Interestingly, a high proportion of mice demonstrated a measurable virus-neutralizing antibody response, demonstrating that neutralizing antibody titres do not necessarily correlate with the outcome of infection via the intracerebral route. Assessing the ability of existing rabies vaccines to protect against novel divergent lyssaviruses is important for the development of future public health strategies.

Utilisation of Chimeric Lyssaviruses to Assess Vaccine Protection against Highly Divergent Lyssaviruses.

Lyssaviruses constitute a diverse range of viruses with the ability to cause fatal encephalitis known as rabies. Existing human rabies vaccines and post exposure prophylaxes (PEP) are based on inactivated preparations of, and neutralising antibody preparations directed against, classical rabies viruses, respectively. Whilst these prophylaxes are highly efficient at neutralising and preventing a productive infection with rabies virus, their ability to neutralise other lyssaviruses is thought to be limited. The remaining 15 virus species within the lyssavirus genus have been divided into at least three phylogroups that generally predict vaccine protection. Existing rabies vaccines afford protection against phylogroup I viruses but offer little to no protection against phylogroup II and III viruses. As such, work involving sharps with phylogroup II and III must be considered of high risk as no PEP is thought to have any effect on the prevention of a productive infection with these lyssaviruses. Whilst rabies virus itself has been characterised in a number of different animal models, data on the remaining lyssaviruses are scarce. As the lyssavirus glycoprotein is considered to be the sole target of neutralising antibodies we generated a vaccine strain of rabies using reverse genetics expressing highly divergent glycoproteins of West Caucasian Bat lyssavirus and Ikoma lyssavirus. Using these recombinants, we propose that recombinant vaccine strain derived lyssaviruses containing heterologous glycoproteins may be a suitable surrogate for wildtype viruses when assessing vaccine protection for the lyssaviruses.

Correction: Molecular Subtypes in Head and Neck Cancer Exhibit Distinct Patterns of Chromosomal Gain and Loss of Canonical Cancer Genes.

[This corrects the article DOI: 10.1371/journal.pone.0056823.].

Integrated RNA and DNA sequencing reveals early drivers of metastatic breast cancer.

Breast cancer metastasis remains a clinical challenge, even within a single patient across multiple sites of the disease. Genome-wide comparisons of both the DNA and gene expression of primary tumors and metastases in multiple patients could help elucidate the underlying mechanisms that cause breast cancer metastasis. To address this issue, we performed DNA exome and RNA sequencing of matched primary tumors and multiple metastases from 16 patients, totaling 83 distinct specimens. We identified tumor-specific drivers by integrating known protein-protein network information with RNA expression and somatic DNA alterations and found that genetic drivers were predominantly established in the primary tumor and maintained through metastatic spreading. In addition, our analyses revealed that most genetic drivers were DNA copy number changes, the TP53 mutation was a recurrent founding mutation regardless of subtype, and that multiclonal seeding of metastases was frequent and occurred in multiple subtypes. Genetic drivers unique to metastasis were identified as somatic mutations in the estrogen and androgen receptor genes. These results highlight the complexity of metastatic spreading, be it monoclonal or multiclonal, and suggest that most metastatic drivers are established in the primary tumor, despite the substantial heterogeneity seen in the metastases.