Approaches to demonstrating the effectiveness of filovirus vaccines: Lessons from Ebola and COVID-19.

Zaire ebolavirus (EBOV), Sudan ebolavirus (SUDV) and Marburg virus (MARV), are members of the Filoviridae family that can cause severe disease and death in humans and animals. The reemergence of Ebola, Sudan and Marburg virus disease highlight the need for continued availability of safe and effectives vaccines as well as development of new vaccines. While randomized controlled trials using disease endpoints provide the most robust assessment of vaccine effectiveness, challenges to this approach include the unpredictable size, location, occurrence and duration of filovirus disease outbreaks. Thus, other approaches to demonstrating vaccine effectiveness have been considered. These approaches are discussed using examples of preventive vaccines against other infectious diseases. In addition, this article proposes a clinical immunobridging strategy using licensed EBOV vaccines as comparators for demonstrating the effectiveness of filovirus vaccine candidates that are based on the same licensed vaccine platform technology.

Multiple Biliary Stones: a Rare Long-term Complication after Whipple Procedure / Múltiplos cálculos biliares: uma complicação rara a longo prazo após o procedimento de Whipple

A hypertensive, diabetic woman underwent a successful Whipple procedure at the age of 84 due to carcinoma of the ampulla of Vater. She presented an extremely rare complication 24 months after the surgery, consisting of acute cholangitis due to multiple biliary lithiases associated with a bilioenteric anastomotic stricture. The diagnosis was confirmed with computed tomography, magnetic resonance cholangiopancreatography, and cholangiography. The patient was successfully treated with multiple percutaneous transhepatic cholangioplasties (AU)

Uma mulher hipertensa e diabética foi submetida a um procedimento bem-sucedido de Whipple aos 84 anos devido a um carcinoma da ampola de Vater e apresentou uma complicação extremamente rara 24 meses após da cirurgia, consistindo em colangite aguda devido à presença de litíase biliar múltipla associada com estenose da anastomose bilio-entérica. O diagnóstico foi confirmado com tomografia computadorizada, colangiopancreatografia por ressonância mag-nética e colangiografia.O paciente foi tratado com sucesso com múltiplas colangioplastias transhepáticas percutâneas (AU)

A Single Point Mutation in the Mumps V Protein Alters Targeting of the Cellular STAT Pathways Resulting in Virus Attenuation.

Mumps virus (MuV) is a neurotropic non-segmented, negative-stranded, enveloped RNA virus in the Paramyxovirus family. The 15.4 kb genome encodes seven genes, including the V/P, which encodes, among other proteins, the V protein. The MuV V protein has been shown to target the cellular signal transducer and activator of transcription proteins STAT1 and STAT3 for proteasome-mediated degradation. While MuV V protein targeting of STAT1 is generally accepted as a means of limiting innate antiviral responses, the consequence of V protein targeting of STAT3 is less clear. Further, since the MuV V protein targets both STAT1 and STAT3, specifically investigating viral antagonism of STAT3 targeting is challenging. However, a previous study reported that a single amino acid substitution in the MuV V protein (E95D) inhibits targeting of STAT3, but not STAT1. This provided us with a unique opportunity to examine the specific role of STAT 3 in MuV virulence in an in vivo model. Here, using a clone of a wild type MuV strain expressing the E95D mutant V protein, we present data linking inhibition of STAT3 targeting with the accelerated clearance of the virus and reduced neurovirulence in vivo, suggesting its role in promoting antiviral responses. These data suggest a rational approach to virus attenuation that could be exploited for future vaccine development.

Identification and quantification of defective virus genomes in high throughput sequencing data using DVG-profiler, a novel post-sequence alignment processing algorithm.

Most viruses are known to spontaneously generate defective viral genomes (DVG) due to errors during replication. These DVGs are subgenomic and contain deletions that render them unable to complete a full replication cycle in the absence of a co-infecting, non-defective helper virus. DVGs, especially of the copyback type, frequently observed with paramyxoviruses, have been recognized to be important triggers of the antiviral innate immune response. DVGs have therefore gained interest for their potential to alter the attenuation and immunogenicity of vaccines. To investigate this potential, accurate identification and quantification of DVGs is essential. Conventional methods, such as RT-PCR, are labor intensive and will only detect primer sequence-specific species. High throughput sequencing (HTS) is much better suited for this undertaking. Here, we present an HTS-based algorithm called DVG-profiler to identify and quantify all DVG sequences in an HTS data set generated from a virus preparation. DVG-profiler identifies DVG breakpoints relative to a reference genome and reports the directionality of each segment from within the same read. The specificity and sensitivity of the algorithm was assessed using both in silico data sets as well as HTS data obtained from parainfluenza virus 5, Sendai virus and mumps virus preparations. HTS data from the latter were also compared with conventional RT-PCR data and with data obtained using an alternative algorithm. The data presented here demonstrate the high specificity, sensitivity, and robustness of DVG-profiler. This algorithm was implemented within an open source cloud-based computing environment for analyzing HTS data. DVG-profiler might prove valuable not only in basic virus research but also in monitoring live attenuated vaccines for DVG content and to assure vaccine lot to lot consistency.

Differential durability of immune responses to measles and mumps following MMR vaccination.

The development and wide-spread use of mumps vaccine resulted in a dramatic and sustained decrease in the incidence of mumps disease; however, since 2000, an increase in the size and number of mumps outbreaks in the United States and other countries has sparked renewed interest in the durability of mumps-specific immunity elicited by mumps vaccination. The most likely explanation for mumps cases in previously immunized persons may be secondary vaccine failure, or waning immunity. In the current study, we examined changes in markers of measles and mumps immunity at two timepoints, approximately 7 and 17 years after two-dose MMR-II® vaccination, in a cohort of 98 healthy adults. Our results indicate that mumps IgG titers exhibited a large and significant decline during this time period, while mumps neutralizing Ab titers were relatively stable. There was a similar discrepancy with measles-specific immune responses. For both pathogens, neutralizing antibody titers were fairly low and, given the length of time since vaccination, may have already declined. These data suggest that specific immune outcomes may wane at different rates and highlight our currently incomplete understanding of protective immune responses to mumps and measles.

Entry, Replication, Immune Evasion, and Neurotoxicity of Synthetically Engineered Bat-Borne Mumps Virus.

Bats harbor a plethora of viruses with an unknown zoonotic potential. In-depth functional characterization of such viruses is often hampered by a lack of virus isolates. The genome of a virus closely related to human mumps viruses (hMuV) was detected in African fruit bats, batMuV. Efforts to characterize batMuV were based on directed expression of the batMuV glycoproteins or use of recombinant chimeric hMuVs harboring batMuV glycoprotein. Although these studies provided initial insights into the functionality of batMuV glycoproteins, the host range, replication competence, immunomodulatory functions, virulence, and zoonotic potential of batMuV remained elusive. Here, we report the successful rescue of recombinant batMuV. BatMuV infects human cells, is largely resistant to the host interferon response, blocks interferon induction and TNF-α activation, and is neurotoxic in rats. Anti-hMuV antibodies efficiently neutralize batMuV. The striking similarities between hMuV and batMuV point at the putative zoonotic potential of batMuV.

Hereditary diffuse gastric cancer: One family's story.

Hereditary diffuse gastric cancer (HDGC) is an inherited form of gastric cancer that carries a poor prognosis. Most HDGCs are caused by an autosomal dominant genetic mutation in the CDH1 gene, which carries a 70%-80% lifetime risk of gastric cancer. Given its submucosal origin, endoscopic surveillance is an unreliable means of early detection, and prophylactic gastrectomy is recommended for CDH1 positive individuals older than age 20 years. We describe the case of a male with recurrent gastric cancer who was diagnosed with HDGC secondary to the CDH1 mutation, and we also describe the patient's pedigree and outcomes of recommended genetic testing.

Mumps Virus Nucleoprotein and Hemagglutinin-Specific Antibody Response Following a Third Dose of Measles Mumps Rubella Vaccine.

BACKGROUND: Recent mumps outbreaks among 2-dose measles mumps rubella (MMR) vaccine recipients have raised questions regarding the potential benefits of a third dose of vaccine (MMR3). If MMR3 provides a sustained elevation in mumps antibody, it may be beneficial for certain at-risk groups or as an outbreak control measure. METHODS: Sera were collected immediately prior to MMR3 and at 1 month and 1 year post-MMR3 from 656 healthy adults aged 18-28 years in a nonoutbreak setting. Immunoglobulin G (IgG) was measured by enzyme-linked immunosorbent assay (ELISA) using whole mumps virus (commercial ELISA), hemagglutinin (HN; major neutralizing target), and nucleoprotein (NP; immunodominant) antigens. ELISA measurements were compared with in vitro plaque reduction neutralization (PRN) titers, and baseline antibody was compared with post-MMR3 levels. RESULTS: There were modest but statistically significant (P < .05) increases in mumps antibody at 1 month post-MMR3 by all 3 ELISA methods and by PRN titer. At 1 year post-MMR3, mumps antibody declined toward baseline but remained elevated (P < .05). The correlation between PRN titers and ELISA measurements was poor (r2 = .49), although sera with the highest amount of HN IgG also had the highest PRN titers. CONCLUSIONS: Individuals with the lowest baseline PRN titers had the largest increase in frequency of samples that became positive for HN and NP by ELISA. A third dose of MMR may benefit certain individuals with a low level of mumps virus-neutralizing antibody, especially in the context of an outbreak or other high-risk setting. Additionally, poor correlation among serologic tests does not allow effective prediction of PRN titer by ELISA.

Mechanisms of temperature sensitivity of attenuated Urabe mumps virus.

Temperature sensitivity is a phenotype often associated with attenuation of viruses. Previously, we purified several mumps variants from an incompletely attenuated Urabe strain live attenuated vaccine. Here we characterize one isolate that is sensitive to growth at high temperature. This virus was attenuated in a small animal model of mumps virulence, and we identified unique coding substitutions in the hemagglutinin-neuraminidase (HN), the viral polymerase (L) gene, and a non-coding substitution close to the anti-genome promoter sequences. At the non-permissive temperature, transcription of viral mRNAs and production of the replication intermediate were reduced compared to events at the permissive temperature and to a non-ts virulent Urabe virus. As well, synthesis of viral proteins was also reduced at the higher temperature. While the actual sequence substitutions in the ts virus were unique, the pattern of substitutions in HN, L and genome end sequences is similar to another attenuated Urabe virus previously described by us.

Recombinant mumps viruses expressing the batMuV fusion glycoprotein are highly fusion active and neurovirulent.

A recent study reported the detection of a bat-derived virus (BatPV/Epo_spe/AR1/DCR/2009, batMuV) with phylogenetic relatedness to human mumps virus (hMuV). Since all efforts to isolate infectious batMuV have reportedly failed, we generated recombinant mumps viruses (rMuVs) in which the open reading frames (ORFs) of the fusion (F) and haemagglutinin-neuraminidase (HN) glycoproteins of an hMuV strain were replaced by the corresponding ORFs of batMuV. The batMuV F and HN proteins were successfully incorporated into viral particles and the resultant chimeric virus was able to mediate infection of Vero cells. Distinct differences were observed between the fusogenicity of rMuVs expressing one or both batMuV glycoproteins: viruses expressing batMuV F were highly fusogenic, regardless of the origin of HN. In contrast, rMuVs expressing human F and bat-derived HN proteins were less fusogenic compared to hMuV. The growth kinetics of chimeric MuVs expressing batMuV HN in combination with either hMuV or batMuV F were similar to that of the backbone virus, whereas a delay in virus replication was obtained for rMuVs harbouring batMuV F and hMuV HN. Replacement of the hMuV F and HN genes or the HN gene alone by the corresponding batMuV genes led to a slight reduction in neurovirulence of the highly neurovirulent backbone strain. Neutralizing antibodies inhibited infection mediated by all recombinant viruses generated. Furthermore, group IV anti-MuV antibodies inhibited the neuraminidase activity of bat-derived HN. Our study reports the successful generation of chimeric MuVs expressing the F and HN proteins of batMuV, providing a means for further examination of this novel batMuV.

Evidence that a polyhexameric genome length is preferred, but not strictly required, for efficient mumps virus replication.

Mumps virus (MuV) is postulated to adhere to the "rule of six" for efficient replication. To examine the requirement for MuV, minigenomes of nonpolyhexameric length (6n-1 and 6n+1) were analyzed. Expression of the reporter gene CAT was significantly reduced with minigenomes of nonpolyhexameric length compared to the wild type 6n genome, and reduction was more pronounced for the 6n-1 than for the 6n+1 minigenome. That 6n-1 genomes are impacted by nonconformance with the rule of six to a greater degree as compared to 6n+1 genomes was also suggested with MuV derived from cDNA coding for 6n+1 or 6n-1 genomes. While viruses recovered from 6n+1 cDNAs maintained a nonpolyhexameric genome length over multiple replication cycles, viruses rescued from the 6n-1 cDNAs acquired length correcting mutations rapidly following rescue. Our data indicate that polyhexameric genomes are the preferred template for the MuV RNA polymerase, but that this requirement is not absolute.

Purification of mumps virus particles of high viability.

Mumps is a highly infectious viral disease of humans with a wide array of clinical manifestations ranging from painful swelling of the salivary glands to meningitis and encephalitis. Despite the clinical importance of mumps virus, most of what is known of its biological properties comes from studies using supernatants from virus infected cell cultures, which contain substantial levels of host cell derived debris and biologically active substances such as cytokines, transcription factors and secreted virus proteins. These contaminants complicate interpretation of studies of virus replication, virus-host interactions and in vivo virulence. Here we describe a protocol for concentration of the virus from cell culture supernatants followed by gradient purification, resulting in attainment of high titer live virus of high purity.

Esotropia in Children with Ventricular-Peritoneal Shunts.

PURPOSE: Compared with the general population, patients with hydrocephalus are more likely to have strabismus. This study was undertaken to examine characteristics and outcomes of children with esotropia and ventricular-peritoneal shunt placement due to hydrocephalus. METHODS: This is a retrospective chart review of all pediatric patients with esotropia and a history of ventricular-peritoneal shunt placement seen by our pediatric ophthalmology service between January 2000 and December 2010. RESULTS: Sixteen patients between the age of 3 months and 5.6 years met study criteria. Nine were premature and all but one of the patients had developmental delay. Although all patients had a ventricular-peritoneal shunt, the diagnosis leading to shunt placement was intraventricular hemorrhage or congenital hydrocephalus in 75% of the patients. In all but 3 patients the hydrocephalus was diagnosed before the esotropia. Ten children had congenital esotropia and 6 had acquired esotropia. Eleven of the 16 children required glasses: 5 had a myopic prescription and 6 had a hyperopic prescription. Treatment of the esotropia resulted in 9 patients (56%) with successful ocular alignment (<10 prism diopters) on their last visit: 7 underwent strabismus surgery and 2 were treated with glasses only. Of the 9 patients who had strabismus surgery, 6 had congenital esotropia and 3 had acquired esotropia. Among patients who underwent strabismus surgery, 78% had successful ocular alignment at their last visit. CONCLUSIONS: While acquired accommodative esotropia is more common in the general population, children with ventricular-peritoneal shunts may be more likely to have congenital esotropia. Although developmental delay is very frequent, successful ocular alignment may be possible in this patient population.

Generation and propagation of recombinant mumps viruses exhibiting an additional U residue in the homopolymeric U tract of the F gene-end signal.

As a member of the family paramyxoviridae, subfamily paramyxovirinae, the genome of mumps virus (MuV) is postulated to be polyhexameric in length in order to be able to replicate efficiently. While all natural MuV strains sequenced so far obey to this "rule of six," we describe here the isolation of recombinant MuVs that appeared to contain an additional U residue in the homopolymeric tract of the F gene-end signal, resulting in a genome length of 6n + 1. Sequencing of several plaque-purified viruses from these preparations did not reveal the existence of length-correcting mutations, suggesting that they are violators of the rule of six. Employing high-throughput sequencing technology, we provide evidence that the insertion of an additional U residue is mainly the result of the rescue system used that relies on T7 RNA polymerase. Limited in vitro and in vivo testing of the viruses did not reveal any significant impact of the longer genome on virus replication or virulence, suggesting that the rule of six is not a strict requirement for MuV replication.

A phase II evaluation of the potent, highly selective PARP inhibitor veliparib in the treatment of persistent or recurrent epithelial ovarian, fallopian tube, or primary peritoneal cancer in patients who carry a germline BRCA1 or BRCA2 mutation - An NRG Oncology/Gynecologic Oncology Group study.

BACKGROUND: Veliparib is a potent small molecule inhibitor of PARP-1/2, which is cytotoxic in tumor cells with deficiencies in BRCA1 or BRCA2. We studied the clinical activity and toxicity of veliparib in ovarian cancer patients carrying a germline BRCA1 or BRCA2 mutation (gBRCA). METHODS: Eligibility included three or fewer prior chemotherapy regimens, measurable disease and no prior use of a PARP inhibitor. Veliparib was administered at 400mg orally BID with one cycle being 28days. The two-stage Simon design was capable of detecting a 25% response probability with 90% power while controlling alpha=10% (at a 10% assumed null response probability). RESULTS: The median age of the 50 eligible patients was 57years (range 37-94) and 14, 18, and 18 patients had 1, 2, and 3 prior therapies respectively. Thirty patients (60%) were platinum-resistant. The median number of cycles administered was 6 (1-27). There was one grade 4 thrombocytopenia. Grade 3 adverse events were: fatigue (n=3), nausea (2), leukopenia (1), neutropenia (1), dehydration (1), and ALT (1). Grade 2 events >10% were: nausea (46%), fatigue (26%), vomiting (18%), and anemia (14%). The proportion responding was 26% (90% CI: 16%-38%, CR: 2, PR: 11); for platinum-resistant and platinum-sensitive patients the proportion responding was 20% and 35%, respectively. The most common reason for treatment discontinuation was progression (62%). Twenty-nine patients are alive; two with SD remain on veliparib. The median PFS is 8.18months. CONCLUSIONS: The single agent efficacy and tolerability of veliparib for BRCA mutation-associated recurrent ovarian cancer warrants further investigation.

Functional properties and genetic relatedness of the fusion and hemagglutinin-neuraminidase proteins of a mumps virus-like bat virus.

UNLABELLED: A bat virus with high phylogenetic relatedness to human mumps virus (MuV) was identified recently at the nucleic acid level. We analyzed the functional activities of the hemagglutinin-neuraminidase (HN) and the fusion (F) proteins of the bat virus (batMuV) and compared them to the respective proteins of a human isolate. Transfected cells expressing the F and HN proteins of batMuV were recognized by antibodies directed against these proteins of human MuV, indicating that both viruses are serologically related. Fusion, hemadsorption, and neuraminidase activities were demonstrated for batMuV, and either bat-derived protein could substitute for its human MuV counterpart in inducing syncytium formation when coexpressed in different mammalian cell lines, including chiropteran cells. Cells expressing batMuV glycoproteins were shown to have lower neuraminidase activity. The syncytia were smaller, and they were present in lower numbers than those observed after coexpression of the corresponding glycoproteins of a clinical isolate of MuV (hMuV). The phenotypic differences in the neuraminidase and fusion activity between the glycoproteins of batMuV and hMuV are explained by differences in the expression level of the HN and F proteins of the two viruses. In the case of the F protein, analysis of chimeric proteins revealed that the signal peptide of the bat MuV fusion protein is responsible for the lower surface expression. These results indicate that the surface glycoproteins of batMuV are serologically and functionally related to those of hMuV, raising the possibility of bats as a reservoir for interspecies transmission. IMPORTANCE: The recently described MuV-like bat virus is unique among other recently identified human-like bat-associated viruses because of its high sequence homology (approximately 90% in most genes) to its human counterpart. Although it is not known if humans can be infected by batMuV, the antigenic relatedness between the bat and human forms of the virus suggests that humans carrying neutralizing antibodies against MuV are protected from infection by batMuV. The close functional relationship between MuV and batMuV is demonstrated by cooperation of the respective HN and F proteins to induce syncytium formation in heterologous expression studies. An interesting feature of the glycoproteins of batMuV is the downregulation of the fusion activity by the signal peptide of F, which has not been reported for other paramyxoviruses. These results are important contributions for risk assessment and for a better understanding of the replication strategy of batMuV.

Molecular biology, pathogenesis and pathology of mumps virus.

Mumps is caused by the mumps virus (MuV), a member of the Paramyxoviridae family of enveloped, non-segmented, negative-sense RNA viruses. Mumps is characterized by painful inflammatory symptoms, such as parotitis and orchitis. The virus is highly neurotropic, with laboratory evidence of central nervous system (CNS) infection in approximately half of cases. Symptomatic CNS infection occurs less frequently; nonetheless, prior to the introduction of routine vaccination, MuV was a leading cause of aseptic meningitis and viral encephalitis in many developed countries. Despite being one of the oldest recognized diseases, with a worldwide distribution, surprisingly little attention has been given to its study. Cases of aseptic meningitis associated with some vaccine strains and a global resurgence of cases, including in highly vaccinated populations, has renewed interest in the virus, particularly in its pathogenesis and the need for development of clinically relevant models of disease. In this review we summarize the current state of knowledge on the virus, its pathogenesis and its clinical and pathological outcomes.

Cardiopulmonary arrest following administration of Cyclomydril eyedrops for outpatient retinopathy of prematurity screening.

Eyedrops used for mydriasis and cycloplegia can be systemically absorbed, causing serious side effects, including oxygen desaturation, apnea, bradycardia, transient hypertension, delayed gastric emptying, and transient paralytic ileus. These effects can be more serious in infants because of their lower body mass and immature cardiovascular and nervous systems. We report a case of a 27-week-old infant who suffered a cardiopulmonary arrest after the administration of only Cyclomydril eyedrops (Alcon Laboratories, Fort Worth, TX) during an outpatient retinopathy of prematurity examination.