Principles and practical applications of structure-based vaccine design.

Viral proteins fold into a variety of structures as they perform their functions. Structure-based vaccine design aims to exploit knowledge of an antigen's architecture to stabilize it in a vulnerable conformation. We summarize the general principles of structure-based vaccine design, with a focus on the major types of sequence modifications: proline, disulfide, cavity-filling, electrostatic and hydrogen-bond substitution, as well as domain deletion. We then review recent applications of these principles to vaccine-design efforts across five viral families: Coronaviridae, Orthomyxoviridae, Paramyxoviridae, Pneumoviridae, and Filoviridae. Outstanding challenges include continued application of proven design principles to pathogens of interest, as well as development of new strategies for those pathogens that resist traditional techniques.

Phosphatidylinositol-3-kinase-Akt pathway in negative-stranded RNA virus infection: a minireview.

The PI3K/Akt signalling pathway is a crucial signalling cascade that regulates transcription, protein translation, cell growth, proliferation, cell survival, and metabolism. During viral infection, viruses exploit a variety of cellular pathways, including the well-known PI3K/Akt signalling pathway. Conversely, cells rely on this pathway to stimulate an antiviral response. The PI3K/Akt pathway is manipulated by a number of viruses, including DNA and RNA viruses and retroviruses. The aim of this review is to provide up-to-date information about the role of the PI3K-Akt pathway in infection with members of five different families of negative-sense ssRNA viruses. This pathway is hijacked for viral entry, regulation of endocytosis, suppression of premature apoptosis, viral protein expression, and replication. Although less common, the PI3K/Akt pathway can be downregulated as an immunomodulatory strategy or as a mechanism for inducing autophagy. Moreover, the cell activates this pathway as an antiviral strategy for interferon and cytokine production, among other strategies. Here, we present new data concerning the role of this pathway in infection with the paramyxovirus Newcastle disease virus (NDV). Our data seem to indicate that NDV uses the PI3K/Akt pathway to delay cell death and increase cell survival as a means of improving its replication. The interference of negative-sense ssRNA viruses with this essential pathway might have implications for the development of antiviral therapies.

Rol del virus sincicial respiratorio en una cohorte de adultos mayores / Respiratory Syncytial Virus in the elderly adults

El rol de los virus respiratorios distintos de influenza en las infecciones respiratorias agudas en los adultos mayores ha sido probablemente subestimado. En los últimos años, los avances en técnicas moleculares de diagnóstico han hecho posible la identificación rápida del virus sincicial respiratorio humano (HRSV). Realizamos un estudio prospectivo observacional para evaluar el rol del HRSV en mayores de 65 años que se hospitalizaron por infecciones respiratorias en nuestra institución, ubicada en la ciudad de La Plata, provincia de Buenos Aires. Fueron reclutados 124 pacientes y el HRSV se detectó en 13, influenza B en 9 e influenza A en 8. La presentación clínica más frecuente de los The role of respiratory viruses other than influenza in acute respiratory tract infections among elderly adults has probably been underestimated. Recent advances in molecular diagnosis have made the rapid identification of human respiratory syncitial virus HRSV infection possible. We conducted a prospective observational study to evaluate the role of HRSV in elderly patients (>65 years of age) hospitalized for acute respiratory infections. A total of 124 patients were recruited, HRSV infection was identified in 13 patients, Influenza B in 9 patients and influenza A in 8 patients. The most frequent clinical presentation was bronchospasm and the infection was prevalent in patients with comorbidities. HRSV infections accounted for an important number of hospital admissions and has been associated with high mortality rates (23%). pacientes con HRSV fue el broncoespasmo y afectó principalmente a personas con comorbilidades. HRSV fue responsable de un número importante de internaciones por enfermedad respiratoria aguda en mayores de 65 años en nuestra institución y se asoció a mortalidad elevada (23%).

The role of respiratory viruses other than influenza in acute respiratory tract infections among elderly adults has probably been underestimated. Recent advances in molecular diagnosis have made the rapid identification of human respiratory syncitial virus HRSV infection possible. We conducted a prospective observational study to evaluate the role of HRSV in elderly patients (>65 years of age) hospitalized for acute respiratory infections. A total of 124 patients were recruited, HRSV infection was identified in 13 patients, Influenza B in 9 patients and influenza A in 8 patients. The most frequent clinical presentation was bronchospasm and the infection was prevalent in patients with comorbidities. HRSV infections accounted for an important number of hospital admissions and has been associated with high mortality rates (23%).

Interferon-Induced Transmembrane Protein 1 Restricts Replication of Viruses That Enter Cells via the Plasma Membrane.

The acute antiviral response is mediated by a family of interferon-stimulated genes (ISGs), providing cell-intrinsic immunity. Mutations in genes encoding these proteins are often associated with increased susceptibility to viral infections. One family of ISGs with antiviral function is the interferon-inducible transmembrane proteins (IFITMs), of which IFITM3 has been studied extensively. In contrast, IFITM1 has not been studied in detail. Since IFITM1 can localize to the plasma membrane, we investigated its function with a range of enveloped viruses thought to infect cells by fusion with the plasma membrane. Overexpression of IFITM1 prevented infection by a number of Paramyxoviridae and Pneumoviridae, including respiratory syncytial virus (RSV), mumps virus, and human metapneumovirus (HMPV). IFITM1 also restricted infection with an enveloped DNA virus that can enter via the plasma membrane, herpes simplex virus 1 (HSV-1). To test the importance of plasma membrane localization for IFITM1 function, we identified blocks of amino acids in the conserved intracellular loop (CIL) domain that altered the subcellular localization of the protein and reduced antiviral activity. By screening reported data sets, 12 rare nonsynonymous single nucleotide polymorphisms (SNPs) were identified in human IFITM1, some of which are in the CIL domain. Using an Ifitm1-/- mouse, we show that RSV infection was more severe, thereby extending the range of viruses restricted in vivo by IFITM proteins and suggesting overall that IFITM1 is broadly antiviral and that this antiviral function is associated with cell surface localization.IMPORTANCE Host susceptibility to viral infection is multifactorial, but early control of viruses not previously encountered is predominantly mediated by the interferon-stimulated gene (ISG) family. There are upwards of 300 of these genes, the majority of which do not have a clearly defined function or mechanism of action. The cellular location of these proteins may have an important effect on their function. One ISG located at the plasma membrane is interferon-inducible transmembrane protein 1 (IFITM1). Here we demonstrate that IFITM1 can inhibit infection with a range of viruses that enter via the plasma membrane. Mutant IFITM1 proteins that were unable to localize to the plasma membrane did not restrict viral infection. We also observed for the first time that IFITM1 plays a role in vivo, and Ifitm1-/- mice were more susceptible to viral lung infection. These data contribute to our understanding of how ISGs prevent viral infections.

Human metapneumovirus in Southern Brazil

INTRODUCTION Infections caused by respiratory viruses are important problems worldwide, especially in children. Human metapneumovirus (hMPV) is a respiratory pathogen and causes severe infections with nonspecific symptoms. This study reports the hMPV occurrence and dissemination in southern Brazil and compares the frequency of occurrence of this virus and the human respiratory syncytial virus (hRSV) in the epidemiological weeks in a three-year period (2009-2011). METHODS: In total, 545 nasopharyngeal (NP) specimens from individuals with Severe Acute Respiratory Syndrome (SARS) who were negative for other seven respiratory viruses were analyzed for the presence of hMPV. Human metapneumovirus was detected by direct immunofluorescence and real-time reverse transcription polymerase chain reaction. RESULTS: hMPV was detected in 109 patients from the main geographic regions of the southernmost state of Brazil, presenting similar overall prevalence in males (46.8%) and females (53.2%). Among children who were less than six years old, hMPV was detected in 99 samples of all age groups, with a higher frequency in infants who were less than one year old (45.7%) compared to all other age groups until six years. hMPV and hRSV infection occurred in almost the same epidemiological weeks (EWs) of each year, with peaks of incidence between EW 31/37 and EW 26/38 for the years 2009 and 2011, respectively. hMPV was further detected in several cases of SARS and it was the only virus detected in three deaths. CONCLUSIONS These findings indicate that hMPV is in circulation in southern Brazil and highlight the importance of diagnosing hMPV for influenza-like illness in the population. (AU)

Vaccines for the Paramyxoviruses and Pneumoviruses: Successes, Candidates, and Hurdles.

Human parainfluenza viruses (family Paramyxoviridae), human metapneumovirus, and respiratory syncytial virus (family Pneumoviridae) infect most infants and children within the first few years of life and are the etiologic agents for many serious acute respiratory illnesses. These virus infections are also associated with long-term diseases that impact quality of life, including asthma. Despite over a half-century of vaccine research, development, and clinical trials, no vaccine has been licensed to date for the paramyxoviruses or pneumoviruses for the youngest infants. In this study, we describe the recent reclassification of paramyxoviruses and pneumoviruses into distinct families by the International Committee on the Taxonomy of Viruses. We also discuss some past unsuccessful vaccine trials and some currently preferred vaccine strategies. Finally, we discuss hurdles that must be overcome to support successful respiratory virus vaccine development for the youngest children.

GS-5734 and its parent nucleoside analog inhibit Filo-, Pneumo-, and Paramyxoviruses.

GS-5734 is a monophosphate prodrug of an adenosine nucleoside analog that showed therapeutic efficacy in a non-human primate model of Ebola virus infection. It has been administered under compassionate use to two Ebola patients, both of whom survived, and is currently in Phase 2 clinical development for treatment of Ebola virus disease. Here we report the antiviral activities of GS-5734 and the parent nucleoside analog across multiple virus families, providing evidence to support new indications for this compound against human viruses of significant public health concern.

Generation of monoclonal antibodies specific of the postfusion conformation of the Pneumovirinae fusion (F) protein.

Paramyxovirus entry into cells requires fusion of the viral and cell membranes mediated by one of the major virus glycoproteins, the fusion (F) glycoprotein which transits from a metastable pre-fusion conformation to a highly stable post-fusion structure during the membrane fusion process. F protein refolding involves large conformational changes of the protein trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) from each protomer into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of the Pneumovirinae F proteins, and as extension of previous work (Palomo et al., 2014), a general strategy was designed to obtain polyclonal and particularly monoclonal antibodies specific of the 6HB motif of the Pneumovirinae fusion protein. The antibodies reported here should assist in the characterization of the structural changes that the F protein of human metapneumovirus or respiratory syncytial virus experiences during the process of membrane fusion.

The Pneumovirinae fusion (F) protein: A common target for vaccines and antivirals.

The Pneumovirinae fusion (F) protein mediates fusion of the virus and cell membrane, an essential step for entry of the viral genome in the cell cytoplasm and initiation of a new infectious cycle. Accordingly, potent inhibitors of virus infectivity have been found among antibodies and chemical compounds that target the Pneumovirinae F protein. Recent developments in structure-based vaccines have led to a deeper understanding of F protein antigenicity, unveiling new conformations and epitopes which should assist in development of efficacious vaccines. Similarly, structure-based studies of potent antiviral inhibitors have provided information about their mode of action and mechanisms of resistance. The advantages and disadvantages of the different options to battle against important pathogens, such as human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) are summarized and critically discussed in this review.

Molecular detection of respiratory viruses.

Over the past several years a wide variety of molecular assays for the detection of respiratory viruses has reached the market. The tests described herein range from kits containing primers and probes detecting specific groups of viruses, to self-contained systems requiring specialized instruments that extract nucleic acids and perform the polymerase chain reaction with little operator input. Some of the tests target just the viruses involved in large yearly epidemics such as influenza, or specific groups of viruses such as the adenoviruses or parainfluenza viruses; others can detect most of the known respiratory viruses and some bacterial agents.

[Morbilliviruses].

The genus Morbillivirus in the family Paramyxoviridae contains many pathogens, which are important for medicine or veterinary medicine. Because each morbillivirus has restricted host range and serologically monotypic, the virus infection and transmission is effectively controlled by vaccinations and surveillance. Rinderpest virus has been eradicated in 2011, and elimination of measles virus progresses worldwide. Recently, a new cell receptor for measles virus, nectin4 was identified. Both SLAM, a molecule expressing on immune cells, and nectin4, a molecule expressing on epithelial cells, are important to infectivity and pathogenicity of the virus.

Generation of Tioman virus nucleocapsid-like particles in yeast Saccharomyces cerevisiae.

Tioman virus (TioV) was isolated from a number of pooled urine samples of Tioman Island flying foxes (Pteropus hypomelanus) during the search for the reservoir host of Nipah virus. Studies have established TioV as a new virus in the family Paramyxoviridae. This novel paramyxovirus is antigenically related to Menangle virus that was isolated in Australia in 1997 during disease outbreak in pigs. TioV causes mild disease in pigs and has a predilection for lymphoid tissues. Recent serosurvey showed that 1.8% of Tioman Islanders had neutralizing antibodies against TioV, indicating probable past infection. For the development of convenient serological tests for this virus, recombinant TioV nucleocapsid (N) protein was expressed in the yeast Saccharomyces cerevisiae. High yields of recombinant TioV N protein were obtained. Electron microscopy demonstrated that purified recombinant N protein self-assembled into nucleocapsid-like particles which were identical in density and morphology to authentic nucleocapsids from paramyxovirus-infected cells. Different size nucleocapsid-like particles were stable and readily purified by CsCl gradient ultracentrifugation. Polyclonal sera raised in rabbits after immunization with recombinant TioV N protein reacted reliably with TioV infected tissues in immunohistochemistry tests. It confirmed that the antigenic properties of yeast derived TioV N protein are identical to authentic viral protein.

Sensitive and broadly reactive reverse transcription-PCR assays to detect novel paramyxoviruses.

We have developed a set of reverse transcription-PCR assays for the detection and identification of known and novel paramyxoviruses in clinical specimens. Primers were designed from the conserved motifs of the polymerase pol gene sequences to detect members of the Paramyxovirinae or Pneumovirinae subfamily or groups of genera within the Paramyxovirinae subfamily. The consensus-degenerate hybrid oligonucleotide primer design and seminested or nested PCR assay design were used to enhance the breadth of reactivity and sensitivity of the respective assays. Using expressed RNA and 10-fold dilution series of virus-infected tissue culture isolates from different members of the family or genera, these assays were able to detect on average between 100 and 500 copies of template RNA. The assays were specific to the respective group of genera or subfamily viruses. This set of primers enhances our ability to look for novel viruses in outbreaks and diseases of unknown etiology.

Recovery of avian metapneumovirus subgroup C from cDNA: cross-recognition of avian and human metapneumovirus support proteins.

Avian metapneumovirus (AMPV) causes an acute respiratory disease in turkeys and is associated with "swollen head syndrome" in chickens, contributing to significant economic losses for the U.S. poultry industry. With a long-term goal of developing a better vaccine for controlling AMPV in the United States, we established a reverse genetics system to produce infectious AMPV of subgroup C entirely from cDNA. A cDNA clone encoding the entire 14,150-nucleotide genome of AMPV subgroup C strain Colorado (AMPV/CO) was generated by assembling five cDNA fragments between the T7 RNA polymerase promoter and the autocatalytic hepatitis delta virus ribozyme of a transcription plasmid, pBR 322. Transfection of this plasmid, along with the expression plasmids encoding the N, P, M2-1, and L proteins of AMPV/CO, into cells stably expressing T7 RNA polymerase resulted in the recovery of infectious AMPV/CO. Characterization of the recombinant AMPV/CO showed that its growth properties in tissue culture were similar to those of the parental virus. The potential of AMPV/CO to serve as a viral vector was also assessed by generating another recombinant virus, rAMPV/CO-GFP, that expressed the enhanced green fluorescent protein (GFP) as a foreign protein. Interestingly, GFP-expressing AMPV and GFP-expressing human metapneumovirus (HMPV) could be recovered using the support plasmids of either virus, denoting that the genome promoters are conserved between the two metapneumoviruses and can be cross-recognized by the polymerase complex proteins of either virus. These results indicate a close functional relationship between AMPV/CO and HMPV.

A wild goose metapneumovirus containing a large attachment glycoprotein is avirulent but immunoprotective in domestic turkeys.

The genomic structure and composition of an avian metapneumovirus (aMPV) recently isolated from wild Canada geese (goose 15a/01) in the United States, together with its replication, virulence, and immunogenicity in domestic turkeys, were investigated. The sizes of seven of the eight genes, sequence identity, and genome organization of goose aMPV were similar to those of turkey aMPV subtype C (aMPV/C) strains, indicating that it belonged to the subtype. However, the goose virus contained the largest attachment (G) gene of any pneumovirus or metapneumovirus, with the predicted G protein of 585 amino acids (aa) more than twice the sizes of G proteins from other subtype C viruses and human metapneumovirus and more than 170 aa larger than the G proteins from the other aMPV subtypes (subtypes A, B, and D). The large G gene resulted from a 1,015-nucleotide insertion at 18 nucleotides upstream of the termination signal of the turkey aMPV/C G gene. Three other aMPV isolates from Canada geese had similarly large G genes, whereas analysis of recent aMPV strains circulating in U.S. turkeys did not indicate the presence of the goose virus-like strain. In vitro, the goose virus replicated to levels (2 x 10(5) to 5 x 10(5) 50% tissue culture infective dose) comparable to those produced by turkey aMPV/C strains. More importantly, the virus replicated efficiently in the upper respiratory tract of domestic turkeys but with no clinical signs in either day-old or 2-week-old turkeys. The virus was also horizontally transmitted to naïve birds, and turkey infections with goose 15a/01 induced production of aMPV-specific antibodies. Challenging day-old or 2-week-old turkeys vaccinated with live goose aMPV resulted in lower clinical scores in 33% of the birds, whereas the rest of the birds had no detectable clinical signs of the upper respiratory disease, suggesting that the mutant virus may be a safe and effective vaccine against aMPV infection outbreaks in commercial turkeys.

Accessory genes of the paramyxoviridae, a large family of nonsegmented negative-strand RNA viruses, as a focus of active investigation by reverse genetics.

The Paramyxoviridae, a large family of nonsegmented negative-strand RNA viruses, comprises several genera each containing important human and animal pathogens. They possess in common six basal genes essential for viral replication and, in addition, a subset of accessory genes that are largely unique to each genus. These accessory genes are either encoded in one or more alternative overlapping frames of a basal gene, which are accessed transcriptionally or translationally, or inserted before or between the basal genes as one or more extra genes. However, the question of how the individual accessory genes contribute to actual viral replication and pathogenesis remained unanswered. It was not even established whether they are dispensable or indispensable for the viral life cycle. The plasmid-based reverse genetics of the full-length viral genome has now come into wide use to demonstrate that most, if not all, of these putative accessory genes can be disrupted without destroying viral infectivity, conclusively defining them as indeed dispensable accessory genes. Studies on the phenotypes of the resulting gene knockout viruses have revealed that the individual accessory genes greatly contribute specifically and additively to the overall viral fitness both in vitro and in vivo.

Animal pneumoviruses: molecular genetics and pathogenesis.

Pneumoviruses are single-stranded, negative-sense, nonsegmented RNA viruses of the family Paramyxoviridae, subfamily Pneumovirinae, and include pathogens that infect humans (respiratory syncytial virus and human metapneumovirus), domestic mammals (bovine, ovine, and caprine respiratory syncytial viruses), rodents (pneumonia virus of mice), and birds (avian metapneumovirus). Among the topics considered in this review are recent studies focused on the roles of the individual virus-encoded components in promoting virus replication as well as in altering and evading innate antiviral host defenses. Advances in the molecular technology of pneumoviruses and the emergence of recombinant pneumoviruses that are leading to improved virus-based vaccine formulations are also discussed. Since pneumovirus infection in natural hosts is associated with a profound inflammatory response that persists despite adequate antiviral therapy, we also review the recent experimental treatment strategies that have focused on combined antiviral, anti-inflammatory, and immunomodulatory approaches.

Nucleotide and predicted amino acid sequence-based analysis of the avian metapneumovirus type C cell attachment glycoprotein gene: phylogenetic analysis and molecular epidemiology of U.S. pneumoviruses.

A serologically distinct avian metapneumovirus (aMPV) was isolated in the United States after an outbreak of turkey rhinotracheitis (TRT) in February 1997. The newly recognized U.S. virus was subsequently demonstrated to be genetically distinct from European subtypes and was designated aMPV serotype C (aMPV/C). We have determined the nucleotide sequence of the gene encoding the cell attachment glycoprotein (G) of aMPV/C (Colorado strain and three Minnesota isolates) and predicted amino acid sequence by sequencing cloned cDNAs synthesized from intracellular RNA of aMPV/C-infected cells. The nucleotide sequence comprised 1,321 nucleotides with only one predicted open reading frame encoding a protein of 435 amino acids, with a predicted M(r) of 48,840. The structural characteristics of the predicted G protein of aMPV/C were similar to those of the human respiratory syncytial virus (hRSV) attachment G protein, including two mucin-like regions (heparin-binding domains) flanking both sides of a CX3C chemokine motif present in a conserved hydrophobic pocket. Comparison of the deduced G-protein amino acid sequence of aMPV/C with those of aMPV serotypes A, B, and D, as well as hRSV revealed overall predicted amino acid sequence identities ranging from 4 to 16.5%, suggesting a distant relationship. However, G-protein sequence identities ranged from 72 to 97% when aMPV/C was compared to other members within the aMPV/C subtype or 21% for the recently identified human MPV (hMPV) G protein. Ratios of nonsynonymous to synonymous nucleotide changes were greater than one in the G gene when comparing the more recent Minnesota isolates to the original Colorado isolate. Epidemiologically, this indicates positive selection among U.S. isolates since the first outbreak of TRT in the United States.