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The onset of illness begins with generic flu-like symptoms; a characteristic high fever (typically 39–40 oC), severe headache, chills, myalgia, prostration, and malaise. For many patients (50–75%) this is followed by rapid debilitation characterized by gastrointestinal symptoms including anorexia, abdominal pain, severe nausea, vomiting, and watery diarrhea. Starting on day four to five patients commonly develop enanthem, dysphasia, and pharyngitis. Additionally, a characteristic maculopapular rash is typically the first distinctive feature indicating a filovirus infection versus influenza or malaria. Other common symptoms include lymphadenopathy, leukopenia, and thrombocytopenia.
Many of the initial symptoms may persist in the early organ phase, and patients may sustain a high fever. They may additionally display neurological symptoms including encephalitis, confusion, delirium, irritability, and aggression. Patients can also develop dyspnea and abnormal vascular permeability, particularly conjunctival injection and edema. During the latter part of this phase more than 75% of patients present with some form of clear hemorrhagic manifestation such as petechiae, mucosal bleeding, melena, bloody diarrhea, hematemesis, and ecchymoses. Due to the unusualness of hemorrhagic symptoms, diseases caused by filoviruses have sometimes been referred to as hemorrhagic fevers (Marburg Hemorrhagic Fever (MHF) and Ebola Hemorrhagic Fever (EHF)), although these terms are currently disfavored since not all patients display hemorrhagic symptoms. At this stage, multiple organs are affected including the pancreas, kidney, and liver. Elevated serum activity of a number of liver enzymes including SGOT and SGPT have been observed in most patients sampled.
Viruses are obligate intracellular pathogens that require host cells in order to replicate and produce infectious progeny. Virus entry into host cells is followed by capsid uncoating, genome transcription and replication, synthesis of viral proteins, assembly of progeny virions, and egress. For most viruses, genome replication and assembly take place in specialized intracellular compartments known as viral factories or inclusions, which are often composed of membranous scaffolds, viral and cellular factors, and mitochondria. Viral inclusions (VIs) serve multiple purposes during infection, including the concentration of viral and host factors to ensure the high efficiency of replication, sequestration of viral nucleic acids and proteins from innate immune responses, and the spatial coordination of consecutive replication cycle steps. Most double-stranded RNA (dsRNA) viruses form cytoplasmic inclusions with a characteristic morphology. These neoorganelles constitute sites of genome replication and virion assembly, and contain abundant viral RNA and proteins.
The combination of ultrastructural and functional studies has enhanced our knowledge about VI biogenesis. However, for many viruses, it is still not known how these structures form and mediate functions in viral replication. Here, we describe the current understanding of the morphogenesis and function of reovirus inclusions and compare these neoorganelles with the replication factories formed by other members of the Reoviridae family.
The family Filoviridae includes two accepted genera, Ebolavirus and Marburgvirus. The genus Ebolavirus includes five species (each represented by a single virus): Zaire ebolavirus (Ebola virus, EBOV), Sudan ebolavirus (Sudan virus, SUDV), Reston ebolavirus (Reston virus, RESTV), Taï Forest ebolavirus (Tai Forest virus, TAFV), and Bundibugyo ebolavirus (Bundibugyo virus, BDBV). The genus Marburgvirus includes a single species, Marburg marburgvirus, which has two members: Marburg virus (MARV) and Ravn virus (RAVV). In 1967, the first cases of filoviral infection were documented in three simultaneous outbreaks in West Germany and Yugoslavia. The virus responsible for the outbreaks was named “Marburg virus” after the German city of Marburg in which it was first recognized. From documented instances of infection, it seems that the members of the filovirus genera may exist in quite opposite climates of Africa with marburgvirus infections occurring more frequently in the dry woodlands, while ebolavirus infections occur more frequently in rain forests. More than 40 years of effort have focused on the search for the reservoir of these viruses in Africa, and while the search is still ongoing, recent evidence indicates that bats may be reservoirs for both marburgviruses and ebolaviruses. However, the recent outbreak of RESTV in domestic pigs in the Philippines demonstrated the potential for animals other than primates and bats to be infected and potentially spread or amplify outbreaks.
Filoviruses are named for their long, filamentous shape which can been seen on the order of micrometers in length, while their width is more narrow (usually around 80 nm) with little fluctuation. Contained within this filamentous virus is a single, 19-kb negative-sense RNA genome that encodes seven proteins. The seven filoviral proteins are the glycoprotein (GP), the polymerase (L), the nucleoprotein (NP), a secondary matrix protein (VP24), the transcriptional activator (VP30), the polymerase cofactor (VP35), and the matrix protein (VP40). Homotrimers of the viral GP cover the surface of the virion, and this viral GP is believed to be the sole host attachment factor for filoviruses. Candidates for filoviral receptor and co-factors include transferrin, DC-SIGN, TIM-1, and NPC1. After entry, filoviruses replicate their genomes and viral proteins in the cytoplasm using a RNA-dependent RNA-polymerase which is carried in with the virus.
Wild-type filovirus infection has been associated with severe case fatality rates in humans, as high as 90%. In humans, filovirus infection is characterized by an abrupt onset of flu-like illness, after an initial incubation period of 2–21 days. Following this initial illness, signs and symptoms of disease include anorexia, nausea, vomiting, chest pain, cough, edema, postural hypotension, neurologic complications, petechiae, and mucosal hemorrhage. There have also been several observed wild-type filovirus outbreaks among great apes in Africa that have demonstrated similarly high mortality rates. In an effort to create cost and time-effective models of filoviral disease for the development of vaccines and therapeutics, small animals, such as mice and guinea pigs, are often used. However, these animals usually demonstrate significant resistance to wild-type filovirus infection, and only demonstrate mortality rates similar to primates when the filovirus in question has been adapted to the model species. Due to the difficulties in evaluating wild-type filovirus infection in small animals and the generally high level of immune protection correlates derived from non-human primate (NHP) models of infection, therapeutics and vaccines are ultimately evaluated in NHP species for efficacy against filovirus. Of the NHP models available for filovirus study, rhesus and cynomolgus macaques have been the most highly characterized and utilized for therapeutic and vaccine development, respectively. However, the starting point of vaccine and therapeutic development remains small animal models due to the cost, ethical, and time-associated benefits. This review will highlight the current research into filovirus vaccines and therapeutics.
Filoviruses (family Mononegavirales, genera Ebolavirus (EBOV) and Marburgvirus (MARV)) are single-stranded, negative-sense RNA viruses that exhibit a unique heterogeneous filamentous structure. Both EBOV and MARV infect a wide variety of mammals and this wide tropism has complicated the identification of cellular proteins required for viral entry. A hemorrhagic fever is caused by these viruses in humans, non-human primates and perhaps other mammals and is associated with high morbidity and mortality during outbreaks. No therapeutic drugs or vaccines are currently available to treat or prevent filoviral infection. Because of this and the high lethality associated with infection, filoviruses are considered Category A Priority Pathogens by NIAID and, in recent years, much research has focused on understanding how these viruses bind to and enter permissive cells.
The genus Ebolavirus of the Filoviridae family includes five species: Bundibugyo ebolavirus, Reston ebolavirus, Sudan ebolavirus, Tai Forest ebolavirus, and Zaire ebolavirus. Among them, the Zaire ebolavirus, usually called Ebola virus (EBOV), is the main causative agent of human outbreaks, causing the Ebola virus disease (EVD). EVD is a disease of human and non-human primates that is characterized by a high fatality rate (30–90%). EBOV persists in the environment in a still unidentified animal reservoir, most likely the fruit bats, which maintains the virus in an enzootic cycle. Recently, a new ebolavirus, the Bombali virus, has been detected in free-tailed bats in Sierra Leone while in China a new filovirus (Měnglà virus) was identified in rousettus bats further supporting the role of bats in filovirus ecology. Occasionally, EBOV can be transmitted to non-human primates and duikers in an epizootic cycle causing outbreaks with high mortality. Human infection represents a sporadic event taking place in the context of a human animal interface. Transmission is mainly due to the contact with blood or body fluids from infected humans or animals. EVD begins with nonspecific symptoms involving fever, fatigue, and muscle ache, and evolves to a severe condition associated with vomiting, diarrhea, infrequent hemorrhaging, and mental disorder leading to a comatose state and death. The convalescence phase of survivor patients lasts several months and is characterized by fatigue, joint pain as well as loss of appetite and memory. Viral RNA can be detected in specific organs, such as the testis, for more than one year after symptoms resolution.
Until 2014, EVD was considered a neglected disease, causing small outbreaks in remote African villages. EBOV research was focused mainly on biology aspects of viral infection or preparedness due to its potential use as bioweapon, and was limited to few laboratories equipped with biosafety level-4 (BSL-4) facilities. However, the recent large outbreak of EVD (Western Africa, 2013–2016) characterized by 28,616 cases and 11,310 deaths, highlighted the worldwide danger of this disease and its impact on global public health and economy.
Thus, research on the molecular dissection of EBOV life cycle received a strong stimulus and financial support with the ultimate goal of developing effective preventive and therapeutic approaches. In this review, we summarize the current knowledge of a specific step of the EBOV life cycle, the entry process, and the compounds identified so far capable of interfering with it, as well as the molecular models used to these purposes.
Influenza A viruses (IAVs), which belong to the family Orthomyxoviridae, cause highly contagious diseases in a wide variety of avian and mammalian species, including humans, pigs, horses, dogs, and poultry, and are recognized as one of the most important zoonotic pathogens. IAVs have 8 segmented negative-sense RNA genomes and are divided into subtypes based on combination of two viral envelope glycoproteins, hemagglutinin (HA) and neuraminidase (NA). IAVs with H1-16 HA and N1-9 NA subtypes have been identified in wild aquatic birds, especially migratory ducks, the natural reservoir of IAVs1–4. Importantly, due to their propensity for genetic reassortment, a variety of IAV subtypes are distributed in many host species.
Recently, influenza virus-like RNA genomes were detected in fruit bats (Sturnira lilium and Artibeus planirostris) in Central and South America5,6. Since the amino acid sequences of their HA and NA are distinct from those of all previously known IAV subtypes, these bat-derived influenza viruses (BatIVs) were provisionally designated H17N10 and H18N11. However, previous studies have reported that H17 HA does not bind to sialic acids linked to galactose in sugar chains7, which are known as canonical receptors of IAVs, and that N10 NA lacks neuraminidase activity8 which is also a common property of all known influenza viruses. Thus, BatIV glycoproteins have also been called HA-like (HL) and NA-like (NL) (i.e., HL17NL10 and HL18NL11)9. However, information on the biological properties of these BatIVs is limited since infectious virus particles have never been isolated from infected animals.
Bats belonging to the order Chiroptera, which is known as the second largest order of mammalians, are distributed into more than 1,000 species globally10. It has been shown that bats play crucial roles as natural reservoirs of some zoonotic pathogens such as Marburg virus, Hendra virus, Nipah virus, Lyssa virus, SARS, and MERS coronaviruses11–14. Thus, it is important to investigate the ecology of bat-derived pathogens and to clarify their potential risks as zoonotic pathogens.
Our previous study revealed that vesicular stomatitis viruses (VSVs) pseudotyped with BatIV glycoproteins efficiently infected cultured cells derived from particular bat species (e.g., Miniopterus fuliginosus) but not those commonly used for IAV propagation and other bat cells tested, providing key information on cell lines that are potentially susceptible to BatIVs15. Using these bat cell lines and the well-known plasmid-based reverse genetics approach16, we demonstrate the generation of infectious BatIVs and their reassortants in this study.
Bunyamwera virus (BunV) is an enveloped virus with a negative-sense RNA genome (~12 kb) divided among three segments. In infected mammalian cells, BunV infection leads to formation of tubular structures (up to 50 per cell) encompassing the Golgi membranes, actin, myosin I, and viral non-structural protein NSm (Fontana et al., 2008). The tubes are in close contact with mitochondria and rough endoplasmic reticulum (ER), possibly serving as sources of host factors (e.g., translation elongation factor eEF-2 and ribosomal proteins) aiding the virus replication. Transcription and replication of BunV occur inside the “globular domain,” a U-like structure at one end of the tubes. The replicative complexes consisting of BunV nucleoproteins and RNA replicase, concentrate on the inner surface of the globular domain. BunV transcription yields mRNAs that are transferred to rough ER for translation, and replication produces the progeny nucleoproteins transported to the Golgi stacks modified by inserted BunV surface glycoproteins, for particle maturation (Fontana et al., 2008).
The model by Fontana et al. (2008) implies dynamic changes of, and communication between, the cell membranous compartments induced by bunyavirus infection, driven mainly by actin filaments and that the viral NSm. Apparently, the primary transcription of the gene encoding NSm must occur prior to changes in Golgi. The BunV replication-associated globular domains are open structures, unlike the vesicles and spherules induced by positive-sense RNA viruses (see below). This might reflect a nuclease-protected state of the BunV genomic and antigenomic RNA templates, the absence of dsRNA (which might trigger RNA interference in cells) in negative-sense RNA viruses replication, and employment of strategies against host defense mechanisms (Léonard et al., 2006; Habjan et al., 2008).
Ebola virus (EBOV) and Marburg virus (MARV), classified as biosafety level 4 agents, belong to the Family Filoviridae. Whereas MARV consists of a single species, Lake Victoria Marburgvirus, there are four distinct EBOV species, including Zaire ebolavirus (ZEBOV), Sudan ebolavirus (SEBOV), Côte d’Ivoire ebolavirus (CIEBOV), Reston ebolavirus (REBOV), and the proposed new species Bundibugyo ebolavirus (BEBOV) (Sanchez et al., 2007; Towner et al., 2008) (Figure 1 left). Among these, ZEBOV, first identified in 1976, seems to be the most virulent, killing approximately up to 90% of infected individuals, whereas REBOV, which was initially isolated from cynomolgus monkeys imported from the Philippines into the USA in 1989, is less pathogenic in experimentally infected non-human primates (Fisher-Hoch and McCormick, 1999) and has never caused lethal infection in humans (Sanchez et al., 2007).
Ebola virus and Marburg virus are filamentous, enveloped, non-segmented, single-stranded, negative-sense RNA viruses (Figure 2). The viral genome encodes seven structural proteins, nucleoprotein (NP), polymerase cofactor (VP35), matrix protein (VP40), glycoprotein (GP), replication-transcription protein (VP30), minor matrix protein (VP24), and RNA-dependent RNA polymerase (L). EBOV also expresses at least one secreted non-structural glycoprotein (sGP). Figure 3 summarizes filovirus replication in cells. At the first step of replication, viral attachment through interaction between GP and some cellular molecules is followed by endocytosis, including macropinocytosis (Nanbo et al., 2010; Saeed et al., 2010). Subsequent fusion of the viral envelope with the host cell endosomal membrane releases the viral proteins (i.e., NP, VP35, VP30, and L) and RNA genome into the cytoplasm, the site of replication. Transcription of the negative-sense viral RNA by the viral polymerase complex (VP35 and L) yields mRNAs that are translated at cellular ribosomes. During replication, full-length positive-sense copies of the viral genome are synthesized. They subsequently serve as templates for replication of negative-sense viral RNA synthesis. At the plasma membrane, NP-encapsidated full-length viral RNAs and the other viral structural proteins are assembled with VP40 and GP and incorporated into enveloped virus particles that bud from the cell-surface (Noda et al., 2006; Bharat et al., 2011). Though filoviruses show broad tissue tropism, hepatocytes, endothelial cells, dendritic cells, monocytes, and macrophages are thought to be their preferred target cells, and infection of these cells is important for hemorrhagic manifestation and immune disorders (Geisbert and Hensley, 2004).
Influenza C virus was first isolated during an epidemic of respiratory illness in 1947. Since the virus showed no cross reactivity with antisera against influenza A and B viruses it was classified as a new genus of the Orthomyxoviridae, named influenza C virus (Francis et al., 1950; Taylor, 1949, 1951). Influenza C virus usually causes inflammation of the upper respiratory tract, especially in children from two to six years of age. Clinical symptoms, such as cough, fever, malaise are typically mild. Only occasionally the virus spreads to the lower respiratory tract and causes bronchitis, bronchiectasie and broncho-pneumonia (Gouarin et al., 2008; Kauppila et al., 2014; Matsuzaki et al., 2007; Matsuzaki et al., 2006; Muraki and Hongo, 2010). Although influenza C virus infections occur primarily in a pattern of sporadic cases or in limited outbreaks (Joosting et al., 1968; Minuse et al., 1954), serological studies indicated that this virus is widely distributed around the world and that the majority of humans develope antibodies against the virus early in life (Matsuzaki et al., 2006; Salez et al., 2014). In a serological study carried out in France, 60%–70% of the population was found to be previously exposed to the virus, the highest rates of positive samples was found in the 16–30 years age group (Manuguerra et al., 1992). In a 6-year tracking study in hospitalized children in Spain, influenza C infections accounted for 13% of influenza-positive cases (Calvo et al., 2013). The results indicate intense circulation of influenza C virus in the human population.
The primary host and reservoir of influenza C virus are humans, but there is evidence that this virus possesses the ability to also infect animals (Muraki and Hongo, 2010). Serological studies showed that antibodies against influenza C virus are widely present in dogs and especially in pigs (Brown et al., 1995; Horimoto et al., 2014; Manuguerra and Hannoun, 1992; Manuguerra et al., 1993; Ohwada et al., 1987; Yamaoka et al., 1991; Youzbashi et al., 1996). In 1981, a number of influenza C virus strains were isolated from pigs in Beijing and these strains could be transmitted from pig to pig under experimental conditions (Guo et al., 1983).
In 2011, an influenza C-like virus was isolated from clinically ill pigs exhibiting influenza-like symptoms (C/Oklahoma/1334/2011) and also from cattle (D/bovine/Oklahoma/660/2013) which subsequently turned out to be the main reservoir of this newly discovered virus (Collin et al., 2014, 2015; Hause et al., 2013). Phylogenetic analysis showed that these strains have only 50% overall amino acid homology to human influenza C viruses, a divergence similar to that described between influenza A and B viruses (Hause et al., 2013). Accordingly, no cross reactivity was observed between these strains and human influenza C virus antisera. This new strain has a broader cell tropism than human influenza C virus and is capable of infecting and transmitting by direct contact in both pigs and ferrets. It also encodes a novel mechanism for generating the M1 protein and, importantly, is unable to reassort with human influenza C virus and generate viable progeny. Based on these differences to influenza C virus it was suggested that this virus warrants classification as a new genus of influenza virus, named influenza D virus (Collin et al., 2014; Hause et al., 2013).
The bacterial component of the microbiota can directly or indirectly impact the outcome of infection by a range of different viruses. Direct interactions have been observed between bacteria and influenza A virus (IAV) [1, 2] as well as several enteric viruses: picornaviruses (including poliovirus [3, 4]); coxsackieviruses A21, B2, B3, Echovirus 30, Mengo, and Aichi viruses [5, 6]; human noroviruses (HNoV) [7, 8]; and mammalian orthoreovirus (reovirus). Although bacteria can directly impact the outcome of infection by several viruses, the viral factors involved in the interaction between bacteria and viruses are largely undefined.
In many cases, binding of viruses to bacteria is mediated through bacterial envelope components lipopolysaccharide (LPS), the main component of the gram-negative bacterial envelope, and peptidoglycan (PG), the main component of the gram-positive bacterial envelope. Poliovirus binds to LPS and PG from several bacterial species [3–5, 10]. Although the bacterial binding epitopes for poliovirus are unknown, the virus may bind LPS, PG, and chitin through the monosaccharide N-acetyl-glucosamine (GlcNAc). HNoVs use histo-blood group antigens (HBGAs) to attach to eukaryotic cells and can bind bacterial HBGAs. Reovirus thermostability is enhanced by LPS and PG independent of serotype, but lipoteichoic acid, a major component of the gram-positive bacterial envelope, elevates the thermostability of only one reovirus serotype. As different viral strains and serotypes differ in their interactions with bacterial envelope components, specific genetic determinants of norovirus, poliovirus, and reovirus, likely determine the use of specific bacterial components.
Viruses have evolved as genome packaging machines to efficiently transfer nucleic acids between susceptible host cells, ensuring replication. The majority of viruses have hollow, quasi-spherical shells rather than tubular structures, perhaps because this gives the most efficient packaging of nucleic acid with a fixed copy number of coat protein subunits. In non-enveloped viruses, the volume enclosed by the (usually) icosahedral structure is a constraint on the size of the genome, giving a limited capacity to encode capsid proteins, and usually restricts the genome copy number, or ploidy of the virion, to one. Most membrane-enveloped viruses are also quasi-spherical, but their symmetry is frequently less well-ordered, which is usually described as pleomorphic. This feature allows some flexibility in volume, which could accommodate variation in the size of the genome or its copy number. Nevertheless, most viruses, irrespective of their architecture, appear to have evolved to encapsidate only a single copy of their genome within the protein or protein/lipid shell, or a dimeric copy in retroviruses. Notable exceptions are the Paramyxoviridae and the Birnaviridae where particles may contain up to four copies of the RNA genomes,. Although some strains of influenza can produce elongated virions, there is a mechanism that selectively encapsidates only one set of genome segments in each virion.
The Filoviridae family, including the Ebolavirus and Marburgvirus genera, cause haemorrhagic fevers with high mortality in humans, and no effective treatments are currently approved, although candidate vaccines are promising. The 18.9 kb single-stranded negative-sense non-segmented RNA genome of Ebola virus (EBOV) codes for at least eight proteins. The ribonucleoprotein complex is composed of the nucleoprotein (NP), polymerase protein (L), VP24, VP30, and VP35. The trimeric transmembrane glycoprotein (GP) forms surface spikes on the virion envelope and also has a soluble form, while the matrix protein, VP40, is associated with the inner surface.,–. The GP spike, a class I fusion protein, mediates cellular attachment and entry and is extensively glycosylated, especially in the glycan-rich mucin-like domain–. Three proteins, VP24, VP35 and NP are essential for nucleocapsid formation. Although some of the major protein interactions that occur during EBOV morphogenesis have been characterised, , the three-dimensional (3D) structure and molecular arrangements have not been previously determined. Structural details are essential to understand how protection of the genome, cell binding, entry, and immune evasion are achieved in a filamentous animal virus, and to determine how this unique morphology plays a role in pathogenesis.
Research on filoviruses has been hampered by their status as biosafety level 4 pathogens. Previous investigations of filovirus structures within embedded, sectioned and metal-stained cells by electron tomography revealed few details of the high resolution oligomeric structure,. It has been demonstrated that aldehyde-fixation alone, and subsequent cryo-electron microscopic imaging in the frozen-hydrated state preserves structures, at least up to 12 angstroms resolution, and in some cases, fixation improves the resolution achievable. In addition, it has also been shown that high-resolution X-ray structures can also be obtained in the presence of aldehyde fixatives. Therefore, we analyzed purified and isolated EBOV and Ebola virus-like structures using cryo-electron microscopy (cryo-EM), and cryo-electron tomography (cryo-ET). In the current study, the Zaire strain of EBOV was purified and inactivated by paraformaldehyde fixation: excess fixative was then removed by dialysis to reduce beam damage for imaging in the frozen-hydrated state. The flash-freezing at liquid ethane temperatures used in cryo-electron microscopy preserves the structural and molecular detail, avoiding artifacts associated with conventional EM methods, such as dehydration and/or sectioning or staining, that usually prevent detailed structural analysis. Digital image processing reveals the 3D organization of EBOV, including the structural arrangement of component molecules at resolutions of 14–19 Å.
The current clinical standard for filoviral infection is supportive care as there are currently no FDA-approved treatment strategies. Supportive care consists of oral fluid rehydration, oral medication, nutritional supplementation, and psychosocial support. Nasogastric feeding tubes and i.v. administration of both fluids and medication are increasingly considered supportive care where possible during outbreak scenarios to prevent dehydration and facilitate support of blood pressure. However, given the limited equipment and laboratory support during outbreaks, care must be taken to prevent overaggressive fluid administration. Fluid replacement was evaluated briefly in rhesus macaques, and while there was no significant benefit to survival, a less severe renal compromise was observed. While supportive care may (or may not) reduce the overall case fatality rate in humans, the true impact of simple interventions such as fluid management has yet to be fully evaluated and the potential for benefit in combination with direct antiviral measures has yet to be assessed.
Viruses with RNA genomes are major causes of emerging deadly infectious diseases and represent the most ever-present and everlasting changing cellular parasites known. There are many different alterations of genetic elements occurring during RNA genome replication. This plasticity is fundamental and caused by infidelity of the replication process. Evolution of RNA viruses is basically unpredictable due to the stochastic nature of the mutation and recombination events, as well as environmental factors. A fundamental concern regarding RNA viruses is their high mutation rate. RNA viruses exist as quasispecies which are the result of pressure, mutation, and selection, in ways which are poorly understood. These mutations could affect the binding capability of the virus to both neutralizing antibodies and host receptors which could result in emergence of new viral host ranges. The chance that defined molecules may promote selection of escape mutants or a particular detection tool may fail to detect the virus is high.
The mechanisms of viral infection and prevention are the most difficult investigations in virology research. Virus tropism and neutralizing antibodies are the major crucial steps in virus life cycles and vaccine development respectively. To better understand the molecular pathways of infection and prevention, we must utilize synthetic biology. Synthetic biologists would simplify and engineer complex artificial biological systems that investigate natural biological phenomena.
The current generation of synthetic vaccines, diagnostic reagents (monoclonal antibodies (mAbs), peptide antigens, oligonucleotides, etc.) and therapeutics may be very adequate to prevent, detect and cure infections due to DNA viruses. Unfortunately, these fail to address RNA viruses. This is largely due to the vast number of circulating quasispecies associated with RNA pathogens. The dynamics of viral quasispecies mandates careful consideration. In reality, prevention and therapy of infection due to an RNA virus should rely on multicomponent vaccines and antiviral agents that address the complexity of the RNA quasispecies mutant spectra. This can be accomplished using the RNA Qβ coliphage display peptide library.
Phage display technology has been developed, applied extensively and proven to have success in exposing functional peptides on the exterior surface of phage. A novel display system which exploits the high mutation rate of RNA replicase has been developed. A small peptide library has been presented on this RNA phage surface and selected against the SD6 mAbs of the foot-and-mouth disease virus (FMDV). The membrane-proximal external region (MPER) of the human immunodeficiency virus type-1 (HIV-1) was engineered on the surface of Qβ as well. These studies use the Qβ phage for its large population and high mutation rate. The novel pipeline proposed herein takes into consideration the quasispecies nature of these pathogenic RNA viruses. This review will present a detailed structural and functional synopsis of the Qβ platform and emphasize the methodologies and medical applications associated with the system.
The influenza virus belongs to the class known as Orthomyxoviruses [1, 2] and can be divided into three distinct subtypes (influenza A, influenza B or influenza C), depending on its nucleoproteins (NPs) and the antigen determinants of its matrix proteins. The influenza virus is a negative-sense, single-stranded RNA (ssRNA) virus that is segmented into eight (Subtypes A, B) or seven (Subtype C) separate strands. Each segment codes for at least one protein, and based on the extensive studies of the influenza A virus (IAV) in recent years, it seems likely that all subtypes express at least twice as many proteins as its number of segments.
Influenza is an envelope virus, where viral nucleocapsid (genome + NPs) is protected by a lipid bilayer derived from the modified host cell’s membrane. The outer layer of the influenza virus is studded with the glycoproteins hemagglutinin (HA) and neuraminidase (NA). On the virion, the HA is found in the form of a homotrimer (HA-trimer), while the NA forms into a homotetramer (NA-tetramer). The HA-trimers are relatively evenly distributed on the surface of the virion in the lipid bilayer, whereas the NA-tetramers appear to localize at one end of the virion, most likely at the exit point of the budding virion. Recently, the distribution of the HA-trimers into larger conglomerates was observed; however, the biological consequences of these processes are not well understood.
The IAVs are among the fastest evolving families of viruses and are notable for their ability to rapidly change from one season to the next or even within seasons. Over the course of a few generations, the virus can change sufficiently to become virulent. Like many emerging infectious diseases such as Ebola virus or severe acute respiratory syndrome (SARS), IAV depends on animal reservoirs, where they can reside [9–13] and transfer between species that share the same environment. As one major reservoir involves migratory birds, the potential to rapidly spread an infection throughout the world is of major concern and IAV is under constant watch (http://www.who.int/csr/don/archive/year/en/). During the past 100 years, there have been three major epidemics with extensive fatalities and one minor one (mostly because of quick action from the WHO and health practitioners) [17, 18], the Spanish flu (1918, H1N1, 75 million fatalities worldwide), the Asian flu (1957, H2N2, 2 million fatalities), the Hong Kong flu (1968, H3N2, 1 million fatalities) and more recently the swine flu (2009, H1N1, 14 000 fatalities).
Here, we briefly review topics heavily associated with RNA in the IAV, particularly our current knowledge on how the IAV genome is packaged, how the RNA interacts with proteins, what protein products are generated from alternative splicing in the IAV genome and our present knowledge of RNA structure. We also summarize our current understanding of the infection cycle with a focus on how the viral RNA (vRNA) sequences manage to enter the nucleus of the cell, carry out the transcription and replication process and export of the viral genetic material from the nucleus before the final budding and release of the virion. Finally, considering more than one virion can infect a given cell at the same time, it is also possible to shuffle segments between different viral strains in a process known as reassortment. We discuss briefly on the recent discoveries of RNA/RNA interactions between the vRNA segments that help in the packaging of the viral genome. Such interactions would have implications on the likelihood of such reassortment events.
Please see Additional file 1 for translations of the abstract into the six official working languages of the United Nations.
Streptococcus suis serotype 2 (S. suis 2, SS2) is an important zoonotic pathogen that causes severe porcine infectious diseases, including arthritis, meningitis, and pneumonia. Virulent strains of SS2 can also be transmitted to humans (especially abattoir workers and pork handlers) by direct contact, causing meningitis, permanent hearing loss, septic shock, and even death. Two large-scale outbreaks of severe SS2 epidemics occurred in China in 1998 and 2005, causing great economic losses in the swine industry. These two outbreaks also posed serious public health risks from the newly emerging streptococcal toxin shock syndrome (STSS), which claimed 52 lives. Over the past decade, considerable attention has been given to the study of virulence factors (e.g., CPS, MRP, EF, and suilysin) and the pathogen-host interaction in this emerging pathogen. However, comparative studies at the whole-genome level had little done to decipher the evolutionary aspects by which the virulence and environmental adaptation of SS2 are shaped.
To shed light on the evolution of pathogenicity and potential genomic polymorphisms of SS2, several virulent strains were subjected to whole-genome sequencing and comparative genomic studies. Comparative analysis of the whole-genomic DNA sequence of the European S. suis strain P1/7 (by the Sanger Institute) and two representative highly virulent strains (98HAH12 and 05ZYH33) isolated from STSS patients during the two epidemic outbreaks in China uncovered a candidate pathogenicity island (PAI) named 89K, which has been confirmed to undergo horizontal gene transfer (HGT) by our recent work. Further analysis based on PCR amplification revealed that 89K exclusively present in the epidemic strains in these two Chinese SS2 outbreaks but not in other domestic clinical isolates or international virulent strains. However, analysis of the unfinished genomic sequence of SS2 strain 89/1591 (by the DOE Joint Genome Institute) revealed that a partial 89K sequence (~30 kb) is present in this typical North American virulent strain. Similarly, results from a recently published work suggest that S. suis strain BM407, which was isolated from a human meningitis case in Vietnam in 2004, contains two regions with extended similarity to 89K. These findings led us to hypothesize that the genome of SS2 would be highly polymorphic among different strains.
In this study, we employed the comparative genome re-sequencing (CGS) approach developed by Roche NimbleGen Systems to investigate genomic diversity in a collection of 18 SS2 strains, including isolates from the two outbreaks in China, other virulent strains from China (isolated before these outbreaks), virulent strains from European countries, and several avirulent strains. Although CGS cannot identify recently gained genes due to technical limitations, the DNA microarray-based comparative genome sequencing technique allows high resolution detection of sequence polymorphisms based on a reference genome. Using this technology, we identified a number of novel genetic polymorphisms in SS2 strains and several candidate virulence factors that may contribute to STSS. Our results provide new insight into the virulence mechanisms and genome dynamics of SS2, which will help to elucidate the evolution of SS2 strains and better monitor the incidence and spread of epidemic strains.
Human metapneumovirus (HMPV) is a new member of the Paramyxoviridae that was first identified in children with respiratory diseases in Netherlands. The clinical symptoms that are caused by HMPV infections in children are similar to those observed with respiratory syncytial virus (RSV) infection, ranging from upper respiratory tract infection to bronchiolitis and pneumonia. HMPV has become recognised as a major cause of lower respiratory infection in children.
The mature HMPV particle is surrounded by a lipid envelope in which the virus fusion (F) and attachment (G) proteins are inserted. The F protein mediates fusion of the virus and host cell membranes during virus entry, while a primary role for the G protein in virus attachment to susceptible cells has been demonstrated. The virus envelope surrounds a protein layer formed by the matrix (M) protein, and a ribonucleoprotein (RNP) complex that is formed by the viral genomic RNA (vRNA), the nucleocapsid (N) protein, the phosphoprotein (P protein), the M2-1 protein and the large (L) protein. Based on genetic analysis of HMPV genome sequences two major HMPV genotypes, called HMPV A and B, have been identified [7–9].
Much of the current understanding of the biology of the HMPV can be inferred from other closely related viruses e.g. RSV and avian pneumovirus. Primary isolation of HMPV has been achieved in several different cell lines, and some tissue culture adapted isolates have been described. However, their cultivation can require up to 14 days incubation before cytopathic effects are visualised. This low level of virus replication in standard cell culture, particularly low-passaged clinical isolates, and the subsequent recovery of low levels of infectious HMPV, have hampered functional biochemical studies on the virus. These studies usually require higher levels of biological material that can be achieved following a single cycle of HMPV replication.
Visualising the distribution of individual virus structural protein is a prerequisite for understanding the process of HMPV maturation, and in situ imaging of virus-infected cells stained using virus protein specific antibodies is in general the most direct and unambiguous method to do this. Therefore, in this current study we have circumvented the problems associated with low virus replication rates by using imaging to examine HMPV morphogenesis. This has allowed us to visualize the morphogenesis of a low passaged HMPV clinical isolate in mammalian tissue culture, and to suggest a role for lipid-raft microdomains and F-actin in the process of HMPV maturation.
Human metapneumovirus (HMPV) is a new member of Paramyxoviridae that was first identified in children with respiratory diseases in Netherlands. The clinical symptoms that are caused by HMPV infections in children are similar to those observed with respiratory syncytial virus (RSV) infection; ranging from mild symptoms to pneumonia. HMPV is now a globally recognised cause of lower respiratory infection in children). Genetic analysis identified two major genogroups A and B. HMPV expresses two major integral membrane proteins that play a role in virus entry. The attachment (G) protein plays a role in virus attachment and is expressed as a single polypeptide chain, which subsequently undergoes extensive N- and O-linked glycosylation. The fusion (F) protein mediates fusion of the virus and host-cell membranes, and is initially synthesised as a single polypeptide chain (F0) that undergoes proteolytic cleavage to generate the mature and active form of the protein, consisting of F1 and F2 protein subunits. The virus also expresses a third membrane-associated protein called the matrix (M) protein, which is analogous to the M protein of RSV and is a major determinant of virus morphology.
Primary isolation of HMPV has been achieved in several different cell lines, however tissue culture adapted isolates can require up to 21 days incubation before cytopathic effects are visualised. This low level of virus replication and the subsequent recovery of low levels of infectious HMPV in standard cell culture have hampered biochemical studies on the virus. These experimental methodologies usually require higher levels of biological material than can be achieved following HMPV infection.
Virus-like particle (VLP) formation following the co-expression of specific virus structural proteins has been demonstrated in several paramyxoviruses. These studies have allowed the identification of essential virus proteins that are required for virus particle assembly. Although a central role for the M protein in VLP formation has been reported for human parainfluenza type 1 virus and Newcastle disease virus, the expression of the M protein alone was insufficient for VLP production in simian virus type 5 and avian pneumovirus type C. The use of recombinant HMPV protein expression to drive the formation of similar HMPV VLPs can potentially overcome the problems associated with the poor cultivation of HMPV in tissue culture. In addition, by direct cloning of the virus genes from clinical material the expression of gene sequences that have not been subjected to extensive tissue culture adaptation can be examined. This therefore affords a relatively simple experimental system with which to examine HMPV morphogenesis. In this study we have examined the capacity of the HMPV F, G and M proteins to form VLPs in mammalian cells, and to further examine the minimal virus protein requirements that lead to VLP formation.
The members of the filovirus family, Ebola virus (EBOV) and Marburg virus (MARV), cause a severe hemorrhagic fever in infected humans with high fatality rates. Infected individuals who go on to succumb to filovirus infection exhibit dysregulated immune responses (reviewed in two other articles in this issue). This appears to result from several factors, including viral mediated impairment of early innate immune responses and consequent dysregulation of innate immunity. Some reports have suggested that adaptive immune responses may occur [3–6], but it is evident that these fail to clear the disease. Lymphopenia resulting from apoptosis as the infection progresses has also been suggested to contribute to the failure to clear the infection. Studies of both human survivors and murine model systems suggest that a well-regulated cytokine response early in the course of the infection may be critical to the outcome of the disease.
Ebolaviruses are currently subdivided into four distinct species, Zaire ebolavirus (ZEBOV), Sudan ebolavirus, Tai Forest ebolavirus, and Reston ebolavirus (REBOV), while there is only a single MARV species (Lake Victoria marburgvirus) (ICTV virus taxonomy 2009). Since the Bundibugyo isolate is genetically distinct from the known Ebola viruses, a suggestion has been made to classify it as a new EBOV species, Bundibugyo ebolavirus. The different EBOV species not only show significant molecular differences, they also vary in terms of virulence and pathogenicity. The most pathogenic species in humans is ZEBOV with a case fatality rate of about 80%, followed by Sudan with a case fatality rate of about 50%, and Bundibugyo with a fatality rate of about 30%. To date, there are two reported non-fatal human cases of Tai Forest ebolavirus and several asymptomatic human cases of REBOV infection [15–17].
The first reported MARV outbreak occurred in Germany and Yugoslavia in 1967 and was caused by infected African green monkeys imported from Uganda. Since this outbreak was associated with a case fatality rate of 22%, it was believed for a long time that MARV was less pathogenic than EBOV. However, recent outbreaks of MARV in the Democratic Republic of the Congo in 1998–2000 and in Angola in 2004 were associated with fatality rates up to 90%, indicating that MARV can be as virulent as EBOV [20–22].
Despite the severity of the disease, filoviruses have been regarded as exotic pathogens with fatal outbreaks restricted to Central Africa, and with no major health threat outside of the endemic areas. Knowledge on their biology and pathogenicity consequently remained limited. However, there has been renewed interest given the potential for using filoviruses in bioterrorism attacks and the possibility for infected, asymptomatic persons for bringing the disease to other countries. Indeed, two cases of MARV have been reported in the Netherlands and in the United States, both tourists returning from trips to Uganda. Together, the potential for spread outside central Africa has reignited research endeavors to elucidate the biology of the filoviruses and to develop effective therapeutic strategies.
In this review we will describe how filoviruses enter their target cells, replicate their genomes and assemble progeny viruses by exploiting cellular machineries. We will also briefly touch upon the interaction of filoviruses with cellular signaling pathways. Finally, we will discuss the current understanding of the fate of infected and non-infected cells in filovirus infection. In addition, we will present ultrastructural data of infected and non-infected cells, demonstrating the morphological changes in filovirus infection.
Streptococcosis is regarded as a leading infectious disease in the swine industry, that clinically features with meningitis, septicemia, or arthritis and annually results in significant economic loss worldwide.1
Streptococcus suis (S. suis) that was initially reported in 19542 has been demonstrated as an etiological agent for this kind of frequently-occurring bacterial infection.1,3 Indeed, S. suis, a complex population consisting of heterogeneous strains,4 can be classified into 35 serotypes (1–34, 1/2) based on the differentiation of capsule antigens.1,3 Based on the varied virulence of these bacteria, they may be categorized into highly-pathogenic, weakly-pathogenic (hypo-virulent), and nonpathogenic (avirulent) strains.1 Generally, serotype 2 of S. suis (SS2) is considered to be the most virulent, and is frequently isolated from clinically-diseased piglets.1 In fact, serotype 9 of S. suis is also one of the most important serotypes in several countries. Of particular note, SS2 seems to be a previously neglected but recently emerging human pathogen,5 whose infection has become increasingly potent, especially in the southeast Asian countries like Thailand,6 Vietnam,7 and China.8,9
As the primary agent of meningitis, septicemia, arthritis and as an opportunistic pathogen in the case of pneumonia,1,5
S. suis have been reported to have spread over 30 countries and/or regions (Fig. 1) and has claimed no less than 1600 human cases, some of which were fatal.2 Also, similar clinical symptoms including bacterial meningitis, septicemia, and arthritis are frequently observed in human SS2 infections.2,3 Occasionally, serotypes other than SS2, including SS1,10 SS4,10 SS5,11,12 SS14,13,14 SS16,15,16 and SS2411 can also be found to function as the causative agents responsible for sporadic cases of human S. suis infection.3 Of note, two big outbreaks of human SS2 endemics which occurred in China, in 1998 and 2005, respectively,9,17,18 have raised serious concerns in public health and have challenged the conventional opinion that human SS2 infections are only present in sporadic cases.2,8,19 Unfortunately, no specific/effective human therapeutics or vaccine against SS2 infections is available thus far. Considering the severity (high mortality and modality) of SS2 infection in humans,5,8 it is important to develop a method for convenient and quick diagnosis, which can be applied toward local SS2 detection.4,18
Over the past four decades, significant progress has been made toward better understanding the highly infectious clones of S. suis. At the time of formulating this review, 1104 articles were available in PubMed regarding S. suis (http://www.ncbi.nlm.nih.gov/pubmed/?term=Streptococcus+suis).Totally, over 20 bacterial virulence-associated factors have been identified that include capsular polysaccharides (CPS),20 Muramidase-released protein (MRP),21 and Suilysin (SLY).22 To date, genomic sequences of a collection of S. suis strains are available (Fig. 2), the majority of which are derived from SS2 species,23,24 except two newly-released genomes which correspond to SS325 and SS14,26 respectively. Genomic mining combined with bacterial genetics have elucidated that Chinese epidemic strains of highly pathogenic S. suis 2 carry a specific 89K PAI (pathogenicity island).23,27 Further studies suggested that 89K PAI with a transposon-like essence can undergo GI-type T4SS-mediated horizontal transfer in epidemic SS2 species.28 The systematic elucidation of the of S. suis pathogenesis in the Omics Era was illustrated by functional definition of a collection of other new genes or putative orthologs (such as Zur, a zinc uptake regulator,29 CovR, an orphan response regulator,30 and Rgg-like transcription factor31) following the release of the genome sequence of SS2 (e.g., 05ZYH33).23 Although we have gained a partial glimpse of the molecular mechanism underlying the high pathogenicity of SS2 itself, we are still lacking further insights into the interface between the SS2 pathogen and the host it infects.3,8
In this review, we aim to describe an updated but partial picture of SS2 as an emerging infectious agent, which centers on five aspects: global epidemiology/distribution, clinical diagnostics/typing, pathogenesis, protective antigen/candidate vaccine, and zoonotic potential.
The distal gastrointestinal tract of all animals is colonized by a diverse array of bacterial, fungal, and protozoan species. An animal host maintains a mutualistic relationship with its microbial inhabitants in which the microbes provide protection from pathogenic bacteria through competing for ecological niches and fostering a stable environment for the development of host immunity (Bäckhed et al., 2005). Many vertebrate intestines (such as mice, rats, chickens, humans, and turkeys) harbor commensal organisms named segmented filamentous bacteria (SFB) that bind specifically to the host intestinal epithelium. These organisms are closely related to the genus Clostridium, and appear as long, segmented filaments that bind tightly to the host epithelium via a specialized structure (Chase and Erlandsen, 1976). These bacteria were initially detected through microscopic examination of the gastrointestinal epithelium of mice (Davis and Savage, 1974). SFB drew the attention of researchers due to their unique morphology, life cycle and binding location (Schnupf et al., 2013). Since their discovery, a large body of research has been generated to characterize these bacteria and understand their role in the host-microbiome relationship. Through examining mouse and rat models, it has been revealed that these bacteria play an important role in adaptive and innate immunity of the host.
The interferon (IFN) system is an integral component of innate immunity and an important first-line defense against invading viruses. The IFN system is triggered by sensors that recognize pathogen-associated molecular patterns and, upon ligand binding, induce signaling cascades that trigger the production of IFN (1, 2). Binding of IFN to IFN receptors then induces the expression of IFN-stimulated genes, several of which encode proteins with antiviral activity (3). Understanding how these antiviral effectors block viral spread may allow devising novel antiviral strategies and is thus the focus of many current research efforts.
The family of IFN-induced transmembrane proteins (IFITMs) comprises five members (in humans), including the antivirally active proteins IFITM1, -2, and -3 (4). These proteins inhibit host cell entry driven by the glycoproteins of many enveloped viruses, including influenza A viruses (FLUAV), coronaviruses, and filoviruses (5–12). Expression of IFITMs blocks entry at the stage of glycoprotein-driven fusion of viral and cellular membranes, specifically during hemifusion or the formation of fusion pores (13, 14). This blockade might be due to IFITMs modifying the physical properties of cellular membranes, potentially via IFITM-IFITM interactions (15) or by altering membrane cholesterol levels (16).
The IFITM-mediated blockade of viral entry seems to be restricted largely to viruses that enter target cells via fusion with endo- or lysosomal membranes, although IFITM1 can be expressed at the cell surface (8). Thus, one would assume that entry of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), which is believed to proceed mainly at the plasma membrane, is not inhibited. The laboratories reporting the identification of IFITMs as antiviral factors indeed failed to detect an IFITM-dependent blockade of HIV-1 infection (4, 6). Nevertheless, subsequent studies reported that IFITMs restrict HIV and SIV entry (11, 17, 18). More recently, it was reported that IFITMs are incorporated into progeny HIV and SIV virions and that IFITM expression in infected cells reduces the infectivity of progeny virions (19, 20). The negative impact on infectivity might be due to IFITM interactions with Env, which result in reduced Env processing and incorporation into virions (21). However, the HIV-1 and SIV sensitivity to IFITM is isolate specific and the reasons why some isolates are efficiently inhibited while others are not are unknown (17, 21).
Here, we addressed the question of whether determinants other than the Env protein could impact sensitivity of viral entry to inhibition by IFITM proteins. Such a scenario might account, at least in part, for the strain-specific differences in IFITM sensitivity discussed above and might explain why IFITM sensitivity of HIV/SIV entry was not universally observed. For this, we used previously described vector systems that allow for sensitive detection of viral entry and for robust and comparable expression of IFITM proteins in transduced cells (22, 23). We report that the virion context in which viral envelope proteins are presented as well as the efficiency of Env incorporation into particles can impact sensitivity to IFITM, suggesting that the determinants controlling inhibition of viral entry by IFITMs are more complex than initially appreciated.
This retrospective descriptive study took place at the paediatric wards of Hamad General Hospital (HGH), a 603- bed, tertiary-care facility in Doha, for two consecutive years, 2010 and 2011. The institutional review board at Hamad Medical Corporation approved the study, IRB number 12054.
Bronchiolitis is considered one of the earliest and most common causes of hospitalisation among young children during their first 2 years of life. Although the causative agents include bacteria, fungi and viruses, this acute infection is mainly attributed to respiratory syncytial virus, accounting for 50–90% of the cases [2, 3]. With the current utilisation of reverse transcriptase real-time polymerase chain reaction (PCR), the detection of specific viral nucleic acids facilitated a better understanding of the viral aetiology of the infection. The 2014 American Academy of Paediatrics bronchiolitis guideline recommends against the routine use of radiographic or laboratory studies on the basis that knowing the infecting pathogen would rarely alter the clinical management. However, a growing body of literature has identified an association between specific infecting pathogens with short and long-term outcomes. Association between viral co-infection and disease severity have been assessed in several studies but with conflicting findings [5–7]. Although bronchiolitis is a self-limiting condition, hospitalisation rate has increased during the last two decades. Assessment of bronchiolitis severity, through a combination of clinical symptoms and physical signs, remains a standard measure in daily practice though its impact on clinical outcomes, such as length of hospital stay, has yet to be confirmed. Data has been reported on seasonal variation of viral activity with conflicting evidence on its significance on disease severity and clinical outcomes [8, 9]. In Qatar, viral aetiology of bronchiolitis in children has been limited. Therefore the aims of this retrospective, descriptive study were: to determine the frequency and seasonal trends of viral pathogens causing acute bronchiolitis, and to explore the association between specific viral pathogens, disease severity and length of stay (LOS).