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Central nervous system (CNS) infections including meningitis and encephalitis are important causes of significant mortality and morbidity in the developing nations. Viruses are considered as important etiological agents of CNS infections, causing diseases ranging from febrile illness to myelitis to meningoencephalitis. However, in most cases, the etiology of CNS infection is not known due to lack of diagnostic capacity, standard clinical case definitions, or low levels of surveillance. Specific diagnosis for CNS infection is rarely made and usually categorized empirically as only “viral” or “bacterial”. There have been few reports on the viral etiologies of CNS infections in Indonesia except for Japanese encephalitis virus (JEV), a leading cause of acute encephalitis in children and young adults in the Southeast Asian region [2–5]. Still, JEV is significantly underreported in Indonesia. Furthermore, in endemic provinces like Bali where encephalitis is often suspected to be JEV, there is lack of laboratory capability to accurately determine the disease burden of JEV and other CNS viruses. The objective of this study was therefore to detect and identify the pathogens responsible for viral CNS infections amongst in-patients at a referral hospital in Manado, North Sulawesi, Indonesia.
Viruses in the genus Alphavirus belong to the group IV Togaviridae family and include nearly 30 virus species. Alphaviruses are able to infect humans and various vertebrates via arthropods, such as mosquitoes. The 11–12 kb Alphavirus genome is a single-stranded positive sense RNA flanked by a 5’ terminal cap and 3’ poly-A tail, and composed of four non-structural proteins genes (nsP1 to nsP4) and five structural proteins gene (C (nucleocapsid), E3, E2, 6 K, and E1 proteins). Getah virus (GETV) is a mosquito-borne enveloped RNA virus belonging to the Semliki Forest virus (SFV) complex in the genus Alphavirus. To date, 10 strains of GETV have been isolated in China: M1, HB0234, HB0215-3, YN0540, YN0542, SH05-6, SH05-15–17 and GS10-2. GETV has been shown to cause illnesses in humans and livestock animals and antibodies to GETV have been detected in many animal species worldwide.
The identification of novel virus species is important for the identification and characterization of disease. However, present research methods are mostly applicable for known viruses but few methods exist to characterize unknown viruses. Current molecular biological techniques for the identification of new virus species are troublesome since some viruses do not replicate in vitro but some may cause a cytopathic effect. Furthermore, specific techniques that require sequence identification are not applicable. To overcome these limitations, we developed a new method for virus discovery: Virus-Discovery-cDNA RAPD (VIDISCR), based on the cDNA-random amplified polymorphic DNA technique (cDNA-RAPD). VIDISCR includes two key steps. First, the virus genome nucleic acid must be isolated without cellular RNA and DNA contamination. Second the RAPD analysis using the virus genome cDNA or DNA. Using this method, we tested known viruses (SV40 and SV5) and identified a new Getah virus YN08 strain. Virus nsP3, capsid protein genes, and 3’-UTR sequences were cloned, sequenced, and compared. The phylogenetic analysis indicated that the virus YN08 isolate is more closely related to Hebei HB0234 strain than the YN0540 strain, and genetically distant to the MM2021 Malaysia primitive strain.
This study was approved by the Medical Research Ethics Committee of R.D. Kandou General Hospital (Ethical Approval No. 066/EC-UPKT/III/2016) and Eijkman Institute Research Ethics Commission (Ethical Approval No. 78). Written informed consent for participating in the study was obtained from all of the patients, guardians, or accompanying close relatives.
Vaccine development against infectious diseases has classically been based on live attenuated or inactivated infectious agents. Recently, the approach of vaccination with recombinantly expressed antigens and immunogens from viral and non-viral delivery systems has been introduced to the repertoire. In this context, immunization with surface proteins and antigens has elicited strong humoral and cellular immune responses and vaccinated animals showed protection against challenges with lethal doses of infectious agents or tumor cells.
The types of non-viral vectors applied include liposomes, immunostimulatory complexes (ISCOMs) composed of adjuvant Quil A and peptides, and multiple antigen peptides (MAPs) also known as dendrimers. A number of viral vectors based on adenoviruses, alphaviruses, avipoxiviruses, enteroviruses, flaviviruses, measles viruses (MV), rhabdoviruses, and vaccinia viruses have been engineered for vaccine development. In this context, self-replicating RNA virus vectors have proven highly efficient for immunization studies in various animal models. Among RNA viruses, rabies virus (RABV) and vesicular stomatitis virus (VSV) belonging to the rhabdovirus family carry a single-stranded RNA (ssRNA) genome of a negative polarity. Likewise, MV possess a negative-sense ssRNA genome. In contrast, flaviviruses and alphaviruses are of positive polarity. West Nile virus and Kunjin virus are the most common flaviviruses applied for immunization studies. Similarly, expression vectors have been engineered for alphaviruses such as Semliki Forest virus (SFV), Sindbis virus (SIN) and Venezuelan equine encephalitis virus (VEE).
In this review, various self-replicating RNA virus vectors are described and their applications as recombinant virus particles, RNA replicons and layered DNA plasmids are compared. Moreover, examples are given of utilization of self-replicating RNA virus systems for immunization studies in various animal models to elicit humoral and cellular immune responses and to generate neutralizing antibodies, as well as protection against challenges with pathogens and tumor cells. Finally, a summary of clinical trials already conducted or in progress that apply self-replicating RNA viruses is presented. However, due to the large number of publications available, it is only possible to present key findings and examples of vaccine development for self-replicating viral vectors.
Bats and the viruses they harbor have been of interest to the scientific community due to the unique association with some high consequence human pathogens in the absence of overt pathology. Virologic and serologic reports in the literature demonstrate the exposure of bats worldwide to arboviruses (arthropod-borne viruses) of medical and veterinary importance. However, the epidemiological significance of these observations is unclear as to whether or not bats are contributing to the circulation of arboviruses.
Historically, a zoonotic virus reservoir has been considered a vertebrate species which develops a persistent infection in the absence of pathology or loss of function, while maintaining the ability to shed the virus (e.g., urine, feces, saliva). Haydon et al. extended this definition of a reservoir to include epidemiologically-connected populations or environments in which the pathogen can be permanently maintained and from which infection is transmitted to the defined target population. The significance of the relative pathogenicity of the infectious agent to the purported reservoir host has been debated. In the case of bats as a reservoir species, rigorous field and experimental evidence now exist to solidify the role of the Egyptian rousette bat (Rousettus aegyptiacus) as the reservoir for Marburg virus. Considering arboviruses, additional criteria must be met in order to consider a particular vertebrate species a reservoir. Reviewed by Kuno et al., these criteria include the periodic isolation of the infectious agent from the vertebrate species in the absence of seasonal vector activity, and the coincidence of transmission with vector activity. Further, the vertebrate reservoir must also develop viremia sufficient to allow the hematophagous arthropod to acquire an infectious bloodmeal in order for vector-borne transmission to occur. Bats have long been suspected as reservoirs for arboviruses, but experimental data that would support a role of bats as reservoir hosts for certain arboviruses remain difficult to collect. Here we synthesize what information is currently known regarding the exposure history and permissiveness of bats to arbovirus infections, and identify knowledge gaps regarding their designation as arbovirus reservoirs.
Alphaviruses are single-stranded RNA viruses with an envelope structure belonging to the family of Togaviridae. Certain alphaviruses have been associated with pathogenicity, resulting in global fever epidemics, such as observed recently for Chikungunya virus. Furthermore, Semliki Forest virus (SFV) and Venezuelan equine encephalitis (VEE) virus have been identified as the causes of an outbreak of febrile illness in Central Africa and an epidemic in horses and humans in South America, respectively. Despite this potential concern, several alphaviruses, including SFV, Sindbis virus (SIN) and VEE have been subjected to the engineering of vectors for heterologous gene expression. In these cases, attenuated strains have been employed.
Several types of vector systems have been engineered. There are three types of replication-deficient vectors consisting of naked RNA, recombinant particles and layered DNA vectors (Figure 1). The application of naked RNA vectors involves the use of in vitro transcribed RNA from an expression vector consisting of the viral nonstructural replicase genes and the foreign gene of interest downstream of the strong subgenomic promoter. The production of recombinant particles requires the co-transfection of in vitro transcribed RNA from an expression vector (as described above) and a helper vector supplying the viral structural genes into mammalian cell lines (for example, baby hamster kidney (BHK) cells). The generated particles are capable of one round of infection of a broad range of host cells, but due to the selective packaging of only expression vector RNA, no further virus production occurs. The layered DNA vector system consists of delivery of a DNA vector providing foreign gene expression from a CMV promoter. Furthermore, the engineering of vectors with an additional subgenomic promoter to the full-length genome allows for the generation of replication-proficient particles, which can provide improved delivery and extended gene expression. All alphavirus vectors described take advantage of the extremely efficient RNA replication, resulting in some 200,000 RNA copies from each RNA molecule. The essential question is: which vector system to use? Obviously, replication-proficient particles can provide efficient delivery, but suffer from potential insufficiency related to safety aspects. Although replication-deficient particles provide a higher level of safety, there is still a marginal risk of the generation of replication-proficient particles through non-homologous recombination. To minimize any unwanted recombination events, a split helper vector system with capsid and envelope genes expressed from separate helper vectors has been engineered.
So far, alphaviruses have been applied for the expression of a number of topologically different recombinant proteins. Particularly, the use of SFV particles has resulted in high expression levels of integral membrane proteins in various mammalian host cell lines, in primary neurons and in vivo. For vaccine development, vectors based on SFV, SIN and VEE have been applied as naked RNA, recombinant virus particles and layered DNA vectors. In this context, viral and tumor antigens have been administered in various animal models to elicit neutralizing antibodies and protection against challenges with tumor cells or lethal doses of viruses. Moreover, non-viral pathogens have been subjected to vaccine development. Replicon particles derived from VEE have furthermore demonstrated activity as safe and potent systemic, mucosal and cellular adjuvants when co-administered with antigen. Finally, as alphaviruses have been identified as the cause of viral epidemics in animals and humans, a number of approaches have been initiated for immunization against alphavirus-based infections. In this review, the latest development on alphavirus vectors for vaccine production is summarized.
Vesicular stomatitis is a viral disease which primarily affects cattle, horses, and swine. It occurs in enzootic and epizootic forms in the tropical and subtropical areas. The disease is rarely life-threatening but can have a significant financial impact on the horse industry. Vesicular stomatitis virus (VSV) is the prototype of the genus Vesiculovirus in family Rhabdoviridae. The virus has two serologically distinct serotypes, VSV-New Jersey (NJ) and VSV-Indiana (IND). The neutralizing antibodies generated by these two serotypes are not cross-reactive. The IND serogroup has three subtypes IND-1 (classical IND) IND-2 (cocal virus) and IND-3 (alagoas virus) The virus is endemic in South America, Central America, Southern Mexico, Venezuela, Colombia, Ecuador and Peru but the disease has been reported in South Africa in 1886 and 1897 and France in years 1915 and 1917.
The disease has been reported across continents in Belize, Bolivia, Brazil, Colombia, Costa Rica, Ecuador, El Salvador, Guatemala, Honduras, Mexico, Nicaragua, Pakistan, Panama, Peru, USA and Venezuela [91, 92]. Outbreaks historically occurred in all regions of the USA but have been limited to western states in 1995, 1997, 1998, 2004, 2005, 2006, 2009, 2010, and 2012 [93, 94]. While VS has been reported in horses at about 800 premises in eight states. VSV spread to Europe during the First World War and periodically appears in South Africa. The Chandipura virus, a Vesiculovirus caused encephalitis outbreaks in different states of India leading to mortalities in children. Isfahan another virus in this genus is endemic in Iran [89, 97]. The countries with incidence/serological evidence of vesicular stomatitis are presented in Fig. (2).
Clinical disease has been observed in cattle, horses, pigs and camels whereas sheep, goats and llamas tend to be resistant. White-tailed deer and numerous species of small mammals in the tropics are considered as wild hosts. Many species, including cervids, nonhuman primates, rodents, birds, dogs, antelope, and bats have shown serological evidence of infection. Experimentally different animals like mice, rats, guinea-pig, deer, raccoons, bobcats, and monkeys can be infected.
The virus is zoonotic and causes flu-like symptoms characterized by fever, chills, nausea, vomiting, headache, retrobulbar pain, myalgia, sub-sternal pain, malaise, pharyngitis, conjunctivitis, and lymphadenitis in humans. Vesicular lesions may be present in the pharynx, buccal mucosa, or tongue. Encephalitis is rare but may occur in children [107, 108].
The transmission is more likely by trans-cutaneous or transmucosal route. The virus can be transmitted through direct contact with infected animals having lesions of the disease or by blood-feeding insects. In endemic areas, Lutzomyia sp. (sand fly) is proven biologic vectors. Black flies (Simulidae) are the most likely biologic insect vector in USA. Other insects may also act as mechanical vectors. Saliva, exudates and epithelium from open vesicles are sources of virus. Plants and soil are also suspected as the source of virus.
Horses of all ages appear equally susceptible but lesions do not appear in all susceptible horses. The lesions of the disease resemble foot-and-mouth disease in cattle and the other viral vesicular diseases in pigs. The horses are resistant to foot and mouth disease and susceptible to VS. VSV is the only viral vesicular disease of livestock that infects horses. VSV is also the most important of these four viruses as a zoonotic agent for humans. When vesicular stomatitis occurs in horses, blanched raised or broken vesicles or blister-like lesions develop on the tongue, mouth lining, nose and lips. In some cases, lesions also develop on the udder or sheath or the coronary bands of horses. Animals may become anorectic, lethargic and have pyrexia. One of the most obvious clinical signs is drooling of saliva or frothing at the mouth. The rupture of the blisters creates painful ulcers in the mouth. The surface of the tongue may slough. Excessive salivation is often mistaken as a dental problem or colic. There may be weight loss due to mouth ulcers as animal finds it too painful to eat. The lesions around the coronary band may cause lameness and laminitis. In severe cases, the lesions on the coronary band may cause the hoof to slough. Animals usually recover completely within two weeks. Morbidity rates vary between 5 and 70% but mortality is rare. Vesicular stomatitis like disease disabled 4000 horses during the Civil War in 1862. Major epidemics in the US occurred in 1889, 1906, 1916, 1926, 1937, 1949, 1963, 1982, and 1995, with minor outbreaks during many other years. No specific treatment is available for the disease. Anti-inflammatory medications as supportive care help to minimize swelling and pain. Dressing the lesions with mild antiseptics may help avoid secondary bacterial infections. If fever, swelling, inflammation or pus develops around the sores, treatment with antibiotics may be required. The animals should be quarantined at least for 21 days after recovery of the last case before moving to other places. Vaccines for livestock are available in some Latin American countries.
Viral encephalomyelitis is an important cause of morbidity and mortality worldwide, and many encephalitic viruses are emerging and re-emerging due to changes in virulence, spread to new geographic regions, and adaptation to new hosts and vectors. The term encephalomyelitis refers to inflammation in the brain and spinal cord that results from the immune response to virus infection. In humans, the viruses most commonly identified as causes of viral encephalomyelitis are herpesviruses and RNA viruses in the enterovirus (e.g., polio, enterovirus 71), rhabdovirus (e.g., rabies), alphavirus (e.g., eastern equine, Venezuelan equine, and western equine encephalitis), flavivirus (e.g., West Nile, Japanese encephalitis, Murray Valley, and tick-borne encephalitis), and bunyavirus (e.g., La Crosse) families. Other virus families with members that can cause acute encephalitis are the paramyxoviruses (e.g., Nipah, Hendra) and arenaviruses (e.g., lymphocytic choriomeningitis, Junin). However, this is certainly not a complete list, because for most cases of human viral encephalitis the etiologic agent is not identified, even when heroic attempts are made.
The primary target cells for most encephalitic viruses are neurons, although a few viruses attack cerebrovascular endothelial cells to cause ischemia and stroke or glial cells to cause demyelination, encephalopathy, or dementia–. Widespread infection of neurons may occur or viruses may display preferences for particular types of neurons in specific locations in the central nervous system (CNS). For instance, herpes simplex virus (HSV) type 1 often infects neurons in the hippocampus to cause behavioral changes, while poliovirus preferentially infects motor neurons in the brainstem and spinal cord to cause paralysis and Japanese encephalitis virus infects basal ganglia neurons to cause symptoms similar to those of Parkinson’s disease.
Because infections with encephalitic viruses are initiated outside the CNS (e.g., with an insect bite, skin, respiratory, or gastrointestinal infection), innate and adaptive immune responses are usually mounted rapidly enough to prevent virus entry into the CNS. Therefore, most viruses that can cause encephalitis more often cause asymptomatic infection or a febrile illness without neurologic disease, and encephalomyelitis is an uncommon complication of infection.
Eastern equine encephalitis (EEE) commonly called triple E or, sleeping sickness is a rare but serious viral disease affecting horses and man. The disease is transmitted through mosquitoes and man and horses are dead-end hosts.
EEEV belongs to the genus Alphavirus of the family Togaviridae. It is closely related to Venezuelan equine encephalitis (VEE) virus and Western equine encephalitis (WEE) virus. This virus has North American and South American variants. The North American variant is more pathogenic. EEE is capable of infecting a wide range of animals including mammals, birds, reptiles and amphibians. The virus has been reported to cause disease in poultry, game birds and ratites. The disease has also been reported to occur in cattle, sheep, pigs, deer, and dogs though sporadically. The disease is present in North, Central and South America and the Caribbean. EEE was first recognized in the USA in 1831 from an outbreak where 75 horses died of encephalitic illness and EEE virus (EEEV) was first isolated from infection horse brain in 1933. The serological evidence and outbreaks of the disease have also been reported from horses in Canada and Brazil [119, 120]. Countries with incidence/serological evidence are presented in Fig. (3). EEEV infection in horses is often fatal. The human cases were identified first time in 1938 in the north-eastern United States. Thirty children died of encephalitis in this outbreak. The fatality rate in humans was 35%. The outbreaks of the disease also occurred in horses simultaneously in the same regions. A total of 19 human cases of the disease were reported in children between 1970-2010 in Massachusetts and New Hampshire. As per the CDC reports 220 confirmed human cases of the disease occurred in the U.S. from 1964 to 2004. In 2007, a citizen of Livingston, West Lothian, Scotland became the first European victim of this disease after infected with EEEV from New Hampshire. EEE has been diagnosed in Canada, the United States of America (USA), the Caribbean Islands and Mexico [122, 123]. Eighteen cases of Eastern equine encephalomyelitis occurred in six Brazilian states between 2005 and 2009.
Alternate infection of birds and mosquitoes maintains these viruses in nature. Culiseta melanura and Cs. morsitans species are primarily involved. Transmission of EEEV to mammals occurs via other mosquitoes which are primarily mammalian feeders and called as bridge vectors. Infected mammals do not circulate enough viruses in their blood to infect additional mosquitoes. The virus is introduced by mosquitoes, but feather picking and cannibalism also contribute towards the transmission of the disease within the flocks. Most people bitten by an infected mosquito do not develop any symptoms. The symptoms generally appear 3 to 10 days after the bite of an infected mosquito. The clinically affected patients may have pyrexia, muscle pains, headache, photophobia, and seizures. EEEV is one of the potential biological weapons. The disease in horses is characterized by fever, anorexia, and severe depression. Symptoms appear one to three weeks post-infection, and begin with a fever that may be as high as 106ºF. The fever usually lasts for 24–48 hours. In severe cases, the disease in horses progresses to hyper-excitability, blindness, ataxia, severe mental depression, recumbency, convulsions, and death. The nervous symptoms may appear due to brain lesions. This may be followed by paralysis, causing the horse to have difficulty raising its head. The horses usually suffer complete paralysis and die two to four days after symptoms appear. Mortality rates among horses range from 70 to 90%.
There is no cure for EEE. Severe illnesses are treated by supportive therapy consisting of corticosteroids, anticonvulsants, intravenous fluids, tracheal intubation, and antipyretics. Vaccines containing killed virus are used for prevention of the disease. These vaccinations are usually given as combination vaccines, most commonly with WEE, VEE, and tetanus. Elimination of mosquito breeding sites and use of insect repellents may help in control of the disease.
Chikungunya virus (CHIKV) is a mosquito-borne virus belonging to the genus alphavirus, family Togaviridae and spread by Aedes mosquitoes. CHIKV is the aetiological agent of chikungunya fever (CF) and was first isolated in 1952 from the serum of a febrile patient during an outbreak in Tanzania, Africa. CHIKV infection is characterized by an abrupt onset of fever lasting two to five days, frequently accompanied by arthralgia. The disease is usually self-limiting, but joint pain symptoms can persists for weeks up to years [2–4]. Other common symptoms include muscle pain, headache, nausea, fatigue and rash similar to the dengue virus infection. Besides CHIKV, the Alphavirus genus includes viruses such as O'nyong'nyong virus (ONNV), Ross River virus (RRV), Barmah Forest virus (BFV), Sindbis virus (SNV) and Mayaro virus (MAYV).
Since its discovery in Africa, CHIKV has repeatedly caused outbreaks in Africa, India, Southeast Asia, the Middle East and Europe with irregular intervals [6–10]. Phylogenetic analysis of CHIKV showed that the virus has evolved into three distinct genotypes: Asian, West African and Eastern/Central /South African (ECSA). A single-base mutation E1-A226V in a strain of the ECSA genotype enhanced replication of the virus in Ae. albopictus, and led to a large-scale epidemic on La Reunion in 2005. This ECSA genotype was subsequently associated with epidemics in the Indian Ocean region, and the Asian genotype has been associated with recent outbreaks in the Pacific region.
The first local transmission of CF in the Americas was reported from the Caribbean islands in December 2013. The local transmission of the disease has been reported in 45 countries or territories throughout world with more than 1.7 million suspected cases (CCDR October 20, 2015, http://www.cdc.gov/chikungunya/geo/index.html). It appears to have been introduced twice to Brazil, once from the Pacific region and once from Africa.
Current diagnosis of CHIKV is based on three main laboratory methods: virus isolation, reverse transcription-polymerase chain reaction (RT-PCR) and serological tests such as plaque reduction neutralizing test (PRNT), enzyme-linked immunosorbent assays (ELISA) or immunofluorescence test (IFT). Commonly, blood and serum samples are used as specimens for CHIKV diagnosis. Depending on the type of sample and the time of sample collection relative to symptoms (acute or convalescent phase of disease), an appropriate diagnostic method is applied to the samples.
A pronounced viraemia of up to 109 viral genomes can be observed mainly on days 1–6 after onset of disease and in some cases for longer, therefore, virus isolation and RT-PCR are performed on acute phase specimens collected during the first week after onset of symptoms. Several RT-PCR assays have been published for the detection of CHIKV RNA in clinical specimens and mosquito samples [17–23]. Real-time RT-PCR based assays are suitable for clinical diagnosis due to the closed tube assay format, the option for quantification of viral load, high sensitivity and specificity. Serological tests are applied to either acute or convalescent phase samples for the detection of IgM and IgG anti-CHIKV antibodies. Serological diagnosis is confirmed by direct detection of IgM anti-CHIKV antibodies or by determining a four-fold increase in CHIKV-specific antibody titers in acute and convalescent samples by ELISA, IFT or PRNT tests.
Since the clinical picture of CHIKV is similar to that of DENV and Zika virus, a simple and rapid molecular assay would be needed to select the best treatment approach. The recombinase polymerase amplification (RPA) assay utilizes enzymes and proteins in order to allow the amplification of the DNA at a constant temperature (38–42°C). The presence of the amplicon is detected via the exo-probe, which include an internal abasic site mimic (tetrahydrofuran, THF) flanking fluorophore and Quencher as well as pathogen-specific 30 and 15 bases at both the 5´ and 3´ prime ends, respectively. Upon the hybridization of the exo-probe to the complementary sequence the Exonuclease III cleaves at the THF site and the fluorescence signal is generated. RPA assay was successfully developed for the detection of DENV and YFV as well as other human and veterinary pathogens [25–32]. Moreover, a mobile suitcase laboratory was established for allowing the deployment of the RPA in the field for identifying Ebola virus infected case.
In this study, we developed and evaluated a reverse-transcription recombinase polymerase amplification (RT-RPA) assay as potential point-of-care (POC) diagnostic tool for rapid detection of CHIKV. The RT-RPA assay was designed by targeting the non-structure protein 1 (nsP1). The sensitivity and specificity of the method was evaluated using strains of the three genotypes of CHIKV and compared to reference RT-PCR methods. Finally, the performance of the RT-RPA assay was evaluated on acute-phase serum samples for clinical diagnosis of CHIK.
Flavivirus is a genus of the family Flaviviridae that contains a large number of viral agents capable of causing encephalitis and jaundice. Most flaviviruses are arboviruses and transmitted to the human population by a bite from infected mosquitoes or ticks. Flaviviruses typically contain a positive sense single-stranded RNA genome of approximately 10-11kb in length. The genome encodes 3 structural proteins (Capsid, prM, and Envelope) and 8 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5 and NS5B). The viruses are enveloped with a diameter of around 50nm, and appear icosahedral or spherical when observed under the electron microscope. Individual members such as dengue (DENV), yellow-fever virus (YFV), Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV) and West Nile virus (WNV) cause significant morbidity and mortality worldwide.
DENV is a major public health concern on a global scale with an estimated 400 million infections and 100 million clinical cases in 2010. Most of these patients will carry the disease asymptomatically. However, around 5% of infected individuals will progress to severe dengue, an illness characterized by plasma leakage leading to hypovolemic shock, hemorrhage, and potentially death. The case-fatality rate for individuals with severe dengue can be as high as 10% if untreated, or 0.1% with appropriate clinical management.
Alphaviruses are a diverse group of viruses that are classified as belonging to the group IV Togaviridae family of viruses. There are over thirty members in the alphavirus group that are able to infect a wide range of vertebrates including humans, rodents, fish, birds, and horses. At the genomic level alphaviruses consist of a positive sense, single-stranded RNA genome 11 to 12kb in length with a 5’ cap, and 3’ poly-A tail. Alphavirus particles are enveloped, have a size of around 70 nm in diameter under the electron microscope and appear to be spherical with a 40 nm isometric nucleocapsid. Like flaviviruses the main mode of transmission to the human population is via bites from infected mosquitoes. Notable viruses that infect the human population include chikungunya (CHIKV), Barmah Forest virus (BFV), Mayaro virus (MAYV), O'nyong'nyong virus (ONNV), Ross River virus (RRV), Una virus and Tonate virus.
Epidemics of flavivirus and alphavirus occur globally on an annual basis with different degrees of severity. Table 1 shows a small selection of recent flavivirus/alphavirus outbreaks worldwide.
The global distribution and severity of flavivirus and alphavirus infection requires accurate surveillance tools and timely diagnosis to ensure infected patients obtain the best medical treatment options and alert authorities to possible outbreaks of disease.
The most accurate method to diagnose viral agents is real time Polymerase Chain Reaction (RT-PCR). Primer and probe sequences complementary to the viral RNA are designed and cycled through a series of steps with positive samples seen as amplification curves on a RT-PCR instrument. This process can be completed in less than 1 hour, which significantly assists in patient management.
However, members of the flavivirus and alphavirus families are quite heterogeneous at the RNA level, therefore it can be difficult to design a single set of primers and probe sequences that can detect each of the families at the genus and species level. An example of this is DENV that contains four serotypes, each being quite diverse at the genomic level. Like most current dengue RT-PCR assays, the CDC DENV-1-4 RT-PCR Assay detects serotypes 1–4 using an individual primer pair and probe for each type. Assays that can universally detect all DENV serotypes have been described but these assays still employ more than 2 primers to detect all subtypes.
In order to simplify and improve the detection of alphaviruses and flaviviruses in clinical samples, we developed a commercially available 3base assay that is able to detect the presence of the target alphavirus or flavivirus using a single primer and probe set for each type. 3base assays use chemical modification to reduce the complexity of genomes from 4 to 3 base, which enable screening primers and probes with fewer mismatches to be developed so that bias in amplification efficiency across species is greatly reduced. (Fig 1).
The 3base protocol (Fig 2) deaminates all cytosine residues in nucleic acids to a uracil intermediate. This process makes closely related species more similar at the genomic level. This novel method ultimately means that primers and probe sets can be designed that have fewer mismatches and are able to hybridise to previously heterogeneous target regions with higher efficiency, thus improving PCR amplification of species that contain large numbers of individual pathogens.
The modification process of the genomic nucleic acids to a 3base form does not sacrifice specificity and individual typing primers can be constructed to detect the exact organism responsible for disease.
The method has been used to successfully detect the presence of high risk HPV in clinical samples and the presence of pathogens, including Norovirus, in patients with gastrointestinal disease.
We have utilised the method to produce pan-species assays for the detection of all flavivirus, alphavirus and dengue serotypes 1–4 and successfully applied these assays to screen samples in the 2016/17 Vanuatu dengue outbreak.
The role of the small interfering RNA (siRNA) and P-element induced wimpy testis (PIWI)-interacting RNA (piRNA) pathways in controlling virus infections in mosquitoes has been extensively studied and are considered to be a major part of the antiviral innate immune response. Although several studies have indicated that cellular microRNAs (miRNA) are involved in mosquito antiviral immunity, the miRNA-viralRNA (vRNA) direct interaction and its effect on virus replication in mosquitoes are still unclear. The cellular miRNAs of the mosquito Aedes aegypti, a vector for many arboviral diseases, may directly interact with three major arboviruses: chikungunya, dengue, and Zika viruses. By using the miRanda and TargetSpy tools (http://bioinfo5.ugr.es/srnatoolbox), several miRNAs were predicted to have potential binding sites that are common to multiple viral genotypes or lineages. Further analysis was carried out on miRNA-vRNA interactions that required a low energy threshold to form a complex. This study shows a broad picture of possible interactions between mosquito cellular miRNAs and the viral RNA of different genotypes/lineages of arboviruses, providing a list of mosquito cellular miRNAs candidates for experimental validations in future studies.
Acute encephalitis syndrome (AES) was observed in suckling mouse with growth retardation, panting, abdominal breathing, and arthritis (data not shown). Negative-staining electron microscopy (EM) of the supernatant from infected suckling mouse brain (named YN08) revealed virus-like particles (Figure 1). These particles were spherical in shape, with an envelope, and approximately 50–70 nm in diameter, consistent in size and morphology with that of Togaviruses or Flaviviruses.
Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus genus within the Togaviridae family and was first isolated from horses in the end of the 1930s. These viruses have a natural transmission cycle between rodents and mosquitos. Millions of horses were affected by this arbovirus with a fatality rate of up to 80% in epidemics in Central and South America.
Several species of this family are pathogenic to humans and are recognized as potential biological warfare agent (BWA). VEEV is classified as Bioterrorism Agent Category B by the center of Disease Control (CDC). Alphaviruses do not only have the potential for illness and transmission, but they can also be produced in large quantities and are moderately easy to disseminate. Furthermore, these virus species have the capacity to cause human epidemics. VEEV causes disease symptoms ranging from mild febrile reactions to fatal encephalitic zoonoses. Outcomes are significantly worse for young and elderly patients, with case fatalities ranging from 4 to 35%. These viruses are highly infectious as aerosols and an intentional release of sufficient quantities as inhalable small-particle aerosol is expected to infect a high percentage of individuals within an area of a least 10,000 km2. They can replicate in cell culture to very high titers and are relatively stable to environmental influences.
For the surveillance of possible bioterrorism targets and endangered populations, rapid detection and diagnosis of VEEV are of crucial importance. In the past, the generation of monoclonal murine antibodies has improved the fast identification of VEEV infections to locate human and equine outbreaks of encephalitis. On the other hand, monospecific diagnostic monoclonal antibodies (mAbs) against VEEV are either hardly available on the market or too expensive for extensive use. In view of these current limitations the generation of specific high affinity recombinant antibodies may significantly improve the current situation and can make the rapid immunological detection widely available.
A promising method to generate recombinant antibodies against human pathogenic viruses like VEEV is the antibody phage display technology. Using antibody phage display, genotype and phenotype of an antibody fragment are linked by fusing the antibody gene fragment to the minor coat protein III gene of the filamentous bacteriophage M13. The resulting antibody fragment::pIII fusion protein is displayed on the surface of the phage particles. The most common antibody formats used for this technology are the Fragment antigen binding (Fab) and the single chain Fragment variable (scFv). In comparison to the Fab, that is consisting of the Fragment determining (Fd) of the heavy chain and the light chain linked by a disulphide bond, the scFv simply consists of the variable region of the heavy chain (VH) and the variable region of the light chain (VL), connected by a short peptide linker. The selection of antibody fragments from antibody gene libraries is performed by an in vitro selection process, that is also referred to as "panning".
In this study, we demonstrated the selection of human antibody fragments from a naïve antibody gene library specific for the detection of VEEV. We describe their immunological properties and discuss their possible application of these antibodies for diagnosis and detection of VEEV after a potential bioterrorism assault or natural outbreak of VEEV.
The Flavivirus includes 70 members, etc., many of which, including Japanese encephalitis virus, dengue virus, West Nile virus, yellow fever virus, and Zika virus, which can cause some human severe diseases. Flaviviruses have introduced a substantial disease burden in humans and animals around the world, and, as mosquito-borne viruses, can spread widely [6, 7].
Yokose virus (YOKV) belongs to the Flavivirus, which was first isolated from bats (Miniopterus fuliginosus) collected in Yokosuka, Japan, in 1971, and the first YOKV strain isolated was Oita-36 (Fig. 1). Sequencing results indicate that the YOKV genome is 10,857 nucleotides (nt) in total. Like other flaviviruses, YOKV has only one open reading frame (ORF), which encodes 3425 amino acids. The genome encodes three structural proteins, the capsid (C), membrane (M), and envelope (E) proteins, as well as seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). There are 5′ and 3′ non-coding regions at the ends of the genome. Molecular genetic analysis indicated that YOKV is a new member of the family Flaviviridae, genus Flavivirus. Furthermore, YOKV is genetically related to other flaviviruses, including Japanese encephalitis virus, dengue virus, yellow fever virus, and West Nile virus. YOKV is closely related to yellow fever virus, which both clustered in the same clade. Additionally, YOKV was neutralized by antisera to individuals inoculated with yellow fever virus vaccine. The above results suggested that this new member of flavivirus is genetically related to yellow fever virus. A serological survey of YOKV in the Philippines and Malaysia showed that the YOKV antibody was detected in the serum of bats (Rousettus leschenaultii), suggesting that the distribution of YOKV was not limited to Yokosuka Island, Japan (Fig. 1).
In this study, we isolated a virus (XYBX1332) from a serum sample of Myotis daubentonii (order Chiroptera, family Vespertilionidae) collected in the Yunnan-Guizhou Plateau Region (average altitude 2000–4000 m) in the southwest of China in July 2013. XYBX1332 was identified as YOKV using whole-genome sequencing. However, our study indicated some clear differences in molecular biology between XYBX1332 and the original isolate of YOKV.
Both BHK-21 and Vero E6 cells inoculated with serum specimen numbered XYBX1332 exhibited obvious CPEs, which included rounding up, aggregation, and exfoliation (Fig. 2).
Disease incidence for any given island was compared to the average incidence for the whole country, using the z-score test for two population proportions available at https://www.socscistatistics.com/tests/ztest/default2.aspx. P values < 0.05 were considered statistically significant.
Members of the genus Orthonairovirus of medical and veterinary significance include Crimean Congo hemorrhagic fever virus (CCHFV) and Nairobi sheep disease virus (NSDV). CCHFV is transmitted by ticks in genera Rhipicephalus and Hyalomma. While neither live virus nor nucleic acid of CCHFV has been detected from bats, serologic evidence suggests past infection of populations of bats across a diverse geographic range. Further, bats are often parasitized by both soft and hard ticks, which occupy a diverse range of ecological niches in endemic countries. A 2016 seroprevalance study by Müller and colleagues examining 16 African bat species (n = 1,135) found that the prevalence of antibodies against CCHFV was much higher in cave-dwelling bats (3.6%–42.9%, depending on species) than foliage-living bats (0.6%–7.1%). They also screened 1,067 serum samples by RT-PCR, but all were negative for CCHFV nucleic acid. Experimental studies to assess the ability of bats to support replication of CCHFV have not been published.
Developing a safe and effective vaccine against CHIKF—as well as against other reemerging diseases, such as Ebola, Lassa, or Nipah—is important for several reasons. The impact of CHIKF in terms of burden of disease, work and school absenteeism, and other financial costs is particularly high, especially given its formidable epidemic potential. A paradigmatic example is provided by the 2006 epidemic wave that occurred in India, in which more than 1,500,000 cases of CHIKF were reported (http://www.who.int/denguecontrol/arbo-viral/other_arboviral_chikungunya/en/). Furthermore, the global impact of CHIKV is constantly growing, due to the introduction and spread of the virus into new continents in which it finds optimal conditions for its expansion, including, in some cases, a completely naïve population (Fig 1). The capacity of CHIKV to adapt to a new mosquito vector has been demonstrated during the Indian Ocean epidemic, when a series of mutations increased fitness for transmission by A. albopictus [14, 15], a mosquito that can survive at higher latitudes than A. aegypti. This may lead to the occurrence of outbreaks in temperate climates, as seen in Europe [8, 9].
Secondly, although the case–fatality rate of CHIKF is relatively low—usually well below 1% [1, 2]—it may be underestimated in small outbreaks and in epidemic waves that occur in resource-poor countries. Excess mortality was investigated during an outbreak in Mauritius, and the case–fatality rate was estimated to be around 2.3 per 1000. Although “Old World” alphaviruses are not considered neurotropic sensu strictu, cases of meningoencephalitis have been documented, especially during Indian outbreaks, and fatal encephalitis has been reported with CHIKV infection both in Italy and in La Reunion, where it was observed in patients mostly below 1 year or over 65 years of age. Relatively high case–fatality rates (17%) and persistent disability (30% to 45%) were documented among patients with CHIKV-associated encephalitis. An increased incidence of other neurological syndromes, such as Guillain-Barré syndrome, was also reported during a CHIKF outbreak in French Polynesia. These findings provide evidence of severe disease associated with CHIKV infection, which may have a high impact in terms of hospitalization and mortality during large outbreaks.
Thirdly, persistent arthralgia and joint swelling are common long-term manifestations of CHIKF. Unlike other mosquito-borne viruses such as dengue, Zika, and even yellow fever, CHIKV typically causes symptomatic infection, and consequently outbreaks are accompanied by high attack rates. Chronic joint pain, along with asthenia and mood changes, is a common cause of quality-of-life impairment. Ninety-four percent of symptomatic travelers infected in La Reunion complained of joint or bone pain 6 months after the epidemic peak; this pain was continuous in 41% of the cases. The effect of chronic symptoms on the quality of life was defined as totally disabling or important in almost half of the patients, whereas only 16% reported a normal mood. A study conducted in La Reunion on 147 individuals over the age of 16 found that 84 confirmed cases (57%) self-reported rheumatic symptoms; of these, 63% reported permanent pain, whereas 37% had recurrent symptoms. An age of over 45 years (odds ratio [OR] 3.9), severe initial joint pain (OR 4.8), and the presence of underlying osteoarthritic comorbidity (OR 2.9) were independent predictors of nonrecovery. An assessment of 173 individuals with CHIKF conducted in Mauritius found that 79% reported persisting musculoskeletal symptoms 27.5 months after infection, associated with older age, female gender, and a baseline symmetrical distribution of arthralgia. A study conducted on Italian patients found that over 66% of those with CHIKF developed long-lasting rheumatic disorders, leading to functional impairment affecting daily living activities up to one year after infection. Therefore, the long-term impact of CHIKF is far from negligible in terms of suffering, need for care, impairment of work ability, psychological problems, diminished quality of life, and associated economic costs.
Unfortunately, little information is available on the economic costs of CHIKV infection. Healthcare costs during the La Reunion in 2005 to 2006 epidemic were estimated: the medical management of CHIKF was associated with a major economic burden, with 60% of the CHIKF-related expenditure attributable to direct medical costs, such as medical consultation (47%), hospitalization (32%), and drug consumption (19%). The cost of analgesics accounted for 80% of the CHIKF-related drug expenditures. Loss of productivity, measured as absenteeism costs, was also high. Studies conducted in 2006 in Gujarat, India, estimated an immediate cost to household of CHIKF and dengue around 3.8 billion Indian rupees (ca. US$55 million) per annum, whereas another study conducted in Ahmedabad, a city of 3.5 million people in the State of Gujarat, found that the disease affected primarily working-age adults, with an immediate cost of the outbreak due to lost wages and treatment of approximately US$1.7 million based only on officially reported cases; these figures are probably underestimated, because only 23% of the cases seek treatment within public facilities. Another study estimated a total of about 40 million cases in the Americas, with a burden of 24 million disability life years (DALYs) lost and about US$185 billion in societal cost. Therefore, although CHIKF is in most cases a relatively mild illness, its burden during epidemic waves may be impressive. Finally, when CHIKF outbreaks occur in tourist destinations, economic loss due to decreased numbers of visitors may cause further economic impacts. The under-recognition of the potential impact of CHIKF led to undesired political consequences during the epidemic waves on the tourist destination island of La Reunion, with the French government being accused of negligence and delays in outbreak response.
Therefore, for the reasons reported above, the development of a safe and effective vaccine against CHIKF would have a significant impact on the global burden of this disease with important health, economic, and ethical implications: (i) because of the high infection rates during epidemic waves, the disease burden may be relatively high. An effective vaccine would reduce the number of cases and hospitalizations worldwide, producing economic benefits through the reduction of absenteeism, lower costs for care and hospitalizations, and reduced loss of income associated with tourism; (ii) countries outside the tropics might also benefit from the development of a CHIKF vaccine, because the virus may be introduced from endemic and/or epidemic areas or enzootic circulation and eventually spread to regions where competent urban vectors are present; (iii) protection of travelers and military personnel may be another positive outcome of vaccine development; (iv) there are clear ethical implications in the development of a vaccine against an emerging neglected disease that primarily affects resource-limited parts of the globe.
The nervous system is a target for acute viral infections, as well as a reservoir of latent and persisting viruses. In general, the absence of overt neurological deficits or pathology indicates effective immune control of persisting viruses in immunocompetent individuals. However, this balance is highly tenuous, as indicated by cases of JC virus-mediated progressive multifocal leukoencephalopathy (PML) in immunocompromised individuals with acquired immune deficiency syndrome or those receiving treatment for multiple sclerosis (MS) or lymphoma. Similarly, the activation of herpes simplex virus (HSV) and cytomegalovirus in the nervous system can be devastating in immunocompromised individuals. Moreover, senescent immune responses in an increasingly aging population enhance disease susceptibility to both reactivating persistent viruses in the central nervous system (CNS), as well as to acute encephalitic arboviral infections. Numerous human infections involving the CNS, including those caused by measles, rubella, polio, varicella zoster, mumps, HSV and Japanese encephalitis virus (JEV), as well as lyme neuroborreliosis are characterized by intrathecal antibody (Ab) in the cerebral spinal fluid (CSF) consistent with the presence of local Ab-secreting cells (ASC). Although the causative agent still remains unknown in many cases of suspected viral encephalitis, detection of virus-specific immunoglobulin (Ig) in the CSF can be a reliable diagnostic tool to confirm a suspected viral encephalitis indicated by molecular analysis. For example, acute poliomyelitis or encephalitis mediated by insect-borne viruses such as JEV are associated with virus-specific IgM and IgG in CSF within ~2 weeks of clinical presentation. While Ab persists over several months in the case of JEV, they appear more transient in poliomyelitis. Overall, Ab detection may be more transient in cases of acute encephalitis, while it persists during chronic disease such as measles virus-associated subacute sclerosing panencephalitis. A specific protective or detrimental role is often difficult to infer due to difficulties in obtaining longitudinal serum and CSF samples. Even when available, the role of serum versus intrathecal Ab cannot be readily distinguished. Overall, intrathecal humoral responses appear to be associated with protective rather than pathogenic functions. Thus, a beneficial outcome of JEV encephalitis is correlated with intrathecal IgG. Similarly, intrathecal Ab synthesis may be an indicator of protection during CNS retrovirus infection. Ab also correlates with reduced CNS viral load and milder clinical disease course in patients with tropical spastic paraparesis/HTLV-I-associated myelopathy. An inverse correlation between intrathecal-neutralizing Ab and macrophage-tropic SIV was also observed in the SIV encephalitis model of HIV. Lastly, the CSF of MS patients harbors Ab to multiple viruses prevalent in the Western population, e.g. varicella zoster, rubella, HSV-1 and JC viruses. These Ab appear to be markers of MS and are not indicative of active disease due to virus infection. Nevertheless, the potential danger of losing control of persisting CNS viruses became apparent by the development of PML following rituximab (anti-CD20 monoclonal Ab) reduction of circulating B cells during therapy for rheumatoid arthritis and MS.
During experimental CNS infections, particularly by RNA viruses such as Sindbis, rabies and corona viruses, ASC play a vital local protective role. The reliance on local ASC for sustained Ab output provides a potent complement-independent non-lytic mechanism of immune control within the CNS, potentially regulating a variety of neurotropic infections. Despite constituting a critical component controlling viral persistence, little is known about the origin and maintenance of ASC in the CNS or other specialized microenvironments. This review focuses on insights gained throughout the last decade on humoral immune responses within the CNS during encephalitis and persistent infections mediated by RNA viruses.
Chikungunya virus (CHIKV) is a member of the Alphavirus genus of the Togaviridae family1,2. It is responsible for chikungunya fever (CHIKF), a disease characterized by the presence of incapacitating arthralgia3. CHIKV is transmitted by arthropod vectors, such as the Aedes aegypti and Aedes albopictus mosquitoes, with the latter being implicated in the transmission of CHIKV during the 2005–2006 Indian Ocean outbreak and in Europe4. For the past decade, re-emergence of CHIKV has led to numerous outbreaks in different parts of the world: Asia5–12, Europe4,13,14 and islands in the Indian Ocean15,16. Outbreaks of CHIKV infections have also been reported in the Caribbean islands17,18 and CHIKV has since successfully invaded North, Central and South America19.
Enhancement of arbovirus infections via antibodies was first demonstrated in 196420. This is a paradoxical phenomenon of antibodies forming complexes by binding to viruses, which then interact with cell surface receptors and promote entry into susceptible host cells, subsequently increasing virus replication21,22. This was observed for rabies virus23, influenza virus24, dengue virus (DENV)25,26, Ross River virus (RRV)27, human immunodeficiency virus (HIV)28 and Marburg virus29. Among alphaviruses, although virus enhancement was documented only in RRV infections27,30–32, most of these studies were conducted using in vitro murine cell line-based systems27,31,32. The development of a suitable infection system with primary human cells and an in vivo model allows the study of antibody enhancement in clinically important viruses, such as the recently emerged Zika virus (ZIKV), which infection is enhanced with cross-reactive anti-DENV antibodies33.
Here, we demonstrate antibody-mediated enhancement of CHIKV attachment and infection in primary human monocytes and B cells and a relevant murine cell line in the presence of sub-neutralizing levels of anti-CHIKV antibodies obtained from CHIKV-infected patients or animals. This enhancement was further demonstrated to mediate through the Fc receptors (FcγRs), with FcγRII being the key mediator. Importantly, two complementary animal models demonstrated enhanced CHIKV infections in the presence of sub-neutralizing levels of anti-CHIKV antibodies, with severe disease outcome and increase lethality. This study brings also caution to the importance of such undesired effects in anti-CHIKV vaccine designs.
Although drug development has strongly contributed to finding superior therapeutic efficacy, there is still space and need for further improvement. In addition to classic drug screening of small molecules, innovative modern approaches in biotechnology and genomics research have contributed to new therapeutic possibilities in the areas of vaccine development and gene and immunotherapy. In this context, RNA-based therapeutics have become an interesting alternative. RNA-based drugs have been classified by mechanisms of action including antisense approaches of inhibition of mRNA translation, gene silencing with RNA interference, catalytically active ribozymes, protein binding RNA molecules, and aptamers for diagnostic and therapeutic applications. In this context, lipid-encapsulated nanoparticles containing double-stranded small interfering RNA (siRNA) have been applied for binding to transthyretin (TTR) mRNA causing degradation of TTR deposits present in patients with hereditary TTR-mediated amyloidosis. This novel RNA interference-based drug, Patisiran (ONPATTRO™), has recently been approved in both the US and Europe as a single intravenous infusion. Recently, messenger RNAs (mRNAs) generated by in vitro transcription have become attractive targets for drug and vaccine development. Two approaches for mRNA-based drugs have been taken based on ex vivo transfection of cells from patients or direct mRNA administration. In this context, preclinical and clinical studies have been conducted in the areas of cancer immunotherapy, vaccine development against infectious diseases, protein replacement, and gene editing.
Related to mRNA-based drug approaches, the employment of self-replicating RNA viruses has provided an interesting and attractive alternative to further enhance delivery and efficacy. The unique feature of high-rate cytoplasmic replication combined with extreme transgene expression has made these RNA viruses the system of choice for RNA therapeutics. In this review, self-replicating vectors are described and their applications for preclinical studies and clinical trials are discussed.
For 20 years, alphavirus researchers studied a 6 kilodalton protein aptly named 6K. This protein was identified to play a role in virus exit and was also determined to function as an ion channel. The 6K protein was not required for virus infection. In 2008, a bioinformatics team discovered that what had been termed the 6K protein was actually two proteins. A heptanucleotide slippery sequence within the 6K gene directs ribosomal frameshifting and the consequent translation in the (−1) open reading frame produces the transframe protein, or TF. The 6K and TF proteins have identical N-termini, but unique C-termini. When TF was identified, the accumulated literature was reevaluated to disentangle the results that applied specifically to 6K, to TF, or to both. Within the last nine years, several reports have emerged addressing the specific activities of TF and 6K. Here we address the current state of our knowledge about 6K and TF and explore the most cogent questions remaining to determine their function in an alphavirus infection.
Positive-sense RNA (+RNA) viruses including the Flaviviruses, enteroviruses of the Picornaviridae family, Alphaviruses, and Coronaviruses all dramatically modify cellular membranes to serve as platforms for replication and assembly of new virions. The biogenesis of these replication compartments is a complex interplay of interactions between virus and host proteins. Although considerable progress has been made in identifying host proteins that interact with virus-encoded proteins, much remains to be learned regarding the significance of these interactions. Despite morphological differences in the replication complexes formed by members of each viral family, these viruses have evolved to use common cellular pathways to complete biogenesis. Some of the shared pathways highlighted in this review include lipid metabolism, autophagy, signal transduction and proteins involved in intracellular trafficking (Table 1). Remarkably, even within the higher order of shared pathways, differences within members of specific families (such as Flaviviridae) exist, highlighting that the assembly and function of viral replication complexes (VRCs) varies considerably. As such, this review focuses on a broad view of host factors in which there is significant functional evidence linking them to VRCs in effort to highlight commonalities or differences and further advance the understanding of virus-host interactions.
Technology for detecting biothreat agents requires accurate identification of a broad array of bacterial and viral organisms that can cause severe disease and/or death, whether they occur as a result of a biological attack or from a natural source in the environment. The National Institute of Allergy and Infectious Diseases (NIAID) has compiled a list of priority pathogens for biodefense (http://www.niaid.nih.gov) and several of these are also defined as select agents (http://www.selectagents.gov/) by various agencies such as Health and Human Services (HHS) and the United States Department of Agriculture (USDA) (some of the vaccine and live attenuated strains are, however, excluded from the select agents list: http://www.selectagents.gov/Select%20Agents%20and%20Toxins%20Exclusions.html). These bioagents are often virtually indistinguishable from a group of phylogenetically related species or subspecies often referred to as “near neighbors”. Near neighbors to biothreat agents may be human pathogens or harmless environmental organisms. The biothreat agent along with its near neighbors can be thought of as a biothreat cluster or biocluster for short. When monitoring for biothreat agents, it is important to determine whether any organisms from the biothreat clusters are present and to precisely identify the organism as a biothreat agent or a near neighbor. In some cases, the near neighbors are commonly found in the environment, and it is possible that a pathogenic near neighbor of a biothreat agent might deliberately be chosen for use in a biological attack. Thus, effective biosensor technology must be capable of identifying a broad array of biothreat agents and distinguishing these threats from their near neighbors unambiguously.
This requirement presents a problem for conventional molecular methods where specific PCR is used in conjunction with probes to detect specific bioagents. Not only are potentially pathogenic near neighbors present in a specimen often not distinguished, but the near neighbors sometimes react to produce false positives for the biothreat agent. To overcome these limitations, we have developed a new strategy for biothreat identification that couples biothreat cluster-specific PCR amplification to electrospray ionization/mass spectrometry (PCR/ESI-MS)–. The biothreat assay is performed on a hardware platform with prototypes known as TIGER and as the Ibis T5000, that is now marketed commercially as the Abbott PLEX-ID. In the PCR/ESI-MS approach, PCR primers are designed to amplify regions of the genomes of all species from the entire biothreat cluster, encompassing groups of organisms that include the biothreat and the associated near-neighbor organisms. The primers are designed to target genomic regions sufficiently conserved such that amplification occurs comprehensively within a biothreat cluster, but not outside of the cluster. The amplification products are then analyzed by mass spectrometry, which weighs the amplicons with sufficient mass accuracy that the base composition of A, G, C, and T nucleotides that make up the amplicon can be accurately counted. The base composition serves as a signature of each organism and enables identification and discrimination of the biothreat agents and their near neighbors with equal facility. In addition, previously undiscovered or newly emerging organisms from within these biothreat clusters are also detected. The database of signatures against which multiple additional pathogens could be identified increases over time as newer strain variants are archived and tested. An example of this was the discovery of the 2009 H1N1 virus by the Naval Health Research Center,; this offered the first characterization of a previously unrecognized influenza strain, demonstrating the capability of the PLEX-ID in identification of a real-world case of novel pathogen emergence. Because the mass spectrometer weighs all amplicons presented to it, the amplicons from unexpected or new organisms are detected and identified,,,.
Using this strategy, we designed a comprehensive assay to detect ten bacterial and four viral biothreat clusters. The assay identified the major biothreat organisms and differentiated these from their near neighbors and from thousands of other bacteria and viruses, providing a seamless net of biosurveillance for these clusters in a comprehensive biothreat assay (Figure 1). In this manuscript, we provide a detailed description of the methodology and the results of formal validation experiments with a variety of biothreats, near neighbors, and specimen types. We also describe several examples of how the assay has been used in real-world biothreat scenarios.