An unprecedented number of Ebola virus infections among healthcare workers and patients have raised questions about our understanding of Ebola virus transmission. Here, we explore different routes of Ebola virus transmission between people, summarizing the known epidemiological and experimental data. From this data, we expose important gaps in Ebola virus research pertinent to outbreak situations. We further propose experiments and methods of data collection that will enable scientists to fill these voids in our knowledge about the transmission of Ebola virus. The 2014 outbreak of Ebola virus disease (EVD) in West Africa has claimed more lives than all previous EVD outbreaks combined [Zaire ebolavirus, one of five species of viruses in the genus Ebolavirus, has been identified as the etiological agent of the 2014 outbreak of Ebola virus disease (EVD). In humans, EBOV has been found in variety of bodily fluids, including saliva, blood, breast milk, stool, and semen [Ebola virus (EBOV), species nd semen . There and semen . InfectiAssessing the potential routes of EBOV transmission, the United States Centers for Disease Control and Prevention (CDC) has communicated the scientific consensus that EBOV spreads only through direct contact with mucous membranes or through broken skin with infected blood or bodily fluids, contaminated objects such as needles, and contact with infected animals . AdditioOur knowledge about human-to-human EBOV transmission is based mainly on epidemiological evidence from previous outbreaks . While aAirborne or aerosol transmission of a virus occurs when small, virus-laden droplets evaporate before settling on surfaces, leaving behind infectious droplet nuclei that can travel long distances. The small droplets that can form these droplet nuclei are often called aerosols, but aerosols are also generally defined as any small liquid or solid particles that are suspended in air . In ordeCurrently no data exist for whether EBOV forms droplet nuclei. EBOV particles have been found in human alveoli , yet it While there is no evidence of airborne transmission of EBOV in humans, experiments in NHPs have shed light on this mode of infection. Early experiments examining routes of infection of EBOV demonstrated that the virus could be aerosolized to small droplet or droplet nuclei size, and cause lethal disease in rhesus macaques after inhalation of at least 400 pfu . More reResearchers have also tested the stability of mechanically generated aerosols (1–3 µm diameter) that contain EBOV. These stability studies have shown that 99% of the initial virus decays after 104 min at 50%–55% relative humidity and 19–25 degrees Celsius [Ebolavirus, Reston virus, indirectly infected naïve NHPs in distant cages [Initial concerns about the possibility of aerosol transmission of EBOV were raised when NHPs infected with another virus in the genus nt cages . Indirecnt cages ,9. Howevnt cages .Overall, differences in experimental design make it difficult to know what we can infer from these experiments about traditional airborne transmission. Measures such as the plexiglass barrier may prevent the exchange of droplets, but they may also impede aerosols. Furthermore, all EBOV transmission experiments take place in biosafety level 4 (BSL-4) laboratories that have pressure driven airflow, which could interfere with the path of the virus in the air. In order to definitely prove or disprove the possibility of EBOV airborne transmission in NHPs, rigorous experimental design is necessary to make sure droplet or fomite transmission cannot occur, while a positive control must also demonstrate that aerosol transmission is possible in the experimental setup.Droplet transmission, or large droplet transmission, refers to contact with infectious droplets that do not evaporate or travel long distances. Although these droplets do travel through the air, they are defined differently from aerosols that evaporate in the air to form droplet nuclei. While some viruses are not stable as droplet nuclei, they may remain stable inside droplets and can infect individuals exposed to them. It has been generally assumed that droplet transmission can occur up to a meter from an infected individual, but this varies depending on the droplet size, environment, and stability of the virus . EBOV-coOther ways that EBOV-containing droplets and aerosols could be created are through aerosol-generating procedures (AGPs). Known AGPs in clinical settings are those that stimulate coughing or involve the manipulation of the patient’s airway, including activities such as intubation, manual ventilation, and broncoscopy. Although health care workers may not perform AGPs in ETUs, it is possible that those working in hospital settings could generate EBOV aerosols when using AGPs to treat an EVD patient.Additional AGPs include spraying and pressure washing infected materials. For example, the previously mentioned indirect EBOV infections in NHPs are believed to have occurred from activities done by the researchers, such as pressure washing cages, or from activities by the NHPs themselves that could have formed EBOV-containing droplets that infected other animals. Although such AGPs might not occur outside of a laboratory setting, it would be helpful to experimentally determine the types of research and clinical activities that generate infectious EBOV droplets and aerosols. Air sampling while washing the cages of EBOV infected animals or when intubating an infected animal could reveal whether these activities generate EBOV droplets or aerosols. Additionally, this sampling could be performed during other experiments, thereby limiting the use of animals.Experimentally testing the stability of the virus in bodily fluids such as vomit, saliva, stool, and blood could help us determine how long EBOV can persist in droplets generated from infected patients. Similar environmental stability experiments to those we described for small aerosols could also be done with large droplets to determine how factors such as temperature, humidity, and particle diameter influence the viability of EBOV. By determining the viability of the virus in different size aerosols and droplets, we could better predict the range and modes of EBOV transmission. Additionally, conducting air sampling in Ebola treatment units (ETUs) could reveal whether EBOV aerosols or droplets are generated by patients. These data could inform us whether personal protective equipment (PPE) guidelines and protective behaviors in ETUs are sufficient. For example, the CDC currently recommends that healthcare workers wear a minimum of a face shield, surgical mask, gown, and two pairs of gloves when working with EBOV patients and that a purifying respirator (PAPR) or N95 respirator be used during an AGP with an EVD patient . HoweverA fomite refers to any surface that a pathogen is able to persist on, and fomite transmission can occur when an individual comes into contact with that infected surface. Potential routes of EBOV fomite transmission include touching objects such as bed sheets or gurneys that have been in contact with EBOV patients or other surfaces contaminated with EBOV-containing bodily fluids.10 after 5.9 days when placed on glass and in the dark at 24 °C and 40% relative humidity [Little is known about the stability of EBOV on surfaces. One experiment showed that EBOV viral load is reduced by 4 loghumidity . Anotherhumidity . Both poFurther experiments testing the stability of EBOV on surfaces found in outbreak locations and in tropical conditions could determine whether EBOV persists in the environment and if it is possible to acquire infection through contact with contaminated surfaces. In these laboratory experiments it is also important to accurately mimic the conditions one would find in real outbreak settings. While previous studies tested the stability of EBOV in dried guinea pig sera and cell culture media at low temperature and humidity, it would be insightful to test the stability of EBOV in dried human bodily fluids and at a higher temperature and humidity.In addition to experimentally testing the stability and viability of EBOV in human bodily fluids, both as liquid and dried on different surfaces, it would be useful to test the limits of EBOV stability in other environments, such as water. Because of the reduced public health infrastructure in countries afflicted by EVD outbreaks, it is common to find open sewage canals or waste-containing buckets. Because EBOV is shed in feces and other bodily secretions that come into contact with water, it would be helpful to know how long the virus is stable in water. Contaminated water could come into contact with the eyes, nose, or mouth of individuals, leading to possible infection through contact with mucous membranes.Flaviviridae, persists for 19–30 days in water [Other viruses that are also shed in vomit and diarrhea can cause infection among people who come into contact with sewage contaminated water . Many ofin water . Yet HIVin water . In factin water . TherefoMany biological factors could influence the stability of EBOV, such as whether the virus is cell-associated or protected by organic matter such as fecal waste. Because there are so many permutations for testing the virus in different environments and at varying temperatures, humidities, pH’s, and salinities, it is most reasonable to test the virus in conditions that one is likely to find in outbreak settings. Designing experiments that accurately simulate the environments where one might encounter EBOV, such as on clinical surfaces, corpses, and disposed waste, and are at the same temperature and humidity as outbreak locations, would provide the environmental boundaries for EBOV transmission.Of all the possible modes of transmission, direct contact with bodily fluids remains the most likely way of transmitting EBOV between people. Circumstantial evidence from previous outbreaks, epidemiological data, and experiments in NHPs all demonstrate that contact with EBOV infected fluids via mucous membranes, injection, or open wounds can lead to infection.The use of unsterilized needles was linked to the spread of EBOV in hospital wards during the first recorded EVD outbreak in the Democratic Republic of the Congo in 1976 . AccidenDespite contact with bodily fluids being a confirmed route of EBOV transmission, it is not clear when and which fluids contain infectious virus. EBOV has been isolated from blood, saliva, breast milk and semen , while ROne limitation of sampling the viral load in patients is that we may be missing asymptomatic or subclinical infected individuals. These asymptomatic individuals may have been recently infected and are in the pre-clinical stages of EVD, or they may have been infected for a while and have not developed any symptoms, remaining subclinical. EBOV replication has been identified in patients who never developed any symptoms after being exposed to the virus , but it While evidence from previous outbreaks has enabled public health personnel to make informed decisions during the current outbreak, we do not have a complete understanding of all of the potential routes of EBOV transmission. If we continue to piece together our understanding of transmission from primarily epidemiological data, we will miss understanding the biological phenomena that lead to transmission events.Fortunately, BSL-4 research facilities are ideally equipped to help answer these underlying questions about EBOV environmental stability and transmission. Experiments testing different modes of transmission in animal models and the limits of EBOV viability in different environments could answer many of the remaining questions about EBOV transmission, such as whether the virus can undergo droplet or fomite transmission.As we consider the current EBOV epidemic in regards to potential outbreaks of other viral diseases, it is important to think about how both epidemiology and experimental research can be used together to inform our understanding of virus transmission. Epidemiology allows us to infer how transmission is occurring on a population level, while experiments enable researchers to determine the biological limits of transmission. Therefore, the experiments that we propose for EBOV could also be used to fill gaps in our knowledge about other viruses as well.In summary, sparse epidemiological and experimental evidence exist for mechanisms of EBOV transmission between people. Currently we know that humans shed the virus in a variety of bodily fluids, are infected by multiple routes, and only need a small amount of virus for infection. This creates a high risk of exposure, so all infection control measures must be followed rigorously in order to prevent further infections. From epidemiological evidence it appears that these infections primarily occur through direct contact with bodily fluids, yet more indirect modes of EBOV transmission have yet to be thoroughly studied experimentally. While contact with bodily fluids from EVD patients remains the most likely route of transmission, further experiments regarding the generation of EBOV in the respiratory tract and during AGPs, as well as the stability of EBOV in droplets and on surfaces, could help us define all routes of infection and improve infection control policies.


High-profile epidemics such as Ebola, avian influenza, and severe acute respiratory syndrome (SARS) repeatedly thrust infectious diseases into the limelight. Because the emergence of diseases involves so many factors, the need for interdisciplinary approaches to studying emerging infections, particularly those originating from animals , is frequently discussed –4. HowevZaire ebolavirus (EBOV), the virus responsible for the 2014 Ebola outbreak in West Africa, causes a deadly haemorrhagic disease in humans with case fatality rates ranging from 60%–88% . AlthougUsing an epidemiological model, Bellan et al.  showed tThe Ebola outbreak in West Africa and punctuated outbreaks in Central Africa since the 1970s raise fundamental questions about what drives disease spillover to humans. Ebola outbreaks are not limited to human populations. Wildlife die-offs occur routinely before or during human epidemics, indicating that the virus circulates in a range of other mammal species, including great apes and forest antelopes –16. EvenAlthough unknown for EBOV, the idea that virus circulation in wild species can drive changes that impact human–virus interactions has support for other RNA viruses such as SARS coronavirus and influenza A virus see  19,20].,20.19,20The Ebola outbreak in West Africa reminds us that zoonotic diseases continue to be a major threat. The benefits of cross scale research are evident for several high-profile zoonoses . Neverth


Synthetic Thyroxine (T4) is the treatment of choice for the correction of hypothyroidism.We report a case of drug rash induced by levothyroxine and currently oral desensitization.A 31 year-old woman had been diagnosed as Hashimoto´s Thyroiditis. She was having levothyroxine for 9 years when she begun to present bilateral eyelids maculopapular rash. Blood exams and physical examination were normal except for the eyelids´ rash.As she stopped using levothyroxine, the rash has disappeared.Her endocrinologist prescribed her other brands of levothyroxine, and the patient reported the same reaction.Therefore, we performed an oral desensitization with multiple doses of the drug preparation. The procedure was started at a dose of 1,00µg. Every 30 minutes the dose was increased until a total dose of 127,00µg.After 2 months, she currently tolerates 100,00µg/d.It is unusual that biological substances induce allergic reactions when given exogenously.Once it happens, drug desensitization can be performed at a hospital by experienced professionals with satisfactory response.Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.


Seadornavirus is a genus of viruses in the family Reoviridae, which consists of Banna virus, Kadipiro virus, and Liao ning virus. Banna virus is considered a potential pathogen for zoonotic diseases. Here, we describe a newly discovered Seadornavirus isolated from mosquitos (Culex tritaeniorhynchus) in Yunnan Province, China, which is related to Banna virus, and referred to as Mangshi virus.Mangshi virus was isolated by cell culture in Aedes albopictus C6/36 cells, in which it replicated and caused cytopathic effects, but not in mammalian BHK-21 or Vero cells. Polyacrylamide gel analysis revealed a genome consisting of 12 segments of double-stranded RNA, with a “6–4–2” pattern in which the migrating bands were different from those of the Banna virus. Complete genome sequencing was performed by full-length amplification of cDNAs. Sequence analysis showed that seven highly conserved nucleotides and three highly conserved nucleotides were present at the ends of the 5′- and 3′-UTRs in each of 12 genome segments. The amino acid identities of Mangshi virus shared with Balaton virus varied from 27.3% (VP11) to 72.3% (VP1) with Banna virus varying from 18.0% (VP11) to 63.9% (VP1). Phylogenetic analysis based on amino acid sequences demonstrated that Mangshi virus is a member of the genus Seadornavirus and is most closely related to, but distinct from, Balaton virus and Banna virus in the genus Seadornavirus of the family Reoviridae.The Mangshi virus isolated from mosquitoes (C. tritaeniorhynchus) was identified as a newly discovered virus in the genus Seadornavirus and is phylogenetically close to Banna virus, suggesting that there is genetic diversity of seadornaviruses in tropical and subtropical areas of Southeast Asia. Seadornavirus within the family Reoviridae including Banna virus (BAV), Kadipiro virus (KDV), and Liao ning virus (LNV), whose genomes consist of 12 segments of double-stranded RNA (dsRNA) [Seadornavirus and was first isolated in 1987 from patients with encephalitis in southern China, Yunnan Province, Xishuangbanna Prefecture [There are currently three identified species of the genus efecture . Since tefecture ,5, Indonefecture , and VieBalaton virus, representing a novel Seadornavirus species, was identified in the intestinal contents of freshwater carp (Cyprinus carpio) in Hungary using viral metagenomics [Mangshi virus, was isolated from mosquitoes (C. tritaeniorhynchus) collected in the southwest of Yunnan Province, China. The virus was found to have a genome composed of 12-segmented dsRNA. Here, we describe the molecular properties of the virus and its phylogenetic relationship with members of Seadornavirus. Our data indicate that Mangshi virus is a new Seadornavirus species akin to Balaton virus and BAV.genomics . HoweverMangshi Virus: A Newly Discovered Seadornavirus of the Reoviridae Family Found in Yunnan Province, China” considering that the target population lives in rural areas and they receive little education. The purpose of the survey was explained to district administrative authorities, and then to village leaders and a veterinarian in the location before sample collection, and their agreement were obtained. Before mosquito collections, the investigators explained the objectives of mosquito collection to the head of each household and they provided their informed oral consent with the assistance of the village leaders and the rural veterinarian. No clinical investigation was conducted and no personal identifiers were included; therefore, no written informed consent was sought for this study.Authorization for the collection of mosquitoes was obtained from the Institute for Yunnan Animal Science and Veterinary Institute, Kunming, China . The institution approved oral consent for collection before initiating the project entitled “Isolation and Genetic Characterization of 2. C6/36  cells were propagated and maintained in 60% Dulbecco’s modified Eagle’s medium (HyClone) plus 30% Roswell Park Memorial Institute 1640 (HyClone) with Hanks’ salt solution supplemented with 10% FBS, 100 U/ml of penicillin, and 100 μg/ml of streptomycin, at 28°C under an atmosphere of 5% CO2.BHK-21 and Vero cells were grown in minimal essential medium  with a balanced salt solution supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin, and 100 μg/ml of streptomycin. The mammalian cells were propagated and maintained at 37°C under an atmosphere of 5% COpm to 7:00 am until the next morning, i.e., from sunset to sunrise. Captured mosquitoes were killed by freezing at –20°C for at least 30 minutes and were collected on a chilled plate. Mosquito species were identified by using morphologic characteristics. Each ≤100 mosquitoes of the same species were pooled in a cryogenic vial and stored in liquid nitrogen until virus isolation was conducted.Mosquito samples were collected from caprine and bovine shelters at night using light traps  in a suburb of Menga Town  in Mangshi City, Dehong Prefecture, southwestern Yunnan Province during July 2013. Light traps were set on overnight from 7:00 g for 10 min, and then 200 μl supernatant was inoculated into each well containing a monolayer of C6/36, Vero, or BHK-21 cells in 24-well plates respectively for 7 days. Further blind passages were repeated three times. The cells were observed daily for cytopathic effects (CPE) and the cell culture supernatants were collected and stored at –80°C for further analysis.Pools of mosquitoes were homogenized in 1 ml grinding fluid  in a sterile glass grinder and centrifuged in an Eppendorf tube at 5000Viral RNA was extracted from infectious C6/36 cells using RNAiso Plus  according to the manufacturer’s instructions. The RNA was subjected to electrophoresis at room temperature on a standard discontinuous 7%, 10%, or 15% acrylamide  slab gel (18 cm × 16 cm × 0.075 cm)  with a 3.5% acrylamide stacking gel in Tris–glycine buffer  (Bio-Rad Laboratories). After electrophoresis, virus dsRNA was visualized by staining with silver nitrate as described previously .® Reverse Transcriptase (TaKaRa) according to the manufacturer’s instructions. The viral cDNA was subjected to polymerase chain reaction (PCR) amplification with primers targeting the 12th segment of BAV [First-strand cDNA was synthesized using PrimeScript2 tissue culture flasks. Total RNA was extracted from the infected cells with greater than 90% CPE using RNAisoPlus (TaKaRa) according to the manufacturer’s instructions. Single-stranded RNA (ssRNA) was removed by precipitation with 2 M LiCl (Sigma) at 4°C for 16 h and the dsRNA was purified by addition of 2.5 volumes of isopropanol and 1 volume of 7.5 M ammonium acetate, followed by two more washes with 70% ethanol, and resuspended in RNase-free water. The dsRNA was subjected to 1% agarose gel electrophoresis (AGE)  in TAE buffer and purified from the agarose gel using a MinElute gel extraction kit (Qiagen).Genome sequencing was performed by full-length amplification of cDNAs (FLAC) as described previously . BrieflyEscherichia coli DH5α cells (TakaRa). The selected transformed colonies were cultured and sequenced by the Huada Genome Institute .An “anchor primer” PC3-T7 loop , similar to that described by Maana et al. , was usehttp://www.ncbi.nlm.nih.gov/BLAST/). Identity and alignment analysis were analyzed by using the CLUSTAL X (version 2) and MegAlign . MEGA 5.1 was used for phylogenetic analysis and tree construction based on the neighbor-joining assay. The bootstrap value (the number of replications) was 1,000. The potential glycosylation sites of the amino acid sequence were determined by an online analysis using NetNGlyc 1.0 (http://www.cbs.dtu.dk/services/).Initial sequence assembly and analysis were conducted by using the ATGC software package . Sequences were identified by BLAST analysis (Mangshi virus DH13M041 strain was isolated from one pool of mosquitoes (C. tritaeniorhynchus) captured in Dehong Prefecture, Yunnan Province, China in 2013. The virus was found to cause CPE in second-blind passage in C6/36 cells at 72 h after inoculation. The characteristics of the CPE include cell shrinking, shedding, or cytolysis with eventual detachment from the growth surface   . These fSeadornavirus genus, the Mangshi virus has a genome consisting of 12 dsRNA segments. The length of 1–12 segments varies from 788bp (Seg12) to 3740bp (Seg1) and their corresponding encoding proteins and obtained GenBank accession numbers were presented in Mangshi virus shares a stretch of seven highly conserved nucleotides and three highly conserved nucleotides at the ends of the 5′- and 3′-UTRs  in each of the 12 gene segments. The first two nucleotides of the 5′-UTRs end are the reverse compliments to the last two nucleotides of the 3′-UTRs end. Each gene segment contains a unique open reading frame and encodes its corresponding protein (VP1–VP12).Like other members of the Balaton virus isolated from Europe is from 27.3% (VP11) to 72.3% (VP1) and the identity of amino acid sequence between the Mangshi virus and BAV varies from 18.0% (VP11) to 63.9% (VP1). Much lower levels of amino acid identity of the Mangshi virus were shared with the KDV and LNV of Seadornavirus genus, which vary from 17.3% (LNV VP9) to 40.6% (LNV VP1)  . The ideMangshi virus 1–12 genome segments were matched with proteins encoded by seadornaviruses by analyzing the deduced amino acid sequence. However, the putative function of each gene segment of the Mangshi virus is not consistent with that of the BAV, in which only six proteins encoded by Seg-1, S eg-2, Seg-3, Seg-4, Seg-9, and Seg-10 of the Mangshi virus are similar to that of the BAV, and another six proteins encoded by Seg5, Seg6, Seg7, Seg8, Seg11, and Seg12 of the Mangshi virus matched the proteins encoded by Seg-6, Seg-5, Seg-8, Seg-7, Seg-12, and Seg-11 of BAV respectively  and GDD (positions 755–757) in VP1 of Mangshi virus and BAV varies from 49.9 to 50.7%. Compared with VP3 of the Mangshi virus and VP3 of other seadornaviruses, the Motif Kx(I/V/L)S related to guanylyltransferases is mutated, in which the first aa K is changed into L . The identity of amino acids in VP4 between the Mangshi virus and BAV is 47.3%–48.3%, and is 34.8%–36.5% in VP9, which suggests that glycosylation sites and numbers of Mangshi virus are distinct from BAV.The host range and virulence of virus as well as host immune response against virus are affected by glycosylation of virus proteins . AnalysiMangshi virus and the corresponding sequences from seadornaviruses  were generated. The results indicate that Mangshi virus belongs to the genus Seadornavirus. A more detailed analysis indicates that the Mangshi virus is most closely related to, but distinct from, Balaton virus and BAV within cluster , a new 12-segmented RNA virus genus, Seadornavirus genus, was added into the Reoviridae family [Mangshi virus isolated from C. tritaeniorhynchus in Dehong Prefecture, Yunnan Province, is similar to BAV, an important pathogen in viral encephalitis [Mangshi virus are distinct from those of BAV, including time to develop CPE in C6/36 cells , migration patterns of viral genome, motif Lx(I/V/L)S in VP3, motif in the ends of the 5′-UTRs as well as glycosylation sites in VP4 and VP9. The gene in segment 11 of the Mangshi virus could not be amplified by the specific primers based on the conserved sequence of BAV segment 12 because of the low sequence identity. These data together suggest that the Mangshi virus is significantly different from BAV.According to the 8Reoviridae (VP1 identity is more than 30% at amino acid level) [Seadornavirus species, the identity of VP1 at the amino acid level is from 93% to 99% in BAV [Mangshi virus and seadornaviruses including Balaton virus, BAV, LNV, and KDV is from 40.1% to 72.3% , suggesting that the Mangshi virus is a newly discovered species of Seadornavirus. In addition, the amino acid identity observed in T2 protein (used to classify serotypes within the Orbivirus genus) encoded by segment 2 of Mangshi virus shares only 34%–66.9% with that of Balaton virus, BAV, LNV, and KDV, which is well below the 91% threshold for virus serotype classification according to previously defined criteria of the Orbivirus genus [Mangshi virus clusters into an independent evolutionary branch in seadornaviruses, indicating that the Mangshi virus is a new member of the Seadornavirus genus.Viral RdRp polymerase protein (VP1) encoded by segment 1 is a highly conserved and an important marker for species identification within the family d level) ,19. With% in BAV . In thisus genus ,20,21. TMangshi virus VP8 and VP9 to homologous proteins of Balaton virus from Europe are only 49.5% and 43%, and are only 39.9%–43.6% and 34.8%–36.5% to that of BAV, in which all are far less than 72% and 54% (differences between two genotypes of BAV), respectively. The amino acid identity of the other segments  is also much less than the threshold (83%) for classification of two genotypes [Mangshi virus is a newly discovered virus species that is related to Balaton virus and BAV.BAV is divided into genotype A (BAV-Ch and BAVIn6423) and genotype B (BAVIn6969 and BAV-In7043) by whole-genome sequence analysis. The identity of amino acid sequences of Seg-7 and Seg-9 between two genotypes are 72% and 54%, respectively, and the identity of amino acid sequences among the rest of segments are from 83% to 98% . The amiBalaton virus were isolated in various areas of China, Vietnam, Indonesia, and Hungary [Culex spp. found at the borders of China, Laos, and Myanmar [Mangshi virus are similar to that of the MX6 virus, but the identity of amino acid sequences of Seg9 between the MX6 and Mangshi virus is only 81%, suggesting that the Mangshi virus and MX6 virus may be the same virus of different genotypes.BAV was firstly isolated from cerebrospinal fluid and sera from patients with viral encephalitis in Banna prefecture of Yunnan Province in 1987. Subsequently, BAV, LNV, KDV, and  Hungary . In 2005 Myanmar . ElectroC. tritaeniorhynchus and demonstrated that it is a new virus member of Seadornavirus genus, in the Reoviridae family, related to Balaton virus and BAV. This is the first report of isolation of a new Seadornavirus from C. tritaeniorhynchus in the southwest border area of Yunnan Province, China. The present study suggests that a number of Seadornavirus species and genotypes are prevalent. This should therefore prompt further investigations for a better understanding of the origin and replication of the Mangshi virus and the association between the Mangshi virus and human public and domesticated animal health.In summary, we isolated a 12-segmented dsRNA virus from


By incorporating the time delay due to the period that computers use antivirus software to clean the virus into the SLBRS model a delayed SLBRS computer virus model is proposed in this paper. The dynamical behaviors which include local stability and Hopf bifurcation are investigated by regarding the delay as bifurcating parameter. Specially, direction and stability of the Hopf bifurcation are derived by applying the normal form method and center manifold theory. Finally, an illustrative example is also presented to testify our analytical results. For the purpose of studying the prevalence of computer viruses and posing effective strategies of controlling their spread across the Internet and considering the similarity between the propagation of computer virus across the Internet and that of biological viruses across a population, many scholars have proposed computer virus models by properly modifying some epidemiological models –6. In 22, Han anS(t) denotes the percentage of computers that have no immunity at time t; L(t) denotes the percentage of infected computers that are latent at time t; B(t) denotes the percentage of infected computers that are breaking out at time t; and R(t) denotes the percentage of uninfected computers that have temporary immunity at time t. μ is the constant rate for the external computers connecting to the Internet and that for the internal computers disconnecting from the Internet. β is the rate of contact for the virus-free computer. α is the rate coefficient from class R to class S, ε is the rate coefficient from class L to class B, and γ is the rate coefficient from class B to class R. Yang et al. τ=τ0−1 ≠ 0. According to the Hopf bifurcation theorem in  such thatϕ ∈ C, R4), we defineA* of A asη(θ) = η.By the Riesz representation theorem, there is a 4 × 4 matrix function with bounded variation components , system  is equivρ(θ) = Teiτ0ω0θ be the eigenvector of A(0) corresponding to the eigenvalue +iτ0ω0 and let ρ*(θ) = Deiτ0ω0s be the eigenvector of A*(0) corresponding to the eigenvalue −iτ0ω0. Then, from the definition of A(0) and A*(0), we obtainρ*, ρ〉 = 1 and Let ω0.From , we can E1 and E2 can be determined by the following equations, respectively,Following the algorithms given in  and usinμ2 > 0  (μ2 < 0), then the Hopf bifurcation is supercritical . If β2 < 0  (β2 > 0), then the bifurcating periodic solutions are stable (unstable). If T2 > 0  (T2 < 0), then the period of the bifurcating periodic solutions increases (decreases).For system , If μ2 R0 = 9.944 > 1 and the unique positive equilibrium D∗ of system , the positive equilibrium D∗ of system  of system dt= dS(t)dt= dS(t)dt= 6dS(t)dt=μ2 = −0.8143 < 0, β2 = −0.9001 < 0, and T2 = 0.0466 > 0. Therefore, we can conclude that the Hopf bifurcation of system , the computer virus model is locally asymptotically stable. In such case, the propagation of the computer virus can be easily controlled. However, once the delay passes though the critical value τ0, a Hopf bifurcation occurs and a family of periodic solutions bifurcates from the positive equilibrium of the model. This is not welcome in networks because the computer virus will be out of control in this case. Further, direction and stability of the Hopf bifurcation have also been determined by using the normal form method and center manifold theory. Numerical simulations are also included to verify the analytical predictions.A delayed SLBRS computer virus model has been studied in this paper based on the model proposed in . Compare


Bunyavidae family is the largest grouping of RNA viruses and arguably the most diverse. Bunyaviruses have a truly global distribution and can infect vertebrates, invertebrates and plants. The majority of bunyaviruses are vectored by arthropods and thus have the remarkable capability to replicate in hosts of disparate phylogeny. The family has provided many examples of emerging viruses including Sin Nombre and related viruses responsible for hantavirus cardiopulmonary syndrome in the Americas, first identified in 1993, and Schmallenberg virus which emerged in Europe in 2011, causing foetal malformations in ruminants. In addition, some well-known bunyaviruses like Rift Valley fever and Crimean-Congo haemorrhagic fever viruses continue to emerge in new geographical locations. In this short review we focus on newly identified viruses associated with severe haemorrhagic disease in humans in China and the US.The  Current Opinion in Virology 2014, 5:50–57Emerging virusesThis review comes from a themed issue on Christopher F Basler and Patrick CY WooEdited by Issue and the EditorialFor a complete overview see the Available online 28th February 20141879-6257/$ – see front matter, © 2014 The Authors. Published by Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.coviro.2014.01.011Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus and Tospovirus) on serological, morphological and biochemical features [Bunyaviruses are characterised by the possession of a tri-segmented negative-sense RNA genome. Viruses are enveloped, replicate in the cytoplasm and mature by budding at the Golgi. Virions are about 100 nm in diameter and are composed of just four proteins: two glycoproteins termed Gn and Gc that are embedded in the Golgi-derived viral membrane and two internal proteins, the nucleocapsid (N) protein that encapsidates each of the three genome segments in the form of ribonucleoprotein complexes, and the L protein (RNA dependent RNA polymerase). The largest RNA segment, L, encodes the L protein, the medium segment, M, the glycoproteins, and the smallest segment, S, the N protein. In addition some viruses encode up to three nonstructural proteins . The morfeatures . There aPhlebovirus genus contains 70 viruses, which comprise 9 species and 33 tentative species, and can be divided into two groups, the sandfly fever virus group and the Uukuniemi-like virus group (Uukuvirus), but the similar molecular biological characteristics along with some weak serological cross reactivity shared with viruses in the Phlebovirus genus resulted in their reclassification [In the latest report of the International Committee on Taxonomy of Viruses (ICTV)  the Phleus group . Before fication . There afication .A characteristic of all phleboviruses is the ambisense coding strategy of the S genome segment: the N protein is encoded in the negative-sense orientation on the S segment while the NSs protein is encoded in the positive-sense. However, both proteins are translated from separate subgenomic mRNAs that are transcribed from the genomic or antigenomic RNA as shown in The best-known phlebovirus is Rift Valley fever virus, a serious pathogen of ruminants but also capable of causing disease, sometimes fatal, in man . Other hBetween 2007 and 2010 cases of an unknown infectious disease were reported in Henan and Hubei Provinces, China, with patients presenting gastrointestinal symptoms, chills, joint pain, myalgia, thrombocytopenia, leukocytopenia and some haemorrhagic manifestations, resulting in a case fatality rate of 12–30% ••. The dPhlebovirus genus, roughly equidistant from the already established sandfly fever and Uukuniemi groups [Comparison of SFTSV viral proteins showed 33%, 30–36%, 30–41% and 11–13% similarity to either RVFV or UUKV RNA dependent RNA polymerase, glycoprotein precursor, N or NSs proteins respectively ••. Notabi groups ••  and virus has been isolated from 8 of the pools, implicating A. americanum as the likely primary vector for transmission of the virus within the USA [Heartland virus was isolated from two patients in Missouri, USA •• who ex the USA .Bunyaviridae but were not further classified into a genus. BHAV was isolated in India in 1954 from a tick on a paralysed goat, and causes fever and signs of central nervous system involvement in young ruminants but not in adult animals. A few cases of febrile illness in humans have also been described. BHAV has been reported in southern and central Asia, Africa, and southern Europe  [Haemaphysalis ticks, like SFTSV, whereas the African strains have been isolated from a wider range of tick species.Bhanja virus (BHAV) is not a newly emerged virus but recent molecular characterisation of this and related viruses is pertinent to the above discussion of SFTSV and HRTV. The Bhanja virus antigenic complex  comprises tick-borne viruses that were assigned to the family Romania) . Two recRomania) •,36• andA. americanum (the lone star tick) in Kentucky in 1967 [Bunyaviridae. The sequence of the viral genome has recently been determined by deep sequencing and shown to be in the same clade as BHAV [Lone Star virus was originally isolated from  in 1967 , and lik as BHAV • (FigureAedes aegypti mosquitoes [The number of known phleboviruses has increased markedly in recent years with the emergence of SFTSV and HRTV, and the genetic characterisation of the previously unassigned BHAV, LSV and related viruses. These viruses form clades  that aresquitoes  as a delsquitoes  NSm regusquitoes  and has squitoes , mediatesquitoes . WhetherIt is likely that more relatives of these tick-borne phleboviruses will be discovered in the future, aided by advances in metagenomic analysis and perhaps heightened surveillance, as has been implemented in China. Of great importance is to understand the molecular basis for the different pathogenicity of these viruses in man, a task that will keep bunyavirologists occupied for years to come.• of special interest•• of outstanding interestPapers of particular interest, published within the period of review, have been highlighted as:


Thus, exercises to increase the ROM of the ankle dorsiflexion, hip flexion, and dorsiflexor strength can be recommended to improve squat performance. Future studies should assess an increased ROM of the ankle dorsiflexion, hip flexion, or dorsiflexor strength effect on deep squat performance.The purpose of this study was to determine which variables of the range of motion (ROM) and strength of the hip, and ankle are associated with squat depth. In total, 101 healthy subjects  participated in the study. Outcome measures consisted of the ROM of hip flexion, hip internal rotation, external rotation, ankle dorsiflexion with an extended and flexed knee joint, and strength of the hip flexor and ankle dorsiflexor. Squat depth was measured using SIMI motion analysis software. Pearson correlation was used to determine the relationship between variables and squat depth. Multiple stepwise regression analysis was performed to determine variables associated with squat depth. The multiple regression model indicated that ankle dorsiflexion with a flexed knee and the hip flexion ROM were significantly associated with squat depth in male subjects (R Squatting is a common and popular exercise among athletes and the general public . In partThe squat is defined as a sitting posture with dorsiflexed ankles, a deeply flexed knee and hip  and is oPrevious investigators reported that decreased strength of the hip and ankle musculature reduced the ability to stabilize the lower extremity, resulting in faulty alignment of the lower extremity such as adduction and rotation of the hip and knee valgus . During Squatting performance requires strength as well as mobility of the hip, knee, and ankle, because squatting involves multiple joints . Macrum Understanding how joint mobility and strength can affect squat depth may help in evaluating lower extremity function and direct exercise strategies for individuals with limitations in squatting. Thus, the purpose of this study was to determine variables associated with deep squat depth. We hypothesized that factors such as the ROM and strength of the hip and ankle would explain deep squat depth. We anticipated that the results of this research could be used to enhance the evaluation of the squat depth performance and highly correlated factors with squatting could be targeted as part of an intervention program to improve squat performance.One hundred-one subjects  were recruited as a convenience sample from students enrolled at the Yonsei University. Characteristics of the 101 subjects and descriptive statistics for the ROM and strength of the hip and ankle are presented in A universal goniometer (UG) with a double-armed full-circle protractor was used to measure the active ROM of the hip, and ankle joint. The UG is a useful and convenient device for ROM measurement. Passive ROM movements of the hip and ankle were performed three times prior to data collection for warm-up and familiarization purposes. For ROM measurement of hip flexion, the subject laid on a table . The axiAll strength measurements were performed using a hand-held dynamometer . The test procedure followed the isometric “make” test of The subject was required to stand with legs apart at pelvic width, with both hands clasped and held on the back of his/her head. Subjects squatted to reach the greatest depth of the squat while continuing to look straight ahead. The squat was performed as low as possible without heel-off and the maximal squatting position was maintained for 5 s. The maximum squatting position was captured using a digital camera. To collect photographs of the sagittal plane view, a digital camera  was placed at 2.4 m from the sagittal plane of the subjects at the height of the knee and perpendicularly to the floor using a water-based level.Collected images were imported and analyzed using the SIMI software , and squat depth was measured vertically between the hip and the floor. The squat depth of each subject was normalized using their leg length and presented as % leg length. All data were collected three times, and the average of the three trials was used for further analysis.2) shows variation in the squatting ability that is explained by the regression variables. Pearson correlation and multiple regression were conducted separately for each gender. Data analysis was conducted using the SPSS software (ver. 12.0) and the significance level was set at p = 0.05.Pearson correlation matrices were constructed to examine the relationships between the ROM and strength of the hip, ankle and squat depth in the squatting position. To investigate which lower limb variables contributed most significantly to squatting ability, multiple regression models with a stepwise selection procedure were performed for the active ROM of the hip flexion, internal and external rotation, and ankle dorsiflexion and strength of the hip flexor and ankle dorsiflexor at the dominant leg as independent variables, while squat depth (% leg length) was the dependent variable. The determination coefficient (Rp<0.05). In females, the ROM of the dorsiflexion with an extended and flexed knee was significantly negatively correlated with squat depth , while there was a positive correlation between strength of the dorsiflexor and squat depth . No significant correlation between the ROM of the hip external rotation or strength of the hip flexor and squat depth was found in the squatting position (p>0.05).p<0.001). For females, the ankle dorsiflexion ROM with an extended knee and strength of the dorsiflexor were included as predictor variables, accounting for 32.4 % (p<0.001).Stepwise multiple-regression analyses were performed to identify variables that contributed significantly to deep squat depth in healthy subjects. In the results of the regression analyses for males, ankle dorsiflexion with a flexed knee and the hip flexion ROM were included as predictor variables, accounting for 43.5% of the variance in squat depth ; p<0.001r 32.4 % ; p<0.001We investigated factors associated with squat depth in healthy subjects. Deep squatting ability is important for activities on the ground and efficient exercise to increase muscle strength of the lower extremities. However, to our knowledge, there has been no previous report as to which factors are associated with squat depth. In this study, we demonstrated that the ROM of the ankle dorsiflexion with a flexed knee and hip flexion were important factors for deep squatting in males, and dorsiflexion with an extended knee and dorsiflexor strength were important factors in females. Thus, these results may help in designing exercise programs to improve deep squatting depth in subjects with poor performance.In the resulting model, the ankle dorsiflexion ROM with flexed and extended knee correlated significantly with squat depth, accounting for 38.8 and 23.7% of the variance in male and female subjects, respectively (p<0.05). One of the primary roles of the hip joint is to provide a pathway for the transmission of forces between the lower extremities and pelvis during activities such as squats. During a squat, the mean hip ROM has been reported to be 95.4 ±26.6° of flexion to reach a maximal squat . If hip Ankle dorsiflexor muscle strength explained an additional 8.7% of the variance in squat depth. The ankle plays an important role in the closed kinematic chain during the deep squat. Our study has several limitations. The first limitation is the lack of generalizability to other age groups; all the subjects participating in this study were in their twenties. Second, we only focused on the ROM and muscle strength in the lower extremity. Lumbar stability and thoracic mobility are also important factors to decrease compressive and shearing force of the lumbar region during deep squatting . Third, Squatting has been regarded as an exercise to increase the strength of the lower extremity musculatures. Although squatting performance is commonly used in training or clinical settings, there may still be unknown factors that determine squat depth. This is the first reported study to investigate factors associated with squat depth. The results of this study show that squat depth is negatively related to the ROM of the hip flexion, internal rotation, and ankle dorsiflexion with an extended and flexed knee in males and dorsiflexion with an extended and flexed knee in females. Additionally, strength of the ankle dorsiflexor is positively related to squat depth in females. The ROM of the ankle dorsiflexion is a major factor affecting squat depth in both genders, followed by the hip flexion ROM in male and ankle dorsiflexor strength in female subjects. According to sex, different factors influenced squat ability, what suggests that clinicians and athletes should approach the improvement of squat depth depending on gender. Based on our results, we recommend that in training for the deep squat, it is important to increase the ankle dorsiflexion and hip flexion ROM in male, and the ankle dorsiflexion ROM and strength in female subjects if they are limited. Improved mobility and strength of the ankle and hip joint can help in preventing injuries related to squatting and help to ensure its safe performance.


Interestingly and in contrast with previous results, we found that the bursting mode of virus production generally results in a higher probability of viral extinction than the budding mode of virus production. Second, this mathematical model was not able to accurately describe the change in experimentally determined probability of host infection with increasing viral doses. Third and finally, the model was also unable to accurately explain the decline in the time to virus detection with increasing viral dose. These results suggest that, in order to appropriately model early HIV/SIV dynamics, additional factors must be considered in the model development. These may include variability in monkey susceptibility to infection, within-host competition between different viruses for target cells at the initial site of virus replication in the mucosa, innate immune response, and possibly the inclusion of several different tissue compartments. The sobering news is that while an increase in model complexity is needed to explain the available experimental data, testing and rejection of more complex models may require more quantitative data than is currently available.Upon infection of a new host, human immunodeficiency virus (HIV) replicates in the mucosal tissues and is generally undetectable in circulation for 1–2 weeks post-infection. Several interventions against HIV including vaccines and antiretroviral prophylaxis target virus replication at this earliest stage of infection. Mathematical models have been used to understand how HIV spreads from mucosal tissues systemically and what impact vaccination and/or antiretroviral prophylaxis has on viral eradication. Because predictions of such models have been rarely compared to experimental data, it remains unclear which processes included in these models are critical for predicting early HIV dynamics. Here we modified the “standard” mathematical model of HIV infection to include two populations of infected cells: cells that are actively producing the virus and cells that are transitioning into virus production mode. We evaluated the effects of several poorly known parameters on infection outcomes in this model and compared model predictions to experimental data on infection of non-human primates with variable doses of simian immunodifficiency virus (SIV). First, we found that the mode of virus production by infected cells (budding  Acute infection with human immunodeficiency virus 1 (HIV-1 or simply HIV) consists of several well-defined phases. The majority of transmissions occur through vaginal or rectal intercourse, when the virus is able to cross the epithelial layer of the genital tract or rectal mucosa . For appTo better study early events of virus replication, a useful experimental model for HIV infection is simian immunodeficiency virus (SIV) infection of nonhuman primates of Asian origin ,6,7,8,9.Many of the interventions to halt HIV epidemics, including vaccines and post-exposure antiviral treatment, target virus replication at a very early stage of infection. Vaccines inducing high levels of neutralizing antibodies have been shown to reduce the probability of infection with SIV in vaccinated monkeys , while vTo date, mathematical models of different levels of complexity have been proposed to study early HIV/SIV dynamics ,22,23,24early virus dynamics, and evaluate whether it can describe available experimental data . In ouru et al. ,31. SpecThe paper is structured as follows. We first describe the model and properties of its asymptotic behavior, and then discuss constraints on the model parameters imposed by experimental data . We thenSeveral previous studies have described HIV dynamics during the first weeks post infection using mathematical models ,23,24,30IE, and cells that are actively producing the virus, I , due to the activation of DNA-dependent protein kinase during integration of viral DNA into host chromosome or due to accumulation of DNA intermediates in the cell’s cytoplasm  used 32 infected cells as a threshold for an established infection. We chose instead to track virus numbers, a quantity that is generally measured in experiments. The number of infectious viruses at which the infection becomes detectable was determined by matching model predictions to experimental data for different parameter sets. In some simulations, we used Vdet = 100 or 1000 infectious viruses because at this value the dynamics of the populations in the model became relatively deterministic. At V∼ 103, the dynamics of the populations in our model are nearly deterministic since the coefficient of variation, for example, in virus number is onlydeSolve in R.Pearson χ2 test.To compare the nested models we use the likelihood ratio test ,87. In cin vivo. We believe that our study contributes to a better understanding of the process of transmission and early infection with SIV (and possibly HIV). Our analysis suggests that simple, one-compartment models are not consistent with the data on SIV dynamics during early stages of infection. More detailed models will be needed to provide a better description of early virus dynamics, and testing and rejecting different versions of such models will require very detailed quantitative experimental data.The first week of infection is a crucial time for viral growth and dissemination but is extremely difficult to study


Aedes aegypti infestations throughout most of tropical and subtropical Americas certainly is an important factor and the spread of Aedes albopictus may play a role. However, Aedes vectors have been present in tropical and sub-tropical Americas in sufficient numbers to support dengue and chikungunya epidemics in the Americas for over forty years. An increase in vector prevalence alone does not explain the pandemic. Two other factors may play significant role.The Zika virus, previously known as an uncommon zoonotic virus in Africa, has spread first to Southeast Asia and then to the Pacific islands and to the Americas causing a global health emergency due to the associated increase in children born with microcephaly and an increase in the incidence of Guillain-Barre syndrome. The explanation for what caused this virus, previously confined to a zoonotic niche in Africa, to become a major pandemic threat is as yet unknown but three factors are thought to play a significant role. It is probable that no single factor is responsible but the interaction of several factors has enabled this virus to spread across the globe. The increase in The adaptation of the Zika virus to the human host by changes in codon usage in non-structural proteins has resulted in increased fitness of the virus for replication in the human host . The panAnother major factor is immune enhancement by pre-existing heterologous anti-flavivirus antibodies. Immune enhancement by non-neutralizing cross-reactive antibodies results in virus penetration of cells through the Fc receptor and increased viral replication. Immune enhancement plays a major role in the pathogenesis of severe dengue infections ,3. Zika The above factors proposed as major elements involved in promoting and sustaining the Zika pandemic are not mutually exclusive, indeed they can interact in a synergistic fashion. Both codon optimization and immune enhancement can make this virus a more effective human pathogen and more infectious to the vectors. Acting together they can create the virologic and epidemiologic equivalent of a perfect storm.


This influenza A-derived DI genome protects by two totally different mechanisms: molecular interference with influenza A replication and by stimulating innate immunity that acts against non-influenza A viruses. The review considers what is needed to develop DI genomes to the point of entry into clinical trials.Defective interfering (DI) genomes are characterised by their ability to interfere with the replication of the virus from which they were derived, and other genetically compatible viruses. DI genomes are synthesized by nearly all known viruses and represent a vast natural reservoir of antivirals that can potentially be exploited for use in the clinic. This review describes the application of DI virus to protect from virus-associated diseases  Extin vivo are the negative-sense, single-stranded RNA IAVs [in vivo, even if they interfere strongly in vitro.At the present time the only DI viruses that have given clear evidence of clinical promise RNA IAVs ,6 and thRNA IAVs . There iRNA IAVs . IntranaRNA IAVs . The reain vitro data, DI RNA interferes with infectious IAV in vivo by inhibiting the replication of infectious virus and preferentially packaging the newly synthesized DI RNA into new virions in place of the cognate full-length RNA segment. In addition there is a huge  amplification of the amount of DI RNA present in the respiratory tract by the infecting IAV, and reduction in the amount of new infectious virus [Broadly speaking 244 DI IAV has two different mechanisms of protection. Extrapolating from the us virus ,30,45. Tin vivo stability of DI RNA. 8 or 10−40. In addition there would have to be at least one commensurate mutation in the RNA encoding the polymerase complex  which would reduce the chance of an escape mutant to 10−45, or 1/1000,000,000,000,000,000,000,000,000,000,000,000,000,000,000.With a mutation rate of approximately 10−5)2 or 10−10, which exceeds the yield of infectious virus per cell.The strength of this argument is exemplified by the generation of monoclonal neutralizing antibody escape mutants of IAV. This requires a coding mutation in the gene expressing the haemagglutinin protein such that neutralization is abrogated. The selection is so strong that with one monoclonal antibody a virus population becomes predominantly escape mutant in a single passage in embryonated eggs. However, when virus is incubated simultaneously with two monoclonal antibodies directed against separate and discrete epitopes, no escape mutants are found . The mutThe RNA of 244 DI virus is responsible for stimulating interferon type I since prolonged UV irradiation, sufficient to abrogate protection from influenza A challenge but not to denature virus haemagglutinin and neuraminidase activities, abrogates both interferon production and protection . Proof tde novo. Ideally the DI virus should be generated in the target tissue or cells derived therefrom, as the cell is known to influence both the generation and propagation of DI RNAs [in vivo [Naturally generated DI virus comprises a population of DI genomes that are heterogeneous in sequence and interfering ability, and these continue to evolve as the DI virus is passaged. In order to have a stable product that can be quality controlled it is essential to have a defined DI genome that can be monitored by sequencing. This can be achieved by cloning DI genomes from a heterogeneous population and creating a range of DI viruses each with a unique DI genome sequence. Further passage can be minimized to avoid evolution of the cloned DI genome and the possibility of other DI genomes arising  DI RNAs . These c[in vivo . The cre[in vivo ,52,53,54in trans by a coinfecting helper virus, and ideally this should be removed or inactivated before treating cells or animals, as large amounts of infectious virus can still be present. However, infectious virus is usually a relatively minor component of a DI virus preparation (<1%) and adds little to the total virus mass present. DI and infectious viruses are antigenically identical. Usually DI and infectious viruses cannot be physically separated and even when this can be done (as in the case of DI rhabdoviruses) it is not an efficient process [All DI viruses are replicated  process . As discProtection that involves amplification of the DI genome by a closely related virus can only occur if the genomes of DI and infectious viruses enter the same cell. This is facilitated if they are inoculated in close physical proximity and both use the same cell receptors. This is less relevant if the DI virus acts by stimulating interferon or other mediators of innate immunity, as these can influence cells regardless of virus receptor specificity. However, the amount of interferon stimulated by DI influenza virus is relatively low , so agai0R) of both the DI genome and the infecting virus. (0R is the average number of secondary infections (infected cells) produced by one infected cell when all individual cells are susceptible). Thus to have some chance of success, a ballpark figure for starting an initial titration would be , say, 109 to 1010 particles of DI virus (approximately 1 µg of DI virus protein). Establishing an effective dose is an empirical process.A relatively large amount of DI virus may be needed to protect an animal, as it is necessary for a significant proportion of cells of the target tissue to receive at least one DI genome. In this way, under conditions of prophylaxis, there is a good chance of the DI and infectious virus genomes coming together in the same cell and interacting so that infectious virus can amplify the DI genome before the infectious virus can replicate unimpeded. The number of DI and infectious genomes for successful protection depend on many variables including the amount of DI virus inoculated, the number of susceptible cells, the dose of infecting virus, and the reproductive rate . The example of influenza 244 DI virus is an exciting and promising start and, while no DI virus has yet entered a clinical trial, consideration of how this might come about will inform the route to the clinic for any DI virus candidate.Clinical testing of a DI virus requires convincing and extensive preclinical data. DI virus occupies a unique position in the antiviral lexicon. It is clearly a biological product, not a conventional small molecule antiviral. While a DI virus looks superficially like a vaccine it is clearly very different in its mode of action and therefore in its clinical profile. In particular the aim with a DI virus is to use it at a sub-immunogenic level and avoid any immunity that obviates repeat treatments, so that repeated dosing can be given as required. In addition to this new paradigm can be added consideration of whether or not a DI virus is a live organism: it cannot reproduce itself although in the case of DI 244 it has limited synthetic activity and can transcribe, but not replicate, its RNA. Left on its own in a cell or in the presence of non-IAVs the DI RNA decays, but with an infectious IAV genome in the same cell, the DI RNA is amplified enormously. However, progeny DI virus has the same antigenicity as the infectious virus, and is cleared at the same time as infectious virus at the end of the infection .in vivo have been based on mixed populations of DI genomes, and cannot be relied on as they represent an average of the effects of several different DI molecules. There is a particular dearth of data on viruses that cause systemic infections, although vesicular stomatitis virus was investigated in vivo many years ago [As will be evident from the foregoing discussion there is no “one size fits all” answer for the clinical appraisal of DI viruses. Each disease presents a unique clinical scenario that offers an array of challenges when considering treatment by DI virus. The upside is that there is no shortage of potential candidates, as many, if not most, viruses make DI genomes and DI viruses, although the situation is not fully clear for retroviruses. The ease with which a virus spawns DI genomes/viruses varies hugely ,56,57. Fears ago ,5. This Once a disease is selected for investigation and treatment, the relevant DI genomes have been cloned, and at least one of them shown to have good preclinical efficacy, then the matter of administration has to be decided. Treatment of respiratory tract infections, such as influenza, by DI virus, as discussed above, can be can be carried out intranasally or orally by droplet spray or aerosol administration. However, there appears to be compartmentation of the protective effects of DI virus, as introduction of DI influenza virus into the respiratory tract does not in inhibit viruses that infect systemically. For example, interferon type I stimulated by influenza DI 244 in the respiratory tract was active locally in the respiratory tract, but not against a systemic infection with encephalomyocarditis virus (unpublished data). Presumably the small amount of interferon made locally in the respiratory tract is insufficient to be effective systemically.The majority of known infections affect the respiratory tract, the gut, the urogenital system, or systemically. Viruses can gain entry to the body directly via the respiratory tract; oral intake of virus can lead to respiratory tract or gut infections, and the urogenital system is infected directly. Systemic infections can start in the respiratory tract , oral4–105 infectious units per dose by intranasal instillation  frequently use 10n  to ensun (e.g., . The gre


Cardiovascular disease is the most frequent cause of death in Australia, with an associated cost burden of 11% of Australian annual health expenditure of which 40% is for hospital admissions. We investigated health outcomes and the components of hospital expenditure in the two years after an atherothrombotic disease admission to a tertiary hospital in an Australian setting.Using data linkage we analysed two years of hospitalisation data and death records of all men and women aged 35–84 years with an admission to a Western Australian tertiary hospital for atherothrombotic disease in 2007. Costs were identified by matching the Australian refined diagnostic related group on the admission records to the published schedules of public and private hospital costs for the period of interest, and converted to 2013 Australian dollars.Of 6172 patients studied , 783 (13%) died during follow-up and 174 of these were in hospital case-fatalities at index. Thirty-two percent of patients (n = 1965) accounted for 3172 readmissions to hospital with one in three having multiple hospitalisations. The hazard ratio of atherothrombotic disease readmission was 1.45  in those with more than one vascular territory affected compared to those with only one territory affected after controlling for age, sex, comorbidity, admission type, procedures, and episode length of stay. The total index plus 2-year admission cost for atherothrombotic disease was calculated at $101 million; $71 million for index, and $30 million for readmissions.Among patients hospitalised with atherothrombotic disease, the cost of related rehospitalisations within 24 months is almost a third of the total. Much of the readmission costs fell within the first year. Whether readmissions and cost associated with atherothrombotic disease can be lowered through secondary prevention measures requires further investigation. Cardiovascular disease (CVD) is the most frequent cause of death in Australia, and commands more of the government’s health expenditure than any other single disease. Approximately 11% of Australian annual health expenditure is spent on CVD, of which 40% is for hospital admissions. Better The majority of CVD is attributable to atherothrombotic disease (ATD). ATD describes the shared pathology of atherosclerosis with superimposed thrombosis in ischaemic cerebrovascular disease (CeVD), coronary heart disease (CHD), and peripheral arterial disease (PAD); the combined determinants of CVD. AtherotThe two year medical costs of ATD have been modelled for eight European countries, and 12- The study sample comprised all Western Australian residents aged 35–84 years admitted to a tertiary public hospital in Perth during 2007 with a primary diagnosis of ATD selected using methods previously published. In WestThe first ATD admission to a tertiary hospital in 2007 was identified and considered the index admission (this could be incident or recurrent ATD). Major comorbidities for ATD were also identified. A histoThe data was extracted from two of the state’s administrative databases, the Hospital Morbidity Data Collection (HMDC) and Death Registrations. The HMDC covers all admissions to any public or private hospital in WA, while death registrations include those deaths registered in WA. Both databases are linked by the WA Data Linkage System at the Department of Health. Data liAn episode of care refers to the period from a person’s first admission to the hospital system until they leave the hospital system. This may be an admission and discharge from the same hospital, or a contiguous set of transfers among hospitals. Rehospitalisation or readmission refers to episodes of care occurring after the index event. Rehospitalisation and death data were available for two years after the index event.Ethics approval was obtained from the Human Research Ethics Committees at the WA Department of Health and The University of Western Australia.Costs were identified by matching the Australian refined diagnostic related group (AR-DRG) on the HMDC admission records to the published schedules of public and private hospital costs for the years from 2007 to 2009. We used AR-DRG version 5.1 public and private costs for admissions in 2007 and 2008,, 18 and Individual level costs for ATD episodes of care (defined as an ATD code in the primary diagnosis field at least once during the episode) were calculated on the index admission, and summed for readmissions up to 24 months following the index admission.The linked dataset included an Accessibility/Remoteness Index for Australia (ARIA+) and Socio-Economic Indices For Area (SEIFA) disadvantage score for each admission. ARIA + is based on distance by road to reach service centres. ResidenMean costs per individual for the index admission and for subsequent readmissions to 90 days, from 90 days to 12 months, and 12 to 24 months after index were calculated by 10-year age groups , vascular territory, SEIFA disadvantage quintile, and ARIA + category of the index admission. Mean costs are presented as they reflect the population level costs. Chi-square test was used to compare the proportion of deaths in each age group.Analysis of variance and t-tests were used to compare mean costs by age, sex, metropolitan or rural residence and socioeconomic disadvantage quintile. Cox regression was used to investigate factors associated with time to first ATD readmission within 2 years (and censored if died within 2 years). Age group, sex, rural or metropolitan residence, SEIFA disadvantage quintile, emergency or elective admission, vascular territory , polyvascular disease (more than one territory affected), comorbidity history, and length of stay at the index admission were included in the base Cox model. Forward selection with significance level for entry set to 0.01 was used to check for interactions with age group, sex, polyvascular disease and index vascular territory. Statistical analysis was performed using SAS 9.4.The sample comprised 6172 patients, the characteristics of which are presented in Table There were 783 (13%) deaths in the two years (including those occurring at the index event) and of these 439 had a cardiovascular cause of death code. Most deaths occurred in the first twelve months after the index event . Of these, 174  were in-hospital case-fatalities at the time of the index event and another 85 out-of-hospital fatalities occurred in the 28 days following their index admission. The oldest age group experienced the greatest proportion of deaths with much smaller proportions in the younger age groups .. Fifty seven per cent of readmissions were emergency admissions. Table Thirty-two percent (1965) of patients had at least one ATD readmission in the 2 years after the index admission and 1077 of those patients had a readmission in the first 90 days after the index admissionFigure Having ATD in more than one vascular territory at the index admission was highly significant, with the risk of ATD readmission approximately 1.5 times higher in those with polyvascular disease compared to those with only one vascular territory affected controlling for age, sex, comorbidity, admission type, procedures, and episode length of stay . Rural or metropolitan residence and SEIFA disadvantage quintile were not included in the final model as they were non-significant and missing values were restricting the study sample included in the analysis. The risk of readmission was 2.3 to 2.6 times higher for incident or recurrent PAD than incident CHD for emergency index admissions, but not for elective index admissions . However, in the fully adjusted model , the risk of readmission tends to increase with age .The total index plus 2-year admission cost for ATD was calculated at $101 million; $71 million for index (6172 episodes of care), and $30 million for readmissions, comprising $14 million in the 90 days after the index admission, $9 million from 90 days to 12 months, and $7 million from 12 to 24 months. Hospital admission costs were highly skewed with many patients not contributing readmission costs and a few patients contributing very high costs.Figure Index CeVD admissions had the highest mean cost , whilst patients with a PAD index admission had the highest mean readmission costs at 12 and 24 months . Rural residents have a slightly longer, though not statistically significant, episode length of stay on the index admission. Readmission costs at each follow-up point were also slightly higher for rural residents, but the difference did not reach statistical significance.The most disadvantaged socioeconomic quintile had the highest costs at index, 90 days, and 12 months Table . At indeOf those admitted to a Perth tertiary hospital for ATD in 2007, 13% died and 32% experienced a readmission within 24 months. The 2-year cost to the health system, including index admission, was $101 million . Readmission costs for ATD in 24 months following an index admission were approximately 42% of the total, with more than three-quarters of these costs falling in the first 12 months after the index event.A much greater proportion of those in the youngest age group had no readmissions, but there were remarkable similarities in the four older age groups and this cannot be explained by reduced follow-up in older age groups due to more deaths, as the estimated risks of readmission from the unadjusted Cox model showed a similar age pattern, reflecting the age-specific risks reported by Briffa et al.. In the Those with PAD experienced a high proportion of readmissions. Almost half of the PAD group experienced at least one readmission, and 80% of their readmissions were for PAD. PAD-related hospital admissions are usually for advanced disease requiring costly vascular procedures. Those with PAD are known to be prescribed fewer cardio-protective medications than their counterparts who have experienced CeVD or CHD.The international REACH registry reported that in three years of follow-up 28.4% of those with existing ATD experienced a vascular event or readmission after adjustment for age and sex, with those with CeVD experiencing the lowest readmission rates and PAD the highest. We obseThe ATD hospital admission costs at 12 months for CHD, CeVD, and PAD are slightly higher than the mean hospital costs published by Ademi et al. in their analysis of the Australian REACH registry data in general practice patients. Only 12A European analysis by Levy and colleagues also indicated that the vast majority of costs are incurred in the first year, though the costs varied substantially between the different countries. The proThe current study reports the costs associated with hospital readmissions for ATD in Perth Tertiary Hospitals. AR-DRGs have enabled measurement of the cost of ATD in such a large unselected sample. Indeed, a strength of the study is the bottom-up approach to costing by applying the individual AR-DRG costs to each admission in the dataset, based on the AR-DRG assigned in the HMDC. This means that the cost will best reflect the patient’s hospital admission and care costs. However, the costs assigned to AR-DRGs are based on an average patient across Australia and therefore may not reflect the precise costs incurred as a result of their admission.In conclusion, among patients hospitalised with ATD the total 2-year cost to the health system at $101 million is substantial. The costs of ATD-related rehospitalisation within 90 days of the index admission are also sizable, across age and sex categories, and readmission costs within 24 months is 42% of the baseline admission costs. With 3172 readmissions, and well-known prevention treatment gap, there is a need to further investigate the cost-effectiveness of secondary prevention pharmacotherapy as a potential means for reducing readmissions and costs, in this setting.


By adopting the BioC format, we aim to make iSimp readily interoperable with other applications in the biomedical domain. To examine the utility of iSimp in BioC, we implemented a rule-based relation extraction system that uses iSimp as a preprocessing module and BioC for data exchange. Evaluation on the training corpus of BioNLP-ST 2011 GENIA Event Extraction (GE) task showed that iSimp sentence simplification improved the recall by 3.2% without reducing precision. The iSimp simplification-annotated corpora, both our previously used corpus and the GE corpus in the current study, have been converted into the BioC format and made publicly available at the project’s Web site: Database URL:http://research.bioinformatics.udel.edu/isimp/ With the accelerating growth of biomedical publications, biologists have difficulty in keeping up with the new findings reported in the papers. Natural language processing (NLP) techniques have thus been developed to process the biomedical texts. However, the syntactic complexity of the language poses a challenge in designing and applying NLP systems. One solution is to simplify sentences before applying NLP techniques, thus concealing the syntactic complexity from further NLP steps. For this purpose, we have previously developed iSimp , a senteiSimp can be used as a preprocessing module to provide simplified text to enhance the performance of NLP systems and text mining (TM) applications. To integrate iSimp into wide-ranging applications, we need to design customized adapters for data exchange. Recently, the BioC format has emerged as a community standard for the exchange of text documents and annotations . Based oWe participated in the BioCreative IV Track 1 (BioC: Interoperability) and adopted the BioC format in iSimp. In this article, we report how BioC is used with iSimp, and how iSimp can be integrated with various applications. Overall, this work makes three main contributions.The first contribution is the development of a BioC tag set for annotating simplification constructs. The tag set can be used in conjunction with any sentence simplification system to exchange data with other NLP systems. The standard tag set also serves the purpose of comparing the results among different simplification systems.The second contribution is a mechanism of using the BioC framework. The proposed mechanism denotes simplified sentences in a corpus file, along with the annotation of simplification constructs in the original sentence. It allows simplified sentences to be included in the BioC annotation file so that they can be processed in place of (or in addition to) the original text. Moreover, the annotated phrases within simplified sentences can be mapped back to the original text. This mechanism is important for two reasons. First, it ensures that the output is presented aligned with the original text. Second, it allows the benchmarking of the NLP procedure, where the outputs must be aligned with the gold standard annotation in the original corpus.The third contribution of this work is the construction of an iSimp corpus presented in the BioC format. The corpus, consisting of 130 Medline abstracts annotated with six types of simplification constructs, can be used for the evaluation of the simplifier. In addition to this corpus, we also transform the GENIA Event Extraction (GE) corpora of the BioNLP-ST 2011 to BioC format. The GE corpora were used to evaluate the impact of iSimp in relation extraction (RE) tasks. All these corpora have been made publicly available for evaluating and comparing various simplification systems.To show the wide applicability and good performance of iSimp, we examined its impact on the RE task. We developed a basic rule-based RE system to recognize the BioC format, presented how iSimp could enhance its performance and showed that iSimp was seamlessly added to the RE system with little effort required for the system integration.This section introduces the concepts and related work for sentence simplification and the BioC framework.E1. A third genetic linkage to disease is alpha-synuclein, a protein that is heavily phosphorylated in Lewy bodies and Lewy neuritis, the pathological hallmarks of PD. (PMID-22342821)In this example, we can see coordination (e.g. ‘Lewy bodies and Lewy neuritis’), relative clause (e.g. ‘that is heavily phosphorylated in …’ referring to ‘a protein’) and apposition . These are major syntactic constructs that contribute to the complexity of sentences. After identifying these constructs, the complex sentence can be broken into multiple simple sentences. Here we show only two examples, which require combining the coordination with the relative clause and one of the appositions:E2. Alpha-synuclein is heavily phosphorylated in Lewy bodies.E3. Alpha-synuclein is heavily phosphorylated in Lewy neuritis.Pattern-based or machine-learning approaches will undoubtedly find E2 and E3 much easier to process for the extraction of information/features than the original sentence.Sentence simplification is a technique to detect various types of clauses and constructs contributing to the complexity of sentences, and to produce two or more simple sentences while maintaining both coherence and the communicated message. By reducing the complexity, sentence simplification can ease the development of NLP/TM tools, as well as other tools, such as machine translation tools. To illustrate the usefulness of sentence simplification, consider the following complex sentence from the biomedical literature:The automatic simplification of sentences was first introduced by  to improBioC is a framework that aims to provide an easy and powerful way of integrating TM tools . It usesIn this section, we describe the methodology of iSimp and show how the BioC format is used to enhance the interoperability of iSimp.http://research.bioinformatics.udel.edu/isimp). To make sentence simplification interoperable with other NLP/TM applications, we see a sentence simplifier as a module to be used at the beginning of NLP/TM pipelines. With this in mind, we designed and developed iSimp , we refer the reader to .In comparison with the works using parse trees or dependency graphs, iSimp uses shallow parsing and recursive transition networks to detect all forms of simplifications. iSimp scans the phrase sequence from left to right. Whenever a trigger word of a simplification construct is found (e.g. ‘and’ for coordination or ‘which’ for relative clause), we attempt to identify the simplification construct using transition networks. If a stop state of the network is found, then a simplification construct was detected successfully. We extended the network to address nested constructs. For an in-depth description of this process, we refer the reader to .coordinationphosphorylates and activates] MEK1, [relative clause which in turn [coordinationphosphorylates and activates] the MAP kinases signal regulated kinases, [coordination & appositive ERK1 and ERK2]]. (PMID-8557975)E4. Active Raf-2 [To examine the usefulness of iSimp, we considered a very simple rule-based RE system. The first relation we focused on was the phosphorylation relation between the trigger and the theme (substrate) as defined in the GE corpora. We used straightforward rules, as shown below, where X is a noun phrase in which the protein or protein product appears as a headword:These rules are able to match straightforward mentions of phosphorylation in text. However, they will fail to find mentions of phosphorylation in complex sentences, like the one shown in (E4). However, the first rule can apply to the simplified (E5a)–(E5c) and extract <phosphorylates, MEK1>, <phosphorylates, ERK1> and <phosphorylates, ERK2>. As long as the rules for extraction are precise, the simplification step will help improve the recall of the system, without hurting the precision.We evaluated iSimp in terms of the impact it had on the performance of the RE system. Thus, we compared the results obtained by the RE system when using versus not using iSimp. The BioC XML format and schema described in the previous section were used to transfer the original data to iSimp and the RE system as well as to transfer the enhanced data from iSimp to the RE system. Besides adding and removing iSimp from the pipeline, no additional changes were made to the steps involved in the pipeline. This not only shows the interoperability of iSimp, but also proves that our proposed mechanism of using the BioC framework works as expected.We tested this basic RE system on the BioNLP-ST 2011 GE task training corpus . PrecisiIn this exercise, we did not include agents because the GE corpora did not consider agents. But, because the above rules are most likely to be affected by noun phrase coordination, we believe simplification will benefit the agent extraction as well.This exercise also illustrates the ability of sentence simplification to keep rules simple and yet achieve good results. Because patterns for simplification and RE are orthogonal, we do not need to multiply rules to consider all their combinations. An alternative way, as shown in the above example, is to treat sentence simplification as an independent task, and not for a particular RE. This way, we can focus on simple rules only. Sentence simplification is then applied to increase the recall of the original system.http://research.bioinfor matics.udel.edu/isimp/corpus.html). This corpus can be used by others to evaluate the performance of iSimp or other sentence simplifiers. The corpus consists of 130 Medline abstracts mentioning proteins and genes, with a total of 1199 sentences. The corpus contains three BioC files: (i) Medline abstracts of raw text, (ii) sentences that are split using the OpenNLP sentence detector and (iii) annotations of simplification constructs at the sentence level. Key files are also provided with additional information that describes the meaning of tags used in the BioC files and the annotation schema. The corpus uses the same DTD provided by BioC for validation.We provide a corpus marked with simplification constructs, using the BioC format . The original text files (.txt) are split based on ‘newline’, and the various parts are stored into passage elements. Entities (in files.1) and event triggers (in files.2) are stored into appropriate passages based on their positions in the text files. Target annotations (in files.2), including events, relations, event modifications and equivalences, are recorded at the document level. If the annotation is marked by more than one continuous span of characters, the script creates several location elements. This also shows the generalizability of the BioC format, which allows multi-segmented annotations.We provide a script to convert the BioNLP-ST corpus to the BioC format  and do not necessarily reflect the views of the National Science Foundation.National Library of Medicine of the National Institutes of Health under award number G08LM010720. This material is also based upon work supported by the National Science Foundation under Grant No. DBI-1062520. Funding for open access charge: National Science Foundation (DBI-1062520).Research reported in this article was supported by the Conflict of interest. None declared.


Genetic relationships between bunyaviruses from Australia and pathogenic bunyaviruses from elsewhere indicate emergence potential. Bunyaviridae. Gan Gan and Trubanaman viruses almost perfectly matched 2 recently isolated, purportedly novel viruses, Salt Ash and Murrumbidgee viruses, respectively. Kowanyama and Yacaaba viruses were identified as being related to members of a large clade containing pathogenic viruses. Taggert virus was confirmed as being a nairovirus; several viruses of this genus are pathogenic to humans. The genetic relationships and historical experimental infections in mice reveal the potential for these viruses to lead to disease emergence.To better understand the diversity of bunyaviruses and their circulation in Australia, we sequenced 5 viruses  isolated and serologically identified 4 decades ago as members of the family  Bunyaviridae contains a diverse group of viruses; 100 species have been approved, and many others have yet to be classified , Trubanaman (TRUV), and Kowanyama (KOWV) viruses can infect humans  isolated from ticks collected from royal penguins on Macquarie Island, an Australian Antarctic Territory, indicated that the virus was probably a member of the genus Nairovirus  and Murrumbidgee virus (MURBV), were recently detected in mosquitoes collected in the Australian state of New South Wales  by using methods previously described . We extrVirus was detected in samples by extraction of nucleic acids, followed by RT-PCR amplification with use of the following virus-specific primers: SAVSF1 5′-CATTGAAGTAAACCTACCAAGTGT-3′ and SAVSR1 5′-TCGAATATTGTATTATAATGATGT-3′, SAVMF1 5′-TTGTACAGTTGCTGGAAATTCAGT-3′ and SAVMR1 5′-TCTGGGTATGTTATACATATTTCT-3′, SAVLF1 5′-AGAAATAATCTAAAAAGAAGCTTA-3′and SAVLR1 5′-AAGTATAGGGTCCAATGCTGTCAA-3′ for SASHV small (S) (653 bp), medium (M) (765 bp), and large (L) (744 bp) segment amplification; and MVSF1 5′-CAGTGAGTTGAACCTAGGTAGCCT-3′ and MVSR1 5′-TCTTTTCTCTCTCCTCCTAATTTGAT-3′, MVMF1 5′-ATGCACACCTGCTTTAACTCAAAA-3′ and MVMR1 5′-GTAGGTGTGTTTATGCATATTTCA-3′, MVLF1 5′-AGGAACAATTTTAAACGTTCAATA-3′ and MVLR1 5′-TAAGATAGGATCACATGCAAATAA-3′ for MURBV S (652 bp), M (762 bp), and L (744 bp) segment amplification. We always included a no-template control.We sequenced the viral RNA genomes as described previously (g for 10 min), layered the supernatant on a 20% sucrose cushion, and subjected it to centrifugation . The supernatant was discarded, and the pellet was resuspended in sterile phosphate-buffered saline (20 μL). Virus-enriched resuspension was prepared for transmission electron microscopy on glow-discharged formvar-coated copper grids and negatively stained with 1% uranyl acetate. All images were obtained on a Tecnai F30 FEG-transmission electron microscope  operating at 300 kV.We clarified the tissue culture medium containing cultured virus  of a highly conserved region of the L segment, which is the only region that has been sequenced for many nairoviruses. We generated alignments by using ClustalW implemented in Geneious version 8.1.6  in 1963 and Ae. vigilax (YACV) in 1970. Electron microscopy revealed multiple particles for both viruses, consistent with bunyavirus morphology  from 2001  of the ovarian tumor domain superfamily of proteases, which has been linked to virus virulence , which iAe. vigilax mosquitoes, which are widely distributed in coastal regions and are one of the primary vectors for Ross River virus transmission in Australia (An. annulipes, from which TRUV and KOWV were isolated, only rarely feeds on humans (Ae. vigilax and Culex annulirostris mosquitoes, respectively (This study characterizes several bunyaviruses, originally collected decades ago, that are genetically similar to viruses known to be pathogenic. However, the drivers of infectious disease are not dependent solely on the genetics of the pathogen; they are also dependent on human and environmental factors, in which vector behavior plays a critical role. Previous infections of weanling mice established the neurovirulence potential of GGV, TRUV, and YACV (I. uriae tick vector for TAGV has a circumpolar distribution on both hemispheres (I. uriae ticks, which live at or have migrated through higher latitudes.Intracerebral inoculation of TAGV into “infant” mice caused death, suggesting some level of virulence (Surveillance and characterization of arboviruses are needed to better elucidate virus ecology, understand how viruses evolve, and be better prepared diagnostically if they ultimately emerge. The use of high-throughput sequencing technologies will mean that sequences for the design of diagnostic reagents to determine the virulence of these viruses can now be used. The use of archived material in this sequencing study shows the previously identified genetic stability of the arboviruses (This work highlights an issue arising from the recent use of new high-throughput sequencing technologies, that is, the designation of recently collected virus isolates as novel material and their renaming. This issue arises from the fact that material collected before the 1980s was identified by use of serologic methods, whereas characterization of more recent isolates is primarily based on sequence analysis. To further illustrate this point, since this work was completed, another orthobunyavirus isolate from the Northern Territory in Australia, Buffalo Creek virus, was reported as being the same species as MURBV in the Mapputta group (The risk for emergence of viruses, such as the ones described in this article, increases as population and growth pressures lead to development of previously undisturbed regions and concomitant exposure to native biota. This issue is especially of concern in Australia, where there is a drive to expand into the tropical northern part of the country. An additional consideration is that some persons may be exposed to insect vectors through research, mining, and other occupational activities, thereby increasing their risk for infection. In such instances, one might expect sporadic disease. The degree to which cases of sporadic disease represent a risk for spread to the community will depend on the factors affecting virulence but also on the degree of symptomatic disease and the rate at which these agents adapt to new mammalian hosts—only time will tell. But forewarned is forearmed, and surveillance remains the best preparation for future threats.Phylogenetic trees of orthobunyaviruses and nairoviruses.