Bovine ephemeral fever (or 3-day sickness) is an acute febrile illness of cattle and water buffaloes. Caused by an arthropod-borne rhabdovirus, bovine ephemeral fever virus (BEFV), the disease occurs seasonally over a vast expanse of the globe encompassing much of Africa, the Middle East, Asia and Australia. Although mortality rates are typically low, infection prevalence and morbidity rates during outbreaks are often very high, causing serious economic impacts through loss of milk production, poor cattle condition at sale and loss of traction power at harvest. There are also significant impacts on trade to regions in which the disease does not occur, including the Americas and most of Europe. In recent years, unusually severe outbreaks of bovine ephemeral fever have been reported from several regions in Asia and the Middle East, with mortality rates through disease or culling in excess of 10–20%. There are also concerns that, like other vector-borne diseases of livestock, the geographic distribution of bovine ephemeral fever could expand into regions that have historically been free of the disease. Here, we review current knowledge of the virus, including its molecular and antigenic structure, and the epidemiology of the disease across its entire geographic range. We also discuss the effectiveness of vaccination and other strategies to prevent or control infection.The online version of this article (doi:10.1186/s13567-015-0262-4) contains supplementary material, which is available to authorized users. Ephemerovirus. It causes an acute febrile illness of cattle and water buffalo known as bovine ephemeral fever (BEF) or various other local names such as 3-day sickness, bovine enzootic fever, bovine influenza or stiffseitke. It occurs over a vast expanse of the globe from the southern tip of Africa to the Nile River Delta, across the Middle East through South and South-East Asia, into northern and eastern Australia, and throughout most of China, extending into Taiwan, the Korean Peninsula and southern Japan  an arthropod-borne rhabdovirus which is classified as the type species of the genus n Figure . BEFV doBEFV displays typical rhabdovirus bullet-shaped morphology Figure , althougNS-α1-α2-β-γ]-L-5′ (in negative sense). The GNS gene encodes a 90 kDa class 1 transmembrane glycoprotein that is structurally related to the G protein and appears to have been generated by gene duplication [NS protein is expressed in infected cells but is not incorporated into virions and, unlike the G protein, does not appear to be fusogenic at low pH [The 14.9 kb BEFV genome is much larger and more complex than those of VSV or RABV, possibly explaining the subtle difference in virion morphology Figure . In addilication . The GNSt low pH , 14. Itst low pH . BEFV α1t low pH . The fun487-K503) that is located at the end of the trimerisation domain (DII) just before the C-terminal stalk of the G protein [172–C182) that forms a loop containing a predicted N-glycosylation site. G3 is the major conformational site in the BEFV G protein, occupying most of the base of the pleckstrin homology (PH) domain (DIII). It comprises amino acids from three linear regions of the G protein that are aligned by the fold. It includes two sub-sites (G3a and G3b) defined by either partial or complete inhibition of MAb binding in competition ELISAs [The BEFV G protein is the target of virus-neutralising antibodies , 17. The protein , 21. It The BEFV N protein is also immunogenic in cattle and in mice. It does not induce virus-neutralising antibodies or a protective response , 23 but BEFV is considered to exist as a single serotype worldwide. Various neutralisation tests conducted using isolates from Australia, China, Japan, Kenya, Nigeria and South Africa have demonstrated strong antigenic cross-reactions –29. TherVariations have also been detected in the major neutralisation sites of the G protein. A study of 66 Australian BEFV isolates collected between 1956 and 1992 has indicated that, whilst all isolates were neutralised by MAbs representing antigenic sites G1, G2 and G4, variations have occurred in some epitopes within the major conformational site G3, allowing the identification of four antigenic sub-types , 20. IsoBubalus bubalis). Although cattle are considered to be more susceptible to disease [Bos grunniens) in China and India [Syncerus caffer), waterbuck (Kobus ellipsiprymnus) wildebeest , hartebeest (Alcelaphus buselaphus), topi , tsessebe , blesbok , springbok , impala (Aepycerus melampus), sable antelope (Hippotragus niger), eland (Taurotargus oryx), kudu (Tragelaphus strepsiceros), bushbuck (Tragelaphus scriptus) and giraffe  [Kobus leche), elephant (Loxodonta africana), warthog (Phacochoerus aethiopicus), oryx (Oryx beisa), hippopotamus (Hippopotamus amphibius) and gazelle (Gazella granti) [Dama d. mesopotamica) and gazelle (Gazella g. gazella) in Israel [Sus scrofa) in Korea [Cervus elaphus), Rusa deer (C. timorensis) and Chital deer (Axis axis) in Australia [Camelus dromedaries) in Egypt and Somalia [Clinical BEF has been reported only in cattle and water buffalo  –42. In o granti) . In some granti) , 42. BEFn Israel , pigs  collected in the field were fed blood mixed with Australian and African strains of BEFV. Although BEFV was detected in 18.9% of the midges assayed immediately after feeding, none of the >4000 midges surviving at 10 days post-feeding were found to be infected [Attempts to demonstrate vector competence for BEFV in mosquitoes and biting midges have met with limited success. Artificial membrane-feeding experiments showed no evidence of BEFV replication in  feeding . In othe mixture . It has at 26 °C . In continfected .C. brevitarsis) but is similar to the distribution of Cx. annulirostris mosquitoes [Various other factors also appear to implicate mosquitoes as the principal vectors of BEFV. The observation that direct intravenous injection is required to initiate experimental BEFV infection in cattle suggests that capillary feeders (mosquitoes) rather than pool feeders (midges) would be required for efficient transmission . This issquitoes . The episquitoes . Howeversquitoes . FurtherBovine ephemeral fever has been described in many tropical and sub-tropical regions around the world Figure . It is eThe disease is enzootic in Australia. It was first reported in 1936 when an outbreak occurred 450 km south of Darwin in the Northern Territory . The rem−3 nucleotide substitutions/site/year [Molecular epidemiological studies using G gene ectodomain sequences have shown that all BEFV strains isolated in Australia since 1956 have a single common ancestor indicating that, unless earlier lineages have become extinct, the virus has entered Australia on only a single occasion . The phyite/year . This suite/year  which haBos taurus/Bos indicus cross-bred animals than in local breeds and the highest incidence occurred in older cattle [Although the epidemiology is poorly defined, there is evidence of BEFV infection in an arc of countries in South and South-East Asia extending from the Indian sub-continent and Sri Lanka, eastwards to the Indonesian Archipelago and northwards to the Philippines. An ephemeral fever resembling BEF was first reported in Pakistan (then Punjab) in 1919 as a disease of cattle known locally as “Vil” . It was r cattle . In Nepar cattle . In Sri r cattle , 79.BEF was first reported in Indonesia in 1919 when an outbreak occurred in dairy cattle in West Java. The next reported epizootic occurred from 1928 to 1931 on the east coast of Sumatra . SubsequLess information is available from other countries in the region. A BEF-like illness was reported in water buffalo in the Philippines in 1936 and passed experimentally in water buffalo and cattle, resulting in a brief, non-fatal febrile illness . BEF wasThere have been no confirmed isolations of BEFV from South or South-East Asia and no molecular epidemiological data are available at this time.BEF is enzootic in mainland China . It was Epizootics also occur periodically in Taiwan, Japan and the Korean Peninsula. In Japan, the disease has been known since large epizootics, then called bovine influenza, were recorded in 1889 and 1893 . There wReoviridae, genus Orbivirus) may have occurred in some cattle, contributing to the prolonged nature of the disease and poor prognosis [Although the disease called bovine influenza had long been known to occur in Taiwan, the first confirmed BEF epizootic was in 1967 when an outbreak occurred in the Kaoshiung District in the south of the island , 97, 98.Phylogenetic analysis using G gene ectodomain sequences has shown that isolates from China and East Asia collected during the period from 1966 to 2012 form a single clade that is distinct from the Australia and Middle East clades , 31, 98 Culicoides puncticollis) collected in 1980 from camels in Turkmenistan [There is little published information readily available on the occurrence of BEF in Central Asia. Chunikhin and Alekseev  referredmenistan . There imenistan . A BEF omenistan . EpidemiThere are detailed data on the occurrence of BEF in Egypt, Israel, Turkey and Saudi Arabia. Serological data collected in 2011 has also revealed exposure to BEFV in Jordan, primarily in the Jordan Valley . There are also reports of BEF in Iran , 103 andIn Egypt, BEF was known as “dengue fever” of cattle in the late 19th century but the first detailed report was of an epizootic in 1909 that commenced at Aswan, travelled down the Nile Valley to Cairo and spread across the Delta to the coast . SubsequBEF was first reported in Israel in 1931 . Until 1The first unconfirmed outbreaks of BEF in Saudi Arabia occurred in 1980 . SubsequBEF outbreaks in the Middle East usually commence during the spring (May) or autumn (September) when the rains are ceasing and ambient temperatures rise. In Israel, the most severe outbreaks usually occur in the Jordan Valley were the climate is either Mediterranean or semi-arid while in Saudi Arabia, which is mostly arid, the outbreaks usually occur at oases in which large populations of vectors can emerge . There hBEF is enzootic and seasonally epizootic in Africa. The first recorded epizootic commenced in north-western Zimbabwe (then Rhodesia) in November 1906. From central Zimbabwe, the disease advanced southwards to reach Natal and Transvaal by March 1907 and, by the end of 1907, it was reported near Port Elizabeth in Eastern Cape Province where it remained with intervals of quiescence . IndigenThe disease has also been reported to occur in Sudan, Kenya, Uganda and Tanzania , 119. BEBEF has also been reported historically to occur sporadically or seasonally in many other African countries including Chad, Mauritania, Guinea, Central African Republic, Ethiopia, Rwanda, Burundi, Democratic Republic of Congo, Zambia, Malawi, Angola, Botswana, Lesotho, Swaziland and Madagascar .A significant body of evidence suggests that BEFV dispersal within geographic regions occurs by wind-borne displacement of vectors. Spread of BEF in Australia during the 1968–1969 epizootic was in accordance with a combination of monsoonal influence and an intense low pressure system that developed in inland Queensland . In 2008Wind-borne dispersal of vectors has also been implicated in BEF epizootics in East Asia and the Middle East. BEFV incursion into Japan and Korea has been associated with the wind direction of a low-level jet stream from China , 94 and The phylogenetic clustering of BEFV isolates according to geographic regions suggests that long distance  dispersal of BEFV by animal transport has been rare historically. However, several studies have shown that recent BEFV isolates from Egypt (2005) and Turkey (2012) cluster phylogenetically with isolates from the East Asian clade rather than the Middle East clade , 18, 32.Ephemerovirus. From a clinical perspective, the most significant of these is kotonkan virus (KOTV) which was isolated from biting midges (Culicoides spp.) in Nigeria in 1967 [Kotonkan virus) in the genus Ephemerovirus [Berrimah virus (BRMV), Adelaide River virus (ARV) and Obodhiang virus (OBOV). BRMV was isolated in 1981 from a healthy sentinel steer in the Northern Territory of Australia [Mansonia uniformis) in Sudan with which it cross-reacts weakly in virus-neutralisation tests [Several other viruses that have been isolated from cattle or biting insects are antigenically related to BEFV, some of which have been classified as members of the genus  in 1967 . Serocon in 1967  and mild in 1967 . Based p in 1967 , 122, KOerovirus . Other eustralia . Antigenustralia , 125. Alustralia  and in custralia . ARV wasustralia . It is mon tests . Althougon tests .Ephemerovirus based on serological and phylogenetic relationships, and similarities in genome organisations and host/vector associations. KIMV was first isolated from mosquitoes (Culex annulirostris) collected in Western Australia in 1973 [Culicoides brevitarsis) and healthy sentinel cattle in the Northern Territory and Queensland [Mansonia uniformis) in Sudan in 1963, and these are now considered to be variants of the same virus species [Mansonia uniformis) collected in the Central African Republic. Recent studies have established that YATV clusters phylogenetically with the ephemeroviruses and shares a similar genome organization [Mansonia uniformis mosquitoes and was subsequently shown to cross-react in complement-fixation tests with MALV (strain SudAr-1169) and in indirect immunofluorescence tests with several other ephemeroviruses [Kimberley virus (KIMV), Malakal virus (MALV), Koolpinyah virus (KOOLV), Yata virus (YATV) and Puchong virus (PUCV) have not yet been classified formally but are likely to be assigned to the genus  in 1973  and theneensland , 57, 128 species . KIMV an species  and in c species . KOOLV w species . YATV wanization . PUCV waoviruses . Recent Natural BEFV infection has been reported to result in durable immunity . There hThere is also evidence that innate immunity is involved in both the immune response to infection and the pathology of disease. In an elegant experiment, Young and Spradbrow  challengFour types of BEF vaccine have been developed to date: (1) live-attenuated vaccines; (2) inactivated vaccines; (3) sub-unit G protein-based vaccines; and (4) recombinant vaccines. Live-attenuated, inactivated and subunit vaccines are being used in the field. Vaccination has been adopted to varying extents in Australia, South Africa, Namibia, Japan, South Korea, Taiwan, mainland China, the Philippines, Turkey, Israel, Kuwait, Oman, Bahrain, Saudi Arabia and Egypt. The vaccines differ in the seed virus from which they are prepared, the method of attenuation or inactivation, and the adjuvant formulation.Live-attenuated vaccines have been prepared by serial passage of BEFV in suckling mice and/or in cell cultures, including baby hamster kidney (BHK-21), hamster lung (HmLu-1) or African green monkey kidney (Vero) cells , 142. MaInactivated BEF vaccines have been prepared by treatment with formalin , β-proprInaba et al.  found thIn mainland China, effective vaccination has been achieved by using the BEFV G protein split from a semi-purified virus preparation by using non-ionic detergent . The vacVaccination experiments have also been conducted using the BEFV G protein delivered in recombinant virus vectors. Vaccination with four doses of the Neethling strain of lumpy skin disease virus expressing the BEFV G protein at 0, 3, 6 and 12 weeks induced a specific neutralising antibody and cell-mediated immune responses but failed to protect cattle challenged with BEFV 10 weeks after the last dose . In the In summary, although experimental and commercial BEF vaccines have been developed in various formulations, there are few reports of the evaluation of their efficacy in the field. Protective immunity for most of the vaccines appears to be of limited duration and so their efficacy may be poor unless additional booster doses are administered at intervals of 6 months to 1 year. There remains a need for further research to provide a more informed evaluation of performance in the field and to evaluate slow-release and other advanced technologies that may reduce the required number of doses and extend the duration of protection.As viraemia is brief (3–5 days) and occurs soon after infection, the risks associated with the movement of infected cattle pertains mostly to rapid transport across relatively short distances and a brief quarantine period in a vector-free area should be sufficient to eliminate the risk of introduction of BEFV with imported cattle . HoweverBEF is rare amongst viral diseases in that rationally based treatment is possible . Rest, pBEF is a disease for which the economic and social impacts are not always obvious and are frequently underestimated. Epizootics are now occurring more frequently in some parts of the world, there are increasing reports of alarmingly high case-fatality rates, and there is potential, under the influence of climate change and through the livestock trade, for spread of the disease to regions that are presently free. Although the epizootiology has been studied extensively in some regions, little is known of the distribution, prevalence and impacts over vast areas of Africa and Asia, relatively few virus isolates have been recovered and sequenced, and the specific vectors are not clearly defined anywhere in the world. This severely limits our ability to assess the relative importance and risk of spread by wind-borne dispersal of vectors and translocation through movement of livestock, and to assess the potential for establishment as an enzootic disease in Europe or the Americas through transmission by local vectors. Although a multitude of experimental and commercial vaccines have been developed, usage rates are often poor due to the irregular nature of epizootics and the need for multiple doses, and there are few published reports of the evaluation of vaccines under conditions in the field. The role of related viruses in the epizootiology of BEF is also unclear. This knowledge deficit provides a fertile field for future research.


To the Editor: Zika virus is an arthropodborne member of the genus Flavivirus of the Spondweni serocomplex and is transmitted by Aedes mosquitoes (primarily Ae. aegypti in urban and periurban cycles). Zika virus emerged in Africa and has caused outbreaks of febrile disease that clinically resemble dengue fever and other arboviral diseases  and yellow fever virus. We report Zika virus infection in Colombia and a recent ongoing outbreak in Sincelejo, Colombia, with resulting illness characterized by maculopapular rash, fever, myalgia/arthralgia, and conjunctivitis.The future spread of Zika virus is unpredictable, but the history of the virus has been reminiscent of chikungunya virus (CHIKV), which reemerged in Africa and now circulates on all inhabited continents and is a major global health problem. Zika virus has been found in Colombia and is likely following the path of CHIKV, which reached the country in August 2014  to detect DENV, CHIKV, or Zika virus. Viral RNA was extracted from the serum samples by using the ZR Viral RNA Kit ; reverse transcription was performed by using the Protoscript First Strand cDNA Synthesis Kit . Amplicons were produced by using the OneTaq Quick-Load 2X Master Mix (New England Biolabs) with primers specific to DENV  patients were positive for Zika virus. Among those 9 patients, 7 (78%) were male; median age was 23; and none had a history of international travel. Zika virus was analyzed by sequencing the E gene and NS1 of 2 isolates. Phylogenetic analyses rooted with Spondweni virus showed that the Zika virus sequences (GenBank accession nos. KU646827 and KU646828) belonged to the Asian lineage  and wereAe. aegypti mosquitoes and co-circulation of DENV and CHIKV. These circumstances highlight the need for accurate laboratory diagnostics and suggest that monitoring whether the virus spreads into neighboring countries  is imperative.We report Zika virus infection in Colombia in association with an ongoing outbreak of acute maculoexantematic illness. Since detection of Zika virus in Sincelejo, a total of 13,500 cases have been identified in 28 of the country’s 32 territorial entities (


To the Editor: Zika virus, a member of the Flaviviridae family, is primarily transmitted through Aedes spp. mosquitoes, and evidence of vertical, sexual, and blood transmission of Zika virus has been reported , headache, and dizziness on January 28. He subsequently had rash, chills, retro-orbital pain, and mild diarrhea on February 2, the day on which he departed from Caracas and traveled to Jiangxi Province via Paris and Hong Kong, arriving in Shenzhen, China, on February 5. At the time of hospital admission, the patient had fever (36.7°C), headache, conjunctivae, rash on his back and face, retro-orbital pain, and mild diarrhea. General clinical examination was unremarkable. Results of a complete blood cell count and liver function tests were within reference ranges.4 copies/mL), and Zika virus RNA was detected from urine samples collected on days 10, 11, and 12 .Serum samples were collected at day 9 and day 10 after symptom onset, and urine samples were collected once a day from day 10 through day 14. In the 2 serum samples, no dengue virus (DENV) or chikungunya virus (CHIKV) IgM or IgG were detected by a Panbio IgM and IgG capture ELISA for DENV  or by an indirect immunofluorescence assay slide test kit for CHIKV . Serum and urine samples were negative for DENV nonstructural protein 1 (NS1) antigen on an NS1-ELISA test kit . To detect virus RNA in samples, in-house–designed probe and primers specific to DENV and CHIKV were used  on serum and urine samples. A 1,813-bp of partial genome sequences  was obtained from urine and was used for comparing with selected other strains from GenBank. Phylogenetic analysis showed that the virus was of Asian lineage . PairwisAedes mosquitos, the risk for onward transmission of travel-associated Zika virus should not be overlooked.The clinical findings for the patient were similar to those previously reported among Zika virus–infected patients (


PLOS Neglected Tropical Diseases [in vivo testing and evaluation of candidate vaccines and therapeutics. The authors show that type-1 interferon receptor deficient mice (A129), in contrast to the parental strain (129Sv/Ev), are susceptible to Zika infection.In the latest issue of Diseases , Dowall Diseases ,3, this Aedes (Stegomyia) africanus  mosquitoes from the same region in 1948 [Flavivirus and the family of Flaviviridae. The genus comprises of some 52 other viral species, including more pathogenic viruses such as dengue, yellow fever, St. Louis encephalitis, tick borne encephalitis, and West Nile Viruses. The virions are small, spherical in shape containing a positive single stranded non-segmented RNA of approximately 11kb. The genome contains 2 flanking non-coding regions  a single open reading frame that codes for capsid (C), precursor membrane (PrM), envelope (E) and seven non-structural proteins formed NS1, 2A, 2B, 3, 4A, 4B, and 5.Zika virus was first isolated from the serum of a sentinel rhesus monkey in 1947 in Zika forest, Uganda [ in 1948 . Zika viAedes aegypti mosquitoes, artificially fed with Zika virus, were able to transmit the virus to mice and monkeys [http://www.paho.org/hq/).The significance of mosquito-borne transmission of Zika virus has been experimentally demonstrated in a study in which  monkeys . While t monkeys . The pat monkeys . While Z monkeys  caused c monkeys . The sur6 plaque-forming units (Pfu) in the right and left hind legs, which is reminiscent of the viral dose level of 104–106 per mosquito bite shown for West Nile virus [7 in blood and liver to nearly 1011 in brain are within the range of viral RNA loads detected in human serum and breast milk (106), semen (107), and urine (108) [The manuscript by Dowall et al., utilizes female immuno-deficient mice lacking the IFNα/B receptor A129) and congenic control mice (129SV/EV) for ZIKV infection. The un-adapted viral inocula used was injected at 1029 and coWhile Dowall et al.  utilizedIn many respects, Zika infection may be reminiscent of another significant public health hazard, namely German measles . RubellaFinally, the animal model described in the Dowall and colleagues manuscript  can poteWhile the A129 model lacks a fully competent immune system, it will likely be suitable for testing therapeutics and some vaccine studies [


Distinguishing between hybridization and population structure in the ancestral species is a key challenge in our understanding of how permeable species boundaries are to gene flow. The doubly conditioned frequency spectrum (dcfs) has been argued to be a powerful metric to discriminate between these two explanations, and it was used to argue for hybridization between Neandertal and anatomically modern humans. The shape of the observed dcfs for these two species cannot be reproduced by a model that represents ancient population structure in Africa with two populations, while adding hybridization produces realistic shapes. In this letter, we show that this result is a consequence of the spatial coarseness of the demographic model and that a spatially structured stepping stone model can generate realistic dcfs without hybridization. This result highlights how inferences on hybridization between recently diverged species can be strongly affected by the choice of how population structure is represented in the underlying demographic model. We also conclude that the dcfs has limited power in distinguishing between the signals left by hybridization and ancient structure. Hybridization between different species can play a major role in evolution, both by bringing novel adaptations into species as well as by acting as a barrier to their divergence . HoweverD statistics . Such loci should, in principle, be enriched for mutations that occurred in the Neandertal line and subsequently entered the human line through hybridization, and their relative frequency  should be an informative measure of the strength of hybridization. fig. 2a and b for a graphical representation of this model) predicts a deficit of rare doubly conditioned alleles  compared to the frequencies estimated from real data. Adding hybridization to such a model, however, restored the appropriate shape of the doubly conditioned allele frequency spectrum. Thus, the dcfs seems to be an informative metric to distinguish between hybridization and ancient population structure, and this result has been taken as a confirmation of hybridization between Neandertal and AMHs . Realistic demographic parameters were obtained by fitting the stepping stone to match worldwide patterns of spatial differentiation among modern populations and were further subsetted to focus on parameter combinations that predicted D between Africans and Europeans to be within 0.0020 U of the observed value 0.0457. This simple spatial model, which does not include any hybridization, predicts frequency spectra of doubly conditioned alleles (the dcfs) that are in line with observed values (gray lines and shaded ranges in fig. 3a), matching closely the empirical proportion of rare alleles (giving R2 = 99.2% for the best fit). Some demographic parameter combinations give rise to a slight excess of very common alleles, but there are a large number of combinations that fit the observed dcfs almost perfectly . This spatially explicit model (which has eight free parameters) provides a fit that is comparable (R2 = 99.2 vs. R2 = 99.7%) to the admixture model in fig. 3a). It is also considerably better than the best model fit for ancient population structure presented in R2 = 93.7% (green line in fig. 3a). The large proportion of rare doubly conditioned alleles in our spatially structured model is a consequence of deep splits in gene genealogies, with old, relatively rare lineages being preserved by the fine-grained spatial structure in the model (fig. 3b). In other words, the presence of multiple  populations within Africa prevents lineages from coalescing too quickly, thereby allowing for a few European lineages to merge back with Neandertal before meeting any African lineage. In many cases, such lineages are only represented by one or two individuals, giving an excess of rare doubly conditioned loci.However, it remains to be determined whether the dcfs can distinguish between hybridization and ancient structure when a spatially structured model with multiple populations is used instead of Yang et al.’s representation of ancient structure in the whole Africa continent with only two populations. Such spatially structured models better capture the global genetic clines in within-population genetic diversity observed in AMHs . Here wefig. 2It is beyond the scope of this short letter to provide a formal test for alternative hybridization scenarios with Neandertal. Population structure affects a number of aspects of the similarities between Eurasians and Neandertal. For example, the degree of matching between ancient and derived SNPs in candidate regions for hybridization (SOM 17 in In general, the very different results obtained by a model that represents genetic structure in Africa with two populations  versus o


Acanthamoeba polyphaga mimivirus (APMV) was first described and began to impact researchers around the world, due to its structural and genetic complexity. This virus founded the family Mimiviridae. In recent years, several new giant viruses have been isolated from different environments and specimens. Giant virus research is in its initial phase and information that may arise in the coming years may change current conceptions of life, diversity and evolution. Thus, this review aims to condense the studies conducted so far about the features and peculiarities of APMV, from its discovery to its clinical relevance.In 2003,  Viruses are remarkable organisms that have always attracted scientific interest. The study of unique viral features has been a wellspring of discovery that helped establish the foundations of molecular biology and led to in-depth evolutionary studies -3. In thMimiviridae group deserves special emphasis; they have been the subject of intense research in recent years, which has generated much relevant information [Acanthamoeba polyphaga mimivirus (APMV) was the first known mimivirus, isolated from an amoebal co-culture present in a water sample collected from a cooling tower of a hospital in England. Its characterization revealed surprising characteristics: it was a DNA virus with a diameter of approximately 700 nm and a genome of approximately 1,2 Mb, making it the largest known virus up to then [Mimivirus have challenged virologists and evolutionists alike. The potential existence of many other interesting and unusual unknown viruses in the biosphere makes us realize that the discovery and characterization of giant viruses is in its initial phase and that there is still much to be learned by studying these organisms. Recently, the discovery of Pandoravirus (1 μm in length and with a genome fo 2.8 Mb) and of Pithovirus sibericum  brought even more attention to the prospection and study of giant viruses [Although giant viruses offer deep ecological and clinical importance, the ormation -8,11-14. to then . Five ye to then -19. Data viruses .Acanthamoeba polyphaga cells were named Bradford coccus. Every attempt to isolate this microorganism and amplify its 16S rDNA failed. Moreover, treatment of amoeba cultures with antibiotics to inhibit growth of this microorganism was also unsuccessful, which led doubt whether it was indeed a bacteria [Rickettsia Unit at the School of Medicine  in the early 2000s. After a new series of unsuccessful characterization attempts, electron microscopy of Bradfordcoccus-infected Acanthamoeba polyphaga cells revealed icosahedral-like particles with an astonishing 750 nm diameter size [Acanthamoeba polyphaga mimivirus (APMV), due its ability to infect the free-living amoebae Acanthamoeba polyphaga sp. and mimic a microbe. APMV, also known as mimivirus, had the largest viral genome known up to then, reaching approximately 1.2 Mb. Its characteristics were so different from other viruses that it was not possible to include it into any known viral family, so the Mimiviridae family was created [Mimiviridae family, Mimivirus genus, and its prototype.In 1992, a pneumonia outbreak occurred in a Bradford hospital (England), and water samples from a cooling tower that contained free-living amoebae were investigated to determine the etiological agent of the pneumonia outbreak . At thatbacteria . After aPoxviridae, Asfarviridae, Phycodnaviridae, Ascoviridae and Iridoviridae families) was analyzed in depth. Several common, and supposedly essential, genes were identified, and it was suggested that the origin of these four viral families was monophyletic [Mimiviridae family also share these characteristics, they were added to the NCLDV group as well.Few years before the discovery of APMV, the genomic content of known giant viruses that replicate partly or entirely in the cytoplasm of eukaryotic cells : Mimivirus, with APMV as its only member, and Cafeteriavirus, with Cafeteria roenbergensis virus as its only member (http://www.ictvonline.org). With the increasing number of new giant virus isolates and hypothetical species, a new viral order has been proposed [Megavirales order contains the Mimiviridae family and other NCLDVs, including the newly proposed Marseilleviridae family [After APMV was described, interest in giant viruses grew, several other giant viruses were isolated, and the proposed . The pute family , whose fe family . Mimivire family . All thee family . Nucleo e family .Mimivirus genus contains the best known mimivirus isolates, such as the APMV species [Mimivirus lineage B is represented by Acanthamoeba polyphaga moumouvirus, which was isolated from a water sample in February, 2008; genetic analysis revealed differences that placed this virus into the B lineage [Megavirus chilensis, isolated from a water sample collected off the coast of Chile [The discovery of new isolates of mimiviruses of amoeba revealed the existence of three different lineages . Lineage A of the  species . Mimivir lineage . The firof Chile . Other pof Chile . Other vof Chile ,33, and Acanthamoeba belong to the Protist kingdom and can be part of the normal microbiota of some animals, including humans [As mentioned above, the first mimivirus isolate was discovered from studies of the pathogenic microorganisms associated with amoebae (MPAAs) that were linked to nosocomial pneumonia . Free-lig humans . Amoebasg humans -36. Seveg humans -37.Legionella, Parachlamydia and Mycobacterium genera are MPAAs considered to be causative agents of pneumonia, most of which is associated with many cases of nosocomial lung infection [Free-living amoebae can cause severe and chronic diseases by themselves, such as granulomatous amoebic encephalitis, cutaneous acanthamoebiasis, amoebic keratitis and primary amoebic meningoencephalitis . They canfection ,42,43. Enfection . Studiesnfection -43. The Acanthamoeba genus are the only confirmed APMV hosts; the virus was originally isolated from A. polyphaga, but now is also being cultivated in the laboratory in A. castelannii, A. griffin and A. lenticulata[Up to now, amoebae of the nticulata,44. Howenticulata. The abinticulata. These rnticulata. The mimnticulata,48. Recenticulata. It appenticulata,49. Lastnticulata-57.The remarkable size of mimiviruses and their peculiar features make them unique among viruses. First of all, their enormous size of approximately 700 nm lets them be retained on 0,2 μm filters  Figure -A to D. The particle also contains internal membranes surrounding the genome, likely acquired from endoplasmic reticulum  Figure -A and B.This large and complex particle structure seems to be vital for viral stability and genome integrity under adverse environmental conditions -67. The No previously described viral genomes have shown a genetic arsenal that is able to encode elongation factors, such as tRNA and aaRS; those viruses instead must directly rely on host elongation factors. A substantial proportion of the Mimivirus, and Marseillevirus, ORFs have homologs in bacteria, archaea, eukaryotes or viruses. The large amount of chimeric genes in these viral genomes may have resulted from acquisitions by lateral gene transfer, implying sympatric bacteria and viruses with an intra-amoebal lifestyle .Acanthamoeba castellanii, and instead are correlated with the high adenine and thymine (AT) content of the mimivirus genomes [A. castellanii. Recently, an interesting study evaluated genomic alterations in APMV maintained for 150 successive passages in an axenic amoebal culture in an allopatric system. It was shown that the genome was reduced in size and morphological changes occurred in the viral particle [The genomes of APMV and other mimiviruses have a high adenine-thymine (AT) content, with their most frequent codons being AAA (lysine) and AAT (asparagine). Both the codon and amino acid usages of mimiviruses are highly dissimilar to those of their amoebal host,  genomes ,75. Addiparticle . In the particle . Excludiparticle ,77. In cparticle ,77.Acanthamoeba genus through phagocytosis  led to the discovery of one of the most intriguing and differentiating features of the Mimiviridae family: its close association with other small viruses called the Sputnik virus. Sputnik virus was first identified as a satellite virus [Mimivirus factories inside amoebas, resembled satellite viruses that affect animals and plants. However, Sputnik virus replication appears to impair the normal morphogenesis and production of Mimivirus, a process closer to true parasitism than to the previously known satellite viruses. From a biological view, the infection with Sputnik virus results in ~ 70% reduction of the cytopathic effect of the giant viruses in amoeba and leads to formation of some atypical viral forms, in a way never described for traditional satellite viruses [Sputnik virus is an icosahedral virus 50 nm in diameter and a genome of 18 kb, which contains a mosaic of genes related to bacteriophages, other viruses and amoebae. Its genome is circular double-stranded DNA that is hypothetically able to encode 21 proteins, some of which have no detectable homologues in current databases of nucleotide sequences [The isolation of te virus . Its rep viruses ,12. This viruses ,12. Sputequences ,12.Cafeteria roenbergensis virus[Lentille virus revealed that a virophage, Sputnik 2, is part of this genetic element [Sputnik 2 was integrated into the Lentille virus genome [Lentille virus was sequenced on different platforms, and a sequence related to Sputnik2 was integrated in its genome, thus suggesting that Sputnik2 is a provirophage. These analyses also revealed that Lentille virus  contained an extrachromosomal DNA rich in CG that was called a transpoviron , which can undergo recombination with Sputnik2 and many other organisms [Recently, we have isolated Rio Negro virophage associated with Samba virus, an APMV strain from the Brazilian Amazon . Other dsis virus. On the sis virus. It has  element . It was s genome . This warganisms . Phylogerganisms .Mimiviridae members needs to be considered when interpreting negative PCR tests in several studies because the diversity may have impaired detection of APMV-related DNA [Hospitalized patients are a risk group for nosocomial infections, including those caused by amoeba-associated pneumonia agents ,93. APMVated DNA . An animated DNA . Althougated DNA .. Its characteristics linked it to the Marseilleviridae family, representing the first detection of a marseillevirus from a human sample [The first studies in which a giant virus was successfully isolated from a human specimen were published in 2013 ,52. In an sample . Addition sample .The potential clinical relevance of mimivirus can also be analyzed by studies involving the host-virus relationship. In 2008, it was observed that APMV is internalized by professional phagocytic cells such as macrophages, but not by non-phagocytic cells like fibroblasts, epithelial or neuronal cells . This suLegionella, Parachlamydia, Mycobacterium and also APMV, representing a public health concern. In 2013 we investigated the APMV survival in certain adverse conditions when present in the intracellular environment of A. castellanii[Acanthamoeba spp. may represent a training field for human pathogens, as several micro-organisms resisting these amoebae were concurrently found to resist human macrophages [The probable importance of APMV as a human pathogen in hospital environments has led to the need for investigating the virucidal activity of chemical biocides used for disinfection in hospitals. In a study performed in 2012, it was shown that APMV is especially resistant to alcohols but is sensitive to the action of active chlorine and glutaraldehyde, and it is able to remain stable on inanimate surfaces for 30 days, even in the absence of organic matter; this highlights the need for best strategies to control this putative pneumonia agent in hospital environments . Amoebaestellanii. It was stellanii. In addirophages .An indicator of the growth of related research fields concerning giant viruses is the recent increase in the number of publications about APMV and other giant viruses. In 2013, the number of papers related to these fields was 21-fold greater than to the number of papers published in 2003, the year of APMV discovery Figures . InteresAs described, APMV and other giant viruses have emerged as a fascinating line of research. Each discovery regarding mimiviruses has overwhelmed scientists from different areas of expertise, which may explain why so many outstanding publications are multidisciplinary. The future of mimivirus studies might go beyond the description of bigger and more complex viruses but also may contemplate deep structural, genetic and evolutionary studies. With all this knowledge, we expect it will be possible to understand the exact role of mimiviruses in environmental dynamics and their importance as etiological agents of pneumonia in humans and other animals.The authors declare that they have no competing interests.JSA, FPD, LCS, GMA, PVB, PC, BS and EGK wrote the manuscript. PVB performed analysis in Google Trends and Pubmed. FPD performed electron microscopy analysis. JSA designed the figures. All authors read and approved the final manuscript.


In this paper, we demonstrate the use of 2-pyridinemethanol (2P) aqueous solutions as a refractive index matching liquid. The high refractive index and low viscosity of 2P-water mixtures enables refractive index matching of beads that cannot be index matched with glycerol-water or sucrose-water solutions, such as silica beads that have the refractive index of bulk fused silica or of polymethylmethacrylate beads. Suspensions of beads in a nearly index-matching liquid are a useful tool to understand the response of particle counting instruments to particles of low optical contrast, such as aggregated protein particles. Data from flow imaging and light obscuration instruments are presented for bead diameters ranging from 6 µm to 69 µm, in a matrix liquid spanning the point of matched refractive index. Proper assessment of the concentration of particles in protein drugs within specified diameter limits ,2 requirn ≈ 0.07 as a maximum value; increased solute concentration or a high level of particle hydration or porosity would drive Δn to lower values. In contrast, PSL beads in a water matrix have Δn ≈ 0.25. Calculations of the response of light obscuration counters reveal that as Δn increases above ≈ 0.15, the scattering strength approaches a plateau and is insensitive to further increases in Δn [n << 0.15, the scattering intensity is proportional to Δn2. As a result, calibration of LO counters with PSL beads is primarily a problem for particles with Δn < 0.15, as is the case with protein particles. Consequently, particles with small Δn are reported as being undersized when measured in an instrument calibrated with PSL beads.The two optical particle-counting methods most commonly used to measure particle concentrations in the size range from 2 µm to 200 µm are flow imaging and light obscuration. Flow imaging (FI) systems measure the projected area of particles passing through a flow cell. Light obscuration (LO) instruments measure the attenuation of a light beam caused by the combined light scattering and absorption of a particle. Both aggregated protein and the polystyrene latex (PSL) beads commonly used for instrument calibration are highly transparent, so the light attenuation is predominantly due to light scattering. Consequently, the LO instrument reports the diameter of a PSL bead that scatters the same amount of light as the actual particle. Theoretical calculations  and expees in Δn . For Δn n. Silica beads fabricated by the Stöber-Fink-Bohn (STB) process [ process  have typThe ideal index-matching liquid should have a refractive index up to 1.49, be water soluble to promote ready instrument cleaning, and have a sufficiently low viscosity to allow ready clearing of air bubbles on sample mixing. Aqueous mixtures of sucrose, ethylene glycol, or glycerol have all been used as refractive index matching liquids , but theet al. have used sucrose solutions successfully for refractive index matching of STB silica beads in a similar study [We examine in this paper the use of STB silica, fused silica, and PMMA beads of known size, suspended in a fluid of known refractive index, as a means of characterizing the response of a light obscuration instrument and a flow imaging instrument to particles of low optical contrast. Both 2-pyridinemethanol (2P) and glycerol were used as the high refractive-index diluent for silica beads (refractive index of ≈ 1.43 and ≈ 1.47 for different lots of beads used for this work) and PMMA (refractive index of 1.49). We demonstrate that 2P-water solutions work well at refractive indices up to 1.52 in these instrument counters. Zölls To prepare and use a bead standard of known diameter and concentration, beads of known size are first dispersed in water. The refractive index of water differs sufficiently from that of common bead material to enable accurate calibration of the count of the aqueous suspension against commercial PSL count standards. The bead suspension is then mixed with filtered, water-miscible solvents that increase the refractive index of the liquid and reduce the optical contrast between the beads and liquid in a controlled manner.et al. [Because of the variability of refractive index of silica beads, the refractive index must be measured for each lot of beads. A series of suspensions with fixed bead concentration and known liquid refractive index, spanning the expected refractive index of the bead, is prepared first. Then the relative optical contrast of the beads can be measured in one of two ways. Zölls et al.  measuredPMMA beads are available at larger sizes than silica beads, have good optical homogeneity, and have a predictable refractive index. The refractive index of PMMA is appreciably lower than that of PSL (1.49 versus 1.59) , enablinSilica beads of reported 6.1 µm diameter were obtained from Bangs Laboratories , and of reported 10.3 µm and 25.0 µm diameter from Corpuscular, Inc. .For the matrix fluids, ultrafiltered water  was combined with either 2P  or glycerol . Prior to use, the solutions were filtered through a 0.22 µm, PVDF membrane filter (Millipore GV syringe filter). Particle suspensions were stored in polyethylene sample bottles that had been precleaned by agitation in 0.1 % mass concentration sodium dodecyl sulfate solution, followed by two rinses with ultrafiltered water. Lack of appreciable particles in the water and other solutions was confirmed by measurement by light obscuration and flow imaging. The refractive index of prepared mixtures was measured at the sodium D line (589 nm wavelength), using an Abbe refractometer that had been calibrated using ultrafiltered water and NIST Standard Reference Material 1922, a mineral oil with known refractive index.The prepared samples were measured by light obscuration and by flow imaging. The light obscuration particle counter  had a size range from 1 µm to 200 µm diameter. To minimize the effects of particle settling during the span of each measurement, the measurement volume was only 0.4 mL and the sampling needle was positioned halfway down into the bead suspension.For the flow imaging results, a Micro-Flow Imaging DPA-4200  was used. Because the silica beads settle much more rapidly than PSL beads, the measurement protocol was modified. The instrument illumination was optimized with a particle-free sample of the same liquid used to suspend the particles. A particle-laden sample of 0.42 mL was then added to the instrument inlet, and the measurement commenced without further priming. As a result, the measured volume consisted of the particle-free liquid in the inlet tube between the counting chamber and the inlet, and the added particle-laden sample. The measurement volume was adjusted so that the measurement terminated with no more than 10 µL of liquid remaining above the counting chamber. The particle concentration results were multiplied by a constant factor to account for the volume of particle-free liquid included in the measurement. Although this method accounts for particle settling, there were still difficulties with beads adhering to the sides of the inlet tubing.For each bead type and matrix-fluid refractive index, the particle size distribution was inspected, and upper and lower diameter limits were adjusted to exclude any extraneous particles that were clearly not beads. This adjustment can be done in the data analysis step for flow imaging, but for light obscuration, the instrument settings were adjusted. The reported concentration is the number of particles per unit volume between these diameter limits.Both light obscuration and flow imaging counters were checked for correct operation by measuring nearly particle-free filtered water as a blank and by measuring commercial PSL bead count standards .n, 6.1 µm, 10.2 µm, and 19.9 µm diameter silica beads and 69 µm PMMA beads were measured by flow imaging and light obscuration. The beads were immersed in either glycerol-water or 2P-water mixtures of varying refractive index. Data were obtained for particle diameter and, where not prevented by fast sedimentation, bead concentration.To study how indicated diameter varied with the refractive index difference ΔBoth light obscuration and flow imaging instruments gave results for the particle diameter that agreed with manufacturer’s value for 6.1 µm, 10.2 µm, and 69 um beads, and with the brightfield microscopy value for 19.9 µm beads, when the particles were immersed in water (data not shown).n for silica and PMMA beads ranging in diameter from 6.1 µm to 69 µm.  The dashed vertical lines indicating Δn = 0 is only approximately determined for the silica beads; additional data on solutions with small Δn would be necessary to find the index-matching point accurately. As Δn approaches zero, the light-obscuration diameter of each bead drops. The number of particles, however, remains relatively constant. Once Δn becomes small enough that the particle size distribution of the beads is near the lower limit of the instrument, the particle count drops precipitously. Note that the reported diameter never drops below the lower threshold of the particle counter (1 µm for the counter used here): at very small Δn when no peak is observed in the particle size distribution, the particle counter only reports particles having larger than average scattering.Figures 1n ≈ 0) varied substantially among the measured silica bead lots. For the smaller 6.1 µm beads, the minimum diameter occurs at a fluid refractive index of ≈ 1.43, consistent with the refractive index expected for amorphous silica beads fabricated by the Stöber-Fink-Bohn process [The refractive index of the fluid where the beads have the smallest indicated diameter  were not significant problems in the present measurements. The refractive index of the mixtures was measured with a refractometer rather than being inferred from literature values, so shifts in refractive index due to degradation did not affect the measurements. The optical extinction also did not impair the measurements because the solutions remained largely transparent over the small-path-lengths of the flow cells used for light obscuration and flow imaging.n values smaller than ≈ 0.02. We also evaluated the stability of PMMA beads in the same retained samples. The diameter of nominally 70 µm PMMA beads at the initial measurement and after 24 months was (68.89 ± 0.08) µm (mean and standard error of the mean) and (69.13 ± 0.12) µm, respectively. A t-test confirmed that there was no statistically significant change in the measured diameter. We conclude that PMMA is chemically compatible with 2P.The stability of 2P-water mixtures was evaluated by remeasuring the refractive index of four retained samples . After storage in the dark for 24 months, the refractive index of the mixtures shifted by +0.0064 ± 0.0022 (mean and standard deviation of four samples). The magnitude of this shift is small enough that 2P-water mixtures are sufficiently stable for studying instrument performance; the shift is large enough that year or longer shelf lives of 2P-water mixtures are not attainable for ΔThe use of 2P-water mixtures enables the measurement of refractive-index matched beads over a much broader refractive index range than other aqueous matrix liquids, such as glycerol-water or sucrose-water solutions. This broader range, in turn, allows use of silica beads made from bulk silica glass or PMMA beads, which are available in larger sizes than STB-process silica beads. The low viscosity of the 2P-water mixtures ensured that particles could be resuspended without significant air bubble entrainment.The data obtained for flow imaging and light obscuration instruments demonstrate the significant shifts in measured diameter that occur when the refractive index of the particle is close to that of the matrix liquid. A challenge for future work is to understand how the response of these instruments for a homogeneous bead sample can be related to the response for inhomogeneous particles, such as aggregated protein particles.


Cryptococcus albidus keratitis is a rare and difficult diagnosed disease. Here we report a case of C albidus keratitis early diagnosed by dot hybridization assay and successfully treated with intrastromal injection of Amphotericin B (AB).C albidus keratitis was diagnosed. Despite the identification of C albidus, the clinical condition still worsened due to deep corneal infiltration. After performing intrastromal injection of AB, the corneal infiltration gradually improved.A 45-year-old man presented with left red eye for 2 days. The slit lamp examination exhibited deep corneal infiltrations. Smears and cultures were performed but revealed negative findings. Molecular detection of pathogens was performed by dot hybridization assay, and C albidus is a rare cause of diseases in humans and should be considered as a potential pathogen of corneal ulcer. The prognosis of C albidus keratitis will improve if the condition is recognized early and treated properly. Cryptococcus spp is a kind of saprophytic yeast usually found in soil, dust, manure, and litter made from bark or leaves from various trees.1Cryptococcus spp had been reported to be responsible for infection in several animal species and caused a wide range of diseases such as meningoencephalitis, pneumonia, osteomyelitis, abscesses in various internal organs, and ocular disorders.3 In human beings, Cryptococcus most commonly presented with meningitis, and the incidence ranges from 2% to 9%.4 In recent decades, the incidence was rising due to the increasing numbers of patients with acquired immunodeficiency syndrome.4 Ophthalmic infections can occur by direct extension along the subarachnoid space of the optic nerve or by hematogenous dissemination.5 The ocular presentations can manifest as papilledema, optic atrophy, extraocular muscle paresis, chorioretinitis, and endophthalmitis;5 however, the corneal involvement was rare. Here we report an unusual case of Cryptococcus albidus keratitis that was early diagnosed by dot hybridization assay and successfully treated by intrastromal injection of Amphotericin B (AB).A 45-year-old man without any ophthalmic or systemic diseases came to our clinic due to pain and redness (OS) for 2 days. He was a farmer and was hit by a plant accidentally while working. The initial best-corrected visual acuity (BCVA) of left eye was 20/400, and the intraocular pressure (OS) was 16 mm Hg. Slit lamp examination revealed corneal infiltrations with satellite lesions . Under aseptic conditions, AB was injected into the corneal stroma using a 1-mL syringe with a 30-gauge needle according to the method described before.7 In brief, the needle was bended, inserted obliquely, and beveled down from the uninvolved, clear area to reach the lesion site at the midstromal level. The procedure was performed under microscope without entering the anterior chamber, and multiple injections were administered in order to hydrate the corneal stroma and form barriers around the infiltrations  for intravenous administration was initially diluted in sterile distilled water to obtain a 0.5% concentration. The preparation was then mixed with balanced salt solution  under sterile conditions and was diluted to a concentration of 100 μg/0.1 mL. Local anesthesia was established with proparacaine hydrochloride 0.5% .6 In addition, a probe designed from the 5.8S rRNA gene was used as a positive control and a digoxigenin-labeled universal bacterial primer 6R was used as a position marker.6 The dot hybridization method was proved to have high sensitivity and specificity for laboratory diagnosis of FK11 and can be completed in 16 to 24 hours, regardless of the infecting species.6 No additional time was required for mixed cultures. Conversely, identification for yeast species by conventional methods requires 1 week and takes 2 weeks or longer for molds.6 Therefore, the dot hybridization assay provided timely and accurate diagnosis in this case.Microbial keratitis is an important cause of monocular blindness worldwide.Cryptococcus spp received therapeutic penetrating keratoplasty or even enucleation due to diffuse corneal involvement.8 Since intrastromal injection of antifungal agents was effective in deep recalcitrant FK,17 we used instratromal injection of AB to treat our patient (Figure C albidus corneal ulcer is an effective treatment.Despite the early diagnosis by dot hybridization assay, the treatment for deep corneal infection was very difficult. In previous reports, patients infected by t Figure A, and tht Figure B. It shot Figure B, we sugCryptococcus keratitis early diagnosed by dot hybridization assay and successfully treated by intrastromal injection of AB. We suggest that in recalcitrant corneal infections, using diagnostic tool such as dot hybridization assay is a quick, sensitive, and specific method to identify the pathogen.11 Additionally, intrastromal injections of therapeutic agents may offer a less invasive, in-office alternative to penetrating keratoplasty.In conclusion, to our knowledge, this is the first report of


Silicon has persevered as the primary substrate of microelectronics during last decades. During last years, it has been gradually embracing the integration of ferroelectricity and ferromagnetism. The successful incorporation of these two functionalities to silicon has delivered the desired non-volatility via charge-effects and giant magneto-resistance. On the other hand, there has been a numerous demonstrations of the so-called magnetoelectric effect (coupling between ferroelectric and ferromagnetic order) using nearly-perfect heterostructures. However, the scrutiny of the ingredients that lead to magnetoelectric coupling, namely magnetic moment and a conducting channel, does not necessarily require structural perfection. In this work, we circumvent the stringent requirements for epilayers while preserving the magnetoelectric functionality in a silicon-integrated device. Additionally, we have identified an in-plane tunnelling mechanism which responds to a vertical electric field. This genuine electroresistance effect is distinct from known resistive-switching or tunnel electro resistance. Electric modulation of the magnetic properties of a nanoscale ferromagnetic semiconductor (FM-SC) at room temperature, in a system integrated with Si is a challenge for the scientific community4569101−xSrxMnO3 as a channel material, which showed the largest modulation on the magnetic properties induced by ferroelectric polarization reversal19c) for the ferroelectrically-gated SCM is commonly much larger than the one measured for a FM-SC channel  bilayer directly grown on as-received Si substrate as sketched in c, as well as a change of absolute resistance and magnetoresistance, despite the polycrystalline nature of the structure. Our experimental data allow us to infer a possible distinct mechanism  that accounts for the observed transport modulation. The results presented here show the advantages of the use of a SCM as a channel, with large effects and high order temperature, in a structure grown on Si.Here we report on a LaCross-sectional high-resolution Transmission Electron Microscopy (TEM) image is shown in r ≈ 25 μC/cm2). The Pr value is comparable to that obtained for good crystalline ferroelectric films on buffered SiAs shown in up-turn) and its origin is discussed in detail below. The distinctive peak at 216 K, at which LSMO undergoes an insulating to metallic transition  marks the onset of the ferromagnetic orderdown (with +9 V), all along the LSMO bar. Complementary PFM characterization to crosscheck the polarization switch can be seen in MI shifts from 216 (Pup) to 200 (Pdown). After switching to Pup (−9 V) the measured R(T) curve overlaps to the one obtained in the as-grown state. The latter finding is expected, since P in the as-grown state is mainly Pup , that hinders the possibility to obtain the fully polarized Pdown state. Note that the characterized bilayer grown on Si allows disregarding strain as the parameter that mediates the found magnetoelectric effect between PZT and LSMO because of the substrate clamping effect that forbids the LSMO lattice modulation.The influence of the polarization of the PZT layer on the electronic transport of the underneath LSMO layer is revealed by the dependence on temperature of the LSMO resistance acquired for three different polar states of PZT. In inly Pup . Upon suWe now focus on the transport and magnetotransport properties of LSMO at low temperatures, where LSMO behaves as semiconductor and it is ferromagnetic. The current versus voltage (I-V) characteristics recorded for the three polar states at 5 K are depicted in MI, and the change in the magnetoresistance amplitude upon ferroelectric switching univocally point to the fact that the magnetism is modulated by the field effect in the studied system. However, the peculiar crystalline character and the rather unexplored LSMO composition make the presented results not really comparable to those obtained in similar epitaxial structures. This raises the question whether the conduction mechanism at low temperature in this system is the same and how the different structural and morphological characteristics of our system can affect the obtained electric field effect as compared to the single-crystalline ones.The remarkable change in the metal-insulator transition temperature Tc can play an important role, explaining the high resistivity found at T < Tup-turn and the dR/dT change of the sign at Tup-turn−1/4 plot is shown. The data have been fitted to N(EF) for the density of states at the Fermi level, β is a constant, and KB is the Boltzmann’s constant. T0 from the linear fit is extracted and a non-significant difference between the Pdown (T0 = 13 K) and Pup (T0 = 16 K) states is obtained. Further analysis shows that these results are at odds with precedent works, where it was concluded that transport is modulated by a change in the number of carriers upon ferroelectric switchingdown sets the electronic state of depletion at LSMO (accumulation for Pup) with a subsequent decrease of charge carriers (transport in LSMO is governed by holes); therefore, T0 must be larger for Pdown than for Pup. In fact, in epitaxial bilayer films of the same LSMO as the one analysed here, the modulation takes place and it is remarkably large (see N(EF).It has been already proposed that a phase coexistence of metallic and insulating regions at T < Targe see . This poxA1−x)MnO3 family has been exhaustively studiedup-turn can be influenced by the tunnelling current between different grains; therefore grain boundaries act as an insulating layer30310 + G1 + G2V4/3 + G3V5/2 after Glazman and MatveevxA1−x)MnO3 films as demonstrated in detail in ref. down the contribution of the tunnelling current is smaller than for Pup. Interestingly, in the ohmic regime (low V), where G(V) ≈ G0 + G1, the tunnelling resistivity shows a large change from ≈3 (for Pup) to ≈27 (for Pdown) kΩ·cm (near 1000%).The failure to explain the obtained resistance modulation by VRH at low temperature suggests that another transport mechanism can play an important role. The particular influence of the grain boundaries on the conductivity in the  as already introduced in ref. Tunnelling is a temperature independent effect; therefore the found modulation must be temperature independent. This is confirmed by the dependence of the electro-resistance up  on commercial as-received Si(001) wafers in a two steps process, following the same procedure described elsewhereTEM investigation has been performed by using FEI TITAN 80–300 operated at 300 kV.2 Cu top electrodes ex-situ evaporated.Macroscopic ferroelectric characterization was performed by measuring the dynamic P-V hysteresis loops at 10 Hz using a TFAnalyzer 2000  using 60 × 60 μmTransport measurements have been performed by using an electrometer (6517B Keithley Co.). The resistance of LSMO is measured using a two points contact method at 50 V .


Arthropod borne viruses have developed a complex life cycle adapted to alternate between insect and vertebrate hosts. These arthropod-borne viruses belong mainly to the families Togaviridae, Flaviviridae, and Bunyaviridae. This group of viruses contains many pathogens that cause febrile, hemorrhagic, and encephalitic disease or arthritic symptoms which can be persistent. It has been appreciated for many years that these viruses were evolutionarily adapted to function in the highly divergent cellular environments of both insect and mammalian phyla. These viruses are hybrid in nature, containing viral-encoded RNA and proteins which are glycosylated by the host and encapsulate viral nucleocapsids in the context of a host-derived membrane. From a structural perspective, these virus particles are macromolecular machines adapted in design to assemble into a packaging and delivery system for the virus genome and, only when associated with the conditions appropriate for a productive infection, to disassemble and deliver the RNA cargo. It was initially assumed that the structures of the virus from both hosts were equivalent. New evidence that alphaviruses and flaviviruses can exist in more than one conformation postenvelopment will be discussed in this review. The data are limited but should refocus the field of structural biology on the metastable nature of these viruses. Evidence exists that arboviruses from the alphavirus lineage evolved from plant viruses , fl, flT = 4, , PE2, 6K, and E1  with the host cell. Thus, the infectious virion is a metastable intermediate that assumes sequentially different conformations depending on the pH, temperature, and host environments it encounters (discussed below). The plasticity of the virion structure underscores the flexibility of the viral proteins. This flexibility is most likely not due to global changes in the structural proteins but rather local changes in the metastable domains of the proteins . For strT = 4,9, 16,25, and 36 [P = 1 of icosadeltahedra. It has been suggested that the T = 4 structure of the nucleocapsid organizes the outer glycoprotein layer [The first example is that of the Sindbis virus capsid protein mutant Y180S/E183G. Sindbis virus is a macromolecular structure composed of RNA, protein, and lipid. The inner protein shell, the nucleocapsid, is bound to the outer protein shell via interactions to the E2 endodomain. Genetic and structural evidence suggest that the nucleocapsid interacts with the E2 endodomain by aromatic amino acid interactions between Y420 in the E2 protein and Y180 and W 247 in the capsid protein , 50. Mut, and 36 –53. All in layer . These oin layer . While tA second example of a Sindbis virus structural protein mutant able to organize into nonnative structures is that of the furin mutants in the glycoproteins E1 and E2 shown in These mutant virus structures help us to understand the metastable structure of alphaviruses by illustrating that assembly of the virion proceeds through structural intermediates. In the case of the C mutant, this protein can fold into native or the mutant conformations and assemble multiple forms. For this change to occur, the mutant protein must be able to assume an energetically stable intermediate. This is thought to happen during the folding of the protein and assembly, in this case, of the nucleocapsid. The E1-392/E2-341 mutant demonstrates that the metastability of the virus extends to both shells and can trap the glycoproteins into a conformation that only allows the formation of the hexagonal lattice.http://www.ncbi.nlm.nih.gov/). The metastable state of the virion, however, suggests caution in interpreting structures of the isolated structural proteins. Virus particles are macromolecular machines poised to deliver their genomic cargo once the appropriate stimuli are encountered. These particles must also shield the genome from the adverse effects of the surrounding environment. These requirements are accomplished by the virion assembling into intermediates which refold into a highly energy rich infectious conformation, which is a sufficiently stable structure [T-number virus without distorting the X-ray structure in order to make the fit. For Sindbis virus, a single alphavirus E1 structure is commonly used in virion structural reconstruction analysis, at least four other conformations of the E1 protein are known to exist as the protein is folding and unfolding [Protein conformations have been reported for many of these virus structural proteins as single proteins and in protein aggregates  of the microscope so that images do not require CTF correction. The phase plate technology is still under development because those that work well contaminate quickly when used –73. Addi in vitro or outside the context of the virus. Soluble E protein X-ray structures have been solved for many of the flaviviruses [As seen in iviruses –91. The iviruses . Domain iviruses . This 3Diviruses . Unlike iviruses . E proteiviruses . It is wiviruses , 97–99, T = 4 icosahedron surrounding a T = 3 core [T = 4 [As electron optics and electron cryomicroscopy in general have improved, it has become possible to take images of frozen hydrated viruses from electron microscopes and use them to reconstruct the three-dimensional structures of infectious virus to ever higher resolutions by cryoEM (reviewed in ). Electr= 3 core ; howevere [T = 4 . The fire [T = 4 . This ree [T = 4 –107. Thee [T = 4 , 108. See [T = 4 . The alpe [T = 4 . The pose [T = 4 . The gene [T = 4 .In a 1990 study by Flynn et al., conformational changes in Sindbis virus E1 and E2 were observed as virus engaged the plasma membrane at neutral pH in cells that were not acidified . These r de novo modeling. The final VEEV model was compared to a pseudomodel in which CHIKV E1 and E2 X-ray structures were fit separately into the VEEV model. The results indicate that E2 (residues 1–341) had a higher RMSD value (4.2) than E1 (residues 1–391) with an RMSD of 1.8 Å, representing the difference from identical molecules with an RMSD of 0.0 [ de novo. In addition, the structure of the capsid protein as determined by cryoEM reveals a predicted α helix at residues 115–124 that is missing in the capsid structure as revealed by X-ray crystallography. Thus, this cryoEM reconstruction has served to refine the structures of viral components by independently using improved methods to define the protein structures. Advances in single-particle cryo-EM have pushed the limit to near atomic resolution of ~3.3 Å [ in situ cryo-EM has the potential of looking at virus proteins without the need for crystallization. Unlike X-ray crystallography, this method ensures that the viral proteins imaged in this manner are in their native conformations. While this method yields lower resolution images than single particle reconstructions, advancements in this technology will undoubtedly improve the resolution.A high resolution alphavirus structure was recently reported by Zhang et al. and is shown in D of 0.0 . The lowf ~3.3 Å –119. MetA low pH study of Sindbis virus was undertaken by Paredes et al. in 2004 which hypothesized that low pH triggers the same or similar conformational rearrangements as does contact of the virus with the cell receptor. Sindbis virus treated at low pH was investigated by cryoEM. SVHR is a laboratory strain selected for heat resistance (Sindbis virus heat resistant)  which alThat low pH inactivates Sindbis virus in solution is consistent with the observation that treated virus does not return to its infectious conformation –124. ForT = 4 structure. This is surprising since this pH should have induced a global conformational change in both E1 and E2 as was shown previously with SVHR displaying ~100% infectivity [A recent study by Cao and Zhang reported an early-stage fusion intermediate of Sindbis virus using cryoEM to reconstruct the low pH intermediate . The strectivity . This laectivity . When thectivity . Fusogenectivity , 128–130ectivity , 131, thectivity  rather tectivity .Cell-mediated endosomal uptake of alpha and flaviviruses followed by acidification and membrane fusion with the virus membrane is currently the favored model of alphavirus penetration and entry –136. Thi a priori belief that enveloped virus structures must encode a fusion loop and penetrate cells by fusion, now dominates the field to the exclusion of alternate modes of virus entry. E1 of the Alphaviruses and E of the flaviviruses are referred to as group II fusion proteins [ Flaviviridae, do not encode a fusion loop [As early as 1978, Fan and Sefton proposed that virus entry for Sindbis and VSV involved a mechanism which did not require fusion . This evproteins , 155. Inion loop , 157 andion loop . The Bunion loop . The ability of alpha and flaviviruses to fuse membranes is not disputed; however, this may not result in virus penetration and infection.A large volume of work has focused on the ability of alpha and flaviviruses to fuse artificial membranes and to elucidate the mechanism of low pH-mediated fusion of virus. The model of infection for these two families posits that membrane-containing viruses infect cells via low pH-mediated fusion within cell endosomes. The membrane-fusion mechanism of virus infection has been studied extensively for the influenza (flu) hemagglutinin and has been shown by direct evidence to form structural intermediates involved in virus penetration , 160–162Our work on virus penetration has shown that Sindbis, West Nile virus (WNV), and dengue virus can penetrate cells at temperatures that do not allow membrane fusion , 164. UsRecent data show that Sindbis virus can penetrate mosquito C7-10 cells even more quickly than what has been seen in BHK cells. By 60 min. postinfection at 4°C, 90% of the virus was empty as compared to the 75% seen in BHK cells see . Cultureα-sarcin (17 kDa) [Strong biochemical support for the model of direct penetration at the cell surface comes from studies showing that cells become permeable to ions and small molecules as they are infected with alphaviruses , 151. Vi(17 kDa) . These r(17 kDa) , 169. This point is crucial for the development of prophylactics for pathogenic arboviruses.Evidence of direct penetration has been presented for both mammalian and insect cells , 166. ThRg (radius of gyration) indicated that the BHK-grown virus is less compact than that grown from mosquito cells. The diameter of the BHK grown virus was found to be 689 Å, compared to that of the insect grown virus which was 670 Å. The mass at the center of the BHK particles was less centrally distributed than that seen in the C7-10 virus. It was also determined that while the radial position of the lipid bilayer did not change significantly, the membrane had significantly more cholesterol when the virus was grown in mammalian cells than in insect cells. This property has been shown to affect the virus stability and, with it, infectivity [In the paper by He et al. , we usedectivity . Distribectivity  reportedDengue virus particles are structurally classified as (1) mature, (2) partially mature, or (3) immature, depending on the surface morphology seen in cryoEM preparations prior to averaging of the particles . The claperature . In eachperature , 180. Coperature .The structure by Zhang et al. is similar to the one observed by Fibriansah et al, in which E and M density is seen to move away from the core of the virus after heating insect grown virus to 37°C. The former structure was solved to 35 Å, while the latter had a resolution of 14 Å. In the work by Zhang et al. the same experiment is performed; insect grown virus is heated to 37°C. Both virus structures were found to be equally infectious. These authors reconstructed the heated form to a fit with E protein into trimers, however, unlike Fibriansah who fit E into the traditional herringbone pattern. In the Zhang study, the authors propose that this larger 37°C structure represents the prefusogenic form of the virus that was previously just a predicted structure . In Zhan in vitro, and it was previously not understood how known neutralizing Ab (NAb) anti-DV2 epitopes on the virus bound Ab to sequences that were occluded in the virus structure. This anomaly was explained by proposing that superficially hidden epitopes could be unmasked during thermal flux or “breathing” of the virus [ in vivo mammalian infection because the more compact 28°C structure will only be exposed to the host for one round of replication, after which the second, 37°C structure will be adopted. Because both compact and expanded forms of the viruses from both hosts are infectious, these virions represent metastable structural intermediates primed for infection, and these two distinct infectious intermediates may represent the optimal form of the structure from its specific host biochemical environment.Both these flavivirus studies speculated that the change in structure may affect the response of the mammalian host by revealing previously unexposed epitopes in virus from insect cells. This is important for mapping neutralizing epitopeshe virus . This quOne interpretation of these observations is that the mammalian virus is “born” in the prefusogenic-like structure, and the mosquito virus is not. While there is no direct evidence for any function of these predicted intermediate models, mosquito cells were speculated to be infected by virus adopting the fusogenic form via exposure to low pH or contact with the receptor, thus triggering the rearrangement. Again, as in the former study, the Zhang study demonstrated that virus incubated at both temperatures is infectious. This becomes an important point because arboviruses do not always cycle between insect and vertebrate hosts. For mosquito-borne viruses, there exists a “mosquito only” zoonotic cycle in the natural transmission cycle, during which mosquito and virus would only experience ambient temperature , 183. ArIn addition to interpreting the high temperature structures, these papers modeled crystal structures of isolated E into the cryoEM reconstructions. Both studies used E from Tick Borne Encephalitis virus PDB 1SVB . Zhang eT = 3 symmetry is assembled in the flaviviruses, and T = 4 for the alphaviruses, representing an approximate 200 Å increase in virion size while the genome lengths are not significantly larger. As discussed for the Sindbis virus C mutant, Y180S/E183G, even larger T numbers are possible. Not discussed, but pertinent to this point, is the T = 12 structure found in the bunyaviruses that deviate to pleomorphic structures in the other members of the Bunyaviridae. These viruses are also presumed to have arisen from an ancient common fold. The relationship of this adaptation is not understood because the T = 12 structure genome size ranges from 10.5 to 22.7 kbp and is composed of a tripartite, single-stranded, negative-sense RNA genome that could theoretically be packaged in a much smaller virion [As has been discussed, the structural proteins of the icosahedral arboviruses display a remarkable amount of plasticity. From the same conserved folds, r virion . Plasticr virion . This waIt has been a long quest to solve the atomic structure of a membrane-containing virus. As technology improves, atomic details will certainly be resolved. However, there are certain impediments to the process that are not directly related to the technology. First, it has become a canon of some investigators that crystal structures of proteins and their multimeric states represent a native structure. This is a huge assumption for metastable virus proteins because in the absence of control structures or direct functional assays this assumption cannot be tested. Most virus samples also contain a large particle to pfu ratio. The most rigorous experiments would not require the dissolution of the virus particle , 187. Th


Fulminant hepatitis is a rare outcome of infection with hepatitis E virus. Several recent reports suggest that virus variation is an important determinant of disease progression. To critically examine the evidence that virus-specific factors underlie the development of fulminant hepatitis following hepatitis E virus infection.Published sequence information of hepatitis E virus isolates from patients with and without fulminant hepatitis was collected and analysed using statistical tests to identify associations between virus polymorphisms and disease outcome.Fulminant hepatitis has been reported following infection with all four hepatitis E virus genotypes that infect humans comprising multiple phylogenetic lineages within genotypes 1, 3 and 4. Analysis of virus sequences from individuals infected by a common source did not detect any common substitutions associated with progression to fulminant hepatitis. Re-analysis of previously reported associations between virus substitutions and fulminant hepatitis suggests that these were probably the result of sampling biases.Host-specific factors rather than virus genotype, variants or specific substitutions appear to be responsible for the development of fulminant hepatitis. The aetiology of hepatitis E virus infection (HEV) is complex. Many individuals exposed to HEV infection remain asymptomatic while others go on to develop an acute hepatitis of varying severity that usually resolves within 2–3 months [reviewed in ]. ChroniSeveral previous studies have suggested that the pathogenicity of HEV infection might be genotype or strain dependent. For example, a bias towards genotype 4 has been noted in Japanese patients with FH or severe disease –4, whileHowever, an unacknowledged problem with some of these studies is the common geographical origin of the variants studied so the reported associations between particular virus substitutions and FH might simply occur because of epidemiological relationships amongst the viruses rather than because such substitutions were involved in the development of FH. This article re-examines published evidence for an association between FH and particular HEV genotypes, lineages or particular nucleotide substitutions.Complete HEV genome sequences downloaded from Genbank were as follows: Genotype 1: JF443726, JF443725, JF443724, JF443723, JF443722, FJ457024, X98292, M73218, JF443721, JF443720, JF443719, JF443718, JF443717, AF459438, AF076239, X99441, AF051830, DQ459342, M80581, D10330, AF185822, L08816, D11092, D11093, L25595, AY204877, AY230202, AB720034, AB720035, JQ655734, AF444002, AF444003, L25547, M94177, Genotype 3: AB291955, Genotype 4 human isolates: AB291967, AB291959, AB193176, AB220971, AB220972, AB220973, AB091395, JQ740781, AB291966, AB291965, AB291968, AB220974, AB291964, AB220975, AB220976, AB220977, AB220978, AB220979, AJ272108, AB108537, AB097812, FJ763142, KC492825, AB698654, JQ655735, JQ655733, HQ634346, HM439284, AB369690, AB369688, AB197674, AB197673, AB193178, AB193177, AB099347, AB074917, AB074915, AB080575.P < 0.01 in a two-tailed test as implemented in an R script available upon request from the authors. The same data sets were analysed using meta-CATS (http://www.viprbrc.org) that uses both the chi-squared test of independence and Pearson's chi-squared test  following which at least one individual developed FH. Of two men who consumed uncooked boar liver, one developed FH while the other individual had acute hepatitis . SimilarWe tested possibility that particular substitutions are responsible for the development of FH by tabulating sequence differences in two of the common source outbreaks for which complete genome sequences were available Table . None ofP < 0.01) associated with FH, but 136 sites if only the five FH isolates from Pune were included, or four sites if only one of the Pune FH isolates was used. These four sites  were all synonymous, although position 6312 lies within a proposed secondary structure that is essential for virus replication (P = 0.06). Together, these observations suggest that the previous reports of sites significantly associated with FH amongst genotype 1 viruses may be actually reflect the common geographical and temporal origin of 5/7 FH isolates.Previous studies have used statistical methods to identify substitutions that are associated with isolates derived from FH patients infected with HEV genotype 4 ,11 or gelication . When wen = 8) and non-FH (n = 30) patients revealed only two sites significantly associated with FH . These FH-associated substitutions were also present in >41% of the non-FH sequences and differed from the two sites reported from analysis of 22 isolates (P = 0.031); a previous study reported a much lower P value of 0.0042    that useIn contrast to several previous studies ,7,9–12, The identification of virus lineages or substitutions associated with the development of FH would be more difficult if this trait had incomplete penetrance. However, in this case, many more isolates from FH patients would be required. For example, a recent survey of virus determinants of FH following infection with hepatitis B virus that included 50 cases of FH with age- and sex-matched controls was able to demonstrate an association with the G1896A pre-core mutation , a substAlthough FH can develop following infection with any of the HEV genotypes known to infect humans, evidence that FH might be associated with particular genotypes comes from a survey of HEV in Japan over the last decade . Of 199 Alternatively, the development of FH consequent to HEV infection could be because of patient-specific factors. A precedent for this comes from previous studies showing a correlation between the severity of HEV infection (including FH) and pregnancy ,33, pre-


Susceptibility to infection varies by species, and asymptomatic birds could be carriers.  In January 2014, an outbreak of infection with highly pathogenic avian influenza (HPAI) A(H5N8) virus began on a duck farm in South Korea and spread to other poultry farms nearby. During this outbreak, many sick or dead wild birds were found around habitats frequented by migratory birds. To determine the causes of death, we examined 771 wild bird carcasses and identified HPAI A(H5N8) virus in 167. Gross and histologic lesions were observed in pancreas, lung, brain, and kidney of Baikal teals, bean geese, and whooper swans but not mallard ducks. Such lesions are consistent with lethal HPAI A(H5N8) virus infection. However, some HPAI-positive birds had died of gunshot wounds, peritonitis, or agrochemical poisoning rather than virus infection. These findings suggest that susceptibility to HPAI A(H5N8) virus varies among species of migratory birds and that asymptomatic migratory birds could be carriers of this virus. Highly pathogenic avian influenza (HPAI) A virus infection of gallinaceous birds  is associated with high morbidity and mortality rates  have occurred in South Korea (during winter and spring); migratory birds were identified as putative vectors , and influenza viruses were identified by using a hemagglutination assay and reverse transcription PCR. Virus identification was confirmed by sequence analysis, as described previously (Paraffin-embedded sections were cut (5 μm), dewaxed, and stained with hematoxylin and eosin. Duplicate sections were immunohistochemically analyzed to determine the distribution of influenza virus antigens in individual tissues. Briefly, sections were stained with a mouse monoclonal antibody against influenza A virus nucleoprotein , followed by a biotinylated goat anti-mouse IgG secondary antibody. Bound antibodies were detected with an avidin-biotin detection system . The RedMap kit  served as the substrate chromogen.Escherichia coli, Staphylococcus aureus, and Salmonella Typhimurium), parasitic , and fungal infections were diagnosed for 29, 9, and 3 birds, respectively. We found that 73% of birds died of noninfectious causes. Agrochemicals, including monocrotophos, phosphamidon, carbofuran, diazinon, carbosulfan, endosulfan, parathion, dichlorvos, and methomyl, were found in the stomach contents of 222 birds; gunshot wounds, trauma (road kill or fracture), or miscellaneous  were the cause of death for 12, 103, and 32 birds, respectively. For 194 wild birds, the cause of death could not be determined because of postmortem autolysis, putrefaction, or both. Of a total of 771 wild birds, HPAI A(H5N8) viruses were isolated from 167. For the other 604 birds, test results for other avian influenza viruses were negative . BacteriDuring January–March 2014, a total of 167 wild birds of 8 species were infected with HPAI A(H5N8) virus. All 148 infected Baikal teals showed evidence of multifocal necrosis in the pancreas and liver, pulmonary congestion and edema, subepicardial hemorrhage, and myocarditis . All H5NHistologic examination revealed lesions in the pancreas, kidney, brain, and lung of all 8 birds examined. The pancreas showed moderate to severe, multifocal to confluent acinar necrosis, and virus antigen was detected in necrotic cells .In 2 of 3 bean geese examined, major histopathologic lesions were found in the same organs as in the Baikal teals. Moderate multifocal pancreatic necrosis was also observed. Myocardial myofibers showed evidence of segmental necrosis, and mildly swollen nuclei, focal necrosis, and virus antigen were detected in the heart .In the 1 bird examined, lesions were found mainly in the pancreas, kidney, and brain. The distribution of the lesions and the antigenic staining patterns were similar to those observed for bean geese.Of the 2 mallard ducks examined, a heterotopic parasite was observed in the pancreas of 1 and fibrinous peritonitis affecting the pancreas and intestine was observed in the other. No virus antigen was detected in the intestine or pancreas of either bird ,3.The 2014 outbreak of HPAI A(H5N8) in South Korea was unexpected because the H5N8 subtype is uncommon in this area. A genetic characterization study suggests that this H5N8 virus (clade 2.3.4.6) was introduced into South Korea by migratory birds and spread from there to poultry farms , no evidence of lesions was found; however, the H5N8 virus was identified in the trachea and kidneys. Thus, the H5N8 virus did not cause sudden death in these waterfowl, despite their infection with the virus. This finding suggests that the infection is not peracute during the early stages.By contrast, although mallard ducks and white-fronted geese were asymptomatically infected with H5N8 HPAI, these birds died of other causes, including gunshot wounds or peritonitis. Experimental infection studies show that some wild ducks, geese, and swans shed H5N1 virus despite being asymptomatic  infects the tubular epithelium in the kidneys of various waterfowl, no evidence of gross or histopathologic lesions has been found in the kidneys  virus (clade 2.3.4.6) in various species of waterfowl in South Korea. Baikal teals, bean geese, and whooper swans are susceptible to this virus, which causes high mortality rates; however, infection in mallard ducks is asymptomatic. Although many questions regarding HPAI A(H5N8) virus pathogenesis remain, the results reported herein suggest that susceptibility to HPAI A(H5N8) virus differs among different species of migratory birds. Thus, these birds may be susceptible to or carriers of this infectious virus.


We used Ebola virus–infected macaques to model humans who died of Ebola virus disease. Viable virus was isolated  The ongoing outbreak of Ebola virus (EBOV) infection in West Africa highlights several questions, including fundamental questions surrounding human-to-human transmission and stability of the virus. More than 20,000 cases of EBOV disease (EVD) have been reported, and >8,000 deaths have been documented  , swab and tissue samples were obtained and used for EBOV titration on Vero E6 cells to quantify virus or for quantitative reverse transcription PCR (qRT-PCR) (40 cycles) to measure viral RNA, as reported  swab samples and tissue biopsy specimens from the liver, spleen, lung, and muscle. Swabs were placed in 1 mL of culture medium and tissue samples were placed in 500 μL of RNAlater , or an empty vial for titration, before freezing at −80°C. Carcasses were placed in vented plastic containers in an environmental chamber at 27°C and 80% relative humidity throughout the study to mimic conditions in West Africa  and longest-lasting isolatable virus (7 days posteuthanasia)  , panel Athanasia , panel B<3 weeks postmortem from a monkey carcass that was viremic at the time of death, in environmental conditions similar to those during current outbreak  would compromise the ability of the virus to replicate. Thus, the ability to isolate replicating virus in cell culture from postmortem materials was much less sensitive than detection of viral RNA by qRT-PCR. The sensitivity for quantitating infectious virus is probably lowered because of limitations in isolation efficiency on cell culture and necessary dilutions of tissues for homogenization for titration. Nonetheless, we detected viable virus ,Humans who die of EVD typically have high levels of viremia, suggesting that most fresh corpses contain high levels of infectious virus, similar to the macaques in this study  would be beneficial for sample integrity assessment, which is a limitation of this study.>7 days on surfaces of bodies, confirming that transmission from deceased persons is possible for an extended period after death. These data are also applicable for interpreting samples collected from remains of wildlife infected with EBOV, especially nonhuman primates, and to assess risks for handling these carcasses.In summary, we present postmortem serial sampling data for EBOV-infected animals in a controlled environment. Our results show that the EBOV RT-PCR RNA target is highly stable, swabbing upper respiratory mucosa is efficient for obtaining samples for diagnostics, and tissue biopsies are no more effective than simple swabbing for virus detection. These results will directly aid interpretation of epidemiologic data collected for human corpses by determining whether a person had EVD at the time of death and whether contact tracing should be initiated. Furthermore, viable virus can persist for


Food products containing raw pork liver are suspected to be vehicles for transmission of hepatitis E virus. Four categories of food products, comprising 394 samples, were analyzed to determine hepatitis E virus prevalence. Virus was detected in 3%–30% of the different categories. Phylogenetic analysis showed high identity with human and swine sequences. In humans, hepatitis E virus (HEV) is responsible for an acute, entero-transmissible form of hepatitis, similar to that caused by hepatitis A. In most cases, it is a self-limited infection with rapid viral clearance, but it can evolve into more severe forms, including fatal fulminant hepatitis. Chronic hepatitis E also has occurred in solid-organ transplant recipients and has progressed to more serious conditions, such as fibrosis or liver cirrhosis and liver failure . In a national survey in 2009, the presence of HEV in the swine reservoir was characterized and 65% of pig farms were found to have infected animals; 4% of pork livers entering the food chain were contaminated by the virus  figatellu and fitone, 2) dried salted liver, 3) quenelle and quenelle paste, and 4) dried or fresh liver sausages. HEV can be heat-inactivated by thorough cooking at 71°C for 20 min (2–106 copies of HEV RNA/g of food (http://www.r-project.org). HEV RNA prevalence was high in figatelli (30% [95% CI 23%–38%]), liver sausages (29% [95% CI 22%–36%]), and quenelles (25% [95% CI 15%–37%]). The prevalence of HEV RNA was lower in dried salted liver: 3% (95% CI 0%–10%)  .Four HEV RNA–positive food samples collected in the present study were tested for infectious virus in collaboration with 2 laboratories . Virus growth from 1 HEV-positive sample was observed in a 3-dimensional culture system developed by these laboratories (8 copies of HEV RNA/g)  was mixed with fat and spices. Therefore, even if only 4% of raw livers are infected, as shown in a previous study (http://blast.ncbi.nlm.gov/Blast.cgi) to identify the closest sequences. Thirty-three sequences had >98% nt identity with human and/or swine sequences. The human sequences with the highest identity originated in France, except for 2 sequences from the United Kingdom and 1 from Spain  should be informed about the HEV risk and should avoid eating such pork liver food products without thoroughly cooking them.