Aedes mosquitoes. This paper reviews the status of the Zika virus outbreak, including medical countermeasure options, with a focus on how the epidemiology, insect vectors, neuropathology, virology and immunology inform options and strategies available for medical countermeasure development and deployment.Reports of high rates of primary microcephaly and Guillain–Barré syndrome associated with Zika virus infection in French Polynesia and Brazil have raised concerns that the virus circulating in these regions is a rapidly developing neuropathic, teratogenic, emerging infectious public health threat. There are no licensed medical countermeasures  available for Zika virus infection and disease. The Pan American Health Organization (PAHO) predicts that Zika virus will continue to spread and eventually reach all countries and territories in the Americas with endemic Multiple information sources were employed to support the review. These included publically available literature, patents, official communications, English and Lusophone lay press. Online surveys were distributed to physicians in the US, Mexico and Argentina and responses analyzed. Computational epitope analysis as well as infectious disease outbreak modeling and forecasting were implemented. Field observations in Brazil were compiled and interviews conducted with public health officials. Wh. Wh49]. Uncertainties about Zika virus transmission abound. The degree to which humans, non-human primates, or other animals can amplify and transmit the virus to insect vectors is poorly understood. The typical range and types of insect vectors observed in the past may not be predictive for the virus now circulating in the Americas. Infectivity of the circulating strain, viremia levels, duration, and risk of occult persistence are not yet understood.The highest risk for introduction and establishment of autochthonous Zika transmission is likely to be associated with infected humans traveling by international ground, sea, and air transportation, and with the transport of mosquito larvae by trucks, ships and aircraft. Countering transportation-based introduction is the best immediate strategy available for delaying the spread. Options include more rigorous cargo fumigation at ports and border crossing points, use of larvicides and insecticides, and monitoring ground, sea, and air travel from infected areas. Seaports and the US/Mexico border are the most critical points for reducing the risk of large-scale vector borne viral distribution into the United States and Canada. Cases acquired abroad will continue to be identified in regions that have not reported autochthonous infection, and must be differentiated from local transmission. Rapid identification of infected persons who are subclinical and viremic is nearly impossible.There is a critical need for development and deployment of Zika diagnostics to regional clinical reference laboratories . Obstetricians throughout the Americas must advise their patients on very difficult decisions involving risk to ongoing or planned pregnancies. Neurologists are confronting unprecedented GBS outbreaks. These front-line physicians lack access to critical tests necessary to guide decisions, information concerning infection monitoring after possible exposure, understanding of the window of susceptibility to birth defects, and clear direction and resources for testing, diagnosing, and managing obstetric and neurology patients.Delaying spread of the virus into new regions may buy some time to develop MCM, but will not help those who live in infected areas. During gestation, women of means may choose to leave infected countries for safe zones , 54. Thevia blood products. Procedures for minimizing risk of salivary transmission must be developed [In affected areas, regional surges in GBS may stress medical response capacity. MCM preparation for GBS surges should include sufficient intensive care unit capacity, ventilators, plasma exchange equipment , trainedeveloped . Guidanceveloped , 59, 60In contrast to the relatively slow spread of Ebola virus through West Africa, the Zika outbreak in the Americas appears to be moving very rapidly. While the potential association of Zika virus with teratology and neuropathology place a particular urgency on the development of MCM, strategies for developing and deploying MCM must account for the differences and similarities between the observed epidemiology and that of prior outbreaks. For example, developing, testing and deploying a new vaccine may be feasible for endemic pathogens or slowly moving epidemics, but may not be practical for a rapidly moving infectious disease outbreak. Until the pathogenesis of the disease, nature of vectors and mechanisms of spread are understood, caution must be exercised in making assumptions in the design of MCM.Flaviviruses can appear significantly more pathogenic when introduced into new niches and populations, but as a new virus becomes established, herd immunity effects often attenuate apparent virulence. West Nile virus in birds shifted from a relatively benign profile in the traditional endemic African host range to very high mortality upon introduction in North America in 1999. This change was associated with specific mutations that increased viral reproductive fitness in avian hosts and the North American environment . The rapMany questions about Zika virus epidemiology and transmission remain, but among the most pressing questions are whether the change in disease phenotype correlates to changes in viral genotype, and if current clinical disease is influenced by viral entry into a new population with indigenous confounding or effect modification. Historically restricted to Africa and Asia, outbreaks of autochthonous Zika virus infection were reported in Micronesia beginning in 2007 . As predvia shared ancestry of the sequences. Based on data released as of January 18, 2016 we have focused on two genes to reconstruct the spread and evolution of Zika from Africa to Southeast Asia to the South Pacific and to South America; E (Envelope) and NS5 (RNA-dependent-RNA-polymerase). Results from applying this method for tracking and summarizing sequence accessions, together with associated temporal and geographic metadata, suggest a pattern of stepwise accumulation of sequence changes. The Zika virus circulating in the Americas appears to have acquired mutations while hopping along distant points across the Pacific, and then emerged as a burst of infection by a cohort of closely related viruses upon arrival in Brazil.To summarize these events and to help guide assessment of genetic and immunologic differences between historic and current Zika virus populations, we performed preliminary phylogeographic analyses of available molecular sequence data and metadata (place and time of isolation) from the viruses to connect these incidents The initial introduction of Zika virus into continental South America may have occurred in Brazil during 2014 or very early 2015. Our results suggest entry to Brazil from the Cook Islands  or Easter Island . Some speculation concerning viral introduction into Brazil near Rio de Janeiro has assumed that the virus was imported by infected humans, and has centered on two sporting events which included participants from Polynesia (the 2014 FIFA World Cup and the Va’a World Sprint Canoe World Championships) . These sAfter introduction, Zika virus rapidly spread throughout much of Brazil. In January 2016, there were cases in 14 states in Brazil  and in nAs summarized in 0). Other means of introduction must also be considered, including birds or insects via cargo shipping. Evidence supporting avian infection by Zika virus has been reported [via insect intermediates has not been studied. West Nile virus was rapidly spread throughout North America by birds. Transoceanic movements of arboviruses in insects has been reported [Although human transmission may be a source of initial introduction into Brazil in 2014, the apparent incidence of new infection in the region implies a high reproduction number . During what appears to have been a four month, self-limited outbreak, it is estimated that 73% of Yap residents 3 years of age or older were infected with a Zika virus strain hypothesized to have been brought to the island by an imported non-human primate . This suAedes sp.  appear to be the leading candidate Zika vectors in the outbreak. Potential involvement of other insect vectors including Culex sp. mosquitoes are currently being examined [Ae. hensilli Farner was the predominant vector species [Ae. aegypti and Ae. albopictus mosquito populations reaches around the globe, with remarkable parallels to the global distribution of Zika virus , documented a significant increase in the number of microcephaly cases compared with previous years. Temporal and spatial concordance of the distribution of primary microcephaly with that of Zika virus infection raised public health concerns of a possible causal relationship . These fThe link between high rates of microcephaly and Zika infection was initially greeted with skepticism. Although a possible link between microcephaly and Zika virus infection was first reported in French Polynesia (the apparent source of the virus which seeded Brazil), it has not been reported over the many prior years that Zika has existed in its traditional endemic range , 93, 94.via amniocentesis confirmed presence of Asian-type Zika virus. Ultrasound analysis revealed findings similar to those observed with cytomegalovirus infection (but more severe), and also similar to those previously reported with intrauterine infection by West Nile virus. On the basis of observed ultrasound findings, the authors of the alert speculate that “as with other intrauterine infections, it is possible that the reported cases of microcephaly represent only the more severely affected children and that newborns with less severe disease, affecting not only the brain but also other organs, have not yet been diagnosed.” This has since proven true with the reports of ocular lesions in affected infants [Koch’s postulates concerning infectious disease causality include demonstrating the presence of the pathogen in affected patients. Molecular biologic evidence demonstrating Zika genomes in tissue and amniotic fluid of Brazilian children born with microcephaly “support the conclusion of the rapid risk assessment of 24 November that a causal association between microcephaly in newborns and Zika virus infection during pregnancy is plausible” . More re infants . A recen infants . To our  infants .Concurrent with growing evidence from Brazil of a correlation between Zika infection microcephaly, on November 24, 2015 French Polynesian public health authorities published a report documenting an increase of at least 17 cases of primary microcephaly relative to background incidence during 2014–2015. Based on the timing of the Zika outbreak in French Polynesia, this report hypothesizes peak sensitivity to teratogenic effects during the first or second trimester . These fStrength of epidemiologic association and evidence indicating a biological gradient correlating exposure and disease are also key criteria for establishing causation . Fig 3 iThe state of Pernambuco (located in northeastern Brazil) was the first to identify an increase of microcephaly, and has reported 1,236 cases up to January 09 , followed by Paraíba (569), Bahia (450), Ceará (192), Rio Grande do Norte (181), Sergipe (155), Alagoas (149), Mato Grosso (129) and Rio de Janeiro (122) . In the In the typical initial infection event, Zika virus is transmitted to a bitten human host after skin injection of a mixture of insect saliva, virus, and blood components from the most recent feeding during female mosquito blood meals. Probability of viral particle transmission is related to the volume of fluid held in the proboscis from a prior blood meal, viral replication levels and volume of insect salivary glands, and the viral infectious titer of the preceding host . In the The infection and host response cascade triggered by initial infection with Zika virus has yet to be characterized. Dengue infection in humans may provide a model until further information becomes available. In the case of dengue, the infection then spreads to both lymphatic and non-lymphatic tissues; fever, arthralgia and myalgia ensue. Viral titers peak with fever onset, are stable for one to two days, and then decline as adaptive immune responses begin to control the infection (T and B cells), with IgM and IgG levels increasing rapidly as viremia drops. The CD8+ T cell responses to dengue infection are primarily directed to nonstructural protein epitopes including NS3 and NS5. Human infection by dengue provides one of the most classic examples of antibody dependent enhancement of disease by pre-existing non-neutralizing antibody, resulting in dengue hemorrhagic fever , 108. Thvia FcRn receptors on the placenta. Epitopes with dengue or YF could result in preexisting antibodies to these viruses binding Zika and enhancing initial virus replication or placental cell infection, or transplacental viral transfer.The duration of viremia, infectivity, and persistence of Zika virus, is not known for either post-partum or intrauterine infection. Nor is the route of fetal infection, or the degree of neurotropism. Related flaviviruses may cause persistent infection despite the presence of serum antibodies . West NiRapid immunoinformatic analysis of the envelope protein of Zika, from Brazilian Zika SPH2015 (KU321639), indicates predicted B and T cell epitopes in peptides that are consistent to those reported for dengue, YFYF and Japanese encephalitis. The envelope Domain II B cell epitope, to which much dengue non-neutralizing cross reaction is attributed , is alsoOpportunities and strategies for Zika medical management and countermeasure development will benefit from answers to key questions concerning the virology and immunology of Zika infection in the human host. A better understanding of natural immune responses and viral infection may clarify the potential role of Zika in eliciting GBS or microcephaly. Targeted identification and design of antivirals, neutralizing antibody preparations and immunotherapeutics still require understanding of the underlying biology. Critical priorities for early characterization include duration and levels of viremia and transmissibility, whether circulating non-neutralizing antibody complexes contribute to either primary infection or fetal pathology, and the potential for interaction with pre-existing immunity elicited by other flaviviruses or flavivirus vaccines.in vitro tests and animal challenge models. Once identified, testing of medical products may be expedited by focusing on high-risk populations (pregnant women and those wishing to become pregnant); risk/benefit ratios in these populations may be more compelling, and clinical safety and efficacy testing may be more efficient when subpopulations with higher risk for clearly defined disease outcomes, rather than general populations, are selected for clinical study enrollment. Pregnant women are typically the last “special population” to be clinically tested when developing a MCM, but this outbreak represents a special case where the fetus is apparently at highest risk.Over the short term, development and testing of antiviral drugs, neutralizing antibody preparations, and medicines designed to interfere with Fc receptor interactions  are amonDevelopment of a general use prophylactic vaccine for Zika virus-induced disease will require considerable time and careful evaluation of safety, effectiveness, and risk/benefit ratio for the population at large. This is particularly true for a vaccine designed to protect against a virus apparently associated with both neurologic teratogenic effects and neurologic autoimmune disease (GBS), and which belongs to a genus notorious for antibody-mediated enhancement of infection , 125. FoIn the Yap island outbreak of 2007, 73% of the residents of Yap were infected by Zika within four months . By the in vitro testing has demonstrated inhibition of dengue virus infection via induction of reactive oxygen species and mitochondrial antiviral signaling protein [via inhibition of autophagy [in situ inhibition of Ebola pathogenicity using this compound has been demonstrated at clinically relevant doses [In vitro, ADE has been demonstrated with Zika virus [in levitro and in vivo experiments [In the absence of currently available vaccines, the likely long timeline for vaccine development, and the open questions about the basic pathogenesis of Zika virus infection, parallel development of other prophylactics and therapeutics must be explored. Regarding drugs, the Assistant Director General of the World Health Organization has indicated that preventive therapies, similar to those for malaria, seem like a faster and more workable option than treatments . Current protein . Of inte protein . Amodiaqutophagy , is safeutophagy , and in nt doses . In prelnt doses , 140, anka virus . A US paeriments .De novo antibodies may be generated which target Zika-specific epitopes. Further study of the role of transplacental immunoglobulin in Zika teratology will be needed.The potential for monoclonal antibody based therapies for arbovirus infections was recently reviewed , concludMAbs which have been appropriately engineered and de-risked have the potential to protect against Zika infection, but a mAb product must have high potency if it is to provide an adequate number of doses at reasonable cost. For example, the adult dose of ZMapp that may reduce the spread of Ebola within the body requires nearly 200 x 10ml vials at 100mg/ml of three antibodies that recognize distinct epitopes of the Ebola Zaire glycoprotein. New tools such as affinity maturation to create a comprehensive map of the paratope sequence space to allow identification of beneficial, neutral, and detrimental amino acid substitutions at each complementarity determining region (CDR) position, as well as use of phage displays, may lead to improved manufacturability  and lead to faster testing of each antibody variant in a cost-effective manner. A similar strategy for Zika, combining two or three mAbs binding non-overlapping specific epitopes, would increase the chances of neutralization by first pass hepatic clearance of the immune complexes. In the absence of dose-response information in humans, a reliable estimate can be obtained from LD50 exposure animal studies where the level of protection may be titrated.Zika infection is rapidly spreading throughout the Americas. To keep up with this outbreak, surveillance tracking, outbreak tracking and threat analysis will necessarily involve a combination of methods, both traditional and modern. Traditional methods include case reporting, vector sampling, reservoir animal sampling, and sentinel systems. A number of tools can be added to this list. These include human networking, reporting signature pattern recognition and forecasting, social media tracking and bioinformatics, including geospatial analysis of isolates and immunoinformatics. The West African Ebola virus outbreak of 2014–2015 revealed serious deficiencies in global surveillance, threat identification and management capabilities for infectious disease epidemics. The various lessons-learned exercises which followed may help guide a more effective response to the threats associated with the current outbreak.Physicians and their patients are asking for practical information to guide routine decisions, and are expressing frustration about public health communication and availability of the clinical tests required to manage important reproductive health decisions. To better understand the questions and issues which medical caregivers and patients need to have addressed, informal on-line surveys were distributed to physicians in the US, Mexico and Argentina. In an initial sample of 56 responses addressing the question “What are the key questions you or your patients might ask about Zika virus?”. The top two responses were “How long does a woman need to wait to get pregnant following potential exposure to Zika virus?” (30%) and “What is the likelihood that a pregnant woman who is exposed to Zika virus will have an infant with a severe defect?” (23%). Many comments focused on frustrations associated with the absence of necessary clinical diagnostic laboratory tests. However, the most telling initial finding involved a question distributed to physicians outside of the United States. In response to “Do you think your health system is prepared for the Zika Virus?”, 79% (343/472) of physicians responded “No”, and 21% (99/472) responded “Yes”.Prompt and effective public health communications have also been a challenge during both the H1N1 outbreak and the West African Ebola outbreak. In an initiative specifically designed to apply lessons learned from the Ebola experience concerning the importance of rapidly disseminating key information, the International Severe Acute Respiratory and Emerging Infection Consortium (ISARC) in cooperation with Fundação Oswaldo Cruz (Fiocruz), WHO, Institute Pasteur, and the German Centre for Infection Research and others have established an internet-based resource for sharing and developing public health research and response information concerning Zika virus, under the coordination of Fernando Bozza of Fiocruz .PAHO is working to provide timely access to the information which physicians and the public require, and has published a statement on Zika Virus Transmission and Prevention which included the following comments :Aedes mosquitoes—the main vector for Zika transmission—are present in all the region's countries except Canada and continental Chile.There are two main reasons for the virus's rapid spread: (1) the population of the Americas had not previously been exposed to Zika and therefore lacks immunity, and (2) Aedes mosquitoes are found.PAHO anticipates that Zika virus will continue to spread and will likely reach all countries and territories of the region where https://disaster.nlm.nih.gov/dimrc/zikavirus.html#a6)).The National Library of Medicine has established a Disaster Information Research Center website listing resources providing links :2536–43.Lanciotti RS, Kosoy OL, Laven JJ, Velez JO, Lambert AJ, Johnson AJ, et al. Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007. Emerg Infect Dis. 2008;14(8):1232–9.Musso D, Nhan T, Robin E, Roche C, Bierlaire D, Zisou K, et al. Potential for Zika virus transmission through blood transfusion demonstrated during an outbreak in French Polynesia, November 2013 to February 2014. Euro Surveill. 2014;19(14).Faye O, Freire CC, Iamarino A, Faye O, de Oliveira JV, Diallo M, et al. Molecular evolution of Zika virus during its emergence in the 20(th) century. PLoS Neglected Tropical Diseases. 2014;8(1):e2636.Musso D, Roche C, Robin E, Nhan T, Teissier A, Cao-Lormeau VM. Potential sexual transmission of Zika virus. Emerg Infect Dis. 2015;21(2):359–61.Hamel R, Dejarnac O, Wichit S, Ekchariyawat P, Neyret A, Luplertlop N, et al. Biology of Zika Virus Infection in Human Skin Cells. J Virol. 2015;89(17):8880–96.Oliveira Melo AS, Malinger G, Ximenes R, Szejnfeld PO, Alves Sampaio S, Bispo de Filippis AM. Zika virus intrauterine infection causes fetal brain abnormality and microcephaly: tip of the iceberg? Ultrasound Obstet Gynecol. 2016;47(1):6–7.Mlakar J, Korva M, Tul N, Popović M, Poljšak-Prijatelj M, Mraz J, et al. Zika Virus Associated with Microcephaly. N Engl J Med. 2016 Feb 10.


H5N1 highly pathogenic avian influenza virus (HPAIV) of clade 2.3.2 has been circulating in waterfowl in Southern China since 2003. Our previous studies showed that certain H5N1 HPAIV isolates within clade 2.3.2 from Southern China had high pathogenicity in different birds. Guinea pigs have been successfully used as models to evaluate the transmissibility of AIVs and other species of influenza viruses in mammalian hosts. However, few studies have reported pathogenicity and transmissibility of H5N1 HPAIVs of this clade in guinea pigs. In this study, we selected an H5N1 HPAIV isolate, A/duck/Guangdong/357/2008, to investigate the pathogenicity and transmissibility of the virus in guinea pigs. The virus had high pathogenicity in mice; additionally, it only replicated in some tissues of the guinea pigs without production of clinical signs, but was transmissible among guinea pigs. Interestingly, virus isolates from co-caged guinea pigs had the D701N mutation in the PB2 protein. These mutant viruses showed higher pathogenicity in mice and higher replication capability in guinea pigs but did not demonstrate enhanced the transmissibility among guinea pigs. These findings indicate the transmission of the H5N1 virus between mammals could induce virus mutations, and the mutant viruses might have higher pathogenicity in mammals without higher transmissibility. Therefore, the continued evaluation of the pathogenicity and transmissibility of avian influenza virus (AIVs) in mammals is critical to the understanding of the evolutionary characteristics of AIVs and the emergence of potential pandemic strains. Orthomyxoviridae (Avian influenza viruses (AIVs) are enveloped RNA viruses with an eight-segmented, single-stranded, negative-sense genome, and belong to the family oviridae . They aroviridae . At presoviridae . To dateoviridae .H5N1 highly pathogenic avian influenza virus (HPAIV) isolates derived from the goose/Guangdong/1/96 (Gs/GD) lineage have been found in over sixty countries in Europe, Asia, and Africa . They noThe most commonly used mammalian model in the study of the pathogenesis of AIVs and the evaluation of vaccines and antiviral drugs is the mouse . One cleUnlike mice, ferrets are suitable for both transmission and pathogenesis studies, but the higher cost and large amount of space required for housing this species severely limit the number of animals that can be included in individual studies . In an aOur previous studies demonstrated that a large number of H5N1 HPAIVs within clade 2.3.2 from Southern China had high pathogenicity in different birds . Some st50) was calculated according to the method published by The A/duck/Guangdong/357/2008 (DK357) virus used in this study was isolated from ducks in the Guangdong Province of China in 2008 and identified as H5N1 AIV by means of HA inhibition and NA inhibition tests. The virus was purified and propagated in the allantoic cavity of 10-day-old, specific-pathogen-free (SPF), embryonated hens’ eggs. The allantoic fluid from multiple eggs was pooled, clarified by centrifugation, and frozen in aliquots at -70°C. The 50% egg infectious dose  and served as the sham control group. Three mice in each group were euthanized at 3 days post-inoculation (DPI), and the lungs, kidneys, spleens, and brains were collected for virus titration in eggs as described previously  of each guinea pig in the experimental groups. Five guinea pigs were inoculated with 0.3 ml PBS and acted as the sham control group. Three guinea pigs in each group were euthanized at 3 DPI, and the nasal turbinates, tracheas, lungs, brains, kidneys, spleens, livers, and colons were collected for virus titration in eggs following the previously described method  and xylazine (1 mg/kg) were used to anesthetize guinea pigs. To determine morbidity and mortality, groups of eight animals were anesthetized, and a 0.3 ml volume of inoculum containing 10d method . The rem6 EID50 of the test virus and housed in a cage. Five naive animals were introduced into the same cage 24 h later. Three naive animals were euthanized at 5 days post contact (DPC), and their organs were collected for virus titration in eggs following the previously described method , and the manufacturer’s instructions were followed. Reverse transcription polymerase chain reaction (RT-PCR) was conducted using the Superscript III  and Uni12 (5′-AGCAAAAGCAGG-3′) primer. Eight genes were amplified using universal primers , and theFigure 1). For the three mice euthanized on 3 DPI, the mean virus titer was 5.8, 3.8, 3.3, and 3.9 log10EID50 in the lung, spleen, kidney, and brain, respectively (Table 1). Infection with the DK/357 virus caused 23.1% loss of body weight by 7 DPI, while the sham control group inoculated with PBS increased in body weight by an average of 11.4% by 14 DPI. These findings illustrated that DK/357 virus had high pathogenicity in mice.In the mouse experiment, clinical signs of illness were observed in the experimental group as early as 3 DPI, and all mice exposed to the virus were dead by 7 DPI . No virus was isolated from any of the other collected guinea pig tissues. These results indicated that DK/357 virus only replicated in some tissues of the guinea pigs without production of clinical signs.In the mice studies, DK/357 was HP, i.e., causing 100% morbidity and mortality. In contrast, in guinea pig model, infection with the virus did not cause morbidity. What’s more, infection with the DK/357 virus only caused slight weight loss in guinea pigs during the initial DPI. In the evaluation of the organ tropism of the virus in the guinea pigs, the virus was found in the nasal turbinates, tracheas, and lungs with mean titers of 5.8, 2.6, and 5.6 log10EID50, respectively, at 5 DPC. At 9 DPC, one co-caged guinea pig showed slight clinical symptoms. This guinea pig was euthanized, and samples were collected for virus titration. The virus was isolated from the trachea (named DK/357-T) and the lung (named DK/357-L) with titers of 1.75 and 3 log10EID50, respectively.In the investigation of virus transmission among co-caged guinea pigs, the virus was isolated only from the nasal turbinates of two guinea pigs with titers of 2.75 and 2 logTable 2). In total, the following five amino acid changes between DK/357 and DK/357-L were identified: V96I, F135I, S688F, and D701N in the polymerase PB2 protein and N417S in the NP protein (Table 2).Viral RNA was extracted from the trachea and lung of the co-caged guinea pig, and RT-PCR was conducted. The PCR products of eight genes from the viral RNA extracted from the trachea and lung of the co-caged guinea pig were sequenced and were compared to the sequence of the original virus isolate in order to identify any molecular differences between the two. When comparing DK/357 to the DK/357-T, three amino acids substitutions were found, i.e., V96I and D701N in the polymerase PB2 protein and N417S in the nucleoprotein (NP) protein . The DK/357-T virus replicated systemically to the mean titers of 7, 3.9, 3.6, and 4.3 log10EID50 in the lung, spleen, kidney, and brain, respectively (Table 1). The virus titers in the detected tissues were all higher than that of the original DK/357 virus. The DK/357-L virus was replicated to the mean titers of 6.6, 3.4, 2.9, and 3.9 logEID50 in the lung, spleen, kidney, and brain, respectively (Table 1). The mean virus titers were higher than that in the lung and brain after infection with the original DK/357 virus but lower in the spleen and brain than that after infection with the original DK/357 virus.All mice exposed to the DK/357-T and DK/357-L were dead by 4 and 6 DPI, respectively . The virus was even found in the brain of one guinea pig with a titer of 2.7 log10EID50. After infection with the DK/357-L virus, the virus was also found in the nasal turbinates, tracheas, and lungs of the sacrificed animals with mean titers of 6, 2.2, and 6.3 log10EID50, respectively (Table 1). Overall, the replication ability of the two viruses was higher than the original virus isolate; however, the mean titer value in the trachea of the guinea pigs infected with DK/357-L was lower than that of the guinea pigs infected with the original DK/357 isolate.To investigate the morbidity and mortality of DK/357-T and DK/357-L in guinea pigs, the previously described intranasal inoculation procedures were performed using DK/357-T and DK/357-L for the virus inoculum with groups of eight guinea pigs for each. As observed in the original DK/357 virus, neither of the viruses caused morbidity in guinea pigs. In the organs of the three sacrificed animals from each group on 3 DPI, the DK/357-T virus had replicated in the nasal turbinates, tracheas, and lungs with mean titers of 6.8, 2.8, and 6 log50. In comparison, the virus was only detected in the nasal turbinates of one guinea pig co-caged with the guinea pigs inoculated with the DK/357-L with a mean titer 1.8 log10EID50.In order to investigate the transmission ability of DK/357-T and DK/357-L in guinea pigs, the previously described guinea pig inoculation and co-cage procedures were repeated using DK/357-T and DK/357-L. At 5 DPC, the virus was only detected in the nasal turbinates of two of the guinea pigs co-caged with the guinea pigs inoculated with the DK/357-T with a mean titer 2.1 logEIDhttp://www.who.int/). Most patients who had confirmed infections of H5N1 HPAIV died of respiratory failure complicated by acute respiratory distress syndrome; in most instances, the viral infection resulted from direct exposure to poultry or poultry products infected with H5N1 HPAIV . Our findings indicated that the pathogenicity of the DK/357 virus in mice and guinea pigs was different.Mice have previously been used as a mammalian animal model when evaluating the pathogenicity of the influenza virus . In thisTable 2). Previous researches have reported that V96I in the PB2 protein and N417S in the NP protein also existed in low pathogenic human and/or AIV, and these mutations did not enhance their pathogencity to mice  influenza virus isolates, develop an upper respiratory tract infection, and transmit the virus to other guinea pigs in both contact and non-contact situations . In addi to mice . The D70 to mice . These pTable 1), they were not lethal to guinea pigs. In addition, when guinea pigs were inoculated with DK/357-T, the virus was found in the brain of one guinea pig. The DK/357-T and DK/357-L isolates manifested an increased pathogenicity in mice; indeed, they caused more severe weight loss and demonstrated stronger replication capability than DK/357. Although the replication capability of DK/357-T and DK/357-L was a little higher than that of DK/357 in guinea pigs (In summary, our findings illustrated that the H5N1 duck-origin influenza virus that belongs to the 2.3.2 clade demonstrated high pathogenicity in mice. This virus only replicated in some tissues of the guinea pigs without production of clinical signs but could be transmitted among guinea pigs. Interestingly, the viruses isolated from the co-caged guinea pigs had a D701N mutation in the PB2 protein. These mutant viruses showed higher pathogenicity in mice and higher replication capability in guinea pigs; however, these viruses did not show enhanced transmissibility among guinea pigs. Therefore, the continued evaluation of the pathogenicity and transmissibility of AIVs in mammals is critical to the understanding of the evolutionary characteristics of AIVs and the emergence of potential pandemic strains.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Quercus robur, are organized into two rDNA loci, the major (NOR-1) and the minor (NOR-2) locus. We present the first cytogenetic evidence indicating that the NOR-1 represents the active nucleolar organizer responsible for rRNA synthesis, while the NOR-2 probably stays transcriptionally silent and does not participate in the formation of the nucleolus in Q. robur, which is a situation resembling the well-known phenomenon of nucleolar dominance. rDNA chromatin topology analyses in cycling root tip cells by light and electron microscopy revealed the minor locus to be highly condensed and located away from the nucleolus, while the major locus was consistently associated with the nucleolus and often exhibited different levels of condensation. In addition, silver precipitation was confined exclusively to the NOR-1 locus. Also, NOR-2 was highly methylated at cytosines and rDNA chromatin was marked with histone modifications characteristic for repressive state. After treatment of the root cells with the methylation inhibitor 5-aza-2′-deoxycytidine, we observed an increase in the total level of rRNA transcripts and a decrease in DNA methylation level at the NOR-2 locus. Also, NOR-2 sites relocalized with respect to the nuclear periphery/nucleolus, however, the relocation did not affect the contribution of this locus to nucleolar formation, nor did it affect rDNA chromatin decondensation, strongly suggesting that NOR-2 has lost the function of rRNA synthesis and nucleolar organization.Around 2200 copies of genes encoding ribosomal RNA (rRNA) in pedunculate oak,  In plants, NOR (nucleolar organizer region) locus is composed of multiple repeats of ribosomal (rRNA) genes that encode the 45S pre-ribosomal transcript, which is processed to generate mature 18S, 5.8S and 26–28S rRNA molecules. The redundancy of the information allows for a fraction of rRNA genes within a locus to be transcriptionally active while others stay silent, and the proportion of silent and active genes is determined by cellular needs for protein synthesis Arabidopsis, the “off” switch in rRNA gene regulation involves cytosine methylation, histone deacetylation, H3K9 dimethylation (H3K9me2) and chromatin condensation, while the “on” switch involves loss of cytosine methylation, histone H3 and H4 hyperacetylation, H3K4 trimethylation (H3K4me3) and chromatin decondensation Methyltransferase 1) and siRNA-guided DRM2 (Domains Rearranged Methyltransferase 2) directed DNA methylation followed by histone deacetylation are involved in rRNA gene silencing in hybrid A. suecicaMethyl Binding Domain MBD1 and MBD6 proteins and further recruitment of Histone Deacethylases (HDACs) needed to silence rRNA genes.Epigenetic mechanisms regulating the transcriptional state of rRNA genes associated with rDNA chromatin organization have been well studied in plant hybrid species exhibiting a phenomenon known as nucleolar dominance. In these hybrids, rDNA loci of only one parent are transcriptionally active while rDNA loci of the other parent stay silent in situ hybridization) and S1 nuclease protection data, it has been shown that partial rDNA chromatin decondensation is a cytogenetic manifestation of active rRNA gene transcription of a given locus Active rRNA genes associate with silver-reducing argyrophylic proteins and can be visualized at the cytogenetic level by a simple staining technique The position of competent, i.e. transcriptionally active, rDNA locus/loci determines the position of the nucleolus/nucleoli. This was initially observed by Heitz and McClintock in the early cytogenetic era Quercus robur, there are around 2200 copies of rRNA genes organized as two rDNA loci – the major (NOR-1) and the minor locus (NOR-2) – and maximally two nucleoli, with a high frequency of fusing into a single large nucleolus, are present in the nuclei of root tip cycling cells versus decondensed rDNA chromatin state). Therefore, it appears that only a subset of rRNA copies within the NOR-1 locus is transcriptionally active and this subset is not random since the rDNA chromatin topology is mitotically inherited. Although the inhibition of DNA methylation by 5-aza-2′-dC relocated NOR-2 locus closer to the nucleolus/nucleoli, we found no cytological evidence of its transcriptional activity, suggesting that rRNA genes residing within this locus probably exist as pseudogenes.In pedunculate oak, Q. robur, L. (Fagaceae) used in this study were collected on location Jasterbarsko with the permission of Croatian Forest Research Institute by their employees and given to the authors to grow under laboratory conditions. The authors therefore required no special field permit for obtaining the samples. The species used in this study are not endangered or protected. Both oak acorns and seeds of triticale were germinated on moist cotton in Petri dishes at room temperature. For silver staining, fluorescence in situ hybridization (FISH) and immunodetection of 5-methylcytosine (5-mC), root tips were fixed in freshly prepared ethanol-acetic acid (3∶1) overnight at 4°C, transferred to 70% ethanol and stored at −20°C until use. Prior to fixation, oak root tips were treated with 2 µM 8-hydroxiquinoline for 4 h at 18°C. Root tips of triticale were pre-treated with ice-cold water for 24 h and fixed in ethanol-acetic acid (3∶1) overnight at 4°C, transferred to 70% ethanol and stored at −20°C until use. Root tips for immunofluorescence (IF) experiments were fixed in 2% (w/v) paraformaldehyde, 1% (w/v) PVP, 0.5% (v/v) Triton X-100 in 1×PBS for 20 min at room temperature and then washed extensively in 1×PBS .The acorns of 2O and squashed. After freezing, the coverslips were removed and slides transferred to 1×PBS.Oak nuclei suspensions for silver staining, FISH and immunodetection of 5-mC were prepared by incubating root tips in the enzymatic mixture  for 1.5–3 h at 37°C. Triticale root tips were digested in the same enzymatic mixture for 30 min at 37°C. Root tips were then macerated in a droplet of 60% acetic acid, the obtained macerate was transferred into a 1.5 mL Eppendorf tube and nuclei were collected by centrifugation at 800×g for 3 min. The supernatant was discarded and the pellet was washed twice with freshly prepared fixative. Finally, nuclei were resuspended in an appropriate volume of the fixative and 2–3 µl of the suspension were dropped on clean slides and air-dried. For immunodetection of histones, meristems were digested with the enzymatic mixture  for 90 min at 37°C, washed in 1×PBS for at least 15 min, transferred into a droplet of dH2HPO4, 0.05 M KH2PO4) for 1 h at room temperature. Material was then dehydrated in a graded ethanol series, embedded in LR White (medium) resin (London Resin Company LTD) and polymerized at 52°C overnight. Ultra-thin sections (0.1 µm) were cut with a glass knife on an ultra-microtome (Reichert OmU3). Sections were picked up on gold 100 mesh grids with a carbon-coated support Formvar film. Grids with sections were incubated overnight at 37°C prior to in situ hybridization (ISH).Root tips were fixed in 1% (v/v) glutaraldehyde and 0.25% (v/v) saturated aqueous picric acid solution in phosphate buffer  was dissolved in 10 mM PBS and stored at 4°C and −20°C until use. Acorns were germinated on moist cotton in Petri dishes in a humid place at room temperature. After 7 days of germination, root tips of the seedlings were immersed in a Petri dish containing 50 mL of 10 µM 5-aza-2′-dC and left to grow for 10 days at room temperature. Water solution containing 5-aza-2′-dC was replaced every 2 days keeping the concentration effective. Seedlings for the control experiment were grown in sterile water in the same environmental conditions. Meristematic tissue was isolated from root tips and then fixed as previously described Triticum aestivum) specific probe was added to the hybridization mix. The plasmid containing the 2.4 kb fragment of 18S rRNA gene from Cucurbita pepoBamHI fragment of 5S rRNA gene and spacer region of wheat 5′-GATCCACTGAGATTCAGCCC-3′) and a primer specific for wheat IGS -TIGS (5′-TGAATCCACTTGCCTCAAATAGT-3′) - were used to amplify a 508 bp fragment of wheat intergenic spacer (IGS) and label it with digoxigenin. The PCR mixture consisted of 1×PCR buffer, 2 mM MgCl2, 0.2 mM dNTP, 1 mM digoxigenin-dUTP, 0.2 mM of each primer and 1U of Taq Polymerase (Promega). The cycling conditions were as follows: 95°C for 5 min and 35 cycles of 30 s at 94°C, 30 s at 55°C, 1.5 min at 72°C with a final elongation step of 5 min at 72°C. The oak and triticale chromosomes together with the probes were denatured simultaneously for 5 min at 80°C or 2.5 min at 70°C, respectively, and were then left to hybridize overnight in a humid chamber at 37°C. After post-hybridization stringent washes, the slides were counterstained with 0.5 µg/mL DAPI for 8 min and mounted in antifade (DAKO Fluorescent Mounting Medium). In case of digoxigenin labelled rDNA probe, the slides were washed in 2×SSC, blocked for 30 min in 5% BSA (w/v) in 4×SSC/0.01% Tween (v/v) and incubated in 1∶125 dilution of FITC-conjugated anti-digoxigenin antibody (Roche), washed in 4×SSC/0.01% Tween (v/v), counterstained and mounted as previously described.Slides were treated with RNase A solution (50 µg/mL in 2×SSC) for 1 h at 37°C, washed in 2×SSC and treated with Proteinase K (0.1 mg/mL) for 15 min. After washing in 2×SSC, the slides were dehydrated through ethanol series and air-dried. The hybridization mixture consisted of 50% (v/v) formamide, 10% (w/v) dextran sulphate, 10% SDS, 1.5–2 ng/µL Cy3 labelled 18S rDNA probe in 2×SSC. In case of triticale samples, an additional 1.5–2 ng/µL of wheat .For immunodetection of 5-mC, the slides were denatured in 70% formamide in 2×SSC at 70°C for 2 min and dehydrated through an ice-cold ethanol series. The IF of 5-mC was done as described previously Cucurbita pepo. pRZ18 is a clone of the 2.4 kb HindIII fragment containing the whole 18S rRNA gene together with the ITS1 and 400 bp of the intergenic spacer. pRZ25 is a clone of the 2.9 kb HindIII fragment containing the 5.8S rRNA gene and ITS2, while the 25S rRNA gene lacks 200 bp of the 3′ end Two different probes were used for ISH, both containing ribosomal genes of 2 and then re-fixed in freshly depolymerized 4% (w/v) paraformaldehyde for 5 min, followed by rinsing in 2×SSC. For hybridization, grids with ultra-thin sections were put into a drop of the denatured probe (10 min incubation at 70°C), covered with a coverslip and then put again at 72°C for 10 min to denature DNA on ultra-thin sections. Grids were then transferred into a humid chamber at 37°C to allow hybridization overnight. Post-hybridization stringent washes were performed as follows: 1×5 min in 2×SSC, 1×10 min in 50% formamide, and 2×5 min in 2×SSC at 40°C and then 2×5 min in 2×SSC and 1×5 min in PBS/Tween at room temperature. Grids were then treated with a BSA blocking solution for 5 min at room temperature and incubated in streptavidin/horseradish peroxidase  in PBS/Tween buffer (1∶500) for 1h at 37°C. After incubation, the grids were rinsed in PBS/Tween 3×5 min. The signal was visualized by diaminobenzidine (DAB) reduction; grids were incubated for 20 min at 4°C in 50 mM Tris-HCl, pH 7.4, 0.05% (w/v) DAB and 0.015% (v/v) fresh hydrogen peroxide. Reaction was stopped by rinsing with water several times and air-drying. Ultra-thin sections on grids were contrasted with a 4% (w/v) aqueous solution of uranyl-acetate and examined at 60 kV using a Zeiss EM 10A electron microscope.Prior to hybridization, grids were treated with 100 µg/mL of RNase for 1 h at 37°C and with 5 µg/mL proteinase K (Sigma) for 10 min at 37°C, washed in 2×SSC supplemented with 50 mM MgClGTTCTGCTGTGCAGGTTTCG; PIGS2cr- CTACCTGGTTGATCCTGCCA) and the Oligo(dT)12–18 Primer (Invitrogen) for Actin 3. The obtained cDNA was further amplified by PCR using the same primer pair for amplification of 18S rRNA and Act3F (GATTTGGCATCACACTTTCTACAATG) and Act3R (GTTCCACCACTGAGCACAATG) for Actin 3 amplification. The cycling conditions were as follows: 94°C for 2 min, 35 cycles of 30 s at 95°C, 1 min at 55°C and 2 min at 72°C for amplification of 18S cDNA; and 94°C for 3 min, 35 cycles of 45 s at 94°C, 45 s at 58°C and 1.5 min at 72°C for amplification of Actin 3. After electrophoresis of PCR products in 1% agarose gel and staining with ethidium bromide, gel image was captured using the Kodak EDAS 290 system with an Uvitec UV transilluminator. Bands were quantitated using the ImageJ software. Background was removed by the “rolling ball” method, which was followed by densitometry using the gel analysis module. All bands (18S rRNA and Actin3) were from the same gel, so the comparison was done by estimating uncalibrated integrated optical density expressed in arbitrary units. Direct analysis of the bands using the “region of interest” measurement was conducted to double-check and confirm the results of the gel analysis module.To determine the expression levels of 18S rRNA genes and Actin 3 following treatment with 5-aza-2′-dC, RNA from the oak root tips was extracted according to Manickavelu For microscopy analysis, FISH images were captured using an epi-fluorescence microscope Axiovert 200 M , while IF and IFF images were captured using a confocal microscope Leica Microsystems TCS SP2 AOBS , both coupled with CCD cameras. Optical sections were acquired by confocal microscope using integrated software. Images were processed and merged in ImageJ. Prior to co-localization analyses, images were deconvolved using 3D Parallel Iterative Deconvolution plug-in for ImageJ. Co-localization analyses were performed on 6-stack images by measuring the Pearson’s coefficient (PC) using an ImageJ-based plug-in JACoP Confocal Z-stacks from the co-localization study were used for size comparison between the major (NOR-1) and the minor (NOR-2) rDNA locus. In total, 81 loci were analyzed (37 NOR-1 and 44 NOR-2). Images were analyzed using the ImageJ software NOR size data were processed using the R statistical software Four FISH signals representing two 45S rDNA loci, referred to as the major (NOR-1) and the minor (NOR-2) locus, were identified in interphase nuclei of the common oak root tip cycling cells, consistent with our previous findings The mean integrated OD by area  of the largest cross-section of NOR-1 was 139.34, while it was 22.11 for NOR-2 and the difference in measured values was statistically significant (p<0.0001) . The obsThe mean integrated OD by volume  of NOR-1 was 150.91, while it was 18.59 for NOR-2 , the difWhile rDNA chromatin of the minor locus was always highly condensed . The disTwo homologous NOR-1 sites showed dimorphism regarding chromatin topology and the type of chromatin organization was not fully random as metaphase NOR-1 sites showed the same dimorphism in majority of the cells within an individual . Even thWe tried to determine relative abundance of each type of rDNA chromatin fraction - condensed (rDNA knob) and decondensed  - on confocal Z-stacks as described in the Materials and methods section. Of the six nuclei where the decondensation fraction could be measured, two nuclei showed one fully condensed NOR-1 site, while the second site was 75.3% and 52.7% decondensed. The sum of the decondensed and the condensed fraction closely matched the fully condensed NOR-1 site (within 25%). The other four measured nuclei had a single visible NOR-1 site, which was partially decondensed. The decondensed fraction was 61.9%, 45.3%, 62.7% and 57.7%. The measured decondensation corresponded to approximately half of an NOR-1 site. In cases when there was only a single NOR-1 site visible, it was unclear whether the decondensed material was a part of the visible NOR-1 site, or represented the other which could not be seen due to complete decondensation. Examples of Z-stacks showing partial decondensation are shown in the supplementary The sites of 18S rDNA probe hybridization were visualized on ultra-thin sections by electron-opaque deposits of reduced DAB. ISH signal corresponding to condensed rDNA chromatin (rDNA knob) was seen outside of the nucleolus  sites , arrows Secale cereale) and wheat (Triticum aestivum), with clearly differentiated NOR activities. The expression of 45S rDNA locus originating from rye is suppressed, while the two wheat-derived 45S rDNA loci are expressed To test whether the proximity of an rDNA locus to the nucleolus is indicative of its transcriptional activity, we performed an additional analysis in triticale, a hybrid between rye . Interestingly, we could observe a clear re-localization of the NOR-2 sites towards the nuclear center and the nucleoli, however it appeared that they never associated with the nucleolus/nucleoli  and closed  conformation of rDNA chromatin We performed multiple IFF experiments using antibodies against histone marks shown in In order to see the effect of a change in global DNA methylation level on the distribution of the analyzed histone marks within the rDNA chromatin, we repeated the IFF analyses after treatment of oak seedlings with 5-aza-2′-dC . Again, A global decrease in DNA methylation following 5-aza-2′-dC treatment resulted in rDNA loci redistribution and an increase in nucleoli volume/number, suggesting a potential increase in rRNA gene activity. To address this question, we performed a RT-PCR analysis. In 3 independent experiments we found more than a twofold increase in 18S rRNA transcription following the treatment, confirming that the observed epigenetic changes go hand in hand with the changes in transcriptional activity . HoweverSilver staining of oak chromosomes revealed a prominently stained nucleolus and two regions of silver precipitation with position and size corresponding to rDNA chromatin knobs of the NOR-1 sites. Therefore, silver staining enabled positive identification of NOR-1 sites positioned adjacent to the nucleolus  arrows. The aim of this study was to deduce the transcriptional status of the major and the minor rDNA locus (NOR-1 and NOR-2) in root tip cycling cells of pedunculate oak by: 1) analyzing the association between a NOR and the nucleolus; (2) observing the rDNA chromatin topology; (3) staining NORs with silver; (4) comparing the presence/absence of DNA methylation and histone modifications at rDNA loci and (5) comparing levels of rRNA transcripts before and after the treatment with DNA methylation inhibitor 5-aza-2′-dC. Our data suggest that only rRNA genes residing within the major rDNA locus (NOR-1) are transcriptionally active under normal physiological conditions and the minor locus (NOR-2) probably stays transcriptionally silent. This situation resembles a phenomenon known as nucleolar dominance, where epigenetic differences determine and maintain the dominant and repressed sets of rRNA genes in hybrid species Q. robur nuclei models, constructed on the basis of measured distances of NORs from the nucleolus/nuclear periphery, shows that thedistance from the nucleolus can be considered as an indication of NOR activity in pedunculate oak. In triticale, we have found that only NORs of wheat origin associate with the nucleolus, indicating a strong correlation between a NOR locus position and its activity. Therefore, positioning of the oak’s NOR-1 locus close to the nucleolus would suggest that rRNA genes within this locus are transcriptionally active, while NOR-2 sites that are closer to the nuclear periphery can be considered transcriptionally silent. Treatment with the methyltransferase inhibitor 5-aza-2′-dC causes global hypomethylation and reactivation of silent NORs in triticale, A. thaliana and Brassica napusComparison of the triticale and Arabidopsis, a mutant for AtHDA6 (histone deacetylase gene), that hyperacetylation is not concomitant with increased rRNA transcription rDNA chromatin within NOR-2 locus was highly methylated under normal physiological conditions of root tip cycling cells, and chromatin was marked with H3K9me and moderately with H3K27me2, both histone modifications characteristic for repressive rDNA chromatin. Treatment of the cells with 5-aza-2′-dC resulted in a decrease in cytosine methylation and H3K9me1 within NOR-2, both signs of a change from the repressive to the permissive chromatin state. Also, the treatment caused repositioning of NOR-2 locus much closer to the nucleolus. Nevertheless, we found no cytological evidence of NOR-2 transcriptional activity, i.e. no silver precipitation and/or rDNA decondensation. In fact, the presence/absence of an epigenetic modification itself in rDNA chromatin is not sufficient for determination of its transcriptional status, as it has been shown for Q. robur after the treatment with 5-aza-2′-dC is rather due to up-regulation of the active fraction of genes in the NOR-1 locus or possibly reactivation of the silent portion of that locus, than reactivation of the NOR-2 locus. Muir and co-workers suggested that only a single rDNA family of the three divergent rDNA families identified in Q. robur is functional Q. robur genome, but rather that these sequences have lost their function. The results from this study also suggest that NOR-2 associated rRNA genes have lost their function. In fact, Muir et al. Q. robur through an ancient hybridization process, which prevented concerted evolution to homogenize these sequences Q. robur). Since nucleolar dominance could be maintained over generations Q. robur) could have evolved as a pseudogene.Our results suggest that the increase in total transcription levels of rRNA genes in A. arenosa NOR in the hybrid A. suecicaTwo different structural domains – a highly condensed (rDNA knob) and a dispersed heterogeneous rDNA chromatin fraction of peri- and intranucleolar position - co-exist within the major rDNA locus (NOR-1). The same has been shown for the active NOR in diploid rye A. suecica, which shows nuclear dominance, the dominant A. arenosa rRNA genes associate with both active and silent histone marks. The same situation is found in non-hybrid A. thaliana in which a fraction of rRNA genes within the same locus associates with H3K4me3 and another fraction associates with H3K9me2 According to the presence of epigenetic marks, silver staining and deposits of reduced DAB, it can be concluded that active rRNA copies reside within the dispersed rDNA chromatin fraction of the NOR-1 locus in root tip cycling cells of the common oak. Interestingly, even though the condensed rDNA knob of the NOR-1 locuswas enriched with the repressive histone mark H3K9me1 and poor in the active histone mark H3K4me3, it showed a low level of 5-mC and a mild co-localization with the H3K9ac mark, both hallmarks of permissive chromatin. In addition, the rDNA knob showed mild silver precipitation. One plausible explanation for this would be that rRNA genes within the rDNA knob of NOR-1 locus stay hypomethylated to be accessible for rapid reactivation, which can be achieved by a change in histone modifications, mostly demethylation of Lys 9 of histone H3 and acetylation at the same position. Cytosine methylation is a more stable epigenetic modification than histone marks and gradually and slowly changes on a larger scale, i.e. along a longer chromosome region occupied by rRNA genes The dispersed rDNA chromatin fraction of NOR-1 locus comprised smaller peri- and intra-nucleolar rDNA chromatin knobs (FISH dots), as visible in LM, interconnected with thin threads of chromatin suggesting that both transcriptionally silent and active rRNA genes are possibly present in this fraction. ISH using 18S probes on ultra-thin sections of oak root tips revealed condensed sites on the nucleoplasm-nucleolar boundary that sometimes protruded into the nucleolar mass while decondensed rDNA chromatin was clearly spread in the volume of the nucleolus. It is possible that the condensed rDNA chromatin on the nucleoplasm-nucleolar boundary represents an intermediate heterochromatin state, which under appropriate conditions might become transcriptionally active Q. robur root tip led to a conclusion that the active fraction of rRNA genes within the NOR-1 is not fully random, as the (pro)metaphase NOR sites always show the same condensation pattern at the same position of the NOR-1 sites. In addition, stable dimorphism was observed for this locus: while dispersed rDNA chromatin fraction was located at the terminal part of one site, at another site it connected the proximal rDNA knob with the condensed rDNA chromatin on the satellite, i.e. dispersed rDNA chromatin corresponded to SC. The mechanisms underlying this stable dimorphism of NOR-1 sites, reflecting the choice of rRNA copies to be transcribed in oak root tip cycling cells, remains unknown. We hypothesize that epigenetic mechanisms responsible for cell memory are involved.Study of chromatin topology of the NOR-1 locus in different cells of Figure S1Reduction of the global level of 5-mC in root tip cycling cells of Q.robur following treatment with 5-aza-2′-dC. Immunolocalization of 5-mC before (A) and following the epigenetic treatment (B). C. Integrated fluorescence intensity analysis confirmed reduction of global 5-mC levels following treatment. Number of asterisks indicates p<0.0001; error bars represent the standard error of the mean. Scale bar is 5 µm.(TIF)Click here for additional data file.Figure S2Examples of full Z-stacks for measurement of NOR-1 and NOR-2 size. Size was measured only when features were fully included in the confocal image stack. Two nuclei are given (upper panel. lower panel). Green channel represents chromatin while the red channel corresponds to 18S rDNA signal. Upper and lower panel are at different scales.(TIF)Click here for additional data file.Figure S3Examples of full Z-stacks for measurement of decondensation percentage. Two nuclei are shown: one with both NOR-1 sites visible (upper panel), and another with only one NOR-1 site visible (lower panel). Green channel represents chromatin while the red channel corresponds to 18S rDNA signal. Upper and lower panel are at slightly different scales.(TIF)Click here for additional data file.Table S1Measured maximum cross-section (Area) and integrated volume (Volume) for NOR-1 and NOR-2 loci. All values are in arbitrary OD units normalized by pixel area or voxel volume to make them directly comparable across images.(XLSX)Click here for additional data file.


The orbital reconstruction induced antiferromagnetic phase enhances the exchange bias in the LSMO/YMO heterostructures, lightening an effective way for electric-field modulated magnetic moments in multiferroic magnetoelectric devices.The exchange bias in ferromagnetic/multiferroic heterostructures is usually considered to originate from interfacial coupling. In this work, an orbital reconstruction enhanced exchange bias was discovered. As La The reasons that we used YMO and LSMO in the heterostructures were based on the following considerations. Firstly, both of them are manganites, a strong exchange coupling can be expected due to the Mn3+–O2−–Mn4+ double exchange interaction at the interface. Secondly, both the orthorhombic YMO and LSMO films can be synthesized under similar conditions by applying proper substrate strain, so LSMO and YMO based heterostructures with different growth orders can then be achieved. Finally, the symmetric exchange striction mode3 systems, but also makes the magnetic field modulated polarization feasible, which will provide new perspectives to exploit the exchange coupling in multiferroic heterostructures17To investigate the physical picture of EB in FM/MF heterostructures, the multiferroic orthorhombic YMnO3 (STO) single crystal substrates were fabricated by radio-frequency magnetron sputtering. The YMO and LSMO layer thicknesses were ∼50 and ∼12 nm, respectively. Details for the epitaxial growth of YMO and LSMO layers were referred to our previous work20k-mesh was used for the Brillouin-zone integrations.By strain engineering, the YMO/LSMO and LSMO/YMO heterostructures with different lattice orientations on SrTiO3+–O2−–Mn4+ bond angles at the interfaceB/Mn in the LSMO/YMO/STO heterostructures is much smaller than ~3.2 μB/Mn in the YMO/LSMO/STO heterostructures; (2) the coercivity and EB field in 233+–O2−–Mn4+ bond angle which leads to the different strength of interfacial coupling in the heterostructuresHEB (ΔHEB = HEB(LSMO/YMO)−HEB(YMO/LSMO)) is given in 25M-T curve of the LSMO(001) single layer. The Curie temperature of ~280 K and magnetization of ~2.2 μB/Mn measured under 200 Oe at 5 K are close to the YMO/LSMO(001)/STO heterostructure with the values of ~275 K and ~2.3 μB/Mn. From the ZFC and FC curves, it is clear to see that not only the Curie temperature (~200 K) of the LSMO layers in the LSMO/YMO heterostructures are much lower than that (~300 K) of the LSMO layers in the YMO/LSMO/STO heterostructures, but also the magnetization of the LSMO/YMO/STO heterostructures is greatly suppressed. The reduction of magnetization probably originates from several factors, such as oxygen vacancies6 octahedra that strongly suppresses the FM Curie temperatureOur previous study on the magnetic properties of the YMO/LSMO/STO heterostructures with different lattice orientations showed that EB strongly depends on the lattice orientations with different Mnan that ~0 K of thHEB = −JEB/μoMFtFJEB is the interfacial exchange coupling energy, tF and MF the thickness and saturation magnetization of the ferromagnetic layer. As shown in The LSMO and YMO thickness dependent EB in the LSMO(001)/YMO/STO heterostructures are shown in The YMO thickness dependence of EB is shown in To characterize the effect of strain on magnetic properties, the high resolution transmission electron microscopy (HRTEM) was employed to investigate the microstructure of the LSMO/YMO/STO and YMO/LSMO/STO heterostructures. a and b are 3.98(2) Å and 3.75(2) Å. This strongly indicates a rectangular growth of LSMO on the YMO layer. Compared to the bulk value of 3.87 Å, the LSMO layer elongates in a,b plane and shrinks along c-axis.Together with the analyses of reciprocal space mappings, HRTEM images and SAED patterns, the lattice parameters of YMO and LSMO are listed in 3+ 3d4 orbital configuration. In spherical symmetry, the 3d orbitals are five-folds degenerate. For the unstrained perovskite manganites, as shown in the top panel of 3+ ion is surrounded by six O2− with the octahedral symmetry. The wave function of eg orbital stretches along the <100> axes on which the nearest neighbor O2− is located, so that the eg orbital is increased with doubly degenerate because of the strong Coulomb interaction between the negatively charged electron and the O2−. When the MnO6 octahedral suffers a tensile (a > c) strain, the Coulomb interaction is suppressed and the x2 − y2 orbital shifts to the low energy orbital. Thus the eg electron tends to occupy it and becomes localized. Due to the orbital reconstruction of the eg electron, the double exchange interaction is suppressed and the layer-typed antiferromagnetic order is formed163738How does the tensile strain affect the EB effect? In manganites, the key parameter governing the physical properties is the Mnz2 − r2 and x2 − y2 orbitals exhibits a similar occupancy, indicating a doubly degenerate orbital. However, for the LSMO grown on the YMO layer, the tensile strain shifts the x2 − y2 orbital to the low energy level, leading to a localized and splited eg orbital.The results were further confirmed by the DFT calculation. The calculated densities of states (DOS) of Mn ions are shown in the bottom panel of Therefore, the AFM phase was introduced into the LSMO layers by tensile strain induced orbital reconstruction when they were grown on YMO. The coupling of orbital reconstruction induced AFM order with the intrinsic FM order leads to an enhanced magnetic anisotropy, suppressed saturation magnetization, reduced FM Curie temperature and enhanced EB. Similarly, for the LSMO(011)/YMO/STO and LSMO(111)/YMO/STO heterostructures, the strain will also bring AFM order to this system and contribute to the EB.x2 − y2 orbital of LSMO shifts to the low energy level, and the eg orbital was splited. The eg electron occupies the low energy x2 − y2 orbital and becomes localized, thus an AFM order is formed. The coupling of FM order with the formed AFM order enhances the magnetic anisotropy and EB effect.An enhanced EB in the LSMO/YMO/STO as compared to the YMO/LSMO/STO heterostructures was discovered, which can be ascribed to the strain induced orbital reconstruction. Consistent with the first principle calculation results, when the LSMO layer suffers a tensile strain from the YMO layer, the How to cite this article: Zheng, D. et al. Orbital Reconstruction Enhanced Exchange Bias in La0.6Sr0.4MnO3/Orthorhombic YMnO3 Heterostructures. Sci. Rep.6, 24568; doi: 10.1038/srep24568 (2016).


Living organisms can be conveniently classified into three domains, Archaea, Bacteria, and Eukarya  that can replicate in both its insect vector (Delphacidae) and flowering plants  and plants (viridiplantae), when a virus adapts to an established consortium of ecological partners. The same virus can sometimes infect both plant and animal cells when these are linked by their mode of life. One example is the Fiji disease virus (Aeropyrum pernix), each from a different family . Moreover, only 9 virion morphologies have been observed in bacterioviruses (compared to 16 in archaeoviruses)  is the largest known ssDNA virus and displays unique coil-shaped virion morphology  in their major capsid proteins and homologous packaging ATPases  encode segmented genomes and infect mostly Pseudomonas species  in terms of life cycle and homologous RNA-dependent-RNA-polymerase gene sequences  . These viruses are amongst the simplest and smallest known viruses, and historically yielded useful insights into mRNA function  bacterioviruses could be identified. In turn, none of the ssRNA(−) and retrotranscribing viruses associated with bacterial hosts. Among the RNA bacterioviruses, dsRNA viruses (Viruses with all possible types of replicons infect eukaryal organisms. RNA viruses are predominant and cover the entire taxonomic range within Eukarya Figure . EukaryoThe diversity of eukaryoviruses is intriguing, both in terms of genome structure and virion morphology (see Figure To conclude, the distribution of viral lineages follows an ancient, highly dynamic and ongoing process that impacts the evolution of organisms. New viral lineages often arise from existing ones and may cross species barriers to infect new hosts (e.g., parvovirus; Shackelton et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The frequency and global impact of infectious disease outbreaks, particularly those caused by emerging viruses, demonstrate the need for a better understanding of how spatial ecology and pathogen evolution jointly shape epidemic dynamics. Advances in computational techniques and the increasing availability of genetic and geospatial data are helping to address this problem, particularly when both information sources are combined. Here, we review research at the intersection of evolutionary biology, human geography and epidemiology that is working towards an integrated view of spatial incidence, host mobility and viral genetic diversity. We first discuss how empirical studies have combined viral spatial and genetic data, focusing particularly on the contribution of evolutionary analyses to epidemiology and disease control. Second, we explore the interplay between virus evolution and global dispersal in more depth for two pathogens: human influenza A virus and chikungunya virus. We discuss the opportunities for future research arising from new analyses of human transportation and trade networks, as well as the associated challenges in accessing and sharing relevant spatial and genetic data. In a highly mobile world, with over half a million travellers in the air at any one moment, viruses have more opportunities than ever before to disseminate globally. Growth in the reach, volume and speed of human mobility over the past century has connected pathogens with new and growing host populations, and contributed to a boom in emerging and re-emerging epidemics ,2.The increasing connectivity of our world affects transmission in many ways. Greater mobility, through business travel, tourism and labour movement, leads to more pathogen introductions, while social and ecological changes in recipient locations may raise the likelihood that introductions will become entrenched rather than die out. The establishment of new travel routes between previously unconnected locations also contributes. For example, direct air travel between South America, Africa and southeast Asia now links tropical continental regions, where infectious disease burdens are higher and year-round transmission is more common. Further, the increasing volume of global trade through shipping and air freight can spread contaminated goods or introduce disease vectors such as mosquitoes to new locations through accidental carriage (e.g. ).Despite the importance of geography for infectious disease epidemiology, the effects of global mobility upon the genetic diversity and molecular evolution of pathogens are under-appreciated and only beginning to be understood; indeed, a recent monograph on the spatial epidemiology of infectious disease makes no reference to pathogen genetic variation . PatternIn this article, we explore each of these themes. We first review how spatial and genetic data are combined in empirical studies of viral transmission. Later we discuss in depth two human pathogens, influenza A virus (IAV) and chikungunya virus (CHIKV), whose global dynamics depend critically on the reciprocal interplay between virus evolution, spatial ecology and host mobility.2.Since the contemporary spatial distribution of a fast-evolving virus is the result of interacting ecological and evolutionary processes, consideration of spatial incidence or genetic data in isolation may provide only partial insight into the underlying transmission dynamics ,9. ConseSeveral trends in technology and data availability over the last decade have spurred innovation in this area. The advent of cheap, mobile global positioning systems and their widespread adoption in disease surveys has revolutionized the geospatial recording and analysis of infectious disease incidence and prevalence, especially when combined with geographical information systems (GIS) and pervasive electronic communication . FurtherThe term ‘phylogeography’ is commonly applied to studies that use evolutionary trees to combine genetic data with spatial information . Other s−3–10−4 substitutions per site per year and a genome 10 000–20 000 nucleotides long [Helicobacter pylori genes reveal the global spread of the bacterium over more than 50 000 years [Methods that attempt to combine viral genetic and geographical information will be worthwhile only if the spatial epidemiology of the pathogen population is recorded in its genome sequences. The degree to which that occurs for the pathogen in question will depend on its relative rates of spatial movement and molecular evolution. A pair of typical RNA virus genomes will diverge genetically from each other on average at a rate of 1–20 nucleotide changes per year (assuming 10des long ). Hence,des long ,29). It des long ). In gen00 years .Several of the most popular phylogeographic methods for reconstructing epidemic spatial spread from genetic data e.g. ,32–34) t–34 t32–3However, it is not always fully recognized that the estimated locations of ancestors can be highly uncertain, particularly those that are only distantly related to the sampled cases. Consequently, viral phylogeography is far more informative when applied to datasets that contain genetic sequences sampled sequentially through time, and which include genomes situated close to the root of the sample phylogeny. A second under-appreciated aspect of phylogeographic analysis is the importance of sample composition . Althoug3.The simplest way to combine viral spatial and genetic data is through the mapping of infections attributable to different viral strains. This creates a link to genetic variation because RNA viruses are classified into genotypes and subtypes using analysis of their gene sequences. In recent years, the global geographical distribution of strains of HIV-1 , dengue Evolutionary analysis of viral genes can be used to validate the putative source of an emerging viral outbreak that has been identified through epidemiological surveillance and contact tracing. For example, the proposed index case of the 2007 outbreak of CHIKV in northeast Italy had hosted a relative from Kerala, India (where the virus was epidemic), and phylogenetic analysis of virus E1 gene sequences from the Italian outbreak showed it to be very closely related to strains previously isolated in India . IndepenIn addition to its confirmatory role, analysis of virus genomes can answer questions of relevance to infectious disease control that cannot be addressed using incidence reports alone. For example, viral phylogenies can indicate if an outbreak in a specific region is the result of a single introduction followed by onward transmission within the host population of that region, or is composed of multiple independent chains of transmission, each initiated by a separate introduction from elsewhere or from a zoonotic reservoir species. For example, analysis of viral genomes from the Ebola epidemic in west Africa that began in Guinea in early 2014 indicated that it developed from a single introduction from the virus's reservoir in central Africa, and that the epidemic in Sierra Leone arose from the transmission of two distinct viral lineages from Guinea . By contIf transmission is predominantly local and movement unimpeded by geographical barriers then the genetic and geographical distances among sampled infections are expected to be positively correlated. This principle, known as isolation by distance , forms aHighly heterogeneous dispersal may be a common feature of all ecological invasions . This vaA key goal of viral phylogeography is to help predict future pathogen spread by indicating those social or environmental factors that are associated with virus movement. This is often achieved by qualitative comparison of virus genetic diversity or dispersal history with geographical data. For example, the early spread of HIV-1 in east Africa was explored by combining phylogenetic analyses with regional data on road network architecture and population density, obtained using GIS techniques . More re4.In addition to generating information essential for vaccine design, the global surveillance of influenza viruses has resulted in an unparalleled collection of virus genome sequences sampled through space and time, providing an opportunity to explore the processes that underpin the global dynamics of this important pathogen . AlthougThe emergence of pandemic H1N1 (pH1N1) influenza in 2009 was the first influenza pandemic in the post-genomic era. Genetic analysis of the pandemic in its early stages was aided by pre-planned and intensive virus sequencing in some countries, and by the immediate and open sharing of the resulting data through online databases. Consequently, the molecular epidemiology of the virus could be tracked in ‘real time’ as the epidemic unfolded ,58. ThisLocal persistence of transmission chains also raises questions about the mobility processes that drive IAV spread at sub-national scales. Mathematical analyses of mortality and physician visit statistics have suggested different drivers for the spread of seasonal ,68 and pThe spatial dynamics of influenza are also critical in assessing the evolution of anti-viral drug resistance. The global cycling of IAV lineages and low levels of local persistence mean that resistance mutations can spread worldwide, and can quickly erode any association at the local level between rates of drug usage and viral resistance. Recent examples of anti-viral drug resistance evolution include the rapid spread in oseltamivir resistance in seasonal H1N1 influenza from 2007 to 2009 and the global rise of adamantane-resistant H3N2 influenza during 2003–2006. An investigation of the former that used a stochastic model of international air travel concluded that the oseltamivir-resistant strain rose to global dominance because it exhibited a transmission advantage in untreated hosts, probably conferred by genetic hitchhiking . Phyloge5.CHIKV is a mosquito-borne alphavirus that, while rarely fatal, causes a debilitating fever and sometimes persistent arthralgia, so is of some public health concern. In the 50 years that followed the virus's discovery in 1952 in Tanzania, sporadic outbreaks were reported in central, west and east Africa, and in south and southeast Asia . HoweverThe worldwide expansion of CHIKV has left a clear footprint in the genomic diversity of the virus, despite the fact that its rate of molecular evolution is somewhat slower than that of viruses like influenza and HIV . PhylogeAedes aegypti and Ae. albopictus. The collapse of Ae. aegypti elimination efforts in the Americas [Ae. albopictus to expand its range from southeast Asia to large parts of the rest of the world [Aedes mosquitoes are prevalent, including at times of the year when the vectors are highly active in both places [Multiple genetic and ecological factors are thought to have contributed to the global emergence of CHIKV. The two mosquito species principally responsible for human CHIKV transmission are Americas  and growhe world . Furtherh places ,77.Ae. albopictus mosquitoes [Ae. albopictus was the sole or dominant vector species [Ae. albopictus mosquitoes.In addition to these ecological factors, there is strong evidence that, as the geographical range of CHIKV expanded, the virus evolved and adapted to local variation in the distribution of vector species. Specifically, a single amino acid change (A226V) in the viral E1 protein has been shown to increase transmission and infectivity in squitoes . This mu species , and thu6.Our understanding and evaluation of the risks of infectious disease spread are being refined by access to growing geographically referenced databases of disease prevalence, detailed satellite-based imagery and unprecedented information about patterns of human mobility. Successful integration of these sources of information with viral genetic data will be technically and intellectually challenging, yet holds great promise for our response to emerging viruses.Recent modelling work indicates that pathogen diffusion becomes highly regular when measured against a so-called ‘effective distance’ along the relevant mobility or transport network . Concepthttp://gisaid.org) and other initiatives in facilitating the timely sharing of influenza virus genomes during the 2009 H1N1 pandemic has unfortunately not been repeated in subsequent outbreaks. We strongly support the recent call for an international and inter-disciplinary consensus towards the open sharing and release of pathogen genetic information during epidemics [A significant obstacle to further progress is the availability and expense of some of the most powerful and relevant datasets. For air travel, origin–destination data derived from air ticket sales are available, but are highly expensive for research purposes, and their use may require legal and confidentiality agreements, resulting in a reliance on modelled datasets . Moreovepidemics .New outbreaks of infectious disease, especially those caused by viruses, are a common phenomenon in the twenty-first century, and future trends in global mobility and trade seem likely to maintain or even accelerate their rate of appearance. Techniques and data to describe, explain and predict such occurrences can help to measure and mitigate the risks from novel and re-emerging pathogens. Statistical and mathematical models that integrate spatially explicit data on pathogen evolution with information on human movement and environmental variability have much to contribute to epidemic management, as well as deepening our understanding of fundamental evolutionary and ecological processes.et al. [Since this review was written, the Asian genotype of CHIKV has spread from the Caribbean to Mexico, Brazil and Columbia, and local transmission has been observed in mainland France and Florida, USA. A second CHIKV genotype (ESCA) appears to have been introduced to Brazil from central Africa . In addiet al.  reported