Although it has been >30 years since the eradication of smallpox, the unearthing of well-preserved tissue material in which the virus may reside has called into question the viability of variola virus decades or centuries after its original occurrence. Experimental data to address the long-term stability and viability of the virus are limited. There are several instances of well-preserved corpses and tissues that have been examined for poxvirus viability and viral DNA. These historical specimens cause concern for potential exposures, and each situation should be approached cautiously and independently with the available information. Nevertheless, these specimens provide information on the history of a major disease and vaccination against it. Illness is characterized by 3 phases: incubation, prodrome, and rash. The incubation phase is ≈10–14 days. During the prodromal period, which lasts 2–4 days, persons with smallpox typically have fever, malaise, vomiting, headache, backache, and myalgia. The rash phase can be moderate or severe and is characterized by a centrifugal distribution of lesions in the same stage of development  announced worldwide eradication of smallpox in 1980. Successful eradication was accomplished by vaccinating populations and contacts of ill persons with live vaccinia virus, a closely related orthopoxvirus that confers immunity to variola virus. Once smallpox was eradicated, WHO recommended that routine vaccination be discontinued and that the vaccine be used only for select groups at risk for exposure to orthopoxviruses. Thus, persons born after 1980 are likely to not have residual immunity (Present-day stocks of variola virus are maintained at 2 WHO reference laboratories: the Centers for Disease Control and Prevention (CDC)  and the State Research Center of Virology and Biotechnology (VECTOR) . There is concern that if variola virus is present outside these 2 laboratories, its accidental or intentional release could cause illness in a population increasingly composed of unvaccinated persons. Anecdotal reports and formal scientific evidence have not ruled out the possibility that the virus may survive prolonged periods in preserved skin and tissue material, such as those that might be on display in museums, or in unearthed human remains. For example, permafrost is an environment that closely mirrors laboratory freezer storage of live virus, and the maintenance of viable smallpox virus in human remains found in such an environment has been debated . Studies or reports on historical specimens were included if there was suspicion of variola virus.Existing specimens at the CDC Poxvirus Laboratory  were reexamined by using modern molecular techniques. In addition, we examined newer relic specimens  for molecular signatures of poxviruses. Non-variola orthopoxvirus DNA signatures were amplified by using real-time PCR  was stored for ≤8 years before successful use (In the past 10 years, suspected variola crusts have been discovered in the United States on 3 occasions. In Virginia, a crust labeled as a smallpox scab was on display at a museum and was accompanied by a letter describing its origin , panel AA second incident of suspected smallpox scabs on display at a local museum occurred in Arkansas , panel BIn 2003, a librarian in New Mexico opened a book and an envelope containing lesion crusts fell out of the book , panel CHistorical specimens come to the attention of public health authorities when there is a perception that they may constitute a potential risk to those who are handling or may have handled the artifacts. This concern extends to specific groups of persons who might work routinely with historical specimens, including archeologists and museum archivists, as well as those who may stumble upon these specimens on an irregular basis, such as construction workers or the general public. Although live variola virus has never been isolated from historical tissues, this finding does not eliminate the possibility of live variola virus resurfacing from well-preserved tissue material (,,,The discovery of a series of corpses and mummies with suspected smallpox lesions in the late 1970s and 1980s sparked a series of commentaries over the risks to archeologists and anthropologists and the potential need for vaccination of workers (If a suspected smallpox relic or body of a person who died of smallpox has been discovered, local and state public health departments are an excellent resource. Public health officials can work closely with those who have handled any suspect artifacts, determine risks, help mitigate concern, and arrange for appropriate testing. Testing can be performed on a suspected specimen to definitively determine if live virus is present. The WHO smallpox reference laboratories can perform these tests and have successfully participated in inquiries involving historical specimens .Aside from immediate public health concerns surrounding a suspected smallpox specimen, historical cases help highlight disease history in terms of the society and patient in question. Historical specimens might also help explain the history of smallpox epidemics and vaccine development. Recent exhumation of a corpse from permafrost in Siberia led to sequence characterization of an older strain of variola virus, which shed light on the evolutionary history of the virus (Today, the smallpox vaccine consists of an intradermal inoculation with vaccinia virus, and the premise and method of this vaccination has not changed since the time of Jenner (Modern molecular approaches can be used with historical specimens to confirm the presence of variola or another orthopoxvirus and elucidate the evolutionary history of the virus. Full genome, gene, or partial gene sequencing of isolates enables investigating the history of 1 virus compared with others. Long-term stability of smallpox virus DNA is not well characterized. However, constant low temperatures, such as those in crypts and permafrost, are believed to be key to the stability of DNA molecules. Theoretically, DNA can survive up to ≈1 million years in cold environments (Archival specimens offer opportunities to delve into the past and capture a glimpse of the history of an eradicated disease. There are no published reports of residual live microbes found in archeologic relics. Furthermore, on the basis of experiences in the past several decades, risks for transmission of live organisms from such relics would seem to be nonexistent; nevertheless, archeologic specimens should be handled with caution. Each situation should be approached independently and with vigilance and attention. Special attention to the scientific value of a specimen will yield useful data about smallpox and vaccination history that might provide useful information about the virus and affected populations.


Cryptococcus gattii have caused an outbreak in the US Pacific Northwest since 2004. Outbreak-associated infections occur equally in male and female patients (median age 56 years) and usually cause pulmonary disease in persons with underlying medical conditions. Since 2009, a total of 25 C. gattii infections, 23 (92%) caused by non–outbreak strain C. gattii, have been reported from 8 non–Pacific Northwest states. Sixteen (64%) patients were previously healthy, and 21 (84%) were male; median age was 43 years (range 15–83 years). Ten patients who provided information reported no past-year travel to areas where C. gattii is known to be endemic. Nineteen (76%) patients had central nervous system infections; 6 (24%) died. C. gattii infection in persons without exposure to known disease-endemic areas suggests possible endemicity in the United States outside the outbreak-affected region; these infections appear to differ in clinical and demographic characteristics from outbreak-associated C. gattii. Clinicians outside the outbreak-affected areas should be aware of locally acquired C. gattii infection and its varied signs and symptoms.Clonal VGII subtypes (outbreak strains) of  Cryptococcus gattii, a fungal pathogen found in the environment, is associated with soil and decaying organic debris. Infection in humans results from inhalation of spores from the environment and typically causes pneumonia or meningitis , 2 of which are uncommon outside this region and 1 (VGIIc) that is unique to the region  disease  to collect surveillance data on C. gattii infections from states outside that region. Whether these reported cases are an effect of the US Pacific Northwest outbreak on other areas of the United States and the implications for clinical care and broader surveillance have not been addressed. We summarize C. gattii cases reported to CDC from non–US Pacific Northwest states and discuss their implications.The outbreak in the US Pacific Northwest has increased interest in C. gattii infection in a resident of the United States outside of Oregon or Washington who had no known recent travel history (within 1 year) to these states and had illness onset after January 1, 2009. Cases were reported to CDC by state health departments or physicians who were treating patients with suspected C. gattii infection . Isolates were sent to CDC for confirmation; isolates were plated on differential canavanine-glycine-bromothymol blue medium (www.megasoftware.net). State and local health department staff and treating physicians completed standardized case report forms with demographic and clinical information on case-patients and submitted these reports to CDC. Sites of infection were determined as follows: for pulmonary, blood, cerebrospinal fluid (CSF), or tissue specimens, culture was considered evidence of infection at that body site. Histopathologic examination results demonstrating Cryptococcus yeasts in tissue also was considered evidence of infection at the tissue site. Cryptococcal antigen in the CSF was considered evidence of CNS infection; however, serum cryptococcal antigen in the absence of a bloodstream cryptococcal isolate was not considered evidence of a bloodstream infection.A case was defined as a culture-confirmed Twenty-five cases for which descriptive data were available were identified in Alabama (1 case), California (13), Florida (1), Georgia (5), Hawaii (1), Michigan (1), Montana (1), and New Mexico (2). Some cases have been published as individual case reports ; CD4 counts, available for 4 patients, ranged from 92 cells/mm3 to 838 cells/mm3 (median 595 cells/mm3). Twenty-three (92%) patients were hospitalized for a median of 23 days (range 1–88 days); 10 (48%) of 21 patients with data required admission to the intensive care unit. Results of lung imaging were abnormal for 19 (83%) of 23 patients with images; 14 (61%) had documented lung cryptococcomas. Results of head imaging were abnormal in 15 (75%) of 20 patients with images; 10 (50%) patients had documented brain cryptococcomas. Nineteen (76%) patients had culture, histopathologic, or serologic evidence of CNS infection, either alone or with pulmonary, blood, or tissue infections . The most common symptoms were headache (67%), blurred vision (62%), and nausea (46%) (3), diabetes and liver transplant, an unspecified immunocompromising condition, active lung cancer (patient receiving chemotherapy), and congenital hyper-IgE syndrome (Job syndrome). Four (16%) additional patients had nonimmunocompromising underlying disease at diagnosis. For the 12 patients with documented HIV test results, all results were negative.Sixteen (64%) patients were otherwise healthy at diagnosis (C. gattii infections (2O) showed evidence of yeast; she was given liposomal amphotericin B and 5-flucytosine and was transferred to a referral hospital, where she received a lumbar puncture to drain CSF and endotracheal intubation for mechanical ventilation in the intensive care unit. Despite intensive supportive care, she died 2 days later of brain stem herniation caused by persistent elevated intracranial pressure (ICP).Six patients  died, all from their In addition to this patient, 4 patients died before (or on the day of) their diagnoses, and 1 died 263 days after diagnosis (from shunt blockage). Three of the 4 who died before diagnosis had bloodstream infections. Three of the 4 female patients died; female sex  and having cryptococci isolated from blood  were significantly associated with increased risk for death. Death was not associated with other clinical or demographic variables.Of the 21 patients surviving to diagnosis, treatment data were available for 19 (90%). All 17 who survived to diagnosis with bloodstream or CNS infections were initially given amphotericin B or liposomal amphotericin B; 16 also were given 5-flucytosine or fluconazole, and 1 was given amphotericin B alone. Of the 4 with isolated pulmonary infections who survived to diagnosis, 1 was given fluconazole alone, and 1 received amphotericin B and flucytosine; for 2, no treatment data were available.Seven patients were given corticosteroids to treat their infection. One patient was given interferon. Twelve (57%) of 21 patients surviving to diagnosis had diagnostic or interventional surgeries, including 6 who needed a ventriculoperitoneal shunt or lumbar drain for refractory elevated ICP, 4 who needed brain or lung biopsies, 1 requiring placement of a chest tube for pleural effusion, and 1 requiring leg surgery at the infection site (C. gattii isolates from patients were typed as VGI (11 [44%]), VGIII (11 [44%]), VGII , and VGIIb (2 [8%]) ((2 [8%]) . Fifteen(2 [8%]) . One of (2 [8%]) . Of the (2 [8%]) . Ten pat(2 [8%]) ; 2 of thC. gattii infections in the United States, many probably acquired in states outside the outbreak-affected Pacific Northwest. Most of these cases differed clinically and genetically from outbreak strains of C. gattii that are causing disease in the US Pacific Northwest, and they are unlikely to be associated with the outbreak. Ten patients with travel history had not traveled to areas where C. gattii is known to be endemic for at least 1 year before illness onset, suggesting that C. gattii may be endemic in some areas of the United States outside the Pacific Northwest, and infections might be going unrecognized in these areas. California is increasingly recognized as an area where C. gattii is endemic, and the 13 patients from California could have acquired their infections locally or, perhaps in the case of the California and New Mexico patients with travel to Mexico, in Mexico. The high case-fatality rate indicates a serious public health issue, the scope of which is unknown.We describe severe C. gattii molecular types VGI or VGIII who had CNS disease and positive CSF cultures; fewer patients had positive pulmonary cultures or respiratory symptoms. Many patients had visual disturbances, cranial nerve palsies, and hydrocephalus when seeking treatment or that developed during treatment. Several patients required surgical interventions to mediate persistent elevated ICP. Several patients were treated with corticosteroids, possibly to manage their elevated ICP; clinical treatment guidelines for cryptococcosis recommend judicious use of therapeutic corticosteroids under specific circumstances  persons living in comparable environments and circumstances  and non–outbreak-associated (other molecular types) C. gattii surveillance. Thus, the distribution of reported C. gattii isolates might not represent the national distribution of C. gattii isolates outside the Pacific Northwest. Second, most infections in this report came from physicians specifically interested in obtaining species information for cryptococcal infections in their patients, usually because they were HIV uninfected; thus, we cannot make inferences about the frequency of C. gattii infections in HIV-infected persons nor the clinical course of C. gattii disease in HIV-infected persons from this report. Case ascertainment among patients in this group was also likely to differ from ascertainment in the Pacific Northwest, and comparisons between patients in each group should be interpreted with care. Additionally, co-existing pulmonary disease among these patients might be underreported. Although clinicians are likely to try to rule out CNS infection in patients with pulmonary cryptococcosis, such as those in the Pacific Northwest, patients with CNS cryptococcosis might be less likely to have pulmonary sites cultured because identifying the site will not influence treatment. Finally, we might have underestimated travel-associated infections; although most C. gattii infections are considered to have an incubation of 2–13 months (C. gattii incubation period data are based on persons exposed in the Pacific Northwest outbreak, and the outbreak is still relatively nascent, longer incubation periods might manifest as the time since exposure to the outbreak-affected region lengthens.Our report has limitations. First, case reporting was carried out only in areas where laboratory staff, clinicians, or state health departments were aware of and interested in participating in CDC C. gattii infections have been documented, albeit rarely, in at least some of these areas of the United States: in addition to the few cases identified in southern California, where C. gattii previously has been thought to be endemic, and Hawaii (C. gattii, primarily based on cases from Australia and Papua New Guinea, suggest that C. gattii infections might require more aggressive or longer treatment than C. neoformans infections (Although this case series might represent an emerging public health issue in states outside the US Pacific Northwest, it also might represent previously unrecognized disease in these states. Individual clinicians submitted multiple cases for this report, which suggests hotspots of environmental colonization, hotspots of individual interest, or both. However, C. gattii infection may be warranted in some areas of the United States; recognition of species gattii infection and early diagnosis might improve outcomes. Species identification of cryptococcal isolates requires specialized agar; however, most clinical laboratories do not identify isolates to the species level. Routine surveillance and species and molecular type identification for C. gattii outside the Pacific Northwest would enable identification of the true range of infections, and shed light on where diagnostics for C. gattii would be most useful.An increased index of suspicion for


Most pathogens gain access to the host through surfaces of the body that are exposed to the surrounding environment and rife with resident microorganisms, termed microbiota. Microbiota play an integral role in modulating host health. One significant benefit of the microbiota is that they provide protection against incoming bacterial pathogens Aedes aegypti indirectly mitigate Dengue virus transmission Because the microbiota are present at the sites used by viruses to gain entry to their host, they can potentially alter the outcome of infection. For example, the commensal microbiota of the insect vector It should be stressed that both of the aforementioned studies used antibiotics to alter the microbiota of the host. However, antibiotic treatment can lead to changes in the host's physiology that are independent of microbiota disturbance. Additionally, antibiotic treatment may not eliminate all microorganisms from the host—many microbes are known to be resistant to antimicrobial therapies Although microbiota can help the host fight viral infections, as in the case of influenza, it may also enhance viral infection, either indirectly or directly. One example of the indirect beneficial effects of microbiota on virus replication is the promotion of viral infection by stimulating the proliferation or activation of target cells . This isTo date, two studies, including our own, indicate that viruses from three distinct families rely on commensal organisms for efficient replication/transmission We discovered that Mouse Mammary Tumor Virus (MMTV), a retrovirus transmitted through the milk, utilizes the innate immune Toll-like receptor TLR4 to induce tolerance to itself, and thus to evade the antiviral response  [13]. TrLike the viruses described in the preceding section, HIV-1 is also transmitted across mucosal surfaces, which are rich in microbiota. This prompts the question—do the microbiota contribute to infection with HIV-1? People chronically infected with HIV exhibit raised plasma levels of LPS With the advent of the Human Microbiome Project, we are now aware of the number and diversity of microbes that make the human body their primary place of residence. Consequently, the microbiota can no longer be ignored when studying host–pathogen interactions. The influences of microbiota on virus infections could be either protective or detrimental for the host. Whereas the microbiota positively regulate adaptive immune responses against influenza, they suppress antivirus adaptive responses against MMTV and facilitate replication of poliovirus and reovirus by enhancing virus attachment to target cells. Thus, microbiota play a dual role in virus–host interactions. An open question that currently drives research related to microbiota is how the microbiota can be manipulated so that the host is protected from deleterious infections. In the case of pathogens that take advantage of the microbiota, one can hope to find a way to ablate these interactions, thus preventing pathogen spread/propagation. This could be done either by manipulating the composition of the microbiota (ablation of a specific microbe exploited by a virus) or by blocking interactions between the viral pathogen and specific bacterial compounds that benefit the pathogen. Future discoveries in the area of microbiota–pathogen interactions will undoubtedly unveil new opportunities for therapeutic interventions in infectious disease.


When the World Health Organization declared smallpox eradicated in 1979, smallpox vaccination was discontinued worldwide. Although cessation of smallpox vaccination is well justified, given the risks associated with complications from the vaccine, lack of vaccination nevertheless creates a growing population of persons now susceptible to infection by a few poxviruses previously covered by the smallpox vaccine. These include the orthopoxviruses monkeypox; cowpox; and, ironically, vaccinia, the virus used for smallpox vaccination. Although few persons die from these infections, they are nevertheless a public health nuisance and expense. Thus, understanding the epidemiology of these viruses is in the interest of public health.The most common vaccine-preventable poxvirus infections in humans are cowpox and monkeypox (Vaccinia, the virus used in the live smallpox vaccine, was originally isolated in the late 18th century from persons with illness that clinically resembled cowpox. However, modern genomics have shown that the vaccinia virus strains used for smallpox control in the 20th and 21st centuries are genetically distinct from cowpox viruses currently circulating. In fact, with 2 notable exceptions, vaccinia virus is not found in nature. However, vaccinia infection has been documented in India and Brazil (In this issue of Emerging Infectious Diseases, Abrahão et al. (These finding suggest a remarkably high incidence of vaccinia infection in the Brazilian wilderness and in a host, namely monkeys, not normally considered as an active reservoir for orthopoxviruses. Although Abrahão et al. do not specifically identify monkeys as a primary reservoir for vaccinia virus and do not address the mode of transmission of the virus among these animals, the results suggest a substantial repository of vaccinia virus in the Brazilian wilderness. Especially given the broad host range of vaccinia, these findings warrant a substantial effort to characterize further the circulation of vaccinia virus in this region. If the results described in the article by Abrahão et al. (


More than 25 years have passed since the discovery of a filovirus, Marburg virus, which caused an epidemic of hemorrhagic fever among laboratory workers in Marburg, Germany, in 1967. The persons affected had contact with the blood or tissues of monkeys or with other infected persons. Marburg virus has reappeared only three times since its discovery, with the largest and most recent outbreak occurring in 1999 in Durba, Democratic Republic of the Congo. Ebola virus, another filovirus, was first described in 1976 during two hemorrhagic fever epidemics in Zaire and Sudan. Since then, Ebola virus has caused large hospital outbreaks of hemorrhagic fever in Kikwit, Zaire, in 1995, and Gulu, Uganda, in 2000. Ebola virus has also been implicated in small chains of transmission among persons with direct contact with intermediary hosts, mostly nonhuman primates in the central African countries of Gabon and Republic of the Congo.The reservoirs for both viruses are still unknown, and the rarity of outbreaks and the remote location of human outbreaks make it difficult, if not impossible, to study the pathogenesis of the human disease. Thus, animal models have been the best, and often only, approach available for studying the progression of disease caused by Marburg and Ebola viruses. Dr. Ryabchikova, the principal author of this book, and her laboratory group have studied the pathogenesis of filoviruses for several decades by using animal models and electron microscopy, a unique approach that has made her one of the few filovirus experts in the world. This book is a compilation not only of her work but of all the information available on Marburg and Ebola viruses.The first three chapters of the book provide a general review of filovirus history, laboratory methods (with an emphasis on electron microscopy), viral structure, morphology, and replication. Chapters 4 and 5 provide more specific details on infection of the target cells  in different organs and during the course of filoviral infection. In chapter 6, the authors deal with the "hemorrhagic" side of Ebola and Marburg virus infection. Not all patients infected with these viruses bleed, and when bleeding disorders do occur, no correlated infection of endothelial and hematopoietic cells occurs. Dr. Ryabchikova has found that changes in the microcirculation system, such as the appearance of hemorrhages, clotting, and fibrin deposits, vary by virus and by animal species. Chapter 7, which describes pathologic changes in the organs during the course of filoviral infection, could have been combined with chapter 5. Likewise, the last chapter, which covers immunopathology, appears more like a discussion of the previous chapters.Much of the data have already been published in the Russian or Western literature. However, this book provides one source for all information available on Marburg and Ebola viruses and has a great advantage over other sources. The number and the quality of the illustrations are impressive, and a comprehensive index is provided. The book will prove useful to clinicians and researchers interested in understanding the pathogenesis of hemorrhagic fevers, and it will provide researchers working with other viruses a lesson in the benefits of using electron microscopy technology.


C. intestinalis has become an important model organism for the study of cis-regulation. This is largely due to the technology that has been developed for assessing cis-regulatory activity through the use of transient reporter transgenes introduced into fertilized eggs. This technique allows the rapid and inexpensive testing of endogenous or altered DNA for regulatory activity in vivo. This review examines evidence that C. intestinaliscis-regulatory elements are located more closely to coding regions than in other model organisms. I go on to compare the organization of cis-regulatory elements and conserved non-coding sequences in Ciona, mammals, and other deuterostomes for three representative C.intestinalis genes, Pax6, FoxAa, and the DlxA-B cluster, along with homologs in the other species. These comparisons point out some of the similarities and differences between cis-regulatory elements and their study in the various model organisms. Finally, I provide illustrations of how C. intestinalis lends itself to detailed study of the structure of cis-regulatory elements, which have led, and promise to continue to lead, to important insights into the fundamentals of transcriptional regulation.The ascidian (sea squirt)  Ciona intestinalis has emerged as an important model organism for the study of genomic regulation in the last 15 years. C. intestinalis is an ascidian - a member of the class Urochordata, which along with the cephalochordates are considered basal chordates Fig. (1). They have a simple chordate body plan, with a dorsal nervous system, axial skeleton (notochord), ventral heart, and an elaborate pharyngial apparatus. The developmental biology of ascidians has been the subject of detailed study for over 100 years, in part because of their stereotyped cell lineage, which enables experimental study of the relative roles of cytoplasmic inheritance and cell-cell signaling in development [C. intestinalis has particular advantages as a laboratory model organism, since it is a common marine organism, and thousands of synchronously, and rapidly, developing fertilized embryos can be obtained easily by in vitro fertilization on a daily basis.elopment . C. inteCiona for several reasons [Drosophila, which facilitated the release of whole genome sequences of the congeneric species C. intestinalis and C. savignyii, by 2003. The genome is compact, with only about 7.5 kb. per gene, making it relatively easy to capture significant portions of transcriptional units in readily cloneable pieces. Significantly, ascidians lack the genome duplications present in vertebrates, so they have only one paralog of many genes that have been duplicated in vertebrates. This lack of duplicate genes reduces the amount of functional overlap and redundancy, simplifying functional genomic study. These characteristics of Ciona, among others, have enabled the generation of a host of bioinformatic resources, as summarized in Table 1. This review concentrates on the progress in cis-regulatory study in C. intestinalis. It does not cover the large and growing literature on other aspects of genomic research in Ciona, such as the elicidation of gene regulatory networks  variants at low cost [Perhaps the most important reason for the interest in  zygotes -11. Thislow cost . HoweverC. intestinalis is a chordate, its genome has some significant differences with those of vertebrates. It is very AT rich, with only about 35% GC content. The proportion of repetitive sequence has not been determined precisely, but it is much lower than in many vertebrates, such as humans. It does however, harbor several transposon types [C. intestinalis [. Like other invertebrates, C. intestinalis has a very high level of nucleotide polymorphism, around 1.2% SNPs and indels between alleles. The high allelic variation makes genome assembly a difficult task [While on types . Methylastinalis . Like otult task , 15.1). Since cis-regulatory experiments are more difficult in other basal chordates, such as amphioxus and lamprey, Ciona has become a major model organism for the functional study of the evolution of chordate genomic regulation and the evolution of development [Recent phylogenomic studies have placed the ascidian urochordates as the sister group to the Vertebrata , 17  in Ciona are enhancers, which activate transcription in a more or less tissue-specific manner. This is probably due to the fact that other elements, such as insulators or silencers, are more difficult to identify. Table 2 is a compilation of most, if not all, transcription factor and signaling molecule enhancer elements reported to date for C. intestinalis.  A 2005 review of ascidian cis-regulatory elements listed 11 genes of these types with experimentally determined CREs [2, several with multiple CREs determined. Many other CREs have been reported in Ciona in other functional catagories.Nearly all reported experimentally confirmed ned CREs . As of tCiona enhancers listed in Table 2 were all found within 12 kb. upstream or 7.5 kb. downstream of their respective estimated transcription start sites (TSSs). Consistent with the compact genome, most enhancers are found within 1.5 kb. upstream of the TSS [2A. Fig. 2B shows the contrasting condition in the mouse. Out of a random selection of 79 experimentally verified mouse enhancers from 30 different transcription factor and cell-cell signaling genes, those within the total range of the Ciona enhancers were much less concentrated close to the TSS, and more likely to be located downstream. In fact, in this sample, mouse enhancers ranged as far as 123 kb downstream and 93 kb upstream. .The 83  the TSS , as showC. intestinalis . In this sample the median locations of C. intestinalis CREs was 630 bp upstream from the TSS and 2.4 kb downstream, while in the mouse the medians were 4.1 kb upstream from the TSS, and 6.3 kb downstream. Because CREs more than 12 kb upstream or 8 kb downstream of the mouse TSSs were ignored in constructing Fig. 3C, the actual median distance to CREs in the mouse is even further than shown here.There is clearly a much greater spread in the locations of mouse CREs as compared with those of C. intestinalis CREs means that investigators have a good chance of finding major CREs by examining only a few kb upstream of the TSS. However, an important caveat to this axiom is that it is possible that searches for CREs in C. intestinalis are biased towards elements proximal to the TSS. The kinds of long-range CRE searches that have been done in mouse, using BAC reporter transgenes, or Drosophila using P-element transgenes, for example, have not been done in C. intestinalis, so the presence of CREs acting at more than a few kb cannot be ruled out.The close position to the TSS of most C. intestinalis reflect the extreme compactness of the Ciona haploid genome, which is only 160 Mb, as opposed to 2700 Mb in the mouse. However, genome size doesn't necessarily correlate with compactness of CRE architecture, as seen in the large distances to some CREs found in Drosophila, which has a similar genome size to C. intestinalis[. For example, in a very incomplete look at D. melanogaster CREs in the Redfly database [lab and Scr are 1-2 kb 5' of the TSS, and for Dfd about 4 kb 5'. However, numerous other enhancers for each gene are found further from the promoter, as much as 40 kb 5' and 25 kb 3' of the TSS for Scr. For the fly Dll gene, the most proximal enhancer found is about 5 kb 5' of the TSS, with longer range enhancers up to 15 kb 5' and 40 kb 3'. In C. intestinalis, on the other hand, the enhancers for Ci-Hox1 and Ci-Hox3 are within 2 kb 5' and 2.5 kb 3' of the TSS, and for Ci-DllA and DllB, major enhancers are within 0.5 kb 5', without longer range enhancers having been found. Thus, the ascidian may have a constraint on CRE distance that is not present in some other metazoans with small genomes, such as Drosophila.This difference in the distances to CREs between the mammal and estinalis. For exadatabase , the mosCiona genome. For example, the Hox and ParaHox genes, which have conserved cluster arrangements in many other animals, are dispersed and rearranged in C. intestinalis [. If the C. intestinalis genome is subject to frequent rearrangement, there may be a selective advantage to having enhancers located close to TSSs, since there would be a lower probability of a rearrangement event between the enhancer and basal promoter that might render the enhancer non-functional. In this scenario, enhancers located more distally would be lost to purifying selection if rearranged away from their appropriate basal promoter.While this apparent constraint on distance of enhancers from transcription start sites may be an artifact of the lack of longer range enhancer searches, if the constraint turns out to be real it may be related to the apparent prevalence of rearrangements in the stinalis , 26. If C. intestinalis for CRE study is the fortuitous genetic distance to its congener C. savignyi, also sequenced, which allows for reliable prediction of CRE positions by finding conserved non-coding elements (CNEs) in alignments of the genomes [C. savignyi and C. intestinalis are capable of producing viable hybrid offspring by in-vitro fertilization, they exhibit very low similarity in most non-coding DNA sequences. Genomic comparisons indicate that the genetic distance between the two congeneric ascidians is in the range of human-chicken or human-frog [Another advantage of  genomes . Even thman-frog , 28. The5 map experimentally determined CRE locations onto same-scale plots of sequence conservation for 3 transcription factor genes in C. intestinalis, mouse, and certain other taxa. These comparisons were chosen because of the availability of published experimental CRE data along with complete genome sequences. Each comparison provides different insights into CRE characteristics.In order to visualize the relationship between CNEs and CREs, Figs. 3, data for Pax6 are plotted. Here an alignment for amphioxus vs. frog is also included, as a CRE has been reported for this other invertebrate chordate. In the alignment of C. intestinalis vs. C. savignyi , each of the four CNEs tested by reporter gene assay had some cis-regulatory activity, although the I1 and I4 regions were not dissected in enough detail to exclude the possibility that some individual CNE peaks within those regions are not involved in cis-regulation. The mouse Pax6 gene is clearly orthologous to Pax6 in C. intestinalis. The alignment shown  compares the mouse genome with that of chicken, as the chicken-mouse overall genetic distance is similar to C. intestinalis-C. savignyi. More individual CREs have been found in Mm-Pax6, than in Ci-Pax6, as would be expected given the much greater anatomical complexity of the mammal.  Interestingly, although 5 of the 7 CREs identified map to mouse-chicken CNEs, 2 of the CREs (P0 and P1) do not. In fact, even in the mouse-human comparison, these CREs correspond to regions much less conserved than several others in this region that were not found to have CRE activity. Included in this catagory are 3 upstream and two intronic CNEs conserved with chicken. An unusual case of an active CRE is the "D/α. The less sensitive AVID or LAGAN alignment programs implemented in VISTA, and used for the Fig. (5) plots, failed to show an amphioxus CNE shared with frog, lamprey, zebrafish or human genomes at the empirically identified CRE. For amphioxus, either alignment algorithms more sophisticated than those commonly used, or a more closely related comparison genome, will be required to discover more CREs using sequence analysis.In the case of amphioxus, a survey of CNEs found in an exhaustive and sensitive search  located C. intestinalis, mouse, and sea urchin. In the case of C. intestinalis, CREs were carefully mapped on a VISTA plot of CNEs. This mapping shows that 3 of the experimentally identified CREs correspond with CNEs, but two significant CREs do not . In fact, two CNEs fall just adjacent to CREs. For the mouse Foxa2 gene 2 of 3 CREs coincide with CNEs in the mouse-human comparison. However, once again there are equally or more conserved sequence elements in the mouse-human comparison that failed to show CRE activity in the published studies. As for a more distant comparison, most of the mouse Foxa2 locus fails to align with the chicken genome, so the comparison here is with the frog, X. tropicalis. None of the 3 identified CREs show up as CNEs in the frog alignment.Empirical data on FoxA CREs are available for Strogylocentrotus purpuratus FoxA gene, which revealed 4 CREs . All 4 correspond with CNEs showing up in an alignment with the urchin Lytechinus variegatus, the species the study authors used for comparison [4C), on the other hand, only about half of the CNEs correspond with CREs that show up in the reporter assays. Interestingly, from the work published on these gene loci to date, it appears that the complexity in terms of numbers of CREs regulating each gene is similar in the three taxa, even though anatomical complexity varies widely from C. intestinalis to urchin to mouse, and the genes are expressed both early and late in development.A study has also been published of the sea urchin mparison . While 9C. intestinalis have Dlx homeobox genes arranged in convergently transcribed 2-gene clusters, which are linked to Hox clusters on the same chromosomes [C. intestinalis cluster have not been able to be robustly determined. Because the most CRE data are available for the mouse Dlx1-2 cluster, that locus has been used here  for comparison. For C. intestinalis the major enhancer elements are located proximal to the respective TSSs and correspond with the largest regions of conserved sequence with C. savignyi . For Ci-DllB, additional upstream sequence in which four CNEs are located was tested, only one of which had an effect (attenuation) on expression.Both vertebrates and omosomes . MammalsDlx1-2 cluster, the most broadly acting CREs are located in the large intergenic region. Other CREs activating Mm-Dlx1 (URE1 and 2) have been found far upstream in the introns of the Metap1d gene model. The number of CREs found to date, 3 in C. intestinalis and 4 in mouse, suggest a comparable level of cis-regulatory complexity. However, it is possible that more mouse CREs would be found with more extensive searching, and that the individual mouse cis-regulatory modules have more internal complexity than those in the ascidian.For the mouse C. intestinalis-C. savignyi and mouse-chicken genomic comparisons do a good job of correlating CNEs with CREs. Once again though, several CNEs are found that do not correspond with the CREs uncovered in reporter gene assays.For the Dlx clusters the et al. (2004) [C. intestinalis CREs correspond to non-coding sequences conserved with C. savignyi.  However, even in the small sample of 3 genes examined here, one case - Ci-FoxAa - has limited conserved sequence showing up in the commonly used VISTA analysis. Here, a more sensitive conservation-finding method might have located more of the discovered CREs. On the other hand, it may be the case that there is too much rearrangement or reorganization of the CREs since the split of C. intestinalis and C. savignyi to be able to use sequence analysis as a CRE finding tool. It is likely that this would be the case for a significant minority of both individual CREs, or the complement of CREs for particular genes.As found by Johnson . (2004) , in manyFoxa2 gene exhibits a somewhat similar situation to that of Ci-FoxAa. In the intermammalian comparison, there are many CNEs with the same or greater degree of conservation to the few that have been empirically shown to have CRE activity, while no sequence conservation with the phylogenetically closest non-mammalian sequenced genome, X. tropicalis, was found. In other words, for this gene the consensus of mammalian alignments have too much sequence conservation to efficiently identify CREs, but not enough sequence conservation to the next closest taxon.Interestingly, the mouse et al. [Alternative approaches for identifying CREs rely on various ways of searching for shared non-coding motifs, in, for example, coexpressed genes, or searching for combinations of known transcription factor binding sites (TFBSs) (reviewed in ). Even tet al. .C. intestinalis system for cis-regulatory study. First, non-coding sequence conservation comparisons with C. savignyi, are at a fortuitous genetic distance to have a high probability of locating functional CREs. These C. intestinalis-C. savignyi comparisons consistently identify CNEs that correspond with CREs. Mammals and other chordates, such as amphioxus, do not have such phylogenetically well-positioned sequenced comparison taxa for this purpose. Second, the close proximity of C. intestinalis CREs to the transcription start site means that only a few kb of DNA must be scanned experimentally to have a high probability of recovering the major CREs for any gene. This is certainly not the case for the mouse, and to some extent even for the sea urchin.Taken together, these comparative observations point out 2 major advantages of the C. intestinalis genes shown here  the correspondance is much better, 67%, although there are many distal CNEs that were not tested for cis-regulatory activity. In addition, two Ci-FoxAa CREs did not align with a CNE, and would have been missed if only testing conserved sequence elements.Even though phylogenetic or binding site sequence analysis can point the way to locating CREs, many, if not most, CNEs do not correspond with CREs identified in empirical experiments, such as reporter gene assays, or ChIP-Seq. In a study of human conserved sequence elements tested in mouse , only 29cis-regulation, but does not have activation activity that would drive reporter gene expression. These elements could be repressors, insulators or modifiers that have negative regulatory function, and can only be detected by their effect on another cis-regulatory element. A CRE may only have activity in concert with another element, so the individual element shows no activity on its own eg. [There are, of course, a number of reasons that a conserved non-coding sequence might be functional, and therefore constrained by purifying selection, but not show CRE activity in a reporter assay . One set own eg. . The CNE own eg. , 36. ChaApart from these genuine but undetected CREs, are other non-coding functional elements, such as matrix attachment regions, or transcribed non-coding RNAs, such as micro RNAs, or long non-coding RNAs. Identification of the function of these CNEs will rely on completely different types of experimental and bioinformatic approaches than those used for CRE discovery.Troponin I gene in C. intestinalis [Ci-DllB, a CNE was identified that accounted for the majority of the endogenous expression pattern , but an adjacent non-conserved sequence was capable of acting with a portion of the conserved region to activate a similar expression pattern [As mentioned above, some CREs found through reporter gene assays do not correspond with CNEs . In addition, in cases where CRE dissections have been done in some detail, even if major CRE activity is found in conserved sequences there are sequences outside those conserved that contribute to the expression pattern. For example, in a detailed study of the stinalis , 4 conse pattern .cis-regulatory architecture of individual genes, C. intestinalis lends itself to both broader and deeper investigations of cis-regulation. Two studies may serve to demonstrate the particular advantages of reporter gene assays in this animal. Brown and colleagues [cis-regulation of co-expressed muscle-specific genes in C. intestinalis. They found that though similar DNA-binding proteins cluster in muscle gene CREs, their arrangement and relative contributions to activation of expression vary greatly. The authors also compared the cis-regulation of six orthologous muscle-specific genes in C. intestinalis vs. C. savignyi, and found large variations between the relative activity conferred by individual binding sites. The simplicity and rapidity of transient in vivo transgenic reporter experiments in Ciona contributed to the feasibility of performing the large number of assays required for this study. Other workers have also used the C. intestinalis system for study of groups of co-expressed genes e.g. [Apart from the dissection of the lleagues  producednes e.g. , 40.Otx enhancer that confers early neural expression [in silico analysis to find 55 occurances of this motif in the C. intestinalis genome, and then tested each of these in in vivo reporter assays. 19 of the sequences showed enhancer activity, and examination of these CREs revealed a nucleosome exclusion motif that was found to be conserved between Ciona and Drosophila [. Once again, the ability to test a large number of reporter transgenes rapidly, along with the compactness of the C. intestinalis genome and CREs, enhanced the practicality of this study.Another study by Lemaire and colleagues built on a previous finding of paired ETS and GATA binding sites in an pression . They thosophila . Once agCis-regulatory elements in C. intestinalis tend to be located relatively close to the transcription start sites of genes, which may be associated with the small size of its genome. This fact combined with the ease and economy of producing transient transgenic embryos in large numbers, have made it an attractive model system for the study of cis-regulation. Comparison of experimentally verified CREs with those found in other animals, such as the mouse, suggests that C. intestinalis transcriptional regulation relies on a similar complexity of CREs given its anatomical simplicity. Recent work touched on here makes increasingly sophisticated use of the extensive bioinformatic resources for Ciona, coupled with analysis of large numbers of in vivo reporter gene experiments, to study fundamental aspects of transcriptional regulation. Future work promises to further exploit the C. intestinalis model to advance understanding of the function and evolution of cis-regulation.


Torque teno virus is a nonenveloped single-stranded DNA virus infecting humans and nonprimate species. We report the complete genome sequence of a pigeon torque teno virus isolated from pigeons in Jiangsu Province, China, in 2012. This genome sequence will be useful for viral diagnostics and disease control. Pigeon torque teno virus (PTTV) is a novel DNA virus that was first discovered in pigeons in China. PTTV is a species-specific virus, and there is little information about how it affects different pigeons or about its molecular characteristics and evolutionary process.Anelloviridae (Torque teno virus (TTV) is a small, nonenveloped, single-stranded, negative-sense circular DNA virus  belonginoviridae . In 1997oviridae . TTV is oviridae . Analysioviridae , 6. In hIn this study, a total of 144 pigeon blood serum or pigeon disease samples were collected from six pigeon markets in Jiangsu Province, China. PTTV was detected in 103 out of 144 (72%) samples using specific PCR methods with the primer pair NG343 and NG344 . Six repThe complete genome of PTTV is a single-stranded DNA molecule comprising 1,584 or 1,585 bp. It has two putative open reading frames (ORFs) encoding Rep replication protein (ORF V1) and Cap coat protein (ORF C1). There is a small overlapping part between the two ORFs, and translation begins in the single-stranded DNA. The ORF V1 of Jiangsu/PTTV is located at nucleotides 464 to 923 in the genome, encoding 154 amino acids, and ORF C1 was located at nucleotides 788 to 1189 in the genome, encoding 134 amino acids. However, unlike genomes of TTVs infecting mammals, the untranslated region (UTR) region of PTTV shows no significant G+C-rich region . Phylogenetic analyses based on the genome indicated that six Jiangsu PTTVs are closely related to one another. Sequence analysis showed that the genomes of six Jiangsu PTTVs are 96.4% to 100% homologous to that of chicken anemia virus (CAV), while they show significant difference in their genetic relationship with TTVs infecting mammals, including humans, apes, pigs, and cats.This is believed to be the first investigation of PTTV infection in pigeons, and five complete genome sequences have been amplified. Pigeons have great commercial value worldwide; therefore, more research on the molecular characteristics and evolutionary process of PTTV is needed.KF477315 to KF477319.The complete genome sequences of A/pigeons/Jiangsu/PTTV/2012 were deposited in GenBank under accession no.


To attain immortality, he performed, as penance for his misdeeds, arduous service to the community, using his unparalleled strength to support his fellow humans.The thick marsh habitat of the Stymphalian birds worked against Herakles. His bow and arrows failed, for he could neither see nor reach the birds through the dense vegetation. Only asked to drive them away, he abandoned efforts to eliminate the birds; instead, he conned them into leaving the area on their own. With a pair of krotala  made by Hephaestus, god of the forge, Herakles frightened the birds out of their refuge and chased them as they flew east to the Isle of Ares in the Black Sea.The detail on this month's cover comes from a black-figured amphora, a ceramic vase popular in ancient Athens in the 6th century BC. Such vases were made of iron-rich clay and decorated with black silhouettes in mythical heroic scenes. Illustrations were incised and painted with a slip (liquid clay), which turned black during firing without oxygen  have changed little. Waterfowl, a benign species, were demonized in the Stymphalian myth, their hideous mien likely borne of human fear and helplessness, for who knows what pestilence they had inflicted on the community around the lake. And each time those birds flew to a new place, they had contact with other birds and opportunities for genetic reassortment, redistribution, and modification of pathogens throughout the migratory route.,Resistant to slings and arrows and prone to long-distance migrations, birds such as the ones on this cover's amphora persist beyond our ancestors' morbid imaginations. Not because of mythical metal paraphernalia but for their explosive potential as natural reservoirs and amplifying hosts of pathogens. Viremic migratory birds acting as introductory hosts may have brought West Nile virus to the Western Hemisphere, perhaps by infecting ornithophilic mosquitoes, which may have infected amplifying hosts and eventually humans  also carry flu viruses in their intestines and shed them in their secretions and excretions. As these waterfowl migrate around the globe, they introduce new flu strains into domestic poultry and swine. These strains can then amplify and mutate close to human populations, increasing the risk that the virus will recombine with local human strains to form a new virus with pandemic potential. Like the legendary harpies, these new strains, half human half avian, pose an immense public health challenge. We now know more about bird pestilence. West Nile virus infection and avian flu are just as ominous as razor-edged feathers. And while Herakles had krotala from the gods, we must work with human tools: repellants and pesticides, vaccines (


Since 2006, canine distemper outbreaks have occurred in rhesus monkeys at abreeding farm in Guangxi, People’s Republic of China. Approximately10,000 animals were infected (25%–60% disease incidence); 5%–30%of infected animals died. The epidemic was controlled by vaccination. Amino acidsequence analysis of the virus indicated a unique strain. Paramyxoviridae  was reported (Canine distemper is a highly contagious infectious disease of canine and feline speciescaused by canine distemper virus (CDV), a member of familyMacaca mulatta) at a breeding farm in the Guangxi ZhuangAutonomous Region in southern China. The farm, the largest in China, comprised31,260 monkeys, of which ≈1 in 5 was unweaned. Approximately 10,000 monkeyscontracted the disease, and 4,250 died. The morbidity rate in young monkeys was 60%,with an ≈30% death rate . In 2007,surviving monkeys were vaccinated with an inactivated suspension made from thelivers and lungs of dead animals. After vaccination, the number of cases decreasedduring 2007 and 2008 to ≈100–200 per year.In 2006, an unidentified respiratory disease occurred in rhesus monkeys. Additionally, anti-CDV serum seemedto help infected animals recover more rapidly from the infection.Infected monkeys initially displayed measles-like signs, including respiratory signs;anorexia; fever; and red rashes over the entire body, with reddening and swelling ofthe footpads; conjunctivitis; and thick mucoid nasal discharge. Coma preceded death.Postmortem examination demonstrated discrete purple or rosy rashes on the body, withmacules of 2–4 mm , panel ABecause of the signs of a measles-like infection, identification of the etiologicagent first focused on measles virus and later on CDV. Reverse transcription PCRamplification of lung specimens by using virus-specific primers was negative formeasles virus but positive for CDV. The supernatant of ground liver samples also waspositive for CDV by immunochromatographic analysis . Lung specimens alsoinfected tree shrews (1–2 months old) after subcutaneous injection, resultingin excitement and death from encephalitis and enterohemorrhage.The full-length viral genome was amplified and sequenced (GenBank accession no.HM852904). We constructed phylogenetic trees by the neighbor-joining method, usingthe full sequence of this virus and others available in GenBank. The full genome ofthis isolate shared the highest homology with a ferret isolate (accession no.AY386316) from the United States, a raccoon isolate (accession no. AY649446) fromthe United States, and a dog isolate (accession no. AB474397) from Japan , panel AReasons for the epidemic remain unclear. The first monkey to contract the infectionwas in the farm in Guangxi; however, the source of the infection is unknown becausethere were no dogs or other fur-bearing animals at the farm. Breeding facilitieswere self-contained, with no introduction of external animals and no other farmsnearby. Food for the monkeys had no animal content except fish powder. One possiblesource of infection is contact by the monkeys at the farm with local wild monkeys.Another possibility is spillover of the virus from a stray dog carrying CDV thatbecame adapted to the new host.Large-scale breeding and caging of the monkeys might have contributed to increasingsusceptibility to canine distemper infection. Reared in groups of 20–30within fenced-off areas or in pens of several hundred, close contact between animalswas inevitable. CDV appears to have been transmitted by droplets derived from fecesor other body discharges, all of which contained CDV.This canine distemper outbreak poses a threat to monkey populations. Because theGuangxi farm routinely supplies monkeys for animal facilities throughout China,monitoring for canine distemper in its monkeys, including those shipped to otheranimal facilities, as well as human handlers, is advisable to prevent possiblesecondary spread and interspecies transmission.Although CDV spread has been largely controlled by inactivated and live caninedistemper vaccines, sporadic cases still occur and the high number of mutations inthe virus makes future transmission unpredictable. Therefore, surveillance forcanine distemper should be considered among monkey populations and among humans whohave close contact with them.


Schistosoma (S.) haematobium infection is a common cause of genital morbidity in adult women. Ova in the genital mucosal lining may cause lesions, bleeding, pain, discharge, and the damaged surfaces may pose a risk for HIV. In a heterogeneous schistosomiasis endemic area in South Africa, we sought to investigate if young girls had genital symptoms and if this was associated with urinary S. haematobium.In a cross-sectional study of 18 randomly chosen primary schools, we included 1057 schoolgirls between the age of 10 and 12 years. We interviewed assenting girls, whose parents had consented to their participation and examined three urines from each of them for schistosome ova.One third of the girls reported to have a history of genital symptoms. Prior schistosomal infection was reported by 22% (226/1020), this was associated with current genital symptoms (p<0.001). In regression analysis the genital symptoms were significantly associated both with urinary schistosomiasis (p<0.001) and water contact (p<0.001).Even before sexually active age, a relatively large proportion of the participating girls had similar genital symptoms to those reported for adult genital schistosomiasis previously. Anti-schistosomal treatment should be considered at a young age in order to prevent chronic genital damage and secondary infections such as HIV, sexually transmitted diseases and other super-infections. Urogenital schistosomiasis (Bilharzia) is a common cause of gynecological disease in adult women. Reports to date indicate that genital lesions in adults become chronic and that the damages make women susceptible to HIV. This is the first study on urogenital schistosomiasis in pre-pubertal girls. We interviewed girls aged 10 to 12 years of age for urinary and gynecological symptoms. The research assistants did not know the schistosomiasis infection status in the school or the individuals. We collected three urines that were examined for schistosome eggs. We found that a significantly increased number of girls with urinary schistosomiasis have stinking, bloody discharge, ulcers, tumors and a burning sensation in their genitals. This indicates that gynecological damages due to schistosomiasis start before sexual activity, and before menstruation. By preventing urogenital schistosomiasis in girls we may have an innovative opportunity to reduce teenage HIV transmission and gynecological disease. This study presents a new aspect of a neglected disease affecting more than 100 million females, long overdue for mass intervention. Schistosoma (S.) haematobium is primarily known for its effect on the urinary tract, but in endemic areas schistosomiasis may be the most common cause of genital morbidity and mucosal lesions Urogenital schistosomiasis causes gynecological morbidity in adult women S. haematobium ova when deposited in the female reproductive tract seem to be equally distributed in the different genital parts, but are most commonly identified in the cervix and the vagina Previous studies on urogenital schistosomiasis have been conducted in adult women of childbearing age. S. haematobium in young girls.It has been hypothesized that female genital schistosomiasis poses an increased risk to secondary infections such as human papillomavirus and other STDs. Most importantly, these women have been found to have significantly more HIV We carried out a cross-sectional study in 18 primary schools, which were randomized for inclusion from 309 primary schools in the area. We invited all girls aged 10–12 years in the included schools. Girls who were absent on the days of the invitations were excluded, as were girls with serious illnesses, or if their guardians or they refused.S. haematobium endemic area, which covers 5866 km2  and Education (2009) in Ugu District. The Helsinki Declaration was followed. All members of the group, including students and research assistants had passed exams in Good Clinical Practice and signed Declarations of Confidentiality. The interviewers did not know the study subjects beforehand. Prior to the study there were information meetings for the parents, principals, school governing bodies and/or teachers of each school. Informed consent was given by each girl, and the parents/guardians signed consent forms. Identifying information was stored separate from the interview information (in separate towns). All were informed about their right to withdraw and to abstain from answering questions without negative consequences. In order to protect girls from stigmatization the disease was discussed in general terms as urinary schistosomiasis. Treatment for schistosomiasis was offered to all, and all were informed about possible side effects. A private psychologist was hired by the project to take care of referred cases as felt necessary; for psychological, practical and legal issues. When other medical help was required, the girls were referred to a government clinical facility, or offered private care if government services were unavailable. For ethical and community liaison reasons the project staff was not involved in any physical or psychological examinations after referral.The interviews (30 minutes duration) were conducted face to face in isiZulu  by trained female fieldworkers. Questions were asked about recent (the last week) or previous urogenital symptoms of itch, burn, ulcers or tumors  in the genitals, malodorous discharge, color of discharge and feeling of a burning sensation in the genitals, as well as red urine, dysuria, urge and stress incontinence Ascaris lumbricoides, Ancylostoma duodenale, Taenia solium, Trichuris trichiura and S. mansoni. If there was at least one ovum in a specimen it was defined as positive.The researchers aimed to visit each school at least three times. We obtained urine samples from each girl on three consecutive days between 10 am and 2 pm. After gentle tilting we deposited 10 ml of urine into a container with 1 ml methylate-formalin solution and the same week we investigated the specimens by microscopy Based on data from studies in adults we estimated the prevalence of genital schistosomiasis to be 30% and urinary schistosomiasis 40% Schools that were randomized for inclusion were visited in no particular order. We invited all pupils aged 10 to 12 years. All schools were visited several times in order to collect guardian consent forms and to find as many students as possible. The parents of 1241/1948 (64%) pupils in 18 schools provided consent. On the days we were in the schools we were able to include 1057 assenting girls. In the first 13 schools, where there was adequate time before exams, the consent forms were returned and signed by 92% (1109/1201) of the parents. The pupils were recruited from grades 1 to 7, median grade 5.Answers were recorded as missing if the child chose not to answer or did not understand the topics. Seven percent of the girls (71/1019) had started menstruating. Only 5% (51/981) said they had been tested for HIV. Three out of 980 (0.3%) knew they had HIV, as many as 495 said they did not know and 77 girls did not reply to this question. Less than one percent (7/1017) reported to have had intra-vaginal sex. However, two percent (22/953) reported to have been sexually abused. They were referred to psychosocial follow-up. All in all 24 of 1019 young girls had experienced voluntary or involuntary vaginal sex.S. haematobium eggs were found in 32% (312/970) of the girls. High-intensity urinary infection was found in 28% (88/312) of these. In those who had ova in the urine, the mean intensity of infection was 52 eggs/10 ml urine (range 1–624/10 ml). Among the 658 girls with negative urine specimens, 79% (522) submitted three negative urine samples. There was neither any difference in urinary schistosomiasis infection intensity nor presence of symptoms between those who had submitted three urine samples versus one sample.Out of the 1057 girls who were interviewed, 970 submitted at least one urine for examination, and out of these 791 submitted three urine samples. S. mansoni. The discharge color was white in 51% (84/164) of the cases; cream color in 35% (58/164) and yellow in 9% (14/164). The discharge had streaks/traces of red in 13% (11/87) of the cases, light red in 66% (57/87) of the cases and an even lighter shade of red (light pink) in 9% (8/87). Patients with symptoms were referred but not investigated by the project.Thirty five percent reported to have had genital symptoms (356/1018), and as many as 17% (172/1008) reported genital symptoms the last week. Eighteen girls reported having a genital tumor or an ulcer this last week. In order to explore the urinary negative girls in more detail they were first divided into two groups , those iS. haematobium ova in urine. Among the girls with three negative urines, 54% (281/522) reported water contact. These reported significantly more genital symptoms the last week than their peers without water contact (p = 0.001).We found a significant association between water contact and all the listed symptoms (data not shown). However, among the 364 who denied water contact, 21% (76) had urinary schistosomiasis. Sixty three percent of the girls reported water contact (667/1057), among these 606 submitted urines and 39% (236/606) had Twenty two percent of the young girls (226/1020) reported having had urinary schistosomiasis previously. This was significantly associated with current genital symptoms such as bloody discharge , malodorous discharge (p<0.001), genital itch (p<0.017) and genital ulcer (p = 0.001). Furthermore 29% (251/853) knew of a family member who had had urinary schistosomiasis or red urine and these factors too were associated with all the queried symptoms (p≤0.002), except genital tumor (p = 0.08).Twelve percent (129/1057) reported that they had been treated for schistosomiasis previously. Girls who said that they had not been treated had significantly more urinary schistosomiasis than those who had been treated (p<0.001), but not more symptoms .S. haematobium endemic area girls aged 10 to 12 years with schistosomiasis had significantly more often unpleasant symptoms such as genital ulcers, bloody discharge, malodorous white to yellow cultured discharge, genital itch or tumors than those without this infection. Even before sexual debut and independent of menstruation more than 40% of girls with S. haematobium ova in urine reported having had gynecological symptoms previously, one third reported having it the last week or ‘always’. Girls living in endemic areas without urinary schistosomiasis also had significantly more genital symptoms than their peers in low-endemic schools. This study shows that urinary schistosomiasis, water contact, history of red urine and family history of schistosomiasis (‘Isichenene’ in Zulu) are also associated with the full range of symptoms. As shown in adults previously, the history of water contact was an excellent predictor for genital symptoms also in girls In an S. haematobium infection and are significantly associated with discharge S. haematobium in the lower genital tract may make the genital mucosa more susceptible to super-infections by other agents such as bacterial infections, which in turn may cause the reported symptoms. Likewise, the association between water contact and symptoms may be influenced by social and other practices. Poor perinea hygiene may be more common in a group that has limited access to water; and cultural cleansing rituals may also be hypothetical reasons for the association between water contact and symptoms In adults the grainy sandy patches have been found to be diagnostic of In this study three urines were collected and the presence of schistosome ova defined the urinary schistosomiasis positive group. However, this study confirms that even urinary negative cases in endemic areas have gynecological morbidity. Hence the prevalence is most likely higher in this population. A more sensitive diagnostic method, such as antigen detection or PCR, would likely have made the reported finding more apparent, though this was not possible in our study S. haematobium ova in urine. Girls may be shy, worried about repercussions or not be able to differentiate between urinary and genital symptoms. Some girls were ignorant of some phenomena in the questionnaire such as menstruation or discharge. The interviewers – all female – were trained to explain the differences, however information sessions using dolls followed by more thorough questioning of genital symptoms could perhaps have produced more reliable answers and less under-reporting. This was not done in the present study. Further, the most reliable method to determine water contact is by direct observation, although many schistosomiasis studies have used self-reported water body data Some girls denied having had water contact, but were found to still have S. haematobium ova in the genitals of adults, even after correcting for sexually transmitted diseases It is well documented that many adult women may have genital schistosomiasis even without having detectable schistosome ova in the urine S. haematobium ova S. haematobium infection for several years The girls in this study report the same symptoms as adult women in previous studies At the present time there are no suitable tools for the diagnosis of genital schistosomiasis in girls. Abnormal malodorous or bloody genital discharge are mucosal symptoms S. haematobium endemic areas have gynecological symptoms as a result of schistosoma infection. Mucosal damages may be present as these young girls enter into their first sexual relationships, making them particularly susceptible to HIV or human papillomavirus infection For the rural clinician history taking and urine analyses are simpler sources of information than gynecological examinations, and especially so in virgins. The findings in this study suggest that young girls in Checklist S1Enclosed is a Strobe checklist for cross-sectional studies.(DOC)Click here for additional data file.


N-glycosylation pattern analysis was combined with in-depth pyrosequencing analysis of the virus genomic RNA. Forward and backward adaptation from Madin-Darby Canine Kidney (MDCK) cells to African green monkey kidney (Vero) cells was investigated for two closely related influenza A virus PR/8/34 (H1N1) strains: from the National Institute for Biological Standards and Control (NIBSC) or the Robert Koch Institute (RKI). Furthermore, stability of HA N-glycosylation patterns over ten consecutive passages and different harvest time points is demonstrated. Adaptation to Vero cells finally allowed efficient influenza A virus replication in Vero cells. In contrast, during back-adaptation the virus replicated well from the very beginning. HA N-glycosylation patterns were cell line dependent and stabilized fast within one (NIBSC-derived virus) or two (RKI-derived virus) successive passages during adaptation processes. However, during adaptation new virus variants were detected. These variants carried “rescue” mutations on the genomic level within the HA stem region, which result in amino acid substitutions. These substitutions finally allowed sufficient virus replication in the new host system. According to adaptation pressure the composition of the virus populations varied. In Vero cells a selection for “rescue” variants was characteristic. After back-adaptation to MDCK cells some variants persisted at indifferent frequencies, others slowly diminished and even dropped below the detection limit.The genome of influenza A viruses is constantly changing (genetic drift) resulting in small, gradual changes in viral proteins. Alterations within antibody recognition sites of the viral membrane glycoproteins hemagglutinin (HA) and neuraminidase (NA) result in an antigenetic drift, which requires the seasonal update of human influenza virus vaccines. Generally, virus adaptation is necessary to obtain sufficiently high virus yields in cell culture-derived vaccine manufacturing. In this study detailed HA  Influenza A virus is a highly virulent human and animal pathogen, particularly due to its ability to cause severe disease. Hygiene measures and vaccination still represent the most efficient prevention strategies. Since several years cell culture-based processes are being established to overcome problems of traditional egg-based vaccine manufacturing such as egg supply shortages and difficulties in rapid scale-up during pandemics, as well as to increase manufacturing capacities. The need for the seasonal reformulation of influenza vaccines is attributed to the virus' ability to rapidly adapt to changing environments. Virus adaptation is one of the most important processes in virus evolution, and a crucial factor to be taken into account for seasonal and pandemic vaccine production. On the one hand, adaptation allows the virus to cross species boarders, evade immune or therapeutic pressures and optimize its replication in a given host system N-glycosylation, reflecting variations in glycosylation site occupancy (macroheterogeneity) and in structure of sugar residues (microheterogeneity). This complexity on the glycomic level is increased additionally by a co-existence of related virus subpopulations on the genomic and therefore also on the proteomic level, so-called quasispecies Glycoproteins such as HA can be considered as a collection of different glycoforms or glycosylation variants N-glycosylation pattern over 10 successive virus passages in Madin-Darby canine kidney (MDCK) cell culture. In addition, HA-stability was characterized for MDCK and Vero (African green monkey kidney) cells for up to 96 hours post infection (hpi) and 360 hpi, respectively. Furthermore, the impact of adaptation from MDCK to Vero cells and back on HA N-glycosylation and composition of the virus population was studied. Therefore, two closely related MDCK cell-adapted influenza A virus Puerto Rico/8/34 (H1N1) strains were adapted to growth on Vero cells. Resulting Vero cell-adapted viruses were subsequently back-adapted to replicate in MDCK cells. Finally, the consequences on HA level of different virus adaptation processes from MDCK cell to Vero cell based production and back were monitored. Therefore, detailed HA N-glycosylation pattern analysis of this major vaccine antigen was combined with in-depth pyrosequencing analysis of the HA encoding RNA segment 4. This allowed a characterization of quasispecies' composition.In this study we investigated the stability of the HA 2 and 37°C or in roller bottles with closed caps at 37°C. For cell growth GMEM  was supplemented with 5.5 g/L glucose , 2 g/L peptone , 10% FCS Invitrogen/Gibco, #10270-106, Darmstadt, Germany) and 4 mg/mL NaHCO3 .Adherent MDCK (No. 84121903) or Vero (No. 88020401) cells were purchased from ECACC . The cells were either grown in T75- and T175- flasks at 5% COH1N1) from either the National Institute for Biological Standards and Control  or the Robert Koch Institute  according to Genzel et al. Infections were performed with influenza A/Puerto Rico/8/34 . In order to monitor the impact of harvest time points, MDCK  and Vero  cell-adapted virus seed was propagated in MDCK and Vero cells, respectively and isolated at different time points within the time frame of interest for MDCK as well as Vero cell-derived virus. All controls were performed in roller bottles using PR/8/34 (RKI).All infections were performed when cells were confluent using 0.2 mL virus seed for T75 flasks, 0.4 mL for T175 flasks or 1 mL for roller bottles . Furthermore, Genzel et al. showed that the multiplicity of infection (moi) has no effect on final HA virus titers, only on the time needed to reach this maximum HA value 0.5 in PBS resulting in a final volume of 100 µL using round-bottomed 96-well plates. 1.9–2.1×106 red blood chicken cells in a total volume of 100 µL in PBS were added and incubated for 3 h to 4 h at room temperature. Light extinction was measured at 700 nm and plotted over the logarithm of the inversed dilutions. The end point was defined as the point of inflection - the last dilution (d) showing complete hemagglutination - corresponding to 1 HAU (HAU  =  HA Units  =  -log10 d/100 µL). A sample's HA-titer is calculated by considering the dilution factor of the end point. Consequently, if e.g. the end point is reached at a dilution of 1/64 (1/101.8), this corresponds to an HA-titer of 1.8 HAU for the undiluted sample.Influenza virus from cell culture supernatant was titrated by hemagglutination based on the method by Kalbfuss et al. N-glycan analysis and data evaluation were performed according to Schwarzer et al. (aq) with 50 mM ammonium hydrogen carbonate (aq) during enzymatic in-gel-deglycosylation and N-glycan extraction as well as the reduction of the final sample volume for PNGaseF-digestion to 60 µL, which resulted in an increased enzyme concentration. Labeled samples were desalted by size exclusion chromatography as described by Schwarzer et al. N-glycan pool of an HA sample was separated by capillary gel electrophoresis, resulting in capillary electropherograms, which – after migration time normalization - corresponds to the N-glycosylation pattern, in which relative fluorescence units (RFU) are plotted over the normalized migration time (tmig) in base pairs (bp). In N-glycosylation patterns, one peak corresponds to at least one distinct N-glycan structure. The total peak height (TPH) is calculated as the sum of all peaks and is set to 100%. Differences in relative abundances are expressed in percentage of relative peak height (RPH). Accordingly, overlays of multiple N-glycosylation patterns allow a direct comparison of different HA N-glycan pools. In the following peaks are annotated as shown in Virus purification by gradient step centrifugation, sample preparation, www.gisaid.org), the accession numbers are compiled in the supplementary (Segment 4 (HA) of influenza A was sequenced using the Genome Sequencer FLX instrument . For DNA preparation the protocol of Höper et al. ementary . For quaIn the following, amino acid substitutions during the adaptation processes will be annotated by the one letter amino acid code for the original residue (in passage 1), followed by the position number of the residue, followed by the one letter code of the substituting amino acid in the later passage. Insertions and deletions are indicated by a minus sign (-) at the first or last position, respectively.N-glycans for glycosylation pattern analysis were stored at −20°C. Sealed Coomassie-stained polyacrylamide gels with HA bands  were stored at 4°C.Virus containing cell culture supernatants as well as virus isolates were stored at −80°C; HA H1N1) to different host cell lines is the stability of the HA N-glycosylation pattern over multiple virus passages in one cell line. For MDCK cell-derived PR/8/34 (RKI), HA-titers of 10 successive virus passages ranged between 2 and 2.4 HAU at 24 hpi (data not shown). Furthermore, all HA N-glycosylation patterns feature the same 15 main peaks  throughout all passages . With differences in replication dynamics during adaptation to a new cell line, i.e. slow replication dynamics during initial adaptation steps and fast progress of infection towards the end, different time points for sampling were required to achieve a minimum of 1.8 HAU. Hence, the impact of harvest time point on HA N-glycosylation patterns was investigated for Vero cell-adapted PR/8/34 (RKI) virus in Vero cells, and for MDCK cell-adapted virus in MDCK cells. For all sampling points  during MDCK cell-based virus replication, the HA N-glycosylation pattern was dominated by the same 15 main peaks  and the RPH for peaks 16, 20, 22 to 25 steadily decreased over time. However, even despite those changes of RPH, the HA N-glycosylation pattern itself was stable over 360 h.In contrast, the pattern from Vero cell-derived HA was defined by 16 main peaks , the relative amounts  of single structures present in the glycan pool, were dependant on the harvest time point of the virus-containing supernatant.Overall, results demonstrated a highly reproducible HA In 2010 Genzel et al. showed that viruses often require adaptation to new host cells to optimize yields and time of harvest. In order to further characterize this adaptation process and to investigate possible biological mechanisms MDCK cell-adapted virus was propagated for 24 h in MDCK cells (passage 1). As a result HA-titers of 1.9 HAU and 2.2 HAU were obtained for the strains from RKI and NIBSC, respectively (data not shown). Supernatant of this passage served as the virus seed for a first infection of Vero cells (passage 2). For the RKI-strain virus-release was not detected before 288 hpi 0.8 HAU,  and the N-glycosylation patterns of MDCK cell-derived virus samples were similar. The same applied to the Vero cell-derived virus samples  compared to the same peaks of the time series experiment  compared to the respective time series and biological reproducibility experiments  with the SD observed during adaptation experiments is shown in periment . Closer eriments . Of thesN-glycosylation pattern during virus adaptation to different host cell lines was demonstrated for both PR/8/34 influenza virus strains. Obviously, it is mainly the host cell line which determines the HA N-glycosylation patterns of a specific virus strain while the harvest time point or the viral adaptation status have only a minor impact on the respective RPH of glycan structures. Finally, the discrepancy of the HA N-glycosylation pattern for NIBSC- as well as the RKI-virus between the first passage in Vero cells (passage 2) and the patterns of all subsequent Vero cell adaptation passages (passages 3 – 6) can not completely be explained by differing harvest time points and remains to be further investigated.Overall, however, a clear trend during the forward and backward adaptation process was not evident . FurtherN-glycosylation sites. The consensus sequences of segment 4 of the initial, MDCK cell-derived virus seeds from passage 1 are shown in the supplementary , T397A (1.3%), T397S (0.6%), D455Y (21.4%) and N460D (12.2%).To investigate the impact of forward and backward adaptations in both cell lines segment 4 coding for the HA was sequenced for passage 1, 6 and 11. Focus was in particular on changes in potential HA ementary . RegardiK460E, where a positively charged lysine was replaced by the negatively charged glutamic acid. Another population of 19% carried the S457L substitution, where polar serine was replaced by non-polar leucine , 80% of the RKI-strain virus population carried amino acid substitution  leucine . These tto 41.1% .K460E substitution in the HA. The minor subpopulation (19% after Vero-adaptation) carrying the S457L substitution decreased to 9% after back-adaptation. The remaining population of 10% represented the initial virus seed sequence from passage 1  strains from NIBSC and the RKI (K459E substitution was detected with a frequency of 44.2%.After back-adaptation of the RKI-strain to MDCK cells the dominating virus population (81%) still carried the assage 1 . This dessage 11 . This T3 the RKI . FurtherH1N1) strains are located in the HA2 chain, neither inside nor in close proximity of any N-glycosylation site. They are, however, located in the inside of the HA trimer within or in close proximity to the fusion peptide pocket: within the subunits' contact site for the RKI-strain . In 2001, Schickli et al. T146N, K147-). Furthermore, they described a second substitution D204E present in this virus strain (passage 1). The third substitution was located at position 158, where lysine was substituted by glutamic acid (K158E).An alignment of the segment 4 sequence of the initial, MDCK cell-derived virus PR/8/34 (RKI) from passage 1 with the viral sequence published in 1981 N-glycosylation sites or amino acids in their close neighborhood were affected. In contrast, the initial virus seed population (passage 1) of the NIBSC-strain of the adaptation series comprises more subpopulations concerning the HA-encoding RNA segment 4 than the RKI virus seed. Previously, these two  influenza virus seeds have been described to differ significantly in infection characteristics such as interferon (IFN) and apoptosis induction, expression of interferon stimulated genes, final virus yields In summary, the genome sequences of RNA segment 4, coding for HA, are homogenous in the initial virus seed of the RKI-strain (passage 1). This suggests clearly that the virus seed consisted of only one population. The adaptation processes resulted in additional virus variants, due to mutations in the HA stem region. However, no potential HA K459E variant from NIBSC that carries a deletion in the HA1 chain (K147-) corresponds to the K460E variant from the RKI. Moreover, also the substitutions D455H and N460D of the NIBSC strain are located in the same region of the HA2 subunit as the substitutions S457L and K460E of the RKI-strain.A comparison of results of both adaptation experiments with influenza virus strains from NIBSC and from RKI shows one interesting similarity, the substitution of lysine by glutamic acid. In particular, the N-glycosylation pattern with the change of host cell system. Additionally, both experiments demonstrate that only a small sequence adaptation is required for successful infection and fast growth to high titers in new host cell lines. These findings are in agreement with results of Wagner et al. and Klenk et al. N-glycans attached to the stem domain of HA efficiently regulate influenza A virus replication. The authors showed that a loss of N-glycans in the stem region results in increased pH-sensitivity of the virus and that these viruses are also temperature sensitive.For both influenza virus strains the amino acid substitutions in HA during virus adaptation are located exclusively in the HA2 chain . Rott etK460E (RKI) replicate well and infect either cell line while others grow only poorly in one cell line (e.g. S457L (RKI) in MDCK cells). Whether the substitutions that occurred during the adaptation processes alter acid stability N-glycosylation on residues 28 and/or 40 in the stem region of HA for achieving a low-pH conformation, remains to be further elucidated. Finally it should be pointed out, that other factors besides the adaptation status to a host cell line contribute to HA N-glycosylation. Besides the host cell itself - as demonstrated by Schwarzer et al. N-glycosylation patterns during vaccine production processes allows not only to investigate the impact of process modifications on antigen quality, but also offers a sensitive tool to evaluate consequences of unwanted process variations or process failures.Taken together, our data show that a MDCK cell-adapted virus seed that had not been exposed to Vero cells before, hardly produces any virus progeny in Vero cells. Nevertheless, amino acid substitutions within the stem region of HA can rescue the virus population and ensure efficient virus replication in the new host. Interestingly, for both RKI- and NIBSC-derived virus strains the glycan pattern stabilized with the first passage in the new cell line, with all passage to passage SDs values below 8.5%. The faster release of virus particles and to begin with the further increase in HA-titers during passages two to six indicates that other factors are as well involved in the adaptation process, i.e. changes in the sequence of the viral genome resulting in improved fitness of virus subpopulations. In agreement with Schwarzer et al. the HA glycan profile of Vero cell-adapted virus shows a tendency towards smaller glycan structures compared to MDCK cell-derived viruses Figure S1Stability of HA N-glycosylation patterns of influenza A/PR/8/34 . (A) The shifted overlays of normalized capillary electropherograms demonstrate reproducible HA N-glycosylation patterns over 10 successive virus passages in MDCK cells. The relative fluorescence units (RFU) are plotted over the normalized migration time in basepairs (bp). All 10 patterns exhibit the same 15 numbered main peaks between 300 bp and 420 bp. (B) Relative peak heights (RPH) of the 15 main peaks. Standard deviations (error bars) for 10 successive virus passages, range between 0.25% and 1.14%. The corresponding relative standard deviations (RSD) of low abundant peaks, ; numbered 1, 7, 9, 14, 16) range between 8.16% and 23.64%, while the RSD for all high abundant peaks (each representing more than 5% of TPH) range between 4.16% and 7.65%.(TIF)Click here for additional data file.Figure S2Impact of harvest time point on the HA N-glycosylation pattern of MDCK cell-derived influenza A PR/8/34 . (A) Relative fluorescence units (RFU) are plotted over the normalized migration time in basepairs (bp). The shifted overlay of normalized capillary electropherograms demonstrates an overall stability of the HA N-glycosylation pattern within the range from 24 hpi to 96 hpi. Both harvest time points exhibited the same 15 numbered main peaks Click here for additional data file.Figure S3Impact of harvest time point on the HA N-glycosylation pattern of Vero cell-derived influenza A PR/8/34 . (A) Relative fluorescence units (RFU) are plotted over the normalized migration time in basepairs (bp). The shifted overlay of normalized capillary electropherograms demonstrates an overall stability of the HA N-glycosylation pattern from 48 hpi to 360 hpi. The harvest time point has an impact on the RPH of the 16 numbered main peaks , exhibiting normalized migration times between 220 bp and 380 bp. (B) For each harvest time point, the relative peak heights (RPH) of all 16 dominating peaks  are represented by a column. Corresponding average values (⁃) and standard deviations (error bars) are indicated in grey.(TIF)Click here for additional data file.Figure S4Localization of substitutions during adaptation within the 3D HA-stucture. Structures are disolayed in a cartoon diagram with potential HA N-glycosylation sites highlighted by red space filled residues.  Influenza A/PR/8/34 ,  influenza A/PR/8/34 . Trimeric  and monomeric  HA molecules. (A-D) The HA1 chains are colored in pink, green and brown; the HA2 chains are colored in blue, grey and orange. The K460E mutation is highlighted in yellow, the S457L substitution by white space-filled residues. (A) Bottom (B) side and (C) top view. (D) indicates the close proximity within the monomer of these two substitutions, which are one helix turn apart from each other. (E-H) For the isolate from NIBSC the HA1 chains are colored in pink, purple and green; the HA2 chains are colored in turquoise, yellow and orange. The substitutions already present in the virus seed are highlighted by grey (Y24H), yellow (D455Y) and pink (N460D) space-filled residues. Substitutions occurring during virus adaptation are indicated by white  or yellow (D455H) space-filled residues. (E) Top,  side, (G) bottom view. The PDB entry 1RU7 and PyMOL  software was used for structure display.(TIF)Click here for additional data file.Figure S5Alignment of HA amino acid consensus sequences of two closely related influenza viruses A/PR/8/34 (H1N1). The virus seed of RKI (Amp. 3138) corresponds to a homogeneous population. Substitutions in the sequence during the virus adaptation processes are indicated in red. In contrast, the virus seed from NIBSC (#06/114) comprises various virus variants; substitutions in the sequence are indicated in green. The positions of substitutions, acquired during the adaptation processes are indicated in blue. The amino acid assembly was performed at http://services.uniprot.org/clustalw.(TIF)Click here for additional data file.Table S1Accession numbers for influenza A virus PR/8/34 sequences during adaptation from MDCK to Vero cells and back. The original virus seed was either purchased from the National Institute for Biological Standards and Control (NIBSC) or the Robert Koch Institute (RKI). The first virus passages, produced in MDCK cell culture (M1), served as virus seed for the first pasages in Vero cells. The last of five consecutive Vero cell-derived virus passages (V5) served as seed for five consecutive MDCK cell-derived virus passages, of which M6 represents the last. All sequences were generated by pyrosequencing and deposited in the GISAID EpiFlu database (www.gisaid.org).(PDF)Click here for additional data file.Table S2Overview of relative peak heights (RPH) averages and according standard (SD) and relative standard deviations (RSD). The average RPH and the respective SD and RSD of each peak (no. 1 - 25) within each experiment  are listed. The factors indicate x–fold increase (>1) or dercease (<1) of respective deviations observed during adaptation compared to the maximal deviation during controls. Factors of > 3 are defined as significantly influenced during adaptation and highlighted in blue bold numbers.(PDF)Click here for additional data file.