We describe a case of acquired infection of a strain of hepatitis E virus (HEV)with a 100% amino acid identity to the analogous region in strains of HEV circulating in a United Kingdom pig herd. This case further supports the theory that autochthonous HEV infection in industrialized countries is zoonotic. Hepatitis E virus (HEV) is a common cause of acute hepatitis in many developing countries  M; no serologic evidence indicated active infection with Epstein-Barr virus, cytomegalovirus, or hepatitis A, B, or C. Results of a liver ultrasound were normal, and serologic evidence did not indicate autoimmune or metabolic liver disease. One month later, she felt better, was no longer jaundiced, and results of her liver function tests were generally normal. A diagnosis of acute hepatitis E was made.Total RNA was extracted in Trizol  from a blood sample drawn from the patient at the time she sought treatment. Template cDNA from the blood extraction was prepared by a reverse transcriptase step, according to standard protocols. A nested PCR was used to detect HEV from the prepared cDNA samples. This assay used degenerate oligonucleotide primers to amplify a 348-bp fragment of open reading frame 2 (ORF2) of HEV as described by Meng et al. (1997) .Amplicons of correct size were excised from 2% agarose gels, purified with QIAquick Gel Extraction Kit , and sequenced with Big Dye Terminator Cycle Sequencing Ready Reaction . Sequences comprising 304 bp of HEV ORF2 were assembled by SEQMAN (DNASTAR) and phylogenetic analysis was performed using PHYLIP (ver 3.6) with NEIGHBOR. Bootstrap confidence values were calculated by using SEQBOOT and CONSENSE (available from Analysis of the 304-bp nucleotide sequence obtained between the primer sequences showed 16 and 32 nucleotide differences from the two U.K. pig strains 1-40 and 14-P354, respectively. However, all of these differences were silent when the gene sequence was translated into amino acids; the putative amino acid sequence of the virus detected in the patient had 100% identity with that of the two U.K. pig strains. Based on the nucleotide data the phylogeny of the three strains was determined , 2, showHEV was discovered in pigs as recently as 1997 (


As a model for HTLV, reverse genetics can be used in the bovine leukemia virus (BLV) system to identify important mechanisms of viral persistence and pathogenesis. The question addressed here pertains to the role of glycans bound to the BLV envelope glycoprotein (SU gp51). Addition of carbohydrates to the BLV SU potentially creates a structure called « glycan shield » that confers resistance to the virus against the host immune response. On the other hand, glycosylation can also modulate attachment of the virus to the cell membrane.To unravel the role of SU glycosylation, three complementary strategies were developed: pharmacological inhibition of different glycosylation pathways, interference with glycan attachment and site-directed mutagenesis of N-glycosylation sites in an infectious BLV provirus. Collectively, our results demonstrate that glycosylation is important for the Gp51 maturation process, for virus infection in vitro and for infectivity in vivo.


Plant leucine-rich repeat receptor-like kinases (LRR-RLKs) are receptor kinases that contain LRRs in their extracellular domain. In the last 15 years, many research groups have demonstrated major roles played by LRR-RLKs in plants during almost all developmental processes throughout the life of the plant and in defense/resistance against a large range of pathogens. Recently, a breakthrough has been made in this field that challenges the dogma of the specificity of plant LRR-RLKs.We analyzed ~1000 complete genomes and show that LRR-RK genes have now been identified in 8 non-plant genomes. We performed an exhaustive phylogenetic analysis of all of these receptors, revealing that all of the LRR-containing receptor subfamilies form lineage-specific clades. Our results suggest that the association of LRRs with RKs appeared independently at least four times in eukaryotic evolutionary history. Moreover, the molecular evolutionary history of the LRR-RKs found in oomycetes is reminiscent of the pattern observed in plants: expansion with amplification/deletion and evolution of the domain organization leading to the functional diversification of members of the gene family. Finally, the expression data suggest that oomycete LRR-RKs may play a role in several stages of the oomycete life cycle.In view of the key roles that LRR-RLKs play throughout the entire lifetime of plants and plant-environment interactions, the emergence and expansion of this type of receptor in several phyla along the evolution of eukaryotes, and particularly in oomycete genomes, questions their intrinsic functions in mimicry and/or in the coevolution of receptors between hosts and pathogens. Receptor-like kinases (RLKs) are plant-specific transmembrane (TM) receptor kinases (RKs) that are closely related to the Pelle proteins, a family of animal cytoplasmic kinases. These RLK/Pelle proteins are involved in host defense against a range of pathogens and are also key regulators of many developmental processes in both plants and animals -4. In anIn this report, we show that LRR-KD subfamilies have been reinvented in several eukaryotic genomes outside plants. Moreover, the evolutionary history of these LRR-RKs is comparable to the one described for the LRR-RLK plant subfamilies.Monosiga brevicollis (a choanoflagellate), Chlorella variabilis NC64A  and several stramenopiles   contain up to 34 LRR-RK genes per genome. The step-by-step procedure used to detect the LRR-RK genes is described in Additional file As LRRs and KDs are present in all genomes, we searched for the presence of structurally related LRR-RLKs in non-plant lineages. We analyzed 884 bacterial, 50 archaeal and 77 eukaryotic genomes to identify LRR-containing RKs that were structurally related to plant LRR-RLKs Figure . Additio) Figure . Some ofEctocarpus, Chlorella, Monosiga and oomycete LRR-RK genes form four separate lineage-specific clades within the eukaryotic RKG. Because we did not observe incongruence between the phylogenetic and the species trees, we excluded the hypothesis of lateral transfer of the LRR-RK genes between species. However, because the tree is not fully resolved, we cannot rule out the possibility that the KDs of the LRR-RKs are more closely related to one of the RKG subfamilies. Nevertheless, with regard to the combination of the three modules  to form new genes that are structurally related to LRR-RLKs, the most parsimonious hypothesis, based primarily on the species tree  in the public databases  from 2 sources (JGI and NCBI). We also downloaded all of the available bacterial and archaeal genomes from NCBI. When LRR-RK sequences were found in one genome, we downloaded all of the available complete genomes in that phylum. Thus, the complete proteomes of 77 eukaryotic, 50 archaeal and 884 bacterial species have been downloaded from their respective databases. See Additional file http://www.cbs.dtu.dk/services/SignalP/ and TMHMM http://www.cbs.dtu.dk/services/TMHMM/ websites, respectively, hosted at the Center for Biological Sequence Analysis, Technical University of Denmark ; AtCDC2a ; AtCPK7 ; AtCKA1 ; AtCTR1_Raf ; AtAME2 ; AtMKK3 ; AtMEKK1 ; AtNAK ; AtNPH1 ; AtPVPKlikePK5 ; AtGSK3b ; AtGSK3i ; AtSnRK2 ; AtMPK1 ; AtS6KlikePK1  and AtTousled . The accession numbers for Homo sapiens are as follows: hAXL ; hRYK ; hTRKalpha ; hMuSK ; hKLGlikePTK7 ; hIR ; hLTK ; hRET ; hTIE1 ; hPDGFRbeta ; hVGFR1 ; hTousledLK1 ; hMAPKKK1 ; hCLK1 ; hMAPK1 ; hCDK3 ; hCKIalpha2 ; hCaMK1 ; hCK2a ; hGRK6 ; hEGFR ; hFGFR2 ; hHGFR ; hEPH ; hDDR ; hRaf1 ; TGF beta receptors, hTGFbRI  and hTGFbRII ; hIRAK1  and hMAPKK1 . Additional accession numbers are as follows: mCKIalpha  for Mus musculus; xtPELLE  for Xenopus tropicalis; drIRAK1  for Danio rerio and dmPELLE  for Drosophila melanogaster. The accessions of representative Arabidopsis thaliana LRR-RLK genes in each subfamily are as follows: [TAIR: AT4G29180] for LRRI, AtNIK1 for LRRII [TAIR: AT5G16000], AtIMK3 for LRRIII [TAIR: AT3G56100], [TAIR: AT2G45340] for LRRIV, AtSCM_SUB for LRRV [TAIR: AT1G11130], [TAIR: AT1G14390] for LRRVI-1, [TAIR: AT5G41180] for LRRVI-2, [TAIR: AT2G24230] for LRRVII, [TAIR: AT1G06840] for LRRVIII-1, [TAIR: AT3G14840] for LRRVIII-2, AtTMK1 for LRRIX [TAIR: AT1G66150], [TAIR: AT3G28450] for LRRXa, AtBRI1 for LRRXb , AtFLS2 for LRRXII [TAIR: AT5G46330], AtFEI1 for LRRXIIIa [TAIR: AT1G31420], AtER for LRRXIIIb [TAIR: AT2G26330], [TAIR: AT3G14840] for LRRXIV and AtRPK1 for LRRXV [TAIR: AT1G69270]. We followed the subfamily nomenclature of a previous report [The accession numbers for LRR: leucine-rich repeat; RLK: receptor-like kinases; RK: receptor kinase; LRR-RLK: leucine-rich repeat receptor-like kinase; LRR-RK: leucine-rich repeat receptor kinase; TM: transmembrane; KD: kinase domain; ECD: extracellular domain; RKG: receptor kinase group; RTK: receptor tyrosine kinase; RSK: receptor serine/threonine kinase; EST: expressed sequence tag; TM: transmembrane domain; SP: signal peptide; hpi: hours post inoculation.The authors declare that they have no competing interests.AD, NG and CP designed the experiments and discussed the results. AD and GD performed the bioinformatics analyses. AA and MG performed the qRT-PCR experiments. AD, NG, EG and CP drafted the manuscript. All of the authors have read and approved the final manuscript.Genomes analyzed.Click here for fileStep-by-step procedure to determine the number of LRR-RKs per genome. The last column of the table shows the number of proteins that we considered to be LRR-RKs in our analysis. The accession number of each gene is listed in Additional file Click here for fileMonosiga, Chlorella and Ectocarpus LRR-RKs and closely related receptors lacking LRRs and/or TM domainsStructural features of the oomycete, . All sequences except one sequence of Phytophthora ramorum (Pr81779) and one sequence of Phytophthora sojae (Ps136026) have been used for the phylogenetic analysis presented in Figure Monosiga brevicollis (Mb34608) and one sequence of Chlorella variabilis NC64A (Ch136834) produced inconsistent results in the phylogenetic analysis and do not appear in the tree. These sequences may represent misannotated proteins or pseudogenes. Abbreviations: S, Saprolegnia parasitica; Pi, Phytophthora infestans; Pr, Phytophthora ramorum; Ps, Phytophthora sojae; Pu, Pythium ultimum; Mb, Monosiga brevicollis; Ch, Chlorella variabilis NC64A; Esi, Ectocarpus siliculosus; SP, signal peptide; KD, kinase domain; LRRs, leucine-rich repeats; +, presence; -, absence. See Materials and Methods for details about predicted SPs, LRR numbers, TM domains and KDs.ae Ps13606 have beClick here for fileAlignment of LRR-RKs and reference eukaryotic KDs. The KDs of LRR-RKs from Ectocarpus siliculosus (Esi), Phytophthora ramorum (Pr), Phytophthora sojae (Ps), Phytophthora infestans (Pi), Saprolegnia parasitica (S), Pythium ultimum (Pu), Chlorella variabilis NC64A (Ch) and Monosiga brevicollis (Mb) were aligned with reference eukaryotic kinases from human (h), Arabidopsis thaliana (At), Drosophila melanogaster (dm), Danio rerio (dr), mouse (m) and Xenopus tropicalis (Xt). The positions of the 12 kinase subdomains are shown in roman numerals.Click here for fileDetails of the maximum likelihood tree representing the phylogenetic relationships among the LRR-RK KDs and other eukaryotic kinases. A. General view of the tree presented in Figure Monosiga brevicollis ; Chlorella variabilis NC64A ; Ectocarpus siliculosus  and oomycetes . Oomycete LRR-RKs, Monosiga LRR-RKs, Chlorella LRR-RKs and Ectocarpus LRR-RKs are included in the monophyletic receptor kinase group  consisting of the plant receptor-like kinase (RLK) and animal cytoplasmic Pelle , receptor serine/threonine kinase , receptor tyrosine kinase  and Raf  proteins. The RKG members are distinct from other the eukaryotic kinases. Note that the tree is not fully resolved. The addition of more RKs lacking LRRs to this phylogenetic analysis did not improve the resolution of the tree (data not shown); similarly, neither did the addition of non-RKs from the oomycete species (data not shown). Branch support values are shown at the nodes. B. A detailed view of the oomycete clade. In the oomycete LRR-RK subfamily, some subdivisions are Saprolegnia or Phytophthora specific (black boxes), suggesting the lineage-specific amplification by duplications in the Saprolegnia and Phytophthora genomes. Three subgroups contain both of these lineages with or without Pythium (orange boxes), suggesting that at least three genes were present in the last common ancestor of these species. Oomycete genes with evidence of expression (expressed sequence tags) are indicated with a plus sign (details in Additional file C. A detailed view of other clades.Click here for fileNumber of gains and losses of oomycete LRR-RK genes in orthologous groups. We counted the number of genes gained and lost for each orthologous group defined in the oomycete clades of the phylogenetic tree (Additional file A. Orthologous groups containing Saprolegnia, Pythium and Phytophthora species, B. orthologous groups containing only Pythium and Phytophthora species and C. orthologous groups containing only Phytophthora species. The number on the first branch represents the number of genes present in the last common ancestor. Numbers preceded by a plus sign are the number of genes gained; numbers preceded by a minus sign are the number of genes lost.Click here for fileOomycete LRR-RK subgroups with evidence of expression. Abbreviations: S, Saprolegnia parasitica; Pi, Phytophthora infestans; Ps, Phytophthora sojae; Pp, Phytophthora parasitica; Pp, Phytophthora parasitica; EST, expressed sequence tags.Click here for filePhytophthora parasitica LRR-RKs with evidence of expression. Abbreviations: Pi, Phytophthora infestans; EST, expressed sequence tags.Click here for file


In January 12, 2010, a 7.0 magnitude earthquake shook Port-au-Prince, Haiti. The massive disaster made it difficult for local Haitian community officials to respond immediately, leaving the country reliant on foreign aid and international and non-governmental relief organizations. This study explores the effectiveness of various supports that were made available to health and social service providers in Haiti, by focusing on their lived experiences pre-deployment, on-site and post-deployment. The paper provides a qualitative exploration of participant perceptions with respect to the success of their performance in response, and relevant literature describing the various supports provided to health and social service providers responding to disasters. Methods: A single, semi-structured interview was conducted with Canadian health professionals (n=21) who deployed to Haiti during the time of, or after, the 2010 earthquake. The study uses Strauss and Corbin’s structured approach to grounded theory to identify main themes and relationships in the interviews. Results: The interviews indicate that training, and psychological and emotional supports for health and social service providers require improvement to enhance the experience and effectiveness of their work. Conclusions: Findings indicate that supports are most effective when they are tailored to the volunteers. The paper highlights future research stemming from the grounded theory findings. Specifically, the purpose of this research is to understand and evaluate the types of supports provided to health and social service workers deployed to Haiti in response to the 2010 earthquake, and to learn from these experiences the means by which to design and improve support systems for foreign aid workers.Participants were recruited from a variety of organizations including non-governmental organizations, universities, hospitals, the Canadian government, as well as faith-based organizations. Independent responders, and those in the military, were also interviewed, to provide contrast to the predominantly NGO-based sample and test the boundaries of the proposed findings. The inclusion criteria for the study required the individual to have served as a health or social service responder in Haiti during, or following, the 2010 earthquake. The research team interviewed health and social service responders deploying to Haiti up to 1 year after the disaster. Following university ethics approval, n=21 participants were recruited via email notices using purposeful and snowball sampling. The interviews took place between December 2011 and February 2012. The research team observed that many individuals responding within the first 3 months of the disaster were required to stay for a period of 2-3 weeks (which is considered a standard length of deployment through many large NGOs). To determine how the supports varied following the acute crisis of the earthquake, participants who deployed later in the year, as well as those who deployed for longer periods of time, were also recruited. These interviews were used to test the boundaries of the experiences of those who deployed immediately following the disaster in 2010.Participant demographics are outlined in Table 1. Each participant was asked to participate in a single open-ended interview, 1 hour in duration, conducted by the primary author. The interview guide included questions pertaining to: training and preparation, systems and relationships in organized disaster response, stressors, roles, and the maintenance of an organized response system. A copy of the interview guide is attached in Appendix 1.NVivo9 qualitative software. The use of “thick descriptions”, detailed descriptions that present the context and emotion of the quote, were crucial to the validity of this study,,All data were transcribed, coded and analyzed using Contextual and Intervening Conditions1) Causal Conditions2) Strategies3) Consequences4) The interview analysis process included the development of higher level pattern codes from the interview data to determine relationships between the categories and identify emergent themes. Tables 2 and 3 list all supports available to health responders. To determine which supports were most common, frequency scores for each individual type of support, by organization, were calculated. These tables were used as a supplement to the grounded theory analysis to highlight the supports available, as well as identify gaps in the individual supports. “Our first priority is not to our staff, as awful as that sounds, our first priority is to the work and to the end beneficiaries ...the staff care is important but often it comes second and third...when we get around to it...” (Canadian Government)Need for Supports for Canadian Health and Social Service Providers who responded to the disaster in Haiti, as well as the contextual, causal and consequential conditions that stem from these supports are illustrated in Figure 1. The Context refers to the societal, organizational and personal attributes and conditions that were present during the response. The first contextual factor, the country context, refers to the context within Haiti and the Haitian society at the time. Secondly, the context of the various relief organizations was explored to determine the organizational and coordinating factors that affected the response context. Finally, the personal context of the individuals responding to this disaster was analyzed.The study results are summarized below according to the aforementioned categories. The central, or core, phenomenon of the study is identified as the Causes of the central phenomenon emerged and were integrated with the need for supports along two main themes. The first cause for support related to unclear expectations, which led to feelings of uncertainty and confusion, fear, lack of communication and awareness, and Post-Traumatic Stress Disorder. These feelings stemmed from a variety of causes, including unclear roles on-site, disorganization within teams and between organizations, and insufficient safety protocols and resources, training, and psychological and physical supports. The second main cause of the need for support relates to participants’ perceptions that influence their experiences, attitudes and personal initiatives. In response to the problems identified in the context and causes, organizations employed the use of various strategies to support their delegates. Analysis of the data resulted in the identification of two main categories of support in Haiti, Instrumental Supports, and Communication and Leadership Supports.As seen in Figure 1, the Consequences of the response refer to the impacts, as perceived by the participants. These consequences were developed in relation to the context, causes and strategies, to highlight the strengths and weaknesses of the various supports present.Finally, the Contextual and Intervening ConditionsAlready known as the ‘nation of NGOs’, the country context in Haiti was unstable prior to the earthquake, making the 2010 disaster both unique and challenging for responders. The earthquake created a barrier to food security, road access and phone communication, making it difficult for responders to assess the situation on the ground and report back to appropriate authorities. During the first few weeks after the earthquake, the ground was also unstable, leading to aftershocks that further destroyed the country’s already weakened infrastructure. As a result, responders were faced with the challenge of responding in a situation where they were forced to perform emergency procedures with little supplies, while coping with crumbling infrastructure, frequent tremors in the ground and harsh conditions, such as extreme heat. This context made logistical challenges a major frustration for the responders. Because much of the work was carried out in field-like hospitals, the logistical capacity of organizations fluctuated, depending on size and experience.Finally, the culture of the Haitian people also brought uniqueness to the country context. Many of the participants interviewed were amazed at the positive attitudes and resilient spirits held by Haitians during this difficult time.Upon arrival, many of the delegates witnessed a system in chaos, void of strong leadership of coordinating and government bodies, which had become compromised in the earthquake. Because there was no clear chain of command during the response, the initial result was disorganization and a lack of coordination. There were over 200 organizations that were registered in the Pan American Health Organization (PAHO) health cluster; however, project coordinators witnessed that there were few large organizations that had representatives and a strong voice at these ‘health cluster’ meetings. Some of the participants who attended the cluster meetings found the atmosphere to be, at times, hostile and uncoordinated. The notion of “egocentrism” came up repeatedly in the interviews. Larger organizations were described as territorial and preferring to work alone, perceiving smaller response organizations as peripheral. Some participants indicated there was pressure among independent volunteers, small NGOs, and faith based organizations to conform to the practices of larger organizations. Causal ConditionsThe aforementioned context caused a need for a variety of physical and logistical supports. In addition to dealing with environmental challenges and crumbling infrastructure, many delegates fell sick while in Haiti, with some only recovering near the end of their deployment. Many participants stated that their roles were not clearly outlined pre-departure. Undefined roles often led to negative experiences, particularly when delegates felt their expertise was not being utilized. Some of the most commonly reported challenges included lack of clarity of roles, disorganization, and lack of leadership.Finally, there was a need for psychological and emotional supports. One general attitude shared by many of the delegates was a feeling of ‘invincibility’. The study found that many healthcare professionals tend not to complain or ask for help, as they are accustomed to being ‘okay’ and being ‘the boss’. As a result, they were reluctant to admit when they were in need of emotional or psychological supports. This often led to stress, fatigue, frustration, and burn-out.StrategiesStrategies refer to the various supports that were provided to the responders in Haiti. These were classified into two major categories 1) Instrumental Supports and 2) Communication & Leadership Supports. The first of these categories, Instrumental Supports, refers to: (i) training strategies, (ii) safety and security supports, (iii) psychological and emotional supports, as well as (iv) physical supports. The second category, Communication & Leadership Supports, refer to (i) team dynamics and effective team leaders, (ii) communication between the field and headquarters, and (iii) communication with the media.The Instrumental Support StrategiesAccording to one delegate from a large NGO, the pre-deployment period to Haiti was very short. Some organizations chose to waive their typical two-week training period, due to the extent of damage from the earthquake, and the extreme need for immediate help. As a result, many delegates were sent to Haiti quickly, sometimes making the decision to leave within 24 hours. As a result, their training and briefing, particularly within the first few days, was not extensive. The interviews highlighted some forms of training that would have been an effective pre-departure strategy. The first of these was training during times of ‘normalcy’, to produce well-prepared responders that could be quickly deployed in an emergency. In addition, medical training would have been an effective pre-departure strategy, as many physicians and surgeons struggled to perform procedures uncommon to their specialty or rare to Canada. For the purposes of this paper, 'medical training' refers to training for specific medical procedures pertinent to the affected country (example: 'tenting' for cholera). Another type of training that was prominent in larger NGOs was Emergency Response Unit (ERU) training. This type of training provided responders with hands-on skills that allowed them to build and effectively run field-hospitals in a disaster zone. Larger organizations and NGOs, particularly those with previous experience in disaster response, seemed to be more regulated in their safety protocols. According to one participant from a large NGO,“You make zero decisions about that [safety], you’re just told what to do by [organization name] and they’re quite rigid about that, as opposed to many other organizations where people are independent.” (NGO)One of the most essential tools for maintaining safety was the provision of communication devices to ensure the location and safety of the delegate was not compromised. Other security supports included transportation in specialized vehicles. It was crucial that logistics and timing were accurate in order to avoid accidents and safety threats. Organizations needed to ensure that they were able to remove their delegates from the country if there was a need to do so.There were many strategies available to address the third category of instrumental supports, focused on emotional and psychological needs. The context of Haiti made the need for these supports even greater. Supports varied greatly, depending on the individual’s organization, team leader and colleagues. One of the on-site strategies employed was the appointment of chaplains or counsellors who lived and worked with the delegates. These individuals were available to address any issues regarding stress or trauma experienced by the delegates, and often provided services in an ad-hoc fashion. Other organizations chose to employ on-the-spot critical incident clinical debriefings or morale and welfare officers when delegates had a difficult time with the country context or with patients. Program managers found that many responders did not ask for emotional and psychological supports. Other informal psychological and emotional health supports took the form of regular communication with family and loved ones back home. As stated by one participant, “For them [delegates] to be able to call their families...or just to hear their kids, was really important, so whoever you go with needs to be able to set up some kind of email or voiceover or satellite phone communication with home and make it available to their workers and their volunteers...if you go with a group that doesn’t have the infrastructure resources to set that up, it can be really tough.” (Faith-based organization)The context of Haiti was very difficult, and many larger NGOs chose to utilize shorter deployment times, approximately 2-3 weeks, to protect their responders. Relief coordinators began to notice this trend, and attempted to adjust their deployment periods accordingly. When shorter deployment periods were not feasible, alternative strategies organizations chose to employ to combat burnout were rest and relaxation breaks (R&R). Some larger organizations provided delegates with the resources for respite in the Dominican Republic or Canada.Finally there was the need to provide physical supports for the delegates. Larger organizations with greater experience in disaster response were typically well prepared to provide their delegates with the appropriate physical resources, such as food, water, and shelter.Communication and Leadership Support StrategiesParticipants stated the importance of having teams on site. As stated in the following participant quote,“You go with this team and they become as close as your family when you’re there...they’re all you have and it’s amazing the relationships that develop, are like family.” (NGO)make sure the novice, the rookies, have got a mentor.” Some organizations chose to apply this technique pre-departure so the support would carry out both on-site and post-deployment. Organizations that were able to introduce delegates pre-departure found this to be an asset on-site, as complementary skills emerged within the teams. In addition to these more formalized strategies, there were ‘unwritten’ rules that were present on-site, such as being inclusive of new volunteers.One of the team strategies was the implementation of a ‘buddy system’. As stated by one nurse, “The second crucial element in response teams was the presence of an effective leader who possessed strong communication skills. In addition to providing encouragement and instruction, the team leader was responsible for ensuring that all of the delegates were aware of their roles and surroundings and were prepared to deal with emergency situations.One of the strategies for effective communication included having lines of communication between individuals at headquarters and those in Haiti. Having individuals of equal rank and position communicate allowed for the establishment of clear expectations for those at headquarters and support for those on-site. As stated by a physician,“Some administrator in Ottawa can tell me what they think I’m doing, a physician on the ground can tell me exactly what I’m doing. And there’s always a huge difference between what the head office thinks you’re doing and what the people on the ground know you’re doing.” (NGO)Finally, accurate media coverage is very important to the relief response, as it is a driver of whether new delegates will volunteer to deploy to a disaster. The Haitian earthquake received extensive media coverage and attention. However, the situation portrayed in Haiti, particularly once the initial ‘response hype’ had settled, was often inaccurate and downplayed the efforts and effectiveness of the response, which led to much frustration among existing relief workers. Consequences Consequences are used to determine the effects of the central phenomenonneed for supports led to both positive and negative consequences. The Consequences outline the impacts of the various strategies, as perceived by the participants. Positive perceptions arose when an organizational strategy of instrumental or communication and leadership supports was tailored to an individual responder.According to Strauss and Corbin (1990), Organizational Supports and Gaps. The interview information provided feedback from participants on their perceived gaps in organizational support. Table 2 is a summary of all of the supports by type received, and considered as particularly effective by the participants. These included Training Supports , Safety and Security Supports , Psychological and Emotional Supports , Physical Supports , Teams and Team Leaders , Communication between the Field and Headquarters, Communication with the Media and Accurate Media Coverage. Support received, or “SR” was placed next to every support cited in the participant interviews. If a participant found the support to be particularly effective, the mark was replaced by “ES” for effective support. Finally, “NM” or not mentioned, was placed in the table if the organization was known to provide a particular support, yet the support was not mentioned in the interview. An overall tally of each provided support was calculated, by organization, in order to distinguish the supports most frequently provided to the responders.According to the data, 67% of participants (n=14 of 21) were given training supports, safety and security supports were provided to 86% of the delegates (n=18 of 21), and some form of psychological and emotional support was provided to 100% of the participants. Physical supports were provided to 81% of the participants (n=17 of 21) and 76% of participants (n=16 of 21) reported having been given team supports and had effective team leaders. Finally, 57% of participants (n=11 of 21) were provided with communication portals between the field and headquarters and 24% (n=5 of 21) reported effective communication with media and accurate media coverage.Table 3 presents a summary of all the supports the participants considered needed improvement and were thus deemed insufficient. The need for improved training supports was the biggest identified gap (71% of participants). In particular, participants highlighted the need for improved pre-departure training and briefing. Improved psychological and emotional supports were also strongly identified as a need by 62% of participants. In particular, the need for debriefings and follow-ups were recommended by 57% of the participants. Another support strongly recommended was the need for logistical support, including organization of visas, passports, etc. Finally, 57% of participants expressed a need for improved teams and team leadership. The consequences of some of these supports are highlighted below.Consequences of Instrumental Support Strategies. Participants found that training during times of normalcy was very effective, in contrast to organizations that waited until the disaster struck to train their delegates. This left a very small window of opportunity for effective training, thereby increasing stress and disorganization. As one participant states,It’s very hard to show someone what to expect even though you try, so it’s a difficult task to brief someone...people can only absorb so much when they come down...it doesn’t translate to being well prepared when you come.” (Faith-based organization)“The second type of instrumental support that was provided by organizations was safety and security. Most delegates were comforted by the presence of safety personnel and high security, and those who deployed with organizations that did not provide these supports cited this as a gap. However, some delegates who entered the response with background experience in disaster response, and who were in Haiti for prolonged periods of time, eventually found these security supports to be stifling and recommended that organizations find a balance in their safety provisions. One program manager recalls,I know volunteers...some of them were a little shocked at the level of security and wondered ‘why do we have so much security, is there something we need to be worried about here?’ And that got them more upset. And then some of them were really relieved to see all of the security.” (Faith-based organization)“need of help’. Team dynamics and emotional support from colleagues were also crucial. As stated by one participant who spoke about using her husband for emotional support regarding her experience with Katrina,The third instrumental strategy of support was psychological and emotional supports. The data indicated that responders had a hard time accepting support from mental health personnel, unless they had developed a personal rapport with them. Many responders shared an attitude of “invincibility”, which amplified the need to develop strategies that would allow delegates to utilize psychological supports without feeling stigmatized as ‘psychologically weak’ or ‘in “I quickly learned that wasn’t a good idea because my husband worried about me. He didn’t know that this [anxiety] was normal and that it would pass...he just wanted to make it go away and his response was ‘you shouldn’t do this stuff anymore’. So I learned not to do that, because it’s not fair to him either, and then you also bring up the issue of secondary traumatisation.” (Faith based organization)She stated the importance of having a friend on-site who was able to relate to the experiences and provide her with that much needed support.A final strategy that was used to promote psychological and mental health was to have defined periods of deployment. Shorter periods of deployment were recommended to those who responded immediately after the disaster. The organizations were faced with choosing a deployment period that was long enough to allow for effective work and continuity, but short enough to ensure that personnel did not burn out whilst in Haiti. This dilemma was highlighted by a few of the participants,“Some [delegates] definitely should have gone home...that’s the catch 22...ten days wasn’t enough to do what you wanted to do. Two or three weeks would have been optimum...but I can see how healthy it would be for people to take a break after two weeks even if it’s heading back to North America for a week and then re-deploying” (Faith based organization)Upon completing their deployment, many participants experienced grief and had difficulty resuming their normal routines. Much of this was attributed to feelings of guilt for leaving the disaster situation, combined with fatigue.“I went back to Canada for a reprieve...I get into the airplane and I look back and I saw the unfinished work and suddenly I had a feeling as if I’m abandoning those people and it was awful. So I spent the whole time flying from Haiti to Ottawa crying, because [I’d] abandoned them” Finally, many of the participants shared the common difficulty of returning to their demanding vocations shortly after their return. One of the organizational strategies used to address these feelings of stress was debriefing and follow-ups. Many participants believed that follow-ups not only allowed them the option of emotional support, but gave organizations the opportunity to improve, learn from their mistakes, and address any gaps in the supports that were provided. These participants recommended that debriefings take place immediately upon return. As stated by one participant,“Most people probably say ‘I don’t need to’ [debrief] but it might be a precaution...when there’s so much death and destruction around you...something like that should be made probably mandatory.” (Canadian Government)The final type of instrumental strategy was the provision of physical supports. Many participants who deployed with smaller organizations recalled having to bring all of their own medical equipment, drinking water and resources to Haiti. Participants who did not have logisticians to organize physical resources and provide guidance and support to medical best practices reported feelings of frustration.Consequences of Communication and Leadership StrategiesTeam dynamics were a critical element of the response. Effective team leaders ensured delegates were provided with a clear sense of purpose and defined roles and tasks, to avoid confusion and frustration. As stated by a participating surgeon, “The day I arrived, they informed me a) there would be no anaesthetist available, and b) there would be no elective surgery to be performed, so the day I got there, they told me that they didn’t need me. I ran out of patients, saw people and advised them...but we didn’t do nearly as much surgery as we could have.” (NGO)Another strategy that was important to delegates was the importance of communication between those on-site and headquarters in Canada. Lack of communication is stressful not only to the delegates on-site but to those working in headquarters. As stated by a program manager,“...all the communications were shut down and it wasn’t possible to contact the [on-site] people in our team...from a head office perspective, it was very hard for us not knowing if they were alive or if they were dead, so it was a very stressful situation.” (NGO),,,,,According to our results, 71% of participants identified a need for improved training supports, particularly pre-deployment training and briefing, making this the biggest identified gap in this study. The literature echoes these findings, indicating that there is currently no universal standard when it comes to the availability and usage of standard training guidelines, making training supports highly varied, dependent on the affiliated organization,,,,,Findings show that while all of the participants reported being provided with some form of psychological and emotional support, it was identified as one the least effective supports. This study supports the literature that suggests that psychological and emotional supports are crucial at the pre-departure, on-site and post-deployment phases of the responsewillingly use or seek out these supports. Therefore, while organizations should be careful not to pressure responders to disclose information, the recommendation is that program managers appoint licensed, experienced psychologists to place follow-up phone calls to delegates upon their return. This follow-up should be used as an opportunity to thank responders and make them aware of any supports available to them post-deployment.Fifty-seven per cent of participants in this study indicated they would have liked a debriefing or follow up almost immediately upon return, which differs from some of the popular literature. According to Suzuki, Nakajima & Kim (2012), “debriefing should not be conducted immediately after disasters because of the risk of making [delegates] relive the experience. Some even get hurt after talking about work that they should have done” (p.18). They recommend that the supports be made available for when a delegate is willing to have this conversationFinally, organizations should recognize the importance of being self-sufficient and ensure that the details of their responders’ deployments are well planned. Organizations that were found to be the most resilient in Haiti typically provided their delegates with field hospitals and housing. They also highlighted the importance of logistics, such as flights, transportation, visas, passports, vaccinations. Finally, these, typically larger, organizations, often had the capacity to allocate budgets for food and water for responders, equipment repair, and emergency medical support for responders.These findings open the door for future researchers to explore the individual components of each of the support categories and further describe the relationships provided in the model. Primarily, researchers should investigate the identified supports that were provided to the responders in Haiti, using the results and consequences to improve gaps in the current systems of support.This study has certain limitations that should be acknowledged. The demographic breakdown of the participants was not equally representative by occupation. A large number of participants in the sample were physicians (n=10) or from large organizations (n=10), which may have affected the findings. Future researchers should use the results of this preliminary study to conduct a follow up study, using a bigger sample size, which would allow organizations to better direct their focus according to their volunteer demographic and generalize findings. Secondly, it is important to remember that Haiti was an extensive disaster with a unique context. Regardless, all of the identified supports can be tailored to suit the particular context of the emergency, as well as the needs of the responders.


The core promoter is the region flanking the transcription start site (TSS) that directs formation of the pre-initiation complex. Core promoters have been studied intensively in mammals and yeast, but not in more diverse eukaryotes. Here we investigate core promoters in oomycetes, a group within the Stramenopile kingdom that includes important plant and animal pathogens. Prior studies of a small collection of genes proposed that oomycete core promoters contain a 16 to 19 nt motif bearing an Initiator-like sequence (INR) flanked by a novel sequence named FPR, but this has not been extended to whole-genome analysis.Phytophthora infestans, the potato blight pathogen. The motifs corresponded to INR, FPR, and a new element found about 25 nt downstream of the TSS called DPEP. TATA boxes were not detected. Assays of DPEP function by mutagenesis were consistent with its role as a core motif. Genome-wide searches found a well-conserved combined INR+FPR in only about 13% of genes after correcting for false discovery, which contradicted prior reports that INR and FPR are found together in most genes. INR or FPR were found alone near TSSs in 18% and 7% of genes, respectively. Promoters lacking the motifs had pyrimidine-rich regions near the TSS. The combined INR+FPR motif was linked to higher than average mRNA levels, developmentally-regulated transcription, and functions related to plant infection, while DPEP and FPR were over-represented in constitutively-expressed genes. The INR, FPR, and combined INR+FPR motifs were detected in other oomycetes including Hyaloperonospora arabidopsidis, Phytophthora sojae, Pythium ultimum, and Saprolegnia parasitica, while DPEP was found in all but S. parasitica. Only INR seemed present in a non-oomycete stramenopile.We used expectation maximization to find over-represented motifs near TSSs of Phytophthora transformants. The association of the INR+FPR motif with developmentally-regulated genes shows that oomycete core elements influence stage-specific transcription in addition to regulating formation of the pre-initiation complex.The absence of a TATA box and presence of novel motifs show that the oomycete core promoter is diverged from that of model systems, and likely explains the lack of activity of non-oomycete promoters in  Growth, development, and responses to environmental signals require the proper level and timing of transcription. In eukaryotes, the DNA binding sites for the transcription machinery are named enhancers, proximal or distal elements, or core promoter motifs depending on their location and function . The corMost of what is known about core promoters comes from yeast and animals . The firArabidopsis and rice were shown to use a TATA box, INR, and plant-specific motifs such as the pyrimidine-rich Y-patch [Trichomonas appears to employ an INR and two novel elements but not a TATA box, while Plasmodium core promoters lack any defined sequence element. In the latter, initiation sites appear to be determined by the physicochemical properties of DNA, which may also play roles in all eukaryotes [In other eukaryotic kingdoms, the nature of core promoters is just starting to be understood. Most is known from plants, where  Y-patch ,7. In thkaryotes ,9.Phytophthora infestans, which suggested divergence of the transcriptional apparatus of oomycetes [′ end followed by an approximately 9 nt sequence named Flanking Promoter Region or FPR [This paper focuses on core promoter elements in oomycetes, a group within the kingdom Stramenopila that includes many important plant and animal pathogens. Although most oomycetes superficially appear fungal-like, they contain many characteristics distinct from fungi which is consistent with the divergence of stramenopiles from opisthokonts early in the eukaryotic radiation . Little omycetes . Researcn or FPR ,13. Somen or FPR ,15.Phytophthora infestans and relatives, by using whole-genome analyses to characterize known and novel motifs and assess their association with gene expression patterns. We report that INR and FPR motifs usually occur separately, but come together as an INR plus FPR “supramotif” in 10-15% of promoters where they are linked to higher than average mRNA levels, an increased propensity towards developmental regulation, and gene functions related to pathogenesis. We also identify and functionally test a new motif, DPEP, which tends to associate with housekeeping genes, but fail to obtain convincing evidence for the presence of a TATA box-like sequence. These data help illuminate how the oomycete transcriptional apparatus has evolved, and may be useful for predicting genes within their genomes and optimizing transgene expression.Here we address the structure of core promoters in P. infestans genes. This involved mapping expressed sequence tags (ESTs) against all 17,797 gene models [′ termini that mapped upstream of the predicted start codon. A “High Confidence” promoter set was developed based on the 121 genes for which two or more ESTs had their 5′ ends upstream of the start codon and within two bases of each other; the upstream EST terminus was inferred to represent the TSS. The distance between these inferred TSSs and the start codon ranged from 32 to 144 nt, with a median of 50 nt. This is just slightly larger than a prior estimate [To identify the appropriate search space for core promoter motifs, we defined the approximate locations of transcription start sites (TSSs) for e models . Of 74,1P. infestans gene models had incorrect 5′ termini, and thus improperly delineated promoters [As described below, both the High Confidence and Expanded promoter sets were used to identify and preliminarily characterize core motifs. Analyses were later expanded to total promoters, most of which lack defined TSSs and may contain more erroneous gene models due in part to less EST support. A prior study found that as many as 15% of romoters . Total promoters .′ untranslated region from P. infestans. Several rounds of searches were performed using MEME with parameters for motif width that ranged from 5 to 18 nt in different iterations.Motifs were predicted by searching for over-represented sequences within 100 nt windows centered on the TSSs. This involved using the motif discovery tool MEME, which employs an expectation maximization technique , on the MEME identified several candidate motifs including separate INR and FPR-like motifs (7 nt each), a 16 nt motif in which INR and FPR were separated by 2 nt, a new motif referred to later as DPEP (7 nt), and a motif similar to the eukaryotic CCAAT box . The CCAp-value for over-representation in that set of 10-9; p-value of 10-23 in the Expanded set). Systematic tests of variants of this pattern also suggested that YCAYTYY was optimal. As shown in Figure +1NWYY.Examination of the INR-like motifs returned by MEME suggested that the definition of the oomycete INR proposed previously, YCATTYY, was too narrow . Our resP. infestans promoters. As shown in Figure p-value for over-representation of 10-3; p-value of 10-13 in the larger Expanded set).The output from MEME that resembled FPR suggested that the motif was best-defined by MWTTTNC. This is similar to the CAWTTTNYY proposed by McLeod et al. , but witPhytophthora promoters considered INR and FPR as adjoining components of an approximately 16 nt conserved block found in most genes, with INR located 5′ of FPR [i.e. within a 16 nt block, in the 100 nt search window. Moreover, as shown in Figure vice versa[Prior studies of ′ of FPR . This isice versa.′ and 3′ ends of the same 16 nt window, we refer to this as the “INR+FPR” supramotif. If INR and FPR are found at more distant sites, these are referred to as separate INR and FPR motifs, i.e. INR alone and FPR alone elements.As a consequence of our observation, if matches to INR and FPR are found at 5Phytophthora. DPEP is unrelated in sequence to the metazoan DPE. As shown in Figure p-value for over-representation of 10-2; p-value of 10-6 in Expanded set). In only 24 cases was it present in the same promoter with an INR or FPR.Our search also identified a new putative core promoter element, which we name DPEP for Downstream Promoter Element Peronosporales, after the taxonomic order within the Oomycota that contains Phytophthora ramorum and Phytophthora sojae. While the distance between INR and FPR is constant in the two species at 2 nt, the space between DPE and FPR ranges from 5 to 12 nt. Variable spacing of the DPE from the INR+FPR block was also noted in alignments of other genes (not shown). It should be noted that while the relative positions of the INR, FPR, and DPE shown in Figure To illustrate the typical spacing between INR, FPR, and DPEP, a promoter containing all three motifs is shown in Figure P. infestans genes, and the resulting matches were used to establish position-specific probability matrices , FPR alone (i.e. without an upstream INR), and DPEP are shown in Figure ′ end of each promoter, within 50-100 nt of the start codon. While the data for these motifs in Figure The spatial distributions of INR+FPR, INR alone  both the INR and FPR. This is illustrated by the contour chart shown in Figure p=7×10-5), followed after 2 nt by a weak match to FPR . An inference is that counting occurrences of the INR+FPR motif based on the overall p-value or score may overestimate the frequency of the supramotif.Close examination of sequences shown by FIMO to match the PSPM for the combined INR+FPR motif, applying the program’s default P. infestans ortholog of TAF1 is PITG_02547 and there are two TAF2 proteins, PITG_14044 and PITG_18882. Other proteins that bind INR include well-conserved transcriptional regulators such as YY1  and taxon-specific proteins such as IBP39 of Trichomonas and USF-1 of metazoans [P. infestans genes. Since INR and FPR are nearly always separated by a 2 nt gap, this spacing is apparently optimal. In other eukaryotes, there are similar examples of synergy between core elements such as TATA and INR, or INR and MTE which must be separated by a certain distance for full effect [Our conclusion that the 16 nt region is not a well-conserved, monolithic motif may contradict previous interpretations, but is consistent with its likely evolutionary history. While FPR has only been described in oomycetes, INR is widely distributed throughout eukaryotes. Studies in model systems have shown that the main proteins that bind INR are the TAF1 and TAF2 subunits of TFIID, which are widely conserved; the etazoans -26. We pl effect ,28.P. infestans are similar, with positions 3-6 of INR resembling positions 2-5 of FPR. The proteins that bind the two regions could be related and work synergistically. In contrast, in other species only a few genes with tandem INR-like core promoter sequences have been described [It is very notable that INR and FPR of escribed ,30.′ of INR in some promoters. We therefore submitted to MEME 241 16-nt sequences that matched the INR well but only weakly matched the FPR. This only yielded a consensus that maintained the INR definition but had low information content in the downstream region  and lacked orientation bias. These are probably enhancers or proximal elements. We also used the High Confidence dataset to search for features near the TSS in promoters lacking an identified core motif. The regions just upstream of such TSSs were enriched for stretches of pyrimidines , but not significantly more than promoters with core motifs.We also searched the High Confidence, Expanded, and total promoter sets for core promoter elements detected before in metazoans, yeast, and plants. These included several variants of the widely distributed TATA box and motifs specific to plants  and aniP. infestans is predicted to encode a TATA-binding protein (TBP) as the product of gene PITG_07312. This 251 amino acid protein shows 66% amino acid identity (83% similarity) to TBP of S. cerevisiae, with an E-value in BLASTP of 10-83. An alignment of the P. infestans protein with orthologs from S. cerevisiae, A. thaliana, human, and five oomycetes is shown in Additional file S. cerevisiae, A. thaliana, and human, nine are different in all five oomycetes. For example, an alanine conserved in the three non-oomycetes (position 191 in S. cerevisiae) is changed to threonine in the oomycetes P. infestans, Phytophthora sojae, Pythium ultimum, and Hyaloperonospora arabidopsidis. Such changes may alter the DNA-binding specificity of the oomycete protein, and may help explain the apparent absence of a TATA box. In contrast, only 2 of 22 residues that contact other components of the preinitiation factor transcription factor complex  are difp-value cut-off of 10-5, with equal numbers of forward and reverse matches . Most were within 150 nt of the start codon 3, (CAA)3, (AGA)3, (AAG)3, and (GAA)3 would include the DPEP pattern. However, only 3% of DPEP hits within promoters fell within such sequences.Due to the large evolutionary distance between oomycetes and systems in which transcription has been well-studied, it is not surprising that oomycetes contain a novel element such as DPEP. In model systems, new core motifs continued to be identified long after promoters were first examined in detail . NeverthP. capsici, P. ramorum, and P. sojae. Stable transformants were obtained using a plasmid containing a 382 nt promoter fragment from PITG_10185, or a version in which the DPEP was mutated, fused to the GUS reporter gene. Quantitative assays indicated that the mutation reduced expression by an average of 5-fold  than the other Peronosporales (12-14%). This difference was not due to the use of the P. infestans PSPM, as similar results were obtained using one based on H. arabidopsidis.Searching for the motifs in other species suggested that the INR+FPR combination was unique to oomycetes Figure . This inBremia lactucae have been shown to effectively drive transgene expression across several genera of oomycetes [Interestingly, INR+FPR PSPMs from each oomycete were nearly identical .Oomycetes belong to the kingdom Stramenopila along with diatoms, and another stramenopile, the diatom R Figure . These mS. parasitica, and thus appeared specific to Peronosporales. DPEP was over-represented in promoters of T. pseudonana compared to randomized sequences, but these were questionable matches. All hits in the diatom involved A and C-containing versions of DPEP, compared to oomycetes where G and C were also common. A large fraction of the hits resided within microsatellites of ACA, CAA, or AAC. In addition, while DPEP in oomycetes occurred mostly in the forward orientation, this was not true in T. pseudonana after correcting for false discovery. While some microsatellites are known to concentrate near TSSs and influence transcription [T. pseudonana lack a common evolutionary history.DPEP was not present at significant levels in cription , we beliC. elegans[Some core promoter elements in model systems have been associated with certain patterns of gene expression. In vertebrates, most housekeeping genes lack a TATA box and instead employ INR sequences near the TSS, while the TATA box is preferentially linked to tissue-specific expression . Houseke. elegans.P. infestans core motifs, we used data from a microarray study that measured mRNA at five sequential life stages [P. infestans genes that are up- or down-regulated by 2-fold or more at each developmental transition.To assess whether relationships existed between expression pattern and the e stages . These wGenes containing the combined INR+FPR in their promoters were much more likely to be differentially expressed. In contrast, genes containing only DPEP or FPR showed the smallest variation. The average percent variance in mRNA levels between each of the five life-stages was 53% higher than average for genes with INR+FPR, 12% higher for INR only-containing genes, and 8% lower for genes in the FPR and DPEP groups.p<10-4. This supports the premise that there is a functional distinction between the canonical INR+FPR supramotif and INR alone, i.e. INR with a weak or absent downstream FPR. Expression patterns of INR+FPR genes during the transition from zoospores to germinated cysts was also distinct from the other classes, as 40% were up-regulated compared to about 25% for the other groups. This difference between the INR+FPR class and each of the others is significant with p<10-3.The most striking difference was observed during the transition from hyphae to sporangia Figure . During CesA family of cellulose synthases, which are induced in the appressorium stage [CesA3, PITG_17007). Moreover, even though relatively few INR+FPR promoters tend to be induced in sporangia, we have shown that inserting binding sites for sporulation-specific transcription factors upstream of a INR+FPR minimal promoter confers sporulation-specific expression [It is important to note that each pattern of transcription can be found for some genes within each core promoter class. This is because other transcription factor proteins, not those that bind core elements, likely play the dominant role in determining developmental regulation. For example, of the four genes comprising the um stage , only onpression .p<10-2. This trend was even seen within the DPEP class, which overall showed little variation in mRNA levels during development. CCAAT boxes in model systems have historically also been associated with housekeeping genes, although tissue-specific promoters containing the motif are also known [The presence of a CCAAT box decreased the tendency of genes to vary, regardless of which core element was present Figure . The preso known ,50.p<10-3).Correlations between absolute mRNA concentrations and motifs were also observed. This is shown in Figure C. elegans transcription increased with the number of T-block core promoter elements [One explanation for this finding is that the INR+FPR motif strongly enhances the efficiency of transcription initiation, even more so than INR alone. This is consistent with the observation of Juven-Gershon et al.  that comelements . There ielements .i.e. having low variance with s2<0.2, median mRNA levels in each motif class were nearly identical  showed higher mRNA levels than average. This was especially true for the INR+FPR class; its mRNA levels were 3.7-fold higher than all promoter types combined, and were significantly different from the other classes with p<0.002.The higher transcript levels of INR+FPR genes are partly related to the fact that mRNAs of developmentally regulated genes are more abundant than average. This was revealed by analyzing genes having high and low variation. These were identified by scoring the variance in per-gene normalized data, which discriminated better than Shannon entropy since data were available for only five tissue types . In moreThe possibility was considered that the INR+FPR genes showed higher mRNA levels because more of those genes were expressed at peak levels in hyphae, where transcripts might have more time to accumulate. In contrast, most spore-related stages persist only for a few hours. This did not appear to be the case, however. Transcripts of INR+FPR genes were more abundant regardless of whether they were mostly up-regulated in hyphae (3.4-fold higher mRNA levels than average), sporangia (2.0-fold higher than average), chilled sporangia (2.2-fold), zoospores (2.5-fold), or germinated cysts (2.7-fold). INR+FPR therefore appears to be a special motif that not only is more associated with stage-specific expression, but also with higher absolute levels of transcripts.All motifs except INR were associated with over-represented Gene Ontology (GO) terms. As shown in Table In contrast, over-represented terms for DPEP, FPR, and CCAAT were related to housekeeping functions. Examples include transcription (GO:0010467), translation , and metabolism . This is consistent with the more constitutive patterns of expression displayed by promoters containing these motifs.Phytophthora, Pythium, Hyaloperonospora, and Saprolegnia contain the supramotif. It is easy to understand why the earlier workers came to their conclusion about its prevalence: they had access to only 35 promoters, from a biased gene set that included multigene families [Our results have led us to re-evaluate the prior belief that most oomycete promoters contain the 16 nt INR+FPR motif. Based on our genome-wide analyses, only a minority (8-15%) of genes in families . More thDrosophila the TATA and MTE elements act synergistically within INR-containing promoters [Regardless of the absolute number of genes containing INR+FPR, the supramotif is interesting since it has a potent effect on transcript levels. Both experimental and bioinformatic studies in other taxa have also shown that combining motifs that otherwise function independently can enhance transcription. For example, in romoters , and theromoters . CombiniP. infestans encodes multiple isoforms of some of these proteins, such as for INR-binding protein TAF2 , which could have distinct effects through the life cycle. It is also possible that a P. infestans transcription factor that does not participate in preinitiation complex formation has evolved to bind the INR+PFR element. This would resemble the case of transcription factor YY1 of metazoans, which is able to bind the Initiator element and interacts with various protein partners to regulate processes such as embryonic development and cell cycle progression [The prevalence of INR+FPR in regulated genes, especially those depressed in sporangia relative to hyphae and related to pathogenesis, implies that the supramotif has evolved to interact with a specific subgroup of preinitiation complex-related proteins. In metazoans, the influence of core promoters on developmental regulation is believed to occur at least in part through their interaction with cell-type-specific TBP-associated factors (TAFs) and TBP-related factors (TRFs) . P. infegression .P. infestans genes lack a recognized core motif. It is possible that the pyrimidine-rich stretches common in many promoters may be sufficient to initiate transcription by inducing a particular DNA conformation, or favoring nucleosome eviction [P. infestans promoters are compact with median intergenic regions of 430 nt, and most known transcription factor binding sites are within 75 to 150 nt of the TSS [Even including the new DPEP motif, about half of eviction ,32. P. i the TSS -60. We h the TSS .http://www.broadinstitute.org) for P. infestans (v. 2) and S. parasitica (v.1), Joint Genome Institute of the United States Department of Energy for P. sojae and T. pseudonana , Michigan State University (http://pythium.plantbiology.msu.edu) for Py. ultimum, and the Virginia Bioinformatics Institute for H. arabidopsidis (v.8.3). These were used to obtain promoter sequences and start codons for each species. To predict TSSs, ESTs from P. infestans[Genome sequences and GFF files were obtained from the Broad Institute of MIT and Harvard  was useP-values associated with the strength of motif matches were taken directly from FIMO. For estimating motif frequencies, we searched upstream from start codons, and estimated false discovery rates using same datasets randomized by three rounds of DNA shuffling using shuffleseq in EMBOSS or Shuffle DNA (http://www.bioinformatics.org). Fisher’s Exact Test was used to calculate p-values for over-representation of a motif within the searched space. For initial analyses of P. infestans, INR+FPR motifs (selected using a p<10-4 cut-off) were distinguished from INR and FPR-alone motifs by splitting the putative INR+FPR in half and searching each against INR and FPR PSPMs, respectively, using FIMO; matches of the INR or FPR components with p>0.05 were assumed to be weak or insignificant.Motifs from MEME or other sources were identified within promoter sets using PERL scripts developed in-house or FIMO using PSPMs for each motif . P-valueP. infestans. After correcting for false discovery based on searches of shuffled datasets, similar results were obtained whether 200, 300, or 400 nt search windows had been used; the data in Results are based on using a 200 nt window. For determining whether a hit against the INR+FPR PSPM contained significant matches to both the INR and FPR components, it was found that results similar to the method described in the preceding paragraph (94% concordance) could be obtained by raising the p-value cut-off for the FIMO search to 5 x 10-5; matches with lower p-values were then analyzed separately for INR and FPR. Since the same location might match the INR and FPR definitions, addresses that recorded hits against both were reanalyzed to identify which motif was the closest match, and estimates of INR and FPR frequency were adjusted accordingly. On average, 23% of INR and FPR hits overlapped and required this correction. The observed DPEP frequencies were also adjusted by discarding potential microsatellite hits that were identified using the Microsatellite Repeats Finder program (http://insilico.ehu.es).Searches for motifs across oomycete species  and then checked for over-represented GO terms using GOSTAT [Annotations were obtained from the Broad Institute and supplemented with published data on effectors . Genes ig GOSTAT .′-ATGGATCCGCGT CATGCTTGATCTG) and 10185R (5′-ATGAATTCGGTTGAAATTAGAAAAGG), which contain BamHI and EcoRI sites at their 5′ end, respectively. The amplicon was digested with those enzymes and inserted upstream of the GUS gene in similarly-digested pNPGUS, which is a derivative of pOGUS [′-TAGAATTCGGTTGAAATTA GAAAAGGAAGG AAGAGATTCTTGCTGATTAGGT).A 398 nt portion of DNA upstream of the PITG_10185 open reading frame was amplified by polymerase chain reaction using primers 10185F 6, 5 mM K3Fe(CN)6, 0.1% Triton X-100. For quantitative assays, mycelia were grown on clarified rye sucrose media, collected after 5 days, dried on absorbent paper, and placed in a 2-ml tube with two 3.2-mm chrome steel beads and one 6.35-mm bead . Samples were frozen in liquid nitrogen, vortexed twice for 30-sec, mixed with 300 μl of extraction buffer , and clarified by centrifugation for 10 min. Supernatants were assayed for protein using the Bradford assay, and then 20 μg of protein were mixed with 100 μl of 2 mM 4-methylumbelliferyl glucuronide in extraction buffer. After 1 hour at 37°C, 25 μl was mixed with 250 μl of 200 mM sodium carbonate in a black 96-well plate, and fluorescence was measured in a Wallac Victor II plate reader  set to 365 nm excitation and 455 nm emission.Transformation of the resulting plasmids was achieved by electroporating zoospores with 30 μg of DNA in a 4-mm cuvette at 550 V, followed by selection on 7 μg/ml G418 . PrelimiThe authors declare that they have no competing interests.SR performed bioinformatics analyses, LP performed functional tests of promoters, HJ planned the experiments and conducted additional bioinformatics analyses, and TJ helped in experimental design. SR and HJ wrote the manuscript. All authors read and approved the final manuscript.PSPMs for the P. infestans motifs.Click here for fileComparison of oomycete and non-oomycete TATA-binding proteins, sequence logos of INR+FPR motif from different oomycetes, and correlation analysis of microarray and qRT-PCR data.Click here for fileList of genes containing motifs associated with over-represented GO terms.Click here for file


While these amino acid-derived compounds were all effective against HSV-1 and HSV-2, CAE and Amilite were the most effective. These two compounds were, however, not as effective against IAV, another enveloped virus, as against HSV. Cytotoxicity of CAE was weak; at 0.012%, only 5% of the cells were killed under the conditions, in which 100% cells were killed by either SDS or BKC. In addition to these direct virucidal effects, CAE inhibited the virus growth in the HSV-1- or PV-1-infected cells even at 0.01%. These results suggest a potential application of CAE as a therapeutic or preventive medicine against HSV superficial infection at body surface.Various amino acid-derived compounds, for example, N Recently there has been an increasing necessity for a multitude of preparedness against infectious diseases. Every year we encounter emerging and reemerging infectious diseases. While vaccination is a primary strategy to overcome viral diseases , vaccine in vivo applications. We have observed that aqueous arginine solution inactivates various enveloped viruses, although relatively high arginine concentration was required -L-arginine hydrochloride (Amisafe LMA-60), L-arginine cocoate (Aminosoap AR-12), potassium Nα-Cocoyl-L-glycinate (Amilite GCK-12K), benzalkonium chloride (BKC), sodium dodecylsulfate (SDS), sodium Nα-dodecanoylsarcosinate (LS), Chlorhexidine gluconate (CHX), ethylalchol (EtOH), povidone iodine (PI), and sodium hypochlorite (NaOCl) were of biochemical research grade and kindly provided by Ajinomoto Co., Inc . The compounds with trademark are used as ingredients for cosmetic products. The pH of each solvent was adjusted to the desired values using pH electrode type 6378 and pH meter type F-54 . A pH meter was manually calibrated every 2 h during the preparation of test solutions.L-arginine hydrochloride (referred as arginine), butyroyl-L-arginine (ButArg), NμL aliquot of the solutions received a 10 μL aliquot of virus preparations (approximately 107 ~ 108 plaque-forming units [PFU]/mL). This virus preparation was incubated at the indicated temperature for 5 min. After incubation, the test mixtures were chilled in ice-water bath, and, immediately, aliquots of these virus samples were 100-fold diluted with ice-cold Dulbecco's phosphate-buffered saline (PBS) without Ca2+ and Mg2+ containing 0.1% BSA for IAV or 0.5% FBS for other viruses to stop the virus inactivation. The viruses were further diluted to yield virus counts suitable for measurements, and the number of infectious virus in the treated preparation was measured by a plaque assay on Vero  or MDCK (for IAV) cells. There was little virus inactivation in PBS, and hence the amount of infectious virus in PBS was used as a control count [All the starting materials were stored on ice prior to the virus inactivation experiments. An excess volume of solutions was mixed with the virus stock so that both the pH and concentration of the test solutions would not be affected: that is, a 190 ol count .μg/mL) was also added to the medium for the proteolytic activation of virus infectivity. At the indicated time, the culture medium was harvested and the amount of total progeny virus in the culture was determined as described previously [Monolayered cells in 35 mm dishes were infected with the virus at an indicated multiplicity of infection (MOI). The infected cells were further incubated at 37°C  or 35.5°C (for PV-1) for the indicated period in the serum-free MEM (pH 7.4) containing 0.1% BSA and the indicated concentrations of the test compounds. The compounds were added at the onset of the incubation. For the experiments with influenza virus, acetylated trypsin (4 eviously –13.Confluent monolayers of Vero cells were incubated in 0.85% sodium chloride solution (pH 5.5) containing 20 mM MES (2-Morpholinoethanesulfonic acid) and varying concentrations of each test compound at room temperature for 15 min. To determine the extents of cell death, monolayered cells were trypsinized to obtain single cell suspension. After the addition of MEM containing 10% calf serum to the suspension to neutralize the trypsin and to stabilize the cells, the number of the living or dead cells was determined by a dye exclusion method with trypan blue as described previously [We examined the virucidal activity of arginine and amino acid derived compounds  on viruses of three completely different kinds; that is, HSV-1 and HSV-2 (Family of Herpesviridae), IAV (Family of Orthomyxoviridae), and PV-1 (Family of Picornaviridae). Both HSVs and IAV are large enveloped viruses and need the cell nucleus for the virus multiplication: the former has a double-stranded DNA genome, and the latter has a segmented negative-stranded RNA genome , 17. In −5 of the control). In the case of Amisafe, the decrease in the virus infectivity was marginally less than that incubated in the PBS. These results clearly show that arginine and amino acid derived compounds (except Amisafe) can efficiently inactivate HSV-1, an enveloped DNA virus, under the experimental conditions. Although pH, ion species, or ionic strength of the solution can affect the degree of virus inactivation [tivation , we havetivation . The res−5 of the control). Arginine, Amisafe, Amilite, SDS, LS, and CHX showed a varying degree of virucidal activities, with Amisafe being significantly less effective, similar to its effect on HSV-1, and with CHX being least effective  or 100-fold (for HSV-2) at pH 4.0 at the same CAE concentration.To further characterize the virucidal effects of CAE, it was compared with SDS, an anionic detergent, and BKC, a cationic detergent, at neutral pH. The above three compounds were then compared for cell toxicity. As shown in in vitro virus inactivation. Whether the same compounds can yield a reduced virus multiplication in in vivo experiments was then examined by treating the infected cells with them. The results in Figures Generally, virus-inactivating agents (disinfectants) show toxicity to cells and tissues, limiting their applications. We have undertaken a systematic screening of amino acid derived compounds in search for effective, nontoxic virus-inactivating agents and found that, against HSV-1 and HSV-2, CAE has notable activities to inactivate the infectivity of extracellular virus particles and to suppress the virus multiplication in the infected cells at the concentration with tolerable cytotoxic effect. These results may support a future potential application of CAE as a therapeutic or preventive medicine against HSV superficial infection at body surface, such as herpetic keratitis and genital herpes.


This study's aim was to develop a first quantification of the frequency and costs of adverse drug events (ADEs) originating in ambulatory medical practice in Germany.The frequencies and costs of ADEs were quantified for a base case, building on an existing cost-of-illness model for ADEs. The model originates from the U.S. health care system, its structure of treatment probabilities linked to ADEs was transferred to Germany. Sensitivity analyses based on values determined from a literature review were used to test the postulated results.For Germany, the base case postulated that about 2 million adults ingesting medications have will have an ADE in 2007. Health care costs related to ADEs in this base case totalled 816 million Euros, mean costs per case were 381 Euros. About 58% of costs resulted from hospitalisations, 11% from emergency department visits and 21% from long-term care. Base case estimates of frequency and costs of ADEs were lower than all estimates of the sensitivity analyses.The postulated frequency and costs of ADEs illustrate the possible size of the health problems and economic burden related to ADEs in Germany. The validity of the U.S. treatment structure used remains to be determined for Germany. The sensitivity analysis used assumptions from different studies and thus further quantified the information gap in Germany regarding ADEs.This study found costs of ADEs in the ambulatory setting in Germany to be significant. Due to data scarcity, results are only a rough indication. Medications are used to cure or slow disease processes, to reduce symptoms and to improve quality of life . HoweverLiterature reviews regarding the number of hospital admissions due to ADRs have reported various results. One review in 2002 reported that ADRs account for 4.9% of hospital admissions  whereas The proportion of preventable ADEs is significant , rangingIn US emergency departments, 1/3 of ADEs treated in persons over 65 were caused by warfarin, insulin and digoxin, all having a narrow therapeutic index and a high risk of overdose or toxicity . SimilarIt is estimated that in the USA, ADEs occur at a rate of 2-7/100 admissions in hospital  and at aCosts associated with drug-related mortality and morbidity in ambulatory care have been estimated using a probability-pathway model for the USA ,16. The Previous studies for Germany have reported the percent of hospitalisations due to ADRs and the associated costs for the hospital admission ,8. HowevParts of the drug-related morbidity and mortality cost-of-illness-model ,16 were In the paper by Ernst et al. , the numTo calculate the hospital costs of ADEs in the year 2007, the adverse drug reactions described in the Schneeweiss paper  were assSince the only available data for Germany is the percent of hospitalisations related to adverse drug events ,8, the mIn order to test estimates based on the model, MEDLINE was searched for studies reporting the frequency of ADEs in adults in the ambulatory setting published after 1990. Studies including chemotherapeutic agents or only certain diseases or specific ADEs were excluded. The studies found in this search are listed and summarized in Additional File The following sensitivity analyses were performed. To test the effect of a higher rate of drug-related hospitalisations, the complete model was calculated based on the rate reported by Dormann . Other oThe base case calculations using German data are shown in Table Expected resource use and cost estimates associated with ADEs are also reported in Table The sensitivity analyses in which the German hospitalisation rate  or only This study uses a probability pathway model to estimate the frequency and costs of ADEs occurring in ambulatory medical practice in Germany over 1 year (2007). The model includes drug-related morbidity and mortality. It calculates ADEs and bases all resource utilisation on the number of hospital admissions attributed to ADEs reported by the German Schneeweiss study . ResultsThe predicted probabilities of ADEs and resource utilisation due to ADEs in the population treated with medications were lower for the German base case than in the original model, but close to the lower end of rates reported in the literature, indicating a limited usefulness of the model in this respect. The calculation of total costs of resource utilisation attributes the highest proportion of costs to hospitalisation, the factor in the model for which there are the most data for Germany. Thus, in this respect, the model is useful in estimating the lower limits of resource utilisation costs which can be expected to be due to ADEs. According to the base case, hospital admission due to an ADE is required by 0.3% of the population taking medications  and emergency visits (0.4%) which are lower than those predicted using Dormann's study (5% and 0.7% respectively), the original model (12% and 2% respectively) and close to the lower end of rates reported in the literature  ), due toADEs pose a significant problem in outpatient treatment which may be preventable by improving communication between pharmacists, physicians and patients. Studies assessing the frequency of ADEs in the ambulatory setting exist for the USA but were not found for Germany. Costs related to ADEs in the ambulatory setting were postulated using a cost-of-illness model. A major proportion of these costs were attributed to hospitalisation and to long-term care. Due to the paucity of information regarding the ambulatory setting, it was difficult to assess whether cases, resource use and costs were valid for Germany. As all estimates in the sensitivity analysis were higher, base case results can be considered a very conservative estimate. To more precisely estimate the population-related costs in Germany, reliable data would be required on the prevalence of ADEs in general as well as for specific medications and the share of ADEs which are preventable.The authors declare that they have no competing interests.RS participated in the design of the study, performed the literature review, performed statistical analyses and drafted the manuscript. JJ participated in its design and helped to draft the manuscript. RL participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/11/9/prepubMean hospital costs calculated for each adverse drug reaction described by Schneeweiss. All possible ICD codes which are associated with an adverse drug reaction as described in the Schneeweiss paper were determined using the German DRG grouper. Using data from the Federal Office of Statistics for patients over 20 years of age, the sum of hospital days reported for the possible ICD codes for each described adverse drug reaction was divided by the total number of patients with the ICD code to calculate an average length of stay. The calculated average length of stay was multiplied with the average cost per hospital day for general hospitals, 437€ [ls, 437€ .Click here for fileMean overall hospital costs calculated using mean costs of adverse drug reactions and weighting these according to their frequency of occurrence in each drug class as described in the paper by Schneeweiss. Mean overall weighted hospital costs were calculated using the average hospital cost per adverse drug reaction (ADR), shown in additional file Click here for fileLiterature review of studies examining the frequency of adverse drug events in ambulatory patients. MEDLINE was searched for studies reporting the frequency of adverse drug events (ADEs) in adults in the ambulatory setting published after 1990. Studies including chemotherapeutic agents or only certain diseases or specific ADEs were excluded. The studies found in this search are listed and summarized regarding their methodology and results.Click here for file


The poxviruses Warsaw Agricultural University 86 (WAU86) and 88-1 (WAU88-1) were isolated in 1986 to 1988 from separate outbreaks in laboratory mice in Poland and described as ectromelia virus isolates. The genome sequences of these poxviruses reveal that they are almost identical and represent a novel variant of the vaccinia virus Lister strain. These viruses were previously described as ectromelia virus (ECTV) isolates based on electron microscopy analysis, neutralization tests, and clinical symptoms in infected outbred white Swiss mice . Pyroseq7 PFU of VACV-WAU86/88-1 did not cause mortalities or symptoms of disease, such us weight loss or skin lesions. This high attenuation of VACV-WAU86/88-1 was similar to that observed after infection of BALB/c mice with VACV Lister from Vestric Limited . The genetic classification of this virus as VACV Lister, together with its high attenuation in mice, questions the implications of WAU86 and WAU88-1 in the outbreaks in Poland. VACV Lister was used as a control in the characterization of these viruses, suggesting that the stocks of the poxvirus that caused the outbreaks may have been cross-contaminated with VACV Lister during their in vitro amplification  and a length of 187,723 bp. Phylogenetic analysis based on the central conserved region of the genome (63 Kbp) revealed that this virus is related to vaccinia virus (VACV) strain Lister, commonly used in Europe during the smallpox eradication program . It has KF866253.The genome sequence of VACV-WAU86/88-1 was submitted to GenBank with the accession no.


We evaluated titers of homotypic and heterotypic neutralizing antibodies (NAbs) to Andes and Sin Nombre hantaviruses in plasma samples from 20 patients from Chile and the United States. All but 1 patient had high titers of NAb. None of the plasma samples showed high titers against the heterologous virus. Sigmodontinae, family Muridae, and the distribution of each virus parallels that of the host (Hantavirus cardiopulmonary syndrome (HCPS), an emergent disease caused by New World hantaviruses, is associated with case-fatality ratios of 30% to 50%. Sin Nombre virus (SNV) and Andes virus are well-characterized hantavirus serotypes responsible for this disease in the southwestern United States and in the south cone of the Americas , respectively . Since tthe host .No specific treatment for HCPS exists. Ribavirin, the only approved antiviral agent that is effective against hantaviruses in vitro , has shoSome evidence shows that neutralizing antibody (NAb) can affect the course of HCPS. Animal studies have shown that NAb confers passive protection from severe disease by Andes virus. Specifically, passive transfer of serum with high NAb titers from rhesus macaques vaccinated against Andes virus protected 100% of Syrian hamsters from lethal disease, even when administered 4–5 days after challenge with Andes virus . In humaAdministering convalescent-phase plasma with a high NAb titer could be therapeutic in HCPS, as it is in other hemorrhagic fevers . In survHantavirus suggests that a monovalent vaccine will not likely confer sufficient protection for all of the pathogenic hantaviruses  or Andes virus  and incubated at 37° for 1 hour (We studied 20 serum samples from survivors of confirmed hantavirus infection, 11 from Chilean patients and 9 from patients in the southwestern United States. Samples were collected from 8 months to 11 years after the patient was hospitalized with HCPS. The neutralizing titer was measured for each sample against SNV and Andes virus by a focus-reduction neutralization assay in Vero E6 cells, as described previously . In brier 1 hour . The mix>1:400 in all but 1 patient. In contrast, 9 of the 11 samples failed to show NAb titers >1:100 against SNV, while the other 2 neutralized SNV only at low titers. Similarly, all North American patients had plasma NAb against SNV at titers >400, and only 1 showed some neutralization against Andes virus, at low titer. No relationship was seen between the endpoint NAb titers against the homotypic virus and time elapsed from acute disease in either Chilean or North American patients, nor did a particularly high homotypic titer predict that neutralizing activity would be present against the heterologous virus.The endpoint plasma NAb titers against Andes virus and SNV from Chilean and North American survivors of hantavirus infection are shown in the In survivors of hantavirus disease who reside in Chile or the United States, we found high titers of plasma NAb against the type of hantavirus that is prevalent in the patient's own region, while substantial titers against the heterologous agent of HCPS were absent. In this small group of participants, NAb titers did not show any readily detectable decline with time elapsed after infection; titers as high as 1:1,600 could be detected 11 years after illness. These results suggest that plasma from patients who survive hantavirus infection is a potential source of NAb and could be used as a therapeutic alternative for patients with acute disease or as a prophylactic intervention for persons who may have been exposed to the virus. The absence of in vitro cross-neutralization makes the alternative of clinically effective cross-protection less likely and discourages the use of convalescent-phase sera to treat patients whose geographic origin is different from that of the plasma donor. Our results suggest that a monovalent vaccine would not elicit protection against different types of hantavirus, even when the viruses are phylogenetically as similar as SNV and Andes virus. The positive results of cross-protection studies in hamster models should be interpreted cautiously, since experimental infection in those studies would tend to favor unusually brisk immune responses that go well beyond eliciting NAb and likely include potent cell-mediated or innate immune responses that cannot be mimicked with passive immunization . Similar