Search

Highlights • Efficient responses to viral threats require a integrated multidisciplinary approach. • Several relatively newly formed initiatives focus on effective response management., Recent advances in the metagenomics field have had huge effects on the identification and characterization of newly emerging viral pathogens. To allow timely and efficient responses to future viral threats, an integrated multidisciplinary approach utilizing expertises in several areas, including clinical assessment, virus surveillance, virus discovery, pathogenesis, and the molecular basis of the host response to infection, is required. It requires the scientific community involved in virus discovery to go one step beyond.

As infectious diseases cause approximately 25% of the annual global mortality, vaccines are found to be a time proven and promising response to infectious disease need. However, like for pharmaceutical small molecules, vaccine development is lengthy, risky and resource demanding. Faced with an attrition rate estimated around 80%, key opinion leaders were interviewed with the question: is there a recipe for success?

Aims To determine the burden of disease in a unique sample of patients with uveitis from a rural South African setting. Methods Data in this cross-sectional study were collected from patients presenting with uveitis (n=103) at the ophthalmology outpatient department of three hospitals in rural South Africa. Demographic and clinical data were collected, and laboratory analysis of aqueous humour, serological evaluation and routine diagnostics for tuberculosis (TB) were performed. Results Sixty-six (64%) participants were HIV infected. Uveitis was predominantly of infectious origin (72%) followed by idiopathic (16%) and autoimmune (12%). Infectious uveitis was attributed to herpes virus (51%), Mycobacterium tuberculosis (24%) and Treponema pallidum (7%) infection. HIV-infected individuals were more likely to have infectious aetiology of uveitis compared with HIV-uninfected individuals (83% vs 51%; p=0.001). Conclusions Microbial aetiology of uveitis is common in areas where HIV and TB are endemic. In these settings, a high index of suspicion for infectious origin of uveitis is warranted.

Unlike most species in the genus Sarcocystis, Sarcocystis canis has a broad intermediate host range. Its life cycle is incompletely known and most reports are from the USA. Here we report fatal hepatitis in a 4 year old male Indo-Pacific bottlenose dolphin (Tursiops aduncus) from Hong Kong associated with a S. canis-like infection. Diagnosis was made based on clinical presentation, histopathology, transmission electron microscopy (TEM), and molecular characterization. Microscopically, S. canis-like like infection was confined to the liver. Immature and mature schizonts were found in hepatocytes and the parasite was associated with generalized hepatic necrosis. By TEM, schizonts divided by endopolygeny, and merozoites lacked rhoptries. Molecular characterization of parasites present in liver and brain tissues at the cox1 gene showed a high degree of identity (97–98%) and clustered together with Sarcocystis canis, S. lutrae, S. arctica, S. speeri, S. turdusi, and S. rileyi in a phylogenetic study. This is the first report of S. canis-like infection from Asia.

The role of the multifunctional accessory Nef protein in the immunopathogenesis of HIV-2 infection is currently poorly understood. Here, we performed comprehensive functional analyses of 50 nef genes from 21 viremic (plasma viral load, >500 copies/ml) and 16 nonviremic (nef alleles from both groups were equally active in modulating CD4, TCR-CD3, CD28, MHC-I, and Ii cell surface expression and in enhancing virion infectivity. Thus, many HIV-2-infected individuals efficiently control the virus in spite of efficient Nef function. However, the potency of nef alleles in downmodulating TCR-CD3 and CD28 to suppress the activation and apoptosis of T cells correlated with high numbers of CD4 + T cells in viremic patients. No such correlations were observed in HIV-2-infected individuals with undetectable viral load. Further functional analyses showed that the Nef-mediated downmodulation of TCR-CD3 suppressed the induction of Fas, Fas-L, PD-1, and CTLA-4 cell surface expression as well as the secretion of gamma interferon (IFN-γ) by primary CD4 + T cells. Moreover, we identified a single naturally occurring amino acid variation (I132T) in the core domain of HIV-2 Nef that selectively disrupts its ability to downmodulate TCR-CD3 and results in functional properties highly reminiscent of HIV-1 Nef proteins. Taken together, our data suggest that the efficient Nef-mediated downmodulation of TCR-CD3 and CD28 help viremic HIV-2-infected individuals to maintain normal CD4 + T cell homeostasis by preventing T cell activation and by suppressing the induction of death receptors that may affect the functionality and survival of both virally infected and uninfected bystander cells.

ObjectiveBarriers to international Ebola preparedness may be elucidated by identifying heterogeneities in arguments to invest in countermeasures during “peace time.”MethodsFor each patent family (related patent documents that differed only by limited alterations to the same invention) concerning Ebola and published until the end of 2014 the oldest patent document was analyzed. Grounded theory coding identified 5 unmet needs for (1) vaccines and therapies, (2) control of outbreaks in endemic areas, (3) detection and control of outbreaks in nonendemic areas, (4) better understanding of filoviruses, and (5) protection against bioterrorism. Odds ratios for unmet needs by geographic regions and institution types were compared by using Pearson’s chi-square test.ResultsStatistically significant heterogeneities in unmet need profiles were found. US applicants combined self-centric and altruistic arguments, focusing on medical unmet needs and bioterrorism protection. Russian and Asian applicants emphasized self-centric motives, specifically, detection and control of nonendemic outbreaks. A clear, statistically significant mismatch between industry and academia was found: whereas industrial applicants focused on bioterrorism and neglected detection and control of nonendemic outbreaks, academic applicants did the opposite.ConclusionsThis research identified heterogeneities in articulated needs between geographic regions and stakeholder types. Structural articulation of unmet needs may form the basis for attuning stakeholder engagement strategies while progression across the demand-driven value chain might necessitate international concordance. (Disaster Med Public Health Preparedness. 2016;10:644–648)

Background Respiratory syncytial virus (RSV) and influenza A viruses are known to cause severe acute respiratory tract infections (SARIs) in children. For other viruses like human rhinoviruses (HRVs) this is less well established. Viral or bacterial co-infections are often considered essential for severe manifestations of these virus infections. Objective The study aims at identifying viruses that may cause SARI in children in the absence of viral and bacterial co-infections, at identifying disease characteristics associated with these single virus infections, and at identifying a possible correlation between viral loads and disease severities. Study Design Between April 2007 and March 2012, we identified children (

International audience; AbstractHerpesvirus infection causes disease of variable severity in many species, including cetaceans. However, little is known about herpesvirus infection in harbor porpoises (Phocoena phocoena), despite being widespread in temperate coastal waters of the Northern Hemisphere. Therefore, we examined harbor porpoises that stranded alive in the Netherlands, Belgium, and Germany between 2000 and 2014 for herpesvirus infection and associated disease. Porpoises that died or had to be euthanized were autopsied, and samples were collected for virological and pathological analyses. We found one known herpesvirus (Phocoena phocoena herpesvirus type 1, PPHV-1)—a gammaherpesvirus—and two novel herpesviruses (PPHV-2 and PPHV-3)—both alphaherpesviruses—in these porpoises. A genital plaque, in which PPHV-1 was detected, occurred in 1% (1/117) of porpoises. The plaque was characterized by epithelial hyperplasia and intranuclear inclusion bodies that contained herpesvirus-like particles, and that stained positive by a PPHV-1-specific in situ hybridization test. PPHV-2 occurred in the brain of 2% (1/74) of porpoises. This infection was associated with lymphocytic encephalitis, characterized by neuronal necrosis and intranuclear inclusion bodies containing herpesvirus-like particles. PPHV-3 had a prevalence of 5% (4/74) in brain tissue, 5% (2/43) in blowhole swabs, and 2% (1/43) in genital swabs, but was not associated with disease. Phylogenetically, PPHV-1 was identical to a previously reported herpesvirus from a harbor porpoise, PPHV-2 showed closest identity with two herpesviruses from dolphins, and PPHV-3 showed closest identity with a cervid herpesvirus. In conclusion, harbor porpoises may be infected with at least three different herpesviruses, one of which can cause clinically severe neurological disease.

Probable epidemic influenza outbreaks have been described as early as the 5th century BC, as part of the Cough of Perinthus associated with the winter solstice, in Hippocrates' Corpus Hippocraticum “Of the Epidemics” [1]. The word “influenza” was first introduced in the 16th century, defining the illness of the cold season that in the early 1930s was shown to be caused by “filterable agents”, since then called influenza viruses. Three types or genera of influenza viruses have been recognised (influenza A, B and C viruses), while a fourth genus has been recently proposed [2]. Today, we distinguish three manifestations of influenza in humans, which may impose from mild to severe morbidity and mortality burdens: zoonotic, pandemic and seasonal influenza, all caused by an infection with an influenza virus. Although our arsenal of intervention strategies for influenza has advanced in the past decades and a growing public conception has arisen of influenza as a usually seasonal inconvenience, threats posed by influenza A viruses, with their different manifestations and associated complications, are continuously knocking on our door., Global surveillance and advances in vaccine technology are essential to answer the threat of influenza pandemics http://ow.ly/Yt3e4

Human respiratory syncytial virus (HRSV) is an important cause of severe respiratory tract disease in immunocompromised patients. Animal models are indispensable for evaluating novel intervention strategies in this complex patient population. To complement existing models in rodents and non-human primates, we have evaluated the potential benefits of an HRSV infection model in ferrets (Mustela putorius furo). Nine- to 12-month-old HRSV-seronegative immunocompetent or immunocompromised ferrets were infected with a low-passage wild-type strain of HRSV subgroup A (105 TCID50) administered by intra-tracheal or intra-nasal inoculation. Immune suppression was achieved by bi-daily oral administration of tacrolimus, mycophenolate mofetil, and prednisolone. Throat and nose swabs were collected daily and animals were euthanized four, seven, or 21 days post-infection (DPI). Virus loads were determined by quantitative virus culture and qPCR. We observed efficient HRSV replication in both the upper and lower respiratory tract. In immunocompromised ferrets, virus loads reached higher levels and showed delayed clearance as compared to those in immunocompetent animals. Histopathological evaluation of animals euthanized 4 DPI demonstrated that the virus replicated in the respiratory epithelial cells of the trachea, bronchi, and bronchioles. These animal models can contribute to an assessment of the efficacy and safety of novel HRSV intervention strategies.

The purpose of this investigation was to determine the clinical and corneal microbial profile of infectious keratitis in a high human immunodeficiency virus (HIV) prevalence setting in rural South Africa. Data in this cross-sectional study were collected from patients presenting with symptoms of infectious keratitis (n = 46) at the ophthalmology outpatient department of three hospitals in rural South Africa. Corneal swabs were tested for herpes simplex virus type 1 (HSV-1) and 2 (HSV-2), varicella zoster virus (VZV) and adenovirus DNA by real-time polymerase chain reaction (PCR) and for bacteria and fungi by culture. Based on clinical history, disease characteristics and laboratory results, 29 (63 %) patients were diagnosed as viral keratitis, including 14 (48 %) viral keratitis cases complicated by bacterial superinfection, and 17 (37 %) as bacterial keratitis. VZV and HSV-1 DNA was detected in 11 (24 %) and 5 (11 %) corneal swabs, respectively. Among clinically defined viral keratitis cases, a negative viral swab was predominantly (93 %) observed in cases with subepithelial inflammation and was significantly associated with an increased duration of symptoms (p = 0.003). The majority of bacteria cultured were Gram-positive (24/35), including Staphylococcus epidermidis and S. aureus. Viral aetiology was significantly associated with a history of herpes zoster ophthalmicus (p

Rapid population aging has become a major challenge in the industrialized world and progressive aging is a key reason for making improvement in vaccination a cornerstone of public health strategy. An increase in age-related disorders and conditions is likely to be seen in the near future, and these are risk factors for the occurrence of a number of vaccine-preventable diseases. An improvement in infectious diseases prevention specifically aimed at adults and the elderly can therefore also decrease the burden of these chronic conditions by reducing morbidity, disability, hospital admissions, health costs, mortality rates and, perhaps most importantly, by improving the quality of life. Among adults, it is necessary to identify groups at increased risk of vaccine-preventable diseases and highlight the epidemiological impact and benefits of vaccinations using an evidence-based approach. This document provides clinical practice guidance on immunization for adults in order to provide recommendations for decision makers and healthcare workers in Europe. Although immunization is considered one of the most impactful and cost-effective public health measures that can be undertaken, vaccination coverage rates among adults are largely lower than the stated goal of >= 95% among adults, and stronger efforts are needed to increase coverage in this population. Active surveillance of adult vaccine-preventable diseases, determining the effectiveness of the vaccines approved for marketing in the last 5 y, the efficacy and safety of vaccines in immunocompromised patients, as well as in pregnant women, represent the priorities for future research.

Infectious diarrheal diseases remain a common cause of morbidity and mortality especially in children younger than 5 years in low-income and middle-income countries.1 The most common viral pathogens known to cause acute gastroenteritis in humans are rotavirus A, norovirus, sapovirus, enteric adenoviruses and astrovirus. In addition to these well-established etiologies, a large number of moderate to severe diarrhea cases go without etiological diagnosis and many unanswered questions surrounding the burden and etiology of diarrhea, and the factors determining clinical expression of infection remain. [...]

Noroviruses are a major cause of acute gastroenteritis worldwide and are a genetically diverse group of viruses. Since 2002, an increasing number of norovirus outbreaks have been reported globally, but it is not clear whether this increase has been caused by a higher awareness or reflects the emergence of new genogroup II genotype 4 (GII.4) variants. The hypothesis that norovirus prevalence has increased post-2002 and is related to the emergence of GII.4 is tested in this study. Sera collected from children aged

We analyzed the varicella-zoster virus (VZV) transcriptome in 43 latently infected human trigeminal ganglia (TG) with postmortem intervals (PMIs) ranging from 3.7 to 24 h. Multiplex reverse transcriptase PCR (RT-PCR) revealed no VZV transcripts with a PMI of P = 0.02) in VZV ORF63 transcript levels but not the virus DNA burden with longer PMI. Overall, both the breadth of the VZV transcriptome and the VZV ORF63 transcript levels in human cadaver TG increased with longer PMI.

Influenza A virus-specific T cells are highly cross-reactive and contribute to heterosubtypic immunity, which may afford protection against novel pandemic strains of influenza virus. However, the magnitude and nature of virus-specific T-cell responses induced by natural infections and/or vaccination in young children is poorly understood. Host factors, such as the development of the immune system during childhood and environmental factors such as exposure rates to influenza viruses and interference by vaccination contribute to shaping the magnitude and specificity of the T-cell response. Here, the authors review several of these factors, including the differences between T-cell responses of young children and adults, the age-dependent frequency of virus-specific T cells and the impact of annual childhood influenza vaccination. In addition, the authors summarize all currently available studies in which influenza vaccine-induced T-cell responses were evaluated. The authors discuss these findings in the light of developing vaccines and vaccination strategies aiming at the induction of protective immunity to seasonal and pandemic influenza viruses of antigenically distinct subtypes.

Highly pathogenic avian influenza A/H5N1 virus can cause morbidity and mortality in humans but thus far has not acquired the ability to be transmitted by aerosol or respiratory droplet ("airborne transmission") between humans. To address the concern that the virus could acquire this ability under natural conditions, we genetically modified A/H5N1 virus by site-directed mutagenesis and subsequent serial passage in ferrets. The genetically modified A/H5N1 virus acquired mutations during passage in ferrets, ultimately becoming airborne transmissible in ferrets. None of the recipient ferrets died after airborne infection with the mutant A/H5N1 viruses. Four amino acid substitutions in the host receptor-binding protein hemagglutinin, and one in the polymerase complex protein basic polymerase 2, were consistently present in airborne-transmitted viruses. The transmissible viruses were sensitive to the antiviral drug oseltamivir and reacted well with antisera raised against H5 influenza vaccine strains. Thus, avian A/H5N1 influenza viruses can acquire the capacity for airborne transmission between mammals without recombination in an intermediate host and therefore constitute a risk for human pandemic influenza.

Human influenza viruses have their ultimate origin in avian reservoirs and may adapt, either directly or after passage through another mammalian species, to circulate independently in the human population. Three sets of barriers must be crossed by a zoonotic influenza virus before it can become a human virus: animal-to-human transmission barriers: virus-cell interaction barriers: and human-to-human transmission barriers. Adaptive changes allowing zoonotic influenza viruses to cross these barriers have been studied extensively, generating key knowledge for improved pandemic preparedness. Most of these adaptive changes link acquired genetic alterations of the virus to specific adaptation mechanisms that can be screened for, both genetically and phenotypically, as part of zoonotic influenza virus surveillance programs. Human-to-human transmission barriers are only sporadically crossed by zoonotic influenza viruses, eventually triggering a worldwide influenza outbreak or pandemic. This is the most devastating consequence of influenza virus cross-species transmission. Progress has been made in identifying some of the determinants of influenza virus transmissibility. However, interdisciplinary research is needed to further characterize these ultimate barriers to the development of influenza pandemics, at both the level of the individual host and that of the population. (c) 2012 Elsevier Ltd. All rights reserved.

We screened 1,200 living heart, lung, liver, and kidney transplant recipients for hepatitis E virus infection by reverse transcription PCR. In 12 (1%) patients, hepatitis E virus infection was identified; in 11 patients, chronic infection developed. This immunocompromised population is at risk for hepatitis E virus infection.

The route by which highly pathogenic avian influenza (HPAI) H5N1 virus spreads systemically, including the central nervous system (CNS), is largely unknown in mammals. Especially, the olfactory route, which could be a route of entry into the CNS, has not been studied in detail. Although the multibasic cleavage site (MBCS) in the hemagglutinin (HA) of HPAI H5N1 viruses is a major determinant of systemic spread in poultry, the association between the MBCS and systemic spread in mammals is less clear. Here we determined the virus distribution of HPAI H5N1 virus in ferrets in time and space—including along the olfactory route—and the role of the MBCS in systemic replication. Intranasal inoculation with wild-type H5N1 virus revealed extensive replication in the olfactory mucosa, from which it spread to the olfactory bulb and the rest of the CNS, including the cerebrospinal fluid (CSF). Virus spread to the heart, liver, pancreas, and colon was also detected, indicating hematogenous spread. Ferrets inoculated intranasally with H5N1 virus lacking an MBCS demonstrated respiratory tract infection only. In conclusion, HPAI H5N1 virus can spread systemically via two different routes, olfactory and hematogenous, in ferrets. This systemic spread was dependent on the presence of the MBCS in HA.

Almost five decades after their first application in diagnostics, dried blood spot (DBS) cards remain to be of key interest in many research areas and clinical applications. The advantages of sample stability during transport and storage, can now be combined with the high sensitivity of novel diagnostic techniques for the measurement and analysis of nucleic acids, proteins and small molecules which may overcome the limitations of the small samples sizes in DBS cards. Here we present a survey of the literature on the use of DBS cards for diagnosis, monitoring and epidemiological studies of virus infections other than HIV, including CMV, HBV, HCV, HAV, HEV, HTLV, EBV, HSV, measles-, rubella- and dengue-virus. The minimal invasiveness of sampling and the relative ease of handling and storing DBS cards is expected to offer additional opportunities to measure and analyze biomarkers of viral disease in resource poor settings or when limited amount of blood can be obtained. Large retrospective studies of virus infections in newborns using stored DBS cards have already been undertaken for screening of congenital infections. In addition, DBS cards have been used prospectively for prevalence studies, outbreak surveillance, mass screening for viral infections, follow-up of chronic infection and its treatment in resource-limited areas. We do not expect that current wet sampling techniques of plasma or serum will be replaced by DBS sampling but it allows extension of sampling in persons and settings that are currently difficult to access or that lack suitable storage facilities. In conclusion, DBS card sampling and storage will aid adequate outbreak management of existing and emerging viral diseases.

Varicella-zoster virus (VZV) causes chickenpox, establishes latency in trigeminal (TG) and dorsal root ganglia (DRG), and can lead to herpes zoster upon reactivation. The VZV proteome expressed during latency remains ill-defined, and previous studies have shown discordant data on the spectrum and expression pattern of VZV proteins and transcripts in latently infected human ganglia. Recently, Zerboni and colleagues have provided new insight into this discrepancy (Zerboni et al. in J Virol 86:578-583, 2012). They showed that VZV-specific ascites-derived monoclonal antibody (mAb) preparations contain endogenous antibodies directed against blood group A1 proteins, resulting in false-positive intra-neuronal VZV staining in formalin-fixed human DRG. The aim of the present study was to confirm and extend this phenomenon to snap-frozen TG (n = 30) and DRG (n = 9) specimens of blood group genotyped donors (n = 30). The number of immunohistochemically stained neurons was higher with mAb directed to immediate early protein 62 (IE62) compared with IE63. The IE63 mAb-positive neurons always co-stained for IE62 but not vice versa. The mAb staining was confined to distinct large intra-neuronal vacuoles and restricted to A1(POS) donors. Anti-VZV mAb staining in neurons, but not in VZV-infected cell monolayers, was obliterated after mAb adsorption against blood group A1 erythrocytes. The data presented demonstrate that neuronal VZV protein expression detected by ascites-derived mAb in snap-frozen TG and DRG of blood group A1(POS) donors can be misinterpreted due to the presence of endogenous antibodies directed against blood group A1-associated antigens present in ascites-derived VZV-specific mAb preparations.

Recently, we and others obtained experimental evidence that highly pathogenic avian influenza virus subtype H5 can acquire the ability to transmit via aerosols between ferrets. Upon submission of manuscripts describing the results of these studies, the US National Science Advisory Board for Biosecurity was consulted and recommended that the main conclusions of the work be published but without the experimental details and mutation data that would enable replication of the experiments. Over the past few months, these events have led to intense discussions. Should this type of experiment be conducted? If so, under what conditions? Do the scientific and public health benefits of the work and its publication outweigh the potential risks? In February 2012, public health and influenza experts discussed these issues during a World Health Organization–organized technical consultation. This perspective article reviews the current state of the field and the recommendations made during the meeting.

In 2011 the Food and Agriculture Organization formally announced that rinderpest was eradicated from the globe. Rinderpest virus had long been associated with huge disease outbreaks among cattle. The disease not only had a devastating effect on cattle herds world-wide, but also on human populations that depended on them. Rinderpest virus - a member of the genus Morbillivirus of the family Paramyxoviridae - is a close relative of measles virus. Both viruses are highly infectious and share many other biological properties. Although no formal goal or timeframe has been set, plans are currently being developed to eradicate measles. Here, we discuss how lessons learned from the global eradication of rinderpest may help in the future eradication of measles.

C-type lectins are important molecules of the innate immune system. These molecules, like surfactant protein D (SP-D) can recognize glycans on pathogens and neutralize these. Also influenza viruses are recognized by SP-D and their susceptibility to neutralization by SP-D is dependent on the number of N-linked glycosylation sites in the hemagglutinin in particular. Porcine SP-D displayed stronger neutralizing activity to human influenza A viruses than to swine influenza A viruses. Although viruses from these species differ with regard to the number of glycosylation sites in the hemagglutinin, the mechanism underlying the differential recognition by porcine SP-D is poorly understood. Here we investigated the molecular basis for the differential recognition of a seasonal H1N1 and a 2009 pandemic H1N1 virus by porcine SP-D. We demonstrated that the number and position of glycosylation sites determine viral susceptibility to the neutralizing activity of porcine SP-D. However, predicting the effect remains difficult as it was shown to be dependent on the strain and the position of the glycosylation sites. (C) 2012 Elsevier B.V. All rights reserved.

The classification of dengue virus-infected patients continues to be a challenge to researchers and clinicians in the field. The accuracy of the 1997 World Health Organization (WHO) dengue case definition has been debated for a decade, because the definition was very stringent, for instance, several researchers showed that apparently severe cases were misclassified as not severe. Therefore the WHO issued revised guidelines in 2009. Here, we retrospectively compared the performance of the WHO case definition of 2009 with the WHO case definition of 1997 in a detailed documented pediatric cohort from Indonesia. Intensive treatment intervention was used as an indicator of severity of disease. In line with our expectations, the 2009 WHO case classification proved to be significantly more specific, albeit less sensitive than the WHO case classification of 1997. We conclude that the revised classification is promising both from research and clinical perspectives, but validation of the classification criteria still needs to be addressed.

Avian A/H5N1 influenza viruses pose a pandemic threat. As few as five amino acid substitutions, or four with reassortment, might be sufficient for mammal-to-mammal transmission through respiratory droplets. From surveillance data, we found that two of these substitutions are common in A/H5N1 viruses, and thus, some viruses might require only three additional substitutions to become transmissible via respiratory droplets between mammals. We used a mathematical model of within-host virus evolution to study factors that could increase and decrease the probability of the remaining substitutions evolving after the virus has infected a mammalian host. These factors, combined with the presence of some of these substitutions in circulating strains, make a virus evolving in nature a potentially serious threat. These results highlight critical areas in which more data are needed for assessing, and potentially averting, this threat.

Background Inactivated paramyxovirus vaccines have been associated with hypersensitivity responses upon challenge infection. For measles and canine distemper virus (CDV) safe and effective live-attenuated virus vaccines are available, but for human respiratory syncytial virus and human metapneumovirus development of such vaccines has proven difficult. We recently identified three synthetic bacterial lipopeptides that enhance paramyxovirus infections in vitro, and hypothesized these could be used as adjuvants to promote immune responses induced by live-attenuated paramyxovirus vaccines. Methods Here, we tested this hypothesis using a CDV vaccination and challenge model in ferrets. Three groups of six animals were intra-nasally vaccinated with recombinant (r) CDV5804PL(CCEGFPC) in the presence or absence of the infection-enhancing lipopeptides Pam3CSK4 or PHCSK4. The recombinant CDV vaccine virus had previously been described to be over-attenuated in ferrets. A group of six animals was mock-vaccinated as control. Six weeks after vaccination all animals were challenged with a lethal dose of rCDV strain Snyder-Hill expressing the red fluorescent protein dTomato. Results Unexpectedly, intra-nasal vaccination of ferrets with rCDV5804PL(CCEGFPC) in the absence of lipopeptides resulted in good immune responses and protection against lethal challenge infection. However, in animals vaccinated with lipopeptide-adjuvanted virus significantly higher vaccine virus loads were detected in nasopharyngeal lavages and peripheral blood mononuclear cells. In addition, these animals developed significantly higher CDV neutralizing antibody titers compared to animals vaccinated with non-adjuvanted vaccine. Conclusions This study demonstrates that the synthetic cationic lipopeptides Pam3CSK4 and PHCSK4 not only enhance paramyxovirus infection in vitro, but also in vivo. Given the observed enhancement of immunogenicity their potential as adjuvants for other live-attenuated paramyxovirus vaccines should be considered.

To identify unknown human viruses in the enteric tract, we examined 105 stool specimens from patients with diarrhea in Bangladesh. A novel calicivirus was identified in a sample from 1 patient and subsequently found in samples from 5 other patients. Phylogenetic analyses classified this virus within the proposed genus Recovirus.

Background: Although in the majority of cases dengue virus (DENV) infection results in a self-limiting febrile disease, it can cause severe plasma leakage in a minority of patients. The appearance of plasma leakage indicates an increased permeability of the vascular wall. In this study we investigated if DENV infection can lead to leakage of lipopolysaccharide (LPS) from the intestine into the blood of the patient, indicative of an increased permeability of the intestinal mucosal barrier. Objectives: The aim of this study was to investigate if LPS levels were elevated in DENV infected patients and if these levels correlated with disease severity. Study design: LPS levels in the blood of DENV infected children were determined using the Limulus Amebocyte Lysate assay. To determine disease severity we used the 1997-WHO criteria, the expert physician's judgement and a score that focused on plasma leakage in particular. Furthermore, the modulatory factors LPS binding protein (LBP) and sCD14, as well as the immune activation marker neopterin were determined. Results: We showed significantly elevated LPS levels in plasma of DENV infected children compared to healthy controls. The plasma leakage severity score had the strongest correlation with levels of LPS. LBP, sCD14 and neopterin were elevated compared to healthy controls. Conclusion: In this study we show evidence of elevated LPS levels during DENV infection. Moreover, a correlation between LPS levels and disease severity was found, especially when disease severity was determined in terms of plasma leakage. (C) 2011 Elsevier B. V. All rights reserved.

Although hepatitis B virus (HBV) infection is hyperendemic in Ethiopia and constitutes a major public health problem, little is known about its genetic diversity, genotypes, and circulation. The aim of this study was to determine the molecular epidemiology and genetic diversity of HBV in Ethiopia, using 391 serum samples collected from HBsAg-positive blood donors living in five different geographic regions. The HBV S/pol gene was amplified, sequenced, and HBV genotypes, subgenotypes, serotypes, and major hydrophilic region (MHR) variants were determined. Phylogenetic analysis of 371 samples (95%) revealed the distribution of genotypes A (78%) and D (22%) in Ethiopia. Further phylogenetic analysis identified one subgenotype (A1) within genotype A, and 4 subgenotypes within genotype D (D1; 1.3%, D2; 55%, D4; 2.5%, and D6; 8.8%). Importantly, 24 isolates (30%) of genotype D formed a novel phylogenetic cluster, distinct from any known D subgenotypes, and two A/D recombinants. Analysis of predicted amino-acid sequences within the HBsAg revealed four serotypes: adw2 (79%), ayw1 (3.1%), ayw2 (7.8%), and ayw3 (11.6%). Subsequent examination of sequences showed that 51 HBV isolates (14%) had mutations in the MHR and 8 isolates (2.2%) in the reverse transcriptase known to confer antiviral resistance. This study provides the first description of HBV genetic diversity in Ethiopia with a predominance of subgenotypes A1 and D2, and also identified HBV isolates that could represent a novel subgenotype. Furthermore, a significant prevalence of HBsAg variants in Ethiopian population is revealed.

Infection with seasonal influenza A viruses induces immunity to potentially pandemic influenza A viruses of other subtypes (heterosubtypic immunity). We recently demonstrated that vaccination against seasonal influenza prevented the induction of heterosubtypic immunity against influenza A/H5N1 virus induced by infection with seasonal influenza in animal models, which correlated with the absence of virus-specific CD8 + T cell responses. Annual vaccination of all healthy children against influenza has been recommended, but the impact of vaccination on the development of the virus-specific CD8 + T cell immunity in children is currently unknown. Here we compared the virus-specific CD8 + T cell immunity in children vaccinated annually with that in unvaccinated children. In the present study, we compared influenza A virus-specific cellular and humoral responses of unvaccinated healthy control children with those of children with cystic fibrosis (CF) who were vaccinated annually. Similar virus-specific CD4 + T cell and antibody responses were observed, while an age-dependent increase of the virus-specific CD8 + T cell response that was absent in vaccinated CF children was observed in unvaccinated healthy control children. Our results indicate that annual influenza vaccination is effective against seasonal influenza but hampers the development of virus-specific CD8 + T cell responses. The consequences of these findings are discussed in the light of the development of protective immunity to seasonal and future pandemic influenza viruses.

The XIII International Symposium on Respiratory Viral Infections was convened by The Macrae Group LLC (NY, USA) in Rome, Italy, on the 13–16 March 2011. This annual symposium provides a forum for public health specialists, vaccinologists, clinicians, virologists and pharmacologists to discuss recent advances in respiratory virus research in an interdisciplinary fashion. Here, we highlight presentations at this conference with a special focus focus on respiratory infections other than influenza.

The XIII International Symposium on Respiratory Viral Infections was convened by The Macrae Group LLC (NY, USA) in Rome, Italy, on the 13–16 March 2011. This annual symposium provides a forum for public health specialists, vaccinologists, clinicians, virologists and pharmacologists to discuss recent advances in respiratory virus research in an interdisciplinary fashion. Here, we highlight presentations at this conference with a special focus on influenza.

The hemagglutination inhibition (HI) assay is used most commonly for the detection of antibodies to influenza viruses. However, for the detection of antibodies to avian influenza viruses of the H5N1 subtype either induced by infection or by vaccination, the HI assay is insensitive. Therefore, the virus neutralization (VN) assay has become the method of choice to detect human serum antibodies directed to these viruses. However, this assay requires a second assay for the detection of residual virus replication, which makes it laborious to perform and less suitable for high throughput testing of large numbers of samples. Here we describe an alternative method for the detection of these antibodies, which is based on the use of reporter viruses that express the green fluorescent protein (GFP) upon infection of target cells. GFP-expressing viruses were generated carrying the HA of a variety of antigenically distinct H5N1 influenza viruses. The method proved easy to perform and could be carried out rapidly. Using a panel of antisera raised against H5N1 influenza viruses, the assay based on GFP expressing viruses was compared with the classical virus neutralization assay and the hemagglutination inhibition assay. In general, the results obtained in these assays correlated well. It was concluded that the assay based on the reporter viruses is an attractive alternative for the classical virus neutralization assay and suitable for large sero-epidemiological studies or for the assessment of vaccine efficacy in clinical trials. (C) 2011 Elsevier Ltd. All rights reserved.

Infection with seasonal influenza viruses induces a certain extent of protective immunity against potentially pandemic viruses of novel subtypes, also known as heterosubtypic immunity. Here we demonstrate that infection with a recent influenza A/H3N2 virus strain induces robust protection in ferrets against infection with a highly pathogenic avian influenza virus of the H5N1 subtype. Prior H3N2 virus infection reduced H5N1 virus replication in the upper respiratory tract, as well as clinical signs, mortality, and histopathological changes associated with virus replication in the brain. This protective immunity correlated with the induction of T cells that cross-reacted with H5N1 viral antigen. We also demonstrated that prior vaccination against influenza A/H3N2 virus reduced the induction of heterosubtypic immunity otherwise induced by infection with the influenza A/H3N2 virus. The implications of these findings are discussed in the context of vaccination strategies and vaccine development aiming at the induction of immunity to pandemic influenza.

To gain insight into the age at which children become infected with influenza viruses for the first time, we analyzed the seroprevalence of antibodies against influenza viruses in children 0 to 7 years of age in the Netherlands. Serum samples were collected during a cross-sectional population-based study in 2006 and 2007 and were tested for the presence of antibodies against influenza A/H1N1, A/H3N2, and B viruses representative of viruses present in previous influenza seasons using the hemagglutination inhibition assay. The seroprevalence of antibodies to influenza virus was higher in children 1 to 6 months of age than in children 7 to 12 months of age, which likely reflects the presence of maternally derived antibodies. The proportion of study subjects >1 year of age with detectable antibodies against influenza viruses gradually increased with age until they reached the age of 6 years, when they all had antibodies to at least one influenza A virus. These findings may have implications for the development of vaccination strategies aiming at the protection of young children against seasonal and/or pandemic influenza virus infection.

Persistent viruses, such as cytomegalovirus or human immunodeficiency virus, cause major perturbations of CD8(+) T-lymphocyte subpopulations. To test whether chronic infection with hepatitis B virus (HBV) could also be responsible for such modifications, we analyzed the expression of CD27, CD28, CCR7, and perforin in blood CD8(+) T lymphocytes. In comparison to healthy subjects and patients recovering from acute hepatitis B, chronic hepatitis B patients showed higher percentages of naive CD8(+) T lymphocytes (CD45RA(+)CD27(+)CD28(+)), and lower percentages of intermediately-differentiated CD27(+)CD28(-)CD8(+) T cells. The late differentiated CD45RA(+)CD27(-)CD28(-) subset was also present in a large percentage in some patients, but no statistically significant difference with healthy controls was observed. Removal from the circulation of intermediately-differentiated CD8(+) T lymphocytes may occur during chronic HBV infection, favoring the recruitment of naive cells. This may result in impairment of the generation of functionally-competent memory cells, and an inability to achieve control of HBV replication.

In the context of an A/H1N1 influenza pandemic situation, this study demonstrates that heterologous vaccination with an AS03-adjuvanted 2008/2009 seasonal trivalent and pandemic H5N1 monovalent split vaccine conferred partial protection in influenza-naive ferrets after challenge with the influenza pandemic H1N1 A/The Netherlands/602/09 virus. Further, unlike saline control and non-adjuvanted vaccine, it was shown that immunization of naive ferrets with an AS03-adjuvanted pandemic H1N1 A/California/7/09 influenza split vaccine induced increased antibody response and enhanced protection against the challenge strain, including significant reduction in viral shedding in the upper respiratory tract and reduced lung pathology post-challenge. These results show the need for vaccination with the adjuvanted vaccine to fully protect against viral replication and influenza disease in unprimed ferrets. (C) 2011 Elsevier Ltd. All rights reserved.

West Nile virus (WNV) is a neurotropic, arthropod-borne flavivirus that is maintained in an enzootic cycle between mosquitoes and birds, but can also infect and cause disease in horses and humans. WNV is endemic in parts of Africa, Europe, the Middle East, and Asia, and since 1999 has spread to North America, Mexico, South America, and the Caribbean. WNV infects the central nervous system (CNS) and can cause severe disease in a small minority of infected humans, mostly immunocompromised or the elderly. This review discusses some of the mechanisms by which the immune system can limit dissemination of WNV infection and elaborates on the mechanisms involved in pathogenesis. Reasons for susceptibility to WNV-associated neuroinvasive disease in less than 1% of cases remain unexplained, but one favored hypothesis is that the involvement of the CNS is associated with a weak immune response allowing robust WNV replication in the periphery and spread of the virus to the CNS.

The paramyxovirus F protein promotes fusion of the viral and cell membranes for virus entry, as well as cell-cell fusion for syncytium formation. Most paramyxovirus F proteins are triggered at neutral pH to initiate membrane fusion. Previous studies, however, demonstrated that human metapneumovirus (hMPV) F proteins are triggered at neutral or acidic pH in transfected cells, depending on the strain origin of the F sequences (S. Herfst et al., J. Virol. 82:8891–8895, 2008). We now report an extensive mutational analysis which identifies four variable residues (294, 296, 396, and 404) as the main determinants of the different syncytial phenotypes found among hMPV F proteins. These residues lie near two conserved histidines (H368 and H435) in a three-dimensional (3D) model of the pretriggered hMPV F trimer. Mutagenesis of H368 and H435 indicates that protonation of these histidines (particularly His435) is a key event to destabilize the hMPV F proteins that require low pH for cell-cell fusion. The syncytial phenotypes were reproduced in cells infected with the corresponding hMPV strains. However, the low-pH dependency for syncytium formation could not be related with a virus entry pathway dependent on an acidic environment. It is postulated that low pH may be acting for some hMPV strains as certain destabilizing mutations found in unusual strains of other paramyxoviruses. In any case, the results presented here and those reported by Schowalter et al. (J. Virol. 83:1511–1522, 2009) highlight the relevance of certain residues in the linker region and domain II of the pretriggered hMPV F protein for the process of membrane fusion.

The 2009 H1N1 influenza pandemic provided an opportunity to study human virus-specific T cell responses after infection with a novel influenza virus against which limited humoral immunity existed in the population. Here we describe the magnitude, kinetics, and nature of the virus-specific T cell response using intracellular gamma interferon (IFN-γ) staining and fluorochrome-labeled major histocompatibility complex (MHC) class I-peptide complexes. We demonstrate that influenza virus-infected patients develop recall T cell responses that peak within 1 week postinfection and that contract rapidly. In particular, effector cell frequencies declined rapidly postinfection in favor of relatively larger proportions of central memory cells.

Influenza viruses continue to cause disease of varying severity among humans. People with underlying disease and the elderly are at increased risk of developing severe disease after infection with an influenza virus. As effective and safe vaccines are available, the WHO has recommended vaccinating these groups against influenza annually. In addition to this recommendation, public health authorities of a number of countries have recently recommended vaccinating all healthy children aged 6–59 months against influenza. Here, we review the currently available data concerning the burden of disease in children, the economical impact of implementing universal vaccination of children, the efficacy of currently available influenza virus vaccines, the theoretical concerns regarding preventing immunity otherwise induced by infections with seasonal influenza viruses, and finally, how to address these concerns.

Since emergence of the pandemic (H1N1) 2009 virus in April 2009, three influenza A viruses-seasonal (H3N2), seasonal (H1N1), and pandemic (H1N1) 2009-have circulated in humans. Genetic reassortment between these viruses could result in enhanced pathogenicity. We compared 4 reassortant viruses with favorable in vitro replication properties with the wild-type pandemic (H1N1) 2009 virus with respect to replication kinetics in vitro and pathogenicity and transmission in ferrets. Pandemic (H1N1) 2009 viruses containing basic polymerase 2 alone or in combination with acidic polymerase of seasonal (H1N1) virus were attenuated in ferrets. In contrast, pandemic (H1N1) 2009 with neuraminidase of seasonal (H3N2) virus resulted in increased virus replication and more severe pulmonary lesions. The data show that pandemic (H1N1) 2009 virus has the potential to reassort with seasonal influenza viruses, which may result in increased pathogenicity while it maintains the capacity of transmission through aerosols or respiratory droplets.

The zoonotic transmissions of highly pathogenic avian influenza viruses of the H5N1 subtype hat occur since 1997 have sparked the development of novel influenza vaccines. The advent of reverse genetics technology, cell culture production techniques and novel adjuvants has improved the vaccine strain preparation, the production process and the immunogenicity of the vaccines respectively and would accelerated the availability of pandemic influenza vaccines. However, there is still room for improvement and alternative vaccine preparations are explored such as recombinant antigens (e.g. baculovirus expression) and viral vectors. Modified Vaccinia virus Ankara (MVA), originally developed as a safe smallpox vaccine can be exploited as a viral vector. It has favourable properties, which makes it an attractive candidate as a pandemic influenza vaccine (for review see reference [1]). Recently we have evaluated a MVA-based vaccine for highly pathogenic influenza virus of the H5N1 subtype in mice and macaques. To this end, recombinant MVA was constructed expressing the gene encoding the hemagglutinin of H5N1 influenza virus A/Vietnam/1194/04 (clade 1) (MVA-HA-VN/04) and used to immunize C57BL/6 mice and cynomolgus macaques (macaca fascicularis). Two immunizations induced strong virus specific antibody responses in both species and protected the animals from the development of severe disease observed in control animals inoculated with empty MVA vector or PBS after challenge infection with the homologous or the antigenically distinct influenza virus A/Indonesia/5/05 (clade 2.1). In vaccinated animals virus replication in the respiratory tract was not detectable and the development of histopathological changes in the lungs was prevented. Furthermore, a MVA-based 2009 pandemic H1N1 vaccine protected against severe disease in a pH1N1 ferret model. The preclinical evaluation of MVA-based candidate vaccines indicated that they have potential as vaccines against highly pathogenic H5N1 and pH1N1 influenza viruses. The MVA-based vaccines proved to be immunogenic and induced broad-protective immune responses. MVA has favourable properties for the production, storage and use as a pandemic influenza vaccine and further clinical development seems warranted.

Vaccination is the most cost-effective way to reduce the considerable disease burden of seasonal influenza. Although seasonal influenza vaccines are effective, their performance in the elderly and immunocompromised individuals would benefit from improvement. Major problems related to the development and production of pandemic influenza vaccines are response time and production capacity as well as vaccine efficacy and safety. Several improvements can be envisaged. Vaccine production technologies based on embryonated chicken eggs may be replaced by cell culture techniques. Reverse genetics techniques can speed up the generation of seed viruses and new mathematical modelling methods improve vaccine strain selection. Better understanding of the correlates of immune-mediated protection may lead to new vaccine targets besides the viral haemagglutinin, like the neuraminidase and M2 proteins. In addition, the role of cell-mediated immunity could be better exploited. New adjuvants have recently been shown to increase the breadth and the duration of influenza vaccine-induced protection. Other studies have shown that influenza vaccines based on different viral vector systems may also induce broad protection. It is to be expected that these developments may lead to more universal influenza vaccines that elicit broader and longer protection, and can be produced more efficiently.