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Angiotensin-converting enzyme 2 (ACE2) has been established as the functional host receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the current devastating worldwide pandemic of coronavirus disease 2019 (COVID-19). ACE2 is abundantly expressed in a variety of cells residing in many different human organs. In human physiology, ACE2 is a pivotal counter-regulatory enzyme to ACE by the breakdown of angiotensin II, the central player in the renin-angiotensin-aldosterone system (RAAS) and the main substrate of ACE2. Many factors have been associated with both altered ACE2 expression and COVID-19 severity and progression, including age, sex, ethnicity, medication, and several co-morbidities, such as cardiovascular disease and metabolic syndrome. Although ACE2 is widely distributed in various human tissues and many of its determinants have been well recognised, ACE2-expressing organs do not equally participate in COVID-19 pathophysiology, implying that other mechanisms are involved in orchestrating cellular infection resulting in tissue damage. Reports of pathologic findings in tissue specimens of COVID-19 patients are rapidly emerging and confirm the established role of ACE2 expression and activity in disease pathogenesis. Identifying pathologic changes caused by SARS-CoV-2 infection is crucially important as it has major implications for understanding COVID-19 pathophysiology and the development of evidence-based treatment strategies. Currently, many interventional strategies are being explored in ongoing clinical trials, encompassing many drug classes and strategies, including antiviral drugs, biological response modifiers, and RAAS inhibitors. Ultimately, prevention is key to combat COVID-19 and appropriate measures are being taken accordingly, including development of effective vaccines. In this review, we describe the role of ACE2 in COVID-19 pathophysiology, including factors influencing ACE2 expression and activity in relation to COVID-19 severity. In addition, we discuss the relevant pathological changes resulting from SARS-CoV-2 infection. Finally, we highlight a selection of potential treatment modalities for COVID-19. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

TIPiCO is an annual expert meeting and workshop on infectious diseases and vaccination. The edition of 2020 changed its name and format to aTIPiCO, the first series and podcasts on infectious diseases and vaccines. A total of 13 prestigious experts from different countries participated in this edition launched on the 26 November 2020. The state of the art of coronavirus disease-2019 (COVID-19) and the responsible pathogen, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), and the options to tackle the pandemic situation were discussed in light of the knowledge in November 2020. Despite COVID-19, the status of other infectious diseases, including influenza infections, respiratory syncytial virus disease, human papillomavirus infection, measles, pertussis, tuberculosis, meningococcal disease, and pneumococcal disease, were also addressed. The essential lessons that can be learned from these diseases and their vaccines to use in the COVID-19 pandemic were also commented with the experts. © 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.

The Ninth Interactive Infectious Disease workshop TIPICO was held on November 22–23, 2018, in Santiago de Compostela, Spain. This 2-day academic experience addressed current and topical issues in the field of infectious diseases and vaccination. Summary findings of the meeting include: cervical cancer elimination will be possible in the future, thanks to the implementation of global vaccination action plans in combination with appropriate screening interventions. The introduction of appropriate immunization programs is key to maintain the success of current effective vaccines such as those against meningococcal disease or rotavirus infection. Additionally, reduced dose schedules might improve the efficiency of some vaccines (i.e., PCV13). New vaccines to improve current preventive alternatives are under development (e.g., against tuberculosis or influenza virus), while others to protect against infectious diseases with no current available vaccines (e.g., enterovirus, parechovirus and flaviviruses) need to be developed. Vaccinomics will be fundamental in this process, while infectomics will allow the application of precision medicine. Further research is also required to understand the impact of heterologous vaccine effects. Finally, vaccination requires education at all levels (individuals, community, healthcare professionals) to ensure its success by helping to overcome major barriers such as vaccine hesitancy and false contraindications FMT and AS research activities received support from the Instituto de Salud Carlos III (Proyecto de Investigación en Salud, Acción Estratégica en Salud): project GePEM ISCIII/PI16/01478/Cofinanciado FEDER, and project ReSVinext ISCIII/PI16/01569/Cofinanciado FEDER; Consellería de Sanidade, Xunta de Galicia (RHI07/2-intensificación actividad investigadora, PS09749 and 10PXIB918184PR), Instituto de Salud Carlos III (Intensificación de la actividad investigadora 2007–2012, PI16/01569), Fondo de Investigación Sanitaria (FIS; PI070069/PI1000540) del plan nacional de I+D+I and ‘fondos FEDER’, and 2016-PG071 Consolidación e Estructuración REDES 2016GI-1344 G3VIP (Grupo Gallego de Genética Vacunas Infecciones y Pediatría, ED341D R2016/021). Research on flaviviruses at the AG-S laboratory is partially funded by NIAID grants R21AI129486, R21AI142337 and U19AI118610 Research on influenza virus vaccines and immunity at the AG-S laboratory is partly funded by NIAID grants U19AI117873, U01AI124297, R01AI127658, P01AI097092, U19AI135972, and R01 AI141226-01, by the Bill and Melinda Gates Foundations (OPP1084518), by a research contract from GSK and by CRIP (Center for Research on Influenza Pathogenesis), an NIAID funded Center of Excellence for Influenza Research and Pathogenesis (CEIRS, contract number HHSN272201400008C) SI

Rabies is a fatal disease of all mammals causing almost 60,000 human deaths every year. To date, there is no effective treatment of clinical rabies once the symptoms appear. Here, we describe the promising effect of combination therapy composed of molecules that target replication of the rabies virus (RV) at different stages of life cycle and molecules that inhibit some pathways of the innate host immune response accompanied by a blood-brain barrier opener on the outcome of RV infection. The study reports statistically significant extension of survival of mice treated with the drug cocktail containing T-705, ribavirin, interferon α/β, caspase-1 inhibitor, TNF-α inhibitor, MAPKs inhibitor and HRIG compared to the survival of mice in the virus control group (p = 0.0312). Furthermore, the study points to the significant impact of interferon α/β on the survival of RV-infected mice. We have shown a significant down regulation of pro-inflammatory molecules (caspase-1 and TNF-a) in the CNS in RV-infected mice treated with a combination of drugs including interferon α/β.

Rabies is a deadly viral disease with an extremely high fatality rate in humans. Previously, it was suggested that an enhancement of the blood-brain barrier (BBB) permeability, which allows immune cells and/or antibodies to enter the central nervous system (CNS) tissue, is critical to clear the infection. In this study, we utilised mannitol to increase BBB permeability in mice infected with highly pathogenic silver-haired bat rabies virus (SHBRV). We found that intraperitoneal injection of mannitol causes a slight, transient increase of BBB permeability in the treated mice. SHBRV-infected mice were treated with intraperitoneally administered mannitol daily from day 3 or day 4 post-infection, but no effect of this treatment on the time of disease onset, clinical signs or survival was observed. This data indicates that the increase of BBB permeability by mannitol is not efficient in promoting CNS virus clearance in SHBRV-infected mice.

Rabies virus infects almost all mammals resulting in lethal disease. To date there is no treatment available for symptomatic rabies and there is an urgent need to develop treatment strategies that would prolong survival, thereby providing a window of opportunity for the host to mount a protective immune response. We hypothesized that both virus and excessive immune response contribute to disease and that interfering with both is necessary to prevent lethal disease. Here, we have inhibited the pro-inflammatory response associated with pyroptosis and showed that inhibition of CASP-1 had a beneficial effect on survival time. Our results confirm that some inflammatory responses may be involved in the pathogenesis of severe disease and the results suggest that effective intervention includes inhibition of virus and host response.

Summary Importin-α adaptor proteins orchestrate dynamic nuclear transport processes involved in cellular homeostasis. Here, we show that importin-α3, one of the main NF-κB transporters, is the most abundantly expressed classical nuclear transport factor in the mammalian respiratory tract. Importin-α3 promoter activity is regulated by TNF-α-induced NF-κB in a concentration-dependent manner. High-level TNF-α-inducing highly pathogenic avian influenza A viruses (HPAIVs) isolated from fatal human cases harboring human-type polymerase signatures (PB2 627K, 701N) significantly downregulate importin-α3 mRNA expression in primary lung cells. Importin-α3 depletion is restored upon back-mutating the HPAIV polymerase into an avian-type signature (PB2 627E, 701D) that can no longer induce high TNF-α levels. Importin-α3-deficient mice show reduced NF-κB-activated antiviral gene expression and increased influenza lethality. Thus, importin-α3 plays a key role in antiviral immunity against influenza. Lifting the bottleneck in importin-α3 availability in the lung might provide a new strategy to combat respiratory virus infections., Graphical Abstract, Highlights • Importin-α3 is the most abundantly expressed isoform in the mammalian lung • Importin-α3 is highly conserved across species • Importin-α3 is one of the major nuclear transporters of NF-κB • Importin-α3 acts as an immune sensor of influenza A virus infections, Thiele et al. show that importin-α3 is one of the major nuclear transporters of NF-κB in the mammalian lung. High-level TNF-α-inducing HPAIVs inhibit importin-α3 mRNA transcription by interfering with its promoter activity. Thus, HPAIVs may evade antiviral immunity in the respiratory tract by generating a bottleneck in importin-α3 availability.

BACKGROUND: High-pathogenicity avian influenza viruses continue to circulate in poultry and wild birds and occasionally infect humans, sometimes with fatal outcomes. Development of vaccines is a priority to prepare for potential pandemics but is complicated by antigenic variation of the surface glycoprotein hemagglutinin. We report the immunological profile induced by human immunization with modified vaccinia virus Ankara (MVA) expressing the hemagglutinin gene of influenza A(H5N1) virus A/Vietnam/1194/04 (rMVA-H5). METHODS: In a double-blinded phase 1/2a clinical trial, 79 individuals received 1 or 2 injections of rMVA-H5 or vector control. Twenty-seven study subjects received a booster immunization after 1 year. The breadth, magnitude, and properties of vaccine-induced antibody and T-cell responses were characterized. RESULTS: rMVA-H5 induced broadly reactive antibody responses, demonstrated by protein microarray, hemagglutination inhibition, virus neutralization, and antibody-dependent cellular cytotoxicity assays. Antibodies cross-reacted with antigenically distinct H5 viruses, including the recently emerged subtypes H5N6 and H5N8 and the currently circulating subtype H5N1. In addition, the induction of T cells specific for H5 viruses of 2 different clades was demonstrated. CONCLUSIONS: rMVA-H5 induced immune responses that cross-reacted with H5 viruses of various clades. These findings validate rMVA-H5 as vaccine candidate against antigenically distinct H5 viruses. CLINICAL TRIALS REGISTRATION: NTR3401.

West Nile virus (WNV) and Usutu virus (USUV) are arboviruses that are maintained in enzootic transmission cycles between mosquitoes and birds and are occasionally transmitted to mammals. As arboviruses are currently expanding their geographic range and emerging in often unpredictable locations, surveillance is considered an important element of preparedness. To determine whether sera collected from resident and migratory birds in the Netherlands as part of avian influenza surveillance would also represent an effective source for proactive arbovirus surveillance, a random selection of such sera was screened for WNV antibodies using a commercial ELISA. In addition, sera of jackdaws and carrion crows captured for previous experimental infection studies were added to the selection. Of the 265 screened serum samples, 27 were found to be WNV-antibody-positive, and subsequent cross-neutralization experiments using WNV and USUV confirmed that five serum samples were positive for only WNV-neutralizing antibodies and seven for only USUV. The positive birds consisted of four Eurasian coots (Fulica atra) and one carrion crow (Corvus corone) for WNV, of which the latter may suggest local presence of the virus, and only Eurasian coots for USUV. As a result, the screening of a small selection of serum samples originally collected for avian influenza surveillance demonstrated a seroprevalence of 1.6% for WNV and 2.8% for USUV, suggesting that this sustained infrastructure could serve as a useful source for future surveillance of arboviruses such as WNV and USUV in the Netherlands.

Emerging infections have threatened humanity since times immemorial. The dramatic anthropogenic, behavioral and social changes that have affected humanity and the environment in the past century have accelerated the intrusion of novel pathogens into the global human population, sometimes with devastating consequences. The AIDS and influenza pandemics have claimed and will continue to claim millions of lives. The recent SARS and Ebola epidemics have threatened populations across borders. The emergence of MERS may well be warning signals of a nascent pandemic threat, while the potential for geographical spread of vector-borne diseases, such as Zika, but also Dengue and Chikungunya is unprecedented. Novel technologies and innovative approaches have multiplied to address and improve response preparedness towards the increasing yet unpredictable threat posed by emerging pathogens.

Hepatitis B virus (HBV) is genetically highly divergent and classified in ten genotypes and forty subgenotypes in distinct ethno-geographic populations worldwide. Ethiopia is a country with high HBV prevalence; however, little is known about the genetic variability of HBV strains that circulate. Here, we characterize the complete genome of 29 HBV strains originating from five Ethiopian regions, by 454 deep sequencing and Sanger sequencing. Phylogenetically, ten strains were classified as genotype A1 and nineteen as genotype D. Fifteen genotype D strains, provisionally named subgenotype D10, showed a novel distinct cluster supported by high bootstrap value and >4% nucleotide divergence from other known subgenotypes. In addition, the novel D10 strains harboured nine unique amino acid signatures in the surface, polymerase and X genes. Seventy-two per cent of the genotype D strains had the precore premature stop codon G1896A. In addition, 63% genotype A and 33% genotype D strains had the basal core promoter mutations, A1762T/G1764A. Furthermore, four pre-S deletion variants and two recombinants were identified in this study. In conclusion, we identified a novel HBV subgenotype D10 circulating in Ethiopia, underlining the high genetic variability of HBV strains in Africa.

Background Chikungunya virus (CHIKV) is a re-emerging arbovirus capable of causing chronic arthralgia, which can last for months to years. Although neutralizing antibodies have been shown to be important for viral clearance, is it not clear whether the quantitative and qualitative nature of antibodies play a role in progression to chronic disease. Objectives To characterize and compare the antibody responses in acute and chronic patients in a prospective observational CHIKV study in Curacao during the 2014-2015 outbreak. Study design We performed virus neutralization tests and ELISA on plasma samples collected from a prospective observational chikungunya study in Curacao to compare the complement-dependent and –independent neutralization capacity, as well as the antibody avidity index of acute and chronic patients. Results We found that there was no significant difference in the virus neutralization titers between patients with acute and chronic chikungunya infection. Furthermore, we found that complement increased the neutralization capacity when large amounts of virus was used. Moreover, we found that patients with acute chikungunya disease had a significantly higher antibody avidity index compared to those with chronic disease. Conclusions This study suggests that virus neutralization titers in late convalescent sera do not play a role in chronic chikungunya. However, the median antibody avidity was lower in these patients and may therefore suggest a role for antibody avidity in the development of chronic disease.

Ferret coronaviruses (FRCoVs) exist as an enteric and a systemic pathotype, of which the latter is highly lethal to ferrets. To our knowledge, this study provides the first full genome sequence of a FRCoV, tentatively called FRCoV-NL-2010, which was detected in 2010 in ferrets in The Netherlands. Phylogenetic analysis showed that FRCoV-NL-2010 is most closely related to mink CoV, forming a separate clade of mustelid alphacoronavirus that split off early from other alphacoronaviruses. Based on sequence homology of the complete genome, we propose that these mustelid coronaviruses may be assigned to a new species. Comparison of FRCoV-NL-2010 with the partially sequenced ferret systemic coronavirus MSU-1 and ferret enteric coronavirus MSU-2 revealed that recombination in the spike, 3c and envelope genes occurred between different FRCoVs.

SummaryCompanion animals comprise a wide variety of species, including dogs, cats, horses, ferrets, guinea pigs, reptiles, birds and ornamental fish, as well as food production animal species, such as domestic pigs, kept as companion animals. Despite their prominent place in human society, little is known about the role of companion animals as sources of viruses for people and food production animals. Therefore, we reviewed the literature for accounts of infections of companion animals by zoonotic viruses and viruses of food production animals, and prioritized these viruses in terms of human health and economic importance. In total, 138 virus species reportedly capable of infecting companion animals were of concern for human and food production animal health: 59 of these viruses were infectious for human beings, 135 were infectious for food production mammals and birds, and 22 were infectious for food production fishes. Viruses of highest concern for human health included hantaviruses, Tahyna virus, rabies virus, West Nile virus, tick-borne encephalitis virus, Crimean–Congo haemorrhagic fever virus, Aichi virus, European bat lyssavirus, hepatitis E virus, cowpox virus, G5 rotavirus, influenza A virus and lymphocytic choriomeningitis virus. Viruses of highest concern for food production mammals and birds included bluetongue virus, African swine fever virus, foot-and-mouth disease virus, lumpy skin disease virus, Rift Valley fever virus, porcine circovirus, classical swine fever virus, equine herpesvirus 9, peste des petits ruminants virus and equine infectious anaemia virus. Viruses of highest concern for food production fishes included cyprinid herpesvirus 3 (koi herpesvirus), viral haemorrhagic septicaemia virus and infectious pancreatic necrosis virus. Of particular concern as sources of zoonotic or food production animal viruses were domestic carnivores, rodents and food production animals kept as companion animals. The current list of viruses provides an objective basis for more in-depth analysis of the risk of companion animals as sources of viruses for human and food production animal health.

Coronaviruses in the Middle East Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe acute respiratory illness and kills about a third of people infected. The virus is common in dromedary camels, which can be a source of human infections. In a survey for MERSCoV in over 1300 Saudi Arabian camels, Sabir et al. found that dromedaries share three coronavirus species with humans. Diverse MERS lineages in camels have caused human infections, which suggests that transfer among host species occurs quite easily. Haagmans et al. made a MERS-CoV vaccine for use in camels, using poxvirus as a vehicle. The vaccine significantly reduced virus excretion, which should help reduce the potential for transmission to humans, and conferred cross-immunity to camelpox infections. Science , this issue p. 81 , p. 77

Genotype 3 (gt3) hepatitis E virus (HEV) infections are emerging in Western countries. Immunosuppressed patients are at risk of chronic HEV infection and progressive liver damage, but no adequate model system currently mimics this disease course. Here we explore the possibilities of in vivo HEV studies in a human liver chimeric mouse model (uPA +/+ Nod-SCID-IL2Rγ −/− ) next to the A549 cell culture system, using HEV RNA-positive EDTA-plasma, feces, or liver biopsy specimens from 8 immunocompromised patients with chronic gt3 HEV. HEV from feces- or liver-derived inocula showed clear virus propagation within 2 weeks after inoculation onto A549 cells, compared to slow or no HEV propagation of HEV RNA-positive, EDTA-plasma samples. These in vitro HEV infectivity differences were mirrored in human-liver chimeric mice after intravenous (i.v.) inoculation of selected samples. HEV RNA levels of up to 8 log IU HEV RNA/gram were consistently present in 100% of chimeric mouse livers from week 2 to week 14 after inoculation with human feces- or liver-derived HEV. Feces and bile of infected mice contained moderate to large amounts of HEV RNA, while HEV viremia was low and inconsistently detected. Mouse-passaged HEV could subsequently be propagated for up to 100 days in vitro . In contrast, cell culture-derived or seronegative EDTA-plasma-derived HEV was not infectious in inoculated animals. In conclusion, the infectivity of feces-derived human HEV is higher than that of EDTA-plasma-derived HEV both in vitro and in vivo . Persistent HEV gt3 infections in chimeric mice show preferential viral shedding toward mouse bile and feces, paralleling the course of infection in humans. IMPORTANCE Hepatitis E virus (HEV) genotype 3 infections are emerging in Western countries and are of great concern for immunosuppressed patients at risk for developing chronic HEV infection. Lack of adequate model systems for chronic HEV infection hampers studies on HEV infectivity and transmission and antiviral drugs. We compared the in vivo infectivity of clinical samples from chronic HEV patients in human liver chimeric mice to an in vitro virus culture system. Efficient in vivo HEV infection is observed after inoculation with feces- and liver-derived HEV but not with HEV RNA-containing plasma or cell culture supernatant. HEV in chimeric mice is preferentially shed toward bile and feces, mimicking the HEV infection course in humans. The observed in vivo infectivity differences may be relevant for the epidemiology of HEV in humans. This novel small-animal model therefore offers new avenues to unravel HEV's pathobiology.

Natural influenza A virus infections elicit both virus-specific antibody and CD4 + and CD8 + T cell responses. Influenza A virus-specific CD8 + cytotoxic T lymphocytes (CTLs) contribute to clearance of influenza virus infections. Viral CTL epitopes can display variation, allowing influenza A viruses to evade recognition by epitope-specific CTLs. Due to functional constraints, some epitopes, like the immunodominant HLA-A*0201-restricted matrix protein 1 (M1 58–66 ) epitope, are highly conserved between influenza A viruses regardless of their subtype or host species of origin. We hypothesized that human influenza A viruses evade recognition of this epitope by impairing antigen processing and presentation by extraepitopic amino acid substitutions. Activation of specific T cells was used as an indication of antigen presentation. Here, we show that the M1 58–66 epitope in the M1 protein derived from human influenza A virus was poorly recognized compared to the M1 protein derived from avian influenza A virus. Furthermore, we demonstrate that naturally occurring variations at extraepitopic amino acid residues affect CD8 + T cell recognition of the M1 58–66 epitope. These data indicate that human influenza A viruses can impair recognition by M1 58–66 -specific CTLs while retaining the conserved amino acid sequence of the epitope, which may represent a yet-unknown immune evasion strategy for influenza A viruses. This difference in recognition may have implications for the viral replication kinetics in HLA-A*0201 individuals and spread of influenza A viruses in the human population. The findings may aid the rational design of universal influenza vaccines that aim at the induction of cross-reactive virus-specific CTL responses. IMPORTANCE Influenza viruses are an important cause of acute respiratory tract infections. Natural influenza A virus infections elicit both humoral and cellular immunity. CD8 + cytotoxic T lymphocytes (CTLs) are directed predominantly against conserved internal proteins and confer cross-protection, even against influenza A viruses of various subtypes. In some CTL epitopes, mutations occur that allow influenza A viruses to evade recognition by CTLs. However, the immunodominant HLA-A*0201-restricted M1 58–66 epitope does not tolerate mutations without loss of viral fitness. Here, we describe naturally occurring variations in amino acid residues outside the M1 58–66 epitope that influence the recognition of the epitope. These results provide novel insights into the epidemiology of influenza A viruses and their pathogenicity and may aid rational design of vaccines that aim at the induction of CTL responses.

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.

Large outbreaks of zoonotic influenza A virus (IAV) infections may presage an influenza pandemic. However, the likelihood that an airborne-transmissible variant evolves upon zoonotic infection or co-infection with zoonotic and seasonal IAVs remains poorly understood, as does the relative importance of accumulating mutations versus re-assortment in this process. Using discrete-time probabilistic models, we determined quantitative probability ranges that transmissible variants with 1–5 mutations and transmissible re-assortants evolve after a given number of zoonotic IAV infections. The systematic exploration of a large population of model parameter values was designed to account for uncertainty and variability in influenza virus infection, epidemiological and evolutionary processes. The models suggested that immunocompromised individuals are at high risk of generating IAV variants with pandemic potential by accumulation of mutations. Yet, both immunocompetent and immunocompromised individuals could generate high viral loads of single and double mutants, which may facilitate their onward transmission and the subsequent accumulation of additional 1–2 mutations in newly-infected individuals. This may result in the evolution of a full transmissible genotype along short chains of contact transmission. Although co-infection with zoonotic and seasonal IAVs was shown to be a rare event, it consistently resulted in high viral loads of re-assortants, which may facilitate their onward transmission among humans. The prevention or limitation of zoonotic IAV infection in immunocompromised and contact individuals, including health care workers, as well as vaccination against seasonal IAVs—limiting the risk of co-infection—should be considered fundamental tools to thwart the evolution of a novel pandemic IAV by accumulation of mutations and re-assortment.

The population of ring-necked pheasants (Phasianus colchicus) is decreasing all over Germany since the years 2008/2009. Besides impacts of habitat changes caused by current rates of land conversion, climatic influences or predators, a contribution of infectious pathogens needs also to be considered. Infectious and non-infectious diseases in free-living populations of ring-necked pheasants have been scarcely investigated so far. In the present study, carcasses of 258 deceased free-ranging pheasants of different age groups, predominantly adult pheasants, collected over a period of 4 years in the states of Lower Saxony, North Rhine–Westphalia and Schleswig-Holstein, were examined pathomorphologically, parasitologically, virologically and bacteriologically, with a focus set on infectious pathogens. A periocular and perinasal dermatitis of unknown origin was present in 62.3% of the pheasants. Additional alterations included protozoal cysts in the skeletal musculature (19.0%), hepatitis (21.7%), enteritis (18.7%), gastritis (12.6%), and pneumonia (11.7%). In single cases, neoplasms (2.6%) and mycobacteriosis (1.7%) occurred. Further findings included identification of coronaviral DNA from trachea or caecal tonsils (16.8%), siadenoviral DNA (7.6%), avian metapneumoviral RNA (6.6%), and infectious bursal disease viral RNA (3.7%). Polymerase chain reaction (PCR) on herpesvirus, avian influenza virus (AIV), paramyxovirus type 1 (PMV-1), avian encephalomyelitis virus (AEV), and chlamydia were negative. Based on the present results, there is no indication of a specific pathogen as a sole cause for population decline in adult pheasants. However, an infectious disease can still not be completely excluded as it may only affect reproduction effectivity or a certain age group of pheasants (e.g., chicks) which were not presented in the study.

Hepatitis B virus (HBV) is a major cause of human hepatitis. There is considerable uncertainty about the timescale of its evolution and its association with humans. Here we present 12 full or partial ancient HBV genomes that are between approximately 0.8 and 4.5 thousand years old. The ancient sequences group either within or in a sister relationship with extant human or other ape HBV clades. Generally, the genome properties follow those of modern HBV. The root of the HBV tree is projected to between 8.6 and 20.9 thousand years ago, and we estimate a substitution rate of 8.04 × 10−6–1.51 × 10−5 nucleotide substitutions per site per year. In several cases, the geographical locations of the ancient genotypes do not match present-day distributions. Genotypes that today are typical of Africa and Asia, and a subgenotype from India, are shown to have an early Eurasian presence. The geographical and temporal patterns that we observe in ancient and modern HBV genotypes are compatible with well-documented human migrations during the Bronze and Iron Ages1,2. We provide evidence for the creation of HBV genotype A via recombination, and for a long-term association of modern HBV genotypes with humans, including the discovery of a human genotype that is now extinct. These data expose a complexity of HBV evolution that is not evident when considering modern sequences alone. Phylogenies reconstructed using 12 hepatitis B virus genomes, which were recovered from ancient human genome data, reveal a complex history of hepatitis B evolution that is not evident when using only modern samples.

Several human pathogens exhibit distinct patterns of seasonality and circulate as pairs of discrete strains. For instance, the activity of the two co-circulating influenza A virus subtypes oscillates and peaks during winter seasons of the world’s temperate climate zones. These periods of increased activity are usually caused by a single dominant subtype. Alternation of dominant strains in successive influenza seasons makes epidemic forecasting a major challenge. From the start of the 2009 influenza pandemic we enrolled influenza A virus infected patients (n = 2,980) in a global prospective clinical study. Complete hemagglutinin (HA) sequences were obtained from 1,078 A/H1N1 and 1,033 A/H3N2 viruses and were linked to patient data. We then used phylodynamics to construct high resolution spatio-temporal phylogenetic HA trees and estimated global influenza A effective reproductive numbers (R) over time (2009-2013). We demonstrate that R, a parameter to define host immunity, oscillates around R = 1 with a clear opposed alternation pattern between phases of the A/H1N1 and A/H3N2 subtypes. Moreover, we find a similar alternation pattern for the number of global virus migration events between the sampled geographical locations. Both observations suggest a between-strain competition for susceptible hosts on a global level. Extrinsic factors that affect person-to-person transmission are a major driver of influenza seasonality, which forces influenza epidemics to coincide with winter seasons. The data presented here indicate that also cross-reactive host immunity is a key intrinsic driver of global influenza seasonality, which determines the outcome of competition between influenza A virus strains at the onset of each epidemic season.Significance statementAnnual influenza epidemics coincide with winter seasons in many parts of the world. Environmental factors, such as air humidity variation or temperature change, are commonly believed to drive these seasonality patterns. Interestingly, three out of the four latest pandemics (1918, 1968 and 2009) did not spread in winter initially, but during summer. This questions to what extent other factors could also impact virus spread among humans. We demonstrate that cross-reactive host immunity is a key factor. It drives the well-known seasonal patterns of virus activity oscillation and alternation of the dominant influenza virus subtype in successive seasons. Furthermore, this factor may also explain the efficient spread of pandemic viruses during summer when cross-reactive host immunity is relatively low.

The ability of Middle East respiratory syndrome coronavirus (MERS-CoV) to infect small animal species may be restricted given the fact that mice, ferrets, and hamsters were shown to resist MERS-CoV infection. We inoculated rabbits with MERS-CoV. Although virus was detected in the lungs, neither significant histopathological changes nor clinical symptoms were observed. Infectious virus, however, was excreted from the upper respiratory tract, indicating a potential route of MERS-CoV transmission in some animal species.

Since 2013, avian influenza viruses of subtype H7N9 have been transmitted from poultry to humans in China and caused severe disease. Concerns persist over the pandemic potential of this virus and further understanding of immunity and transmission is required. The isogenic guinea pig model uniquely would allow for investigation into both. Eighteen female isogenic guinea pigs 12-16 weeks were inoculated intratracheally with either A/H7N9 virus (n=12) or PBS (n=6) and sacrificed on days 2 and 7 post-inoculation. Nasal and pharyngeal swabs were taken daily to assess viral replication kinetics and necropsies were performed to study pathogenesis. All animals showed peak virus titers in nasal secretions at day 2 post-inoculation and by day 7 post-inoculation infectious virus titers had decreased to just above detectable levels. At day 2, high virus titers were found in nasal turbinates and lungs and moderate titers in trachea and cerebrum. At day 7, infectious virus was detected in the nasal turbinates only. Histology showed moderate to severe inflammation in the entire respiratory tract and immunohistochemistry (IHC) demonstrated large numbers of viral antigen positive cells in the nasal epithelium at day 2 and fewer at day 7 post-inoculation. A moderate number of IHC positive cells was observed in the bronchi(oli) and alveoli at day 2 only. This study indicates that isogenic guinea pigs are a promising model to further study immunity to and transmission of H7N9 influenza virus. (C) 2015 Elsevier Ltd. All rights reserved.

BACKGROUND: Modified vaccinia virus Ankara (MVA) is a promising viral vector platform for the development of an H5N1 influenza vaccine. Preclinical assessment of MVA-based H5N1 vaccines showed their immunogenicity and safety in different animal models. We aimed to assess the safety and immunogenicity of the MVA-haemagglutinin-based H5N1 vaccine MVA-H5-sfMR in healthy individuals.METHODS: In a single-centre, double-blind phase 1/2a study, young volunteers (aged 18-28 years) were randomly assigned with a computer-generated list in equal numbers to one of eight groups and were given one injection or two injections intramuscularly at an interval of 4 weeks of a standard dose (10(8) plaque forming units [pfu]) or a ten times lower dose (10(7) pfu) of the MVA-H5-sfMR (vector encoding the haemagglutinin gene of influenza A/Vietnam/1194/2004 virus [H5N1 subtype]) or MVA-F6-sfMR (empty vector) vaccine. Volunteers and physicians who examined and administered the vaccine were masked to vaccine assignment. Individuals who received the MVA-H5-sfMR vaccine were eligible for a booster immunisation 1 year after the first immunisation. Primary endpoint was safety. Secondary outcome was immunogenicity. The trial is registered with the Dutch Trial Register, number NTR3401.FINDINGS: 79 of 80 individuals who were enrolled completed the study. No serious adverse events were identified. 11 individuals reported severe headache and lightheadedness, erythema nodosum, respiratory illness (accompanied by influenza-like symptoms), sore throat, or injection-site reaction. Most of the volunteers had one or more local (itch, pain, redness, and swelling) and systemic reactions (rise in body temperature, headache, myalgia, arthralgia, chills, malaise, and fatigue) after the first, second, and booster immunisations. Individuals who received the 10(7) dose had fewer systemic reactions. The MVA-H5-sfMR vaccine at 10(8) pfu induced significantly higher antibody responses after one and two immunisations than did 10(7) pfu when assessed with haemagglutination inhibition geometric mean titre at 8 weeks against H5N1 A/Vietnam/1194/2004 (30·2 [SD 3·8] vs 9·2 [2·3] and 108·1 [2·4] vs 15·8 [3·2]). 27 of 39 eligible individuals were enrolled in the booster immunisation study. A single shot of MVA-H5-sfMR 10(8) pfu prime immunisation resulted in higher antibody responses after the booster immunisation than did two shots of MVA-H5-sfMR at the ten times lower dose.INTERPRETATION: The MVA-based H5N1 vaccine was well tolerated and immunogenic and therefore the vaccine candidates arising from the MVA platform hold great promise for rapid development in response to a future influenza pandemic threat. However, the immunogenicity of this vaccine needs to be compared with conventional H5N1 inactivated non-adjuvanted vaccine candidates in head-to-head clinical trials.FUNDING: European Research Council.

Summary Zoonotic influenza viruses that are a few mutations away from pandemic viruses circulate in animals, and can evolve into airborne-transmissible viruses in human beings. Paradoxically, such viruses only occasionally emerge in people; the four influenza pandemics that occurred in the past 100 years were caused by zoonotic viruses that acquired efficient transmissibility. Emergence of a pandemic virus in people can happen when transmissible viruses evolve in individuals with zoonotic influenza and replicate to titres allowing transmission. We postulate that this step in the genesis of a pandemic virus only occasionally occurs in human beings, because the immune response triggered by zoonotic influenza virus also controls transmissible mutants that emerge during infection. Therefore, an impaired immune response might be needed for within-host emergence of a pandemic virus and replication to titres allowing transmission. Immunocompromised individuals—eg, those with comorbidities, of advanced age, or receiving immunosuppressive treatment—could be at increased risk of generating transmissible viruses and initiating chains of human-to-human infection.

West Nile virus (WNV) and chikungunya virus (CHIKV) are arboviruses that are constantly (re-)emerging and expanding their territory. Both viruses often cause a mild form of disease, but severe forms of the disease can consist of neurological symptoms, most often observed in the elderly and young children, respectively, for which the mechanisms are poorly understood. To further elucidate the mechanisms responsible for end-stage WNV and CHIKV neuroinvasive disease, we used transcriptomics to compare the induction of effector pathways in the brain during the early and late stage of disease in young mice. In addition to the more commonly described cell death pathways such as apoptosis and autophagy, we also found evidence for the differential expression of pyroptosis and necroptosis cell death markers during both WNV and CHIKV neuroinvasive disease. In contrast, no evidence of cell dysfunction was observed, indicating that cell death may be the most important mechanism of disease. Interestingly, there was overlap when comparing immune markers involved in neuroinvasive disease to those seen in neurodegenerative diseases. Nonetheless, further validation studies are needed to determine the activation and involvement of these effector pathways at the end stage of disease. Furthermore, evidence for a strong inflammatory response was found in mice infected with WNV and CHIKV. The transcriptomics profile measured in mice with WNV and CHIKV neuroinvasive disease in our study showed strong overlap with the mRNA profile described in the literature for other viral neuroinvasive diseases. More studies are warranted to decipher the role of cell inflammation and cell death in viral neuroinvasive disease and whether common mechanisms are active in both neurodegenerative and brain infectious diseases.

Background Recently Chikungunya virus (CHIKV) outbreaks have been reported in the Carribean. There is no data regarding the outbreak in Curacao. In addition, to date there is no biomarker that could be used to predict chronic infection. Objectives To characterize the first CHIKV outbreak in Curacao and to identify potential biomarkers for chronic infection. Study design A serological test and quantitative polymerase chain reaction (qPCR) were used on samples collected in Curacao to confirm infection. Subsequently, six samples with high viral load were selected for phylogenetic analysis. Furthermore we investigated the association of macrophage-related biomarkers during CHIKV infection with chronic arthralgia/arthritis. Results 116 patients in Curacao were diagnosed with CHIKV infection based on ELISA and 77% were tested positive for CHIKV by qPCR. Phylogenetic analysis showed that an Asian genotype was the cause of the outbreak. Elevated levels of ferritin and CRP were significantly associated with viraemia. In addition, elevated ferritin levels were significantly associated with chronic arthralgia. Conclusions The results showed that the presence of an Asian genotype of CHIKV in Curacao for the first time. Moreover, we found an association between ferritin levels with chronic arthralgia.

Current standard-of-care treatment of chronically infected hepatitis C virus (HCV) patients involves direct-acting antivirals (DAA). However, concerns exist regarding the emergence of drug -resistant variants and subsequent treatment failure. In this study, we investigate potential natural drug-resistance mutations in the NS5B gene of HCV genotype 1b from treatment-naïve patients. Population-based sequencing and 454 deep sequencing of NS5B gene were performed on plasma and liver samples obtained from 18 treatment- naïve patients. The quasispecies distribution in plasma and liver samples showed a remarkable overlap in each patient. Although unique sequences in plasma or liver were observed, in the majority of cases the most dominant sequences were shown to be identical in both compartments. Neither in plasma nor in the liver codon changes were detected at position 282 that cause resistance to nucleos(t)ide analogues. However, in 10 patients the V321I change conferring resistance to nucleos(t)ide NS5B polymerase inhibitors and in 16 patients the C316N/Y/H non-nucleoside inhibitors were found mainly in liver samples. In conclusion, 454-deep sequencing of liver and plasma compartments in treatment naïve patients provides insight into viral quasispecies and the pre-existence of some drug-resistant variants in the liver, which are not necessarily present in plasma.

Since the first reports in early 2013, >440 human cases of infection with avian influenza A(H7N9) have been reported including 122 fatalities. After the isolation of the first A(H7N9) viruses, the nucleotide sequences became publically available. Based on the coding sequence of the influenza virus A/Shanghai/2/2013 hemagglutinin gene, a codon-optimized gene was synthesized and cloned into a recombinant modified vaccinia virus Ankara (MVA). This MVA-H7-Sh2 viral vector was used to immunize ferrets and proved to be immunogenic, even after a single immunization. Subsequently, ferrets were challenged with influenza virus A/Anhui/1/2013 via the intratracheal route. Unprotected animals that were mock vaccinated or received empty vector developed interstitial pneumonia characterized by a marked alveolitis, accompanied by loss of appetite, weight loss, and heavy breathing. In contrast, animals vaccinated with MVA-H7-Sh2 were protected from severe disease.

Vaccines used against seasonal influenza are poorly effective against influenza A viruses of novel subtypes that may have pandemic potential. Furthermore, pre(pandemic) influenza vaccines are poorly immunogenic, which can be overcome by the use of adjuvants. A limited number of adjuvants has been approved for use in humans, however there is a need for alternative safe and effective adjuvants that can enhance the immunogenicity of influenza vaccines and that promote the induction of broad-protective T cell responses. Here we evaluated a novel nanoparticle, G3, as an adjuvant for a seasonal trivalent inactivated influenza vaccine in a mouse model. The G3 adjuvant was formulated with or without steviol glycosides (DT, for diterpenoid). The use of both formulations enhanced the virus-specific antibody response to all three vaccine strains considerably. The adjuvants were well tolerated without any signs of discomfort. To assess the protective potential of the vaccine-induced immune responses, an antigenically distinct influenza virus strain, A/Puerto Rico/8/34 (A/PR/8/34), was used for challenge infection. The vaccine-induced antibodies did not cross-react with strain A/PR/8/34 in HI and VN assays. However, mice immunized with the G3/DT-adjuvanted vaccine were partially protected against A/PR/8/34 infection, which correlated with the induction of anamnestic virus-specific CD8(+) T cell responses that were not observed with the use of G3 without DT. Both formulations induced maturation of human dendritic cells and promoted antigen presentation to a similar extent. In conclusion, G3/DT is a promising adjuvant formulation that not only potentiates the antibody response induced by influenza vaccines, but also induces T cell immunity which could afford broader protection against antigenically distinct influenza viruses. (C) 2014 Elsevier Ltd. All rights reserved.

Efficient detection of human respiratory viral pathogens is crucial in the management of patients with acute respiratory tract infection. Sequence-independent amplification of nucleic acids combined with next-generation sequencing technology and bioinformatics analyses is a promising strategy for identifying pathogens in clinical and public health settings. It allows the characterization of hundreds of different known pathogens simultaneously and of novel pathogens that elude conventional testing. However, major hurdles for its routine use exist, including cost, turnaround time, and especially sensitivity of the assay, as the detection limit is dependent on viral load, host genetic material, and sequencing depth. To obtain insights into these aspects, we analyzed nasopharyngeal aspirates from a cohort of 81 Thai children with respiratory disease for the presence of respiratory viruses using a sequence-independent next-generation sequencing approach and routinely used diagnostic real-time reverse transcriptase PCR (real-time RT-PCR) assays. With respect to the detection of rhinovirus and human metapneumovirus, the next-generation sequencing approach was at least as sensitive as diagnostic real-time RT-PCR in this small cohort, whereas for bocavirus and enterovirus, next-generation sequencing was less sensitive than real-time RT-PCR. The advantage of the sequencing approach over real-time RT-PCR was the immediate availability of virus-typing information. Considering the development of platforms capable of generating more output data at declining costs, next-generation sequencing remains of interest for future virus diagnosis in clinical and public health settings and certainly as an additional tool when screening results from real-time RT-PCR are negative.

We sampled 7,511 black-headed gulls for influenza virus in the Netherlands during 2006–2010 and found that subtypes H13 and H16 caused annual epidemics in fledglings on colony sites. Our findings validate targeted surveillance of wild waterbirds and clarify underlying factors for influenza virus emergence in other species.

Mass bird mortality has been observed in North America after the introduction ofWest Nile virus(WNV), most notably massive die-offs of American crows (Corvus brachyrhynchos). In contrast, WNV epidemic activity in Europe has been characterized by very low incidences of bird mortality. As the general susceptibility of European corvids to strains of WNV remains in question, European jackdaws (Corvus monedula) were inoculated with WNV strains circulating currently in Greece (Greece-10), Italy (FIN and Ita09) and Hungary (578/10), as well as a North American (NY99) genotype with a demonstrated corvid virulence phenotype. Infection with all strains except WNV-FIN resulted in mortality. Viraemia was observed for birds inoculated with all strains and virus was detected in a series of organs upon necropsy. These results suggested that jackdaws could potentially function as a sentinel for following WNV transmission in Europe; however, elicited viraemia levels might be too low to allow for efficient transmission of virus to mosquitoes.

We obtained the full genome of Middle East respiratory syndrome coronavirus (MERS-CoV) from a camel in Qatar. This virus is highly similar to the human England/Qatar 1 virus isolated in 2012. The MERS-CoV from the camel efficiently replicated in human cells, providing further evidence for the zoonotic potential of MERS-CoV from camels.

Chikungunya virus (CHIKV) causes acute illness characterized by fever and long-lasting arthritic symptoms. The need for a safe and effective vaccine against CHIKV infections is on the rise due to on-going vector spread and increasing severity of clinical complications. Here we report the results of a comparative vaccination-challenge experiment in mice using three different vaccine candidates produced in insect cells by recombinant baculoviruses: (i) secreted (s)E1 and (ii) sE2 CHIKV glycoprotein subunits (2 μg/immunization), and (iii) CHIKV virus-like particles (VLPs) (1 μg E2 equivalent/immunization). These experiments show that vaccination with two subsequent administrations of 1 μg of Matrix M adjuvanted CHIKV VLPs completely protected AG129 mice from lethal CHIKV challenge. Vaccination with E1 and E2 subunits provided partial protection, with half of the mice surviving but with significantly lower neutralizing antibody titres as compared to the VLP vaccinated mice. This study provides evidence that even a modest neutralizing antibody response is sufficient to protect mice from CHIKV infections. Neutralization was the prominent correlate of protection. In addition, CHIKV VLPs provide a superior immune response and protection against CHIKV-induced disease in mice as compared to individual CHIKV-sE1 and -sE2 subunits.

To the Editor: Avian influenza A viruses occasionally cross the species barrier; influenza A(H5N1) virus and the recently emerged influenza A(H7N9) virus are prime examples of bird-to-human transmission (1,2). In addition, avian influenza A viruses can cross to various other mammalian species, including pinnipeds (e.g., seals) (3,4). Recently, mass deaths have occurred among harbor seals (Phoca vitulina); hundreds of carcasses washed up the shores of Sweden (March 2014), Denmark (July 2014), and Germany (October 2014). Approximately 1,400 dead harbor seals were seen in the coastal waters of Schleswig-Holstein in Germany alone, where the population is ≈12,000 animals. We report the investigation of the deaths of 17 seals from different age groups that were found dead on the islands of Helgoland and Sylt, Germany, during the second week of October 2014. Complete necropsies were performed on the carcasses, which were in variable nutritional conditions, ranging from very poor to good. Necropsy results showed consistently poorly retracted lungs with severe congestion, occasional diffuse consolidation, and multifocal firm nodular areas of gray-yellow discoloration with varying numbers of metazoic parasites. Histologic examinations showed acute necrotizing bronchitis and adenitis of bronchial glands with sloughing of epithelial cells (Figure, panel A). Occasionally, mild interstitial pneumonia was found. Multifocal pyogranulomatous to necrotizing pneumonia was associated with parasite infestation. A few animals had suppurative to necrotizing or nonsuppurative rhinitis and tracheitis. Figure Histopathologic and phylogenetic analyses of necropsy samples from harbor seals infected with avian influenza A(H10N7) virus, Germany, 2014. A) Lung of harbor seal showing marked necrosis and sloughing of epithelial cells in bronchial glands (arrows); ... Because mass deaths among seals were caused by phocine distemper virus in the same area in 1988 and 2002, we tested lung and throat swab samples for morbillivirus using reverse transcription PCR (RT-PCR) and immunohistochemical analysis (5). In addition, real-time RT-PCR targeting the influenza A virus matrix gene was performed (6). No indications for the morbillivirus were detected by RT-PCR and immunohistochemistry; however, in lung lesions and throat swab samples of 11 animals, a positive signal was observed by the influenza A matrix gene real-time RT-PCR (cycle threshold values 15.0–33.9). Influenza A virus (A/harbor seal/Germany/1/2014) was subsequently isolated from lung and throat swab samples; the virus replicated to high titers in 11-day-old embryonated chicken eggs and on MDCK cells. By PCR using specific primers and subsequent Sanger sequencing of the hemagglutinin and neuraminidase genes, this virus was characterized as an influenza A virus of the H10N7 subtype, commonly found in migratory waterfowl (6). In addition, genetic analyses of all other gene segments indicated that the influenza virus A/harbor seal/Germany/1/2014 was most closely related to various influenza A viruses detected in wild birds. Specifically, the hemagglutinin and neuraminidase genes were genetically most closely related to subtype H10N7 viruses recently found in migratory ducks in Georgia, Egypt, and the Netherlands (Figure, panel C) (7). Genetic analyses were based on BLAST analyses using public databases available as of October 17, 2014 (http://www.ncbi.nlm.nih.gov, http://www.gisaid.com) and supplemented with H10 and N7 sequences from the international wild bird surveillance program of Erasmus Medical Center (Technical Appendix Table). A maximum-likelihood phylogenetic tree of the hemagglutinin gene was generated by using PhyML version 3.1 (8) with the general time reversible +I+Γ model of nucleotide substitution; a full heuristic search and subtree pruning and regrafting searches were performed. The tree was visualized by using Figtree version 1.4.0 (http://tree.bio.ed.ac.uk/software/figtree). To further elucidate the role of influenza A(H10N7) virus in the pathogenesis of the disease causing deaths among the seals, we conducted immunohistochemical analysis on the lungs using an influenza A virus nucleoprotein-specific monoclonal antibody (9). Evaluation of the lung tissues of the dead seals showed viral antigen in cytoplasm and nuclei of epithelial cells of bronchi and bronchial glands of affected lung areas (Figure, panel B), which suggests that this virus played a major role in the deaths. Immunohistochemical analysis performed on various organs (including brain and olfactory bulb) indicated that viral antigen was restricted to the respiratory tract. Although avian influenza A virus infections previously have caused mass deaths in seals (3,4,10), subtype H10N7 has not been associated with such events. We can speculate that the ongoing deaths could eventually affect all harbor seal populations of northwestern Europe and have consequences for wildlife management and seal rehabilitation activities. In addition, preliminary analysis of the hemagglutinin sequence of the influenza A(H10N7) virus suggests the presence of molecular determinants that indicate mammalian adaptation. Various analyses are ongoing to answer questions about the route of transmission among seals and possible transmissibility to humans. Note added in proof: Zohari et al. also recently reported the involvement of avian influenza A(H10N7) virus in mass deaths of harbor seals in Sweden (Euro Surveill. 2014;19:pii: 20967). Technical Appendix: Details of hemagglutinin sequences shown in the Figure. Click here to view.(22K, pdf)

We here report a 7 year old acute myeloid leukemia patient with persistent spiking fever likely caused by chronic echovirus 20 infection. After immunoglobulin substitution fevers subsided and the virus was cleared. Enterovirus infection should be considered in immunocompromised patients with unexplained persistent fever.

Background: Acyclovir (ACV) is the antiviral drug of choice to treat patients with herpes simplex virus type 1 (HSV-1) uveitis. The prevalence of intra-ocular ACV-resistant (ACVR) HSV-1 in herpetic uveitis is unknown and may have clinical consequences. In addition to its predictive value on ACV susceptibility, the polymorphic HSV-1 thymidine kinase (TK) gene facilitates differentiation between HSV-1 strains. Objectives: The objective of this study was to determine the genetic composition and ACV susceptibility of the causative virus in intra-ocular fluid samples (IOF) of HSV-1 uveitis patients. Study design: The intra-ocular HSV-1 pool from 11 HSV-1 uveitis patients was determined by sequencing IOF-derived viral TK genes. The ACV susceptibility profile of the cloned intra-ocular TK variants was defined by mass spectrometry. In addition, the ganciclovir (GCV) susceptibility of the ACVR HSV-1 TK variants was defined. Results: Intra-ocular fluid samples of HSV-1 uveitis patients contain HSV-1 quasispecies, principally consisting of one major and multiple genetically related minor patient-specific TK variants. Four of 10 patients analyzed had an intra-ocular ACVR HSV-1 of which 3 were cross-resistant to GCV. The ACVR profile of intra-ocular HSV-1 did not correlate with symptomatic ACV treatment. Conclusions: Affected eyes of HSV-1 uveitis patients are commonly infected with a patient-specific HSV-1 quasispecies, including one major and multiple genetically related minor variants. A relatively high prevalence of intra-ocular ACVR HSV-1, mainly ACV/GCV cross-resistant viruses, was detected in HSV-1 uveitis patients. (C) 2013 Elsevier B. V. All rights reserved.

Primary herpes simplex virus 1 (HSV-1) and varicella-zoster virus (VZV) infection leads to a life-long latent infection of ganglia innervating the oral mucosa. HSV-1 and VZV reactivation is more common in immunocompromised individuals and may result in viral shedding in saliva. We determined the kinetics and quantity of oral HSV-1 and VZV shedding in HSV-1 and VZV seropositive individuals infected with HIV and to assess whether HSV-1 shedding involves reactivation of the same strain intra-individually. HSV-1 and VZV shedding was determined by real-time PCR of sequential daily oral swabs (n=715) collected for a median period of 31 days from 22 individuals infected with HIV. HSV-1 was genotyped by sequencing the viral thymidine kinase gene. Herpesvirus shedding was detected in 18 of 22 participants. Shedding of HSV-1 occurred frequently, on 14.3% of days, whereas solely VZV shedding was very rare. Two participants shed VZV. The median HSV-1 load was higher compared to VZV. HSV-1 DNA positive swabs clustered into 34 shedding episodes with a median duration of 2 days. The prevalence, duration and viral load of herpesvirus shedding did not correlate with CD4 counts and HIV load. The genotypes of the HSV-1 viruses shed were identical between and within shedding episodes of the same person, but were different between individuals. One-third of the individuals shed an HSV-1 strain potentially refractory to acyclovir therapy. Compared to HSV-1, oral VZV shedding is rare in individuals infected with HIV. Recurrent oral HSV-1 shedding is likely due to reactivation of the same latent HSV-1 strain.

Middle East respiratory syndrome coronavirus (MERS-CoV) has recently emerged as a causative agent of severe respiratory disease in humans. Here, we constructed recombinant modified vaccinia virus Ankara (MVA) expressing full-length MERS-CoV spike (S) protein (MVA-MERS-S). The genetic stability and growth characteristics of MVA-MERS-S make it a suitable candidate vaccine for clinical testing. Vaccinated mice produced high levels of serum antibodies neutralizing MERS-CoV. Thus, MVA-MERS-S may serve for further development of an emergency vaccine against MERS-CoV.

Influenza A viruses cause annual epidemics and occasionally pandemics. Antibodies directed to the conserved viral nucleoprotein (NP) may play a role in immunity against various influenza A virus subtypes. Here, we assessed the immunological significance of a human monoclonal antibody directed to NP in vitro . This antibody bound to virus-infected cells but did not display virus-neutralizing activity, complement-dependent cell cytotoxicity, or opsonization of viral antigen for improved antigen presentation to CD8 + T cells by dendritic cells.

Hepatitis E virus (HEV) is increasingly acknowledged as a cause of hepatitis in healthy individuals as well as immunocompromised patients. Little is known of HEV infection in recipients of allogeneic hematopoietic stem cell transplantation (alloHSCT). Therefore, we set out to study the incidence and sequelae of HEV as a cause of hepatitis in a recent cohort of 328 alloHSCT recipients. HEV RNA was tested in episodes of liver enzyme abnormalities. In addition, HEV RNA and HEV serology were assessed pre- and post-alloHSCT. We found 8 cases (2.4%) of HEV infection, of which 5 had developed chronic HEV infection. Seroprevalence pre-alloHSCT was 13%. Four patients died with HEV viremia, with signs of ongoing hepatitis, having a median time of infection of 4.1 months. The 4 surviving patients cleared HEV after a median period of 6.3 months. One patient was diagnosed with HEV reactivation after a preceding infection prior to alloHSCT. Although the incidence of developing acute HEV post-alloHSCT is relatively low, the probability of developing chronic hepatitis in severely immunocompromised patients is high. Therefore, alloHSCT recipients should be screened pretransplantation by HEV serology and RNA. Furthermore, a differential diagnosis including hepatitis E is mandatory in all alloHSCT patients with severe liver enzyme abnormalities.

Influenza A viruses from animal reservoirs have the capacity to adapt to humans and cause influenza pandemics. The occurrence of an influenza pandemic requires efficient virus transmission among humans, which is associated with virus attachment to the upper respiratory tract. Pandemic severity depends on virus ability to cause pneumonia, which is associated with virus attachment to the lower respiratory tract. Recently, a novel avian-origin H7N9 influenza A virus with unknown pandemic potential emerged in humans. We determined the pattern of attachment of two genetically engineered viruses containing the hemagglutinin of either influenza virus A/Shanghai/1/13 or A/Anhui/1/13 to formalin-fixed human respiratory tract tissues using histochemical analysis. Our results show that the emerging H7N9 virus attached moderately or abundantly to both upper and lower respiratory tract, a pattern not seen before for avian influenza A viruses. With the caveat that virus attachment is only the first step in the virus replication cycle, these results suggest that the emerging H7N9 virus has the potential both to transmit efficiently among humans and to cause severe pneumonia.

Several viruses of the family of Adenoviridae are associated with disease in birds. Here we report the detection of a novel adenovirus in the cloacal bursa of herring gulls (Laws argentatus) and lesser black-backed gulls (Larus fuscus) that were found dead in the Netherlands in 2001. Histopathological analysis of the cloacal bursa revealed cytomegaly and karyomegaly with basophilic intranuclear inclusions typical for adenovirus infection. The presence of an adenovirus was confirmed by electron microscopy. By random PCR in combination with deep sequencing, sequences were detected that had the best hit with known adenoviruses. Phylogenetic analysis of complete coding sequences of the hexon, penton and polymerase genes indicates that this novel virus, tentatively named Gull adenovirus, belongs to the genus Aviadenovirus. The present study demonstrates that birds of the Laridae family are infected by family-specific adenoviruses that differ from known adenoviruses in other bird species. (C) 2013 Elsevier Inc. All rights reserved.

Flu Drift Limited Five antigenic sites in the virus surface hemagglutinin protein, which together comprise 131 amino acid positions, are thought to determine the full scope of antigenic drift of influenza A virus. Koel et al. (p. 976 ) show that major antigenic change can be caused by single amino acid substitutions. These single substitutions substantially skew the way the immune system “sees” the virus. All substitutions of importance are located next to the receptor-binding site in the hemagglutinin. Because there are few positions of importance for antigenic drift, there are strict biophysical limitations to the substitutions at these positions, which restricts the number of new antigenic drift variants at any point in time. Thus, the evolution of influenza virus may be more predictable than previously thought.

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

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