Expression of the hepatitis C virus structural proteins in mammalian cells induces morphology similar to that in natural infection

Like many positive-strand RNA viruses, replication of the hepatitis C virus (HCV) is associated with cytoplasmic membrane rearrangements. However, it is unclear which HCV Proteins induce these ultrastructural features. This work examined the morphological changes induced by expression of the HCV structural proteins, core, E1 and E2, expressed from a Semliki Forest Virus (SFV) recombinant RNA replicon. Electron microscopy of cells expressing these proteins showed cytoplasmic vacuoles containing membranous and electron-dense material that were distinct from the type I cytoplasmic vacuoles induced during SFV replicon replication. Immunogold labelling showed that the core and E2 proteins localized to the external and internal membranes of these vacuoles. At times were also associated with some of the internal amorphous material. Dual immunogold labelling with antibodies raised against the core protein and against an endoplasmic reticulum (ER)-resident protein (protein disulphide isomerase) showed that the HCV-induced vacuoles were associated with ER-labelled membranes. This report has identified an association between the HCV core and E2 proteins with induced cytoplasmic vacuoles which are morphologically similar to those observed in HCV-infected liver tissue, suggesting that the HCV structural proteins may be responsible for the induction of these vacuoles during HCV replication in vivo.

Clinical patterns and seasonal trends in respiratory syncytial virus hospitalizations in São Paulo, Brazil

The respiratory viruses are recognized as the most frequent lower respiratory tract pathogens for infants and young children in developed countries but less is known for developing populations. The authors conducted a prospective study to evaluate the occurrence, clinical patterns, and seasonal trends of viral infections among hospitalized children with lower respiratory tract disease (Group A). The presence of respiratory viruses in children's nasopharyngeal was assessed at admission in a pediatric ward. Cell cultures and immunofluorescence assays were used for viral identification. Complementary tests included blood and pleural cultures conducted for bacterial investigation. Clinical data and radiological exams were recorded at admission and throughout the hospitalization period. To better evaluate the results, a non- respiratory group of patients (Group B) was also constituted for comparison. Starting in February 1995, during a period of 18 months, 414 children were included- 239 in Group A and 175 in Group B. In Group A, 111 children (46.4%) had 114 viruses detected while only 5 children (2.9%) presented viruses in Group B. Respiratory Syncytial Virus was detected in 100 children from Group A (41.8%), Adenovirus in 11 (4.6%), Influenza A virus in 2 (0.8%), and Parainfluenza virus in one child (0.4%). In Group A, aerobic bacteria were found in 14 cases (5.8%). Respiratory Syncytial Virus was associated to other viruses and/or bacteria in six cases. There were two seasonal trends for Respiratory Syncytial Virus cases, which peaked in May and June. All children affected by the virus were younger than 3 years of age, mostly less than one year old. Episodic diffuse bronchial commitment and/or focal alveolar condensation were the clinical patterns more often associated to Respiratory Syncytial Virus cases. All children from Group A survived. In conclusion, it was observed that Respiratory Syncytial Virus was the most frequent pathogen found in hospitalized children admitted for severe respiratory diseases. Affected children were predominantly infants and boys presenting bronchiolitis and focal pneumonias. Similarly to what occurs in other subtropical regions, the virus outbreaks peak in the fall and their occurrence extends to the winter, which parallels an increase in hospital admissions due to respiratory diseases.

The 2009 Pandemic Influenza A (H1N1) Infection: Renal Histopathology Findings in Two Infected Patients

Background: Acute kidney injury in the pandemic swine origin influenza A virus (H1N1) infection has been reported as coursing with severe illness, although renal pathogenic mechanisms and histologic features are still being characterised. Case Report: We present two patients admitted with H1N1 pneumonia, sepsis, acute respiratory distress syndrome and need for invasive mechanical ventilation who developed acute kidney injury and became dialysis-dependent. In both cases a kidney biopsy was performed to establish a definitive diagnosis. Severe acute tubular necrosis was identified, with no further abnormalities. Conclusion: This report seems to confirm that the acute kidney injury in H1N1 infection is focused on the tubular cells. Our cases corroborate the renal histopathologic findings of other studies, highlighting the central role of the tubular cell. We bring new evidence of the histopathology of AKI in H1N1 infection since our data were collected in living patients and not via post-mortem studies.

Influenza A(H1N1)pdm09 Resistance and Cross-Decreased Susceptibility to Oseltamivir and Zanamivir Antiviral Drugs

Neuraminidase inhibitors (NAIs) oseltamivir and zanamivir are currently the only effective antiviral drugs available worldwide for the management of influenza. The potential development of resistance is continually threatening their use, rationalizing and highlighting the need for a close and sustained evaluation of virus susceptibility. This study aimed to analyze and characterize the phenotypic and genotypic NAIs susceptibility profiles of A(H1N1)pdm09 viruses circulating in Portugal from 2009 to 2010/2011. A total of 144 cases of A(H1N1)pdm09 virus infection from community and hospitalized patients were studied, including three suspected cases of clinical resistance to oseltamivir. Oseltamivir resistance was confirmed for two of the suspected cases. Neuraminidase (NA) H275Y resistant marker was found in viruses from both cases but for one it was only present in 26.2% of virus population, raising questions about the minimal percentage of resistant virus that should be considered relevant. Cross-decreased susceptibility to oseltamivir and zanamivir (2-4 IC50 fold-change) was detected on viruses from two potentially linked community patients from 2009. Both viruses harbored the NA I223V mutation. NA Y155H mutation was found in 18 statistical non-outlier viruses from 2009, having no impact on virus susceptibility. The mutations at NA N369K and V241I may have contributed to the significantly higher baseline IC50 value obtained to oseltamivir for 2010/2011 viruses, compared to viruses from the pandemic period. These results may contribute to a better understanding of the relationship between phenotype and genotype, which is currently challenging, and to the global assessment of A(H1N1)pdm09 virus susceptibility profile and baseline level to NAIs.

Influenza surviellance in Rio de Janeiro between 1980-1981: a virological and serological study

Laboratory surveillance of influenza has shown a low virus activity in Rio de Janeiro during 1980 and 1981. A few influenza A (H3N2) viruses were isolated in both years during the winter months. Serological investigations showed that this subtype has circulated mostly among children under 10 years of age. No H1N1 virus was isolated but an increase in the proportion of adults with antibody to his virus was noted in sera collected in 1981. Influenza B virus was isolated from children in the spring of 1981 and again an increase was noted in the proportion of adults with antibody to this virus.

Pandemic Influenza Background (PDF 99 KB)

The Presence of highly pathogenicH5N1 avian influenza has been confirmed in birds in Turkey, Romania, Russia and Kazakhstan. There have been a total of 4 confirmed cases of human infection in Eastern/South Eastern Turkey that has resulted in 2 deaths. Background Avian influenza naturally circulates in wild waterfowl such as ducks and geese often causing little or no symptoms. Many other bird species are susceptible to infection with these influenza viruses and in many of these species it may cause severe disease associated with high mortality. Outbreaks associated with high bird mortality are called Highly Pathogenic Avian Influenza (HPAI) to distinguish them from less pathogenic influenza. In January 2004 avian influenza in poultry was confirmed in Vietnam. Subsequently, there have been very substantial outbreaks of avian influenza associated with high mortality affecting poultry in various countries throughout Asia including Vietnam, Thailand, China, Malaysia, Mongolia, North & South Korea, Cambodia, Indonesia, Laos and Japan. These outbreaks are caused by H5N1 subtype of influenza A virus, the same subtype (but not identical to the virus) that caused the outbreak in Hong Kong in 1997. åÊ

Infección por virus pandémico (H1N1) 2009 en Andalucía

In April 2009, in response to the WHO's alert due to the existence of human infection cases with a new AH1N1 influenza virus, known as swine flu, Andalusian Health Authorities trigger an specific action plan. The surveillance actions developped provided us with appropriate clinical, epidemiological and virological characteristics of the disease. During the first few days, contingency plans were set up based on epidemiological surveillance and outbreak control measures were adopted through early alert and rapid response systems. After phase 6 was declared, influenza sentinel and severe cases surveillance were used in order to plan healthcare services, to reduce transmission and to identify and protect the most vulnerable population groups. Behaviour of pandemic influenza in Andalusia was similar to that observed in the rest of the world. Atack rate was similar to a seasonal flu and the peak was reached at the 46th/2009 week. Most of them were mild cases and affected particularly to young people. The average age of hospitalised patients was 32. Prior pulmonary disease, smoking and morbid obesity (BMI>40) were the most common pathologies and risk factors in severe cases. An impact scenario of pandemic wave in Andalusia, with an expected attack rate from 2 to 5%, was prepared considering watt observed in the southern hemisphere. Characteristics of the epidemic concerning its extent, severity and mortality rate were adjusted to this scenario.

Pandemic influenza control in Europe and the constraints resulting from incoherent public health laws.

Background With the emergence of influenza H1N1v the world is facing its first 21st century global pandemic. Severe Acute Respiratory Syndrome (SARS) and avian influenza H5N1 prompted development of pandemic preparedness plans. National systems of public health law are essential for public health stewardship and for the implementation of public health policy[1]. International coherence will contribute to effective regional and global responses. However little research has been undertaken on how law works as a tool for disease control in Europe. With co-funding from the European Union, we investigated the extent to which laws across Europe support or constrain pandemic preparedness planning, and whether national differences are likely to constrain control efforts. Methods We undertook a survey of national public health laws across 32 European states using a questionnaire designed around a disease scenario based on pandemic influenza. Questionnaire results were reviewed in workshops, analysing how differences between national laws might support or hinder regional responses to pandemic influenza. Respondents examined the impact of national laws on the movements of information, goods, services and people across borders in a time of pandemic, the capacity for surveillance, case detection, case management and community control, the deployment of strategies of prevention, containment, mitigation and recovery and the identification of commonalities and disconnects across states. Results Results of this study show differences across Europe in the extent to which national pandemic policy and pandemic plans have been integrated with public health laws. We found significant differences in legislation and in the legitimacy of strategic plans. States differ in the range and the nature of intervention measures authorized by law, the extent to which borders could be closed to movement of persons and goods during a pandemic, and access to healthcare of non-resident persons. Some states propose use of emergency powers that might potentially override human rights protections while other states propose to limit interventions to those authorized by public health laws. Conclusion These differences could create problems for European strategies if an evolving influenza pandemic results in more serious public health challenges or, indeed, if a novel disease other than influenza emerges with pandemic potential. There is insufficient understanding across Europe of the role and importance of law in pandemic planning. States need to build capacity in public health law to support disease prevention and control policies. Our research suggests that states would welcome further guidance from the EU on management of a pandemic, and guidance to assist in greater commonality of legal approaches across states.

Cytokine levels in bronchoalveolar lavage and serum in 3 patients with 2009 influenza A(H1N1)v severe pneumonia.

Introduction: Pandemic Influenza A (H1N1)v pneumonia has led to a notable increase of admissions to intensive care units. A cytokine-mediated inflammatory response has been well documented in pneumonia and acute respiratory distress syndrome. However, few studies have focused on the role of these inflammatory mediators in infections caused by the Influenza A (H1N1)v. In this study, we assess the inflammatory response mediated by cytokines at the local and systemic levels in three cases of severe pneumonia caused by Influenza A (H1N1) virus. Methodology: Serum and bronchoalveolar lavage samples were obtained from three mechanically ventilated patients diagnosed with Influenza A (H1N1) virus pneumonia by bronchoscopic bronchoalveolar lavage. Levels of interleukin 6 (IL-6), interleukin 8 (IL-8), tumour necrosis factor alpha (TNFα) and interleukin 1 beta (IL-1ß) were meassured in these samples by enzyme-linked immunosorbent assay (ELISA). Results: High levels of C Reactive Protein, Procalcitonin below 1 ng/ml and absence of leukocytosis were common findings in all patients. TNF α and IL-1ß were not detected in the serum. IL-6 levels in serum were (94, pg/ml, 77 pg/ml and 84 pg/ml) respectively in the three patients, while IL-8 levels were (30,2 pg/ml, 128 pg/ml and 40,5 pg/ml). In the BAL samples, only one of the analysed cytokines, IL-1ß was present at detectable levels in two patients (21 pg/ml and 11 pg/ml respectively). Conclusions: Our results support previous findings which suggest that high levels of IL-6 and IL-8 in serum somehow participate in the inflammatory response in severe cases of pandemic influenza pneumonia.

Necrotising pneumonia due to influenza A (H1N1) and community-acquired methicillin-resistant Staphylococcus aureus clone USA300: successful management of the first documented paediatric case.

Necrotising pneumonia in young, previously healthy patients due to Panton–Valentine leucocidin (PVL) producing Staphylococcus aureus has been increasingly recognised. PVL pneumonia is often associated with influenza co-infection and high mortality. This case report describes the successful management of the first documented paediatric case of a previous healthy adolescent who developed necrotising pneumonia due to community-acquired methicillin-resistant (CA-MRSA) clone USA300 with pandemic influenza A (H1N1) co-infection, and highlights the importance of early recognition and initiation of appropriate therapy for this potentially fatal co-infection. PCR remains the gold standard to diagnose pandemic H1N1 since it may not be detected by rapid antigen tests. Bacterial necrotising pneumonia should be suspected in those presenting with worsening flu-like symptoms and clinical and/or radiological evidence of PVL infection (multifocal infiltrates, effusion and cavitation). These patients may benefit from the administration of toxin neutralising agents. In light of the current H1N1 pandemic, healthcare professionals will be increasingly confronted with this clinical scenario.

Low-dose intradermal versus intramuscular trivalent inactivated seasonal influenza vaccine in lung transplant recipients.

BACKGROUND: In this study we compared the immunogenicity of influenza vaccine administered intradermally to the standard intramuscular vaccination in lung transplant recipients. METHODS: Patients were randomized to receive the trivalent inactivated seasonal 2008-9 influenza vaccine containing either 6 μg (intradermal) or 15 μg (intramuscular) of hemagglutinin per viral strain. Immunogenicity was assessed by measurement of geometric mean titer of antibodies using the hemagglutination-inhibition (HI) assay. Vaccine response was defined as a 4-fold or higher increase of antibody titers to at least one vaccine antigen. RESULTS: Eighty-five patients received either the intradermal (n = 41) or intramuscular (n = 44) vaccine. Vaccine response was seen in 6 of 41 patients (14.6%) in the intradermal vs 8 of 43 (18.6%) in the intramuscular group (p = 0.77). Seroprotection (HI ≥1:32) was 39% for H1N1, 83% for H3N2 and 29% for B strain in the intradermal group vs 28% for H1N1, 98% for H3N2 and 58% for B strain in the intramuscular group (p = 0.36 for H1N1, p = 0.02 for H3N2, p < 0.01 for B). Mild adverse events were seen in 44% of patients in the intradermal group and 34% in the intramuscular group (p = 0.38). CONCLUSIONS: Immunogenicity of the 2008-9 influenza vaccine given intradermally or intramuscularly was overall poor in lung transplant recipients. Novel strategies for influenza vaccination in this population are needed.

Th1 and Th17 hypercytokinemia as early host response signature in severe pandemic influenza.

INTRODUCTION Human host immune response following infection with the new variant of A/H1N1 pandemic influenza virus (nvH1N1) is poorly understood. We utilize here systemic cytokine and antibody levels in evaluating differences in early immune response in both mild and severe patients infected with nvH1N1. METHODS We profiled 29 cytokines and chemokines and evaluated the haemagglutination inhibition activity as quantitative and qualitative measurements of host immune responses in serum obtained during the first five days after symptoms onset, in two cohorts of nvH1N1 infected patients. Severe patients required hospitalization (n = 20), due to respiratory insufficiency (10 of them were admitted to the intensive care unit), while mild patients had exclusively flu-like symptoms (n = 15). A group of healthy donors was included as control (n = 15). Differences in levels of mediators between groups were assessed by using the non parametric U-Mann Whitney test. Association between variables was determined by calculating the Spearman correlation coefficient. Viral load was performed in serum by using real-time PCR targeting the neuraminidase gene. RESULTS Increased levels of innate-immunity mediators (IP-10, MCP-1, MIP-1beta), and the absence of anti-nvH1N1 antibodies, characterized the early response to nvH1N1 infection in both hospitalized and mild patients. High systemic levels of type-II interferon (IFN-gamma) and also of a group of mediators involved in the development of T-helper 17 (IL-8, IL-9, IL-17, IL-6) and T-helper 1 (TNF-alpha, IL-15, IL-12p70) responses were exclusively found in hospitalized patients. IL-15, IL-12p70, IL-6 constituted a hallmark of critical illness in our study. A significant inverse association was found between IL-6, IL-8 and PaO2 in critical patients. CONCLUSIONS While infection with the nvH1N1 induces a typical innate response in both mild and severe patients, severe disease with respiratory involvement is characterized by early secretion of Th17 and Th1 cytokines usually associated with cell mediated immunity but also commonly linked to the pathogenesis of autoimmune/inflammatory diseases. The exact role of Th1 and Th17 mediators in the evolution of nvH1N1 mild and severe disease merits further investigation as to the detrimental or beneficial role these cytokines play in severe illness.

Early estimates of the effectiveness of the 2011/12 influenza vaccine in the population targeted for vaccination in Spain, 25 December 2011 to 19 February 2012.

We present early estimates of influenza vaccine effectiveness (VE) in the population targeted for vaccination, during 25 December 2011 to 19 February 2012. The adjusted VE was 55% (95% CI: 3 to 79) against any type of influenza virus and 54% (95% CI: 1 to 79) against influenza A(H3N2) virus. This suggests a moderate protective effect of the vaccine in the targeted population in a late influenza epidemic with limited match between vaccine and circulating strains.

Comparison of adverse events following immunization with pandemic influenza A (H1N1)pdm09 vaccine with or without adjuvant among health professionals in Rio de Janeiro, Brazil

A vaccination campaign against pandemic influenza A (H1N1)pdm09 was held in Brazil in March 2010, using two types of monovalent split virus vaccines: an AS03-adjuvanted vaccine and a non-adjuvanted vaccine. We compared the reactogenicity of the vaccines in health professionals from a Clinical Research Institute in Rio de Janeiro, Brazil and there were no serious adverse events following immunization (AEFI) among the 494 subjects evaluated. The prevalence of any AEFI was higher in the AS03-adjuvanted vaccine at 2 h and 24 h post-vaccination [preva-lence ratio (PR): 2.05, confidence interval (CI) 95%: 1.55-2.71, PR: 3.42, CI 95%: 2.62-4.48, respectively]; however, there was no difference between the vaccines in the assessments conducted at seven and 21 days post-vaccination. The group receiving the AS03 post-adjuvanted vaccine had a higher frequency of local reactions at 2 h (PR: 3.01, CI 95%: 2.12-4.29), 24 h (PR: 4.57, CI 95%: 3.29-6.37) and seven days (PR: 6.05, CI 95%: 2.98-12.28) post-vaccination. We concluded that the two types of vaccines caused no serious AEFI in the studied population and the adjuvanted vaccine was more reactogenic, particularly in the 24 h following vaccination. This behaviour must be confirmed and better characterised by longitudinal studies in the general population.

Reactivation of transcription from a vaccinia virus early promoter late in infection.

We have studied the kinetics of RNA synthesis from the vaccinia virus 7,500-molecular-weight gene (7.5K gene) which is regulated by early and late promoters arranged in tandem. Unexpectedly, after a first burst of RNA synthesis early in infection, transcription was reactivated late in infection. Reactivation was not dependent on the location of the promoter in the genome or on the presence of the upstream late regulatory sequences. The mRNA synthesized from the reactivated promoter in the late phase had the same 5' and 3' ends as the molecules transcribed in the early phase. Interestingly, these molecules were efficiently translated despite the absence of the poly(A) leader characteristic of late mRNAs. Reactivation appears to be dependent on virus assembly since it is prevented by rifampin, a specific inhibitor of morphogenesis. Finally, analysis of various other early genes showed that reactivation is not unique to the 7.5K early promoter.