Esa Rönkkö

Pandemic H1N1 2009 Influenza A Virus Induces Weak Cytokine Responses in Human Macrophages and Dendritic Cells and Is Highly Sensitive to the Antiviral Actions of Interferons

ABSTRACT In less than 3 months after the first cases of swine origin 2009 influenza A (H1N1) virus infections were reported from Mexico, WHO declared a pandemic. The pandemic virus is antigenically distinct from seasonal influenza viruses, and the majority of human population lacks immunity against this virus. We have studied the activation of innate immune responses in pandemic virus-infected human monocyte-derived dendritic cells (DC) and macrophages. Pandemic A/Finland/553/2009 virus, representing a typical North American/European lineage virus, replicated very well in these cells. The pandemic virus, as well as the seasonal A/Brisbane/59/07 (H1N1) and A/New Caledonia/20/99 (H1N1) viruses, induced type I (alpha/beta interferon [IFN-α/β]) and type III (IFN-λ1 to -λ3) IFN, CXCL10, and tumor necrosis factor alpha (TNF-α) gene expression weakly in DCs. Mouse-adapted A/WSN/33 (H1N1) and human A/Udorn/72 (H3N2) viruses, instead, induced efficiently the expression of antiviral and proinflammatory genes. Both IFN-α and IFN-β inhibited the replication of the pandemic (H1N1) virus. The potential of IFN-λ3 to inhibit viral replication was lower than that of type I IFNs. However, the pandemic virus was more sensitive to the antiviral IFN-λ3 than the seasonal A/Brisbane/59/07 (H1N1) virus. The present study demonstrates that the novel pandemic (H1N1) influenza A virus can readily replicate in human primary DCs and macrophages and efficiently avoid the activation of innate antiviral responses. It is, however, highly sensitive to the antiviral actions of IFNs, which may provide us an additional means to treat severe cases of infection especially if significant drug resistance emerges.

Severe Acute Respiratory Syndrome Coronavirus Fails To Activate Cytokine-Mediated Innate Immune Responses in Cultured Human Monocyte-Derived Dendritic Cells

ABSTRACT Activation of host innate immune responses was studied in severe acute respiratory syndrome coronavirus (SCV)-infected human A549 lung epithelial cells, macrophages, and dendritic cells (DCs). In all cell types, SCV-specific subgenomic mRNAs were seen, whereas no expression of SCV proteins was found. No induction of cytokine genes (alpha interferon [IFN-α], IFN-β, interleukin-28A/B [IL-28A/B], IL-29, tumor necrosis factor alpha, CCL5, or CXCL10) or IFN-α/β-induced MxA gene was seen in SCV-infected A549 cells, macrophages, or DCs. SCV also failed to induce DC maturation (CD86 expression) or enhance major histocompatibility complex class II expression. Our data strongly suggest that SCV fails to activate host cell cytokine gene expression in human macrophages and DCs.

Genetic Diversity of the 2009 Pandemic Influenza A(H1N1) Viruses in Finland

Background In Finland, the first infections caused by the 2009 pandemic influenza A(H1N1) virus were identified on May 10. During the next three months almost all infections were found from patients who had recently traveled abroad. In September 2009 the pandemic virus started to spread in the general population, leading to localized outbreaks and peak epidemic activity was reached during weeks 43–48. Methods/Results The nucleotide sequences of the hemagglutinin (HA) and neuraminidase (NA) genes from viruses collected from 138 patients were determined. The analyzed viruses represented mild and severe infections and different geographic regions and time periods. Based on HA and NA gene sequences, the Finnish pandemic viruses clustered in four groups. Finnish epidemic viruses and A/California/07/2009 vaccine virus strain varied from 2–8 and 0–5 amino acids in HA and NA molecules, respectively, giving a respective maximal evolution speed of 1.4% and 1.1%. Most amino acid changes in HA and NA molecules accumulated on the surface of the molecule and were partly located in antigenic sites. Three severe infections were detected with a mutation at HA residue 222, in two viruses with a change D222G, and in one virus D222Y. Also viruses with change D222E were identified. All Finnish pandemic viruses were sensitive to oseltamivir having the amino acid histidine at residue 275 of the neuraminidase molecule. Conclusions The Finnish pandemic viruses were quite closely related to A/California/07/2009 vaccine virus. Neither in the HA nor in the NA were changes identified that may lead to the selection of a virus with increased epidemic potential or exceptionally high virulence. Continued laboratory-based surveillance of the 2009 pandemic influenza A(H1N1) is important in order to rapidly identify drug resistant viruses and/or virus variants with potential ability to cause severe forms of infection and an ability to circumvent vaccine-induced immunity.

Respiratory viruses in laryngeal croup of young children

Objectives To determine the viral cause of laryngeal croup by use of highly sensitive methods, and including recently recognized viruses in the analysis. Study design One hundred forty-four consecutive children with hoarse voice and inspiratory stridor attending the emergency department were enrolled. Age- and season-matched children presenting with a wheezing illness served as control subjects (n = 76). Nasopharyngeal swabs were analyzed by polymerase chain reaction for rhinovirus and enterovirus, coronavirus, respiratory syncytial virus (RSV), parainfluenza virus (PIV), influenza A and B virus, human bocavirus, human metapneumovirus, adenovirus, and Mycoplasma pneumoniae. Results Virus infection was documented in 80% of patients with croup and 71% of control subjects. Children with croup had significantly more positive test results for PIV 1 and 2 (31% vs 4% and 6% vs 0%, respectively) and significantly fewer positive test results for RSV (15% vs 28%) than wheezing children. Rhinoviruses and enteroviruses were present equally in both groups (21% vs 25%). There was no significant difference in the frequency of influenza A virus or human bocavirus. Few subjects with adenovirus or M. pneumoniae were detected. Conclusion Acute laryngeal croup is most often associated with PIV, RSV, rhinovirus, and enterovirus. Rhinovirus and enterovirus appeared equally often in croup and in wheezing illness. During late fall, they were found in 39% and 40%, respectively, of the tested samples.

Specific Viruses Detected in Nigerian Children in Association with Acute Respiratory Disease

Occurrence of different viruses in acute respiratory tract infections of Nigerian children was examined. Respiratory swabs were collected from 246 children referred to hospital clinics because of acute respiratory symptoms from February through May 2009. Validated real-time RT-PCR techniques revealed nucleic acids of at least one virus group in 189 specimens (77%). Human rhinoviruses and parainfluenza viruses were present each in one third of the children. Adenoviruses, enteroviruses, human metapneumovirus, human bocavirus, and influenza C virus were also relatively common. Possibly due to their seasonal occurrence, influenza A and B virus, and respiratory syncytial virus were detected rarely. We conclude that all major groups of respiratory tract viruses are causing illness in Nigerian children.

The use of the probiotic Lactobacillus rhamnosus GG and viral findings in the nasopharynx of children attending day care

Limited data are available on the effects of probiotics on the nasopharyngeal presence of respiratory viruses in children attending day care. In this substudy of a randomized, double‐blinded, placebo‐controlled 28‐week intervention study, nasopharyngeal swab samples were collected, on visits to a physician due to symptoms of infection, from children receiving control milk (N = 97) and children receiving the same milk supplemented with probiotic Lactobacillus rhamnosus GG (N = 97). The presence of 14 respiratory viruses was assessed by PCR methods, and viral findings were compared with symptom prevalences in the intervention groups. Rhinovirus was identified in 28.6% of 315 swab samples, followed by respiratory syncytial virus (12.4%), parainfluenza virus 1 (12.1%), enterovirus (8.9%), influenza A(H1N1)pdm09 (7.9%), human bocavirus 1 (3.8%), parainfluenza virus 2 (3.2%), adenovirus (2.9%), and influenza A(H3N2) (0.6%). The children in the probiotic group had less days with respiratory symptoms per month than the children in the control group (6.48 [95% CI 6.28–6.68] vs. 7.19 [95% CI 6.98–7.41], P < 0.001). Probiotic intervention did not reduce significantly the occurrence of the examined respiratory viruses, or have an effect on the number of respiratory symptoms observed at the time of a viral finding. Rhinovirus, respiratory syncytial virus, and parainfluenza virus 1 were the most common respiratory viruses in symptomatic children. Children receiving Lactobacillus rhamnosus GG had fewer days with respiratory symptoms than children in the control group, although probiotic intervention was not effective in reducing the amount of viral findings or the respiratory symptoms associated with viral findings. J. Med. Virol. 85:1632–1638, 2013.

Detection of influenza A viruses with a portable real-time PCR instrument

Timely identification of respiratory pathogens is essential for appropriate patient care and cohorting. In order to do rapid identification-technology near the patient we utilized the field-deployable RAZOR EX-thermocycler with a reverse transcription real-time PCR assay that detects all subtypes of influenza A virus. In addition, we developed a RT PCR assay for specific detection of influenza A(H1N1)pdm09 virus. These assays amplified segments of the matrix (M)- and the hemagglutinin (HA)-gene, respectively. Detection limits of the M-gene and the influenza A(H1N1)pdm09-specific HA-gene assays were 0.15 PFU and 8.8 PFU per reaction, respectively. With 18 influenza A viruses of different subtypes and influenza B, C, and 7 other respiratory viruses the RAZOR EX and standard real-time PCR assay results were in total agreement. From 104 clinical samples identical results were obtained by both PCR methods. Additional 21 clinical samples were tested under field conditions with the RAZOR EX instrument. Results were achieved in 90 min, including 45 min for sample preparation and they were in complete agreement with those obtained by standard real-time PCR under laboratory conditions. These methods enable highly sensitive and rapid on-site diagnostics to reliably identify patients infected with influenza A, including the influenza A(H1N1)pdm09-virus.

Influenza C virus infection in military recruits--symptoms and clinical manifestation.

Due to the lack of rapid diagnostic tests, clinical features of Influenza C virus infections are poorly characterized. Respiratory infections in military recruits in eastern Finland were monitored between July 2004 and December 2005 in order to study the epidemiology and clinical picture of infections caused by this virus. Blood samples were obtained at entry and at the end of the military service, and during each episode of respiratory infection to measure antibody responses against 10 viral and 2 bacterial pathogens. If possible, sputum samples were collected during the acute phase of respiratory infection episodes. Symptoms of the episodes were recorded for comparison of the clinical picture caused by various infectious agents. Infection with influenza C virus was detected in 38 of 892 young men during their service. The virus usually caused a mild upper respiratory tract infection. Most typical clinical features of influenza C virus infection were cough, rhinitis, and hoarseness. A striking difference to infections caused by influenza A virus was the lack of fever. Influenza C virus is an important cause of a respiratory tract infection in army conscripts. Infections with this virus are usually mild but can be complicated in some cases. J. Med. Virol. 86:879–885, 2014.

Influenza C virus infection in military recruits-symptoms and clinical manifestation

Due to the lack of rapid diagnostic tests, clinical features of Influenza C virus infections are poorly characterized. Respiratory infections in military recruits in eastern Finland were monitored between July 2004 and December 2005 in order to study the epidemiology and clinical picture of infections caused by this virus. Blood samples were obtained at entry and at the end of the military service, and during each episode of respiratory infection to measure antibody responses against 10 viral and 2 bacterial pathogens. If possible, sputum samples were collected during the acute phase of respiratory infection episodes. Symptoms of the episodes were recorded for comparison of the clinical picture caused by various infectious agents. Infection with influenza C virus was detected in 38 of 892 young men during their service. The virus usually caused a mild upper respiratory tract infection. Most typical clinical features of influenza C virus infection were cough, rhinitis, and hoarseness. A striking difference to infections caused by influenza A virus was the lack of fever. Influenza C virus is an important cause of a respiratory tract infection in army conscripts. Infections with this virus are usually mild but can be complicated in some cases. J. Med. Virol. 86:879–885, 2014.

Middle East respiratory syndrome coronavirus shows poor replication but significant induction of antiviral responses in human monocyte-derived macrophages and dendritic cells

In this study we assessed the ability of Middle East respiratory syndrome coronavirus (MERS-CoV) to replicate and induce innate immunity in human monocyte-derived macrophages and dendritic cells (MDDCs), and compared it with severe acute respiratory syndrome coronavirus (SARS-CoV). Assessments of viral protein and RNA levels in infected cells showed that both viruses were impaired in their ability to replicate in these cells. Some induction of IFN-λ1, CXCL10 and MxA mRNAs in both macrophages and MDDCs was seen in response to MERS-CoV infection, but almost no such induction was observed in response to SARS-CoV infection. ELISA and Western blot assays showed clear production of CXCL10 and MxA in MERS-CoV-infected macrophages and MDDCs. Our data suggest that SARS-CoV and MERS-CoV replicate poorly in human macrophages and MDDCs, but MERS-CoV is nonetheless capable of inducing a readily detectable host innate immune response. Our results highlight a clear difference between the viruses in activating host innate immune responses in macrophages and MDDCs, which may contribute to the pathogenesis of infection.