The rapid diagnosis and subtyping of influenza is particularly important in areas where avian influenza (H5N1) is present. The ability to recognise both typical and atypical presentations of influenza is also critical in such settings. A six-month-old male child who visited a H5N1-affected area subsequently died from a severe febrile diarrhoeal illness with minimal respiratory symptoms, and was initially diagnosed with influenza A of an unknown subtype. The final microbiological results showed a highly unusual combination of influenza A (H3N2) and Campylobacter jejuni infection.
Matched MeSH terms: Influenza A Virus, H3N2 Subtype*
To determine effects of pandemic (H1N1) 2009 on children in the tropics, we examined characteristics of children hospitalized for this disease in Malaysia. Of 1,362 children, 51 (3.7%) died, 46 of whom were in an intensive care unit. Although disease was usually mild, ≥ 1 concurrent conditions were associated with higher death rates.
Matched MeSH terms: Influenza A Virus, H1N1 Subtype/physiology*
Of the ≈400 cases of avian influenza (H7N9) diagnosed in China since 2003, the only travel-related cases have been in Hong Kong and Taiwan. Detection of a case in a Chinese tourist in Sabah, Malaysia, highlights the ease with which emerging viral respiratory infections can travel globally.
Matched MeSH terms: Influenza A Virus, H7N9 Subtype/genetics*
From 2005 to 2009, the Institute for Medical Research (IMR), Kuala Lumpur received a total of 7,117 respiratory specimens from patients with influenza-like illness (ILI) for influenza screening. Seasonal influenza virus was isolated from 17.3% of patients with ILI in 2005, 31.6% in 2006, 12.8% in 2007, 10.2% in 2008 and 13.5% in 2009. There were one or more influenza A and B virus strains circulating in Malaysia throughout the year, with distinctly a peak in May to August. The predominant circulating strains of seasonal influenza A were A/California/7/2004-like (H3N2) in 2005, A/New Caledonia/20/99-like (H1N1) in 2006, A/ Brisbane/10/2007-like (H3N2) in 2007 and 2008, and A/Perth/16/2009-like (H3N2) virus in 2009. The predominant circulating strains of influenza B were B/Hong Kong/330/2001-like in 2005, B/Malaysia/2506/2004-like in 2006, B/Florida/4/2006-like in 2007 and 2008, and B/Brisbane/60/2008-like in 2009.
Matched MeSH terms: Influenza A virus/isolation & purification*
The development of highly pathogenic avian H5N1 influenza viruses in poultry in Eurasia accompanied with the increase in human infection in 2006 suggests that the virus has not been effectively contained and that the pandemic threat persists. Updated virological and epidemiological findings from our market surveillance in southern China demonstrate that H5N1 influenza viruses continued to be panzootic in different types of poultry. Genetic and antigenic analyses revealed the emergence and predominance of a previously uncharacterized H5N1 virus sublineage (Fujian-like) in poultry since late 2005. Viruses from this sublineage gradually replaced those multiple regional distinct sublineages and caused recent human infection in China. These viruses have already transmitted to Hong Kong, Laos, Malaysia, and Thailand, resulting in a new transmission and outbreak wave in Southeast Asia. Serological studies suggest that H5N1 seroconversion in market poultry is low and that vaccination may have facilitated the selection of the Fujian-like sublineage. The predominance of this virus over a large geographical region within a short period directly challenges current disease control measures.
Matched MeSH terms: Influenza A Virus, H5N1 Subtype/genetics*; Influenza A Virus, H5N1 Subtype/immunology; Influenza A Virus, H5N1 Subtype/isolation & purification; Influenza A Virus, H5N1 Subtype/pathogenicity
Increased reports of oseltamivir (OTV)-resistant strains of the influenza virus, such as the H274Y mutation on its neuraminidase (NA), have created some cause for concern. Many studies have been conducted in the attempt to uncover the mechanism of OTV resistance in H274Y NA. However, most of the reported studies on H274Y focused only on the drug-bound system, so the direct effects of the mutation on NA itself prior to drug binding still remain unclear. Therefore, molecular dynamics simulations of NA in apo form, followed by principal component analysis and interaction energy calculations, were performed to investigate the structural changes of the NA binding site as a result of the H274Y mutation. It was observed that the disruption of the NA binding site due to the H274Y mutation was initiated by the repulsive effect of Y274 on the 250-loop, which in turn altered the hydrogen-bonding network around residue 274. The rotated W295 side chain caused the upward movement of the 340-loop. Consequently, sliding box docking results suggested that the binding pathway of OTV was compromised because of the disruption of this binding site. This study also highlighted the importance of the functional group at C6 of the sialic acid mimicry. It is hoped that these results will improve the understanding of OTV resistance and shed some light on the design of a novel anti-influenza drug.
Matched MeSH terms: Influenza A Virus, H5N1 Subtype/drug effects; Influenza A Virus, H5N1 Subtype/enzymology
Hemagglutinin (HA) protein plays an important role in binding the influenza virus to infected cells and therefore mediates infection. Deposited HA sequences of 86 Asian strains of influenza A (H1N1) viruses during the first outbreak were obtained from the NCBI database and compared. Interaction of the HA protein of influenza A (H1N1) virus with the human sialic acid receptor was also studied using bioinformatics. Overall, not more than three single-point amino acid variants/changes were observed in the HA protein region of influenza A (H1N1) virus from Asian countries when a selected group sequence comparison was made. The bioinformatics study showed that the HA protein of influenza A (H1N1) binds to the sialic acid receptor in human airway receptors, possibly key to air-borne infection in humans.
Matched MeSH terms: Influenza A Virus, H1N1 Subtype/genetics; Influenza A Virus, H1N1 Subtype/metabolism*
The 2009 pandemic influenza A(H1N1) was first detected in Malaysia in May 2009. It quickly spread in the general population and contributed to a number of influenza-like illness. The objective of the study is to characterize genetic changes in early Malaysian isolates of mild and severe illness of the novel influenza, and to compare sequences of viruses circulating in Malaysia to those in other countries between May to September 2009. Viral isolates of 56 mild cases and 10 severe (intensive care unit or fatal) cases were sequenced for haemagglutinin (HA) and neuraminidase (NA). Genome sequencing of the viral RNA was conducted on 5 isolates (3 were from fatal cases). Highly conserved sequences with few sporadic variations were identified in HA and NA. E374K and D222N were identified in 2 viral isolates from patients with severe illness. Phylogenetic analysis showed close genetic relatedness to the vaccine strain A/California/07/09 and other isolates circulating worldwide during the same period. Sporadic variations were identified in the viral isolates, however a larger sample size is required to make associations with disease severity.
Matched MeSH terms: Influenza A Virus, H1N1 Subtype/genetics*; Influenza A Virus, H1N1 Subtype/isolation & purification
Highly pathogenic Avian Influenza (HPAI) virus is able to infect many hosts and the virus replicates in high levels in the respiratory tract inducing severe lung lesions. The pathogenesis of the disease is actually the outcome of the infection as determined by complex host-virus interactions involving the functional kinetics of large numbers of participating genes. Understanding the genes and proteins involved in host cellular responses are therefore, critical for the elucidation of the mechanisms of infection.
Matched MeSH terms: Influenza A Virus, H5N1 Subtype/growth & development; Influenza A Virus, H5N1 Subtype/pathogenicity*
The influenza activity and circulation of influenza viruses in Lombardy (the most populous Italian region) were observed during two consecutive seasons (2005/2006 and 2006/2007) characterized by low influenza activity by the Italian Influenza Surveillance Network. The molecular characteristics of circulating viruses were analyzed to evaluate the introduction of new variants and emergence of vaccine-escape viruses. In both seasons, the epidemic in Lombardy was sustained almost exclusively by influenza A viruses, accounting for 80.5% and 93.6% of total detections, respectively, and the co-circulation of A/H3 viruses belonging to distinct phylogenetic groups was observed. The A/H1N1 viruses isolated during the 2005/2006 season were closely related to A/New Caledonia/20/99, while the hemagglutinin (HA) sequences of the A/H1N1 viruses from the 2006/2007 season exhibited a greater diversity. These viruses were A/Solomon Islands/3/2006-like and showed several variants. All B isolates were similar to B/Malaysia/2506/2004 belonging to the B/Victoria/2/87-lineage. Influenza B virus was the dominant virus in Europe in the 2005/2006 season and accounted for the 20% of total detections in Lombardy. Overall, the viruses studied presented heterogeneity in their HA sequences suggesting the circulation of a miscellaneous set of variants during the two seasons notwithstanding the medium-low activity of influenza. The importance of virological surveillance of influenza viruses is recognized widely and the molecular characterization of the viruses, especially in vaccinated subjects, is of particular importance to evaluate the introduction and circulation of new variants.
Matched MeSH terms: Influenza A virus/classification*; Influenza A virus/isolation & purification*
Avian influenza viruses are highly infectious micro-organisms that primarily affect birds. Nevertheless, they have also been isolated from a number of mammals, including humans. Avian influenza virus can cause large economic losses to the poultry industry because of its high mortality. Although there are pathogenic variants with a low virulence and which generally cause only mild, if any, clinical symptoms, the subtypes H5 and H7 can mutate from a low to a highly virulent (pathogenic) virus and should be taken into consideration in eradication strategies. The primary source of infection for commercial poultry is direct and indirect contact with wild birds, with waterfowl forming a natural reservoir of the virus. Live-poultry markets, exotic birds, and ostriches also play a significant role in the epidemiology of avian influenza. The secondary transmission (i.e., between poultry farms) of avian influenza virus is attributed primarily to fomites and people. Airborne transmission is also important, and the virus can be spread by aerosol in humans. Diagnostic tests detect viral proteins and genes. Virus-specific antibodies can be traced by serological tests, with virus isolation and identification being complementary procedures. The number of outbreaks of avian influenza seems to be increasing - over the last 5 years outbreaks have been reported in Italy, Hong Kong, Chile, the Netherlands, South Korea, Vietnam, Japan, Thailand, Cambodia, Indonesia, Laos, China, Pakistan, United States of America, Canada, South Africa, and Malaysia. Moreover, a growing number of human cases of avian influenza, in some cases fatal, have paralleled the outbreaks in commercial poultry. There is great concern about the possibility that a new virus subtype with pandemic potential could emerge from these outbreaks. From the perspective of human health, it is essential to eradicate the virus from poultry; however, the large number of small-holdings with poultry, the lack of control experience and resources, and the international scale of transmission and infection make rapid control and long-term prevention of recurrence extremely difficult. In the Western world, the renewed interest in free-range housing carries a threat for future outbreaks. The growing ethical objections to the largescale culling of birds require a different approach to the eradication of avian influenza.
Matched MeSH terms: Influenza A virus/genetics; Influenza A virus/pathogenicity*
Influenza A viruses (IAVs) have a remarkable tropism in their ability to circulate in both mammalian and avian species. The IAV NS1 protein is a multifunctional virulence factor that inhibits the type I interferon host response through a myriad of mechanisms. How NS1 has evolved to enable this remarkable property across species and its specific impact in the overall replication, pathogenicity, and host preference remain unknown. Here we analyze the NS1 evolutionary landscape and host tropism using a barcoded library of recombinant IAVs. Results show a surprisingly great variety of NS1 phenotypes according to their ability to replicate in different hosts. The IAV NS1 genes appear to have taken diverse and random evolutionary pathways within their multiple phylogenetic lineages. In summary, the high evolutionary plasticity of this viral protein underscores the ability of IAVs to adapt to multiple hosts and aids in our understanding of its global prevalence.
Matched MeSH terms: Influenza A virus/genetics; Influenza A virus/pathogenicity*
Equine influenza is a major cause of respiratory infections in horses and can spread rapidly despite the availability of commercial vaccines. In this study, we carried out molecular characterization of Equine Influenza Virus (EIV) isolated from the Malaysian outbreak in 2015 by sequencing of the HA and NA gene segments using Sanger sequencing. The nucleotide and amino acid sequences of HA and NA were compared with representative Florida clade 1 and clade 2 strains using phylogenetic analysis. The Florida clade 1 viruses identified in this outbreak revealed numerous amino acid substitutions in the HA protein as compared to the current OIE vaccine strain recommendations and representative strains of circulating Florida sub-lineage clade 1 and clade 2. Differences in HA included amino acids located within antigenic sites which could lead to reduced immune recognition of the outbreak strain and alter the effectiveness of vaccination against the outbreak strain. Detailed surveillance and genetic information sharing could allow genetic drift of equine influenza viruses to be monitored more effectively on a global basis and aid in refinement of vaccine strain selection for EIV.
Matched MeSH terms: Influenza A Virus, H3N8 Subtype/genetics*; Influenza A Virus, H3N8 Subtype/isolation & purification
Numerous lessons have been learned so far in controlling H5N1 avian influenza in Asia. Early detection of incursions of virus prevented establishment of the disease in several countries, notably Japan, South Korea, and Malaysia. In countries where detection of early cases was delayed, infection is endemic and has been for three or more years. Control measures implemented in these countries need to reflect this finding. Vaccination will continue to be one of the key measures used in these endemically infected countries. Used alone, vaccination will not result in elimination of H5N1 viruses from a country, but, if used correctly, it will markedly reduce the prevalence of and susceptibility to infection. Vaccination has already played a valuable role in reducing the adverse effects of H5N1 viruses. Mass culling also reduces the level of infection in infected areas. However, the long-term benefits are limited in endemically infected countries owing to the high probability of reinfection on restocking unless other measures are used in parallel. Full epidemiological studies have not been conducted in many infected countries. Nevertheless, it is recognized that the number of clinical cases does not truly reflect the levels of infection. Domestic ducks and large live poultry markets have played a key role in the persistence of infection, because they can be infected silently. In tackling this disease, countries should adopt integrated control programs using the combination of measures best suited to the local environment. All surveillance data should be shared, both positive and negative, and should include information on cases of infection and disease. Socioeconomic and ecological implications of all control measures should be assessed before implementation, especially the impact on the rural poor.
Matched MeSH terms: Influenza A Virus, H5N1 Subtype/isolation & purification; Influenza A Virus, H5N1 Subtype/physiology*
Malaysia first reported H5N1 poultry case in 2004 and subsequently outbreak in poultry population in 2007. Here, a recombinant gene encoding of peptide epitopes, consisting fragments of HA1, HA2 and a polybasic cleavage site of H5N1 strain Malaysia, was amplified and cloned into pET-47b(+) bacterial expression vector. DNA sequencing and alignment analysis confirmed that the gene had no alteration and in-frame to the vector. Then, His-tagged truncated HA protein was expressed in Escherichia coli BL21 (DE3) under 1 mM IPTG induction. The protein expression was optimized under a time-course induction study and further purified using Ni-NTA agarose under reducing condition. Migration size of protein was detected at 15 kDa by Western blot using anti-His tag monoclonal antibody and demonstrated no discrepancy compared to its calculated molecular weight.
Matched MeSH terms: Influenza A Virus, H5N1 Subtype/genetics*; Influenza A Virus, H5N1 Subtype/isolation & purification
Influenza virus non-structural protein 1 (NS1) counteracts host antiviral innate immune responses by inhibiting Retinoic acid inducible gene-I (RIG-I) activation. However, whether NS1 also specifically regulates RIG-I transcription is unknown. Here, we identify a CCAAT/Enhancer Binding Protein beta (C/EBPβ) binding site in the RIG-I promoter as a repressor element, and show that NS1 promotes C/EBPβ phosphorylation and its recruitment to the RIG-I promoter as a C/EBPβ/NS1 complex. C/EBPβ overexpression and siRNA knockdown in human lung epithelial cells resulted in suppression and activation of RIG-I expression respectively, implying a negative regulatory role of C/EBPβ. Further, C/EBPβ phosphorylation, its interaction with NS1 and occupancy at the RIG-I promoter was associated with RIG-I transcriptional inhibition. These findings provide an important insight into the molecular mechanism by which influenza NS1 commandeers RIG-I transcriptional regulation and suppresses host antiviral responses.
Matched MeSH terms: Influenza A virus/genetics*; Influenza A virus/immunology
Attenuated Salmonella has been used as a carrier for DNA vaccine. However, in vitro and in vivo studies on the bacteria following transfection of plasmid DNA were poorly studied. In this paper, eukaryotic expression plasmids encoding avian influenza virus (AIV) subtype H5N1 genes, pcDNA3.1/HA, NA, and NP, were transfected into an attenuated Salmonella enteric typhimurium SV4089. In vitro stability of the transfected plasmids into Salmonella were over 90% after 100 generations. The attenuated Salmonella were able to invade MCF-7 (1.2%) and MCF-10A (0.5%) human breast cancer cells. Newly hatched specific-pathogen-free (SPF) chicks were inoculated once by oral gavage with 10(9) colony-forming unit (CFU) of the attenuated Salmonella. No abnormal clinical signs or deaths were recorded after inoculation. Viable bacteria were detected 3 days after inoculation by plating from spleen, liver, and cecum. Fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR) were carried out for confirmation. Salmonella was not detected in blood cultures although serum antibody immune responses to Salmonella O antiserum group D1 factor 1, 9, and 12 antigens were observed in all the inoculated chickens after 7 days up to 35 days. Our results showed that live attenuated S. typhimurium SV4089 harboring pcDNA3.1/HA, NA, and NP may provide a unique alternative as a carrier for DNA oral vaccine in chickens.
Matched MeSH terms: Influenza A Virus, H5N1 Subtype/genetics; Influenza A Virus, H5N1 Subtype/immunology