No abstract

Background: Human Respiratory Syncytial Virus (HRSV) is a major viral pathogen associated with acute lower respiratory tract infections (ALRTI) in children. Using monoclonal antibodies against virus proteins, it is categorized into two distinct major groups, A and B. The second hypervariable region of the G protein ectodomain gene provides a reliable surrogate for phylogenetical studies. We carried out a phylogenetic analysis of the HRSV strains isolated from children hospitalized with ALRTI in Malaysia.Methods: Nasopharyngeal aspirates (NPA) were taken from children less than five years of age hospitalized with ALRTI to Hospital Serdang, Malaysia. RT-PCR was used to detect HRSV. The second hypervariable region at the carboxyl-terminal of the G gene was amplified and sequenced using primer sets GPA/F1 and GPB/F1. Neucleotide sequences were edited and aligned with Bioedite software and Clustal X program. The phylogenetic relationships of the samples were determined separately for group A and B using neighbor joining (NJ), maximum parsimony (MP) and Bayesian methods (BI).Results: HRSV was detected in 83 of 165 (50.3%) patients studied. Sequence analysis of 32 isolates showed that multiple lineages of HRSV group A and B serotypes co-circulated. The topologies resulting from the different methods (NJ, MP and BI) congruent with each other. Phylogenetic analysis of nine retrieved sequences showed that all the HRSV-A strains were clustered into the NA1 genotype. All the 23 HRSV-B strains belonged to BA genotypes consisted of a 60-nucleotide duplication region. They were classified into three different genotypes of BA10, BA9 and BA4, respectively.Conclusion: HRSV played a prominent role for hospitalization of children in our study. The sequences of the second hypervariable region of G protein ectodomain gene from HRSV A and B demonstrated remarkable genetic diversity. The present finding seems to be consistent with other studies which found the newly emerged HRSV genotypes of NA1 and BA genotypes are replacing the previously dominant genotypes. This is the first documentation of the phylogenetic relationship and genetic diversity of HRSV isolates among hospitalized children diagnosed with ALRTI in Malaysia.

Interferon-inducible transmembrane protein 3 (IFITM3) is an effector protein of the innate immune system. It confers potent, cell-intrinsic resistance to infection by diverse enveloped viruses both in vitro and in vivo, including influenza viruses, West Nile virus, and dengue virus. IFITM3 prevents cytosolic entry of these viruses by blocking complete virus envelope fusion with cell endosome membranes. Although the IFITM locus, which includes IFITM1, -2, -3, and -5, is present in mammalian species, this locus has not been unambiguously identified or functionally characterized in avian species. Here, we show that the IFITM locus exists in chickens and is syntenic with the IFITM locus in mammals. The chicken IFITM3 protein restricts cell infection by influenza A viruses and lyssaviruses to a similar level as its human orthologue. Furthermore, we show that chicken IFITM3 is functional in chicken cells and that knockdown of constitutive expression in chicken fibroblasts results in enhanced infection by influenza A virus. Chicken IFITM2 and -3 are constitutively expressed in all tissues examined, whereas IFITM1 is only expressed in the bursa of Fabricius, gastrointestinal tract, cecal tonsil, and trachea. Despite being highly divergent at the amino acid level, IFITM3 proteins of birds and mammals can restrict replication of viruses that are able to infect different host species, suggesting IFITM proteins may provide a crucial barrier for zoonotic infections.

Genetic exchange by a process of genome-segment ‘reassortment’ represents an important mechanism for evolutionary change in all viruses with segmented genomes, yet in many cases a detailed understanding of its frequency and biological consequences is lacking. We provide a comprehensive assessment of reassortment in bluetongue virus (BTV), a globally important insect-borne pathogen of livestock, during recent outbreaks in Europe. Full-genome sequences were generated and analysed for over 150 isolates belonging to the different BTV serotypes that have emerged in the region over the last 5 decades. Based on this novel dataset we confirm that reassortment is a frequent process that plays an important and on-going role in evolution of the virus. We found evidence for reassortment in all ten segments without a significant bias towards any particular segment. However, we observed biases in the relative frequency at which particular segments were associated with each other during reassortment. This points to selective constraints possibly caused by functional relationships between individual proteins or genome segments and genome-wide epistatic interactions. Sites under positive selection were more likely to undergo amino acid changes in newly reassorted viruses, providing additional evidence for adaptive dynamics as a consequence of reassortment. We show that the live attenuated vaccines recently used in Europe have repeatedly reassorted with field strains, contributing to their genotypic, and potentially phenotypic, variability. The high degree of plasticity seen in the BTV genome in terms of segment origin suggests that current classification schemes that are based primarily on serotype, which is determined by only a single genome segment, are inadequate. Our work highlights the need for a better understanding of the mechanisms and epidemiological consequences of reassortment in BTV, as well as other segmented RNA viruses.

Bluetongue is a major infectious disease of ruminants that is caused by bluetongue virus (BTV). In this study, we analyzed virulence and genetic differences of (i) three BTV field strains from Italy maintained at either a low (L strains) or high (H strains) passage number in cell culture and (ii) three South African "reference" wild-type strains and their corresponding live attenuated vaccine strains. The Italian BTV L strains, in general, were lethal for both newborn NIH-Swiss mice inoculated intracerebrally and adult type I interferon receptor-deficient (IFNAR ؊/؊ ) mice, while the virulence of the H strains was attenuated significantly in both experimental models. Similarly, the South African vaccine strains were not pathogenic for IFNAR ؊/؊ mice, while the corresponding wild-type strains were virulent. Thus, attenuation of the virulence of the BTV strains used in this study is not mediated by the presence of an intact interferon system. No clear distinction in virulence was observed for the South African BTV strains in newborn NIH-Swiss mice. Full genomic sequencing revealed relatively few amino acid substitutions, scattered in several different viral proteins, for the strains found to be attenuated in mice compared to the pathogenic related strains. However, only the genome segments encoding VP1, VP2, and NS2 consistently showed nonsynonymous changes between all virulent and attenuated strain pairs. This study established an experimental platform for investigating the determinants of BTV virulence. Future studies using reverse genetics will allow researchers to precisely map and "weight" the relative influences of the various genome segments and viral proteins on BTV virulence.