Molecular Characterization of Viruses from Clinical Respiratory Samples Producing Unidentified Cytopathic Effects in Cell Culture

The sequence-independent single primer amplification (SISPA) method was performed to identify a virus in 17 clinical respiratory samples producing uncharacterized cytopathic effects in LLC-MK2 cells. Sequence analysis of 600–1600 bp amplicons allowed the identification of six viruses (one influenza C, two parechovirus-3 and three cardioviruses). Genomic sequences of the cardioviruses showed similarities with those of the recently-described Saffold virus strain although significant variation was present in the viral surface EF and CD loops. These results demonstrate the usefulness of SISPA for identifying emerging viruses and also known viruses not easily identified by standard virological methods

Human Parechovirus 3 and Neonatal Infections

Three case reports expand the clinical spectrum of HPeV-3 infections and highlight the need to characterize this new pathogen

Role of Permissive Neuraminidase Mutations in Influenza A/Brisbane/59/2007-like (H1N1) Viruses

Neuraminidase (NA) mutations conferring resistance to NA inhibitors were believed to compromise influenza virus fitness. Unexpectedly, an oseltamivir-resistant A/Brisbane/59/2007 (Bris07)-like H1N1 H275Y NA variant emerged in 2007 and completely replaced the wild-type (WT) strain in 2008–2009. The NA of such variant contained additional NA changes (R222Q, V234M and D344N) that potentially counteracted the detrimental effect of the H275Y mutation on viral fitness. Here, we rescued a recombinant Bris07-like WT virus and 4 NA mutants/revertants (H275Y, H275Y/Q222R, H275Y/M234V and H275Y/N344D) and characterized them in vitro and in ferrets. A fluorometric-based NA assay was used to determine Vmax and Km values. Replicative capacities were evaluated by yield assays in ST6Gal1-MDCK cells. Recombinant NA proteins were expressed in 293T cells and surface NA activity was determined. Infectivity and contact transmission experiments were evaluated for the WT, H275Y and H275Y/Q222R recombinants in ferrets. The H275Y mutation did not significantly alter Km and Vmax values compared to WT. The H275Y/N344D mutant had a reduced affinity (Km of 50 vs 12 µM) whereas the H275Y/M234V mutant had a reduced activity (22 vs 28 U/sec). In contrast, the H275Y/Q222R mutant showed a significant decrease of both affinity (40 µM) and activity (7 U/sec). The WT, H275Y, H275Y/M234V and H275Y/N344D recombinants had comparable replicative capacities contrasting with H275Y/Q222R mutant whose viral titers were significantly reduced. All studied mutations reduced the cell surface NA activity compared to WT with the maximum reduction being obtained for the H275Y/Q222R mutant. Comparable infectivity and transmissibility were seen between the WT and the H275Y mutant in ferrets whereas the H275Y/Q222R mutant was associated with significantly lower lung viral titers. In conclusion, the Q222R reversion mutation compromised Bris07-like H1N1 virus in vitro and in vivo. Thus, the R222Q NA mutation present in the WT virus may have facilitated the emergence of NAI-resistant Bris07 variants

WU Polyomavirus in Children, Canada

WU polyomavirus was detected in nasopharyngeal aspirates in 2 (2.5%) of 79 children with respiratory infections (both infected with respiratory syncytial virus) and in 5 (6.4%) of 78 asymptomatic children during the same winter season in Canada. The strains were closely related to Australian and American viruses based on analysis of large T antigen (TAg) and VP2 genes. The pathogenic role of WU virus is still uncertain

Human Parechovirus Infections in Canada

These infections are associated with a variety of clinical syndromes, in part related to specific serotype

Respiratory Tract Reinfections by the New Human Metapneumovirus in an Immunocompromised Child

The human Metapneumovirus (HMPV), a new member of the Paramyxoviridae family, has been recently associated with respiratory tract infections in young children. We report the case of a young, immunocompromised child who had severe lower respiratory tract infections during two consecutive winter seasons caused by genetically distinct HMPV strains

E119D Neuraminidase Mutation Conferring Pan-Resistance to Neuraminidase Inhibitors in an A(H1N1)pdm09 Isolate From a Stem-Cell Transplant Recipient

Background. An influenza A(H1N1)pdm09 infection was diagnosed in a hematopoietic stem cell transplant recipient during conditioning regimen. He was treated with oral oseltamivir, later combined with intravenous zanamivir. The H275Y neuraminidase (NA) mutation was first detected, and an E119D NA mutation was identified during zanamivir therapy. Methods. Recombinant wild-type (WT) E119D and E119D/H275Y A(H1N1)pdm09 NA variants were generated by reverse genetics. Susceptibility to NA inhibitors (NAIs) was evaluated with a fluorometric assay using the 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid (MUNANA) substrate. Susceptibility to favipiravir (T-705) was assessed using plaque reduction assays. The NA affinity and velocity values were determined with NA enzymatic studies. Results. We identified an influenza A(H1N1)pdm09 E119D mutant that exhibited a marked increase in the 50% inhibitory concentrations against all tested NAIs (827-, 25-, 286-, and 702-fold for zanamivir, oseltamivir, peramivir, and laninamivir, respectively). The double E119D/H275Y mutation further increased oseltamivir and peramivir 50% inhibitory concentrations by 790- and >5000-fold, respectively, compared with the WT. The mutant viruses remained susceptible to favipiravir. The NA affinity and velocity values of the E119D variant decreased by 8.1-fold and 4.5-fold, respectively, compared with the WT. Conclusions. The actual emergence of a single NA mutation conferring pan-NAI resistance in the clinical setting reinforces the pressing need to develop new anti-influenza strategie

Human Metapneumovirus Infections in Hospitalized Children1

We evaluated the percentage of hospitalizations for acute respiratory tract infections in children <3 years of age attributable to human metapneumovirus (HMPV) and other respiratory viruses in a prospective study during winter and spring 2002. We used real-time polymerase chain assays and other conventional diagnostic methods to detect HMPV, human respiratory syncytial virus (HRSV), and influenza viruses in nasopharyngeal aspirates of children. HMPV was detected in 12 (6%) of the 208 children hospitalized for acute respiratory tract infections, HRSV in 118 (57%), and influenza A in 49 (24%). Bronchiolitis was diagnosed in 8 (68%) and pneumonitis in 2 (17%) of HMPV-infected children; of those with HRSV infection, pneumonitis was diagnosed in 99 (84%) and bronchiolitis in 30 (25%). None of the HMPV-infected children was admitted to an intensive-care unit, whereas 15% of those with HRSV or influenza A infections were admitted. HMPV is an important cause of illness in young children with a similar, although less severe, clinical presentation to that of HRSV

Human Metapneumovirus, Peru

We retrospectively studied 420 pharyngeal swab specimens collected from Peruvian and Argentinean patients with influenzalike illness in 2002 and 2003 for evidence of human metapneumovirus (HMPV). Twelve specimens (2.3%) were positive by multiple assays. Six specimens yielded HMPV isolates. Four of the 6 isolates were of the uncommon B1 genotype