Full factorial analysis of mammalian and avian influenza polymerase subunits suggests a role of an efficient polymerase for virus adaptation.

Amongst all the internal gene segments (PB2. PB1, PA, NP, M and NS), the avian PB1 segment is the only one which was reassorted into the human H2N2 and H3N2 pandemic strains. This suggests that the reassortment of polymerase subunit genes between mammalian and avian influenza viruses might play roles for interspecies transmission. To test this hypothesis, we tested the compatibility between PB2, PB1, PA and NP derived from a H5N1 virus and a mammalian H1N1 virus. All 16 possible combinations of avian-mammalian chimeric viral ribonucleoproteins (vRNPs) were characterized. We showed that recombinant vRNPs with a mammalian PB2 and an avian PB1 had the strongest polymerase activities in human cells at all studied temperature. In addition, viruses with this specific PB2-PB1 combination could grow efficiently in cell cultures, especially at a high incubation temperature. These viruses were potent inducers of proinflammatory cytokines and chemokines in primary human macrophages and pneumocytes. Viruses with this specific PB2-PB1 combination were also found to be more capable to generate adaptive mutations under a new selection pressure. These results suggested that the viral polymerase activity might be relevant for the genesis of influenza viruses of human health concern.

Human monoclonal antibody combination against SARS coronavirus: synergy and coverage of escape mutants.

BACKGROUND: Experimental animal data show that protection against severe acute respiratory syndrome coronavirus (SARS-CoV) infection with human monoclonal antibodies (mAbs) is feasible. For an effective immune prophylaxis in humans, broad coverage of different strains of SARS-CoV and control of potential neutralization escape variants will be required. Combinations of virus-neutralizing, noncompeting mAbs may have these properties. METHODS AND FINDINGS: Human mAb CR3014 has been shown to completely prevent lung pathology and abolish pharyngeal shedding of SARS-CoV in infected ferrets. We generated in vitro SARS-CoV variants escaping neutralization by CR3014, which all had a single P462L mutation in the glycoprotein spike (S) of the escape virus. In vitro experiments confirmed that binding of CR3014 to a recombinant S fragment (amino acid residues 318-510) harboring this mutation was abolished. We therefore screened an antibody-phage library derived from blood of a convalescent SARS patient for antibodies complementary to CR3014. A novel mAb, CR3022, was identified that neutralized CR3014 escape viruses, did not compete with CR3014 for binding to recombinant S1 fragments, and bound to S1 fragments derived from the civet cat SARS-CoV-like strain SZ3. No escape variants could be generated with CR3022. The mixture of both mAbs showed neutralization of SARS-CoV in a synergistic fashion by recognizing different epitopes on the receptor-binding domain. Dose reduction indices of 4.5 and 20.5 were observed for CR3014 and CR3022, respectively, at 100% neutralization. Because enhancement of SARS-CoV infection by subneutralizing antibody concentrations is of concern, we show here that anti-SARS-CoV antibodies do not convert the abortive infection of primary human macrophages by SARS-CoV into a productive one. CONCLUSIONS: The combination of two noncompeting human mAbs CR3014 and CR3022 potentially controls immune escape and extends the breadth of protection. At the same time, synergy between CR3014 and CR3022 may allow for a lower total antibody dose to be administered for passive immune prophylaxis of SARS-CoV infection.

Recurrent mutations associated with isolation and passage of SARS coronavirus in cells from non-human primates.

Four clinical isolates of SARS coronavirus were serially passaged in two primate cell lines (FRhK4 and Vero E6). Viral genetic sequences encoding for structural proteins and open reading frames 6--8 were determined in the original clinical specimen, the initial virus isolate (passage 0) and at passages 5, 10, and 15. After 15 passages, a total of 15 different mutations were identified and 12 of them were non-synonymous mutations. Seven of these mutations were recurrent mutation and all located at the spike, membrane, and Orf 8a protein encoding sequences. Mutations in the membrane protein and a deletion in ORF 6--8 were already observed in passage 0, suggesting these amino acid substitutions are important in the adaptation of the virus isolate in primate cell culture. A mutation in the spike gene (residue 24079) appeared to be unique to adaptation in FRhK4 cells. It is important to be aware of cell culture associated mutations when interpreting data on molecular evolution of SARS coronavirus.

Detection of human influenza A viruses by loop-mediated isothermal amplification.

Here we describe the use of the loop-mediated isothermal amplification (LAMP) method to detect human influenza viruses (H1 to H3). Our results were correlated 100% with results deduced from routine clinical diagnostic tests. In addition, we also developed a LAMP assay specific for human beta-actin cDNA as a quality control test.

A one step quantitative RT-PCR for detection of SARS coronavirus with an internal control for PCR inhibitors.

In this study, we report a one step quantitative RT-PCR assay for severe acute respiratory syndrome (SARS) diagnosis. The overall detection rate for clinical samples collected from Days 1 to 9 of disease onset is 86.2% and the specificity of the assay is 100%. To detect false negative results due to PCR inhibitors or faulty RNA extraction, we have further modified this one step RT-PCR assay for simultaneous detection of severe acute respiratory syndrome (SARS) coronavirus (CoV) and 18S ribosomal RNA (rRNA) as an internal positive control.