Sequence analysis of in vivo defective interfering-like RNA of influenza A H1N1 pandemic virus

Abstract

Influenza virus defective interfering (DI) particles are naturally occurring noninfectious virions typically generated during in vitro serial passages in cell culture of the virus at a high multiplicity of infection. DI particles are recognized for the role they play in inhibiting viral replication and for the impact they have on the production of infectious virions. To date, influenza virus DI particles have been reported primarily as a phenomenon of cell culture and in experimentally infected embryonated chicken eggs. They have also been isolated from a respiratory infection of chickens. Using a sequencing approach, we characterize several subgenomic viral RNAs from human nasopharyngeal specimens infected with the influenza A(H1N1)pdm09 virus. The distribution of these in vivo-derived DI-like RNAs was similar to that of in vitro DIs, with the majority of the defective RNAs generated from the PB2 (segment 1) of the polymerase complex, followed by PB1 and PA. The lengths of the in vivo-derived DI-like segments also are similar to those of known in vitro DIs, and the in vivo-derived DI-like segments share internal deletions of the same segments. The presence of identical DI-like RNAs in patients linked by direct contact is compatible with transmission between them. The functional role of DI-like RNAs in natural infections remains to be established.

Fig 1

Example of mapping of deep-sequence reads on influenza virus segments. The sequencing reads from the GS-FLX (454) were mapped to the respective influenza A (H1N1) genomic segments. The plots depict the number of sequence reads that map to each nucleotide position across the genomic segment. The x axis represents the length of each segment, and each plot represents sequencing data for the indicated influenza virus segment from clinical isolate A/San Diego/INS007/2009 (H1N1).

Fig 2

Alignment of DI-like sequences from deep-sequence data. (A and B) GS-FLX sequence reads of the PB2 segment of the clinical isolates A/San Diego/INS007/2009 (H1N1) (A) and A/District of Columbia/INS047/2009 (H1N1) (B) mapped to their respective reference sequences (CY083837 for INS007 and CY083869 for INS047). Positions are shown in thousands from 100 (0.1k) to 2,300 (2.3k).

Fig 3

Alignment of cloned DI-like sequences. (A and B) Alignment of clone sequences generated from the INSIGHT influenza virus-positive clinical samples mapped to the reference segment 1 (PB2) (A) and segment 2 (PB1) (B).

Fig 4

Graphical representation of the junction sites of cloned DI-like sequences compared to GS-FLX (454) sequences. Nucleotide sequences at the putative junctions between 5′ and 3′ regions of DI-like RNAs are shown. The top sequence is the reference (REF) sequence, whereas the remaining sequences are the cloned PB2 subgenomic RNAs of clinical specimens INS006, INS007, and INS014. The red boxes represent the overlapping sequences at the 5′ end and beginning of the 3′ region of the DI-like sequence. Table 2 shows detailed analysis of various junction sites observed with the INSIGHT samples.

Fig 5

Detection of DI-like RNAs in clinical specimens. (A and B) RNA extracted from selected INSIGHT clinical specimens (INS002 to INS036) was subjected to PB2 segment-specific RT-PCR using SuperScript III (SSIII) first-strand synthesis kit MLV RT (Invitrogen) (A) or AMV RT (New England BioLabs) (B). INS036 is a negative control. The positions of molecular size standards (in kilobases) are shown to the left of the gels.

Fig 6

Gel electrophoresis of genomic RNA from INSIGHT clinical samples. Total RNA (∼30 ng) from the clinical samples was end labeled with ³²P followed by separation of the genomic segments on a 6% native polyacrylamide gel. The autoradiograph was obtained using the Bio-Rad PhosphorImager. INS086 is a control sample for which no subgenomic RNAs were detected in the deep-sequence data. The positions of molecular size standards (M) (from 0.3 to 2.0 kilobases) are shown to the left of the gel.