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Influenza C and D Viruses Package Eight Organized Ribonucleoprotein Complexes

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Influenza C and D Viruses Package Eight Organized Ribonucleoprotein Complexes

Sumiho Nakatsu et al. J Virol.

Abstract

Influenza A and B viruses have eight-segmented, single-stranded, negative-sense RNA genomes, whereas influenza C and D viruses have seven-segmented genomes. Each genomic RNA segment exists in the form of a ribonucleoprotein complex (RNP) in association with nucleoproteins and an RNA-dependent RNA polymerase in virions. Influenza D virus was recently isolated from swine and cattle, but its morphology is not fully studied. Here, we examined the morphological characteristics of D/bovine/Yamagata/10710/2016 (D/Yamagata) and C/Ann Arbor/50 (C/AA), focusing on RNPs packaged within the virions. By scanning transmission electron microscopic tomography, we found that more than 70% of D/Yamagata and C/AA virions packaged eight RNPs arranged in the "1+7" pattern as observed in influenza A and B viruses, even though type C and D virus genomes are segmented into only seven segments. These results imply that influenza viruses generally package eight RNPs arranged in the "1+7" pattern regardless of the number of RNA segments in their genome.IMPORTANCE The genomes of influenza A and B viruses are segmented into eight segments of negative-sense RNA, and those of influenza C and D viruses are segmented into seven segments. For progeny virions to be infectious, each virion needs to package all of their genomic segments. Several studies support the conclusion that influenza A and B viruses selectively package eight distinct genomic RNA segments; however, the packaging of influenza C and D viruses, which possess seven segmented genomes, is less understood. By using electron microscopy, we showed that influenza C and D viruses package eight RNA segments just as influenza A and B viruses do. These results suggest that influenza viruses prefer to package eight RNA segments within virions independent of the number of genome segments.

Keywords: influenza virus.

Figures

FIG 1
FIG 1
D/Yamagata and C/AA virions visualized by negative-staining EM. Both D/Yamagata (A and B) and C/AA (C and D) virions exhibited spherical and filamentous shapes. White arrows indicate virions with hexagonal patterns formed by HEF. Scale bars: A and C, 100 nm; B and D, 1 μm.
FIG 2
FIG 2
ICV and IDV virions visualized by ultrathin section EM. Ultrathin sections (50 nm thick) of D/Yamagata-, D/Oklahoma-, C/AA-, and C/Taylor-infected cells were observed under a transmission electron microscope. Longitudinally (arrowheads) or transversely sectioned D/Yamagata (A to D), D/Oklahoma (E to H), C/AA (I to M), and C/Taylor (N-R) virions were observed. The cord-like structures (CLSs) are indicated by arrows (J and O). Scale bars: A, E, I, J, N, and O, 500 nm; B to D, F to H, K to M, and P to R, 100 nm.
FIG 3
FIG 3
Three-dimensional analysis of D/Yamagata and C/AA virions by STEM tomography. Semithin (250-nm thick) sections were prepared from the same samples as those examined by ultrathin section EM (Fig. 2). (A to D) D/Yamagata; (E to H) C/AA. Digital slices of reconstructed D/Yamagata (A and C) and C/AA (E and G) virions are shown from the top (upper left panel) to the bottom (lower left panel). Panels B, D, F, and H show models of the RNPs packaged within the virions from the top (right) and side (left) views. (I and J) Numbers of RNPs inside each 3-D reconstructed virion. Approximately 10 of each virion were analyzed. (I) D/Yamagata; (J) C/AA.
FIG 4
FIG 4
RT-PCR analysis of virus stocks of D/Yamagata and C/AA. Viral RNAs were extracted from A/WSN-, D/Yamagata-, and C/AA-infected cells and subjected to RT-PCR with strand-specific primers for the NP (A) and M (B) segments of IAV. Reverse transcription (RT) reactions were performed with or without reverse transcriptase.
FIG 5
FIG 5
Urea-PAGE of vRNAs isolated from D/Yamagata and C/AA virions. The RNAs within D/Yamagata (A) and C/AA (B) virions were separated on 7 M urea-containing 4% polyacrylamide gel and stained with SYBR-Gold.

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