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Implementation of reverse genetics for influenza A virus, that is, the DNA-based generation of infectious viral particles in cell culture, opened new avenues to investigate the function of viral proteins and their interplay with host factors on a molecular level. This powerful technique allows the introduction, depletion, or manipulation of any given sequence in the viral genome, as long as it gives rise to replicating virus progeny. Reverse genetics can be used to generate targeted reassortant viruses by mixing segments of different viral strains, thus providing insight into phenotypes of potentially pandemic viruses arising from natural reassortment. It was further instrumental for the development of novel vaccine strategies, allowing rapid and targeted exchange of viral surface antigens on a well-replicating genetic backbone of cell culture-adapted or cold-adapted/attenuated viral strains. Establishment of reverse genetics and rescue of molecular clones of influenza A virus have been extensively described before. Here we give a detailed stand-alone protocol encompassing clinical sampling of influenza A virus specimens and subsequent plasmid-based genetics to rescue, manipulate, and confirm a fully infectious molecular clone. This protocol is based on the combined techniques and experience of a number of influenza laboratories, which are credited and referenced whenever appropriate.

(A) rarefection plot: number of sequences is plotted against Chao1 estimator. Error bars indicate SE. (B) 2D PCoA of SI microbiota mock-treated (blue symbols) or IAV-infected mice (red symbols) at indicated time points post infection. (C) Mean UNIFRAC distances for the comparison of indicated experimental groups. Statistical significance was determined by two-tailed student’s T test. (D) 2D PCoA of SI microbiota mock-treated (blue symbols) or IAV-infected mice (red symbols) at indicated time points post infection. Each symbol represents one mouse. Each symbol type refers to an individual cage. (E) Shannon index, relative abundances of indicated bacterial classes determined by 16S rRNA gene NGS, and relative 16S/18S levels of animals 7 dpi are shown. Each symbol represents one animal. Each symbol type refers to an individual cage. (F) Relative abundance of total bacteria based on qPCR (16S/18S) normalized to median of mock samples is shown for individual mice and indicated time points treated with mock (black symbols) or indicated viruses (red symbols) for for H1N1 7 dpi (n = 8 per group) pH1N1 6 dpi (n = 5 per group). (G) Most abundant OTU on genus level. Median relative abundance (%) is depicted. (PDF 1956 kb)

(A) rarefection plot: number of sequences is plotted against Chao1 estimator. Error bars indicate SE. (B) Most abundant OTU found in baseline, mock or IAV-infected LRT samples 7 dpi on genus level. Median relative abundance (%) is depicted. (C) N-fold changes of 16S/18S DNA levels normalized to mean of mock samples of each day examined. Individual mice and median are depicted (n = 18 per group per time point, two independent experiments, representative data shown). (D) Individual lung titers of mock treated (black circles) and IAV (pH1N1, Neth/602) infected mice (red squares), median titers (pfu/organ) are indicated. Limit of detection (LoD) 50 pfu (n = 5 per group). (E) Relative abundance of total bacteria and Firmicutes based on qPCR (16S/18S) normalized to mean of mock samples is shown for individual mice and indicated time points treated with mock (black symbols) or indicated viruses (red symbols for pH1N1 6 dpi) (n = 5 per group). (F) Bacterial titers of cultivatable URT microbiota 7 dpi grown on indicated agar plates (n = 6 per group). (G) Sequence alignment of Sanger sequencing results of 16S rRNA gene V4 region of Enterobacteriaceae (OTU ID 1111294) colonies. (H) Sequence alignment of Sanger sequencing results of 16S rRNA gene V4 region of Lactobacillaceae (OTU ID 1107027) colonies. (PDF 1691 kb)

(A) Rarefection plot: number of sequences is plotted against Chao1 estimator. Error bars indicate SE. (B) Alpha diversity depicted as shannon index for fecal microbiota of baseline (gray), mock-treated (black) or IAV-infected (red) mice. Individual values per mouse and median are depicted. (C) 2D PCoA plots of fecal microbiota mock-treated (blue) or IAV-infected mice (red) at indicated time points post infection. Each symbol represents one mouse. (D) Most abundant OTU on genus level. Median relative abundance (%) is depicted. (E) Relative abundance (%) of significantly changing OTUs of individual mice and median are depicted (mock red, IAV black). (F) Cultivatable bacterial cfu/fecal pellet of mock-treated (black) or IAV-infected (H5N1 red, H1N1 green) 7 dpi grown under aerobic or anaerobic conditions are depicted (n = 6 per group per time point, two independent experiments). (PDF 1343 kb)

Bunyamwera virus (BUNV) is the prototype virus for both the genus Orthobunyavirus and the family Bunyaviridae . BUNV has a tripartite, negative-sense RNA genome. The coding region of each segment is flanked by untranslated regions (UTRs) that are partially complementary. The UTRs play an important role in the virus life cycle by promoting transcription, replication, and encapsidation of the viral genome. Using reverse genetics, we generated recombinant viruses that contained deletions within the 3′ and/or 5′ UTRs of the L or M segments to determine the minimal UTRs competent for virus viability. We then generated viruses carrying deleted UTRs in all three segments. These viruses were grossly attenuated in tissue culture, being significantly impaired in their ability to produce plaques in BHK cells, and had a reduced capacity to cause host cell protein shutoff. After serial passage in tissue culture, some viruses partially recovered fitness, generating higher titers and producing larger plaques. We determined the complete nucleotide sequence for each virus. The deleted UTR sequences were maintained, and no amino acid changes were observed in the nonstructural proteins (NSs and NSm), the nucleocapsid protein (N), or the Gn glycoprotein. One virus had a single amino acid substitution in Gc. Three viruses contained amino acid changes in the viral polymerase that mostly occurred in the C-terminal domain of the L protein. Although the role of this domain remains unknown, we suggest that those changes might be involved in the evolution of the polymerase to recognize the deleted UTRs more efficiently.

In mammalian cells, nucleolar localization of influenza A NS1 requires the presence of a C-terminal nucleolar localization signal. This nucleolar localization signal is present only in certain strains of influenza A viruses. Therefore, only certain NS1 accumulate in the nucleolus of mammalian cells. In contrast, we show that all NS1 tested in this study accumulated in the nucleolus of avian cells even in the absence of the above described C-terminal nucleolar localization signal. Thus, nucleolar localization of NS1 in avian cells appears to rely on a different nucleolar localization signal that is more conserved among influenza virus strains.

phylum-specific qPCR for streptomycin vs. mock-treated mice. Individual relative levels of Bacteroidetes or Firmicutes normalized to 18S and median are depicted. Pooled data from two independent mouse experiments are shown (n = 5 per group). (PDF 717 kb)

mean copy number of minus strand (vRNA) and plus strand (mRNA/cRNA) RNA copies ± SD per organ sample are depicted as determined by specific RT-qPCR; n.d. not detected. (PDF 215 kb)

mean copy number of minus strand (vRNA) and plus strand (mRNA/cRNA) RNA copies ± SD per organ sample are depicted as determined by specific RT-qPCR; n.d. not detected. (PDF 215 kb)