Inhibition of multiple subtypes of influenza A virus in cell cultures with morpholino oligomers.

Peptide-conjugated phosphorodiamidate morpholino oligomers (P-PMO) are single-stranded nucleic acid-like antisense agents that can reduce gene expression by sterically blocking complementary RNA sequence. P-PMO are water soluble and nuclease resistant, and they readily achieve uptake into cells in culture under standard conditions. Eight P-PMO, each 20 to 22 bases in length, were evaluated for their ability to inhibit influenza A virus (FLUAV) A/PR/8/34 (H1N1) replication in cell culture. The P-PMO were designed to base pair with FLUAV RNA sequences that are highly conserved across viral subtypes and considered critical to the FLUAV biological-cycle, such as gene segment termini and mRNA translation start site regions. Several P-PMO were highly efficacious, each reducing viral titer in a dose-responsive and sequence-specific manner in A/PR/8/34-infected cells. Two P-PMO, one designed to target the AUG translation start site region of PB1 mRNA and the other the 3'-terminal region of nucleoprotein viral genome RNA, also proved to be potent against several other FLUAV strains, including A/WSN/33 (H1N1), A/Memphis/8/88 (H3N2), A/Eq/Miami/63 (H3N8), A/Eq/Prague/56 (H7N7), and the highly pathogenic A/Thailand/1(KAN-1)/04 (H5N1). The P-PMO exhibited minimal cytotoxicity in cell viability assays. High efficacy by two of the P-PMO against multiple FLUAV subtypes suggests that these oligomers represent a broad-spectrum therapeutic approach against a high percentage of known FLUAV strains.

Inhibition, escape, and attenuated growth of severe acute respiratory syndrome coronavirus treated with antisense morpholino oligomers.

The recently emerged severe acute respiratory syndrome coronavirus (SARS-CoV) is a potent pathogen of humans and is capable of rapid global spread. Peptide-conjugated antisense morpholino oligomers (P-PMO) were designed to bind by base pairing to specific sequences in the SARS-CoV (Tor2 strain) genome. The P-PMO were tested for their capacity to inhibit production of infectious virus as well as to probe the function of conserved viral RNA motifs and secondary structures. Several virus-targeted P-PMO and a random-sequence control P-PMO showed low inhibitory activity against SARS coronavirus. Certain other virus-targeted P-PMO reduced virus-induced cytopathology and cell-to-cell spread as a consequence of decreasing viral amplification. Active P-PMO were effective when administered at any time prior to peak viral synthesis and exerted sustained antiviral effects while present in culture medium. P-PMO showed low nonspecific inhibitory activity against translation of nontargeted RNA or growth of the arenavirus lymphocytic choriomeningitis virus. Two P-PMO targeting the viral transcription-regulatory sequence (TRS) region in the 5' untranslated region were the most effective inhibitors tested. After several viral passages in the presence of a TRS-targeted P-PMO, partially drug-resistant SARS-CoV mutants arose which contained three contiguous base point mutations at the binding site of a TRS-targeted P-PMO. Those partially resistant viruses grew more slowly and formed smaller plaques than wild-type SARS-CoV. These results suggest PMO compounds have powerful therapeutic and investigative potential toward coronavirus infection.

Serological Detection of Grapevine Associated Closteroviruses in Infected Grapevine Cultivars.

Western blot immunoassay and enzyme-linked immunosorbent assay using different monoclonal antibodies (MAb) and polyclonal antisera (PA) revealed mixed infections of serologically related and unrelated grapevine leafroll associated viruses (GLRaVs) and grapevine corky bark associated virus (GCBaV) in symptomatic grapevines. A PA designated rootstock-scion incompatibility (RSI)-24 kDa, grapevine corky bark PA, and GLRaV-2b MAb reacted to polypeptides of approximately 24 kDa isolated from grapevines exhibiting rootstock-scion incompatibility, leafroll, and corky bark disease symptoms, suggesting that these isolates are infected with closely related viruses. A PA designated GLRaV-2 US detected virus specific polypeptides of 38, 37, 36, and 24 kDa, while a polyclonal antiserum designated GLRaV-2 FR detected a single virus-specific polypeptide of approximately 24 kDa. The reactivity of GLRaV-2 US to various polypeptides suggests that the immunogen used to produce this antiserum was a mixture of viruses. Apical meristems were excised and cultured to eliminate the infection of viruses in the grapevines showing RSI symptoms and in the cultivar French Colombard infected with GLRaV-1. The elimination of these viruses was confirmed by Western blot assay. These studies show that the Western blot assay can be used to detect and differentiate grapevine disease-associated closteroviruses.