Production and Storage of Virus Simulants

We have examined isolation and identification protocols for three virus simulant candidates to biological warfare agents. MS2 phage, a simulant for yellow fever virus and Hantaan virus, was propagated using as a host an E. coli strain with F pilus. MS2 phage genome was examined by reverse transcription and polymerase chain reaction (RT-PCR). Coat protein of the phage preparation was examined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometric analysis. Cydia pomonella granulosis virus (CpGV) is a virus simulant candidate to smallpox virus. CpGV was isolated from a commercialized CpGV pellet. In this study, we developed new isolation and identification protocols for CpGV. One disadvantage of using CpGV is that it is not easy to determine viability of the virus. Here, we have included T4 phage as an alternative. We established a high titer production protocol and developed an easy genome identification protocol that does not require purified phage DNA. Stability of these virus preparations was also examined under various storage conditions. When the virus preparations were not subjected to freeze drying, MS2 phage was most stable when it was stored in liquid nitrogen but unstable at 4℃. In contrast, T4 phage was most stable when it was stored at 4℃. CpGV was stable at −20℃ but not at 4℃. Stability during or after freeze drying was also investigated. The result showed that 70~80% MS2 survived the freeze drying process. In contrast, only about 15% of T4 phage survived during the freeze drying. CpGV was found to be degraded during freeze drying.

Cysteine-Added Mutants of Turnip Yellow Mosaic Virus

Native turnip yellow mosaic virus (TYMV) is relatively unreactive to maleimide agents, indicating few reactive thiol groups on TYMV. In the present study, we aimed to construct TYMV mutants that have reactive cysteine residues on the surface. To this end, we prepared a library of TYMV mutants where the Thr residue at the C-terminus of coat protein (CP) was replaced by a random sequence of six amino acids that included one cysteine. This library was introduced into Nicotiana benthamiana by agroinfiltration. The CP sequence of the TYMV RNA isolated from inoculated leaves was amplified by reverse transcription-PCR and then used to construct a second library. This process was repeated one more time, and the CP sequences of the TYMV RNA in the inoculated leaves were analyzed. Based on the analysis of over 11,000 CP sequences, the Cys mutants representing most abundant TYMV RNAs were constructed. Analysis of the mutants showed that four Cys mutants were nearly comparable to wildtype with respect to CP and viral RNA levels in N. benthamiana. All these mutants were highly reactive to fluoresceine-5-maleimide. This demonstrates that TYMV can be modified to have additional functional groups on the surface that would be useful for drug delivery.

Replication of Recombinant Flock House Virus RNA Encapsidated by Turnip Yellow Mosaic Virus Coat Proteins in Nicotiana benthamiana

It was previously observed that recombinant flock house virus (FHV) RNA1 was efficiently packaged into turnip yellow mosaic virus (TYMV), provided that the TYMV coat protein (CP) sequence was present at the 3′-end. FHV RNA encapsidated by TYMV CPs also had a four-nucleotide extension at the 5′-end. Since even a short extension at the 5′- and 3′-ends of FHV RNA1 inhibits replication, we examined whether the recombinant FHV RNA is indeed capable of replication. To this end, we introduced constructs expressing recombinant FHV RNAs into the plant Nicotiana benthamiana. Northern blot analysis of inoculated leaves suggested abundant production of recombinant FHV RNA1 and its subgenomic RNA. This demonstrated that recombinant FHV RNA with terminal extensions at both ends was competent for replication. We also showed that the recombinant FHV RNA can express the reporter gene encoding enhanced green fluorescent protein.

N-terminal Extension of Coat Protein of Turnip Yellow Mosaic Virus has Variable Effects on Replication, RNA Packaging, and Virion Assembly Depending on the Inserted Sequence

Turnip yellow mosaic virus (TYMV) is a non-enveloped icosahedral virus composed of 20 kDa single coat proteins. In this study, we modified the TYMV coat protein (CP) ORF by inserting an oligonucleotide linker corresponding to T7, HSV, Tat, (Arg)9, or (RxR)4 peptide at the 5'-end of the CP ORF and examined its effect on replication, RNA packaging, and virion assembly. The results showed that the constructs containing (Arg)9 and (RxR)4 sequences were barely capable of replication. The TYMV constructs containing T7 and Tat peptide produced virions that co-migrated with wild-type virions. However, the insertion of T7 and Tat sequences impaired genomic RNA (gRNA) accumulation and packaging, respectively. When only the CP gene was expressed, CPs with (Arg)9 or (RxR)4 successfully produced virus-like particles whose mobility was comparable to that of wild type. In the case of CP having a HSV tag, the virion band was not detected, although a sufficient amount of CP was produced. This indicates that CP with the HSV tag failed to assemble into virions. Overall, the results suggest that TYMV replication, RNA packaging and virion assembly are strongly influenced by the insertion sequence.