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One inevitable consequence of aging is the gradual deterioration of physical function and exercise capacity, driven in part by the adverse effect of age on muscle tissue. We hypothesized that relationships exist between age-related differentially expressed genes (DEGs) in skeletal muscle and age-associated declines in physical function and exercise capacity. Previously, male C57BL/6mice (6m, months old, 24m, and 28m) were tested for physical function using a composite scoring system (comprehensive functional assessment battery, CFAB) comprised of five well-validated tests of physical function. In this study, total RNA was isolated from tibialis anterior samples (n = 8) randomly selected from each age group in the parent study. Using Next Generation Sequencing RNAseq to determine DEGs during aging (6m vs. 28m, and 6m vs. 24m), we found a greater than five-fold increase in DEGs in 28m compared to the 24m. Furthermore, regression of the normalized expression of each DEG with the CFAB score of the corresponding mouse revealed many more DEGs strongly associated (R ≥ |0.70|) with functional status in the older mice. Gene ontology results indicate highly enriched axon guidance and acetyl choline receptor gene sets, suggesting that denervation/reinnervation flux might potentially play a critical role in functional decline. We conclude that specific age-related DEG patterns are associated with declines in physical function, and the data suggest accelerated aging occurring between 24 and 28 months.

Abstract Japanese encephalitis virus (JEV) is the etiological agent of Japanese encephalitis (JE). The most commonly used vaccine used to prevent JE is the live-attenuated strain SA14-14-2, which was generated by serial passage of the wild-type (WT) JEV strain SA14. Two other vaccine candidates, SA14-5-3 and SA14-2-8 were derived from SA14. Both were shown to be attenuated but lacked sufficient immunogenicity to be considered effective vaccines. To better contrast the SA14-14-2 vaccine with its less-immunogenic counterparts, genetic diversity, ribavirin sensitivity, mouse virulence and mouse immunogenicity of the three vaccines were investigated. Next generation sequencing demonstrated that SA14-14-2 was significantly more diverse than both SA14-5-3 and SA14-2-8, and was slightly less diverse than WT SA14. Notably, WT SA14 had unpredictable levels of diversity across its genome whereas SA14-14-2 is highly diverse, but genetic diversity is not random, rather the virus only tolerates variability at certain residues. Using Ribavirin sensitivity in vitro, it was found that SA14-14-2 has a lower fidelity replication complex compared to SA14-5-3 and SA14-2-8. Mouse virulence studies showed that SA14-2-8 was the most virulent of the three vaccine strains while SA14-14-2 had the most favorable combination of safety (virulence) and immunogenicity for all vaccines tested. SA14-14-2 contains genetic diversity and sensitivity to the antiviral Ribavirin similar to WT parent SA14, and this genetic diversity likely explains the (1) differences in genomic sequences reported for SA14-14-2 and (2) the encoding of major attenuation determinants by the viral E protein.

The genetic diversities of mammalian tick-borne flaviviruses are poorly understood. We used next-generation sequencing (NGS) to deep sequence different viruses and strains belonging to this group of flaviviruses, including Central European tick-borne encephalitis virus (TBEV-Eur), Far Eastern TBEV (TBEV-FE), Langat (LGTV), Powassan (POWV), Deer Tick (DTV), Kyasanur Forest Disease (KFDV), Alkhurma hemorrhagic fever (AHFV), and Omsk hemorrhagic fever (OHFV) viruses. DTV, AHFV, and KFDV had the lowest genetic diversity, while POWV strains LEIV-5530 and LB, OHFV, TBEV-Eur, and TBEV-FE had higher genetic diversities. These findings are compatible with the phylogenetic relationships between the viruses. For DTV and POWV, the amount of genetic diversity could be explained by the number of tick vector species and amplification hosts each virus can occupy, with low diversity DTV having a more limited vector and host pool, while POWV with higher genetic diversities has been isolated from different tick species and mammals. It is speculated that high genetic diversity may contribute to the survival of the virus as it encounters these different environments.

The disease yellow fever was prevented by two live attenuated vaccines, strains 17D and French neurotropic vaccine (FNV), derived by serial passage of wild-type (WT) strains Asibi and French Viscerotropic virus (FVV), respectively. Both 17D and FNV displayed decreased genetic diversity and resistance to the antiviral Ribavirin compared to their WT parental strains, which are thought to contribute to their attenuated phenotypes. Subsequent studies found that only a few passages of WT strain FVV in HeLa cells resulted in an attenuated virus. In the current study, the genome sequence of FVV following five passages in HeLa cells (FVV HeLa p5) was determined through Next Generation Sequencing (NGS) with the aim to investigate the molecular basis of viral attenuation. It was found that WT FVV and FVV HeLa p5 virus differed by five amino acid substitutions: E-D155A, E-K331R, E-I412V, NS2A-T105A, and NS4B-V98I. Surprisingly, the genetic diversity and Ribavirin resistance of the FVV HeLa p5 virus were not statistically different to WT parent FVV. These findings suggest that while FVV HeLa p5 is attenuated, this is not dependent on a high-fidelity replication complex, characterized by reduced genetic diversity or increased Ribavirin stability, as seen with FNV and 17D vaccines.

The yellow fever virus vaccine, 17D, was derived through the serial passage of the wild-type (WT) strain Asibi virus in mouse and chicken tissue. Since its derivation, the mechanism of attenuation of 17D virus has been investigated using three 17D substrains and WT Asibi virus. Although all three substrains of 17D have been sequenced, only one isolate of Asibi has been examined genetically and all interpretation of attenuation is based on this one isolate. Here, we sequenced the genome of Asibi virus from three different laboratories and show that the WT strain is genetically homogenous at the amino acids that distinguish Asibi from 17D vaccine virus.

ABSTRACT The molecular basis of attenuation for live-attenuated vaccines is poorly understood. The yellow fever (YF) 17D vaccine virus was derived from the wild-type, parental strain Asibi virus by serial passage in chicken tissue and has proven to be a very safe and efficacious vaccine. We have previously shown that wild-type Asibi is a typical RNA virus with high genetic diversity, while the 17D vaccine virus has very little genetic diversity. To investigate this further, we treated Asibi and 17D viruses with ribavirin, a GTP analog with strong antiviral activity that increases levels of mutations in the viral genome. As expected, ribavirin treatment introduced mutations into the Asibi virus genome at a very high frequency and decreased viral infectivity while, in contrast, the 17D vaccine virus was resistant to ribavirin, as treatment with the antiviral introduced very few mutations into the genome, and viral infectivity was not lost. The results were confirmed for another YF wild-type parental and vaccine pair, a wild-type French viscerotropic virus and French neurotropic vaccine. Using recombinant Asibi and 17D viruses, ribavirin sensitivity was located to viral nonstructural genes. Thus, two live-attenuated YF vaccine viruses are genetically stable even under intense mutagenic pressure, suggesting that attenuation of live-attenuated YF vaccines is due, at least in part, to fidelity of the replication complex resulting in high genetic stability. IMPORTANCE Live-attenuated viral vaccines are highly safe and efficacious but represent complex and often multigenic attenuation mechanisms. Most of these vaccines have been generated empirically by serial passaging of a wild-type (WT) virus in cell culture. One of the safest and most effective live-attenuated vaccines is yellow fever (YF) virus strain 17D, which has been used for over 80 years to control YF disease. The availability of the WT parental strain of 17D, Asibi virus, and large quantities of clinical data showing the effectiveness of the 17D vaccine make this WT parent/vaccine pair an excellent model for investigating RNA virus attenuation. Here, we investigate a mechanism of 17D attenuation and show that the vaccine virus is resistant to the antiviral compound ribavirin. The findings suggest that attenuation is in part due to a low probability of reversion or mutation of the vaccine virus genome to WT, thus maintaining a stable genotype despite external pressures.