Human Adenovirus Type 55 strains

Human adenoviruses (AdV) are mostly associated with minimal pathology. However, more severe respiratory tract infections and acute respiratory diseases, most often caused by AdV-4 and AdV-7, have been reported. The only licensed vaccine in the United States, live oral AdV-4 and AdV-7 vaccine, is indicated for use in the military, nearly exclusively in recruit populations. The excellent safety profile and prominent antibody response of the vaccine is well established by placebo-controlled clinical trials, while, long-term immunity of vaccination has not been studied. Serum samples collected over 6 years from subjects co-administered live oral AdV-4 and AdV-7 vaccine in 2011 were evaluated to determine the duration of the antibody response. Group geometric mean titers (GMT) at 6 years post vaccination compared to previous years evaluated were not significantly different for either AdV-4 or AdV-7 vaccine components. There were no subjects that demonstrated waning neutralization antibody (NAb) titers against AdV-4 and less than 5% of subjects against AdV-7. Interestingly, there were subjects that had a four-fold increase in NAb titers against either AdV-4 or AdV-7, at various time points post vaccination, suggesting either homotypic or heterotypic re-exposure. This investigation provided strong evidence that the live oral AdV-4 and AdV-7 vaccine induced long-term immunity to protect from AdV-4 and AdV-7 infections.

With the advent of advanced sequencing technology, studies of RNA viruses have shown that genetic diversity can contribute to both attenuation and virulence and the paradigm is that this is controlled by the error-prone RNA-dependent RNA polymerase (RdRp). Since wild-type yellow fever virus (YFV) strain Asibi has genetic diversity typical of a wild-type RNA virus, while 17D virus vaccine has limited diversity, it provides a unique opportunity to investigate RNA population theory in the context of a well-characterized live attenuated vaccine. Utilizing infectious clone-derived viruses, we show that genetic diversity of RNA viruses is complex and that multiple genes, including structural genes and NS2B and NS4B genes also contribute to genetic diversity. We suggest that the replication complex as a whole, rather than only RdRp, drives genetic diversity, at least for YFV.

Zika virus (ZIKV) strains belong to the East African, West African, and Asian/American phylogenetic lineages. RNA viruses, like ZIKV, exist as populations of genetically-related sequences whose heterogeneity may impact viral fitness, evolution, and virulence. Genetic diversity of representative ZIKVs from each lineage was examined using next generation sequencing (NGS) paired with downstream entropy and single nucleotide variant (SNV) analysis. Comparisons showed that inter-lineage diversity was statistically supported, while intra-lineage diversity varied. Intra-lineage diversity was significant for East but not West Africa strains. Furthermore, intra-lineage diversity for the Asian/American lineage was not supported for human serum isolates; however, a placenta isolate differed significantly. Relative entropy values were higher in the pre-membrane/membrane (prM/M) gene of several ZIKV strains. Additionally, the East African lineage contained a greater number of synonymous SNVs, while a greater number of non-synonymous SNVs were identified for American strains. Further, inter-lineage SNVs were dispersed throughout the genome, whereas intra-lineage non-synonymous SNVs for Asian/American strains clustered within prM/M and NS1 gene. This comprehensive analysis of ZIKV genetic diversity provides a repository of SNV positions across lineages. We posit that increased non-synonymous SNV populations and increased relative genetic diversity of the prM/M and NS1 proteins provides more evidence for their role in ZIKV virulence and fitness. GRAPHICAL ABSTRACT

Zika virus (ZIKV) is a mosquito-borne Flavivirus. Previous studies have shown that mosquito-transmitted flaviviruses, including yellow fever, Japanese encephalitis, and West Nile viruses, could be attenuated by serial passaging in human HeLa cells. Therefore, it was hypothesized that wild-type ZIKV would also be attenuated after HeLa cell passaging. A human isolate from the recent ZIKV epidemic was subjected to serial HeLa cell passaging, resulting in attenuated in vitro replication in both Vero and A549 cells. Additionally, infection of AG129 mice with 10 plaque forming units (pfu) of wild-type ZIKV led to viremia and mortality at 12 days, whereas infection with 10³ pfu of HeLa-passage 6 (P6) ZIKV led to lower viremia, significant delay in mortality (median survival: 23 days), and increased cytokine and chemokine responses. Genomic sequencing of HeLa-passaged virus identified two amino acid substitutions as early as HeLa-P3: pre-membrane E87K and nonstructural protein 1 R103K. Furthermore, both substitutions were present in virus harvested from HeLa-P6-infected animal tissue. Together, these data show that, similarly to other mosquito-borne flaviviruses, ZIKV is attenuated following passaging in HeLa cells. This strategy can be used to improve understanding of substitutions that contribute to attenuation of ZIKV and be applied to vaccine development across multiple platforms.