The protein synthesis machineries of two distinct phyla of the Animal kingdom, insects of Arthropoda and mammals of Chordata, have different preferences for how to best encode proteins. Nevertheless, arboviruses (arthropod-borne viruses) are capable of infecting both mammals and insects just like arboviruses that use insect vectors to infect plants. These organisms have evolved carefully balanced genomes that can efficiently use the translational machineries of different phyla, even if the phyla belong to different kingdoms. Using dengue virus as an example, we have undone the genome encoding balance and specifically shifted the encoding preference away from mammals. These mammalian-attenuated viruses grow to high titers in insect cells but low titers in mammalian cells, have dramatically increased LD50s in newborn mice, and induce high levels of protective antibodies. Recoded arboviruses with a bias toward phylum-specific expression could form the basis of a new generation of live attenuated vaccine candidates.

Computer design and chemical synthesis generated viable variants of poliovirus type 1 (PV1), whose ORF (6,189 nucleotides) carried up to 1,297 "Max" mutations (excess of overrepresented synonymous codon pairs) or up to 2,104 "SD" mutations (randomly scrambled synonymous codons). "Min" variants (excess of underrepresented synonymous codon pairs) are nonviable except for P2 Min , a variant temperature-sensitive at 33 and 39.5 • C. Compared with WT PV1, P2 Min displayed a vastly reduced specific infectivity (si) (WT, 1 PFU/118 particles vs. P2 Min , 1 PFU/35,000 particles), a phenotype that will be discussed broadly. Si of haploid PV presents cellular infectivity of a single genotype. We performed a comprehensive analysis of sequence and structures of the PV genome to determine if evolutionary conserved cis-acting packaging signal(s) were preserved after recoding. We showed that conserved synonymous sites and/or local secondary structures that might play a role in determining packaging specificity do not survive codon pair recoding. This makes it unlikely that numerous "cryptic, poliovirus | genome recoding | packaging signal | specific infectivity T he capsid precursor P1 (881 amino acids) of type 1 poliovirus (PV), mapping to the N terminus of the polyprotein (PP) (Fig. 1A), can be encoded in 10 442 ways (1) due to the degenerate genetic code. The tiniest fraction of these possible sequences defines PV, the cause of poliomyelitis. PV occurs in three serotypes, of which the most neurovirulent type 1 Mahoney [PV1(M)], the main viral species analyzed in this study, was isolated in 1941 from pooled feces of three healthy children (2).Genome sequence (3, 4) and gene organization (3) of PV1(M) revealed highly complex structures in its 5'-terminal nontranslated region (5'-NTR), followed by a single ORF encoding the PP, followed by a complex 3'-heteropolymeric region and poly(A) tail (Fig. 1A) (5, 6). The PP (7) is an active molecule that cleaves itself into ∼29 polypeptides by two viral proteinases (2A pro and 3C pro /3CD pro ) and an enzyme-independent maturation cleavage (Fig. 1A) (5,6,8).Capsid domain P1 controls the identity of PV by determining virion structure (9), serotype identity (10), and interaction with the cellular receptor CD155 (10). Since PV replicates as quasispecies at an error rate of ∼10 −4 (11), the following questions arise: How conserved is its synonymous sequence given the astronomical number of alternative possibilities? What encoding could have coevolved that would be optimal to specify 881 capsid residues?If PV, a member of the genus Enterovirus of Picornaviridae, is an evolutionary descendant of C-cluster Coxsackie viruses (C-CAVs) (12), the evolution of PV nucleotide sequences was constrained as it adhered to the basic architecture of C-CAVs, its evolutionary parents (13). A second well-known restriction of sequence variability in ORFs is "codon bias" (14), the unequal use of synonymous codons. Encoding the PV1 P1 domain with an excess of "rarely used" (human) synonymous codons results in a ...

No abstract

Dengue virus (DENV), an arthropod-borne (“arbovirus”) virus, causes a range of human maladies ranging from self-limiting dengue fever to the life-threatening dengue shock syndrome and proliferates well in two different taxa of the Animal Kingdom, mosquitoes and primates. Mosquitoes and primates show taxonomic group-specific intolerance to certain codon pairs when expressing their genes by translation. This is called “codon pair bias”. By necessity, dengue viruses evolved to delicately balance this fundamental difference in their open reading frames (ORFs). We have undone the evolutionarily conserved genomic balance in the DENV2 ORF sequence and specifically shifted the encoding preference away from primates. However, this recoding of DENV2 raised concerns of ‘gain-of-function,’ namely whether recoding could inadvertently increase fitness for replication in the arthropod vector. Using mosquito cell lines and two strains of Aedes aegypti we did not observe any increase in fitness in DENV2 variants codon pair deoptimized for humans. This ability to disrupt and control DENV2’s host preference has great promise towards developing the next generation of synthetic vaccines not only for DENV but for other emerging arboviral pathogens such as chikungunya virus and Zika virus.

Although murine γδ T cells are largely considered innate immune cells, they have recently been reported to form long-lived memory populations. Much remains unknown about the biology and specificity of memory γδ T cells. Here, we interrogated intestinal memory Vγ4Vδ1 T cells generated after foodborne Listeria monocytogenes (Lm) infection to uncover an unanticipated complexity in the specificity of these cells. Deep TCR sequencing revealed that a subset of non-canonical Vδ1 clones are selected by Lm infection, consistent with antigen-specific clonal expansion. Ex vivo stimulations and in vivo heterologous challenge infections with diverse pathogenic bacteria revealed that Lm -elicited memory Vγ4Vδ1 T cells are broadly reactive. The Vγ4Vδ1 T cell recall response to Lm, Salmonella enterica serovar Typhimurium (STm) and Citrobacter rodentium was largely mediated by the γδTCR as internalizing the γδTCR prevented T cell expansion. Both broadly-reactive canonical and pathogen-selected non-canonical Vδ1 clones contributed to memory responses to Lm and STm. Interestingly, some non-canonical γδ T cell clones selected by Lm infection also responded after S Tm infection, suggesting some level of cross-reactivity. These findings underscore the promiscuous nature of memory γδ T cells and suggest that pathogen-elicited memory γδ T cells are potential targets for broad-spectrum anti-infective vaccines.

No abstract

The sequence of Zika virus has evolved as it has spread out of Africa and into the Americas. It is unclear whether American strains of the virus define a new serotype. Here, we have tested the virulence and immunogenicity of three wild-type ZIKV strains in neonatal Swiss Webster mice. We found that all three ZIKV strains (African MR766, 1947; Asian FSS13025, 2010; and American, PRVABC59, 2015) are capable of killing neonatal mice after intracranial injection. Intraperitoneal injection with these viruses did not kill, but produced neutralizing antibodies as measured by a PRNT 50 assay. Sera from mice infected with each virus were tested for neutralizing activity against the infecting virus and also the other two viruses by a PRNT 50 assay. In general, the antibodies induced by each virus were good at neutralizing that virus (the homologous virus), but somewhat poorer at neutralizing the other two viruses (heterologous viruses). Antibodies induced by the African strain MR766 were about 4-fold worse at neutralizing the American strain PRVABC59 than the homologous strain, while antibodies induced by the American strain were about 10-fold worse at neutralizing the African strain than the homologous strain. Because the antibodies are cross-neutralizing at some level, the viruses do not form separate serotypes. Nevertheless, these results raise concern that the immunity conferred by the African virus may protect only relatively poorly against the new American strains. This has implications for the possible spread of the American ZIKV strains to Africa and Southeast Asia, and also for the development of vaccines.

Abstract18 Dengue virus (DENV), an arthropod-borne ("arbovirus") virus causing a range of 19 human maladies ranging from self-limiting dengue fever to the life-threatening 20 dengue shock syndrome, proliferates well in two different taxa of the Animal 21 Kingdom, mosquitoes and primates. Unexpectedly, mosquitoes and primates have 22 distinct preferences when expressing their genes by translation, e.g. members of 23 these taxa show taxonomic group-specific intolerance to certain codon pairs. This is 24 called "codon pair bias". By necessity, arboviruses evolved to delicately balance this 25 fundamental difference in their ORFs. Using the mosquito-borne human pathogen 26 DENV we have undone the evolutionarily conserved genomic balance in its ORF 27 sequence and specifically shifted the encoding preference away from primates.28 However, this recoding of DENV raised concerns of 'gain-of-function,' namely 29 whether recoding could inadvertently increase fitness for replication in the 30 arthropod vector. Using mosquito cell cultures and two strains of Aedes aegypti we 31 did not observe any increase in fitness in DENV2 variants codon pair deoptimized . CC-BY 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.