Deactivation of the antiviral state by rabies virus through targeting and accumulation of persistently phosphorylated STAT1.

Antagonism of the interferon (IFN)-mediated antiviral state is critical to infection by rabies virus (RABV) and other viruses, and involves interference in the IFN induction and signaling pathways in infected cells, as well as deactivation of the antiviral state in cells previously activated by IFN. The latter is required for viral spread in the host, but the precise mechanisms involved and roles in RABV pathogenesis are poorly defined. Here, we examined the capacity of attenuated and pathogenic strains of RABV that differ only in the IFN-antagonist P protein to overcome an established antiviral state. Importantly, P protein selectively targets IFN-activated phosphorylated STAT1 (pY-STAT1), providing a molecular tool to elucidate specific roles of pY-STAT1. We find that the extended antiviral state is dependent on a low level of pY-STAT1 that appears to persist at a steady state through ongoing phosphorylation/dephosphorylation cycles, following an initial IFN-induced peak. P protein of pathogenic RABV binds and progressively accumulates pY-STAT1 in inactive cytoplasmic complexes, enabling recovery of efficient viral replication over time. Thus, P protein-pY-STAT1 interaction contributes to 'disarming' of the antiviral state. P protein of the attenuated RABV is defective in this respect, such that replication remains suppressed over extended periods in cells pre-activated by IFN. These data provide new insights into the nature of the antiviral state, indicating key roles for residual pY-STAT1 signaling. They also elucidate mechanisms of viral deactivation of antiviral responses, including specialized functions of P protein in selective targeting and accumulation of pY-STAT1.

Modulation of acyl-carnitines, the broad mechanism behind Wolbachia-mediated inhibition of medically important flaviviruses in Aedes aegypti.

Wolbachia-infected mosquitoes are refractory to flavivirus infections, but the role of lipids in Wolbachia-mediated virus blocking remains to be elucidated. Here, we use liquid chromatography mass spectrometry to provide a comprehensive picture of the lipidome of Aedes aegypti (Aag2) cells infected with Wolbachia only, either dengue or Zika virus only, and Wolbachia-infected Aag2 cells superinfected with either dengue or Zika virus. This approach identifies a class of lipids, acyl-carnitines, as being down-regulated during Wolbachia infection. Furthermore, treatment with an acyl-carnitine inhibitor assigns a crucial role for acyl-carnitines in the replication of dengue and Zika viruses. In contrast, depletion of acyl-carnitines increases Wolbachia density while addition of commercially available acyl-carnitines impairs Wolbachia production. Finally, we show an increase in flavivirus infection of Wolbachia-infected cells with the addition of acyl-carnitines. This study uncovers a previously unknown role for acyl-carnitines in this tripartite interaction that suggests an important and broad mechanism that underpins Wolbachia-mediated pathogen blocking.

Attenuation of a dengue virus replicon by codon deoptimization of nonstructural genes.

The overwhelming increase of dengue virus (DENV) infections in recent years shows that current strategies to combat dengue do not work. The lack of a highly effective dengue vaccine and the limited effectivity of vector controls exacerbate this situation. To point the way to a novel method of creating DENV vaccine candidates, here we disrupted the codon usage in a DENV-2 reporter replicon to generate variants with different replication characteristics. Six different mutated constructs containing stretches of altered codon usage in the non-structural genes were generated. The mutated sequences were deoptimized to the least favorable codons for human cells. We studied the replication efficiency of these constructs by measuring luciferase reporter activity, relative RNA fold change, and NS1 secretion. Our findings showed that the level of virus attenuation is closely associated with the amount of codon deoptimization. Indeed, replication was completely abolished in extensively-deoptimized constructs D2Rep-6 and D2Rep-5, intermediate with constructs D2Rep-4 (771 bp silent mutations) and D2Rep-3 (756 bp silent mutations) and restored almost to wildtype levels with constructs D2Rep-2 (394 silent mutations) and D2Rep-1 (48 silent mutations). We also determined that the position of codon deoptimization within the genome is crucial to the degree of attenuation observed. Based on our analysis, we propose that the design for an ideal DENV vaccine candidate could include 700-1500 silent mutations within the NS2A and NS3 genes. Our results suggest that codon deoptimization is an ideal strategy that can readily be used to develop a DENV vaccine candidate with "fine-tuned" attenuation.

Dengue subgenomic flaviviral RNA disrupts immunity in mosquito salivary glands to increase virus transmission.

Globally re-emerging dengue viruses are transmitted from human-to-human by Aedes mosquitoes. While viral determinants of human pathogenicity have been defined, there is a lack of knowledge of how dengue viruses influence mosquito transmission. Identification of viral determinants of transmission can help identify isolates with high epidemiological potential. Additionally, mechanistic understanding of transmission will lead to better understanding of how dengue viruses harness evolution to cycle between the two hosts. Here, we identified viral determinants of transmission and characterized mechanisms that enhance production of infectious saliva by inhibiting immunity specifically in salivary glands. Combining oral infection of Aedes aegypti mosquitoes and reverse genetics, we identified two 3' UTR substitutions in epidemic isolates that increased subgenomic flaviviral RNA (sfRNA) quantity, infectious particles in salivary glands and infection rate of saliva, which represents a measure of transmission. We also demonstrated that various 3'UTR modifications similarly affect sfRNA quantity in both whole mosquitoes and human cells, suggesting a shared determinism of sfRNA quantity. Furthermore, higher relative quantity of sfRNA in salivary glands compared to midgut and carcass pointed to sfRNA function in salivary glands. We showed that the Toll innate immune response was preferentially inhibited in salivary glands by viruses with the 3'UTR substitutions associated to high epidemiological fitness and high sfRNA quantity, pointing to a mechanism for higher saliva infection rate. By determining that sfRNA is an immune suppressor in a tissue relevant to mosquito transmission, we propose that 3'UTR/sfRNA sequence evolution shapes dengue epidemiology not only by influencing human pathogenicity but also by increasing mosquito transmission, thereby revealing a viral determinant of epidemiological fitness that is shared between the two hosts.

Dengue subgenomic RNA binds TRIM25 to inhibit interferon expression for epidemiological fitness.

The global spread of dengue virus (DENV) infections has increased viral genetic diversity, some of which appears associated with greater epidemic potential. The mechanisms governing viral fitness in epidemiological settings, however, remain poorly defined. We identified a determinant of fitness in a foreign dominant (PR-2B) DENV serotype 2 (DENV-2) clade, which emerged during the 1994 epidemic in Puerto Rico and replaced an endemic (PR-1) DENV-2 clade. The PR-2B DENV-2 produced increased levels of subgenomic flavivirus RNA (sfRNA) relative to genomic RNA during replication. PR-2B sfRNA showed sequence-dependent binding to and prevention of tripartite motif 25 (TRIM25) deubiquitylation, which is critical for sustained and amplified retinoic acid-inducible gene 1 (RIG-I)-induced type I interferon expression. Our findings demonstrate a distinctive viral RNA-host protein interaction to evade the innate immune response for increased epidemiological fitness.

Detection of porcine circovirus type 2 in pigs imported from Indonesia.

We have detected the presence of porcine circovirus (PCV) type 2 in Indonesian pigs imported to Singapore for food consumption. A total of three viral isolates were identified, and to genetically characterise them further, their full genomes were sequenced. Each genome showed a typical organization of PCV type 2, with the three isolates sharing similar genome lengths of 1767 nucleotide (nt) at high nt identities of 99.8-100%, further indicating that the viral isolates were quite homogeneous. Sequence analysis further revealed that the ORF2 genes contain the nt sequence CCCCGC (from nt position 262 to 267) that was previously reported to be associated with PCV type 2, group 1C. The phylogenetic tree was constructed for the ORF2 genes, and the PCV type 2 isolates distributed into two distinctive groups. The Indonesian PCV type 2 clustered tightly with one China isolate, accession number AY035820, as a sub-cluster in group 1C. The sequence and phylogenetic analyses both confirmed that the three Indonesian PCV type 2 isolates belong to group 1C, and that the genetic changes for the three Indonesian isolates were very stable, possibly due to the low-scale evolution.