Transcriptomic analyses reveal differential gene expression of immune and cell death pathways in the brains of mice infected with West Nile virus and chikungunya virus

West Nile virus (WNV) and chikungunya virus (CHIKV) are arboviruses that are constantly (re-)emerging and expanding their territory. Both viruses often cause a mild form of disease, but severe forms of the disease can consist of neurological symptoms, most often observed in the elderly and young children, respectively, for which the mechanisms are poorly understood. To further elucidate the mechanisms responsible for end-stage WNV and CHIKV neuroinvasive disease, we used transcriptomics to compare the induction of effector pathways in the brain during the early and late stage of disease in young mice. In addition to the more commonly described cell death pathways such as apoptosis and autophagy, we also found evidence for the differential expression of pyroptosis and necroptosis cell death markers during both WNV and CHIKV neuroinvasive disease. In contrast, no evidence of cell dysfunction was observed, indicating that cell death may be the most important mechanism of disease. Interestingly, there was overlap when comparing immune markers involved in neuroinvasive disease to those seen in neurodegenerative diseases. Nonetheless, further validation studies are needed to determine the activation and involvement of these effector pathways at the end stage of disease. Furthermore, evidence for a strong inflammatory response was found in mice infected with WNV and CHIKV. The transcriptomics profile measured in mice with WNV and CHIKV neuroinvasive disease in our study showed strong overlap with the mRNA profile described in the literature for other viral neuroinvasive diseases. More studies are warranted to decipher the role of cell inflammation and cell death in viral neuroinvasive disease and whether common mechanisms are active in both neurodegenerative and brain infectious diseases

Micro-States and Denuclearization in the Pacific Region

Ferrets are widely used as a small animal model for a number of viral infections, including influenza A virus and SARS coronavirus. To further analyze the microbiological status of ferrets, their fecal viral flora was studied using a metagenomics approach. Novel viruses from the families Picorna-, Papilloma-, and Anelloviridae as well as known viruses from the families Astro-, Corona-, Parvo-, and Hepeviridae were identified in different ferret cohorts. Ferret kobu- and hepatitis E virus were mainly present in human household ferrets, whereas coronaviruses were found both in household as well as farm ferrets. Our studies illuminate the viral diversity found in ferrets and provide tools to prescreen for newly identified viruses that potentially could influence disease outcome of experimental virus infections in ferrets

Pairwise amino acid identity in the P1, P2, and P3 regions between ferret kobuvirus 38 (MpKoV38; KF006985) and bovine kobuvirus (BKoV; AB084788), human aichivirus (AIV; FJ890523), porcine kobuvirus (PKoV; GU292559), and klassevirus (Klasse; GQ184145).

<p>Pairwise amino acid identity in the P1, P2, and P3 regions between ferret kobuvirus 38 (MpKoV38; KF006985) and bovine kobuvirus (BKoV; AB084788), human aichivirus (AIV; FJ890523), porcine kobuvirus (PKoV; GU292559), and klassevirus (Klasse; GQ184145).</p

Summary of mammalian viruses found in ferret fecal material.

a<p>Total no. of trimmed reads that were analyzed.</p>b<p>These animals had diarrhea.</p>c<p>NL, Netherlands; SE, Sweden.</p>d<p>FRCoV, ferret coronavirus; MpPV1, ferret papillomavirus; MpKoV, ferret kobuvirus; FRHEV, ferret hepevirus; MpPeV, ferret parechovirus; MpfTTV, ferret anellovirus.</p

Prevalence of ferret viruses.

<p>Percentage coronavirus (A), hepatitis E virus (B) and kobuvirus (<b>C</b>) positive farm versus household ferrets by real time PCR assay. Significant differences (unpaired t-test P<0.05) are indicated by an asterisk.</p

Phylogenetic analysis of ferret anellovirus.

<p>A phylogenetic tree of the partial ORF1 nucleotide sequence of ferret torque teno virus (MpfTTV1) and the corresponding region of representative human and animal anelloviruses was generated using MEGA5, with the neighbor-joining method with p-distance and 1,000 bootstrap replicates. Significant bootstrap values are shown. Genbank accession numbers are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071595#pone.0071595.s001" target="_blank">Table S1</a>.</p

Pairwise amino acid identity in the polyprotein P1, P2, and P3 regions between different parechovirus species.

<p>HPeV1 (JX575746), HPeV2 (AF055846), Ljungan (NC_003976), MpPeV1 (KF006989).</p

Genome organization and phylogenetic analysis of ferret parechovirus.

<p>(A) Predicted genome organization of ferret parechovirus showing amino acid positions of predicted cleavage sites in the polyprotein (numbering based on the ferret parechovirus polyprotein sequence). Sites were predicted by NetPicoRNA analysis and by alignment with other parechoviruses. (B) A phylogenetic tree of the polyprotein sequence of ferret parechovirus (MpPeV1) and representative human (HPeV1-8) and bank vole parechoviruses (Ljungan virus) was generated using MEGA5, with the neighbor-joining method with p-distance and 1,000 bootstrap replicates and human rhinovirus A 86 as an outgroup (HRV-A 86). Significant bootstrap values are shown. Genbank accession numbers are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071595#pone.0071595.s001" target="_blank">Table S1</a>.</p

Genome organization of ferret papillomavirus and nucleotide sequence divergence from other papillomaviruses.

<p>(A) Predicted genome organization of ferret papillomavirus with early (E) and late (L) genes indicated. (B) A phylogenetic tree of the complete ferret papillomavirus (MpPV1) genome and representative human and animal papillomaviruses was generated using MEGA5, with the maximum-likelihood method with Kimura-2 parameter and 1,000 bootstrap replicates. Significant bootstrap values are shown. (C) A phylogenetic tree of the L1 genome region of ferret papillomavirus (MpPV1) and representative human and animal papillomaviruses was generated using MEGA5, with the maximum-likelihood method with Kimura-2 parameter and 1,000 bootstrap replicates. Significant bootstrap values are shown. Genbank accession numbers are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071595#pone.0071595.s001" target="_blank">Table S1</a>.</p

Genome organization of ferret kobuvirus and amino acid sequence divergence from other aichiviruses.

<p>(A) Predicted genome organization of ferret kobuvirus showing amino acid positions of predicted cleavage sites in the polyprotein (numbering based on the ferret kobuvirus polyprotein sequence). Sites were predicted by NetPicoRNA analysis and by alignment with known cleavage sites in aichiviruses. (B) Mean similarity of bovine kobuvirus (AB084788) to ferret kobuvirus (red), porcine kobuvirus (GU292559, green), and human aichivirus (FJ890523; blue) polyprotein-coding nucleotide sequences scaled to the genome diagram in A. (C–E) Phylogenetic trees of the amino acid sequences of ferret kobuvirus (MpKoV32, 38, and 39) with other aichiviruses in the P1 (C), P2 (D), and P3 (E) gene regions were generated using MEGA5, with the neighbor-joining method with p-distance and 1,000 bootstrap replicates. Significant bootstrap values are shown.</p