NiV P gene products inhibit the expression of antiviral chemokines.

<p>(<b>A</b>) RNA extracted from HMVEC-L cells infected at MOI = 5 with individual mutant NiVs at 12 h post-infection was reverse-transcribed into cDNA and used in an antiviral real-time PCR array to measure transcriptional induction of CXCL10 and CCL5. Antiviral chemokine mRNA transcription over mock levels was calculated by the delta delta Ct method (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047790#s4" target="_blank">materials and methods</a>). Differential induction of (<b>B</b>) CXCL10 (gray bars) and (<b>C</b>) CCL5 (white bars) secretion by mutant NiVs. HMVEC-L cells were infected with individual mutant NiVs at MOI = 5 for 2 h before replacement of inoculum with fresh media. At 12 h (left panel), 24 h (center panel), and 48 h (right panel) PI, infected cell supernatants were collected and were subject to Luminex bead analysis to measure levels of CXCL10 and CCL5 induced by each mutant virus. Error bars indicate standard deviation of triplicate samples. ANOVA with Dunnett's multiple comparison test was used to measure the statistical significance of differences between levels of chemokine induced by each mutant compared with the WT virus. * p<0.05; ** p<0.01; *** p<0.001.</p

NiV P gene mRNA editing frequencies in HMVEC-L cells (24 h post-infection).

<p>NiV P gene mRNA editing frequencies in HMVEC-L cells (24 h post-infection).</p

Characterization of recombinant NiVs in HMVEC-L cells.

<p>(<b>A</b>) Comparative viral growth curves. HMVEC-L cells were infected with each indicated recombinant NiV in triplicate wells at an MOI = 2 for 2 h before inoculum was removed and replenished with fresh media. At the time points indicated, infected cell supernatants were serially diluted before being used to infect Vero cells to calculate Log₁₀TCID₅₀/mL. Error bars indicate one standard deviation of the mean of triplicate infections. ANOVA with Dunnett's multiple comparison test was used to measure the statistical significance of differences in peak viral titer at 48 hours PI when compared with WT recombinant virus. * p<0.001. (<b>B</b>) Western blot analysis of P gene products from infected HMVEC-L cell lysates. HMVEC-L cells were infected with each indicated recombinant NiV at an MOI = 2 for 2 h before inoculum was replaced with fresh media. At 24 h PI, infected cell lysates were harvested in NET-BSA buffer and run on a 4–12% Bis-Tris SDS gel. Proteins were transferred onto PVDF membranes, blocked in 5% milk with TBS-T, and probed with mouse antipeptide sera against indicated proteins. NiV C protein could not be detected.</p

Summary of NiV P gene mutation phenotypes in HMVEC-L cells relative to WT.

<p><b>+</b> increased compared to WT; <b>-</b> decreased compared to WT; <b> = </b>approximately equivalent to WT.</p

Multiple NiV P gene encoded elements contribute to limit the antiviral response.

<p>(<b>A</b>) NiV P gene encoded elements limit transcription of antiviral genes. HMVEC-L cells were infected with each recombinant NiV at an MOI = 5 and harvested at 12 h PI for total RNA extraction, reverse-transcription, and ensuing real-time PCR array. Levels of mRNA transcription of each antiviral gene indicated over mock were calculated by the delta delta Ct method (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047790#s4" target="_blank">materials and methods</a>). (<b>B</b>) <i>Left panel</i>: Levels of IFN-β transcription induced by recombinant NiVs at 12 h PI (MOI = 5). Total RNA was extracted and reverse-transcribed as per the above to be used in an antiviral real-time PCR array. Levels of mRNA transcription of IFN-β over mock were calculated by the delta delta Ct method (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047790#s4" target="_blank">materials and methods</a>). <i>Center panel</i>: NiV genome copy numbers from infected HMVEC-L cells at 12 h PI. Total RNA was extracted as mentioned above and reverse-transcribed using a primer against the 3′ leader sequence to only detect genome copies. Primers against the N gene were utilized to amplify viral genome. <i>Right panel</i>: NiV N gene mRNA copy numbers from infected HMVEC-L cells at 12 h post-infection. Total RNA was extracted as mentioned and reverse-transcribed using an oligo-dT primer to detect NiV N gene mRNA copies. Primers against the N gene were utilized to amplify N gene mRNA. (<b>C</b>) Induction of IFN-β secretion by mutant recombinant NiVs. HMVEC-L cells were infected with individual mutant NiVs at MOI = 5. At 12 h (left panel), 24 h (center panel), and 48 h (right panel) post-infection, infected cell supernatants were collected and were subject to IFN-β ELISA to measure levels of IFN-β induced by each mutant virus. Error bars for all panels indicate standard deviation of triplicate samples. ANOVA with Dunnett's multiple comparison test was used to measure the statistical significance of differences in viral replication and transcription, and IFN-β expression for each mutant compared with the WT virus. * p<0.05; ** p<0.01; *** p<0.001.</p

Nipah (NiV) P gene editing, and the establishment of a NiV reverse genetic system.

<p>(<b>A</b>) Schematic of Nipah (NiV) genome with the name of each gene indicated. Black segments represent non-coding regions of the virus genome, while the white regions indicate open reading frames (ORF). Inset magnifies the P gene and indicates the accessible ORFs in the NiV P gene due to mRNA editing (NiV V and W) and to a downstream alternative ORF (NiV C). Approximate location of the mRNA site is indicated by the black arrowhead. (<b>B</b>) Establishment of NiV reverse genetic system. Panel a: mock-infected Vero cells. Panel b: Wild-type recombinant NiV infection of Vero cells at 48 h post infection (PI) (MOI = 1). Panels c–d: light and fluorescence micrographs of Vero cells infected with recombinant red fluorescent (RED2AM) virus, 48 h PI (MOI = 1). Panels e–f: light and fluorescence micrographs of primary human microvascular lung endothelial cells (HMVEC-L) infected with RED2AM virus, 24 h PI (MOI = 2). (<b>C</b>) Generation of recombinant NiVs. Nucleotide changes (indicated by red arrowheads and letters) were incorporated into the NiV P gene to ablate expression of the C, V, and W ORFs, to alter the mRNA editing site, or to prevent STAT-1 binding in the shared N-terminal regions of the P, V, and W proteins. Numbers flanking the nucleotide sequences indicate plus-sense antigenomic position.</p

Characterization of recombinant NiVs in Vero cells.

<p>(<b>A</b>) Comparative viral growth curves. Vero cells were infected with each indicated recombinant NiV in triplicate wells at an MOI = 0.01. At the time points indicated, infected cell supernatants were serially diluted before being used to infect Vero cells to calculate log median tissue culture infectious dose per milliliter (Log₁₀ TCID₅₀/mL). Error bars indicate one standard deviation of the mean of triplicate infections. ANOVA with Dunnett's multiple comparison test was used to measure the statistical significance of differences in peak viral titer at 72 h PI when compared with WT recombinant virus. * p<0.001. (<b>B</b>) Western Blot analysis of Vero cell lysates infected with recombinant NiVs. Vero cells were infected at an MOI = 0.01. At ∼36 hours PI, infected cell lysates were harvested in NET-BSA buffer run on a 4–12% Bis-Tris SDS gel. Proteins were transferred onto PVDF membranes, blocked in 5% milk, and incubated with various antipeptide sera against NiV P gene products as indicated on the left hand side. Names of recombinant NiVs indicated above respective lanes. β-actin served as a loading control.</p

Wild-Type Measles Viruses with Non-Standard Genome Lengths

<div><p>The length of the single stranded, negative sense RNA genome of measles virus (MeV) is highly conserved at 15,894 nucleotides (nt). MeVs can be grouped into 24 genotypes based on the highly variable 450 nucleotides coding for the carboxyl-terminus of the nucleocapsid protein (N-450). Here, we report the genomic sequences of 2 wild-type viral isolates of genotype D4 with genome lengths of 15,900 nt. Both genomes had a 7 nt insertion in the 3′ untranslated region (UTR) of the matrix (M) gene and a 1 nt deletion in the 5′ UTR of the fusion (F) gene. The net gain of 6 nt complies with the rule-of-six required for replication competency of the genomes of morbilliviruses. The insertions and deletion (indels) were confirmed in a patient sample that was the source of one of the viral isolates. The positions of the indels were identical in both viral isolates, even though epidemiological data and the 3 nt differences in N-450 between the two genomes suggested that the viruses represented separate chains of transmission. Identical indels were found in the M-F intergenic regions of 14 additional genotype D4 viral isolates that were imported into the US during 2007–2010. Viral isolates with and without indels produced plaques of similar size and replicated efficiently in A549/hSLAM and Vero/hSLAM cells. This is the first report of wild-type MeVs with genome lengths other than 15,894 nt and demonstrates that the length of the M-F UTR of wild-type MeVs is flexible.</p></div

Detection of indels in the M-F UTR of viral isolates and patient samples.

a<p>Source of importation based on epidemiologic investigation, ^barbitrary outbreak numbers; samples with the same outbreak number are in the same chain of transmission, i: sequence from single amplicon spanning M-F intergenic region, nd: sequence not done, +: indicates insertion and deletion present,: -: neither insertion or deletion present, s: sequence derived from separate amplicons for M and F UTRs, af: amplification failed, bold sample ID: complete genome sequenced.</p

Location of insertions and deletions in the M-F UTR of MVi/New York.USA/26.09/3 and MVi/Florida.USA/19.09.

<p>A. Schematic representation of the MeV genome. The genome is depicted in the positive sense antigenomic orientation. Coding regions of each gene are in shades of grey, non-coding regions are white. Asterisk marks position of M-F intergenic trinucleotide at nucleotides 4872–4874. B. Insertion of 7 nucleotides in cytidine-rich region between nucleotides 4751–4775. C. Deletion of 1 nucleotide between nucleotides 5059–5072. The alignment was generated with MEGA5.10. Numbers under the alignment indicate nucleotide positions in aligned full-length genomes.</p