The emergence and spread of Zika virus (ZIKV) presented a challenge to the diagnosis of ZIKV infections in areas with transmission of dengue (DENV) and chikungunya (CHIKV) viruses. To facilitate detection of ZIKV infections, and differentiate these infections from DENV and CHIKV, we developed the Trioplex real-time RT-PCR assay (Trioplex assay). Here, we describe the optimization of multiplex and singleplex formats of the assay for a variety of chemistries and instruments to facilitate global standardization and implementation. We evaluated the analytical performance of all Trioplex modalities for detection of these three pathogens in serum and whole blood, and for ZIKV in urine. The limit of detection for the three viruses and in different RNA-extraction modalities is near 103 genome copy equivalents per milliliter (GCE/mL). Simultaneous testing of more than one specimen type from each patient provides a 6.4% additional diagnostic sensitivity. Overall, the high sensitivity of the Trioplex assay demonstrates the utility of this assay ascertaining Zika cases.

Different genotypes of avian paramyxovirus serotype-1 virus (APMV-1) circulate in many parts of the world. Traditionally, Newcastle disease virus (NDV) is recognized as having two major divisions represented by classes I and II, with class II being further divided into sixteen genotypes. Although all NDV are members of APMV-1 and are of one serotype, antigenic and genetic diversity is observed between the different genotypes. Reports of vaccine failure from many countries and reports by our lab on the reduced ability of classical vaccines to significantly decrease viral replication and shedding have created renewed interest in developing vaccines formulated with genotypes homologous to the virulent NDV (vNDV) circulating in the field. We assessed how the amount and specificity of humoral antibodies induced by inactivated vaccines affected viral replication, clinical protection and evaluated how non-homologous (heterologous) antibody levels induced by live NDV vaccines relate to transmission of vNDV. In an experimental setting, all inactivated NDV vaccines protected birds from morbidity and mortality, but higher and more specific levels of antibodies were required to significantly decrease viral replication. It was possible to significantly decrease viral replication and shedding with high levels of antibodies and those levels could be more easily reached with vaccines formulated with NDV of the same genotype as the challenge viruses. However, when the levels of heterologous antibodies were sufficiently high, it was possible to prevent transmission. As the level of humoral antibodies increase in vaccinated birds, the number of infected birds and the amount of vNDV shed decreased. Thus, in an experimental setting the effective levels of humoral antibodies could be increased by (1) increasing the homology of the vaccine to the challenge virus, or (2) allowing optimal time for the development of the immune response.

West Nile virus (WNV) recently became endemic in the United States and is a significant cause of human morbidity and mortality. Natural WNV strain infections do not induce stress granules (SGs), while W956IC (a lineage 2/1 chimeric WNV infectious clone) virus infections produce high levels of early viral RNA and efficiently induce SGs through protein kinase R (PKR) activation. Additional WNV chimeric viruses made by replacing one or more W956IC genes with the lineage 1 Eg101 equivalent in the W956IC backbone were analyzed. The Eg-NS4b؉5, Eg-NS1؉3؉4a, and Eg-NS1؉4b؉5 chimeras produced low levels of viral RNA at early times of infection and inefficiently induced SGs, suggesting the possibility that interactions between viral nonstructural proteins and/or between viral nonstructural proteins and cell proteins are involved in suppressing early viral RNA synthesis and membrane remodeling during natural WNV strain infections. Detection of exposed viral double-stranded RNA (dsRNA) in W956IC-infected cells suggested that the enhanced early viral RNA synthesis surpassed the available virus-induced membrane protection and allowed viral dsRNA to activate PKR.

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Oxidative stress activates the cellular kinase HRI, which then phosphorylates eIF2α, resulting in stalled translation initiation and the formation of stress granules (SGs). SG assembly redirects cellular translation to stress response mRNAs and inhibits cap-dependent viral RNA translation. Flavivirus infections were previously reported to induce oxidative stress in infected cells but flavivirus-infected cells paradoxically develop resistance to arsenite (Ars)-induced SG formation with time after infection. This resistance was previously postulated to be due to sequestration of the SG protein Caprin1 by Japanese encephalitis virus capsid protein. However, Caprin1 did not co-localize with West Nile virus (WNV) capsid protein in infected cells. Other stressors induced SGs with equal efficiency in mock- and WNV-infected cells indicating the intrinsic ability of cells to assemble SGs was not disabled. Induction of both reactive oxygen species (ROS) and the antioxidant response was detected at early times after WNV-infection. The transcription factors, Nrf2 and ATF4, which activate antioxidant genes, were upregulated and translocated to the nucleus. Knockdown of Nrf2, ATF4 or apoptosis-inducing factor (AIF), a mitochondrial protein involved in regenerating intracellular reduced glutathione (GSH) levels, with siRNA or treatment of cells with buthionine sulphoximine, which induces oxidative stress by inhibiting GSH synthesis, decreased intracellular GSH levels and increased the number of SG-positive, infected cells. Mitochondria were protected from Ars-induced damage by WNV infection until late times in the infection cycle. The results indicate that the increase in virus-induced ROS levels is counterbalanced by a virus-induced antioxidant response that is sufficient to also overcome the increase in ROS induced by Ars treatment and prevent Ars-induced SG assembly and mitochondrial damage. The virus-induced alterations in the cellular redox status appear to provide benefits for the virus during its lifecycle.

Oas1b was previously identified as the product of the Flv r allele that confers flavivirus-specific resistance to virus-induced disease in mice by an uncharacterized, RNase L-independent mechanism. To gain insights about the mechanism by which Oas1b specifically reduces the efficiency of flavivirus replication, cellular protein interaction partners were identified and their involvement in the Oas1b-mediated flavivirus resistance mechanism was analyzed. Initial difficulties in getting the two-hybrid assay to work with full-length Oas1b led to the discovery that this Oas protein uniquely has a C-terminal transmembrane domain that targets it to the endoplasmic reticulum (ER). Two peptides matching to oxysterol binding protein-related protein 1L (ORP1L) and ATP binding cassette protein 3, subfamily F (ABCF3), were identified as Oas1b interaction partners in yeast two-hybrid assays, and both in vitro-transcribed/translated peptides and full-length proteins in mammalian cell lysates coimmunoprecipitated with T he genus Flavivirus, in the family Flaviviridae, consists of ϳ70 viruses and includes human pathogens such as dengue virus, yellow fever virus, tick-borne encephalitis virus, Japanese encephalitis virus, and West Nile virus (WNV) (20). WNV was first isolated in Uganda in 1937 and was previously reported to be endemic in Africa, Australia, and southern Asia (3); it has recently emerged in the Americas, with over 23,000 human infections reported in the United States as of late 2006 (2, 39). WNV is arthropod borne, with a natural transmission cycle typically between Culex mosquito species and birds, with occasional virus transmission by mosquitoes to horses and humans (3). Usually, WNV infections in humans are asymptomatic or cause mild flu-like symptoms. However, some infections cause more severe disease with symptoms such as meningitis, encephalitis, or paralysis, which can be fatal (3).The 2=-5= oligoadenylate synthetase (OAS) pathway functions as an innate host defense response against viral infections. OAS gene expression is upregulated by the signaling of interferons produced by cells in response to a viral infection (34). Viral doublestranded RNA (dsRNA) binds to and activates OAS, causing it to polymerize ATP into short 2=-5=-linked oligomers (2-5A) (14). These 2-5A oligomers bind to and activate latent endoribonuclease L (RNase L,) which is constitutively expressed in cells. Activated RNase L cleaves viral and cellular single-stranded RNAs.Data from numerous studies indicate that both host factors and virus virulence factors determine the outcome of a virus infection. Genetically controlled resistance to flavivirus-induced central nervous system (CNS) disease in mice was first discovered in the 1920s and rediscovered several times in the 1930s because it was not appreciated that all of the viruses being tested belonged to the same virus genus and family (4). Breeding studies with mice displaying differential susceptibility to flavivirus-induced disease showed that the alleles of a single gene, Flv, control...

BackgroundIn mammals, interleukin 2 (IL-2) has been shown to decrease replication or attenuate pathogenicity of numerous viral pathogens (herpes simplex virus, vaccinia virus, human respiratory syncytial virus, human immunodeficiency virus) by activating natural killer cells (NK), cytotoxic T lymphocytes and expanding subsets of memory cells. In chickens, IL-2 has been shown to activate T cells, and as such it might have the potential to affect replication and pathogenesis of Newcastle disease virus (NDV).MethodsTo assess the effect of IL-2 during NDV infection in chickens, we produced a recombinant virulent NDV strain expressing chicken IL-2 (rZJ1-IL2). The effects of IL-2 expression were investigated in vivo using the intracerebral pathogenicity index (ICPI) in day-old chicks and pathogenesis experiments in 4-week-old chickens. In these studies, rZJ1-IL2 was compared to a control virus expressing the green fluorescent protein (rZJ1-GFP). Assessed parameters included survival curves, detailed histological and immunohistochemical grading of lesions in multiple organs, and virus isolation in blood, spleen and mucosal secretions of infected birds.ResultsAt the site of infection (eyelid), expression of IL-2 was demonstrated in areas of rZJ-IL2 replication, confirming IL-2 production in vivo. Compared to rZJ1-GFP strain, rZJ1-IL2 caused milder lesions and displayed decreased viral load in blood, spleen and mucosal secretions of infected birds. In the rZJ1-IL2-infected group, virus level in the blood peaked at day 4 post-infection (pi) (103.46 EID50 /0.1 ml) and drastically decreased at day 5 pi (100.9 EID50/0.1 ml), while in the rZJ1-GFP-infected group virus levels in the blood reached 105.35 EID50/0.1 ml at day 5. However, rZJ1-IL2-infected groups presented survival curves similar to control birds infected with rZJ1-GFP, with comparable clinical signs and 100 % mortality. Further, expression of IL-2 did not significantly affect the ICPI scores, compared to rZJ1-GFP strain.ConclusionsIncreased expression of chicken IL-2 during virulent NDV replication in naïve chickens decreased viral titers in blood, spleens, oral and cloacal secretions on day 4–5 post infection. This is consistent with the previously described role of IL-2 in enhancing the clearance of viruses in mammals, such as human respiratory syncytial virus.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-015-0353-x) contains supplementary material, which is available to authorized users.

, and produced lower virus yields than WNV strain Eg101. IPS-1 was required for both increased induction of IFN-␤ and decreased yields of W956IC. In Eg101-infected cells, phospho-STAT1/STAT2 nuclear translocation was blocked at all times analyzed, while some phospho-STAT1/STAT2 nuclear translocation was still detected at 8 h after infection in W956IC-infected mouse embryonic fibroblasts (MEFs), and early viral protein levels were lower in these cells. A set of additional chimeras was made by replacing various W956IC gene regions with the Eg101 equivalents. As reported previously, for three of these chimeras, the low early RNA phenotype of Eg101 was restored in BHK cells. Analysis of infections with two of these chimeric viruses in MEFs detected lower early viral RNA levels, higher early viral protein levels, lower early IFN-␤ levels, and higher virus yields similar to those seen after Eg101 infection. The data suggest that replicase protein interactions directly or indirectly regulate genome switching between replication and translation at early times in favor of translation to minimize NF-〉 activation and IFN induction by decreasing the amount of unprotected viral RNA, to produce sufficient viral protein to block canonical type I IFN signaling, and to efficiently remodel cell membranes for exponential genome amplification. West Nile virus (WNV) is a positive-sense, single-stranded RNA, enveloped virus of the family Flaviviridae. The WNV genomic RNA is about 11 kb in length, has a capped 5= end but no poly(A) at the 3= end, and encodes one open reading frame (ORF). The polyprotein precursor produced is posttranslationally processed by virus and host cell proteases to yield three structural proteins, capsid (C), premembrane (prM), and envelope (E), and seven nonstructural (NS) proteins: glycoprotein NS1, membrane anchor NS2A, membrane anchor and protease cofactor NS2B, protease-helicase NS3, membrane anchors NS4A and NS4B, and methyltransferase-RNA-dependent RNA polymerase (RdRp) NS5 (1).The majority of known WNV isolates are classified into two lineages (2-4). Both lineages contain low-and high-virulence strains (5-7). Lineage 1 strains cause epidemics in many parts of the world while lineage 2 strains are endemic to Africa. The lineage II virus 956D117B3 (4, 8) is a laboratory-passaged descendant of the WNV prototype strain B956. Archived 956D117B3 RNA and the 1,496 3= nucleotides (nt) from the lineage 1 strain Eg101 were used to construct the first infectious clone of WNV, SP6WNEg3=/ Xba (also referred to as W956IC) (8). W956IC virus and the parental virus 956D117B3 produce similar growth kinetics and peak titers in both Vero cells and C6/36 cells (8). The full-length W956IC cDNA is stable in bacteria and has been used to efficiently rescue virus with engineered mutations (9, 10). Other studies from our lab have shown that WNV Eg101 and other natural WNV strain infections do not activate eukaryotic translation initiation factor 2-alpha kinase 2 (Eif2ak2, also known as PKR) in BHK cells or mouse embryonic fibro...