Immunostimulatory Motifs Enhance Antiviral siRNAs Targeting Highly Pathogenic Avian Influenza H5N1

Highly pathogenic avian influenza (HPAI) H5N1 virus is endemic in many regions around the world and remains a significant pandemic threat. To date H5N1 has claimed almost 300 human lives worldwide, with a mortality rate of 60% and has caused the death or culling of hundreds of millions of poultry since its initial outbreak in 1997. We have designed multi-functional RNA interference (RNAi)-based therapeutics targeting H5N1 that degrade viral mRNA via the RNAi pathway while at the same time augmenting the host antiviral response by inducing host type I interferon (IFN) production. Moreover, we have identified two factors critical for maximising the immunostimulatory properties of short interfering (si)RNAs in chicken cells (i) mode of synthesis and (ii) nucleoside sequence to augment the response to virus. The 5-bp nucleoside sequence 5′-UGUGU-3′ is a key determinant in inducing high levels of expression of IFN -α, -β, -λ and interleukin 1- β in chicken cells. Positioning of this 5′-UGUGU-3′ motif at the 5′- end of the sense strand of siRNAs, but not the 3′- end, resulted in a rapid and enhanced induction of type I IFN. An anti-H5N1 avian influenza siRNA directed against the PB1 gene (PB1-2257) tagged with 5′-UGUGU-3′ induced type I IFN earlier and to a greater extent compared to a non-tagged PB1-2257. Tested against H5N1 in vitro, the tagged PB1-2257 was more effective than non-tagged PB1-2257. These data demonstrate the ability of an immunostimulatory motif to improve the performance of an RNAi-based antiviral, a finding that may influence the design of future RNAi-based anti-influenza therapeutics

Visualising single molecules of HIV-1 and miRNA nucleic acids

BackgroundThe scarcity of certain nucleic acid species and the small size of target sequences such as miRNA, impose a significant barrier to subcellular visualization and present a major challenge to cell biologists. Here, we offer a generic and highly sensitive visualization approach (oligo fluorescent in situ hybridization, O-FISH) that can be used to detect such nucleic acids using a single-oligonucleotide probe of 19–26 nucleotides in length.ResultsWe used O-FISH to visualize miR146a in human and avian cells. Furthermore, we reveal the sensitivity of O-FISH detection by using a HIV-1 model system to show that as little as 1–2 copies of nucleic acids can be detected in a single cell. We were able to discern newly synthesized viral cDNA and, moreover, observed that certain HIV RNA sequences are only transiently available for O-FISH detection.ConclusionsTaken together, these results suggest that the O-FISH method can potentially be used for in situ probing of, as few as, 1–2 copies of nucleic acid and, additionally, to visualize small RNA such as miRNA. We further propose that the O-FISH method could be extended to understand viral function by probing newly transcribed viral intermediates; and discern the localisation of nucleic acids of interest. Additionally, interrogating the conformation and structure of a particular nucleic acid in situ might also be possible, based on the accessibility of a target sequence

Host genetics determine susceptibility to avian influenza infection and transmission dynamics

Host-genetic control of influenza virus infection has been the object of little attention. In this study we determined that two inbred lines of chicken differing in their genetic background, Lines 0 and C-B12, were respectively relatively resistant and susceptible to infection with the low pathogenicity influenza virus A/Turkey/England/647/77 as defined by substantial differences in viral shedding trajectories. Resistant birds, although infected, were unable to transmit virus to contact birds, as ultimately only the presence of a sustained cloacal shedding (and not oropharyngeal shedding) was critical for transmission. Restriction of within-bird transmission of virus occurred in the resistant line, with intra-nares or cloacal infection resulting in only local shedding and failing to transmit fully through the gastro-intestinal-pulmonary tract. Resistance to infection was independent of adaptive immune responses, including the expansion of specific IFNγ secreting cells or production of influenza-specific antibody. Genetic resistance to a novel H9N2 virus was less robust, though significant differences between host genotypes were still clearly evident. The existence of host-genetic determination of the outcome of influenza infection offers tools for the further dissection of this regulation and also for understanding the mechanisms of influenza transmission within and between birds

Constitutively elevated levels of SOCS1 suppress innate responses in DF-1 immortalised chicken fibroblast cells.

The spontaneously immortalised DF-1 cell line is rapidly replacing its progenitor primary chicken embryo fibroblasts (CEFs) for studies on avian viruses such as avian influenza but no comprehensive study has as yet been reported comparing their innate immunity phenotypes. We conducted microarray analyses of DF-1 and CEFs, under both normal and stimulated conditions using chicken interferon-α (chIFN-α) and the attenuated infectious bursal disease virus vaccine strain PBG98. We found that DF-1 have an attenuated innate response compared to CEFs. Basal expression levels of Suppressor of Cytokine Signalling 1 (chSOCS1), a negative regulator of cytokine signalling in mammals, are 16-fold higher in DF-1 than in CEFs. The chSOCS1 “SOCS box” domain (which in mammals, interacts with an E3 ubiquitin ligase complex) is not essential for the inhibition of cytokine-induced JAK/STAT signalling activation in DF-1. Overexpression of SOCS1 in chIFN-α-stimulated DF-1 led to a relative decrease in expression of interferon-stimulated genes (ISGs; MX1 and IFIT5) and increased viral yield in response to PBG98 infection. Conversely, knockdown of SOCS1 enhanced induction of ISGs and reduced viral yield in chIFN-α-stimulated DF-1. Consequently, SOCS1 reduces induction of the IFN signalling pathway in chicken cells and can potentiate virus replication

Increased Inducible Nitric Oxide Synthase Expression in Organs Is Associated with a Higher Severity of H5N1 Influenza Virus Infection

BACKGROUND: The mechanisms of disease severity caused by H5N1 influenza virus infection remain somewhat unclear. Studies have indicated that a high viral load and an associated hyper inflammatory immune response are influential during the onset of infection. This dysregulated inflammatory response with increased levels of free radicals, such as nitric oxide (NO), appears likely to contribute to disease severity. However, enzymes of the nitric oxide synthase (NOS) family such as the inducible form of NOS (iNOS) generate NO, which serves as a potent anti-viral molecule to combat infection in combination with acute phase proteins and cytokines. Nevertheless, excessive production of iNOS and subsequent high levels of NO during H5N1 infection may have negative effects, acting with other damaging oxidants to promote excessive inflammation or induce apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: There are dramatic differences in the severity of disease between chickens and ducks following H5N1 influenza infection. Chickens show a high level of mortality and associated pathology, whilst ducks show relatively minor symptoms. It is not clear how this varying pathogenicty comes about, although it has been suggested that an overactive inflammatory immune response to infection in the chicken, compared to the duck response, may be to blame for the disparity in observed pathology. In this study, we identify and investigate iNOS gene expression in ducks and chickens during H5N1 influenza infection. Infected chickens show a marked increase in iNOS expression in a wide range of organs. Contrastingly, infected duck tissues have lower levels of tissue related iNOS expression. CONCLUSIONS/SIGNIFICANCE: The differences in iNOS expression levels observed between chickens and ducks during H5N1 avian influenza infection may be important in the inflammatory response that contributes to the pathology. Understanding the regulation of iNOS expression and its role during H5N1 influenza infection may provide insights for the development of new therapeutic strategies in the treatment of avian influenza infection

Highly pathogenic avian influenza virus infection in chickens but not ducks is associated with elevated host immune and pro-inflammatory responses

Highly pathogenic avian influenza (HPAI) H5N1 viruses cause severe infection in chickens at near complete mortality, but corresponding infection in ducks is typically mild or asymptomatic. To understand the underlying molecular differences in host response, primary chicken and duck lung cells, infected with two HPAI H5N1 viruses and a low pathogenicity avian influenza (LPAI) H2N3 virus, were subjected to RNA expression profiling. Chicken cells but not duck cells showed highly elevated immune and pro-inflammatory responses following HPAI virus infection. HPAI H5N1 virus challenge studies in chickens and ducks corroborated the in vitro findings. To try to determine the underlying mechanisms, we investigated the role of signal transducer and activator of transcription-3 (STAT-3) in mediating pro-inflammatory response to HPAIV infection in chicken and duck cells. We found that STAT-3 expression was down-regulated in chickens but was up-regulated or unaffected in ducks in vitro and in vivo following H5N1 virus infection. Low basal STAT-3 expression in chicken cells was completely inhibited by H5N1 virus infection. By contrast, constitutively active STAT-3 detected in duck cells was unaffected by H5N1 virus infection. Transient constitutively-active STAT-3 transfection in chicken cells significantly reduced pro-inflammatory response to H5N1 virus infection; on the other hand, chemical inhibition of STAT-3 activation in duck cells increased pro-inflammatory gene expression following H5N1 virus infection. Collectively, we propose that elevated pro-inflammatory response in chickens is a major pathogenicity factor of HPAI H5N1 virus infection, mediated in part by the inhibition of STAT-3

Role of Position 627 of PB2 and the Multibasic Cleavage Site of the Hemagglutinin in the Virulence of H5N1 Avian Influenza Virus in Chickens and Ducks

Highly pathogenic H5N1 avian influenza viruses have caused major disease outbreaks in domestic and free-living birds with transmission to humans resulting in 59% mortality amongst 564 cases. The mutation of the amino acid at position 627 of the viral polymerase basic-2 protein (PB2) from glutamic acid (E) in avian isolates to lysine (K) in human isolates is frequently found, but it is not known if this change affects the fitness and pathogenicity of the virus in birds. We show here that horizontal transmission of A/Vietnam/1203/2004 H5N1 (VN/1203) virus in chickens and ducks was not affected by the change of K to E at PB2-627. All chickens died between 21 to 48 hours post infection (pi), while 70% of the ducks survived infection. Virus replication was detected in chickens within 12 hours pi and reached peak titers in spleen, lung and brain between 18 to 24 hours for both viruses. Viral antigen in chickens was predominantly in the endothelium, while in ducks it was present in multiple cell types, including neurons, myocardium, skeletal muscle and connective tissues. Virus replicated to a high titer in chicken thrombocytes and caused upregulation of TLR3 and several cell adhesion molecules, which may explain the rapid virus dissemination and location of viral antigen in endothelium. Virus replication in ducks reached peak values between 2 and 4 days pi in spleen, lung and brain tissues and in contrast to infection in chickens, thrombocytes were not involved. In addition, infection of chickens with low pathogenic VN/1203 caused neuropathology, with E at position PB2-627 causing significantly higher infection rates than K, indicating that it enhances virulence in chickens

Sequence-non-specific effects of RNA interference triggers and microRNA regulators

RNA reagents of diverse lengths and structures, unmodified or containing various chemical modifications are powerful tools of RNA interference and microRNA technologies. These reagents which are either delivered to cells using appropriate carriers or are expressed in cells from suitable vectors often cause unintended sequence-non-specific immune responses besides triggering intended sequence-specific silencing effects. This article reviews the present state of knowledge regarding the cellular sensors of foreign RNA, the signaling pathways these sensors mobilize and shows which specific features of the RNA reagents set the responsive systems on alert. The representative examples of toxic effects caused in the investigated cell lines and tissues by the RNAs of specific types and structures are collected and may be instructive for further studies of sequence-non-specific responses to foreign RNA in human cells

Differential lung NK cell responses in avian influenza virus infected chickens correlate with pathogenicity

Infection of chickens with low pathogenicity avian influenza (LPAI) virus results in mild clinical signs while infection with highly pathogenic avian influenza (HPAI) viruses causes death of the birds within 36–48 hours. Since natural killer (NK) cells have been shown to play an important role in influenza-specific immunity, we hypothesise that NK cells are involved in this difference in pathogenicity. To investigate this, the role of chicken NK-cells in LPAI virus infection was studied. Next activation of lung NK cells upon HPAI virus infection was analysed. Infection with a H9N2 LPAI virus resulted in the presence of viral RNA in the lungs which coincided with enhanced activation of lung NK cells. The presence of H5N1 viruses, measured by detection of viral RNA, did not induce activation of lung NK cells. This suggests that decreased NK-cell activation may be one of the mechanisms associated with the enhanced pathogenicity of H5N1 viruses