ADAMTS5 is a critical regulator of virus-specific T cell immunity

The extracellular matrix (ECM) provides physical scaffolding for cellular constituents and initiates biochemical and biomechanical cues that are required for physiological activity of living tissues. The ECM enzyme ADAMTS5, a member of the ADAMTS (A Disintegrin-like and Metalloproteinase with Thrombospondin-1 motifs) protein family, cleaves large proteoglycans such as aggrecan, leading to the destruction of cartilage and osteoarthritis. However, its contribution to viral pathogenesis and immunity is currently undefined. Here, we use a combination of in vitro and in vivo models to show that ADAMTS5 enzymatic activity plays a key role in the development of influenza-specific immunity. Influenza virus infection of Adamts5-/- mice resulted in delayed virus clearance, compromised T cell migration and immunity and accumulation of versican, an ADAMTS5 proteoglycan substrate. Our research emphasises the importance of ADAMTS5 expression in the control of influenza virus infection and highlights the potential for development of ADAMTS5-based therapeutic strategies to reduce morbidity and mortality

Bat lung epithelial cells show greater host species-specific innate resistance than MDCK cells to human and avian influenza viruses

Background With the recent discovery of novel H17N10 and H18N11 influenza viral RNA in bats and report on high frequency of avian H9 seroconversion in a species of free ranging bats, an important issue to address is the extent bats are susceptible to conventional avian and human influenza A viruses. Method To this end, three bat species (Eidolon helvum, Carollia perspicillata and Tadarida brasiliensis) of lung epithelial cells were separately infected with two avian and two human influenza viruses to determine their relative host innate immune resistance to infection. Results All three species of bat cells were more resistant than positive control Madin-Darby canine kidney (MDCK) cells to all four influenza viruses. TB1-Lu cells lacked sialic acid α2,6-Gal receptors and were most resistant among the three bat species. Interestingly, avian viruses were relatively more replication permissive in all three bat species of cells than with the use of human viruses which suggest that bats could potentially play a role in the ecology of avian influenza viruses. Chemical inhibition of the JAK-STAT pathway in bat cells had no effect on virus production suggesting that type I interferon signalling is not a major factor in resisting influenza virus infection. Conclusion Although all three species of bat cells are relatively more resistant to influenza virus infection than control MDCK cells, they are more permissive to avian than human viruses which suggest that bats could have a contributory role in the ecology of avian influenza viruses

An enzyme-linked immunosorbent assay for detection of avian influenza virus subtypes H5 and H7 antibodies

BACKGROUND: Avian influenza virus (AIV) subtypes H5 and H7 attracts particular attention because of the risk of their potential pathogenicity in poultry. The haemagglutination inhibition (HI) test is widely used as subtype specific test for serological diagnostics despite the laborious nature of this method. However, enzyme-linked immunosorbent assays (ELISAs) are being explored as an alternative test method. H5 and H7 specific monoclonal antibodies were experimentally raised and used in the development of inhibition ELISAs for detection of serological response specifically directed against AIV subtypes H5 and H7. The ELISAs were evaluated with polyclonal chicken anti-AIV antibodies against AIV subtypes: H1N2, H5N2, H5N7, H7N1, H7N7, H9N9, H10N4 and H16N3. RESULTS: Both the H5 and H7 ELISA proved to have a high sensitivity and specificity and the ELISAs detected H5 and H7 antibodies earlier during experimental infection than the HI test did. The reproducibility of the ELISA’s performed at different times was high with Pearson correlation coefficients of 0.96-0.98. CONCLUSIONS: The ELISAs are a potential alternative to the HI test for screening of large amounts of avian sera, although only experimental sera were tested in this study

Efficacy of a parainfluenza virus 5 (PIV5)-based H7N9 vaccine in mice and guinea pigs: antibody titer towards HA was not a good indicator for protection.

H7N9 has caused fatal infections in humans. A safe and effective vaccine is the best way to prevent large-scale outbreaks in the human population. Parainfluenza virus 5 (PIV5), an avirulent paramyxovirus, is a promising vaccine vector. In this work, we generated a recombinant PIV5 expressing the HA gene of H7N9 (PIV5-H7) and tested its efficacy against infection with influenza virus A/Anhui/1/2013 (H7N9) in mice and guinea pigs. PIV5-H7 protected the mice against lethal H7N9 challenge. Interestingly, the protection did not require antibody since PIV5-H7 protected JhD mice that do not produce antibody against lethal H7N9 challenge. Furthermore, transfer of anti-H7 serum did not protect mice against H7N9 challenge. PIV5-H7 generated high HAI titers in guinea pigs, however it did not protect against H7N9 infection or transmission. Intriguingly, immunization of guinea pigs with PIV5-H7 and PIV5 expressing NP of influenza A virus H5N1 (PIV5-NP) conferred protection against H7N9 infection and transmission. Thus, we have obtained a H7N9 vaccine that protected both mice and guinea pigs against lethal H7N9 challenge and infection respectively

Polymerase discordance in novel swine influenza H3N2v constellations is tolerated in swine but not human respiratory epithelial cells.

Swine-origin H3N2v, a variant of H3N2 influenza virus, is a concern for novel reassortment with circulating pandemic H1N1 influenza virus (H1N1pdm09) in swine because this can lead to the emergence of a novel pandemic virus. In this study, the reassortment prevalence of H3N2v with H1N1pdm09 was determined in swine cells. Reassortants evaluated showed that the H1N1pdm09 polymerase (PA) segment occurred within swine H3N2 with ∼ 80% frequency. The swine H3N2-human H1N1pdm09 PA reassortant (swH3N2-huPA) showed enhanced replication in swine cells, and was the dominant gene constellation. Ferrets infected with swH3N2-huPA had increased lung pathogenicity compared to parent viruses; however, swH3N2-huPA replication in normal human bronchoepithelial cells was attenuated - a feature linked to expression of IFN-β and IFN-λ genes in human but not swine cells. These findings indicate that emergence of novel H3N2v influenza constellations require more than changes in the viral polymerase complex to overcome barriers to cross-species transmission. Additionally, these findings reveal that while the ferret model is highly informative for influenza studies, slight differences in pathogenicity may not necessarily be indicative of human outcomes after infection

Peak virus titers from plaque isolates with pdm09, swH3N2, swH3N2-huPA, and swH3N2-huPA-PB1 gene constellations.

<p>The cell lines used are: (A) PK-1 cells, (B) NSBE cells, and (C) MDCK cells. Significant (p<0.05) differences in viral titer for swH3N2-huPA and both parental strains is indicated by an asterisk (*). Of note, only one swH3N2 reassortant with huPA-PB1 genes was identified (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110264#pone-0110264-g002" target="_blank">Fig. 2C</a>). All infections used a MOI = 0.01. (D) Plaque diameter were measured 96 h pi in 2% agarose overlay solution. Cumulative plaque total measured from 2–3 separate six-well plates with 10–20 total plaques dilutions per well. * denotes significant difference between pdm09 and swH3N2-huPA.</p

Reassortment findings after eight serial passages.

<p>Co-infection was performed in (A) PK-1 cells and (B) NSBE cells. While pdm09 and swH3N2 parent viruses were evident the only reassortant constellation identified was the PA segment of human pdm09 (light colored, “Hu”) within seven gene segments from swH3N2 (dark colored, “Sw”). Mixed gene segments with Ct values <8 are depicted as hash marks. Serial passaging was done in triplicate with a starting MOI of either 1.0 or 0.1 for each virus as indicated. To the left of (A) PK-1 cells and (B) NSBE cells is the kinetics of PA acquisition and loss for a MOI = 1∶1. Depicted is the first replicate shown from matrix. (C) 12 plaques with PA gene of pdm09 and 5 segments of swH3N2 from reassortant studies (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110264#pone-0110264-g001" target="_blank">Fig. 1</a>) were subjected to qPCR do delineate the final two segments, NP and PB1. Depicted on right, 11 of 12 reassortants were determined to be swH3N2-huPA, and 1 of 12 reassortants to be swH3N2-huPA-PB1.</p

PIV5-H7 and PIV5-NP protected mice against H7N9 challenge.

<p>BALB/c mice in groups of 9–10 were infected IN with PIV5-H7 at a dose of 10⁶ PFU, 10⁶ PFU of PIV5-NP, 10⁶ PFU of PIV5, or PBS. Mice were rested for 8 weeks and were then challenged with 10 50% lethal doses (LD₅₀) of A/Anhui/1/2013 (H7N9) and monitored for (A) survival and (B) weight loss. (*<i>P</i><0.05, log-Rank, compared to PBS or PIV5)</p

Reassortment outcomes following human H1N1 pdm09 and swine H3N2 co-infection.

<p>Segments originating from pdm09 origin (light colored) and swH3N2 (dark-colored) are shown after co-infection in NSBE cells (left) and PK-1 cells. Primer and probes specific to parental strains pdm09 and swH3N2 gene segments were used and from left to right in the matrix correspond to PA, PB2, M, NS, NA and HA. All reassortant plaques identified are categorized by HA and NA backbone type. In total, 121 reassortants for PK-1 cells and 45 reassortants for NSBE cells were evaluated. The number of virus plaques showing 6-gene non-reassortment and the top 6 reassortant types are summarized below profile matrix.</p