Roles of the fusion and hemagglutinin-neuraminidase proteins in replication, tropism, and pathogenicity of avian paramyxoviruses

Virulent and moderately virulent strains of Newcastle disease virus (NDV), representing avian paramyxovirus serotype 1 (APMV-1), cause respiratory and neurological disease in chickens and other species of birds. In contrast, APMV-2 is avirulent in chickens. We investigated the role of the fusion (F) and hemagglutinin-neuraminidase (HN) envelope glycoproteins in these contrasting phenotypes by designing chimeric viruses in which the F and HN glycoproteins or their ectodomains were exchanged individually or together between the moderately virulent, neurotropic NDV strain Beaudette C (BC) and the avirulent APMV-2 strain Yucaipa. When we attempted to exchange the complete F and HN glycoproteins individually and together between the two viruses, the only construct that could be recovered was recombinant APMV-2 strain Yucaipa (rAPMV-2), containing the NDV F glycoprotein in place of its own. This substitution of NDV F into APMV-2 was sufficient to confer the neurotropic, neuroinvasive, and neurovirulent phenotypes, in spite of all being at reduced levels compared to what was seen for NDV-BC. When the ectodomains of F and HN were exchanged individually and together, two constructs could be recovered: NDV, containing both the F and HN ectodomains of APMV-2; and APMV-2, containing both ectodomains of NDV. This supported the idea that homologous cytoplasmic tails and matched F and HN ectodomains are important for virus replication. Analysis of these viruses for replication in vitro, syncytium formation, mean embryo death time, intracerebral pathogenicity index, and replication and tropism in 1-day-old chicks and 2-week-old chickens showed that the two contrasting phenotypes of NDV and APMV-2 could largely be transferred between the two backbones by transfer of homotypic F and HN ectodomains. Further analysis provided evidence that the homologous stalk domain of NDV HN is essential for virus replication, while the globular head domain of NDV HN could be replaced with that of APMV-2 with only a minimal attenuating effect. These results demonstrate that the F and HN ectodomains together determine the cell fusion, tropism, and virulence phenotypes of NDV and APMV-2 and that the regions of HN that are critical to replication and the species-specific phenotypes include the cytoplasmic tail and stalk domain but not the globular head domain.     

The C proteins of human parainfluenza virus type 1 limit double-stranded RNA accumulation that would otherwise trigger activation of MDA5 and protein kinase R

Human parainfluenza virus type 1 (HPIV1) is an important respiratory pathogen in young children, the immunocompromised, and the elderly. We found that infection with wild-type (WT) HPIV1 suppressed the innate immune response in human airway epithelial cells by preventing not only phosphorylation of interferon regulatory factor 3 (IRF3) but also degradation of IκBβ, thereby inhibiting IRF3 and NF-κB activation, respectively. Both of these effects were ablated by a F170S substitution in the HPIV1 C proteins (F170S) or by silencing the C open reading frame [P(C-)], resulting in a potent beta interferon (IFN-β) response. Using murine knockout cells, we found that IFN-β induction following infection with either mutant relied mainly on melanoma-associated differentiation gene 5 (MDA5) rather than retinoic acid-inducible gene I (RIG-I). Infection with either mutant, but not WT HPIV1, induced a significant accumulation of intracellular double-stranded RNA (dsRNA). These mutant viruses directed a marked increase in the accumulation of viral genome, antigenome, and mRNA that was coincident with the accumulation of dsRNA. In addition, the amount of viral proteins was reduced compared to that of WT HPIV1. Thus, the accumulation of dsRNA might be a result of an imbalance in the N protein/genomic RNA ratio leading to incomplete encapsidation. Protein kinase R (PKR) activation and IFN-β induction followed the kinetics of dsRNA accumulation. Interestingly, the C proteins did not appear to directly inhibit intracellular signaling involved in IFN-β induction; instead, their role in preventing IFN-β induction appeared to be in suppressing the formation of dsRNA. PKR activation contributed to IFN-β induction and also was associated with the reduction in the amount of viral proteins. Thus, the HPIV1 C proteins normally limit the accumulation of dsRNA and thereby limit activation of IRF3, NF-κB, and PKR. If C protein function is compromised, as in the case of F170S HPIV1, the resulting PKR activation and reduction in viral protein levels enable the host to further reduce C protein levels and to mount a potent antiviral type I IFN response.     

Subdoligranulum variabile gen. nov., sp. nov. from human feces

During studies on the microflora of human feces we have isolated a strictly anaerobic, non-spore-forming, Gram-negative staining organism which exhibits a somewhat variable coccus-shaped morphology. Comparative 16S ribosomal RNA gene sequencing studies show the unidentified organism is phylogenetically a member of the Clostridium leptum supra-generic rRNA cluster and displays a close affinity to some rDNA clones derived from human and pig feces. The nearest named relatives of the unidentified isolate corresponded to Faecalibacterium prausnitzii (formerly Fusobacterium prausnitzii) displaying a 16S rRNA sequence divergence of approximately 9%, with Anaerofilum agile and A. pentosovorans the next closest relatives of the unidentified bacterium (sequence divergence approximately 10%). Based on phenotypic and phylogenetic considerations, it is proposed that the unusual coccoid-shaped organism be classified as a new genus and species, Subdoligranulum variabile. The type strain of S. variabile is BI 114(T) (=CCUG 47106(T)=DSM 15176(T)).     

Characterisation of complex formation between members of the Mycobacterium tuberculosis complex CFP-10/ESAT-6 protein family: towards an understanding of the rules governing complex formation and thereby functional flexibility

We have previously shown that the secreted M. tuberculosis complex proteins CFP-10 and ESAT-6 form a tight, 1:1 complex, which may represent their functional form. In the work reported here a combination of yeast two-hybrid and biochemical analysis has been used to characterise complex formation between two other pairs of CFP-10/ESAT-6 family proteins (Rv0287/Rv0288 and Rv3019c/Rv3020c) and to determine whether complexes can be formed between non-genome paired members of the family. The results clearly demonstrate that Rv0287/Rv0288 and Rv3019c/3020c form tight complexes, as initially observed for CFP-10/ESAT-6. The closely related Rv0287/Rv0288 and Rv3019c/Rv3020c proteins are also able to form non-genome paired complexes (Rv0287/Rv3019c and Rv0288/Rv3020c), but are not capable of binding to the more distantly related CFP-10/ESAT-6 proteins.     

Salmonella-induced cell death: apoptosis, necrosis or programmed cell death?

Over the past several years, it has become apparent that enteropathogens activate cell death programs. For Salmonella and Shigella species, the induction of cell death is required for pathogenesis, and the mechanisms by which these bacteria induce cell death is an area of intense investigation. Although initial studies suggested that Salmonella induce cell death through an apoptotic pathway, recent studies demonstrate that cell death occurs through a unique caspase 1-dependent mechanism.     

Water deficit in field-grown Gossypium hirsutum primarily limits net photosynthesis by decreasing stomatal conductance,increasing photorespiration,and increasing the ratio of dark respiration to gross photosynthesis

Much effort has been expended to improve irrigation efficiency and drought tolerance of agronomic crops; however, a clear understanding of the physiological mechanisms that interact to decrease source strength and drive yield loss has not been attained. To elucidate the underlying mechanisms contributing to inhibition of net carbon assimilation under drought stress, three cultivars of Gossypium hirsutum were grown in the field under contrasting irrigation regimes during the 2012 and 2013 growing season near Camilla, Georgia, USA. Physiological measurements were conducted on three sample dates during each growing season (providing a broad range of plant water status) and included, predawn and midday leaf water potential (Ψ_PD and Ψ_MD), gross and net photosynthesis, dark respiration, photorespiration, and chlorophyll a fluorescence. End-of-season lint yield was also determined. Ψ_PD ranged from −0.31 to −0.95 MPa, and Ψ_MD ranged from −1.02 to −2.67 MPa, depending upon irrigation regime and sample date. G. hirsutum responded to water deficit by decreasing stomatal conductance, increasing photorespiration, and increasing the ratio of dark respiration to gross photosynthesis, thereby limiting P_N and decreasing lint yield (lint yield declines observed during the 2012 growing season only). Conversely, even extreme water deficit, causing a 54% decline in P_N, did not negatively affect actual quantum yield, maximum quantum yield, or photosynthetic electron transport. It is concluded that P_N is primarily limited in drought-stressed G. hirsutum by decreased stomatal conductance, along with increases in respiratory and photorespiratory carbon losses, not inhibition or down-regulation of electron transport through photosystem II. It is further concluded that Ψ_PD is a reliable indicator of drought stress and the need for irrigation in field-grown cotton.     

The BGN and ACAN genes and carpal tunnel syndrome

The causes of idiopathic carpal tunnel syndrome (CTS) remain unknown and the involvement of the tendons within the carpal tunnel structure in the aetiology of CTS cannot be excluded. Variants within the COL5A1 gene, an important regulator of fibril assembly in tendons, have previously been associated with modulating the risk of CTS. Furthermore, proteoglycans are also important structural components of tendons and variants within the aggrecan gene are associated with musculoskeletal soft tissue injuries. The aim of this study was to determine whether ACAN and BGN variants are associated with CTS.