Initiation of acute pancreatitis in mice is independent of fusion between lysosomes and zymogen granules.

The co-localization of the lysosomal protease cathepsin B (CTSB) and the digestive zymogen trypsinogen is a prerequisite for the initiation of acute pancreatitis. However, the exact molecular mechanisms of co-localization are not fully understood. In this study, we investigated the role of lysosomes in the onset of acute pancreatitis by using two different experimental approaches. Using an acinar cell-specific genetic deletion of the ras-related protein Rab7, important for intracellular vesicle trafficking and fusion, we analyzed the subcellular distribution of lysosomal enzymes and the severity of pancreatitis in vivo and ex vivo. Lysosomal permeabilization was performed by the lysosomotropic agent Glycyl-L-phenylalanine 2-naphthylamide (GPN). Acinar cell-specific deletion of Rab7 increased endogenous CTSB activity and despite the lack of re-distribution of CTSB from lysosomes to the secretory vesicles, the activation of CTSB localized in the zymogen compartment still took place leading to trypsinogen activation and pancreatic injury. Disease severity was comparable to controls during the early phase but more severe at later time points. Similarly, GPN did not prevent CTSB activation inside the secretory compartment upon caerulein stimulation, while lysosomal CTSB shifted to the cytosol. Intracellular trypsinogen activation was maintained leading to acute pancreatitis similar to controls. Our results indicate that initiation of acute pancreatitis seems to be independent of the presence of lysosomes and that fusion of lysosomes and zymogen granules is dispensable for the disease onset. Intact lysosomes rather appear to have protective effects at later disease stages.

Automated quantification of avian influenza virus antigen in different organs.

As immunohistochemistry is valuable for determining tissue and cell tropism of avian influenza viruses (AIV), but time-consuming, an artificial intelligence-based workflow was developed to automate the AIV antigen quantification. Organ samples from experimental AIV infections including brain, heart, lung and spleen on one slide, and liver and kidney on another slide were stained for influenza A-matrixprotein and analyzed with QuPath: Random trees algorithms were trained to identify the organs on each slide, followed by threshold-based quantification of the immunoreactive area. The algorithms were trained and tested on two different slide sets, then retrained on both and validated on a third set. Except for the kidney, the best algorithms for organ selection correctly identified the largest proportion of the organ area. For most organs, the immunoreactive area assessed following organ selection was significantly and positively correlated to a manually assessed semiquantitative score. In the validation set, intravenously infected chickens showed a generally higher percentage of immunoreactive area than chickens infected oculonasally. Variability between the slide sets and a similar tissue texture of some organs limited the ability of the algorithms to select certain organs. Generally, suitable correlations of the immunoreactivity data results were achieved, facilitating high-throughput analysis of AIV tissue tropism.

Phosphatidylinositolmannoside vaccination induces lipid-specific Th1-responses and partially protects guinea pigs from Mycobacterium tuberculosis challenge.

The concept of donor-unrestricted T cells (DURTs) comprises a heterogeneity of lymphoid cells that respond to an abundance of unconventional epitopes in a non-MHC-restricted manner. Vaccinologists strive to harness this so far underexplored branch of the immune system for new vaccines against tuberculosis. A particular division of DURTs are T cells that recognize their cognate lipid antigen in the context of CD1-molecules. Mycobacteria are characterized by a particular lipid-rich cell wall. Several of these lipids have been shown to be presented to T cells via CD1b-molecules. Guinea pigs functionally express CD1b and are hence an appropriate small animal model to study the role of CD1b-restricted, lipid-specific immune responses. In the current study, guinea pigs were vaccinated with BCG or highly-purified, liposome-formulated phosphatidylinositol-hexa-mannoside (PIM6) to assess the effect of CD1-restricted DURTs on the course of infection after virulent Mycobacterium tuberculosis (Mtb) challenge. Robust PIM6-specific T cell-responses were observed both after BCG- and PIM6-vaccination. The cellular response was significantly reduced in the presence of monoclonal, CD1b-blocking antibodies, indicating that a predominant part of this reactivity was CD1b-restricted. When animals were challenged with Mtb, BCG- and PIM6-vaccinated animals showed significantly reduced pathology, smaller necrotic granulomas in lymph node and spleen and reduced bacterial loads. While BCG conferred an almost sterile protection in this setting, compared to control animals' lesions were reduced roughly by two thirds in PIM6-vaccinated. Comprehensive histological and transcriptional analyses in the draining lymph node revealed that protected animals showed reduced transcription-levels of inflammatory cyto- and chemokines and higher levels of CD1b-expression on professional antigen cells compared to controls. Although BCG as a comparator induced by far stronger effects, our observations in the guinea pig model suggest that CD1b-restricted, PIM6-reactive DURTs contribute to immune-mediated containment of virulent Mtb.

Phenotypic effects of mutations observed in the neuraminidase of human origin H5N1 influenza A viruses.

Global spread and regional endemicity of H5Nx Goose/Guangdong avian influenza viruses (AIV) pose a continuous threat for poultry production and zoonotic, potentially pre-pandemic, transmission to humans. Little is known about the role of mutations in the viral neuraminidase (NA) that accompanied bird-to-human transmission to support AIV infection of mammals. Here, after detailed analysis of the NA sequence of human H5N1 viruses, we studied the role of A46D, L204M, S319F and S430G mutations in virus fitness in vitro and in vivo. Although H5N1 AIV carrying avian- or human-like NAs had similar replication efficiency in avian cells, human-like NA enhanced virus replication in human airway epithelia. The L204M substitution consistently reduced NA activity of H5N1 and nine other influenza viruses carrying NA of groups 1 and 2, indicating a universal effect. Compared to the avian ancestor, human-like H5N1 virus has less NA incorporated in the virion, reduced levels of viral NA RNA replication and NA expression. We also demonstrate increased accumulation of NA at the plasma membrane, reduced virus release and enhanced cell-to-cell spread. Furthermore, NA mutations increased virus binding to human-type receptors. While not affecting high virulence of H5N1 in chickens, the studied NA mutations modulated virulence and replication of H5N1 AIV in mice and to a lesser extent in ferrets. Together, mutations in the NA of human H5N1 viruses play different roles in infection of mammals without affecting virulence or transmission in chickens. These results are important to understand the genetic determinants for replication of AIV in mammals and should assist in the prediction of AIV with zoonotic potential.

Systemic Bile Acids Affect the Severity of Acute Pancreatitis in Mice Depending on Their Hydrophobicity and the Disease Pathogenesis.

Acute pancreatitis (AP) is a major, globally increasing gastrointestinal disease and a biliary origin is the most common cause. However, the effects of bile acids (BAs), given systemically, on the pancreas and on disease severity remains elusive. In this study, we have investigated the roles of different circulating BAs in animal models for AP to elucidate their impact on disease severity and the underlying pathomechanisms. BAs were incubated on isolated acini and AP was induced through repetitive injections of caerulein or L-arginine; pancreatic duct ligation (PDL); or combined biliopancreatic duct ligation (BPDL). Disease severity was assessed using biochemical and histological parameters. Serum cholecystokinin (CCK) concentrations were determined via enzyme immunoassay. The binding of the CCK1 receptor was measured using fluorescence-labeled CCK. In isolated acini, hydrophobic BAs mitigated the damaging effects of CCK. The same BAs further enhanced pancreatitis in L-arginine- and PDL-based pancreatitis, whereas they ameliorated pancreatic damage in the caerulein and BPDL models. Mechanistically, the binding affinity of the CCK1 receptor was significantly reduced by hydrophobic BAs. The hydrophobicity of BAs and the involvement of CCK seem to be relevant in the course of AP. Systemic BAs may affect the severity of AP by interfering with the CCK1 receptor.

A Semiquantitative Scoring System for Histopathological and Immunohistochemical Assessment of Lesions and Tissue Tropism in Avian Influenza.

The main findings of the post-mortem examination of poultry infected with highly pathogenic avian influenza viruses (HPAIV) include necrotizing inflammation and viral antigen in multiple organs. The lesion profile displays marked variability, depending on viral subtype, strain, and host species. Therefore, in this study, a semiquantitative scoring system was developed to compare histopathological findings across a wide range of study conditions. Briefly, the severity of necrotizing lesions in brain, heart, lung, liver, kidney, pancreas, and/or lymphocytic depletion in the spleen is scored on an ordinal four-step scale (0 = unchanged, 1 = mild, 2 = moderate, 3 = severe), and the distribution of the viral antigen in parenchymal and endothelial cells is evaluated on a four-step scale (0 = none, 1 = focal, 2 = multifocal, 3 = diffuse). These scores are used for a meta-analysis of experimental infections with H7N7 and H5N8 (clade 2.3.4.4b) HPAIV in chickens, turkeys, and ducks. The meta-analysis highlights the rather unique endotheliotropism of these HPAIV in chickens and a more severe necrotizing encephalitis in H7N7-HPAIV-infected turkeys. In conclusion, the proposed scoring system can be used to condensate HPAIV-typical pathohistological findings into semiquantitative data, thus enabling systematic phenotyping of virus strains and their tissue tropism.

The role of glycosylation in the N-terminus of the hemagglutinin of a unique H4N2 with a natural polybasic cleavage site in virus fitness in vitro and in vivo.

To date, only low pathogenic (LP) H5 and H7 avian influenza viruses (AIV) have been observed to naturally shift to a highly pathogenic (HP) phenotype after mutation of the monobasic hemagglutinin (HA) cleavage site (HACS) to polybasic motifs. The LPAIV monobasic HACS is activated by tissue-restricted trypsin-like enzymes, while the HPAIV polybasic HACS is activated by ubiquitous furin-like enzymes. However, glycosylation near the HACS can affect proteolytic activation and reduced virulence of some HPAIV in chickens. In 2012, a unique H4N2 virus with a polybasic HACS was isolated from quails but was LP in chickens. Whether glycosylation sites (GS) near the HACS hinder the evolution of HPAIV H4N2 remains unclear. Here, we analyzed the prevalence of potential GS in the N-terminus of HA1, 2NYT4 and 18NGT20, in all AIV sequences and studied their impact on H4N2 virus fitness. Although the two motifs are conserved, some non-H5/H7 subtypes lack one or both GS. Both sites were glycosylated in this H4N2 virus. Deglycosylation increased trypsin-independent replication in cell culture, cell-to-cell spread and syncytium formation at low-acidic pH, but negatively affected the thermostability and receptor-binding affinity. Alteration of 2NYT4 with or without 18NGT20 enabled systemic spread of the virus to different organs including the brain of chicken embryos. However, all intranasally inoculated chickens did not show clinical signs. Together, although the conserved GS near the HACS are important for HA stability and receptor binding, deglycosylation increased the H4N2 HA-activation, replication and tissue tropism suggesting a potential role for virus adaptation in poultry.

Genetic incompatibilities and reduced transmission in chickens may limit the evolution of reassortants between H9N2 and panzootic H5N8 clade 2.3.4.4 avian influenza virus showing high virulence for mammals.

The unprecedented spread of H5N8- and H9N2-subtype avian influenza virus (AIV) in birds across Asia, Europe, Africa, and North America poses a serious public health threat with a permanent risk of reassortment and the possible emergence of novel virus variants with high virulence in mammals. To gain information on this risk, we studied the potential for reassortment between two contemporary H9N2 and H5N8 viruses. While the replacement of the PB2, PA, and NS genes of highly pathogenic H5N8 by homologous segments from H9N2 produced infectious H5N8 progeny, PB1 and NP of H9N2 were not able to replace the respective segments from H5N8 due to residues outside the packaging region. Furthermore, exchange of the PB2, PA, and NS segments of H5N8 by those of H9N2 increased replication, polymerase activity and interferon antagonism of the H5N8 reassortants in human cells. Notably, H5N8 reassortants carrying the H9N2-subtype PB2 segment and to lesser extent the PA or NS segments showed remarkably increased virulence in mice as indicated by rapid onset of mortality, reduced mean time to death and increased body weight loss. Simultaneously, we observed that in chickens the H5N8 reassortants, particularly with the H9N2 NS segment, demonstrated significantly reduced transmission to co-housed chickens. Together, while the limited capacity for reassortment between co-circulating H9N2 and H5N8 viruses and the reduced bird-to-bird transmission of possible H5N8 reassortants in chickens may limit the evolution of such reassortant viruses, they show a higher replication potential in human cells and increased virulence in mammals.

Insertion of Basic Amino Acids in the Hemagglutinin Cleavage Site of H4N2 Avian Influenza Virus (AIV)-Reduced Virus Fitness in Chickens is Restored by Reassortment with Highly Pathogenic H5N1 AIV.

Highly pathogenic (HP) avian influenza viruses (AIVs) are naturally restricted to H5 and H7 subtypes with a polybasic cleavage site (CS) in hemagglutinin (HA) and any AIV with an intravenous pathogenicity index (IVPI) ≥ 1.2. Although only a few non-H5/H7 viruses fulfill the criteria of HPAIV; it remains unclear why these viruses did not spread in domestic birds. In 2012, a unique H4N2 virus with a polybasic CS 322PEKRRTR/G329 was isolated from quails in California which, however, was avirulent in chickens. This is the only known non-H5/H7 virus with four basic amino acids in the HACS. Here, we investigated the virulence of this virus in chickens after expansion of the polybasic CS by substitution of T327R (322PEKRRRR/G329) or T327K (322PEKRRKR/G329) with or without reassortment with HPAIV H5N1 and H7N7. The impact of single mutations or reassortment on virus fitness in vitro and in vivo was studied. Efficient cell culture replication of T327R/K carrying H4N2 viruses increased by treatment with trypsin, particularly in MDCK cells, and reassortment with HPAIV H5N1. Replication, virus excretion and bird-to-bird transmission of H4N2 was remarkably compromised by the CS mutations, but restored after reassortment with HPAIV H5N1, although not with HPAIV H7N7. Viruses carrying the H4-HA with or without R327 or K327 mutations and the other seven gene segments from HPAIV H5N1 exhibited high virulence and efficient transmission in chickens. Together, increasing the number of basic amino acids in the H4N2 HACS was detrimental for viral fitness particularly in vivo but compensated by reassortment with HPAIV H5N1. This may explain the absence of non-H5/H7 HPAIV in poultry.

Variable impact of the hemagglutinin polybasic cleavage site on virulence and pathogenesis of avian influenza H7N7 virus in chickens, turkeys and ducks.

Avian influenza viruses (AIV) are classified into 16 hemagglutinin (HA; H1-H16) and 9 neuraminidase (NA; N1-N9) subtypes. All AIV are low pathogenic (LP) in birds, but subtypes H5 and H7 AIV can evolve into highly pathogenic (HP) forms. In the last two decades evolution of HPAIV H7 from LPAIV has been frequently reported. However, little is known about the pathogenesis and evolution of HP H7 from LP ancestors particularly, in non-chicken hosts. In 2015, both LP and HP H7N7 AIV were isolated from chickens in two neighbouring farms in Germany. Here, the virulence of these isogenic H7N7 LP, HP and LP virus carrying a polybasic HA cleavage site (HACS) from HP (designated LP-Poly) was studied in chickens, turkeys and different duck breeds. The LP precursor was avirulent in all birds. In contrast, all inoculated and contact chickens and turkeys died after infection with HP. HP infected Pekin and Mallard ducks remained clinically healthy, while Muscovy ducks exhibited moderate depression and excreted viruses at significantly higher amounts. The polybasic HACS increased virulence in a species-specific manner with intravenous pathogenicity indices of 3.0, 1.9 and 0.2 in chickens, turkeys and Muscovy ducks, respectively. Infection of endothelial cells was only observed in chickens. In summary, Pekin and Mallard were more resistant to HPAIV H7N7 than chickens, turkeys and Muscovy ducks. The polybasic HACS was the main determinant for virulence and endotheliotropism of HPAIV H7N7 in chickens, whereas other viral and/or host factors play an essential role in virulence and pathogenesis in turkeys and ducks.

Potential Biological and Climatic Factors That Influence the Incidence and Persistence of Highly Pathogenic H5N1 Avian Influenza Virus in Egypt.

Highly pathogenic H5N1 avian influenza virus (A/H5N1) of clade 2.2.1 is endemic in poultry in Egypt where the highest number of human infections worldwide was reported. During the last 12 years the Egyptian A/H5N1 evolved into several genotypes. In 2007-2014 vaccinated poultry suffered from antigenic drift variants of clade 2.2.1.1 and in 2014/2015 an unprecedented upsurge of A/H5N1 clade 2.2.1.2 occurred in poultry and humans. Factors contributing to the endemicity or re-emergence of A/H5N1 in poultry in Egypt remain unclear. Here, three potential factors were studied: climatic factors (temperature, relative humidity, and wind speed), biological fitness in vitro, and pathogenicity in domestic Pekin and Muscovy ducks. Statistical analyses using negative binomial regression models indicated that ambient temperature in winter months influenced the spread of A/H5N1 in different geographic areas analyzed in this study. In vitro, at 4 and 56°C 2.2.1.1 and recent 2.2.1.2 viruses were more stable than other viruses used in this study. Further, Pekin ducks were more resistant than Muscovy ducks and the viruses were excreted for up to 2 weeks post-infection assuming a strong role as a reservoir. Taken together, ambient temperature in winter months potentially contributes to increasing outbreaks in some regions in Egypt. Heat stability of clade 2.2.1.1 and recent 2.2.1.2 viruses probably favors their persistence at elevated temperatures. Importantly, asymptomatically infected Pekin ducks may play an important role in the spread of avian and human-like A/H5N1 in Egypt. Therefore, control measures including targeted surveillance and culling of silently infected Pekin ducks should be considered.

Impact of Mutations in the Hemagglutinin of H10N7 Viruses Isolated from Seals on Virus Replication in Avian and Human Cells.

Wild birds are the reservoir for low-pathogenic avian influenza viruses, which are frequently transmitted to domestic birds and occasionally to mammals. In 2014, an H10N7 virus caused severe mortality in harbor seals in northeastern Europe. Although the hemagglutinin (HA) of this virus was closely related to H10 of avian H10N4 virus, it possessed unique nonsynonymous mutations, particularly in the HA1 subunit in or adjacent to the receptor binding domain and proteolytic cleavage site. Here, the impact of these mutations on virus replication was studied in vitro. Using reverse genetics, an avian H10N4 virus was cloned, and nine recombinant viruses carrying one of eight unique mutations or the complete HA from the seal virus were rescued. Receptor binding affinity, replication in avian and mammalian cell cultures, cell-to-cell spread, and HA cleavability of these recombinant viruses were studied. Results show that wild-type recombinant H10N4 virus has high affinity to avian-type sialic acid receptors and no affinity to mammalian-type receptors. The H10N7 virus exhibits dual receptor binding affinity. Interestingly, Q220L (H10 numbering) in the rim of the receptor binding pocket increased the affinity of the H10N4 virus to mammal-type receptors and completely abolished the affinity to avian-type receptors. No remarkable differences in cell-to-cell spread or HA cleavability were observed. All viruses, including the wild-type H10N7 virus, replicated at higher levels in chicken cells than in human cells. These results indicate that H10N7 acquired adaptive mutations (e.g., Q220L) to enhance replication in mammals and retained replication efficiency in the original avian host.