Exploring the Antiviral Potential of Natural Compounds against Influenza: A Combined Computational and Experimental Approach.

The influenza A virus nonstructural protein 1 (NS1), which is crucial for viral replication and immune evasion, has been identified as a significant drug target with substantial potential to contribute to the fight against influenza. The emergence of drug-resistant influenza A virus strains highlights the urgent need for novel therapeutics. This study proposes a combined theoretical criterion for the virtual screening of molecular libraries to identify candidate NS1 inhibitors. By applying the criterion to the ZINC Natural Product database, followed by ligand-based virtual screening and molecular docking, we proposed the most promising candidate as a potential NS1 inhibitor. Subsequently, the selected natural compound was experimentally evaluated, revealing measurable virus replication inhibition activity in cell culture. This approach offers a promising avenue for developing novel anti-influenza agents targeting the NS1 protein.

Influenza NG-34 T cell conserved epitope adjuvanted with CAF01 as a possible influenza vaccine candidate.

Conserved epitopes are targets commonly researched to be part of universal vaccine candidates against influenza viruses (IV). These conserved epitopes need to be cross-protecting against distinct IV subtypes and to have a strong immunogenic potential. Nevertheless, subunit vaccines generally require a strong adjuvant to enhance their immunological effects. Herewith, we compare four different adjuvants differing in their immunological signatures that may enhance efficacy of a conserved hemagglutinin (HA)-epitope from IV, the NG-34, to define the most efficient combination of antigen/adjuvant to combat IV infections. Soluble NG-34 was mixed with adjuvants like aluminium hydroxide (AH) and AddaVax, known to induce Th2 and humoral responses; CAF01 which displays a biased Th1/Th17 profile and Diluvac Forte which augments the humoral response. Combinations were tested in different groups of mice which were subjected to immunological analyses. CAF01 + NG-34 induced a complete immune response with the highest IgG1, IgG2c titers and percentages of activated CD4 T cell promoting IFN-γ, IL-2 and TNF-α producing cells. Furthermore, in NG-34 stimulated mice splenocytes, cytokine levels of IFN-γ, IL-1ß, IL-6, IL-10, IL-17 and TNF-α were also the highest in the CAF01 + NG-34 mouse group. This complete induced immune response covering the humoral and the cellular arms of the adaptive immunity promoted by CAF01 + NG-34 group suggests that CAF01 could be a good candidate as an adjuvant to combine with NG-34 for an efficacious vaccine against IV. However, more studies performed in IV hosts as well as studies with a challenge model are further required.

A genetically engineered H5 protein expressed in insect cells confers protection against different clades of H5N1 highly pathogenic avian influenza viruses in chickens.

The evolution of highly pathogenic H5N1 avian influenza viruses (HPAI-H5N1) has resulted in the appearance of a number of diverse groups of HPAI-H5N1 based on the presence of genetically similar clusters of their haemagglutinin sequences (clades). An H5 antigen encoded by a recombinant baculovirus and expressed in insect cells was used for oil-emulsion-based vaccine prototypes. In several experiments, vaccination was performed at 10 days of age, followed by challenge infection on day 21 post vaccination (PV) with HPAI-H5N1 clades 2.2, 2.2.1, and 2.3.2. A further challenge infection with HPAI-H5N1 clade 2.2.1 was performed at day 42 PV. High haemagglutination inhibition titres were observed for the recH5 vaccine antigen, and lower haemagglutination inhibition titres for the challenge virus antigens. Nevertheless, the rate of protection from mortality and clinical signs was 100% when challenged at 21 days PV and 42 days PV, indicating protection over the entire broiler chicken rearing period without a second vaccination. The unvaccinated control chickens mostly died between two and five days after challenge infection. A low level of viral RNA was detected by reverse transcription followed by a quantitative polymerase chain reaction in a limited number of birds for a short period after challenge infection, indicating a limited spread of HPAI-H5N1 at flock level. Furthermore, it was observed that the vaccine can be used in a differentiation infected from vaccinated animals (DIVA) approach, based on the detection of nucleoprotein antibodies in vaccinated/challenged chickens. The vaccine fulfilled all expectations of an inactivated vaccine after one vaccination against challenge with different clades of H5N1-HPAI and is suitable for a DIVA approach.

The NS segment of H5N1 avian influenza viruses (AIV) enhances the virulence of an H7N1 AIV in chickens.

Some outbreaks involving highly pathogenic avian influenza viruses (HPAIV) of subtypes H5 and H7 were caused by avian-to-human transmissions. In nature, different influenza A viruses can reassort leading to new viruses with new characteristics. We decided to investigate the impact that the NS-segment of H5 HPAIV would have on viral pathogenicity of a classical avian H7 HPAIV in poultry, a natural host. We focussed this study based on our previous work that demonstrated that single reassortment of the NS-segment from an H5 HPAIV into an H7 HPAIV changes the ability of the virus to replicate in mammalian hosts. Our present data show that two different H7-viruses containing an NS-segment from H5-types (FPV NS GD or FPV NS VN) show an overall highly pathogenic phenotype compared with the wild type H7-virus (FPV), as characterized by higher viral shedding and earlier manifestation of clinical signs. Correlating with the latter, higher amounts of IFN-ß mRNA were detected in the blood of NS-reassortant infected birds, 48 h post-infection (pi). Although lymphopenia was detected in chickens from all AIV-infected groups, also 48 h pi those animals challenged with NS-reassortant viruses showed an increase of peripheral monocyte/macrophage-like cells expressing high levels of IL-1ß, as determined by flow cytometry. Taken together, these findings highlight the importance of the NS-segment in viral pathogenicity which is directly involved in triggering antiviral and pro-inflammatory cytokines found during HPAIV pathogenesis in chickens.

Development of a Fully Protective Pandemic Avian Influenza Subunit Vaccine in Insect Pupae.

In this study, we pioneered an alternative technology for manufacturing subunit influenza hemagglutinin (HA)-based vaccines. This innovative method involves harnessing the pupae of the Lepidoptera Trichoplusia ni (T. ni) as natural biofactories in combination with baculovirus vectors (using CrisBio® technology). We engineered recombinant baculoviruses encoding two versions of the HA protein (trimeric or monomeric) derived from a pandemic avian H7N1 virus A strain (A/chicken/Italy/5093/99). These were then used to infect T. ni pupae, resulting in the production of the desired recombinant antigens. The obtained HA proteins were purified using affinity chromatography, consistently yielding approximately 75 mg/L of insect extract. The vaccine antigen effectively immunized poultry, which were subsequently challenged with a virulent H7N1 avian influenza virus. Following infection, all vaccinated animals survived without displaying any clinical symptoms, while none of the mock-vaccinated control animals survived. The CrisBio®-derived antigens induced high titers of HA-specific antibodies in the vaccinated poultry, demonstrating hemagglutination inhibition activity against avian H7N1 and human H7N9 viruses. These results suggest that the CrisBio® technology platform has the potential to address major industry challenges associated with producing recombinant influenza subunit vaccines, such as enhancing production yields, scalability, and the speed of development, facilitating the global deployment of highly effective influenza vaccines.

Genetic diversification patterns in swine influenza A virus (H1N2) in vaccinated and nonvaccinated animals.

Influenza A viruses (IAVs) are characterized by having a segmented genome, low proofreading polymerases, and a wide host range. Consequently, IAVs are constantly evolving in nature causing a threat to animal and human health. In 2009 a new human pandemic IAV strain arose in Mexico because of a reassortment between two strains previously circulating in pigs; Eurasian "avian-like" (EA) swine H1N1 and "human-like" H1N2, highlighting the importance of swine as adaptation host of avian to human IAVs. Nowadays, although of limited use, a trivalent vaccine, which include in its formulation H1N1, H3N2, and, H1N2 swine IAV (SIAV) subtypes, is one of the most applied strategies to reduce SIAV circulation in farms. Protection provided by vaccines is not complete, allowing virus circulation, potentially favoring viral evolution. The evolutionary dynamics of SIAV quasispecies were studied in samples collected at different times from 8 vaccinated and 8 nonvaccinated pigs, challenged with H1N2 SIAV. In total, 32 SIAV genomes were sequenced by next-generation sequencing, and subsequent variant-calling genomic analysis was carried out. Herein, a total of 364 de novo single nucleotide variants (SNV) were found along all genetic segments in both experimental groups. The nonsynonymous substitutions proportion found was greater in vaccinated animals suggesting that H1N2 SIAV was under positive selection in this scenario. The impact of each substitution with an allele frequency greater than 5% was hypothesized according to previous literature, particularly in the surface glycoproteins hemagglutinin and neuraminidase. The H1N2 SIAV quasispecies evolution capacity was evidenced, observing different evolutionary trends in vaccinated and nonvaccinated animals.

Vaccination against swine influenza in pigs causes different drift evolutionary patterns upon swine influenza virus experimental infection and reduces the likelihood of genomic reassortments.

Influenza A viruses (IAVs) can infect a wide variety of bird and mammal species. Their genome is characterized by 8 RNA single stranded segments. The low proofreading activity of their polymerases and the genomic reassortment between different IAVs subtypes allow them to continuously evolve, constituting a constant threat to human and animal health. In 2009, a pandemic of an IAV highlighted the importance of the swine host in IAVs adaptation between humans and birds. The swine population and the incidence of swine IAV is constantly growing. In previous studies, despite vaccination, swine IAV growth and evolution were proven in vaccinated and challenged animals. However, how vaccination can drive the evolutionary dynamics of swine IAV after coinfection with two subtypes is poorly studied. In the present study, vaccinated and nonvaccinated pigs were challenged by direct contact with H1N1 and H3N2 independent swine IAVs seeder pigs. Nasal swab samples were daily recovered and broncho-alveolar lavage fluid (BALF) was also collected at necropsy day from each pig for swine IAV detection and whole genome sequencing. In total, 39 swine IAV whole genome sequences were obtained by next generation sequencing from samples collected from both experimental groups. Subsequently, genomic, and evolutionary analyses were carried out to detect both, genomic reassortments and single nucleotide variants (SNV). Regarding the segments found per sample, the simultaneous presence of segments from both subtypes was much lower in vaccinated animals, indicating that the vaccine reduced the likelihood of genomic reassortment events. In relation to swine IAV intra-host diversity, a total of 239 and 74 SNV were detected within H1N1 and H3N2 subtypes, respectively. Different proportions of synonymous and nonsynonymous substitutions were found, indicating that vaccine may be influencing the main mechanism that shape swine IAV evolution, detecting natural, neutral, and purifying selection in the different analyzed scenarios. SNV were detected along the whole swine IAV genome with important nonsynonymous substitutions on polymerases, surface glycoproteins and nonstructural proteins, which may have an impact on virus replication, immune system escaping and virulence of virus, respectively. The present study further emphasized the vast evolutionary capacity of swine IAV, under natural infection and vaccination pressure scenarios.

VtaA8 and VtaA9 from Haemophilus parasuis delay phagocytosis by alveolar macrophages.

Haemophilus parasuis, a member of the family Pasteurellaceae, is a common inhabitant of the upper respiratory tract of healthy pigs and the etiological agent of Glässer's disease. As other virulent Pasteurellaceae, H. parasuis can prevent phagocytosis, but the bacterial factors involved in this virulence mechanism are not known. In order to identify genes involved in phagocytosis resistance, we constructed a genomic library of the highly virulent reference strain Nagasaki and clones were selected by increased survival after incubation with porcine alveolar macrophages (PAM). Two clones containing two virulent-associated trimeric autotransporter (VtaA) genes, vtaA8 and vtaA9, respectively, were selected by this method. A reduction in the interaction of the two clones with the macrophages was detected by flow cytometry. Monoclonal antibodies were produced and used to demonstrate the presence of these proteins on the bacterial surface of the corresponding clone, and on the H. parasuis phagocytosis-resistant strain PC4-6P. The effect of VtaA8 and VtaA9 in the trafficking of the bacteria through the endocytic pathway was examined by fluorescence microscopy and a delay was detected in the localization of the vtaA8 and vtaA9 clones in acidic compartments. These results are compatible with a partial inhibition of the routing of the bacteria via the degradative phagosome. Finally, antibodies against a common epitope in VtaA8 and VtaA9 were opsonic and promoted phagocytosis of the phagocytosis-resistant strain PC4-6P by PAM. Taken together, these results indicate that VtaA8 and VtaA9 are surface proteins that play a role in phagocytosis resistance of H. parasuis.

Distribution patterns of influenza virus receptors and viral attachment patterns in the respiratory and intestinal tracts of seven avian species.

This study assessed the presence of sialic acid α-2,3 and α-2,6 linked glycan receptors in seven avian species. The respiratory and intestinal tracts of the chicken, common quail, red-legged partridge, turkey, golden pheasant, ostrich, and mallard were tested by means of lectin histochemistry, using the lectins Maackia amurensis agglutinin II and Sambucus nigra agglutinin, which show affinity for α-2,3 and α-2,6 receptors, respectively. Additionally, the pattern of virus attachment (PVA) was evaluated with virus histochemistry, using an avian-origin H4N5 virus and a human-origin seasonal H1N1 virus. There was a great variation of receptor distribution among the tissues and avian species studied. Both α-2,3 and α-2,6 receptors were present in the respiratory and intestinal tracts of the chicken, common quail, red-legged partridge, turkey, and golden pheasant. In ostriches, the expression of the receptor was basically restricted to α-2,3 in both the respiratory and intestinal tracts and in mallards the α-2,6 receptors were absent from the intestinal tract. The results obtained with the lectin histochemistry were, in general, in agreement with the PVA. The differential expression and distribution of α-2,3 and α-2,6 receptors among various avian species might reflect a potentially decisive factor in the emergence of new viral strains.

Sialic acid and sialyl-lactose glyco-conjugates: design, synthesis and binding assays to lectins and swine influenza H1N1 virus.

The terminal parts of the influenza hemagglutinin (HA) receptors α2,6- and α2,3-sialyllactoses were conjugated to an artificial carrier, named sequential oligopeptide carrier (SOC(4) ), to formulate human and avian receptor mimics, respectively. SOC(4) , formed by the tripeptide unit Lys-Aib-Gly, adopts a rigid helicoids-type conformation, which enables the conjugation of biomolecules to the Lys-N(ε) H(2) groups. By doing so, it preserves their initial conformations and functionalities of the epitopes. We report that SOC(4) -glyco-conjugate bearing two copies of the α2,6-sialyllactose is specifically recognized by the biotinylated Sambucus nigra (elderberry) bark lectin, which binds preferentially to sialic acid in an α2,6-linkage. SOC(4) -glyco-conjugate bearing two copies of the α2,3-sialyllactose was not recognized by the biotinylated Maackia amurensis lectin, despite its well-known α2,3-sialyl bond specificity. However, preliminary immune blot assays showed that H1N1 virus binds to both the SOC(4) -glyco-conjugates immobilized onto nitrocellulose membrane. It is concluded that Ac-SOC(4) [(Ac)(2) ,(3'SL-Aoa)(2) ]-NH(2) 5 and Ac-SOC(4) [(Ac)(2) ,(6'SL-Aoa)(2) ]-NH(2) 6 mimic the HA receptors. These findings could be useful for easy screening of binding and inhibition assays of virus-receptor interactions.

Evolution of Swine Influenza Virus H3N2 in Vaccinated and Nonvaccinated Pigs after Previous Natural H1N1 Infection.

Swine influenza viruses (SIV) produce a highly contagious and worldwide distributed disease that can cause important economic losses to the pig industry. Currently, this virus is endemic in farms and, although used limitedly, trivalent vaccine application is the most extended strategy to control SIV. The presence of pre-existing immunity against SIV may modulate the evolutionary dynamic of this virus. To better understand these dynamics, the viral variants generated in vaccinated and nonvaccinated H3N2 challenged pigs after recovery from a natural A(H1N1) pdm09 infection were determined and analyzed. In total, seventeen whole SIV genomes were determined, 6 from vaccinated, and 10 from nonvaccinated animals and their inoculum, by NGS. Herein, 214 de novo substitutions were found along all SIV segments, 44 of them being nonsynonymous ones with an allele frequency greater than 5%. Nonsynonymous substitutions were not found in NP; meanwhile, many of these were allocated in PB2, PB1, and NS1 proteins. Regarding HA and NA proteins, higher nucleotide diversity, proportionally more nonsynonymous substitutions with an allele frequency greater than 5%, and different domain allocations of mutants, were observed in vaccinated animals, indicating different evolutionary dynamics. This study highlights the rapid adaptability of SIV in different environments.

Neuropathogenesis of a highly pathogenic avian influenza virus (H7N1) in experimentally infected chickens.

In order to understand the mechanism of neuroinvasion of a highly pathogenic avian influenza virus (HPAIV) into the central nervous system (CNS) of chickens, specific pathogen free chickens were inoculated with a H7N1 HPAIV. Blood, cerebrospinal fluid (CSF), nasal cavity and brain tissue samples were obtained from 1 to 4 days post-inoculation (dpi) of infected and control chickens. Viral antigen topographical distribution, presence of influenza A virus receptors in the brain, as well as, the role of the olfactory route in virus CNS invasion were studied using different immunohistochemistry techniques. Besides, viral RNA load in CSF and blood was quantified by means of a quantitative real-time reverse transcription-polymerase chain reaction. Viral antigen was observed widely distributed in the CNS, showing bilateral and symmetrical distribution in the nuclei of the diencephalon, mesencephalon and rhombencephalon. Viral RNA was detected in blood and CSF at one dpi, indicating that the virus crosses the blood-CSF-barrier early during infection. This early dissemination is possibly favoured by the presence of Siaα2,3 Gal and Siaα2,6 Gal receptors in brain vascular endothelial cells, and Siaα2,3 Gal receptors in ependymal and choroid plexus cells. No viral antigen was observed in olfactory sensory neurons, while the olfactory bulb showed only weak staining, suggesting that the virus did not use this pathway to enter into the brain. The sequence of virus appearance and the topographical distribution of this H7N1 HPAIV indicate that the viral entry occurs via the haematogenous route, with early and generalized spreading through the CSF.

Pathogenesis and transmissibility of highly (H7N1) and low (H7N9) pathogenic avian influenza virus infection in red-legged partridge (Alectoris rufa).

An experimental infection with highly pathogenic avian influenza virus (HPAIV) and low pathogenic avian influenza virus (LPAIV) was carried out in red-legged partridges (Alectoris rufa) in order to study clinical signs, gross and microscopic lesions, and viral distribution in tissues and viral shedding. Birds were infected with a HPAIV subtype H7N1 (A/Chicken/Italy/5093/1999) and a LPAIV subtype H7N9 (A/Anas crecca/Spain/1460/2008). Uninoculated birds were included as contacts in both groups. In HPAIV infected birds, the first clinical signs were observed at 3 dpi, and mortality started at 4 dpi, reaching 100% at 8 dpi. The presence of viral antigen in tissues and viral shedding were confirmed by immunohistochemistry and quantitative real time RT-PCR (qRRT-PCR), respectively, in all birds infected with HPAIV. However, neither clinical signs nor histopathological findings were observed in LPAIV infected partridges. In addition, only short-term viral shedding together with seroconversion was detected in some LPAIV inoculated animals. The present study demonstrates that the red-legged partridge is highly susceptible to the H7N1 HPAIV strain, causing severe disease, mortality and abundant viral shedding and thus contributing to the spread of a potential local outbreak of this virus. In contrast, our results concerning H7N9 LPAIV suggest that the red-legged partridge is not a reservoir species for this virus.

Pathogenesis of highly pathogenic avian influenza A virus (H7N1) infection in chickens inoculated with three different doses.

To study the pathogenesis of a H7N1 highly pathogenic avian influenza virus strain, specific pathogen free chickens were inoculated with decreasing concentrations of virus: 10(5.5) median embryo lethal dose (ELD(50)) (G1), 10(3.5) ELD(50) (G2) and 10(1.5) ELD(50) (G3). Disease progression was monitored over a period of 16 days and sequential necropsies and tissue samples were collected for histological and immunohistochemical examination. Viral RNA loads were also quantified in different tissues, blood, oropharyngeal swabs, and cloacal swabs using quantitative real-time reverse transcriptase-polymerase chain reaction (RT-qPCR). Clinical signs of depression, apathy, listlessness, huddling and ruffled feathers were recorded in G1 and a few G2 birds, whilst neurological signs were only observed in chickens inoculated with the highest dose. Gross lesions of haemorrhages were observed in the unfeathered skin of the comb and legs, and skeletal muscle, lung, pancreas and kidneys of birds inoculated with 10(5.5) ELD(50) and 10(3.5) ELD(50) doses. Microscopic lesions and viral antigen were demonstrated in cells of the nasal cavity, lung, heart, skeletal muscle, brain, spinal cord, gastrointestinal tract, pancreas, liver, bone marrow, thymus, bursa of Fabricius, spleen, kidney, adrenal gland and skin. Viral RNA was detected by RT-qPCR in kidney, lung, intestine, and brain samples of G1 and G2 birds. However, in birds infected with the lowest dose, viral RNA was detected only in brain and lung samples in low amounts at 5 and 7 days post infection. Interestingly, viral shedding was observed in oropharyngeal and cloacal swabs with proportionate decrease with the inoculation dose. We conclude that although an adequate infectious dose is critical in reproducing the clinical infection, chickens exposed to lower doses can be infected and shed virus representing a risk for the dissemination of the viral agent.

Identification and Characterization of Swine Influenza Virus H1N1 Variants Generated in Vaccinated and Nonvaccinated, Challenged Pigs.

Influenza viruses represent a continuous threat to both animal and human health. The 2009 H1N1 A influenza pandemic highlighted the importance of a swine host in the adaptation of influenza viruses to humans. Nowadays, one of the most extended strategies used to control swine influenza viruses (SIVs) is the trivalent vaccine application, whose formulation contains the most frequently circulating SIV subtypes H1N1, H1N2, and H3N2. These vaccines do not provide full protection against the virus, allowing its replication, evolution, and adaptation. To better understand the main mechanisms that shape viral evolution, here, the SIV intra-host diversity was analyzed in samples collected from both vaccinated and nonvaccinated animals challenged with the H1N1 influenza A virus. Twenty-eight whole SIV genomes were obtained by next-generation sequencing, and differences in nucleotide variants between groups were established. Substitutions were allocated along all influenza genetic segments, while the most relevant nonsynonymous substitutions were allocated in the NS1 protein on samples collected from vaccinated animals, suggesting that SIV is continuously evolving despite vaccine application. Moreover, new viral variants were found in both vaccinated and nonvaccinated pigs, showing relevant substitutions in the HA, NA, and NP proteins, which may increase viral fitness under field conditions.

Sow Vaccination with a Protein Fragment against Virulent Glaesserella (Haemophilus) parasuis Modulates Immunity Traits in Their Offspring.

Glaesserella (Haemophilus) parasuis, an early colonizer of the nasal cavity in piglets, is a highly heterogeneous species, comprising both commensal and virulent strains. Virulent G. parasuis strains can cause fibrinous polyserositis called Glässer's disease. Colostrum is a source of passive immunity for young piglets. When vaccinating sows, protective antibodies are transferred to their offspring through the colostrum. Here, sow vaccination was performed with a protein fragment, F4, from the outer membrane trimeric autotransporters VtaAs exclusively found in virulent G. parasuis. Piglets were allowed to suckle for 3 weeks, following which a challenge with two virulent strains of G. parasuis was performed. A group of nonvaccinated sows and their piglets were included as a control. Antibodies against F4 were confirmed using ELISA in the vaccinated sows and their offspring before the G. parasuis challenge. Compared to the control group, F4-vaccination also resulted in an increased level of serum TGF-ß both in vaccinated sows and in their offspring at early time points of life. After the challenge, a lower body temperature and a higher weight were observed in the group of piglets from vaccinated sows. One piglet from the non-vaccinated group succumbed to the infection, but no other significant differences in clinical signs were noticed. At necropsy, performed 2 weeks after the virulent challenge, the level of surfactant protein D (SP-D) in bronchoalveolar lavage was higher in the piglets from vaccinated sows. Vaccination did not inhibit the nasal colonization of the piglets by the challenge strains.

Immune Responses to Pandemic H1N1 Influenza Virus Infection in Pigs Vaccinated with a Conserved Hemagglutinin HA1 Peptide Adjuvanted with CAF®01 or CDA/αGalCerMPEG.

This study aimed to evaluate the immune response and protection correlates against influenza virus (IV) infection in pigs vaccinated with the novel NG34 HA1 vaccine candidate adjuvanted with either CAF®01 or CDA/αGalCerMPEG (αGCM). Two groups of six pigs each were vaccinated intramuscularly twice with either NG34 + CAF®01 or NG34 + CDA/αGCM. As controls, groups of animals (n = 6 or 4) either non-vaccinated or vaccinated with human seasonal trivalent influenza vaccine or NG34 + Freund's adjuvant were included in the study. All animal groups were challenged with the 2009 pandemic (pdm09) strain of H1N1 (total amount of 7 × 106 TCID50/mL) via intranasal and endotracheal routes 21 days after second vaccination. Reduced consolidated lung lesions were observed both on days three and seven post-challenge in the animals vaccinated with NG34 + CAF®01, whereas higher variability with relatively more severe lesions in pigs of the NG34 + CDA/αGCM group on day three post-infection. Among groups, animals vaccinated with NG34 + CDA/αGCM showed higher viral loads in the lung at seven days post infection whereas animals from NG34 + CAF®01 completely abolished virus from the lower respiratory tract. Similarly, higher IFNγ secretion and stronger IgG responses against the NG34 peptide in sera was observed in animals from the NG34 + CAF®01 group as compared to the NG34 + CDA/αGCM. NG34-vaccinated pigs with adjuvanted CAF®01 or CDA/αGCM combinations resulted in different immune responses as well as outcomes in pathology and viral shedding.

Antibody targeting the ferritin-like protein controls Listeria infection.

The acquisition of iron during the infection process is essential for the growth of pathogenic microorganisms (S. C. Andrews, Adv. Microb. Physiol. 40:281-351, 1998; H. M. Baker, B. F. Anderson, and E. N. Baker, Proc. Natl. Acad. Sci. U. S. A. 100:3579-3583, 2003). Since the solubility of iron is low and it is toxic at low concentrations, following uptake, iron is stored in subcellular microenvironments in the iron storage protein ferritin (C. Cheers and M. Ho, J. Reticuloendothel. Soc. 34:299-309, 1983). Here, we show that ferritin-like proteins (Frl) are highly conserved in the genus Listeria and demonstrate that these proteins are present in both the cytoplasm and cell wall fractions of these bacteria. Even though Frl is expressed under different growth conditions, transcriptional mapping revealed that its regulation is complex. When bacteria are grown in brain heart infusion medium, extracellular expression involves both sigma A (SigA)- and sigma B (SigB)-dependent promoters; however, during intracellular growth, initiation of transcription is additionally SigB dependent. The expression of Frl is greatly enhanced in bacteria grown in the presence of blood, and a mutant strain lacking the frl gene was defective for growth in this medium. Using the monoclonal antibody (MAb) specific for Frl, we demonstrate that administration of anti-Frl MAb prior to infection confers antilisterial resistance in vivo, evidenced in reduced bacterial load and increased survival rates, thereby demonstrating the in vivo significance of upregulated cell surface-associated Frl expression. In vitro studies revealed that the antilisterial resistance is due to increased listerial phagocytosis.

Persistence of highly pathogenic avian influenza virus (H7N1) in infected chickens: feather as a suitable sample for diagnosis.

Selection of an ideal sample is a vital element in early detection of influenza infection. Rapid identification of infectious individuals or animals is crucial not only for avian influenza virus (AIV) surveillance programmes, but also for treatment and containment strategies. This study used a combination of quantitative real-time RT-PCR with an internal positive control and a cell-titration system to examine the presence of virus in different samples during active experimental AIV infection and its persistence in the infected carcasses. Oropharyngeal/cloacal swabs as well as feather pulp and blood samples were collected from 15-day-old chicks infected with H7N1 highly pathogenic AIV (HPAIV) and the kinetics of virus shedding during active infection were evaluated. Additionally, several samples (muscle, skin, brain, feather pulp and oropharyngeal and cloacal swabs) were examined to assess the persistence of virus in the HPAIV-infected carcasses. Based on the results, feather pulp was found to be the best sample to detect and isolate HPAIV from infected chicks from 24 h after inoculation onwards. Kinetic studies on the persistence of virus in infected carcasses revealed that tissues such as muscle could potentially transmit infectious virus for 3 days post-mortem (p.m.), whilst other tissues such as skin, feather pulp and brain retained their infectivity for as long as 5-6 days p.m. at environmental temperature (22-23 degrees C). These results strongly favour feather as a useful sample for HPAIV diagnosis in infected chickens as well as in carcasses.

Identification of a parasitic immunomodulatory protein triggering the development of suppressive M1 macrophages during African trypanosomiasis.

Development of classically activated macrophages (M1 cells) is a prerequisite to controlling parasite growth and therefore resistance to African trypanosomiasis. However, if activation of M1 cells is uncontrolled, including their production of tumor necrosis factor (TNF) and nitric oxide (NO), collateral pathogenic damage to tissues ensues. We report the identification of a novel putative Trypanosoma brucei M1 cell-triggering protein. The recombinant trypanosome-suppressive immunomodulating factor (rTSIF) induced TNF and NO secretion by macrophages. Moreover, M1 cells triggered by rTSIF block T cell proliferation in a manner dependent on NO, interferon gamma, and cell contact. Furthermore, rTSIF could down-regulate type 2-oriented immune responses. Therefore, trypanosome-suppressive immunomodulating factor (TSIF) may represent a new parasite molecule with the potential to modulate the host immune network, whereby it could contribute to the inflammatory response required to control parasite growth and to the pathogenicity of African trypanosomiasis, including immunosuppression. TSIF knock-down trypanosomes died within 2 days, indicating that TSIF may be essential for parasite biology.