Differences in pathogenicity, response to vaccination, and innate immune responses in different types of ducks infected with a virulent H5N1 highly pathogenic avian influenza virus from Vietnam.

In a previous study, we found clear differences in pathogenicity and response to vaccination against H5N1 highly pathogenic avian influenza (HPAI; HA dade 2.3.4) between Pekin (Anas platyrhynchos var. domestica) and Muscovy (Cairina moschata) ducks vaccinated using a commercial inactivated vaccine (Re-1). The objective of the present study was to further investigate the pathogenicity of H5N1 HPAI viruses in different species of ducks by examining clinical signs and innate immune responses to infection with a different strain of H5N1 HPAI virus (HA clade 1) in two domestic ducks, Pekin and Muscovy, and one wild-type duck, mallard (Anas platyrhynchos). Protection conferred by vaccination using the Re-1 vaccine against infection with this virus was also compared between Pekin and Muscovy ducks. Differences in pathogenicity were observed among the virus-infected ducks, as the Muscovy ducks died 2 days earlier than did the Pekin and mallard ducks, and they presented more-severe neurologic signs. Conversely, the Pekin and mallard ducks had significantly higher body temperatures at 2 days postinfection (dpi) than did the Muscovy ducks, indicating possible differences in innate immune responses. However, similar expression of innate immune-related genes was found in the spleens of virus-infected ducks at this time point. In all three duck species, there was up-regulation of IFN-alpha, IFN-gamma, IL-6, CCL19, RIG-I, and MHC class I and down-regulation of MHC class II, but variable expression of IL-18 and TLR7. As in our previous study, vaccinated Muscovy ducks showed less protection against virus infection than did Pekin ducks, as evidenced by the higher mortality and higher number of Muscovy ducks shedding virus when compared to Pekin ducks. In conclusion, infection with an H5N1 HPAI virus produced a systemic infection with high mortality in all three duck species; however, the disease was more severe in Muscovy ducks, which also had a poor response to vaccination. The differences in response to virus infection could not be explained by differences in the innate immune responses between the different types of ducks when examined at 2 days dpi, and earlier time points need to be evaluated.

Effect of age on the pathogenesis and innate immune responses in Pekin ducks infected with different H5N1 highly pathogenic avian influenza viruses.

The pathogenicity of H5N1 highly pathogenic avian influenza (HPAI) viruses in domestic ducks varies between different viruses and is affected by the age of the ducks, with younger ducks presenting a more severe disease. In order to better understand the pathobiology of H5N1 HPAI in ducks including the role of host responses, 2 and 5-week-old Pekin ducks were infected with three different H5N1 HPAI viruses. Virus-induced pathology ranged from no clinical signs to severe disease and mortality, with the 2-week-old ducks being more severely affected by the more virulent viruses. However, these more virulent viruses induced higher body temperatures in the 5-week-old ducks than in the 2-week-old ducks indicating possible differences in innate immune responses. To analyze the ducks host responses to H5N1 HPAI virus infection, expression of innate immune-related genes was measured in the spleens and lungs of infected ducks at the peak of virus infection. IFN-α, RIG-I, and IL-6 RNA levels were increased in spleens regardless of the virus given and the age of the ducks, however differences were observed in the levels of up-regulation of IFN-α and RIG-I between the 2 and the 5-week-old ducks with the more virulent virus. Differences in IL-2 gene expression were also observed. In the lungs, the levels of expression of innate immune-related genes were lower than in the spleen, with mostly up-regulation of RIG-I and IL-6 and down-regulation of IFN-α and IL-2; no significant difference in expression was found between the 2 and the 5-week-old ducks. The differences observed in the innate immune responses to infection with H5N1 HPAI viruses could explain in part the differences in pathogenicity found between the 2 and 5-week-old ducks, however earlier time points after infection and additional innate immune-related genes should be examined.

Regulation of the integrase and cassette promoters of the class 1 integron by nucleoid-associated proteins.

The integrase IntI1 catalyses recombination of antibiotic-resistance gene cassettes in the integron, a widely found bacterial mobile element active in spreading antibiotic multi-resistance. We have previously shown that resistance cassette recombination rate and specificity depend on the amount of intracellular integrase. Here, we used in vivo and in vitro methods to examine convergent expression of the integrase promoter (P(int)) and of the cassette promoters (P(c) and P(2)) in the prototypical plasmid-borne class 1 integron, In2. Highly conserved P(int) has near consensus -10 and -35 hexamers for σ(70) RNA polymerase, but there are 11 naturally occurring arrangements of P(c) alone or combinations of the P(c)+P(2) cassette promoters (note that P(2) occurs with a 14 or 17 bp spacer). Using a bi-directional reporter vector, we found that P(int) is a strong promoter in vivo, but its expression is reduced by converging transcription from P(c) and P(2). In addition to cis-acting convergence control of integrase expression, the regulator site prediction program, prodoric 8.9, identified sites for global regulators FIS, LexA, IHF and H-NS in and near the integron promoters. In strains mutated in each global regulator, we found that: (1) FIS repressed integrase and cassette expression; (2) LexA repressed P(int) and P(2) with the 14 bp spacer version of P(2) and FIS was necessary for maximum LexA repression; (3) IHF activated P(int) when it faced the strong 17 bp spacer P(2) but did not elevate its expression versus LexA-repressed P(2) with the 14 bp spacer; and (4) H-NS repressed both P(int) and the 14 bp P(2) but activated the 17 bp P(2) cassette promoters. Mobility shift assays showed that FIS and IHF interact directly with the promoter regions and DNase I footprinting confirmed extensive protection by FIS of wild-type In2 integron promoter sequence. Thus, nucleoid-associated proteins, known to act directly in site-specific recombination, also control integron gene expression directly and possibly indirectly.

Pekin and Muscovy ducks respond differently to vaccination with a H5N1 highly pathogenic avian influenza (HPAI) commercial inactivated vaccine.

Domestic ducks are key intermediates in the transmission of H5N1 highly pathogenic avian influenza (HPAI) viruses, and therefore are included in vaccination programs to control H5N1 HPAI. Although vaccination has proven effective in protecting ducks against disease, different species of domestic ducks appear to respond differently to vaccination, and shedding of the virus may still occur in clinically healthy vaccinated populations. In this study we compared the response to vaccination between two common domestic duck species, Pekin (Anas platyrhynchos domesticus) and Muscovy (Cairina moschata), which were vaccinated with a commercial inactivated vaccine using one of three different schedules in order to elicit protection to H5N1 HPAI before one month of age. Clear differences in responses to vaccination were observed; the Muscovy ducks developed lower viral antibody titers induced by the same vaccination as Pekin ducks and presented with higher morbidity and mortality after challenge with an H5N1 HPAI virus. When comparing the response to infection in non-vaccinated ducks, differences were also observed, with infected Muscovy ducks presenting a lower mean death time and more severe neurological signs than Pekin ducks. However Pekin ducks had significantly higher body temperatures and higher levels of nitric oxide in the blood at 2 days post challenge than Muscovy ducks, indicating possible differences in innate immune responses. Comparison of the expression of innate immune related genes in spleens of the non-vaccinated infected ducks showed differences including significantly higher levels of expression of RIG-I in Pekin ducks and of IL-6 in Muscovy ducks. Both duck species showed an up-regulation of IFNα and MHC-I expression, and a down-regulation of MHC-II. In conclusion, differences in response to infection and vaccination were observed between the two domestic duck species. This information should be taken into account when developing effective vaccination programs for controlling H5N1 HPAI in different species of ducks.

In vivo transcriptional cytokine responses and association with clinical and pathological outcomes in chickens infected with different Newcastle disease virus isolates using formalin-fixed paraffin-embedded samples.

Little is known about the host response of chickens infected with Newcastle disease virus (NDV) and the relationship between the innate immune response and the severity of clinical disease. Measurement of cytokine responses during infection in vivo can help to elucidate the mechanisms of virus pathogenesis. The transcriptional response of several cytokines from paraffin-embedded, formalin-fixed spleen of chicken naturally infected by NDV velogenic viscerotropic viruses was compared to the responses of atypical velogenic, velogenic neurotropic, and mesogenic strains during the first five days after infection. The RNA expression for IFN-γ and IL-6 was enhanced at day two in the highly virulent velogenic viscerotropic viruses (California and rZJ1 strains) and corresponded with the presence of the virus in tissues. However, in one atypical velogenic viscerotropic virus (Australia strain), two velogenic neurotropic viruses (Turkey ND and Texas GB) and, a mesogenic virus (Anhinga strain) the cytokine responses to infection were delayed or reduced. Increased levels of IFN-ß RNA expression were only detected in the velogenic viscerotropic virus infected chickens (California and rZJ1 strains) at 3 days post-infection and one mesogenic strain (Anhinga) early in infection. The RNA expression levels of IL-2 did not increase upon infection with any of the viruses. A pronounced increase of RNA expression levels of IL-6 and IFN-γ was detected simultaneously with infiltration of macrophages and/or lymphoid necrosis in the histopathological analysis of the spleen and cecal tonsils. The differences in the RNA expression levels may help explain possible underlying mechanisms of clinical disease and/or immune responses in birds infected with strains of APMV-1 that cause distinct pathologic changes.