Raquel Rivas

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.

Influenza A virus subtypes in wild birds in North-Eastern Spain (Catalonia).

Since the spread of H5N1 highly pathogenic avian influenza virus in 2005, many surveillance programmes have been initiated in poultry and wild birds worldwide. This study describes for the first time the detection of different subtypes of avian influenza viruses (AIV) in wild birds in the West Mediterranean area (Catalonia, North-Eastern Spain). During a 3-year period (from mid-2006 to mid-2009), 1374 birds from 16 different families were examined, and a total of 62 AIV were detected by means of a real-time reverse transcriptase PCR assay. AIV were more frequently detected in Anatidae, Phoenicopteridae, Rallidae and Laridae families. Of the 62 positive samples, 28 AIV could be isolated in embryonated eggs. All isolates were subtyped by haemagglutinin and neuraminidase inhibition techniques and 10 different haemagglutinins (HA) and 7 neuraminidases (NA) were found in 13 different subtype combinations. The most common combinations were H4N6 (22.2%) and H1N1 (18.5%). The HA and NA gene sequences of different AIV subtypes were compared and aligned with those available AIV strains from genome databases. Our studies on AIV phylogenetic analysis revealed that all AIV genes sequenced from wild birds in North-Eastern Spain clustered within Eurasian avian clades, including the sequences of H8, N4 and N5 genes analyzed for the first time in Europe. The results contribute to the understanding of AIV in the Mediterranean area and in Europe.

Conserved synthetic peptides from the hemagglutinin of influenza viruses induce broad humoral and T-cell responses in a pig model.

Outbreaks involving either H5N1 or H1N1 influenza viruses (IV) have recently become an increasing threat to cause potential pandemics. Pigs have an important role in this aspect. As reflected in the 2009 human H1N1 pandemia, they may act as a vehicle for mixing and generating new assortments of viruses potentially pathogenic to animals and humans. Lack of universal vaccines against the highly variable influenza virus forces scientists to continuously design vaccines à la carte, which is an expensive and risky practice overall when dealing with virulent strains. Therefore, we focused our efforts on developing a broadly protective influenza vaccine based on the Informational Spectrum Method (ISM). This theoretical prediction allows the selection of highly conserved peptide sequences from within the hemagglutinin subunit 1 protein (HA1) from either H5 or H1 viruses which are located in the flanking region of the HA binding site and with the potential to elicit broader immune responses than conventional vaccines. Confirming the theoretical predictions, immunization of conventional farm pigs with the synthetic peptides induced humoral responses in every single pig. The fact that the induced antibodies were able to recognize in vitro heterologous influenza viruses such as the pandemic H1N1 virus (pH1N1), two swine influenza field isolates (SwH1N1 and SwH3N2) and a H5N1 highly pathogenic avian virus, confirm the broad recognition of the antibodies induced. Unexpectedly, all pigs also showed T-cell responses that not only recognized the specific peptides, but also the pH1N1 virus. Finally, a partial effect on the kinetics of virus clearance was observed after the intranasal infection with the pH1N1 virus, setting forth the groundwork for the design of peptide-based vaccines against influenza viruses. Further insights into the understanding of the mechanisms involved in the protection afforded will be necessary to optimize future vaccine formulations.

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.

Experimental West Nile virus infection in Gyr-Saker hybrid falcons.

West Nile disease (WND) has become a major public and veterinary health concern since the appearance of West Nile virus (WNV) in New York in 1999. The following panzootic spread in the U.S. and the recent WNV outbreaks in Europe and the Mediterranean Basin have increased interest in WND. Despite considerable investigation of WNV infection in birds, the effects of WNV on avian populations are still largely unknown. In Europe, raptors have been found to be particularly susceptible to WNV infection, but studies in birds of prey are lacking. To our knowledge, the present study is the first to report an experimental infection with WNV in Gyr-Saker hybrid falcons. We show that 10-week-old captive-reared Gyr-Saker (Falco rusticolus × Falco cherrug) hybrid falcons are susceptible to WNV infection. Neither morbidity nor mortality was observed after subcutaneous WNV inoculation with mixed extracts of non-infected mosquito salivary glands. Both the macroscopic and microscopic lesions observed were similar to those previously reported in naturally and experimentally infected North American raptors. The results obtained in the present study demonstrate that although Gyr-Saker hybrid falcons do not seem to be a good reservoir for WNV transmission via mosquito, they can become infected with WNV, develop viremia and antibodies, and are able to shed the virus.

Efficacy assessment of an MVA vectored Rift Valley Fever vaccine in lambs.

The present study has evaluated the protection conferred by a single subcutaneous dose of a modified vaccinia virus Ankara (MVA) vectored vaccine encoding the Rift Valley Fever virus (RVFV) glycoproteins Gn and Gc in lambs. Three groups of six to seven lambs were immunized as follows: one group received the vaccine (termed rMVA-GnGc), a second group received an MVA vector (vector control) and a third group received saline solution (non-vaccinated control). Fourteen days later, all animals were subcutaneously challenged with 10(5) TCID50 of the virulent RVFV isolate 56/74 and vaccine efficacy assessed using standard endpoints. Two lambs (one from the vaccine group and one from the vector control group) succumbed to RVFV challenge, showing characteristic liver lesions. Lambs from both the vector control and non-vaccinated groups were febrile from days 2 to 5 post challenge (pc) while those in the rMVA-GnGc group showed a single peak of pyrexia at day 3 pc. RVFV RNA was detected in both nasal and oral swabs from days 3 to 7 pc in some lambs from the vector control and non-vaccinated groups, but no viral shedding could be detected in the surviving lambs vaccinated with rMVA-GnGc. Together, the data suggest that a single dose of the rMVA-GnGc vaccine may be sufficient to reduce RVFV shedding and duration of viremia but does not provide sterile immunity nor protection from disease. Further optimization of this vaccine approach in lambs is warranted.

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: 105.5 median embryo lethal dose (ELD50) (G1), 103.5 ELD50 (G2) and 101.5 ELD50 (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 105.5 ELD50 and 103.5 ELD50 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.

Epidemiological and pathological investigation of fowl aviadenovirus serotypes 8b and 11 isolated from chickens with inclusion body hepatitis in Spain (2011–2013)

ABSTRACT Inclusion body hepatitis caused by different fowl aviadenovirus (FAdV) serotypes has been described in several countries in recent years. In Spain, from the spring of 2011 to 2013, an increased number of outbreaks in broiler and broiler breeder flocks from different regions occurred. The objectives of the present work were to carry out the molecular characterization of FAdV strains from Spanish inclusion body hepatitis cases and to study the pathogenicity and viral dynamics of these strains in specific pathogen-free (SPF) chickens. A total of 52 inclusion body hepatitis clinical cases, including 45 from broiler farms and seven from broiler breeder farms, were analysed by conventional polymerase chain reaction and sequencing targeting the FAdV hexon gene. From these, 37 strains were classified as FAdV type 8b, while the remaining 15 were classified as FAdV types 11 (n = 10), 2 (n = 4) and 8a (n = 1). In addition, two different FAdVs belonging to the genotypes 8b and 11 were used for experimental infection. Specific pathogen-free five-day-old birds were inoculated intramuscularly with a high (106.5 tissue culture infective dose (TCID)50/ml) or low (104 TCID50/ml) dose of the above-mentioned FAdVs. No mortality was observed in any of the experimental groups, and only one bird showed evident clinical signs. However, macroscopic and microscopic hepatic lesions, as well as viral DNA, were detected in birds from all infection groups. Inclusion bodies and viral DNA were also detected in the pancreas and in the small and the large intestine in some birds. Long-lasting shedding and transmission to contact birds were confirmed in all infected groups.

Exposure to a Low Pathogenic A/H7N2 Virus in Chickens Protects against Highly Pathogenic A/H7N1 Virus but Not against Subsequent Infection with A/H5N1

Recent evidences have demonstrated that the presence of low pathogenic avian influenza viruses (LPAIV) may play an important role in host ecology and transmission of avian influenza viruses (AIV). While some authors have clearly demonstrated that LPAIV can mutate to render highly pathogenic avian influenza viruses (HPAIV), others have shown that their presence could provide the host with enough immunological memory to resist re-infections with HPAIV. In order to experimentally study the role of pre-existing host immunity, chickens previously infected with H7N2 LPAIV were subsequently challenged with H7N1 HPAIV. Pre-infection of chickens with H7N2 LAPIV conferred protection against the lethal challenge with H7N1 HPAIV, dramatically reducing the viral shedding, the clinical signs and the pathological outcome. Correlating with the protection afforded, sera from chickens primed with H7N2 LPAIV reacted with the H7-AIV subtype in hemagglutination inhibition assay and specifically with the N2-neuraminidase antigen. Conversely, subsequent exposure to H5N1 HPAIV resulted in a two days-delay on the onset of disease but all chickens died by 7 days post-challenge. Lack of protection correlated with the absence of H5-hemagglutining inhibitory antibodies prior to H5N1 HPAIV challenge. Our data suggest that in naturally occurring outbreaks of HPAIV, birds with pre-existing immunity to LPAIV could survive lethal infections with HA-homologous HPAIV but not subsequent re-infections with HA-heterologous HPAIV. These results could be useful to better understand the dynamics of AIV in chickens and might help in future vaccine formulations.

Lymphoplasmacytic Endotheliitis and Anterior Uveitis in Sheep Infected Experimentally with Rift Valley Fever Virus

Lymphoplasmacytic endotheliitis and anterior uveitis was diagnosed in four lambs infected experimentally with field isolates of Rift Valley fever virus (RVFV). Formalin-fixed and paraffin wax-embedded tissue from these animals was investigated by histopathology and quantitative real time reverse transcriptase polymerase chain reaction. To our knowledge, this is the first pathological description of this ocular manifestation of RVFV infection in ruminants, although these lesions have been described in man.