Poul Henrik Jørgensen

Detection and subtyping (H5 and H7) of avian type A influenza virus by reverse transcription-PCR and PCR-ELISA.

Summary.  Avian influenza virus infections are a major cause of morbidity and rapid identification of the virus has important clinical, economical and epidemiological implications. We have developed a one-tube Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) for the rapid diagnosis of avian influenza A. A panel of reference influenza strains from various hosts including avian species, human, swine and horse were evaluated in a one tube RT-PCR using primers designed for the amplification of a 218 bp fragment of the NP gene. The PCR products were detected by PCR-ELISA by use of an internal catching probe confirming the NP influenza A origin. The PCR-ELISA was about 100 times more sensitive than detection of PCR products by agarose gel electrophoresis. RT-PCR and detection by PCR-ELISA is comparable in sensitivity to virus propagation in eggs. We also designed primers for the detection of the influenza. A subtypes H5 and H7 shown to have pathogenic potential in poultry. The H5 primers cover the cleavage site of the HA gene and specifically amplify influenza A subtype H5. The H7 primers also cover the HA cleavage site and detected all H7 reference strains investigated. In addition, the H7 primers also amplified very weak and/or additional bands on an agarose gel from other subtypes. However, the H7 origin and the pathogenic potential defined by the presence or absence of basic amino acids at the cleavage site can be determined by sequencing of the PCR product. As far as we know this is the first demonstration of RT-PCR detection on a panel of H7 strains using only one primer set.

Identification of Sensitive and Specific Avian Influenza Polymerase Chain Reaction Methods Through Blind Ring Trials Organized in the European Union

Abstract Many different polymerase chain reaction (PCR) protocols have been used for detection and characterization of avian influenza (AI) virus isolates, mainly in research settings. Blind ring trials were conducted to determine the most sensitive and specific AI PCR protocols from a group of six European Union (EU) laboratories. In part 1 of the ring trial the laboratories used their own methods to test a panel of 10 reconstituted anonymized clinical specimens, and the best methods were selected as recommended protocols for part 2, in which 16 RNA specimens were tested. Both panels contained H5, H7, other AI subtypes, and non-AI avian pathogens. Outcomes included verification of 1) generic AI identification by highly sensitive and specific M-gene real-time PCR, and 2) conventional PCRs that were effective for detection and identification of H5 and H7 viruses. The latter included virus pathotyping by amplicon sequencing. The use of recommended protocols resulted in improved results among all six laboratories in part 2, reflecting increased sensitivity and specificity. This included improved H5/H7 identification and pathotyping observed among all laboratories in part 2. Details of these PCR methods are provided. In summary, this study has contributed to the harmonization of AI PCR protocols in EU laboratories and influenced AI laboratory contingency planning following the first European reports of H5N1 highly pathogenic AI during autumn 2005.

First introduction of highly pathogenic H5N1 avian influenza A viruses in wild and domestic birds in Denmark, Northern Europe

BackgroundSince 2005 highly pathogenic (HP) avian influenza A H5N1 viruses have spread from Asia to Africa and Europe infecting poultry, humans and wild birds. HP H5N1 virus was isolated in Denmark for the first time in March 2006. A total of 44 wild birds were found positive for the HP H5N1 infection. In addition, one case was reported in a backyard poultry flock.ResultsFull-genome characterisation of nine isolates revealed that the Danish H5N1 viruses were highly similar to German H5N1 isolates in all genes from the same time period. The haemagglutinin gene grouped phylogenetically in H5 clade 2 subclade 2 and closest relatives besides the German isolates were isolates from Croatia in 2005, Nigeria and Niger in 2006 and isolates from Astrakhan in Russia 2006. The German and Danish isolates shared unique substitutions in the NA, PB1 and NS2 proteins.ConclusionThe first case of HP H5N1 infection of wild and domestic birds in Denmark was experienced in March 2006. This is the first full genome characterisation of HP H5N1 avian influenza A virus in the Nordic countries. The Danish viruses from this time period have their origin from the wild bird strains from Qinghai in 2005. These viruses may have been introduced to the Northern Europe through unusual migration due to the cold weather in Eastern Europe at that time.

The use of serotype 1- and serotype 3-specific polymerase chain reaction for the detection of Marek's disease virus in chickens

A serotype 1- and serotype 3-specific detection of Mareks disease virus (MDV) by polymerase chain reaction (PCR) was developed. The sensitivity of the method when applied to cell culture grown virus was comparable with that of cultivation. The method was applied to various tissue samples from chickens experimentally inoculated with serotype 1 or serotype 3 MDV.The serotype 1 strains CVI988 and RB-1B could be detected in feather follicle epithelium up to 56 and 84 days post-inoculation (p.i.), respectively, while the MDV-3 serotype was detected until 42 days p.i. The purpose of this study was to develop and evaluate a reliable and easy-to-handle method for surveillance of the occurrence of MDV in chicken flocks. We emphasize the development of a method, which can be applied to types of samples conveniently collected in the field, e.g. feather tips and blood samples. In addition, the PCR was applied to samples collected from four commercial table egg layer flocks of young stock or pullets vaccinated with either serotype 1 (CVI988) or serotype 3 (HVT) vaccine. These flocks had various clinical signs of Mareks disease. MDV-1 was detected in buffy-coat cells, spleen, liver, skin, feather tips and ovaries. The detection of MDV in feather tips appeared to be as sensitive as co-cultivation of buffy-coat cells, although an inhibiting factor was observed in extracts from feather tips of non-white chickens. This inhibition could be overcome in most extracts by applying a bovine serum albumen pretreatment. The PCR proved to be a convenient tool for the monitoring of MDV in the poultry population, and feather tips were the most convenient and sensitive samples.

Transcriptional profiles of chicken embryo cell cultures following infection with infectious bursal disease virus

Summary.Infectious bursal disease virus (IBDV) is the causative agent of infectious bursal disease in chickens and causes a significant economic loss for the poultry industry. Little is understood about the mechanism involved in the host responses to IBDV infection. For better understanding the IBDV-host interaction, we measured steady-state levels of transcripts from 28 cellular genes of chicken embryo (CE) cell cultures infected with IBDV vaccine stain Bursine-2 during a 7-day infection course by use of the quantitative real-time RT-PCR SYBR green method. Of the genes tested, 21 genes (IRF-1, IFN 1-2 promoter, IFNAR-1, IRF-10, IFN-γ, 2′,5′-OAS, IAP-1, caspase 8, TRAIL-like, STAT-3, IL-6, IL-8, MIP-3α, MHC-I, MHC-II, TVB, GLVR-1, OTF, IL-13Rα, ST3GAL-VI and PGK) showed an increased expression. The remaining seven genes (IFNAR-2, IFN-α, NF-κB subunit p65, BLRcp38, DDX1, G6PDH and UB) showed a constant expression or only slight alteration. Apparently, the host genes involved in pro-inflammatory response and apoptosis, interferon-regulated proteins, and the cellular immune response were affected by IBDV infection, indicating involvement in the complex signaling pathways of host responses to the infection. This study thus contributes to the understanding of the pathogenesis of IBD and provides an insight into the virus-host interaction.

Detection and strain differentiation of infectious bronchitis virus in tracheal tissues from experimentally infected chickens by reverse transcription-polymerase chain reaction. Comparison with an immunohistochemical technique

Oligonucleotide pairs were constructed for priming the amplification of fragments of nucleocapsid (N) protein and spike glycoprotein (S) genes of avian infectious bronchitis virus (IBV) by reverse transcriptionpolymerase chain reaction (RT-PCR). One oligonucleotide pair amplified a common segment of the N-gene and could detect various strains of IBV in allantoic fluid from inoculated chicken embryos, and in tracheal tissue preparations from experimentally infected chickens. Four pairs of oligonucleotides selectively primed the amplification of the S1 gene of Massachusetts/Connecticut, D1466, D274/D3896 and 793B strains of IBV, respectively. Groups of specific pathogen free chickens were experimentally inoculated with the Massachusetts (H120, M41), the D1466 and the 793B strains of IBV, and tracheal tissue preparations were made from each bird for RT-PCR and for immunohistochemistry (IHC) up to 3 days post-inoculation. The N-gene RT-PCR detected IBV in 82% of the chickens, while IHC only detected IBV in 60%. This difference was significant (P<0.02). The detection rate by N-gene RT-PCR varied from 67 to 100% for the various strains of IBV inoculated. The S1 gene oligonucleotide pairs were applied to the same tissue preparations and they detected specifically the Massachusetts (M41 and H120), the D1466 and the 793B strains of IBV at rates varying between 58 and 92%. When the mixtures of the primers were applied, the detection rate in tissue preparations was reduced to the level of 50 to 67%. It is concluded that the direct detection of IBV in tracheal tissues by RT-PCR is more sensitive than IHC and that the RT-PCR technique is able to distinguish between types of IBV.

Molecular Characterization and Phylogenetic Study of Newcastle Disease Virus Isolates from Recent Outbreaks in Eastern Uganda

ABSTRACT Newcastle disease virus isolates from chickens in eastern Uganda in 2001 were found to be velogenic by fusion protein cleavage site sequence analysis and biological characterization; the intracerebral pathogenicity index was 1.8. Analysis of their hemagglutinin-neuraminidase protein gene sequences revealed a novel genotype unrelated to those that caused previous outbreaks.

Susceptibility of Muscovy (Cairina Moschata) and mallard ducks (Anas Platyrhynchos) to experimental infections by different genotypes of H5N1 avian influenza viruses

It is a fact that in Viet Nam, Muscovy ducks are raised in large populations (approximately 8 million), usually kept in small flocks together with mallards and chickens. As a result, it is a great concern for epidemiologists to elucidate possible differences in relation to these species being exposed to infection with H5N1. To do this, an experimental study on infections with different genotypes of H5N1 in mallards and Muscovy ducks have been conducted, where it was found that the mortality of the inoculated Muscovy ducks was at least 80%, regardless of the virus strain employed. In contrast, the mortality of the mallards ranged from nil to 100%, which suggests that Muscovy ducks are more susceptible to HPAIV H5N1 infection in terms of disease development and mortality. It was also found that higher virus titers developed in vital organs of Muscovy ducks compared to mallards, particularly in the brain. Due to their high susceptibility, it is unlikely that Muscovy ducks act as a silent reservoir. The virus strains used in this study, to a certain degree, differed in their virulence properties to the bird species in question.

Experimental assessment of the pathogenicity of two avian influenza A H5 viruses in ostrich chicks (Struthio camelus) and chickens

Virus excretion, immune response, and, for chickens, deaths were recorded in 3-week-old ostriches and chickens inoculated by either the intramuscular or intranasal route with one of two influenza A viruses of subtype H5. One of the viruses, A/turkey/England/50-92/91 (H5N1) (50/92), was highly pathogenic for chickens causing 5/5 deaths by each route of inoculation. The other virus, A/ostrich/Denmark-Q/72420/96 (H5N2) (72420/96), isolated from ostriches in quarantine in Denmark during 1996, was of low pathogenicity for chickens, causing no clinical signs by either route of inoculation. No significant clinical signs were seen in any of the ostriches infected with either of the viruses by either route of infection. Both viruses were recoverable from both species up to 12 days post-infection, and low serological responses were detected in surviving infected ostriches and chickens at 21 days after inoculation.

Flow cytometric assessment of chicken T cell-mediated immune responses after Newcastle disease virus vaccination and challenge.

The objective of this study was to use flow cytometry to assess chicken T cell-mediated immune responses. In this study two inbred genetic chicken lines (L130 and L133) were subjected to two times vaccination against Newcastle disease (ND) and a subsequent challenge by ND virus (NDV) infection. Despite a delayed NDV-specific antibody response to vaccination, L133 appeared to be better protected than L130 in the subsequent infection challenge as determined by the presence of viral genomes. Peripheral blood was analyzed by flow cytometry and responses in vaccinated/challenged birds were studied by 5-color immunophenotyping as well as by measuring the proliferative capacity of NDV-specific T cells after recall stimulation. Immunophenotyping identified L133 as having a significantly lower CD4/CD8 ratio and a lower frequency of gammadelta T cells than L130 in the peripheral T cell compartment. Furthermore, peripheral lymphocytes from L133 exhibited a significantly higher expression of CD44 and CD45 throughout the experiment. Interestingly, also vaccine-induced differences were observed in L133 as immune chickens had a significantly higher CD45 expression on their lymphocytes than the naïve controls. Immune chickens from both lines had a significantly higher frequency of circulating gammadelta T cells than the naïve controls both after vaccination and challenge. Finally, the proliferative capacity of peripheral CD4+ and CD8+ cells specific for NDV was addressed 3 weeks after vaccination and 1 week after infection and found to be significantly higher in L133 than in L130 at both sampling times. In conclusion, we found the applied flow cytometric methods very useful for the study of chicken T cell biology.