Novel Clade 2.3.4.4b Highly Pathogenic Avian Influenza A H5N8 and H5N5 Viruses in Denmark, 2020.

Since late 2020, outbreaks of H5 highly pathogenic avian influenza (HPAI) viruses belonging to clade 2.3.4.4b have emerged in Europe. To investigate the evolutionary history of these viruses, we performed genetic characterization on the first HPAI viruses found in Denmark during the autumn of 2020. H5N8 viruses from 14 wild birds and poultry, as well as one H5N5 virus from a wild bird, were characterized by whole genome sequencing and phylogenetic analysis. The Danish H5N8 viruses were found to be genetically similar to each other and to contemporary European clade 2.3.4.4b H5N8 viruses, while the Danish H5N5 virus was shown to be a unique genotype from the H5N5 viruses that circulated at the same time in Russia, Germany, and Belgium. Genetic analyses of one of the H5N8 viruses revealed the presence of a substitution (PB2-M64T) that is highly conserved in human seasonal influenza A viruses. Our analyses showed that the late 2020 clade 2.3.4.4b HPAI H5N8 viruses were most likely new incursions introduced by migrating birds to overwintering sites in Europe, rather than the result of continued circulation of H5N8 viruses from previous introductions to Europe in 2016/2017 and early 2020.

Tracing Hepatitis E Virus in Pigs From Birth to Slaughter.

Pigs are considered the main reservoir of genotypes 3 and 4 of the human pathogen hepatitis E virus (HEV). These viruses are prevalent at a high level in swine herds globally, meaning that consumers may be exposed to HEV from the food chain if the virus is present in pigs at slaughter. The aim of this study was to determine the HEV infection dynamics from birth to slaughter using 104 pigs from 11 sows in a single production system. Serum was collected from sows at 2 weeks prior to farrowing, in addition feces and serum samples were collected from the pigs every second week, from week 1 to week 17. Feces and selected organs were also sampled from 10 pigs following slaughter at week 20. All the samples were tested for HEV RNA by real-time RT-PCR and the serum samples were tested for HEV-specific antibodies using a commercial ELISA. Maternal antibodies (MAbs) were only present in pigs from sows with high levels of antibodies and all pigs, except one, seroconverted to HEV during weeks 13-17. In total, 65.5% of the pigs tested positive for HEV RNA at least once during the study (during weeks 13, 15, and/or 17) and significantly fewer pigs with a high level of MAbs became shedders. In contrast, the level of MAbs had no impact on the time of onset and duration of virus shedding. HEV was detected in feces and organs, but not in muscle, in 3 out of 10 pigs at slaughter, indicating that detection of HEV in feces is indicative of an HEV positivity in organs. In conclusion, a high proportion of pigs in a HEV positive herd were infected and shed virus during the finisher stage and some of the pigs also contained HEV RNA in feces and organs at slaughter. The presence of MAbs reduced the prevalence of HEV shedding animals, therefore, sow vaccination may be an option to decrease the prevalence of HEV positive animals at slaughter.

Subtyping of Swine Influenza Viruses Using a High-Throughput Real-Time PCR Platform.

Influenza A viruses (IAVs) are important human and animal pathogens with high impact on human and animal health. In Denmark, a passive surveillance program for IAV in pigs has been performed since 2011, where screening tests and subsequent subtyping are performed by reverse transcription quantitative real-time PCR (RT-qPCR). A disadvantage of the current subtyping system is that several assays are needed to cover the wide range of circulating subtypes, which makes the system expensive and time-consuming. Therefore, the aim of the present study was to develop a high-throughput method, which could improve surveillance of swine influenza viruses (swIAVs) and lower the costs of virus subtyping. Twelve qPCR assays specific for various hemagglutinin and neuraminidase gene lineages relevant for swIAV and six assays specific for the internal genes of IAV were developed and optimized for the high-throughput qPCR platform BioMark (Fluidigm). The qPCR assays were validated and optimized to run under the same reaction conditions using a 48.48 dynamic array (48.48DA). The sensitivity and specificity was assessed by testing virus isolates and field samples with known subtypes. The results revealed a performance of the swIAV 48.48DA similar to conventional real-time analysis, and furthermore, the specificity of swIAV 48.48DA was very high and without cross reactions between the assays. This high-throughput system provides a cost-effective alternative for subtyping of swIAVs.

Identification of swine influenza virus epitopes and analysis of multiple specificities expressed by cytotoxic T cell subsets.

BACKGROUND: Major histocompatibility complex (MHC) class I peptide binding and presentation are essential for antigen-specific activation of cytotoxic T lymphocytes (CTLs) and swine MHC class I molecules, also termed swine leukocyte antigens (SLA), thus play a crucial role in the process that leads to elimination of viruses such as swine influenza virus (SwIV). This study describes the identification of SLA-presented peptide epitopes that are targets for a swine CTL response, and further analyses multiple specificities expressed by SwIV activated CTL subsets. FINDINGS: Four SwIV derived peptides were identified as T cell epitopes using fluorescent influenza:SLA tetramers. In addition, multiple CTL specificities were analyzed using peptide sequence substitutions in two of the four epitope candidates analyzed. Interestingly both conserved and substituted peptides were found to stain the CD4-CD8+ T cell subsets indicating multiple specificities. CONCLUSIONS: This study describes a timely and cost-effective approach for viral epitope identification in livestock animals. Analysis of T cell subsets showed multiple specificities suggesting SLA-bound epitope recognition of different conformations.

Hepatitis E virus variant in farmed mink, Denmark.

Hepatitis E virus (HEV) is a zoonotic virus for which pigs are the primary animal reservoir. To investigate whether HEV occurs in mink in Denmark, we screened feces and tissues from domestic and wild mink. Our finding of a novel HEV variant supports previous findings of HEV variants in a variety of species.

A fast and robust method for full genome sequencing of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Type 1 and Type 2.

PRRSV is a positive-sense RNA virus with a high degree of genetic variability among isolates. For diagnostic sensitivity and vaccine design it is essential to monitor PRRSV genetic diversity. However, to date only a few full genome sequences of PRRSV isolates have been made publicly available. In the present study, fast and robust methods for long range RT-PCR amplification and subsequent next generation sequencing (NGS) were developed and validated on nine Type 1 and nine Type 2 PRRSV viruses. The methods generated robust and reliable sequences both on primary material and cell culture adapted viruses and the protocols performed well on all three NGS platforms tested (Roche 454 FLX, Illumina HiSeq2000, and Ion Torrent PGM™ Sequencer). These methods will greatly facilitate the generation of more full genome PRRSV sequences globally.

Expression of innate immune genes, proteins and microRNAs in lung tissue of pigs infected experimentally with influenza virus (H1N2).

This study aimed at providing a better understanding of the involvement of innate immune factors, including miRNA, in the local host response to influenza virus infection. Twenty pigs were challenged by influenza A virus subtype H1N2. Expression of microRNA (miRNA), mRNA and proteins were quantified in lung tissue at different time points after challenge (24 h, 72 h and 14 d post-infection (p.i.). Several groups of genes were significantly regulated according to time point and infection status including pattern recognition receptors (TLR2, TLR3, TLR7, retinoic acid-inducible gene I, melanoma differentiation associated protein-5), IFN and IFN-induced genes (IFN-ß, IFN-γ, IRF7, STAT1, ISG15 and OASL), cytokines (IL-1 ß, IL-1RN, IL-6, IL-7, IL-10, IL-12A, TNF-α, CCL2, CCL3 and CXCL10) and several acute phase proteins. Likewise, the following miRNAs were differentially expressed in one or more time groups compared with the control pigs: miR-15a, miR-21, miR-146, miR-206, miR-223 and miR-451. At d 1 p.i. lung tissue protein levels of IL-6, IL-12 and IFN-α were significantly increased compared with the control group, and haptoglobin and C-reactive protein were significantly increased at d 3 p.i. Our results suggest that, in addition to a wide range of innate immune factors, miRNAs may also be involved in controlling acute influenza infection in pigs.

Investigation of the presence of human or bovine respiratory syncytial virus in the lungs of mink (Neovison vison) with hemorrhagic pneumonia due to Pseudomonas aeruginosa.

BACKGROUND: Hemorrhagic pneumonia is a disease of farmed mink (Neovison vison) caused by Pseudomonas aeruginosa. The disease is highly seasonal in Danish mink with outbreaks occurring almost exclusively in the autumn. Human respiratory syncytial virus (RSV) has been shown to augment infection with P. aeruginosa in mice and to promote adhesion of P. aeruginosa to human respiratory cells. FINDINGS: We tested 50 lung specimens from mink with hemorrhagic pneumonia for bovine RSV by reverse transcriptase polymerase chain reaction (PCR) and for human RSV by a commercial real-time PCR. RSV was not found. CONCLUSIONS: This study indicates that human and bovine RSV is not a major co-factor for development of hemorrhagic pneumonia in Danish mink.

Hepatitis E virus is highly prevalent in the Danish pig population.

The objective of this study was to examine the prevalence of Hepatitis E virus (HEV) in the Danish pig population. Faecal samples from 97 pigs, 1-5 months of age were analysed for HEV RNA by a new PriProET real time RT-PCR assay. In addition, serum samples from 71 sow herds were screened for the presence of anti-HEV IgG antibodies by ELISA. The genotype of the detected HEV positive samples was estimated based on the melting temperature obtained by the PriProET real time RT-PCR assay. The HEV prevalence of faecal samples was 55.0% and 49.5% for herds and animals, respectively. A HEV IgG prevalence of 91.5% was found for the sow herds which correspond to 73.2% of the sows. The PriProET assay indicated that all HEV positive samples belonged to genotype 3 or 4, which is consistent with the observation of genotype 3 as dominant in European pigs. This is the first study showing that HEV is highly prevalent in the Danish pig population. The abundant presence of HEV in Danish pigs and the known high similarity between HEV isolates from pigs and humans support previous reports indicating possible zoonotic transmission of HEV.

Detection of myxoma viruses encoding a defective M135R gene from clinical cases of myxomatosis; possible implications for the role of the M135R protein as a virulence factor.

BACKGROUND: Myxoma virus is a member of the Poxviridae and causes disease in European rabbits. Laboratory confirmation of the clinical disease, which occurs in the autumn of most years in Denmark, has been achieved previously using antigen ELISA and electron microscopy. RESULTS: An unusually large number of clinically suspected cases of myxomatosis were observed in Denmark during 2007. Myxoma virus DNA was detected, using a new real time PCR assay which targets the M029L gene, in over 70% of the clinical samples submitted for laboratory confirmation. Unexpectedly, further analysis revealed that a high proportion of these viral DNA preparations contained a frame-shift mutation within the M135R gene that has previously been identified as a virulence factor. This frame-shift mutation results in expression of a greatly truncated product. The same frame-shift mutation has also been found recently within an avirulent strain of myxoma virus (6918). However, three other frame-shift mutations found in this strain (in the genes M009L, M036L and M148R) were not shared with the Danish viruses but a single nucleotide deletion in the M138R/M139R intergenic region was a common feature. CONCLUSIONS: It appears that expression of the full-length myxoma virus M135R protein is not required for virulence in rabbits. Hence, the frame-shift mutation in the M135R gene in the nonpathogenic 6918 virus strain is not sufficient to explain the attenuation of this myxoma virus but one/some of the other frame-shift mutations alone or in conjunction with one/some of the thirty two amino acid substitutions must also contribute. The real time PCR assay for myxoma virus is a useful diagnostic tool for laboratory confirmation of suspected cases of myxomatosis.