Epidemiological and Molecular Analysis of an Outbreak of Highly Pathogenic Avian Influenza H5N8 clade 2.3.4.4 in a German Zoo: Effective Disease Control with Minimal Culling.

Outbreaks of highly pathogenic avian influenza A virus (HPAIV) subtype H5N8, clade 2.3.4.4, were first reported in January 2014 from South Korea. These viruses spread rapidly to Europe and the North American continent during autumn 2014 and caused, in Germany, five outbreaks in poultry holdings until February 2015. In addition, birds kept in a zoo in north-eastern Germany were affected. Only a few individual white storks (Ciconia ciconia) showed clinical symptoms and eventually died in the course of the infection, although subsequent in-depth diagnostic investigations showed that other birds kept in the same compound of the white storks were acutely positive for or had undergone asymptomatic infection with HPAIV H5N8. An exception from culling all of the 500 remaining zoo birds was granted by the competent authority. Restriction measures included grouping the zoo birds into eight epidemiological units in which 60 birds of each unit tested repeatedly negative for H5N8. Epidemiological and phylogenetical investigations revealed that the most likely source of introduction was direct or indirect contact with infected wild birds as the white storks had access to a small pond frequented by wild mallards and other aquatic wild birds during a period of 10 days in December 2014. Median network analysis showed that the zoo bird viruses segregated into a distinct cluster of clade 2.3.4.4 with closest ties to H5N8 isolates obtained from mute swans (Cygnus olor) in Sweden in April 2015. This case demonstrates that alternatives to culling exist to rescue valuable avifaunistic collections after incursions of HPAIV.

Introduction and enzootic of A/H5N1 in Egypt: Virus evolution, pathogenicity and vaccine efficacy ten years on.

It is almost a decade since the highly pathogenic H5N1 avian influenza virus (A/H5N1) of clade 2.2.1 was introduced to Egypt in 2005, most likely, via wild birds; marking the longest endemic status of influenza viruses in poultry outside Asia. The endemic A/H5N1 in Egypt still compromises the poultry industry, poses serious hazards to public health and threatens to become potentially pandemic. The control strategies adopted for A/H5N1 in Egyptian poultry using diverse vaccines in commercialized poultry neither eliminated the virus nor did they decrease its evolutionary rate. Several virus clades have evolved, a few of them disappeared and others prevailed. Disparate evolutionary traits in both birds and humans were manifested by accumulation of clade-specific mutations across viral genomes driven by a variety of selection pressures. Viruses in vaccinated poultry populations displayed higher mutation rates at the immunogenic epitopes, promoting viral escape and reducing vaccine efficiency. On the other hand, viruses isolated from humans displayed changes in the receptor binding domain, which increased the viral affinity to bind to human-type glycan receptors. Moreover, viral pathogenicity exhibited several patterns in different hosts. This review aims to provide an overview of the viral evolution, pathogenicity and vaccine efficacy of A/H5N1 in Egypt during the last ten years.

Poultry food products--a source of avian influenza virus transmission to humans?

Global human mobility and intercontinental connectivity, expansion of livestock production and encroachment of wildlife habitats by invasive agricultural land use contribute to shape the complexity of influenza epidemiology. The OneHealth approach integrates these and further elements into considerations to improve disease control and prevention. Food of animal origin for human consumption is another integral aspect; if produced from infected livestock such items may act as vehicles of spread of animal pathogens, and, in case of zoonotic agents, as a potential human health hazard. Notifiable zoonotic avian influenza viruses (AIV) have become entrenched in poultry populations in several Asian and northern African countries since 2003. Highly pathogenic (HP) AIV (e.g. H5N1) cause extensive poultry mortality and severe economic losses. HPAIV and low pathogenic AIV (e.g. H7N9) with zoonotic propensities pose risks for human health. More than 1500 human cases of AIV infection have been reported, mainly from regions with endemically infected poultry. Intense human exposure to AIV-infected poultry, e.g. during rearing, slaughtering or processing of poultry, is a major risk factor for acquiring AIV infection. In contrast, human infections through consumption of AIV-contaminated food have not been substantiated. Heating poultry products according to kitchen standards (core temperatures ≥70°C, ≥10 s) rapidly inactivates AIV infectivity and renders fully cooked products safe. Nevertheless, concerted efforts must ensure that poultry products potentially contaminated with zoonotic AIV do not reach the food chain. Stringent and sustained OneHealth measures are required to better control and eventually eradicate, HPAIV from endemic regions.

Vaccines against influenza A viruses in poultry and swine: Status and future developments.

Influenza A viruses are important pathogens with a very broad host spectrum including domestic poultry and swine. For preventing clinical disease and controlling the spread, vaccination is one of the most efficient tools. Classical influenza vaccines for domestic poultry and swine are conventional inactivated preparations. However, a very broad range of novel vaccine types ranging from (i) nucleic acid-based vaccines, (ii) replicon particles, (iii) subunits and virus-like particles, (iv) vectored vaccines, or (v) live-attenuated vaccines has been described, and some of them are now also used in the field. The different novel approaches for vaccines against avian and swine influenza virus infections are reviewed, and additional features like universal vaccines, novel application approaches and the "differentiating infected from vaccinated animals" (DIVA)-strategy are summarized.

Emergence of a novel cluster of influenza A(H5N1) virus clade 2.2.1.2 with putative human health impact in Egypt, 2014/15.

A distinct cluster of highly pathogenic avian influenzaviruses of subtype A(H5N1) has been found to emergewithin clade 2.2.1.2 in poultry in Egypt since summer2014 and appears to have quickly become predominant.Viruses of this cluster may be associated withincreased incidence of human influenza A(H5N1) infectionsin Egypt over the last months.

Consecutive natural influenza a virus infections in sentinel mallards in the evident absence of subtype-specific hemagglutination inhibiting antibodies.

Dabbling ducks, particularly Mallards (Anas platyrhynchos) have been frequently and consistently reported to play a pivotal role as a reservoir of low pathogenic avian influenza viruses (AIV). From October 2006 to November 2008, hand-raised Mallard ducks kept at a pond in an avifaunistically rich area of Southern Germany served as sentinel birds in the AIV surveillance programme in Germany. The pond was regularly visited by several species of dabbling ducks. A flock of sentinel birds, consisting of the same 16 individual birds during the whole study period, was regularly tested virologically and serologically for AIV infections. Swab samples were screened by RT-qPCR and, if positive, virus was isolated in embryonated chicken eggs. Serum samples were tested by the use of competitive ELISA and hemagglutinin inhibition (HI) assay. Sequences of full-length hemagglutinin (HA) and neuraminidase (NA) genes were phylogenetically analysed. Four episodes of infections with Eurasian-type AIV occurred in August (H6N8), October/November (H3N2, H2N3) 2007, in January (H3N2) and September (H3N8) 2008. The HA and NA genes of the H3N2 viruses of October 2007 and January 2008 were almost identical rendering the possibility of a re-introduction of that virus from the environment of the sentinel flock highly likely. The HA of the H3N8 virus of September 2008 belonged to a different cluster. As a correlate of the humoral immune response, titres of nucleocapsid protein-specific antibodies fluctuated in correlation with the course of AIV infection episodes. However, no specific systemic response of hemagglutination inhibiting antibodies could be demonstrated even if homologous viral antigens were used. Besides being useful as early indicators for the circulation of influenza viruses in a specific region, the sentinel ducks also contributed to gaining insights into the ecobiology of AIV infection in aquatic wild birds.

Towards a new, ecologically targeted approach to monitoring wild bird populations for avian influenza viruses.

Prevalence monitoring of avian influenza in wild bird populations is important to estimate risks for the occurrence of potentially zoonotic and economically disastrous outbreaks of highly pathogenic avian influenza virus (AIV) in poultry worldwide. A targeted, cost-effective monitoring method for AIV in wild birds was developed, which is based on monitoring results for AIV in Germany and information on the distribution and abundance of wild bird species in selected habitat types. Spatial data were combined with virological and outbreak data for the period of 1 January 2006 to 31 December 2010. Using Germany as an example, we identified 11 indicator species. By concentrating monitoring efforts on these species in spatially confined locations, we propose a targeted and more cost-effective risk-based AIV monitoring approach that can be adapted universally for the identification of wild bird indicator species worldwide with the perspective of reducing sample sizes (and costs) without impairing the validity of the results.

Broad spectrum reactivity versus subtype specificity-trade-offs in serodiagnosis of influenza A virus infections by competitive ELISA.

Avian influenza viruses (AIVs) of the H5 and H7 subtypes can cause substantial economic losses in the poultry industry and are a potential threat to public health. Serosurveillance of poultry populations is an important monitoring tool and can also be used for control of vaccination campaigns. The purpose of this study was to develop broadly reactive, yet subtype-specific competitive ELISAs (cELISAs) for the specific detection of antibodies to the notifiable AIV subtypes H5 and H7 as an alternative to the gold standard haemagglutination inhibition assay (HI). Broadly reacting monoclonal competitor antibodies (mAbs) and genetically engineered subtype H5 or H7 haemagglutinin antigen, expressed and in vivo biotinylated in insect cells, were used to develop the cELISAs. Sera from galliform species and water fowl (n=793) were used to evaluate the performance characteristics of the cELISAs. For the H5 specific cELISA, 98.1% test sensitivity and 91.5% test specificity (97.7% and 90.2% for galliforms; 98.9% and 92.6% for waterfowl), and for the H7 cELISA 97.3% sensitivity and 91.8% specificity (95.3% and 98.9% for galliforms; 100% and 82.7% for waterfowl) were reached when compared to HI. The use of competitor mAbs with broad spectrum reactivity within an AIV haemagglutinin subtype allowed for homogenous detection with high sensitivity of subtype-specific antibodies induced by antigenically widely distinct isolates including antigenic drift variants. However, a trade-off regarding sensitivity versus nonspecific detection of interfering antibodies induced by phylo- and antigenically closely related subtypes, e.g., H5 versus H2 and H7 versus H15, must be considered. The observed intersubtype antibody cross-reactivity remains a disturbance variable in AIV subtype-specific serodiagnosis which negatively affects specificity.

Epidemiological and ornithological aspects of outbreaks of highly pathogenic avian influenza virus H5N1 of Asian lineage in wild birds in Germany, 2006 and 2007.

In Germany, two distinct episodes of outbreaks of highly pathogenic avian influenza virus of subtype H5N1 (HPAIV H5N1) in wild birds occurred at the beginning of 2006, and in summer 2007. High local densities of wild bird populations apparently sparked clinically detectable outbreaks. However, these remained restricted in (i) number of birds, (ii) species found to be affected, (iii) time, and (iv) location despite the presence of several hundred thousands of susceptible wild birds and further stressors (food shortage, harsh weather conditions and moulting). Northern and southern subpopulations of several migratory anseriform species can be distinguished with respect to their preference for wintering grounds in Germany. This corroborates viral genetic data by Starick et al. (2008) demonstrating the introduction of two geographically restricted virus subpopulations of Qinghai-like lineage (cluster 2.2.A and 2.2.B) into northern and southern Germany, respectively, in 2006. The incursion of virus emerging in 2007, found to be distinct from the clusters detected in 2006 (Starick et al., 2008), may have been associated with moulting movements. Intensive past-outbreak investigations with negative results of live and dead wild birds and of terrestrial scavengers excluded continued circulation of virus on a larger scale. However, persistence of virus in small pockets of local wild bird populations could not be ruled out resiliently. 1.5% of investigated sera originating from cats sampled at the epicentres of the Ruegen 2006-outbreak contained H5-antibodies. Passive monitoring was found to be highly superior to live bird surveillance when aiming at the detection of HPAIV H5N1 in wild birds (P < 0.0001).

Antigenic characterization of phocine distemper virus causing mass mortality in 2002 and its relationship to other morbilliviruses.

The antigenic relationship between the phocine distemper virus (PDV) strain causing the epidemic in 2002 and the PDV strain of 1988, canine distemper virus from two dogs and one marten, and one measles virus strain was investigated in vivo and in vitro using monospecific polyclonal and monoclonal antibodies directed against five different proteins of canine or phocine distemper virus (N, P, M, F, H). Epitopic mapping revealed no difference between the PDV strains causing the epidemics in 1988 or 2002. However, the use of these antibodies allowed discrimination between different morbilliviruses including a vaccine strain of canine distemper virus. The major differences among the investigated morbilliviruses were found in the H protein.

Pathology of natural infections by H5N1 highly pathogenic avian influenza virus in mute (Cygnus olor) and whooper (Cygnus cygnus) swans.

Mortality in wild aquatic birds due to infection with highly pathogenic avian influenza viruses (HPAIV) is a rare event. During the recent outbreak of highly pathogenic avian influenza in Germany, mortality due to H5N1 HPAIV was observed among mute and whooper swans as part of a rapid spread of this virus. In contrast to earlier reports, swans appeared to be highly susceptible and represented the mainly affected species. We report gross and histopathology and distribution of influenza virus antigen in mute and whooper swans that died after natural infection with H5N1 HPAIV. At necropsy, the most reliable lesions were multifocal hemorrhagic necrosis in the pancreas, pulmonary congestion and edema, and subepicardial hemorrhages. Major histologic lesions were acute pancreatic necrosis, multifocal necrotizing hepatitis, and lymphoplasmacytic encephalitis with neuronal necrosis. Adrenals displayed consistently scattered cortical and medullary necrosis. In spleen and Peyer's patches, mild lymphocyte necrosis was present. Immunohistochemical demonstration of HPAIV nucleoprotein in pancreas, adrenals, liver, and brain was strongly consistent with histologic lesions. In the brain, a large number of neurons and glial cells, especially Purkinje cells, showed immunostaining. Occasionally, ependymal cells of the spinal cord were also positive. In the lungs, influenza virus antigen was identified in a few endothelial cells but not within pneumocytes. The infection of the central nervous system supports the view that the neurotropism of H5N1 HPAIV leads to nervous disturbances with loss of orientation. More investigations are necessary to clarify the mechanisms of the final circulatory failure, lung edema, and rapid death of the swans.

Purification of phocid herpesvirus type 1 glycoproteins B and D and pilot studies of immunogenicity in mice.

In the process of developing a subunit vaccine against phocid herpesvirus type 1, we have cloned and expressed the glycoproteins B and D (gB and gD) of phicid herpesvirus type 1, using an eukaryotic baculovirus expression system. To establish the proof of concept, candidate iscom vaccines based on these affinity-purified proteins either alone or in combination, were tested for their immunogenicity in BALB/C mice. Mice immunised with a combination of gB and gD developed higher antibody and proliferative T cell responses against PhHV-1 than those immunised with gB or gD alone. The corresponding antibody and T cell proliferative responses were higher against PhHV-1 than against FHV. These data favour further testing of these candidate vaccines based on gB and gD in the FHV-cat model.

Seal gammaherpesviruses: identification, characterisation and epidemiology.

Phocid herpesvirus type 2 (PhHV-2), tentatively classified as a gammaherpesvirus, has been isolated from European and American harbour seals (Phoca vitulina). Here we describe the isolation and the molecular as well as biological characterisation of different PhHV-2 isolates from harbour seals and grey seals (Halichoerus grypus). Of 522 harbour seals and 231 grey seals that had been admitted to the seal research and rehabilitation centre in Pieterburen, The Netherlands, between 1992 and 2000, 38 and 18%, respectively, proved to have PhHV-2 neutralising antibodies. PhHV-2 was isolated from peripheral blood mononuclear cells (PBMCs) of 12 and 28% of these seropositive animals, respectively, and 26 and 56% of these cell samples, respectively, were positive by PCR analysis. Analysis of amino acid sequences of PCR products and of the growth characteristics of different PhHV-2 isolates indicated that harbour and grey seals are infected with distinct gamma-herpesviruses, which however, may co-circulate between the two species.

Genetic characterization of the unique short segment of phocid herpesvirus type 1 reveals close relationships among alphaherpesviruses of hosts of the order Carnivora.

To further characterize phocid herpesvirus type 1 (PhHV-1) at the molecular level, a cluster of genes comprising the complete unique short (Us) region of PhHV-1 has been cloned and sequenced. Within this region, ORFs were detected that code for the equivalent of the Us 2- protein of herpes simplex virus (HSV), a putative protein kinase, and for the glycoprotein equivalents gG, gD, gI and gE. In addition, two small ORFs downstream of gE, homologous to the Us 8.5 and Us 9 proteins of HSV were identified. Comparative analysis of the ORF encoding the gD equivalent of PhHV-1 identified the corresponding proteins of the alphaherpesviruses canine herpesvirus and, to lesser degree, feline herpesvirus as the closest relatives.

Rapid molecular discrimination between infection with wild-type varicella-zoster virus and varicella vaccine virus.

Varicella-zoster virus (VZV) infection is immunocompromised patients may cause life-threatening complications. Prevention measures include administration of VZV immuloglobulin, acyclovir and live attenuated varicella vaccine. After vaccination, a mild varicella-like exanthem appears in up to 5% of vaccinees. Morphologically this exanthem cannot be differentiated from wild-type (wt) varicella. The risk of virus transmission after varicella vaccination, in contrast to wt varicella, is low, even in immunocompromised patients. We report on a 2-year-old girl with relapse of cereral anaplastic ependymoma, who received one dose of varicella vaccine. Two weeks later, a maculopapular rash developed while she was an inpatient on the oncology ward. Using VZV-specific PCR and restriction fragment length polymorphism (RFLP) analysis, we were able to diagnose wt varicella infection. Thus, appropriate prevention measures (VZV immunoglobulin and acyclovir) were justified for close contacts to prevent virus transmission. No secondary cases occurred.

Variations in the severity of phocid herpesvirus type 1 infections with age in grey seals and harbour seals.

The data recorded during an outbreak of phocid herpesvirus type 1 infection among 19 harbour seals and 29 grey seals being nursed in a seal rehabilitation centre in The Netherlands in 1998 were used, together with data from similar outbreaks in previous years, to compare the clinical signs observed in the two species at different ages. The severity of the disease was inversely correlated with age in the harbour seals, and the infected harbour seals generally developed more severe clinical signs than the infected grey seals.

A candidate phocid herpesvirus vaccine that provides protection against feline herpesvirus infection.

Phocid herpesvirus type 1 (PhHV-1) causes significant morbidity and mortality among young and immunocompromised harbour seals. Therefore, the availability of an effective PhHV-1 vaccine would be of importance for orphanages and seal rehabilitation centres. Since possibilities to test PhHV-1 candidate vaccines in the target species are limited, a suitable animal model is needed. Given the close genetic and antigenic relationships between PhHV-1 and feline herpesvirus (FHV), the FHV cat system could be considered to test candidate PhHV-1 vaccines. Here we have tested a PhHV-1 based ISCOM vaccine for its protective efficacy against FHV infection in cats. To this end, three groups of cats were vaccinated thrice with ISCOM adjuvanted PhHV-1, FHV, and mock vaccines, respectively. One month after the last vaccination, all cats were challenged with a virulent FHV strain. All PhHV-1 and FHV vaccinated cats were protected from developing severe disease and excreted significantly less FHV than the mock vaccinated cats.

New LightCycler PCR for rapid and sensitive quantification of parvovirus B19 DNA guides therapeutic decision-making in relapsing infections.

Detection of parvovirus B19 DNA offers diagnostic advantages over serology, particularly in persistent infections of immunocompromised patients. A rapid, novel method of B19 DNA detection and quantification is introduced. This method, a quantitative PCR assay, is based on real-time glass capillary thermocycling (LightCycler [LC]) and fluorescence resonance energy transfer (FRET). The PCR assay allowed quantification over a dynamic range of over 7 logs and could quantify as little as 250 B19 genome equivalents (geq) per ml as calculated for plasmid DNA (i.e., theoretically >or=5 geq per assay). Interrater agreement analysis demonstrated equivalence of LC-FRET PCR and conventional nested PCR in the diagnosis of an active B19 infection (kappa coefficient = 0.83). The benefit of the new method was demonstrated in an immunocompromised child with a relapsing infection, who required an attenuation of the immunosuppressive therapy in addition to repeated doses of immunoglobulin to eliminate the virus.

Analysis of the H gene, the central untranslated region and the proximal coding part of the F gene of wild-type and vaccine canine distemper viruses.

This paper summarizes the results of the genetic analysis of several parts of the genome of canine distemper virus (CDV) field isolates and vaccine viruses. The haemagglutinin (H) gene analysis showed that recent viruses did not differ significantly from vaccine strains. The analysis of the long untranslated region between the matrix (M) and fusion (F) gene revealed distinct genetic heterogeneity. The putative F protein start codon AUG461 of vaccine strain Onderstepoort was found to be mutated in all wild-type isolates and in another vaccine strain. The proximal coding part of the F gene was well conserved. Phylogenetic analysis of this segment showed the presence of several cocirculating CDV genotypes.

Rapid detection by reverse hybridization of mutations in the UL97 gene of human cytomegalovirus conferring resistance to ganciclovir.

UNLABELLED: BACKGROUND OF STUDY: Diseases due to human cytomegalovirus (HCMV) infection constitute a major threat in marrow and solid organ transplant recipients. Ganciclovir (GCV) is widely used in prophylaxis and pre-emptive therapy of active HCMV infection. Resistance to ganciclovir (GCV) may arise at variable frequency under GCV therapy and is conferred by mutations (i) in the UL97 gene (codons 460, 520, and 591-607) encoding a phosphotransferase which is essential for monophosphorylation of GCV and, to a lesser extent, (ii) in the UL54 gene coding for the DNA polymerase of HCMV. OBJECTIVE: The purpose was to develop a rapid assay to screen for emerging GCV resistance mutations in the UL97 gene of HCMV whereby avoiding virus isolation and nucleotide sequencing procedures. STUDY DESIGN: A nested PCR (nPCR) amplifying UL97 codons 450-672 was developed. Nested amplicons were subsequently sequenced directly. Oligonucleotides for use in a reverse hybridization assay were designed to detect relevant non-synonymous mutations at codons UL97 460, 520, 603 and 607. Strain AD169 served as a wild-type control. RESULTS: UL97-specific nPCR amplicons were obtained from 18 EDTA blood samples of ten transplant recipients receiving GCV for more than 30 days. In three consecutive samples from a single patient a GCV resistance mutation at codon 603 (C-->W) was detected. In addition, two out of four cell culture-adapted HCMV isolates known to exhibit GCV resistance in vitro revealed mutations at codons 460 (M-->V) and 607 (C-->Y), respectively. By reverse hybridization a discrimination of single nucleotide changes at codons 460, 520, 603 and 607 was possible whereby matching exactly the results of the nucleotide sequence analysis for all 23 amplicons examined. CONCLUSIONS: Reverse hybridization appeared to be a rapid and convenient alternative to nucleotide sequencing when screening the UL97 gene of HCMV for selected markers of GCV resistance.