[Batch testing as an essential pillar of the supply of safe and effective vaccines]. / Chargenprüfung als wesentliche Säule der Versorgung mit sicheren und wirksamen Impfstoffen.

The Paul-Ehrlich Institute (PEI) plays a central role in the release of vaccines in Germany as well as Europe. The experimental testing and release of each vaccine batch is carried out according to the procedures and regulations of the Official Control Authority Batch Release (OCABR) and the German medicine act paragraph 32. The independent testing aims to demonstrate the conformity of quality criteria set in the marketing authorization for each lot produced. This article illustrates both the batch release procedure in general and specifically for the newly developed and approved COVID-19 vaccines during the COVID-19 pandemic.

Animal Welfare Consequences of Organic Boar Fattening and Occurrence of Boar Taint on Five Commercial Farms.

The welfare of male fattening pigs may be improved by refraining from castration, but may be compromised, in turn, by harmful social behaviour in groups of boars. In addition, boar taint may be problematic. This study aimed to evaluate these potential problems in boar fattening under commercial organic conditions. In total, 625 boars were compared with 433 barrows and 83 gilts regarding their social behaviour, lesions and lameness at 80 kg, before and after split marketing. The mixed-model analysis showed that significantly more short agonistic interactions, fights and mounting behaviours were observed in groups of boars. Agonistic interactions were reduced in spring/summer and when boars grew older. Fights and mounts were increased when boars had contact to female pigs in the neighbouring pen. No effect of split marketing, growth rate, homogeneity of groups, group size, feeding space and illumination hours could be detected. Increased interaction frequencies did not result in significantly more skin lesions, lameness, treatments or mortality. Increased space allowance reduced skin lesions. On 9.8% of the dissected boars' penises, wounds were detected; they were absent on two farms with generous litter provision. Boar taint prevalence, as detected by human nose method, was 1.44%. Under the studied organic husbandry conditions, boar fattening appears to be practicable, although penile injuries should be monitored at slaughter.

Novel reassortant swine H3N2 influenza A viruses in Germany.

Analysis of 228 H3N2 swine influenza A virus isolates collected between 2003 and 2015 in Germany revealed important changes in molecular epidemiology. The data indicate that a novel reassortant, Rietberg/2014-like swine H3N2, emerged in February 2014 in Northern Germany. It is comprised of a hemagglutinin gene of seasonal H3N2 (A/Denmark/129/2005-like), a neuraminidase gene of Emmelsbuell/2009-like swine H1N2 and the internal gene cassette of pandemic H1N1 viruses. Together with Danish swine H3N2 strains of 2013-2015 with identical genome layout, the Rietberg/2014-like viruses represent a second swine H3N2 lineage which cocirculates with a variant of the Gent/1984-like swine H3N2 lineage. This variant, named Gent1984/Diepholz-like swine H3N2, has a Gent/1984-like HA and a Diepholz/2008-like NA; the origin of the internal gene cassette likely derived from avian-like swine H1N1. The first isolate of the Gent1984/Diepholz reassortant emerged in Northern Germany in September 2011 whereas the last German Gent/1984-like isolate was collected in October 2011.

Cocirculation of Swine H1N1 Influenza A Virus Lineages in Germany.

The genome analysis of 328 H1N1 swine influenza virus isolates collected in a 13-year long-term swine influenza surveillance in Germany is reported. Viral genomes were sequenced with the Illumina next-generation sequencing technique and conventional Sanger methods. Phylogenetic analyses were conducted with Bayesian tree inference. The results indicate continued prevalence of Eurasian avian swine H1N1 but also emergence of a novel H1N1 reassortant, named Schneiderkrug/2013-like swine H1N1, with human-like hemagglutinin and avian-like neuraminidase and internal genes. Additionally, the evolution of an antigenic drift variant of A (H1N1) pdm09 was observed, named Wachtum/2014-like swine H1N1. Both variants were first isolated in northwest Germany, spread to neighboring German states and reached greater proportions of the H1N1 isolates of 2014 and 2015. The upsurge of Wachtum/2014-like swine H1N1 is of interest as this is the first documented persistent swine-to-swine spread of A (H1N1) pdm09 in Germany associated with antigenic variation. Present enzootic swine influenza viruses in Germany now include two or more co-circulating, antigenically variant viruses of each of the subtypes, H1N1 and H1N2.

Displacement of the Gent/1999 human-like swine H1N2 influenza A virus lineage by novel H1N2 reassortants in Germany.

A swine influenza survey was conducted between 2003 and 2015 in Germany. During this period, 8122 snout swabs or other respiratory specimens from pigs of 5178 herds, mainly from Germany, were investigated for the presence of swine influenza A virus (S-IAV). In total, 1310 S-IAV isolates were collected. Of this collection, the complete genome of 267 H1N2 S-IAV isolates was sequenced and phylogenetically analyzed. The data demonstrate the incursion of human-like swine H1N2 viruses (Gent/1999-like) in 2000 and prevalent circulation until 2010. From 2008 onward, a sustained and broad change of the genetic constellation of the swine H1N2 subtype commenced. The Gent/1999-like swine H1N2 viruses ceased and several new swine H1N2 reassortants emerged and became prevalent in Germany. Of these, the upsurge of the Diepholz/2008-like, Emmelsbuell/2009-like and Papenburg/2010-like viruses is notable. The data reveal the importance of reassortment events in S-IAV evolution. The strong circulation of S-IAV of different lineages in the swine population throughout the year underlines that pigs are important reservoir hosts.

Origin of the European avian-like swine influenza viruses.

The avian-like swine influenza viruses emerged in 1979 in Belgium and Germany. Thereafter, they spread through many European swine-producing countries, replaced the circulating classical swine H1N1 influenza viruses, and became endemic. Serological and subsequent molecular data indicated an avian source, but details remained obscure due to a lack of relevant avian influenza virus sequence data. Here, the origin of the European avian-like swine influenza viruses was analysed using a collection of 16 European swine H1N1 influenza viruses sampled in 1979-1981 in Germany, the Netherlands, Belgium, Italy and France, as well as several contemporaneous avian influenza viruses of various serotypes. The phylogenetic trees suggested a triple reassortant with a unique genotype constellation. Time-resolved maximum clade credibility trees indicated times to the most recent common ancestors of 34-46 years (before 2008) depending on the RNA segment and the method of tree inference.

Seroprevalence of hepatitis E virus (HEV) in humans living in high pig density areas of Germany.

An increase in acute autochthonous hepatitis E virus (HEV) infections has been recorded in Germany. These are suspected to be zoonotically transmitted from wild boar, deer and domestic pig. The latter may represent a major reservoir for HEV. In this study, 537 sera from humans living in Westphalia and Lower Saxony, representing areas of high pig density in Germany, were tested for the presence of HEV-specific antibodies. Among them were 302 individuals with occupational, direct contact to pigs and 235 individuals without direct contact to pigs. Two commercial tests and one in-house assay were applied for the detection of HEV-specific immunoglobulin G (IgG) antibodies. Sera were also tested in an assay that detects all classes of HEV-specific antibodies. Depending on the test used, the seroprevalence ranged from 4.1 to 27.9 %. Exposition to pigs was found to be associated with a significantly higher seroprevalence in subjects with contact to pigs (13.2-32.8 %) compared with that in non-exposed humans (7.7-21.7 %). In particular, individuals younger than 40 years with occupational exposure exhibited a markedly higher HEV seroprevalence compared with non-exposed individuals of that age group. In general, HEV seroprevalence increased with age resulting in a similar prevalence level in the age group of ≥ 50 years for exposed and non-exposed individuals. Analysis of all sera by a commercial anti-HEV IgM ELISA revealed 35 positive and 25 borderline samples. However, only one positive serum could be confirmed by an IgM line assay. Selected samples from IgM and/or IgG as well as total HEV antibody-positive individuals were also tested for the presence of HEV RNA. In one of the 78 samples, the only IgM ELISA positive and IgM line assay confirmed sample, RNA of HEV genotype 3 was detected. This sequence has high similarity to HEV sequences obtained from wild boars and domestic pigs from Germany and The Netherlands. This study demonstrates that in addition to the consumption of raw or undercooked meat, direct contact to pigs has to be considered as an additional risk factor for HEV infection.

The genome of an influenza virus from a pilot whale: relation to influenza viruses of gulls and marine mammals.

Influenza virus A/whale/Maine/328B/1984 (H13N2) was isolated from a diseased pilot whale. Since only a partial sequence was available, its complete genome was sequenced and compared to the sequences of subtype H13 influenza viruses from shorebirds and various influenza viruses of marine mammals. The data reveal a rare genotype constellation with all gene segments derived of an influenza virus adapted to gulls, terns and waders. In contrast, the phylogenetic trees indicate that the majority of influenza viruses isolated from marine mammals derived from influenza viruses adapted to geese and ducks. We conclude that A/whale/Maine/328B/1984 is the first record of an infection of a marine mammal from a gull-origin influenza virus.

Circulation of classical swine influenza virus in Europe between the wars?

The complete genomes of two swine influenza viruses from England were sequenced. Phylogenetic analysis revealed classical swine H1N1 viruses, one of which, A/swine/London, is closely related to virus strains of the early 1930s. Both strains are also antigenically related to A/swine/Iowa/15/1930, the strain originally isolated by Richard Shope. The source of A/swine/London is unknown, but its relationship to early classical swine influenza viruses suggests that the emergence of these viruses in Europe has to be antedated by 15-20 years.

Virus isolate from carp: genetic characterization reveals a novel picornavirus with two aphthovirus 2A-like sequences.

Picornaviruses have been isolated from a variety of hosts, mainly mammals and birds. Here, we describe the sequence analysis of carp picornavirus 1 (CPV-1) F37/06 that was isolated from an organ pool (heart, brain, liver) of a common carp (Cyprinus carpio). This carp perished after an accidental discharge of liquid manure into a fish pond and presented without obvious clinical symptoms. Experimental intraperitoneal infection of young carp with CPV-1 revealed no clinical signs, but the virus was re-isolated from various organs. Sequence analysis of almost the complete genome (7632 nt excluding the poly-A tract) revealed a novel picornavirus clade. In phylogenetic trees, the polymerase sequence clusters with parechoviruses, duck hepatitis A virus, eel picornavirus and aquamavirus A. The ORF includes 6807 nt and encodes a polyprotein of 2269 amino acids. CPV-1 has a genome layout like that of picornaviruses except for the presence of two aphthovirus 2A-like NPGP sequence motifs: VPg+5'UTR[1AB-1C-1D-2A1(npgp)/2A2(npgp)-2B-2C(ATPase)/3A-3B(VPg)-3C(pro)-3D(pol)]3'UTR-poly-A. 2A1(npgp) and 2A2(npgp) are separated by 133 amino acids. The proteins 2A2(npgp), 2B, 3A and 3B(VPg) have no significant similarity to the corresponding proteins of other picornaviruses. Amino acid identities of the orthologous proteins P1, 2C, 3C(pro) and 3D(pol) range from 16.4 to 40.8 % in the eel picornavirus/CPV-1 comparison. 3D(pol) shows the closest similarity to eel picornavirus, with an amino acid identity of 40.8 %, followed by human parechovirus (36.5 %), duck hepatitis A virus (32.7 %) and swine pasivirus (29.3 %). Both the unique genome organization and low sequence similarity support the assignment of CPV-1 to a novel picornavirus species within a novel genus.

Prevalence of antibodies to European porcine influenza viruses in humans living in high pig density areas of Germany.

The risk of zoonotic human infection caused by European porcine influenza virus strains was estimated in German regions with a high pig density. Sera from 622 healthy volunteers were collected between April 2009 and November 2011, mainly in Westphalia and western Lower Saxony. These included 362 subjects with occupational contact to pigs and 260 blood donors without any direct exposition to pigs. Samples were analysed by the haemagglutination inhibition (HI) assay against a panel of six swine viruses of subtypes avian-like H1N1 and human-like H3N2 as well as against human H1N1 and H3N2 viruses including the pandemic H1N1 strain of 2009. Reciprocal HI titres ≥20 were quoted as seroreactive. Compared to the control group, a significantly higher proportion of subjects with direct contact to pigs exhibited seroreactivity against porcine antigens of the avian-like H1N1 (37.0 %/7.7 %), the human-like H3N2 (59.7 %/43.1 %), the pandemic H1N1 strain of 2009 (51.7 %/26.5 %) and against a historic seasonal H3N2 strain that is closely related antigenetically to currently circulating human-like H3N2 viruses of European pigs (57.5 %/36.5 %). This trend was also observed when a reciprocal HI titre ≥40 was chosen as cut-off. Particularly, in younger subjects, the differences in seroreactivity against porcine strains between the exposed and non-exposed group were significant. The data indicate a higher risk of infection in the exposed individuals.

Age-related and regional differences in the prevalence of hepatitis E virus-specific antibodies in pigs in Germany.

An increasing number of acute autochthonous human hepatitis E virus (HEV)-infections was noticed in Germany and other developed countries, most likely the result of a zoonotic virus transmission from pig, wild boar and deer. Currently there is still a lack of profound data concerning the actual prevalence of HEV-specific antibodies in domestic pig herds in Germany, in particular for regions with high pig density, and its age-dependency. 2273 domestic pig sera were collected in 2011 mainly from Bavaria, North Rhine-Westphalia and Lower Saxony from areas having a high pig density. Initially, 420 randomly selected pig sera were tested in three commercially available and in two in-house HEV-antibody ELISAs. 43.6% (183/420) to 65.5% (275/420) of the sera were demonstrated to be reactive against human pathogenic HEV genotypes 1 and/or 3. The majority of sera reacted only weakly or not at all with the rat HEV antigen with very few sera showing a stronger reactivity to this antigen compared to the genotype 3 antigen. The results of all three HEV-IgG tests, i.e. the PrioCHECK(®) HEV Ab porcine ELISA kit, the ID Screen(®) Hepatitis E Indirect Multi-species ELISA kit and the genotype 3 in-house ELISA were in good accordance. Therefore, the remaining sera were tested using the PrioCHECK(®) HEV Ab porcine ELISA kit. Samples with a borderline result were finally determined by application of the conjugate-modified recomLine HEV IgG assay. A total of 1065 of the 2273 sera (46.9%) were found to be anti-HEV IgG-positive. While 38.4% (306/796) of fatteners (age between 3 and 9 months) exhibited HEV-specific antibodies, 51.4% (759/1477) of sows (age older than 9 months) exhibited anti-HEV antibodies (P<0.001). Fatteners kept in Southern Germany had a significantly higher HEV IgG prevalence compared to fatteners kept in the high pig density federal states North Rhine-Westphalia and Lower Saxony but also in German federal states with a low pig density. In conclusion, the present study clearly demonstrates that a high percentage of domestic pigs in Germany have had contact with HEV. Seroprevalence depends on the pig's age and herd origin with the most significant regional variations for fatteners. The presence of anti-HEV-free herds may indicate that it is feasible to establish and sustain HEV-free pig herds. HEV seroprevalence still depends on the assay used for testing. This demonstrates an urgent need for test validation.

Reassortants of the pandemic (H1N1) 2009 virus and establishment of a novel porcine H1N2 influenza virus, lineage in Germany.

The incursion of pandemic (H1N1) 2009 virus (pdmH1N1) into the German pig population was investigated in a serosurvey and by virological means between June 2009 and December 2012. Analysis of 23,116 pig sera from a total of 2,666 herds revealed 224 herds that reacted with pdmH1N1 but not with the prevalent avian-like H1N1 swine influenza virus. Sixty-six pdmH1N1 strains and their reassortant derivatives (pdmH1huN2, huH3pdmN1) have been collected since November 2009. Sequencing of three pdmH1N1, 20 pdmH1huN2 and one huH3pdmN1 strains with conventional and next generation sequencing techniques and subsequent phylogenetic analyses with available sequence data revealed the emergence of five distinct reassortant genotypes in Europe. The most frequent genotype emerged at least three times independently, one of which (Papenburg lineage) established a stable infection chain and became more prevalent in pigs than pdmH1N1 in Germany.

Serological response to influenza A H1N1 vaccine (Pandemrix®) and seasonal influenza vaccine 2009/2010 in renal transplant recipients and in hemodialysis patients.

In the present study, antibody response to seasonal influenza vaccination and to the adjuvanted one-shot influenza A H1N1 vaccine (Pandemrix(®)) was investigated in 57 hemodialysis (HD) patients and 48 renal transplant (RT) recipients. Specific antibodies were measured by hemagglutination inhibition (HI) test using a pandemic H1N1 strain and a seasonal H3N2 virus. HI titers of ≥1:40 were considered as protective. Hemodialysis patients showed seroprotection against pandemic H1N1 in 35.1%, against seasonal influenza in 36.8% and against both in 14.0%. In comparison, renal transplant recipients developed protective antibody titers against the pandemic H1N1 virus in 47.9%, against the seasonal H3N2 strain in 31.3% and against both in 18.8%. HD patients and renal transplant recipients younger than 60 years developed protective antibody response to the pandemic influenza H1N1 vaccine in 50.0% of the HD patients and 55.2% of the RT recipients and against seasonal influenza in 45.0/20.7% (HD/RT) of the cases. Patients aged ≥60 years showed seroprotection against pandemic influenza in 27.0/36.8% (HD/RT) and against seasonal influenza in 32.4/47.4% (HD/RT). Side effects were reported in only four patients. In hemodialysis patients and renal transplant recipients, vaccination against pandemic H1N1 and seasonal influenza is well tolerated. However, more than a half of these patients did not develop seroprotective antibody levels. Thus, new vaccines and altered vaccination regimes are likely necessary to achieve relevant antibody levels in these patient groups.

Prevalence of hepatitis E virus-specific antibodies in humans with occupational exposure to pigs.

Due to the increasing number of non-travel-associated hepatitis E virus (HEV) infections observed in several industrialised countries including Germany, there is a substantial interest in the characterisation of risk factors and transmission routes relevant to autochthonous HEV infections. Autochthonous cases are believed to be the result of a zoonotic HEV transmission from pigs, wild boars and deer. Recently, a high prevalence of HEV-specific antibodies in the German domestic pig population has been demonstrated. Thus, one may assume a higher prevalence of HEV-specific antibodies in humans with occupational exposure to pigs. In this study, sera obtained from 24 slaughterers, 14 meat inspectors, 46 pig farmers and 22 veterinarians were tested for the presence of HEV-specific antibodies using a line immunoassay. For comparison, sera obtained from 116 age- and gender-matched blood donors were also included. Twenty eight per cent (28.3%; 30/106) of the swine-exposed humans and 15.5% (18/116) of the blood donors without contact to pigs exhibited IgG-antibodies determined as reactive (i.e. borderline or positive) against HEV. Thus, an increased risk of HEV infection in humans occupationally exposed to pigs and particularly for slaughterers (41.7%; 10/24) was demonstrated.

Prevalence of antibodies to swine influenza viruses in humans with occupational exposure to pigs, Thuringia, Germany, 2008-2009.

The Eurasian lineages of swine influenza viruses are different genetically from classical swine H1N1 influenza viruses and comprise avian-like H1N1 and human-like H1N2 and H3N2 subtypes. Although sporadic isolation of such viruses from human specimens has been reported, the prevalence of human infections is not known. In the present study, the seroprevalence against Eurasian swine influenza viruses was investigated. Sera were collected in Thuringia, Germany, from December 2007 to April 2009. The study group comprised 118 professionals with occupational exposure to pigs (50 pig slaughterers/meat inspectors, 46 pig farmers, 22 veterinarians caring for pig herds). The control group included 118 age- and gender-matched blood donors from Thuringia. As a result, 18 sera of the study group were identified with raised hemagglutination-inhibition titers against a panel of nine swine influenza viruses (three strains/ subtype). For 17/18 sera this finding was confirmed in the neutralization assay. For 11/18 sera the raise of titers was significant, that is, a fourfold increase of hemagglutination-inhibition titers was observed. No gender-specific bias of the high titer sera was observed. Twelve sera of the control group showed increased hemagglutination-inhibition titers against swine influenza viruses. Hemagglutination-inhibition titers of 2/12 control sera were raised fourfold but did not exhibit a significant increase of neutralization titers. All increased hemagglutination-inhibition titers of the control group may be explained by cross-reactivity with seasonal influenza virus strains, as all these sera also reacted with human strains.