Influenza virus subtypes in aquatic birds of eastern Germany.

We report the findings of a 12-year surveillance study (1977-89) of avian influenza A viruses in eastern Germany. Viruses were isolated directly from feral ducks (n = 236) and other wild birds (n = 89); from domestic ducks (n = 735) living on a single farm; and from white Pekin ducks (n = 193) used as sentinels for populations of wild aquatic birds; mainly sea birds. The efficiency of virus isolation was 9.9% overall, with considerable variability noted among species: 8.7% in wild ducks, 0.9% in other feral birds and 38% in Pekin ducks. Use of sentinel ducks in wild pelagic bird colonies improved virus detection rates fivefold, suggesting that this approach is advantageous in ecological studies. Among the 40 different combinations of hemagglutinin (HA) and neuraminidase (NA) subtypes we identified, H6N1 predominated (23.6% for all avian species), followed by H4N6 (11%). Among individual species, the frequency profiles favored H2N3 (20.8%) and H4N6 (20.3%) in feral ducks; H7N7 (22.3%), H4N6 (24.4%) and H2N3 (10.4%) in Pekin ducks used as sentinels; and H6N1 (34.8%) and H6N6 (15.1%) in domestic ducks maintained on a single farm. By relying on sentinel birds for serological assays, it was possible to trace an "influenza season" in feral swan populations, beginning in August and continuing through the winter months. Comparison of subtype distribution of influenza viruses for Europe and North America showed significant differences. This supports the fact of two geographically distinct gene pools of influenza viruses in birds connected with their distinct flyways of each hemisphere. The high frequency of isolation of H2 influenza viruses is of considerable interest to those interested in the recycling of this subtype in humans. Similarly the frequent isolation of H7N7 influenza viruses raises concern about reservoirs of potentially pathogenic influenza virus for domestic poultry. Our results confirm the existence of a vast reservoir of influenza A viruses in European aquatic birds, which possesses sufficient diversity to account for strains that infect lower animals and humans.

Epidemiology and ecology of tick-borne encephalitis in the eastern part of Germany between 1960 and 1990 and studies on the dynamics of a natural focus of tick-borne encephalitis.

From 1960 to 1990, attempts to isolate TBE virus from ticks and small mammals were made and investigations to detect TBE antibody in small mammals, game and humans were performed in the five new federal Länder of Germany. The confirmed TBE cases for which the site of exposure could be determined were also registered. As a result of these epidemiological and ecological investigations, a map is presented showing the natural foci of TBE which are primarily located in the subatlantic and mountainous climatic regions. TBE was endemic in the area of investigation from 1960 to 1990 showing a morbidity of up to 0.7 per 100,000 inhabitants which decreased in recent years to 0.02 per 100,000 inhabitants. The natural foci of TBE virus in eastern Germany showed a high activity between 1960 and 1970. Since that time, there have hardly been any cases of human disease and TBE virus could no longer be detected, neither in ticks nor in small mammals. Taking the natural focus on the Island of Usedom as an example, attempts have been made to elucidate whether such foci have become extinct or whether they have persisted. From 1983 to 1989, a surveillance programme was performed to detect antibodies to TBE virus in small mammals and game and to attempt to culture virus from ticks and small mammals. The attempts to isolate virus from a total of 8200 ticks were negative. Attempts to isolate virus from the brains of small mammals were also negative. Antibody prevalence in 446 small mammals and 500 animals of game was ca. 1%. At one site, the sero-positive reactions converted from 0% (1983-1988) to 4.5% (1989) among small mammals population. The investigations performed on the Island of Usedom have shown that this natural focus has not disappeared but is in a state of endemic latency. Moreover, the seroconversion observed in the small mammals population shows that further surveillance of such foci is necessary. This becomes obvious by the sporadic occurrence of single TBE cases as well as by a low antibody prevalence of 1% in small mammals and game. The epidemiological situation in eastern Germany is thus completely different from that in western Germany where an average of 70-120 TBE cases per year are registered, occurring mainly in Bavaria and Baden-Württemberg.

Properties of rabies strain ("Pasteur Potsdam") adapted to primary dog kidney cells.

The Pasteur strain of fixed rabies virus (Pasteur Institute Paris, passage 2061 in rabbit brain) was adapted by alternate passages to primary dog kidney cells. The adapted rabies virus designated as "Pasteur Potsdam" developed no CPE and yielded four harvests with a titre of 5.5-7.0 (log MICLD50/ml). The strain could be grown in BHK 21/S13, CER and N2a neuroblastoma cells. In the cultures of BHK 21/S13 cells the virus titered 6.0-8.5 (log MICLD50/ml). In SDS PAGE its G protein migrated faster than that of the ERA strain. The inactivated antigen induced interferon in mice. The strain was identified by anti-rabies immunoglobulin. The harvested material showed an antigenic value of 0.4 IU/ml. The virus was not pathogenic after s.c. and i.p. inoculations to mice, rats, Syrian hamsters, and rabbits and after i.m. inoculation to Syrian hamsters and rats.

Studies of some biological and molecular characteristics of avian influenza A/H7 (Hav1) subtypes.

Current isolates of the subtypes H7N3 and H7N7 from 1979 to 1981 were examined and compared with the reference strains with regard to their antigenic variability and to their pathogenicity for birds and mammals in order to establish the potentiality of influenza A/H7 virus (Hav1) transmission from birds to mammals. The analysis of the electrophoretic mobility of virus-induced polypeptides and of the double-stranded RNA segments after hybridization revealed equal, similar and deviating patterns. A substantial drift was determined in the surface antigens, especially in the neuraminidase. The avian strains replicated also in mammalian cells and were pathogenic for mammals. All strains examined were reisolated from the infected mammals; they caused more pronounced inflammatory changes in the trachea and lungs of infected mammals than in those of birds.

[Characterization of RNA polymerase activity of highly purified preparations of influenza virus A/duck/Alberta/48/76]. / Charakterisierung der RNS-polymerase-aktivität hochgereinigter präparationen des influenzavirus a/duck/alberta/48/76.

The influenza virus A/duck/Alberta/48/76 with the antigen formula H7N3 (16) and Hav1 Nav2 (WHO nomenclature from 1971) (15), respectively, as well as a nonpathogenic virus of the subtype Hav1 were purified to a high degree by ultracentrifugation in continuous sucrose gradients (15-40% w/w and 20-60% w/w, respectively). The activity of the RNA polymerase of this virus preparation was determined by incorporating 3H-UMP in acid insoluble material following preincubation of the virus with the nonionic detergens Nonidet P-40 for 15 min at 32 degrees C. The influence of different concentrations was investigated of dinucleotid, NaCl, MgCl2, Nonidet P-40 and different incubation temperatures. Optimal incorporation rates were found at following conditions: 0.2 mM dinucleotid ApG, 150 mM sodium chloride and 8 mM magnesium chloride by concentration of ions, 0.25-0.5% detergens Nonidet P-40 as well as a temperature of incubation of 32 degrees C. The data for optimal polymerase activity for the avian influenza virus A/duck/Alberta/48/76 are generally not different from the conditions described for the Fowl-Plague-Virus and for human strains.

Influenza virus A/swine-outbreaks in domestic pigs and antibody findings in human sera.

In the last years, the occurrence of influenza viruses A/H1N1 (Hsw1N1) in pig stocks of different countries has been increasingly reported. In general, the isolated viruses were related to the influenza virus A/New Jersey/8/76 H1N1 (Hsw1N1). Human infections were not reported in these outbreaks. Since March 1981, very limited influenza outbreaks in several pig stocks of the GDR with high morbidity and very low lethality have been observed. The illness took an uncomplicated path and generally subsided after 3 days. Some of the virus isolates were examined and typed as influenza virus A/swine/Potsdam/81/H1N1 (Hsw1N1). By serological examinations of convalescent pigs the aetiologic importance of the isolates was confirmed. Infection of the contact persons by the influenza virus A/swine/Potsdam/81 is to be regarded as likely according to the serological results.

Surveillance of pelagic birds for influenza A viruses.

Within a 4-year surveillance period for influenza A virus in pelagic birds, 351 influenza A strains were isolated from the trachea or cloaca of 3344 apparently healthy ducks, gulls, swans, terns and geese. The isolated influenza A viruses represent 14 subtypes. Their haemagglutinins (HA) were mainly related to avian HA, but also to the human HA H2 and to the swine HA Hswl. The neuraminidases (NA) were identified as avian, equine and human NA. The isolated influenza A strains include fowl plague-like viruses Havl Neql, strains with the surface antigen Hswl Nav4 and the subtype Hav7 Navl isolated from unconcentrated water samples. A subtype unknown to date, with the antigen formula H2 Nav4, was isolated from ducks. 8.2% of pekin ducks showed dual infections.

Detection of influenza A viruses and influenza epidemics in wild pelagic birds by sentinels and population studies.

Field experiments with domestic ducks as sentinels and virological and serological population observations on wild pelagic birds on a seabird-breeding island in the Baltic sea and on rivers and lakes near Berlin were conducted to detect the influenza A virus circulation and epidemics in wild pelagic bird populations. Influenza A virus isolates were detected in tracheal and cloacal swabs from sentinel ducks and from healthy pelagic birds. Influenza A virus epidemics with different subtypes in an influenza season beginning in July/August occurred approximately monthly. According to serological population studies in wild pelagic birds there were some epidemics with influenza A viruses, also related to Fowl Plaque Virus (Hav1) and Swine influenza virus (Hsw1). It is possible to recognize epidemics among wild birds by sentinels and by periodic serological observations of wild pelagic bird populations. One isolate from sentinel ducks - A/duck/Potsdam/81 (Hav8 Nav5) - had an hitherto undescribed combination of the surface antigens.

[Effect of low pH values on the infectivity and neuraminidase activity of human and animal strains of influenza virus type A]. / Uber den Einfluss niedriger pH-Werte auf die Infektiosität und Neuraminidaseaktivität menschlicher und tierischer Stämme des Influenzavirus Typ A.

The influence of acidic pH on the infectivity and neuraminidase activity of human, equine and avian type A influenza virus strains has been studied. Following exposure to pH 3 human and equine strains lost their infectivity completely, whereas all investigated strains of the subtypes Hav6N2 and Hav7Neq2 retained a certain amount of infectivity. In contrast to human and equine strains the avian strains retained also 38% of their original neuraminidase activity after acidic treatment. Partial retention of infectivity and the relative stability of the neuraminidase following exposure to acidic pH are supposed to be linked together in avian influenza virus strains implicating neuraminidases for their ability to prevent the aggregation of virions.

[Neuraminidase-Inhibiting Antibodies to Avian Influenza Virus Subtypes in Human Sera (author's transl)]. / Neuraminidasehemmende Antikörper gegen aviäre Influenzabirus- Subtypen in Humanseren.

400 human sera were tested both in hemagglutination inhibition (HI) and neuraminidase inhibition (NI) tests for antibodies to avian and animal influenza virus subtypes. In the H1 test we only found antibodies to the avian subtype Hav 7 and the animal subtypes Hsw 1 and Heq 2 whereby the latter was mainly demonstrated in elderly persons 60 to 100 years old. The findings of Hav 7 are due to H 3 antibodies and reflect the relationship between both antigens. In the NI test we obtained positive results in 21.8% of the human sera with the neuraminidase subtype N 3 (Nav 2/3) with a peak in persons who were 60 to 70 years old. 11.0% of the sera contained antibodies to the neuraminidase subtype N 8 (Neq 2) and were found exclusively in people 60 to 100 years old, and 9.3% of sera showed positive reactions with the subtype N5 (Nav 5). Until now an immunological relationship between the neuraminidase subtypes N 1, N 2, and N 3 is not known, and could'nt be found in our own studies. Contaminations of antigens can also be excluded. The possible origin of these antibodies to avian neuraminidase subtypes is discussed.