An investigation of antigenic drift of neuraminidases of influenza A (H1N1) viruses

A newly developed lectin neuraminidase test (LNT) and a panel of mouse monoclonal and post-infection ferret antibodies have been used to analyse antigenic drift in N1 neuraminidases of influenza A viruses isolated between 1933 and 1957 and also between 1977 and 1980. Significant antigenic differences were detected among the 'early' (1933-57) viruses since the NA of viruses isolated one year apart could be distinguished serologically. The NA of the 're-emerged' virus A/USSR/92/77 (H1N1) was antigenically related but not identical to influenza A viruses isolated in 1949 (A/Paris/49 (H1N1), A/Geneva/49 (H1N1) which thus predates the previously observed antigenic similarity of A/USSR/77 with A/FW/50 (H1N1) virus.     

An investigation of antigenic drift of neuraminidases of influenza A (H1N1) viruses.

A newly developed lectin neuraminidase test (LNT) and a panel of mouse monoclonal and post-infection ferret antibodies have been used to analyse antigenic drift in N1 neuraminidases of influenza A viruses isolated between 1933 and 1957 and also between 1977 and 1980. Significant antigenic differences were detected among the ''early'' (1933-57) viruses since the NA of viruses isolated one year apart could be distinguished serologically. The NA of the ''re-emerged'' virus A/USSR/92/77 (H1N1) was antigenically related but not identical to influenza A viruses isolated in 1949 (A/Paris/49 (H1N1), A/Geneva/49 (H1N1) which thus predates the previously observed antigenic similarity of A/USSR/77 with A/FW/50 (H1N1) virus.     

Antibodies to human influenzavirus neuraminidase (the A-Asian-57 H2N2 strain) in sera from Australian pelagic birds

Sera collected from Australian pelagic birds specifically inhibited the neuraminidase of the Asian/57 (H2N2) strain of human influenzavirus. Neuraminidase inhibition titres of some sera were high and the avidity of the inhibitor was comparable to that of specific antibody. The neuraminidase of A/Hong Kong/1/68 (H3N2), which has undergone considerable antigenic drift in man since 1957, was inhibited to a lesser extent by the bird sera, while the neuraminidases of the A/BEL/42 (H0N1) and A/FM/1/47 (H1N1) strains were not inhibited at all. The inhibitor could be removed from the sera by adsorption with A/57 virus particles, but not by particles of A/BEL or A/FM1 viruses. These results suggested that the inhibitor in the bird sera was specific antibody. The antibodies to A/57 neuraminidase were found in sera from wedge-tailed shearwaters (Puffinus pacificus) and noddy terns (Anous minutus) nesting on islands off the north-east coast of Australia. They were not found in sera from bridled terns (Sterna anaetheta) or brown gannets (Sula leucogaster) nesting on the same islands. Antibodies to A/57 neuraminidase were also detected in sera from short-tailed shearwaters (Puffinus tenuirostris), which migrate around the Pacific Ocean, suggesting that these birds may be responsible for bringing avian influenzaviruses from areas in the Northern Hemisphere into Australian coastal waters.     

Studies on relationships between human and porcine influenza. 2. Immunological comparisons of human A-Hong Kong-68 virus with influenza A viruses of porcine origin

Antigenic comparisons were made between the human A/Hong Kong/68 (H3N2) virus and a collection of influenza A viruses of swine origin. Haemagglutination-inhibition and neuraminidase-inhibition tests were used in addition to immunoprecipitin tests with monospecific antisera prepared against purified haemagglutinin and neuraminidase preparations. The antigenic relationships revealed by the studies are summarized as follows: (1) swine/Taiwan/7310/70 virus contained envelope antigens that were antigenically indistinguishable from those of A/Hong Kong/68 virus, (2) “classical” strains of swine influenzavirus related to A/swine/Iowa/15/30 (Hsw1N1) isolated between 1930 and 1967 contained neuraminidase that was antigenically distinct from that of A/Hong Kong/68 virus but related to that of human A0 and A1 viruses, and (3) the haemagglutinins of certain strains of “classical” influenza A virus appeared to show a minor antigenic relationship with the haemagglutinin of A/Hong Kong/68 virus. Immunoprecipitin tests suggested that this relationship was confined to only one of the two antigenic components of the haemagglutinin subunit. The antigenic relationships are discussed in respect of possible epidemiological relationships between human and swine influenza A viruses. It is proposed that the swine/Taiwan isolates be designated A/swine/Taiwan/70 (H3N2), indicating their antigenic identity with the human A/Hong Kong/1/68 virus.     

A pseudo-outbreak of influenza A associated with use of laboratory stock strain

In November 1982, when influenza type A(H3N2) viruses were spreading in the United States, influenza A(H1N1) viruses were reportedly isolated from 10 hospitalized patients in New Mexico, only two of whom had influenza-like illnesses. Reference stock influenza A/Fort Monmouth (FM)/1/47(H1N1) virus had been used to prepare fluorescent antibody test slides in the laboratory reporting the isolates. After investigation, it was concluded that the isolates from the patients' cultures were a result of laboratory contamination. When an unexpected cluster of unusual virus isolates is reported, the possibility of laboratory contamination should be considered.     

Prevalence of human (H1N1) influenza virus-antibody in Japanese swine.

A total of 571 swine sera collected at an abattoir in the city of Obihiro, Hokkaido during the period February-November 1984 were tested for antibody against human (H1N1) influenza virus strains. A high prevalence of antibody was observed for only 3 months from April to June in that year, in 81/180 sera (45.0%) to A/USSR/92/77 strain and in 50/180 sera (27.8%) to a current epidemic strain (A/Hokkaido/1/84). Some cross-reactions were observed between the A/USSR/92/77 and A/Hokkaido/1/84 antibodies (r = 0.75). Only minor relationships were noted between the A/New Jersey/8/76 (swine type H1N1) and A/USSR/92/77 (r = 0.35) or A/Hokkaido/1/84 (r = 0.51) antibodies. Absorption of sera positive for antibody to the A/Hokkaido/1/84 strain with the homologous virus strain removed all detectable antibodies, while the absorption of the sera with the A/New Jersey/8/76 strain produced incomplete absorption in one half of the sera tested. These results strongly suggest that the swine became infected with a human H1N1 virus as piglets during an epidemic of influenza which occurred in the human population during January and February 1984.     

Prevalence of human (H1N1) influenza virus-antibody in Japanese swine

A total of 571 swine sera collected at an abattoir in the city of Obihiro, Hokkaido during the period February-November 1984 were tested for antibody against human (H1N1) influenza virus strains. A high prevalence of antibody was observed for only 3 months from April to June in that year, in 81/180 sera (45.0%) to A/USSR/92/77 strain and in 50/180 sera (27.8%) to a current epidemic strain (A/Hokkaido/1/84). Some cross-reactions were observed between the A/USSR/92/77 and A/Hokkaido/1/84 antibodies (r = 0.75). Only minor relationships were noted between the A/New Jersey/8/76 (swine type H1N1) and A/USSR/92/77 (r = 0.35) or A/Hokkaido/1/84 (r = 0.51) antibodies. Absorption of sera positive for antibody to the A/Hokkaido/1/84 strain with the homologous virus strain removed all detectable antibodies, while the absorption of the sera with the A/New Jersey/8/76 strain produced incomplete absorption in one half of the sera tested. These results strongly suggest that the swine became infected with a human H1N1 virus as piglets during an epidemic of influenza which occurred in the human population during January and February 1984.     

Recycling of H1N1 influenza A virus in man--a haemagglutinin antibody study.

Sera from people born between 1883 and 1930 and collected in 1977 were tested for the presence of HI antibodies to A/FM/1/47 (H1N1) virus and three recently (1977 and 1978) isolated influenza A-H1N1 viruses. The highest frequency of high-titred antibody to the four H1N1 viruses was detected in sera from people born in 1903-4, i.e. 42, 54, 38, and 22% had antibody against A/FM/1/47, A/Hong Kong/117/77, A/Brazil/11/78, and A/Fukushima/103/78 respectively. The birthdate groups 1896-1907 showed a higher percentage of HI antibody titres greater than or equal to 18, greater than or equal to 50, greater than or equal to 100 or greater than or equal to 1600 against the four H1N1 viruses than the birthdate groups 1907-30. This indicates the existence of an era, 1908-18, in which, apart from the H3N2 virus (1900-18), the H1N1 virus was epidemic among the human population.     

Laboratory-based surveillance of influenza virus in the United States during the winter of 1977--1978. II. Isolation of a mixture of A/Victoria- and A/USSR-like viruses from a single person during an epidemic in Wyoming, USA, January 1978

At a time when outbreaks and sporadic cases of influenza caused a A/Victoria/3/75-like and A/Texas/1/77-like H3N2 strain of influenza were occurring in the Rocky Mountain region of the USA, about 60% of the students of a high school in Cheyenne, Wyoming, were involved in an outbreak of influenza-like illness. Six influenza A(H1N1) virus isolates were obtained from throat swabs collected from 12 of these students. Virus isolated from a seventh student, however, contained a mixture of H1 and H3 (A/Victoria/3/75-like) hemagglutinins and N1 and N2 neuraminidases, as shown by the ability to clone from the mixture viruses with antigenic components H1N1, H3N1, and H3N2. An antigenic hybrid virus with H3N1 composition was re-isolated from the original throat swab. The results show that one student was shedding a mixture of A/Victoria/3/75(H3N2)-like and A/USSR/90/77(H1N1)-like viruses at the time his throat swab was taken.     

Antibody response to immunization with influenza A/USSR/77 (H1N1) virus in young individuals primed or unprimed for A/New Jersey/76 (H1N1) virus.

A group of 269 pupils of the Harbour and Transport Training Institute in Rotterdam (group A), aged 13-20 years, and of 109 patients of the Dr Mr Willem van den Bergh Foundation at Noordwijk (group B), aged 11-21 years, were immunized with a whole virus vaccine containing 10, 20, or 40 microgram HA of A/USSR/92/77 (H1N1) influenza virus. A booster vaccination was administered 6 weeks later with 20 microgram HA of the same virus. Many of the participants had been immunized during the two preceding years with a whole virus vaccine containing A/New Jersey/8/76 (H1N1) (A/NJ/76) virus. The side-effects, mostly of a moderate nature, increased with the dose of virus in the vaccine. In group A side effects were least frequent in the vaccinees who had never received A/NJ/76 vaccine. A single dose of A/USSR/77 vaccine did not produce satisfactory levels of homologous antibodies. After booster immunization with 20 microgram HA of A/USSR/77 virus participants showed a higher homologous antibody response in all vaccine-dose groups if they had not been immunized with A/NJ/76 virus in previous years. After primary and especially after booster immunization with A/USSR/77 virus, a very high response against A/NJ/76 virus and adequate levels of A/NJ/76 antibody were found in participants who had been immunized previously with A/NJ/76 virus. Those who had not been immunized with this virus previously showed no or a very low antibody response to A/NJ/76 virus.     

Influenza virus-like particles comprised of the HA, NA, and M1 proteins of H9N2 influenza virus induce protective immune responses in BALB/c mice

Avian influenza viruses represent a growing threat for an influenza pandemic. To develop recombinant vaccine for avian influenza of the H9N2 subtype, we expressed in insect cells virus-like particles (VLPs) consisting of three structural proteins of influenza A/Hong Kong/1073/99 (H9N2) virus. Upon infection of Sf9 cells with recombinant baculoviruses, the hemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins were co-expressed in the infected cells, self-assembled, and released into the culture medium as VLPs of 80–120 nm in diameter. VLPs exhibited functional characteristics of influenza virus including hemagglutination and neuraminidase activities. In BALB/c mice, VLPs elicited serum antibodies specific for influenza A/Hong Kong/1073/99 (H9N2) virus and inhibited replication of the influenza virus after challenge. Thus, VLPs represent a potential strategy for the development of human vaccines against avian influenza H9N2 viruses.     

Cross-protection against a lethal influenza virus infection by DNA vaccine to neuraminidase

Cross-protection against a lethal influenza virus infection was examined in BALB/c mice immunized with plasmid DNAs encoding the neuraminidase (NA) from different subtype A viruses. Each NA-DNA was administered twice, 3 weeks apart, at the dose of 1 μg per mouse by particle-mediated DNA transfer to the epidermis (gene gun) or at a dose of 30 μg per mouse by electroporation into the muscle. Three weeks after the second vaccination, the mice were challenged with lethal doses of homologous or heterologous viruses and the ability of each NA-DNA to protect the mice from influenza was evaluated by determining the lung virus titers, body weight and survival rates. The H3N2 virus NA-DNA conferred cross-protection against lethal challenge with antigenic variants within the same subtype, but failed to provide protection against infection by a different subtype virus (H1N1). The degree of cross-protection against infection was related to titers of the cross-reacting antibodies. These results suggest that NA-DNA can be used as a vaccine component to provide effective protection against infection not only with homologous virus but also with drift viruses.     

Antigenicity in hamsters of inactivated vaccines prepared from recombinant influenza viruses.

Inactivated vaccines prepared form influenza virus strains obtained by the recombination of A/PR/8/34 (H1N1) or A/FM/1/47 (H1N1) viruses with A/Victoria/3/75 (H3N2) virus, were tested for their antigenicity in hamsters. The parental origin of the genes of each cloned recombinant virus was determined by polyacrylamide gel electrophoresis, and vaccines prepared from each strain by concentration, purification on sucrose density gradients and inactivation with formalin. All the recombinant strains used in these studies possessed surface haemagglutinin and neuraminidase antigens derived from the A/Victoria/75 parent strain. On inoculation into hamsters, at equivalent concentrations, these vaccines varied in their ability to induce haemagglutination-inhibiting (HI) antibodies in the serum. This variation was not dependent on concentration and was observed using neutralization and single radial haemolysis, as well as HI. The possible reasons for the findings are discussed.     

Laboratory-based surveillance of influenza virus in the United States during the winter of 1977-1978. I. Periods of prevalence of H1N1 and H3N2 influenza A strains, their relative rates of isolation in different age groups, and detection of antigenic variants

Influenza A (H3N2) viruses were isolated from outbreaks and epidemics of disease during the period December 1977 to March 1978. For the last two months of this period, H1N1 strains of influenza A were also responsible for epidemics. In some regions (e.g., Hawaii) co-circulation of H1N1 AND H3N2 strains occurred, whereas in other regions (e.g., Wisconsin) isolation of H3N2 strains had almost ceased prior to isolation of H1N1 strains. Few influenza B isolates were reported. Analysis of the ages of patients from whom specimens were submitted for influenza virus isolation confirmed that, whereas H3N2 strains were isolated from persons of all ages, greater than 97 per cent of H1N1 isolates in six states analyzed were recovered from patients less than 26 years old, although specimens were tested from older persons who were ill during the period of prevalence of H1N1 influenza. The majority of H3N2 isolates tested by hemagglutinin-inhibition reaction were similar to A/Texas/1/77, and the majority of H1N1 isolates were similar to A/USSR/90/77. Antigenic analysis of isolates, however, identified a small number of variants of H3N2 and H1N1 strains.     

Homologous and heterologous antibody responses to subunit influenza virus vaccine.

In a group of 32 adult volunteers given subunit influenza virus vaccine containing 250 international units (i.u.) of A/Victoria/3/75, 250 i.u. of A/Scotland/840/74 and 300 i.u. of B/Hong Kong/8/73, there were substantial increases in the geometric mean homologous haemagglutination-inhibiting (HI) antibody titres. There was also substantial boosting of the antibodies to the earlier variants of the Hong Kong (H3N2) series and to a later variant of the Asian (H2N2) series. There was no boosting of antibodies to the A/FM/1/47 strain, a representative member of the H1N1 series, but two individuals showed substantial rises to A/PR/8/34 (HON1). There were increases in the HI titre of antibodies cross reactive with two recent isolations, A/Texas/1/77, and A/Victoria/35/77, but the majority of vaccinees failed to reach antibody titres likely to be protective against such strains.     

Clinical,epidemiological and virological characteristics of the first detected human case of avian influenza A(H5N6) virus

A human infection with novel avian influenza A H5N6 virus emerged in Changsha city, China in February, 2014. This is the first detected human case among all human cases identified from 2014 to early 2016. We obtained and summarized clinical, epidemiological, and virological data from this patient. Complete genome of the virus was determined and compared to other avian influenza viruses via the construction of phylogenetic trees using the neighbor-joining approach. A girl aged five and half years developed fever and mild respiratory symptoms on Feb. 16, 2014 and visited hospital on Feb. 17. Throat swab specimens were obtained from the patient and a novel reassortant avian influenza A H5N6 virus was detected. All eight viral gene segments were of avian origin. The hemagglutinin (HA) and neuraminidase (NA) gene segments were closely related to A/duck/Sichuan/NCXN11/2014(H5N1) and A/chicken/Jiangxi/12782/2014(H10N6) viruses, respectively. The six internal genes were homologous to avian influenza A (H5N2) viruses isolated in duck from Jiangxi in China. This H5N6 virus has not gained genetic mutations necessary for human infection and was suggested to be sensitive to neuraminidase inhibitors, but resistant to adamantanes. Epidemiological investigation of the exposure history of the patient found that a live poultry market could be the source place of infection and the incubation period was 2–5 days. This novel reassortant Avian influenza A(H5N6) virus could be low pathogenic in humans. The prevalence and genetic evolution of this virus should be closely monitored.     

Homologous and heterologous antibody responses to subunit influenza virus vaccine

In a group of 32 adult volunteers given subunit influenza virus vaccine containing 250 international units (i.u.) of A/Victoria/3/75, 250 i.u. of A/Scotland/840/74 and 300 i.u. of B/Hong Kong/8/73, there were substantial increases in the geometric mean homologous haemagglutination-inhibiting (HI) antibody titres. There was also substantial boosting of the antibodies to the earlier variants of the Hong Kong (H3N2) series and to a later variant of the Asian (H2N2) series. There was no boosting of antibodies to the A/FM/1/47 strain, a representative member of the H1N1 series, but two individuals showed substantial rises to A/PR/8/34 (HON1). There were increases in the HI titre of antibodies cross reactive with two recent isolations, A/Texas/1/77, and A/Victoria/35/77, but the majority of vaccinees failed to reach antibody titres likely to be protective against such strains.     

Evolution of H5N1 avian influenza viruses in Asia

An outbreak of highly pathogenic avian influenza A (H5N1) has recently spread to poultry in 9 Asian countries. H5N1 infections have caused > or =52 human deaths in Vietnam, Thailand, and Cambodia from January 2004 to April 2005. Genomic analyses of H5N1 isolates from birds and humans showed 2 distinct clades with a nonoverlapping geographic distribution. All the viral genes were of avian influenza origin, which indicates absence of reassortment with human influenza viruses. All human H5N1 isolates tested belonged to a single clade and were resistant to the adamantane drugs but sensitive to neuraminidase inhibitors. Most H5N1 isolates from humans were antigenically homogeneous and distinct from avian viruses circulating before the end of 2003. Some 2005 isolates showed evidence of antigenic drift. An updated nonpathogenic H5N1 reference virus, lacking the polybasic cleavage site in the hemagglutinin gene, was produced by reverse genetics in anticipation of the possible need to vaccinate humans.     

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.     

Development of Eurasian H7N7/PR8 high growth reassortant virus for clinical evaluation as an inactivated pandemic influenza vaccine

Avian-to-human transmission of the high pathogenicity (HP) H7N7 subtype avian influenza viruses in the Netherlands during 2003 caused zoonotic infections in 89 people, including a case of acute fatal respiratory distress syndrome. Public health emergency preparedness against H7N7 avian influenza viruses with pandemic potential includes the development of vaccine candidate viruses. In order to develop a high growth reassortant vaccine candidate virus, low pathogenicity (LP) A/mallard/Netherlands/12/2000 (H7N3) and A/mallard/Netherlands/2/2000 (H10N7) strains were selected as donors of the H7 haemagglutinin and N7 neuraminidase genes, respectively. The donor viruses exhibited high amino acid sequence homology with the surface glycoproteins of A/Netherlands/219/03 H7N7 virus (NL219), an isolate recovered from the fatal human case. Adhering to the seasonal influenza vaccine licensure regulations, we generated a H7N7/PR8 reassortant containing desired surface glycoprotein genes from the mallard viruses and internal genes of A/Puerto Rico/8/34 human vaccine strain (H1N1). Antigenic analysis revealed that the vaccine candidate virus confers broad antigenic cross-reactivity against contemporary Eurasian and the North American H7 subtype human isolates. Mice immunized with formalin inactivated (FI) H7N7/PR8 whole virus vaccine with or without aluminum hydroxide adjuvant conferred clinical protection from mortality and reduced pulmonary replication of the NL219 challenge virus. The FI H7N7/PR8 whole virus vaccine also afforded cross-protection in mice at the pulmonary level against antigenically distinct North American LP A/Canada/444/04 (H7N3) human isolate. The vaccine candidate virus satisfied the agricultural safety requirements for chickens, proved safe in mice, and has entered in phase-I human clinical trial in the United States.