WHO collaborative studies on enterovirus reference antisera: second report

This paper summarizes the results of the second part of co-operative studies undertaken by the WHO International Reference Centre for Enteroviruses and a number of WHO Regional Virus Reference Centres, WHO Virus Collaborating Laboratories and other laboratories in a comprehensive testing programme of enterovirus equine antisera. The studies were designed to appraise the specificity of immune serum prepared in horses against 16 representative prototype enteroviruses (polioviruses 2 and 3, coxsackieviruses A7, B1, B2, B4, B5 and B6 and echoviruses 1, 2, 3, 5, 7, 9, 12 and 17). Tests for neutralizing antibody were performed not only against the homologous viruses but also against the homotypic strains available in each laboratory. Tests for heterotypic antibody were made against the entire group of enteroviruses, reoviruses 1-3 and adenoviruses 1-11, 13 and 17. Each serum sample represented a pool of the individual bleedings taken from a group of horses before and after immunization with each virus antigen. The results showed that the homologous geometric mean titres of the immune sera ranged from 7000 to 36 000, whereas the preinoculation sera were negative. Results of the homotypic tests showed the usefulness of the sera. The specificity of the haemagglutination-inhibiting antibody of the sera against coxsackieviruses A7, B1, B5, B6 and echoviruses 3, 7, 12 was demonstrated. The results of the tests performed in tissue culture and mice are discussed. Co-operative testing of other enterovirus equine antisera is now in progress.     

WHO collaborative studies on enterovirus reference antisera

This paper summarizes the results of co-operative studies undertaken by the WHO International Reference Centre for Enteroviruses and a number of WHO Regional Reference Centres for viruses, WHO Virus Collaborating Laboratories, or other laboratories in a comprehensive testing programme of enterovirus equine antisera. The studies were designed to appraise the specificity of immune serum prepared in horses against five representative prototype enteroviruses (poliovirus 1, coxsackieviruses A9 and B3, and echoviruses 4 and 11). Tests for neutralizing antibody were performed not only against the homologous viruses, but also against a battery of heterotypic picornaviruses, comprising 63 enteroviruses, several rhinoviruses, and three reoviruses. Each serum sample represented a pool of the individual bleedings taken from groups of five or six horses before and after immunization with each virus antigen (except echovirus 4, for which three animals were immunized). The results showed that, whereas the homologous titres of the antisera (poliovirus 1, coxsackieviruses A9 and B3, echoviruses 4 and 11) were found to be within a useful range (700-8000), heterotypic antibodies of significant titre were not detected. All corresponding pre-inoculation sera were negative. The specificity of the haemagglutination-inhibiting antibody of the coxsackievirus B3 and echovirus 11 antisera was also demonstrated. This accumulated information played a major role in a decision to prepare in horses antisera to the rest of the enterovirus group. Co-operative testing of the other enterovirus equine antisera is now in progress.     

WHO collaborative studies on enterovirus reference antisera; fourth report

This paper summarizes the results of the fourth part of a comprehensive programme undertaken by the WHO International Reference Centre for Enteroviruses and other laboratories for the testing of enterovirus equine antisera prepared for long-term use as reference antisera. The studies were designed to appraise the specificity of the immune serum of horses inoculated with prototype enteroviruses (coxsackievirus types A2, 4, 8, 10, 11, 14-16, 18-21, and 24, and echoviruses E21, 27, 30, 31, and 33). Tests for neutralizing antibody were performed against the homologous viruses and against available regional homotypic strains. Heterotypic tests were performed against reoviruses 1-3, adenoviruses 1-31, and the entire series of enteroviruses (with the exception of enterovirus 68). The homologous geometric mean titre of the 5 echovirus antisera ranged from 3 000 to 10 000; the titre of 1 coxsackievirus antiserum (A24) was only about 400, but the titres of the others ranged from 1 500 to 14 000. All corresponding preinoculation sera were negative. Heterotypic antibody of significant titre was found in 4 antisera: E31 serum against E5 virus, CA8 serum against CA3 virus, CA13 serum against CA18 virus, and CA15 serum against CA2 virus. Information on other heterotypic antibody titres (where found) is recorded for guidance in the use of the sera. The results of homotypic tests with viruses isolated by the collaborating laboratories, though limited in number, were satisfactory.     

Lyophilized combination pools of enterovirus equine antisera: new LBM pools prepared from reserves of antisera stored frozen for two decades

This paper describes the preparation and test procedures for a second batch of lyophilized LBM combination antiserum pools, A through H, used for identifying 42 enteroviruses. Each pool is selectively composed of 10 or 11 of 42 individual enterovirus equine sera so that it contains 500 antibody units of each serum component per 0.1 ml. The new pools have been constituted from equine monovalent antisera that were prepared during the period 1962-67 and then evaluated and standardized in a series of collaborative international studies. An essential aspect of preparing the new pools was ensuring that the individual sera had retained high antibody titres through the long period of storage. At the time of retesting, the original stocks of these monovalent sera had been stored frozen at -20°C for periods ranging from 16 to 20 years.     

Development of an enzyme-linked immunosorbent assay (ELISA) for the detection of specific antibodies against an H7N7 and an H3N8 equine influenza virus.

This paper describes a solid-phase microtitre plate enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to equine influenza viruses. Using egg-grown influenza viruses as the antigens attached to the solid phase, cross-reactions were observed between an H7N7 equine virus (designated A1) and an H3N8 equine influenza virus (designated A2) when untreated antisera were tested. Absorption of antisera with egg-grown A/Porcine/Shope/1/33 influenza virus eliminated cross-reactive antibodies so that specific detection of anti-equine influenza A1 or A2 antibodies was possible. Examination of horse sera following vaccination with A1 and/or A2 isolates showed that antibodies were produced against antigen associated with egg allantoic fluid as well as against virus. Such antibodies were eliminated following the absorption of antisera with porcine influenza virus. Results using sera from horses with known vaccination histories confirmed that the ELISA preferentially detected antibodies homologous to the antigen attached to the solid phase and methods to evaluate the current serological state of individual horses by relating the titres of specific antibodies against equine influenza A1 and A2 isolates are shown. This ELISA provides a simple and rapid method of assessing specific antibodies from horse sera and offers advantages over the ''routine'' HI and SRH assessments since it gives high precision, is economical of reagents and has the capacity to handle large numbers of serum samples.     

Changes in the antibody status of a population following epidemic infection by influenza virus A2/Hong Kong/1/68

The haemagglutinin of influenza virus A2/Hong Kong/1/68 was shown to be markedly different from that of previously isolated A2 virus strains. No haemagglutination-inhibiting (HI) antibody to A2/Hong Kong/1/68 virus was detected in serum specimens collected in 1966 from persons aged 60 years or less. In contrast, HI antibody tests with 270 sera collected in 1968 indicated that 9·6% had demonstrable HI antibody at low titres, and 35·2% of 454 postepidemic (1969) sera had demonstrable HI antibody at relatively high titres. Most sera from persons aged 80 years and more collected in 1968 and 1969 had demonstrable HI antibody to influenza virus A2/Hong Kong/1/68. No HI antibody to the Hong Kong virus was detected in pre-epidemic sera from children aged 6 months to 3 years, whereas 32% of postepidemic sera had HI antibody. The acquisition of HI antibody to A2/Hong Kong/1/68 was not accompanied by an increase in the incidence or titres of HI antibody to heterotypic A2 influenza viruses. For sera from children aged 4-11 years, an increase of HI titre to heterotypic A2 influenza was found.     

Enterovirus type 71 infection in Melbourne

Between November 1972 and May 1973, 60 strains of a new enterovirus were isolated from 49 patients investigated at Fairfield Hospital for Communicable Diseases, Melbourne. Of these patients 39 were admitted to hospital with aseptic meningitis (which was accompanied by a rash in 6), 5 others had rash alone, 4 had acute respiratory tract infections, and 1 had infective polyneuritis. A representative strain from this outbreak had the physicochemical properties of an enterovirus but could not be identified with antisera prepared against the prototype polio, coxsackie, and echo viruses. Studies, performed in association with the WHO Collaborating Centre for Virus Reference and Research, Houston, TX, USA, showed the outbreak to be due to enterovirus 71. Most of the epidemic strains required sodium deoxycholate treatment before neutralization could be demonstrated.     

Evaluation of enterovirus immune horse serum pools for identification of virus field strains

Immune horse sera to 42 enterovirus immunotypes were pooled according to the Lim Benyesh-Melnick and the ”intersecting serum” schemes. Each serum was diluted in the pools to contain 50 antibody units. After it was established that the pools correctly neutralized prototype virus strains, they were evaluated in tests against 273 enterovirus field strains representing most of the viral types included in the pools. With test virus doses of 10-100 TCD₅₀, most of the poliovirus and coxsackievirus field strains were correctly identified in both schemes, but a number of the echoviruses were neutralized by heterotypic pools, particularly in the Lim Benyesh-Melnick scheme. However, at higher test virus doses of 320-3200 TCD₅₀, little heterotypic neutralization occurred in either scheme, and 93-94% of the virus field strains were correctly identified in each scheme. With these larger virus doses, breakthrough tended to occur in homologous pools by the 7th day, but rarely by the 5th day. Since the Lim Benyesh-Melnick pool scheme employs 8 pools as compared with 13 for the intersecting serum scheme, and since the two schemes were equally satisfactory for identifying virus field strains at test virus doses of 320-3200 TCD₅₀, immune horse sera will be pooled by the former scheme, thus utilizing fewer pools, for distribution to qualified viral diagnostic laboratories.     

A multi-species malaria antigen for use in the indirect fluorescent antibody test

A multi-species thick smear antigen containing equal proportions of P. vivax, P. falciparum, and P. brasilianum schizonts was prepared for use in the indirect fluorescent antibody test for malaria. Tests with 80 sera showed that antibody titres with the multi-species antigen always equalled or exceeded the highest titre obtained when the sera were tested with the constituent mono-species antigens. Use of the multi-species antigen greatly reduces the time and labour required to screen serum specimens for malaria antibody. This study confirms the need for homologous antigens of all species to be detected if maximum sensitivity and reactivity are to be achieved.     

Human antibody response to fragments A and B of diphtheria toxin and a synthetic peptide of amino acid residues 141-157 of fragment A

Examination of a selection of serum samples from adults from two regions of England showed that 50% of men in the 16-24 years and over 55 years age groups had high titres of antibody to diphtheria toxin (DT). In contrast, only 11% of women aged 16 to over 55 years had high titres of antibody to DT. All human antisera with high anti-DT titres reacted with a synthetic peptide (SP) corresponding to the amino acids 141-157 of DT fragment A, with sera from men aged 35 to over 55 years showing the highest titres. High antibody titres to fragment A paralleled those to SP in both sexes. Titres of antibody to DT fragment B were highest in individuals with high titres to DT. In sera from both sexes immunoglobulin G1 was the predominant subclass reactive with all three antigens. However, both IgG1 and IgG4 and to a lesser extent IgG2 and IgG3 were present in immunoglobulin concentrates.     

Report of the Australian National Polio Reference Laboratory. 1 July to 31 December 1999

Since 1994, as part of the global eradication of poliomyelitis, the Australian National Polio Reference Laboratory (NPRL) at the Victorian Infectious Diseases Reference Laboratory (VIDRL) has been responsible for virological testing to confirm the absence of poliomyelitis in Australia. Samples from patients with acute flaccid paralysis are transported to VIDRL for viral culture. Polio and enteroviruses are referred for intratypic differentiation as wild or Sabin (vaccine) strains. A total of 23 faecal specimens from 17 patients were processed for enterovirus culture in the period 1 July to 31 December 1999. Since 1995, 1,078 enterovirus isolates from six states have been tested for the presence of wild poliovirus. To date, 562 strains were confirmed as Sabin vaccine-like, one non Sabin-like strain was identical with a laboratory control virus and the other strains were non-polio enteroviruses or other viruses. A World Health Organization (WHO) workshop in diagnostic polio polymerase chain reaction techniques was held at VIDRL in November 1999. The laboratory was reaccredited as a regional polio reference laboratory for the WHO Western Pacific region and a national laboratory for Australia, the Pacific Island countries and Brunei Darussalam. Planning is proceeding for the polio-free certification and containment of laboratory stocks of wild poliovirus infectious materials in Australia.     

Lyophilized combination pools of enterovirus equine antisera: preparation and test procedures for the identification of field strains of 42 enteroviruses

This paper describes the preparation of 8 dried pools (designated A to H) of sera. Each pool is composed of 10 or 11 of 42 individual enterovirus equine sera and contains 500 antibody units of each serum component per 0.1 ml. Procedures for using the antiserum pools are given, and guidance is provided for interpreting the results of serum neutralization tests in identifying field isolates.     

Influenza virus ISCOMs: antibody response in animals

A monovalent experimental ISCOM vaccine has been prepared with the envelope glycoproteins haemagglutinin and neuraminidase of the equine virus strain A/Solvalla/79 (H3N8). In vaccination trials on BALB/c mice the ISCOM vaccine induced more than ten times higher serum antibody titres measured in ELISA than a corresponding experimental micelle vaccine. Similarly, in guinea-pigs the ISCOMs induced about tenfold higher haemagglutination inhibition (HI) and neuraminidase inhibition (NI) titres than a micelle vaccine or a conventional killed influenza whole virus vaccine. Horses vaccinated with a divalent experimental ISCOM vaccine, containing the equine strains A/Prague/56 (H7N7) and A/Solvalla/79 (H3N8), responded with ELISA antibody titres against haemagglutinin which were higher and lasted considerably longer than those in horses vaccinated with conventional whole virus vaccine. ISCOMs induced complete immunoprotection in mice vaccinated with a dose of 1 microgram envelope glycoproteins of the mouse pathogenic strain A/PR/8/34 (H1N1).     

A virus-like particle based bivalent vaccine confers dual protection against enterovirus 71 and coxsackievirus A16 infections in mice

Enterovirus 71(EV71) and coxsackievirus A16 (CA16) are responsible for hand, foot and mouth disease which has been prevalent in Asia-Pacific regions, causing significant morbidity and mortality in young children. Co-circulation of and co-infection by both viruses underscores the importance and urgency of developing vaccines against both viruses simultaneously. Here we report the immunogenicity and protective efficacy of a bivalent combination vaccine comprised of EV71 and CA16 virus-like particles (VLPs). We show that monovalent EV71- or CA16-VLPs-elicited serum antibodies exhibited potent neutralization effect on the homotypic virus but little or no effect on the heterotypic one, whereas the antisera against the bivalent vaccine formulation were able to efficiently neutralize both EV71 and CA16, indicating there is no immunological interference between the two antigens with respect to their ability to induce virus-specific neutralizing antibodies. Passive immunization with monovalent VLP vaccines protected mice against a homotypic virus challenge but not heterotypic infection. Surprisingly, antibody-dependent enhancement (ADE) of disease was observed in mice passively transferred with mono-specific anti-CA16 VLP sera and subsequently challenged with EV71. In contrast, the bivalent VLP vaccine conferred full protection against lethal challenge by either EV71 or CA16, thus eliminating the potential of ADE. Taken together, our results demonstrate for the first time that the bivalent VLP approach represents a safe and efficacious vaccine strategy for both EV71 and CA16.     

The response of institutionalized Down's syndrome subjects to enterovirus infections

In a study comparing the responses of institutionalized Down's syndrome (DS) and non-Down's (ND) inmates to enterovirus infections, the frequency of wild enteric viruses and the excretion patterns of oral polio vaccine (OPV) viruses were similar in both groups. Antibody titres developed to poliovirus types 2 and 3 following vaccination were similar in DS and ND vaccinees, but the response to type I virus was significantly less in DS vaccinees. As judged by the development of poliovirus antibody in non-vaccinees, the spread of virus from OPV-immunized to unimmunized subjects in the institution was not noticeably different in DS and ND subjects. An unexpected finding was that the excretion patterns of all three serotypes of poliovirus were strikingly similar for each individual, although the patterns varied considerably from individual to individual. The similarity of excretion occurred despite wide differences within an individual in the titres of neutralizing serum antibodies to the three serotypes. It is suggested that the rate at which a given individual eliminates enteroviruses may be largely determined by factors, the activities of which are not reflected in serum antibody titres.     

The response of institutionalized Down's syndrome subjects to enterovirus infections.

In a study comparing the responses of institutionalized Down''s syndrome (DS) and non-Down''s (ND) inmates to enterovirus infections, the frequency of wild enteric viruses and the excretion patterns of oral polio vaccine (OPV) viruses were similar in both groups. Antibody titres developed to poliovirus types 2 and 3 following vaccination were similar in DS and ND vaccinees, but the response to type I virus was significantly less in DS vaccinees. As judged by the development of poliovirus antibody in non-vaccinees, the spread of virus from OPV-immunized to unimmunized subjects in the institution was not noticeably different in DS and ND subjects. An unexpected finding was that the excretion patterns of all three serotypes of poliovirus were strikingly similar for each individual, although the patterns varied considerably from individual to individual. The similarity of excretion occurred despite wide differences within an individual in the titres of neutralizing serum antibodies to the three serotypes. It is suggested that the rate at which a given individual eliminates enteroviruses may be largely determined by factors, the activities of which are not reflected in serum antibody titres.     

Molecular classification of enteroviruses not identified by neutralization tests

We isolated six viruses from patients diagnosed with aseptic meningitis or hand, foot, and mouth disease. The cytopathic effect of these viruses on cultured cells was like that of enteroviruses. However, viral neutralization tests against standard antisera were negative. Phylogenetic analysis with the complete VP4 nucleotide sequences of these 6 viruses and 29 serotypes of enteroviruses classified 3 of the viruses as serotype echovirus type 18 (EV18) and 3 as serotype human enterovirus 71 (HEV71). These results were confirmed by remicroneutralization tests with HEV-monospecific antisera or an additional phylogenetic analysis with the complete VP4 nucleotide sequences. Phylogenetic analysis with complete VP4 genes is more useful than neutralization tests with enterovirus serotype-specific antisera in identifying enterovirus serotypes.     

Evaluation by flow cytometry of antibody-dependent enhancement (ADE) of dengue infection by sera from Thai children immunized with a live-attenuated tetravalent dengue vaccine

Sera from Thai children immunized with a live-attenuated tetravalent dengue virus vaccine or from naturally infected age-matched site-control subjects were examined for immune enhancement capacity by a highly reproducible flow cytometric assay in Fc receptor-bearing K562 human cells. None of the sera under study corresponded to cases of severe dengue disease. In parallel assays employing each dengue virus serotype, we found no or only minimal antibody-dependent enhancement (ADE) when sera from vaccinated or control subjects were used at a low serum dilution [1/12] that approximated the in vivo condition. Among sera that exhibited homotypic neutralizing antibody activity against DV1-3, the level correlated with absence of ADE or infection with the respective serotype. Similarly, a broad heterotypic neutralizing antibody response that included all four serotypes was linked to complete absence of K562 cell infection. In contrast, at higher serum dilutions a correlation between breadth of antibody response and heightened immune enhancement emerged, a pattern identical to that observed among control subjects. These findings support the use of live dengue vaccines and protocols that induce broad serotype-specific neutralizing antibody responses, but they also suggest that clinically relevant immune enhancement may not be likely if this is not uniformly achieved after the first immunization.     

The use of the haemagglutination-inhibition test for detecting antibodies to type SAT 2 foot-and-mouth disease viruses in cattle sera.

Two strains of type SAT 2-foot-and-mouth disease virus which gave high titres of haemagglutinin activity reacted type-specifically in direct haemagglutination-inhibition tests with reference, bovine convalescent antisera. Comparisons of the haemagglutination-inhibition and the serum neutralization tests using cattle sera showed that both were equally specific and sensitive for detecting virus antibody.     

Detection of markers of hepatitis B infection in serum dried on to filter-paper: an application to field studies

In an attempt to find a cheap, reliable, and convenient method for the transportation and storage of serum specimens during seroepidemiological surveys, a technique in which serum was dried on to pieces of filter paper was developed and evaluated. For the evaluation, a total of 382 sera were selected from the extensive serum collection held by the WHO Collaborating Centre for Virus Reference and Research at Fairfield Hospital, Australia. These sera were dried on to pieces of filter-paper, stored at different temperatures and then tested for the presence of the various markers of infection with hepatitis B virus by solid-phase radioimmunoassay. The results were in complete agreement with those obtained on whole serum specimens. In addition, storage at 4 °C, room temperature (22 °C), or 37 °C for up to 30 days did not alter the sensitivity of the test. This technique may be useful in field surveys, not only for the detection of hepatitis B infection, but also in the study of other diseases and metabolic disorders.