Alan J. Hay

Antigenic and genetic diversity among swine influenza A H1N1 and H1N2 viruses in Europe.

Three subtypes of influenza A viruses, H1N1, H1N2 and H3N2, co-evolve in pigs in Europe. H1N2 viruses isolated from pigs in France and Italy since 1997 were closely related to the H1N2 viruses which emerged in the UK in 1994. In particular, the close relationship of the neuraminidases (NAs) of these viruses to the NA of a previous UK H3N2 swine virus indicated that they had not acquired the NA from H3N2 swine viruses circulating in continental Europe. Moreover, antigenic and genetic heterogeneity among the H1N2 viruses appeared to be due in part to multiple introductions of viruses from the UK. On the other hand, comparisons of internal gene sequences indicated genetic exchange between the H1N2 viruses and co-circulating H1N1 and/or H3N2 subtypes. Most genes of the earlier (1997-1998) H1N2 isolates were more closely related to those of a contemporary French H1N1 isolate, whereas the genes of later (1999-2000) isolates, including the HAs of some H1N2 viruses, were closely related to those of a distinct H1N1 antigenic variant which emerged in France in 1999. In contrast, an H3N2 virus isolated in France in 1999 was closely related antigenically and genetically to contemporary human A/Sydney/5/97-like viruses. These studies reveal interesting parallels between genetic and antigenic drift of H1N1 viruses in pig and human populations, and provide further examples of the contribution of genetic reassortment to the antigenic and genetic diversity of swine influenza viruses and the importance of the complement of internal genes in the evolution of epizootic strains.

The NB protein is an integral component of the membrane of influenza B virus

The results of biochemical and immunoelectron microscopic studies provide evidence that the NB protein is an integral component of the influenza B virion. Its glycosylation and orientation in the membrane were shown to be equivalent to that of NB in the plasma membrane of virus-infected cells. Sensitivity to proteinase K showed that the N terminus is exterior to the virion and gold immunolabelling of freeze-fractured replicas showed that the C terminus is located in the interior of the virion. The similarities between NB of influenza B and M2 of influenza A viruses in structural features, their presence in the virion and possession of an ion channel activity suggest that, by analogy with the M2 protein, NB may also have a role in virus entry.

Infection of a child in Hong Kong by an influenza A H3N2 virus closely related to viruses circulating in European pigs

Influenza virus A/Hong Kong/1774/99, isolated from a young child with mild influenza, was shown to be similar in its antigenic and genetic characteristics to H3N2 viruses circulating in pigs in Europe during the 1990s and in particular to be closely related to viruses isolated from two children in the Netherlands in 1993. Similar viruses had previously not been identified outside Europe. Although there is little evidence as to how the child contracted the infection, it appears likely that pigs in southern China were the source of infection. Characteristics shared with the European swine viruses include resistance to the anti-influenza drugs amantadine and rimantadine. Thus not only does this incident once again highlight the potential of pigs as a source of novel human influenza viruses, but also indicates the potential for emergence of amantadine-resistant human viruses.

Human infection by a swine influenza A (H1N1) virus in Switzerland

Summary. The isolation of A/Switzerland/8808/2002 provides further evidence of sporadic human infection by contemporary swine influenza A H1N1 viruses, antigenically and genetically distinct from H1N1 viruses circulating in the human population. Together with the recent emergence of human-swine-avian reassortant viruses in pig populations in Europe and North America, frequent transmission between swine and human populations emphasises the potential for the emergence in pigs of novel subtypes with the capacity to cause major human epidemics.

The specific inhibition of influenza A virus maturation by amantadine: an electron microscopic examination

Amantadine specifically inhibits the release of virus particles from cells infected with the Rostock (H7N1) strain of influenza A virus, apparently as a consequence of a membrane protein M2-mediated conversion of haemagglutinin (HA) to its low pH conformation. Electron microscopic observations, together with immunogold labelling, showed that amantadine action does not alter the distribution of HA on the cell surface nor does it prevent the formation of budding virus particles. It was not possible, however, to discern whether low pH HA inhibited the final stage in virus maturation, i.e. pinching off, or simply prevented release of fully formed particles.

Rapid molecular analysis of the haemagglutinin gene of human influenza A H3N2 viruses isolated in Spain from 1996 to 2000

Summary. A simple molecular technique was used for the rapid preliminary genetic characterization of human influenza A H3N2 viruses isolated in Spain from 1996 to 2000. Subtyping, based on RT-PCR, was followed by subtype-specific restriction enzyme fragment length polymorphism (RFLP) analyses of an amplified region of the HA1 domain of the H3 haemagglutinin (HA) gene to distinguish variants differentiated by common amino acid substitutions in HA1. The approach was tested using 135 Spanish H3N2 isolates and included nucleotide sequencing and phylogenetic analyses of a region of the HA1 domain of 41 representative isolates. The viruses were distinguished by haemagglutination inhibition (HI) assays into two antigenically discernible groups, the A/Wuhan/359/95-like and A/Sydney/5/97-like viruses. The results of PCR-RFLP analysis allowed a finer classification into five genetic variant subgroups, corresponding to those distinguished by phylogenetic analyses. This rapid, simple and variant-specific procedure could, therefore, be used to rapidly screen clinical specimens prior to more detailed antigenic and genetic analyses.

Functional defects of fowl plague virus temperature-sensitive mutant having mutation in the neuraminidase.

SummaryA fowl plague virus (FPV) temperature-sensitive mutantts 5 having mutation lesions in the gene coding for the neuraminidase has been obtained. The mutant induced synthesis of cRNA, vRNA and proteins in cells under non-permissive conditions, but formation of virions including non-infectious ones was defective. The neuraminidase and haemagglutinin synthesized under non-permissive conditions possessed functional activity and could migrate from the rough endoplasmic reticulum into plasma membranes; however, cleavage of the haem-agglutinin was reduced. Ints 5-infected cells under non-permissive conditions the synthesis of segments 5 and 8 of cRNA and vRNA was predominant both early and late in the reproduction cycle, and the synthesis of P1, P2, P3, HA and M proteins was reduced after approximately 3 hours.The data obtained suggest that involvement of the neuraminidase in the formation of infectious virions may have no direct association with the enzymatic activity of this protein, and that the mutation in the neuraminidase may affect regulation of replication and transcription processes.

Stability and function of the influenza A virus M2 ion channel protein is determined by both extracellular and cytoplasmic domains

A series of M2/NB chimeras were used to investigate the ion channel activity of the IAV M2 protein. Replacing the M2 cytoplasmic domain with the equivalent NB domain (AAB chimera) did not influence ion channel activity, while replacement of N-terminal domains (BAA and BAB chimeras) resulted in loss of activity. Extension of the M2 protein N-terminal domain resulted in full restoration of ion channel activity in BAA chimeras but only partial restoration in BAB. While not directly involved in ion channel activity, the N- and C-terminals of M2 are important for stabilization of the transmembrane domain structure.

Antigenic and genetic diversity among swine influenza viruses in Europe

Abstract H3N2 and “avian-like” H1N1 subtypes have circulated in European pigs since the mid- to late 1970s. Following reassortment to acquire six internal genes of the H1N1 viruses in the early 1980s, the H3N2 viruses have evolved to produce antigenically distinguishable variants. Sw/Finistere/127/99, isolated from a pig in January 1999, was shown to be closely related in antigenic and genetic characteristics to contemporary human A/Sydney/5/97-like viruses, and may represent the emergence of another “human-like” virus in European pigs. The H1N1 viruses circulating in pigs in France have evolved gradually over the past 20 years, but are in general closely related to early isolates such as Sw/Finistere/2899/82. An antigenically distinct variant, represented by Sw/Ille et Vilaine/1455/99, represents a significant drift from other recent French H1N1 viruses. Most of the H1N2 reassortant viruses isolated in France and Italy since 1997 were shown to be genetically similar to the H1N2 viruses which have become prevalent in the U.K., but to exhibit significant variation in antigenicity. Genetic reassortment between H1N2 and H1N1 viruses has also contributed to the recent increase in diversity of viruses circulating in pigs in Europe, and emphasizes the potential difficulties in controlling influenza by vaccination. Since most of these swine viruses are resistant to amantadine, they provide an increasing pool of amantadine-resistant M genes for potential incorporation into future human viruses.

Comparison of the activities of BM2 protein and its H19 and W23 mutants of influenza B virus with activities of M2 protein and its H37 and W41 mutants of influenza A virus

Co-expression of the BM2 protein with pH-sensitive HA reduces the conversion of HA to its low-pH conformation during transport to the cell surface in the same way as human M2 proteins. BM2 protein is capable of increasing vesicular pH by as much as 0.4 pH units. Mutation analysis showed that replacement of H19 in BM2 protein by A and L resulted in loss of activity, while M2, with the mutation H37A, remained active, but its severe toxicity was intolerable for cells. Whereas substitution of L or A for W23 abolished detectable activity of the BM2 channel, substitution of L for W41 in the M2 protein resulted in a functional ion channel but with reduced activity. W41 was not essential for functional activity of the M2 protein. Our results show some differences in the nature of the interaction of the histidine and tryptophan in the transmembrane domains of BM2 and M2 ion channels.