H9N2 subtype influenza A viruses in poultry in pakistan are closely related to the H9N2 viruses responsible for human infection in Hong Kong.

Following the outbreak of H5N1 "bird flu" in Hong Kong in 1997, the isolation of H9N2 subtype viruses from patients in southern China and Hong Kong SAR once again raised the spectre of a possible influenza pandemic. H9N2 viruses have recently been responsible for disease in poultry in various parts of the world and preliminary studies of the H9 haemagglutinin (HA) genes of viruses isolated during 1998 and 1999 in Germany, Iran, Pakistan, and Saudi Arabia showed a close relationship to the HA genes of the viruses that infected two children in Hong Kong SAR. Analysis of the complete genome of a Pakistan isolate, A/chicken/Pakistan/2/99, showed that it is closely related in all eight genes (97-99% homology) to the human H9N2 isolates and furthermore that the six genes encoding internal components of the virus are similar to the corresponding genes of the H5N1 viruses that caused 6 (out of 18) fatal cases of human infection. Thus H9N2 viruses similar to those that caused human infections in Hong Kong are circulating more widely in other parts of the world. Whether or not these H9N2 viruses also have features that facilitate avian-to-human transmission is not known. Since avian H9N2 viruses are currently perceived to represent a significant threat to human health it is important to determine whether or not viruses of this subtype circulating in poultry in various parts of the world have the potential to infect people.

Antigenic and genetic characterization of current influenza strains.

Annually the influenza centre receives more than 1000 virus isolates from around the world to monitor the changing pattern of viruses causing influenza throughout the year. These are characterized antigenically using both polyclonal and monoclonal antibodies and selected viruses are subjected to closer scrutiny by nucleotide sequence analyses of their HA genes. This information is used in making the annual recommendation of vaccine composition. As in the last 15 years, influenza A viruses of both H3N2 and H1N1 subtypes and influenza B viruses have been isolated during the recent influenza season. Outbreaks in the northern hemisphere were largely caused by influenza B viruses which are similar to the B/Panama/45/90 reference strain. The proportion of influenza A increased later in the season and was predominantly of the H3N2 subtype, viruses similar to the recent A/Beijing/32/92 variant being most prevalent. The observed changes taking place will be discussed in the context of recent trends.

Herpesvirus saimiri has a gene specifying a homologue of the cellular membrane glycoprotein CD59.

Herpesvirus saimiri (HSV) is a T-lymphotropic tumor virus that causes fulminant lymphomas and leukemias in various New World primates other than its natural host, the squirrel monkey (Saimiri sciureus). In the course of completing the nucleotide sequence of its genome, we identified an open reading frame of 363 nucleotides, designated HVS-15, that has no detectable homology to any other viral sequences to date. HVS-15 encodes a 121-amino-acid protein which shows significant similarities to human CD59, a phosphatidyl-inositol-glycan-anchored glycoprotein involved in T-cell activation and restriction of complement-mediated lysis. The predicted HVS-15 gene product is more similar to human CD59 than to the related murine Ly-6 antigens. A nucleotide sequence identity of 64% was found between HVS-15 and the CD59 reading frame, and a 48% identity exists between the corresponding protein sequences. The comparison of the amino acid sequences revealed a number of conserved structural features such as a similar pattern of hydrophobic termini and an identical cysteine skeleton.

Primary structure of the herpesvirus saimiri genome.

This report describes the complete nucleotide sequence of the genome of herpesvirus saimiri, the prototype of gammaherpesvirus subgroup 2 (rhadinoviruses). The unique low-G + C-content DNA region has 112,930 bp with an average base composition of 34.5% G + C and is flanked by about 35 noncoding high-G + C-content DNA repeats of 1,444 bp (70.8% G + C) in tandem orientation. We identified 76 major open reading frames and a set of seven U-RNA genes for a total of 83 potential genes. The genes are closely arranged, with only a few regions of sizable noncoding sequences. For 60 of the predicted proteins, homologous sequences are found in other herpesviruses. Genes conserved between herpesvirus saimiri and Epstein-Barr virus (gammaherpesvirus subgroup 1) show that their genomes are generally collinear, although conserved gene blocks are separated by unique genes that appear to determine the particular phenotype of these viruses. Several deduced protein sequences of herpesvirus saimiri without counterparts in most of the other sequenced herpesviruses exhibited significant homology with cellular proteins of known function. These include thymidylate synthase, dihydrofolate reductase, complement control proteins, the cell surface antigen CD59, cyclins, and G protein-coupled receptors. Searching for functional protein motifs revealed that the virus may encode a cytosine-specific methylase and a tyrosine-specific protein kinase. Several herpesvirus saimiri genes are potential candidates to cooperate with the gene for saimiri transformation-associated protein of subgroup A (STP-A) in T-lymphocyte growth stimulation.

Analysis of nucleotide sequence of the rightmost 43 kbp of herpesvirus saimiri (HVS) L-DNA: general conservation of genetic organization between HVS and Epstein-Barr virus.

We present an analysis of 43,658 bp of contiguous nucleotide sequence comprising the right terminal region (conventional orientation) of the unique protein-coding component (L-DNA) of the herpesvirus saimiri (HVS) genome. Within this region lie the genes encoding the 160-kDa virion protein, which is homologous to the 140-kDa membrane antigen of Epstein-Barr virus (EBV), thymidylate synthase (TS), and the immediate-early (IE) 52-kDa protein which is homologous to the EBV BMLF1 product. The 160-kDa gene of HVS lies at the right terminus of HVS L-DNA, its homologue in EBV occurring at the left terminus of the EBV genome (conventional orientation). The TS gene of HVS occurs within a group of 5 genes that have no homologues in EBV. The translation product of one of these genes, ECRF3, shows amino acid sequence and hydrophobicity pattern similarities to the HCMV and cellular G-protein-coupled receptor family of proteins. Another, ECLF2, is homologous to the cyclin family of cellular proteins. The 5 nonconserved genes lie adjacent to the 160-kDa gene. In EBV, the region to the right of the 140-kDa gene (BNRF1) contains the latent replication origin (OriP) and the open reading frames BCRF1, BWRF1 (repeated 12 times), BYRF1, BHLF1, and BHRF1, counterparts of which are not present in this position in HVS. The subsequent 18 genes in EBV (BFLF2 to BLRF2, approximate positions 56,000-89,500) are represented in HVS, and the relative positions and orientations of these genes are directly comparable between the two viruses. There then occurs a nonhomologous gene in HVS, and genes BLLF2 to BZLF1 (positions 89,500 to 103,200) in EBV which are not present in this region of HVS, before collinearity resumes. Thus, the HVS sequence presented here shows general collinearity between conserved genes in the right terminal region of HVS and the left terminal region of EBV and reveals the presence of two sets of unique genes which occur in exactly analogous positions in HVS and EBV.

Herpesvirus saimiri encodes homologues of G protein-coupled receptors and cyclins.

Herpesvirus saimiri (HVS) is a T-lymphotropic gammaherpesvirus which establishes asymptomatic infections in its natural host the squirrel monkey (Saimiri sciureus), but which causes fatal lymphoproliferative diseases in other New World primates. Sequencing studies show HVS is closely related to the human B-lymphotropic gammaherpesvirus Epstein-Barr virus (EBV). However, despite the general colinearity between the genomes of HVS and EBV, HVS contains genes not found in EBV or in the genomes of any of the other sequenced herpesviruses. We have identified two genes, occurring in a region of divergence between HVS and EBV, that have cellular homologues. One of these, ECRF3, is homologous to the genes encoding the human cytomegalovirus (HCMV) and cellular G protein-coupled receptor family of proteins. The other HVS gene, ECLF2, is homologous to the genes encoding cellular cyclins and to our knowledge is the first reported example of a viral cyclin. The presence of G protein-coupled receptor and cyclin homologues in HVS suggests that these genes may be important in the regulation of viral and cellular processes during productive and/or latent infection of host cells, and in particular may be of relevance in the transformation and rapid proliferation of T cells during HVS infections of hosts susceptible to HVS-induced lymphoproliferative diseases.

Deviations from expected frequencies of CpG dinucleotides in herpesvirus DNAs may be diagnostic of differences in the states of their latent genomes.

The DNA sequences of genomes from G + C-rich and A + T-rich lymphotropic herpesviruses [i.e. gammaherpesviruses; Epstein-Barr virus and herpesvirus saimiri (HVS)] are deficient in CpG dinucleotides and contain an excess of TpG and CpA dinucleotides relative to frequencies predicted from their mononucleotide compositions. In contrast, for sequences from genomes of G + C-rich and A + T-rich neurotropic herpesviruses (i.e. alphaherpesviruses; herpes simplex virus and varicellazoster virus) and human cytomegalovirus (HCMV; a betaherpesvirus) the mean observed frequencies of these dinucleotides are close to those expected from their mononucleotide compositions. Comparisons between DNA sequences that encode proteins conserved in all these viruses also show that sequences of these lymphotropic viruses are CpG-deficient whereas the homologous genes from the neurotropic viruses and the HCMV are not. Analyses of local variations in dinucleotide frequencies reveal some occurrences of clustered CpG dinucleotides in generally deficient genomes (e.g. upstream of the thymidylate synthase gene of HVS) and locally CpG-deficient regions within some generally non-deficient genomes (e.g. the major immediate early genes of human, simian and murine CMVs). A relative deficiency in CpG and an excess of TpG and CpA dinucleotides is a diagnostic feature of higher eukaryotic DNA sequences that have been subjected to methylation of cytosine residues in CpG doublets with the resulting increase in mutations to give TpG (and thereby its complement, CpA). The available evidence implicates the latent genome as the site of methylation of these herpesviruses. We conclude that in the neurotropic herpesviruses the normal latent precursors to infectious progeny are not methylated whereas there is local methylation of the immediate early locus in the latent genomes of CMVs, and the latent genomes of these lymphotropic herpesviruses are extensively methylated.

The 160,000-Mr virion protein encoded at the right end of the herpesvirus saimiri genome is homologous to the 140,000-Mr membrane antigen encoded at the left end of the Epstein-Barr virus genome.

The sequence of 4.4 kilobase pairs (kbp) from the conventional right terminus of the A + T-rich light-DNA (L-DNA) sequences of the herpesvirus saimiri (HVS) genome contains a leftward-directed open reading frame (ORF) for a 1,299-residue protein. The molecular weight predicted for the protein (143,000) is in good agreement with the estimates of 150,000 to 160,000 for the major nonglycosylated polypeptide of the virion tegument (the 160K polypeptide), previously shown to be encoded by this region of the genome. The first initiation codon of the ORF is only 250 nucleotides from the junction of the L-DNA component with the G + C-rich terminal reiterations (i.e., heavy or H-DNA) of the genome. An unusually A + T-rich sequence (43 of 45 nucleotides are A or T, relative to a mean composition of 40% G + C for the ORF) occurs some 75 bp 5' to this initiation codon, and the first adenylation signal (AATAAA) on this DNA strand occurs 18 bp 3' to the termination codon. The amino acid sequence predicted for the 160K protein of HVS is homologous over most of its length to the 1,318-residue protein encoded by the leftmost major ORF of the G + C-rich genome of Epstein-Barr virus (BNRF1, the 140K nonglycosylated membrane antigen). No homology to either of these proteins is evident among the products predicted from the complete sequence of the alpha herpesvirus varicella-zoster virus. Thus gamma herpesviruses with coding sequences which differ in mean nucleotide composition by some 20% G + C have homologous proteins encoded at similar positions with respect to genome termini, with the right end of HVS being homologous to the left end of Epstein-Barr virus.

The A+T-rich genome of Herpesvirus saimiri contains a highly conserved gene for thymidylate synthase.

Herpesvirus saimiri (HVS) is the prototype member of a distinctive subset of lymphotropic herpesviruses (the gamma 2 subgroup) with A+T-rich coding sequences. In this paper, we show that cells productively infected with HVS contain high concentrations of a virus-specified thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45); we identify the active polypeptide and present the sequence of the virus gene. The predicted amino acid sequence of the 294-residue subunit of the virus enzyme is 70% homologous with the sequence of the human enzyme and about 50% homologous with prokaryotic thymidylate synthases, illustrating the remarkable structural constraints imposed by the thymidylate synthase function. However, the presence of the enzyme is not a conserved property of herpesviruses. We find no evidence for a virus-encoded thymidylate synthase activity (or a homology to a thymidylate synthase sequence) in G+C-rich representatives of alpha 1 (e.g., herpes simplex viruses, 66-68% G+C), beta (i.e., human cytomegalovirus, 58-59% G+C), and gamma 1 (i.e., Epstein-Barr virus, 60% G+C) herpesvirus subgroups. The production of excess thymidylate by a virus thymidylate synthase in cells infected with an A+T-rich herpesvirus would provide one plausible source of biased mutations by the virus-encoded replicative enzymes, which we have previously suggested as the likely general cause of differences in the mean nucleotide compositions of herpesvirus genomes.

Evidence for the persistence of paramyxoviruses in human bone marrows.

Human bone marrow cells derived from multiple sclerosis (MS) and control patients were screened for a number of virus antigens by the fluorescent antibody technique using monoclonal antibodies. The results showed that antigens of the paramyxovirus, simian virus 5, were present in about 60% of MS and 25% of control bone marrows. About 25% of the MS and 50% of control bone marrows were found to contain nucleoprotein antigen of the human parainfluenza types 1 and 3. These experiments demonstrated that paramyxoviruses can persist in human tissues possibly in a defective or repressed state.

Does simian virus 5 infect humans?

Simian virus 5 (SV5) isolates derived after co-cultivation of human bone marrow aspirates of multiple sclerosis (MS) patients were shown by immunoprecipitation, cross-neutralization and haemagglutination inhibition techniques to be similar antigenically but not identical to the prototype strain. Analyses of human sera (MS and control) showed that about 20% contained neutralizing antibodies to SV5 and immunoprecipitated the specific SV5 HN polypeptide. A competition assay using a specific SV5 monoclonal antibody confirmed that a human serum containing such neutralizing activity also blocked a specific SV5 epitope whereas another human serum with demonstrable antibodies to the related human parainfluenza virus type 2 did not block this epitope. These tests therefore suggested that SV5 can infect humans. However, there was no indication, on the basis of these tests, of any aetiological relationship of the SV5 infection to the induction of MS.

The nuclear localization of a putative neurotransmitter receptor.

A high affinity strychnine binding site has been identified within a membrane fraction prepared from partially purified rat brain nuclei. This interaction appears similar in its characteristics to that occurring in the non-nuclear membrane fraction which is thought to occur at the synaptic glycine receptor complex. Both the nuclear and non-nuclear membrane binding of tritiated strychnine is greater within the pons-medulla region than in the cerebral cortex. Nuclear membrane binding sites for dopamine, norepinephrine (beta-adrenergic), acetylcholine (muscarinic), GABA, and diazepam were not detected.

Chemotherapy of herpes simplex virus encephalitis in mice by direct intracerebral injection.

The effects of 9-beta-D-arabinofuranosyladenine-5'-phosphate (ara-A-5'-P) on herpes simplex virus encephalitis in mice were studied. This drug is a monophosphate derivative of adenine arabinoside (ara-A). When administered by the intracerebral (ic) route, ara-A-5'-P was highly effective in virus-infected mice in the 17-20 g weight range provided therapy was begun before clinical symptoms of encephalitis appeared. The most effective dose schedule was found to be three daily ic injections totaling 400 micrograms/day (20 mg/kg) beginning on the third day after injection. Such treatment resulted in a survival rate of 50%-75% among infected mice after six months. In the early stages of many experiments, 90%-100% of the controls died whereas all of the treated animals survived.

Multiple sclerosis-associated agent. Failure of MS brain and serum to depress the polymorph count in normal mice and mice inoculated with cells containing C/type particles.

The groups that originally reported and confirmed the demonstration of a multiple sclerosis associated agent (MSAA) are now, along with others, unable to reproduce this effect. In view of this confusion and the potential importance of this work for multiple sclerosis (MS) we have done a strict double-blind trial using larger groups of mice (10) and counting more cells (900) than in previous reports to offset the high variability of mouse polymorphonuclear neutrophil (PMN) counts. Sera from 5 active MS patients and 4 normal subjects were tested in mice, half of which had previously been injected with PAM line cells (containing C-type particles and subject to reduced cell yield when cultured with MS brain extract). No significant PMN depression was found in either MS or normals on any basis of comparison. However, a significant depression was seen following PAM cell injection irrespective of serum origin. Higher counting accuracy did not reduce PMN variability. A single MS brain specimen was also without effect. consequently we have been unable to confirm the existence of an MSAA as defined by PMN depression in mice.

The effects of ultraviolet and ionizing radiation on herpesviruses, SV40 and adenoviruses in relation to the small-plaque effect.

The small-plaque effect occurs with a wide range of herpesviruses following irradiation with ultraviolet light. The 37 per cent survival (D37) values, or dose required for one lethal hit (e-1), for herpes simplex, pseudorabies and pigeon herpesviruses in different cells indicate a broad spectrum of host-cell repair capacity. Other DNA-containing viruses such as SV40 and adenoviruses, which also replicate in the cell nucleus, show the small-plaque effect. Ionizing irradiation of herpes simplex virus type 1 (HSV-1) showed but little reduction in plaque-size.