Evolutionary dynamics of influenza A nucleoprotein (NP) lineages revealed by large-scale sequence analyses.

Influenza A viral nucleoprotein (NP) plays a critical role in virus replication and host adaptation, however, the underlying molecular evolutionary dynamics of NP lineages are less well-understood. In this study, large-scale analyses of 5094 NP nucleotide sequences revealed eight distinct evolutionary lineages, including three host-specific lineages (human, classical swine and equine), two cross-host lineages (Eurasian avian-like swine and swine-origin human pandemic H1N1 2009) and three geographically isolated avian lineages (Eurasian, North American and Oceanian). The average nucleotide substitution rate of the NP lineages was estimated to be 2.4 × 10(-3) substitutions per site per year, with the highest value observed in pandemic H1N1 2009 (3.4 × 10(-3)) and the lowest in equine (0.9 × 10(-3)). The estimated time of most recent common ancestor (TMRCA) for each lineage demonstrated that the earliest human lineage was derived around 1906, and the latest pandemic H1N1 2009 lineage dated back to December 17, 2008. A marked time gap was found between the times when the viruses emerged and were first sampled, suggesting the crucial role for long-term surveillance of newly emerging viruses. The selection analyses showed that human lineage had six positive selection sites, whereas pandemic H1N1 2009, classical swine, Eurasian avian and Eurasian swine had only one or two sites. Protein structure analyses revealed several positive selection sites located in epitope regions or host adaptation regions, indicating strong adaptation to host immune system pressures in influenza viruses. Along with previous studies, this study provides new insights into the evolutionary dynamics of influenza A NP lineages. Further lineage analyses of other gene segments will allow better understanding of influenza A virus evolution and assist in the improvement of global influenza surveillance.

[Expression and analysis of the nucleoprotein of paramyxovirus Tianjin strain].

Paramyxovirus Tianjin strain is a novel strain of virus causing common cotton-eared marmoset fatal infection. To investigate the relationship between the gene structure and function of nucleoprotein (NP) of Tianjin strain, NP gene of paramyxovirus Tianjin strain was cloned and three domains of NP were expressed. The homologous and phylogenetic analysis of NP sequences among the paramyxovirus Tianjin strain and eight strains of Sendai viruses from GenBank were performed. The results indicated the recombinant proteins NP1, NP2 and NP3 showed the native antigenicity to the polyclonal antiserum of paramyxovirus Tianjin strain, ranking as NP3>NP1>NP2 (precedence order). The homology of NP nucleotide and the deduced amino acid sequences between paramyxovirus Tianjin strain and Sendai virus BB1 strain were 94.5%, 96.2%, respectively, whereas the identity were 85.1% - 88.7% and 92.4% - 94.7% among Tianjin strain and the 7 strains of Sendai viruses from GenBank respectively. There were 15 unique amino acid substitutions in Tianjin strain NP protein and 11 common amino acid substitutions same with BB1 strain. This research confirmed that paramyxovirus Tianjin strain might be a new genotype of Sendai virus and can be helpful in the establishment of detection assay applying recombinant NP as antigen instead of the whole virions.

Evaluation of a nucleoprotein-based enzyme-linked immunosorbent assay for the detection of antibodies against infectious bronchitis virus.

As an immunogen of the coronavirus, the nucleoprotein (N) is a potential antigen for the serological monitoring of infectious bronchitis virus (IBV). In this report, recombinant N protein from the Beaudette strain of IBV was produced and purified from Escherichia coli as well as Sf9 (insect) cells, and used for the coating of enzyme-linked immunosorbent assay (ELISA) plates. The N protein produced in Sf9 cells was phosphorylated whereas N protein from E. coli was not. Our data indicated that N protein purified from E. coli was more sensitive to anti-IBV serum than the protein from Sf9 cells. The recombinant N protein did not react with the antisera to other avian pathogens, implying that it was specific in the recognition of IBV antibodies. In addition, the data from the detection of field samples and IBV strains indicated that using the recombinant protein as coating antigen could achieve an equivalent performance to an ELISA kit based on infected material extracts as a source of antigen(s). ELISAs based on recombinant proteins are safe (no live virus), clean (only virus antigens are present), specific (single proteins can be used) and rapid (to respond to new viral strains and strains that cannot necessarily be easily cultured).

Characterization of H5N2 influenza viruses from Italian poultry.

From October 1997 to January 1998, highly pathogenic H5N2 avian influenza viruses caused eight outbreaks of avian influenza in northern Italy. A nonpathogenic H5N9 influenza virus was also isolated during the outbreaks as a result of virological and epidemiological surveillance to control the spread of avian influenza to neighbouring regions. Antigenic analysis showed that the Italian H5N2 isolates were antigenically similar to, although distinguishable from, A/HK/156/97, a human influenza H5N1 virus isolated in Hong Kong in 1997. Phylogenetic analysis of the haemagglutinin (HA) genes showed that the highly pathogenic Italian viruses clustered with the Hong Kong strains, whereas the nonpathogenic H5N9 virus, despite its epidemiological association with the highly pathogenic Italian isolates, was most closely related to the highly pathogenic A/Turkey/England/91 (H5N1) strain. Like the HA phylogenetic tree, the nonstructural (NS) phylogenetic tree showed that the H5N2 Italian virus genes are clearly separate from those of the H5N9 strain. In contrast, results of the phylogenetic analysis of nucleoprotein (NP) genes indicated a closer genetic relationship between the two Italian virus groups, a finding suggesting a common progenitor. Comparison of the HA, NS and NP genes of the Italian H5 strains with those of the H5N1 viruses simultaneously circulating in Hong Kong revealed that the two groups of viruses do not share a recent common ancestor. No virological and serological evidence of bird-to-human transmission of the Italian H5N2 influenza viruses was found.

Antigenic characterization of the nucleocapsid protein of Newcastle disease virus by means of a new panel of monoclonal antibodies.

Nine monoclonal antibodies (MAB) against nucleocapsid protein (NP) of Newcastle disease virus (NDV) have been prepared and characterized. All the MABs were classified into three groups by means of the competitive binding assay. At least three antigenic sites were delineated on the NP. The 1st site includes two closely located epitopes; the 2nd site includes two related and two distinct epitopes; the 3rd site includes two closely related and one distinct epitopes.

Adelaide River virus nucleoprotein gene: analysis of phylogenetic relationships of ephemeroviruses and other rhabdoviruses.

The nucleotide sequence of the Adelaide River virus (ARV) genome was determined from the 3' terminus to the end of the nucleoprotein (N) gene. The 3' leader sequence comprises 50 nucleotides and shares a common terminal trinucleotide (3' UGC-), a conserved U-rich domain and a variable AU-rich domain with other animal rhabdoviruses. The N gene comprises 1355 nucleotides from the transcription start sequence (AACAGG) to the poly(A) sequence [CATG(A)7] and encodes a polypeptide of 429 amino acids. The N protein has a calculated molecular mass of 49429 Da and a pI of 5.4 and, like the bovine ephemeral fever virus (BEFV) N protein, features a highly acidic C-terminal domain. Analysis of amino acid sequence relationships between all available rhabdovirus N proteins indicated that ARV and BEFV are closely related viruses (48.3% similarity) which share higher sequence similarity to vesiculoviruses than to lyssaviruses. Phylogenetic trees based on a multiple sequence alignment of all available rhabdovirus N protein sequences demonstrated clustering of viruses according to genome organization, host range and established taxonomic relationships.

Nucleotide sequence analysis of the nucleoprotein gene of an avian and a human influenza virus strain identifies two classes of nucleoproteins.

The nucleotide sequences of RNA segment 5 of an avian influenza A virus, A/Mallard/NY/6750/78 (H2N2), and a human influenza A virus, A/Udorn/307/72 (H3N2), were determined and the deduced amino acid sequences of the nucleoprotein (NP) of these viruses were compared to two other avian and two other human influenza A NP sequences. The results indicated that there are separate classes of avian and human influenza A NP genes that can be distinguished on the basis of sites containing amino acids specific for avian and human influenza viruses and also by amino acid composition. The human influenza A virus NP genes appear to follow a linear pathway of evolution with the greatest homology (96.9%) between A/NT/60/68 (H3N2) and A/Udorn/72, isolated only 4 years apart, and the least homology (91.1%) between A/PR/8/34 (H1N1) and A/Udorn/72, isolated 38 years apart. Furthermore, 84% of the nucleotide substitutions between A/PR/8/34 and A/NT/60/68 are preserved in the NP gene of the A/Udorn/72 strain. In contrast, a distinct linear pathway is not present in the avian influenza NP genes since the homology (90.3%) between the two avian influenza viruses A/Parrot/Ulster/73 (H7N1) and A/Mallard/78 isolated only 5 years apart is not significantly greater than the homology (90.1%) between strains A/FPV/Rostock/34 and A/Mallard/78 isolated 44 years apart and only 49% of the nucleotide substitutions between A/FPV/34 and A/Parrot/73 are found in A/Mallard/78. A determination of the rate of evolution of the human influenza A virus NP genes suggested that there were a greater number of nucleotide substitutions per year during the first several years immediately following the emergence of a new subtype in 1968.

[Comparison of the 35S-methionine oligopeptide maps of influenza A virus NP proteins]. / Sravnenie 35S-metioninovykh oligopeptidnykh kart belkov NP virusov grippa A.

Tryptic mapping of radioactive methionine-labeled NP proteins of 15 species of human influenzae A viruses and 11 animal viruses was performed. On the basis of similarities and differences of peptide maps, NP proteins were divided into 4 groups designated A, B, C, and D. Group A included viruses A/WS/33 and A/PR/8/34; Group B viruses H1N1 (apart from those isolated after 1977 and WSN virus), H2N2, H3N2, and 8 species of animal influenza viruses, Group C 4 species of H1N1 viruses isolated in 1977-1979 (A/USSR/90/77, A/USSR/086/79, A/USSR/093/79, A/Brazil/79); Group D three species of animal influenza viruses (A/swine/Iowa/30, A/horse/Praha/56, A/duck/England/56).

[Oligopeptide mapping of the proteins of animal influenza virus nucleoproteins]. / Oligopeptidnoe kartirovanie belkov nukleoproteidov virusov grippa zhivotnykh.

Oligopeptide mapping showed the viruses H1N1 and H3N2 isolated from animals, unlike the majority of animal viruses with "animal" subtypes of the surface antigens, to have NP proteins typical of human H1N1 and H3N2 viruses which confirms their origin from human viruses.

[Electrophoretic and immunological characteristics of non-histone proteins of human skin tumors. I) Methods]. / Caratteristiche elettroforetiche ed immunologiche delle proteine non-istoniche dei tumori cutanei umani. - I) Metodi.

The object of the research is to classify the nuclear proteins of the cellular nucleus of some types of human skin tumours (melanotic and amelanotic melanomas, squamous epitheliomas). Nuclear proteins were extracted from the nuclei with a saline buffer. Nuclear proteins were extracted from a melanoma step by step. After prolonged centrifugation to sediment the DNA and the large ribonucleoprotein complexes, the nuclear proteins were chromatographed on columns of Biorex 70+ to remove proteins were chromatographed on columns of Biorex 70+ to remove the histones. Nuclear proteins from a squamous epithelioma were injected into a rabbit to induce specific antibodies.

[Grouping of influenza A virus NP proteins]. / Gruppirovanie belkov NP virusov grippa A.

A comparative study of influenza A virus NP proteins was carried out using peptide mapping. Thirty-five strains of all main serotypes of human and animal viruses were tested. The greatest diversity was found in NP proteins of human influenza viruses belonging to different serotypes, while within serotypes the variability is less pronounced. Four main groups of NP proteins were distinguished and designated NP0, NP1, NP2, and NP3. The NP0 group includes proteins of viruses of HON1 serotype, NP proteins of all avian viruses with the exception of A/shearwater/Australia/1/71 (Hav6Nav5), NP proteins of A/horse/Prague/56 (Heq1Neq1), A/swine/Iowa/1/30 (Hsw1N1), and A/whale/Pacific/76 (HONav2). The NP1 group comprises NP proteins of viruses of HIN1 serotype (with the exception of A/California/78), A/Singapore/57 (H2N2) and NP protein of A/New Jersey/76 (Hsw1N2) virus. The NP2 group includes NP proteins of H3N2 virus serotype and NP protein of A/California/78 (H1N1) virus. NP proteins of A/horse/Miami/63 (Heq2Neq2) and A/shearwater/Australia/1/71 viruses comprise NP3 group.

[Heterogenicity of influenza A virus NP protein]. / Geterogennost' NP-belka virusov grippa A.

Three classes of NP-proteins were detected by 7% polyacrylamide gel electrophoresis of virion proteins of influenza A various strains. Peptide maps of these proteins vary. The pattern of the discrepancies indicates the presence of two types of influenza A virus NP-protein. Incubation during 30 h at 37 degrees C of the preparations of intact viral particles and particles destroyed by nonion detergent NP-40 does not result in changes of the NP, NP2 and NP3 proteins ratio, that is an evidence of their intracellular origin.