Hemagglutinin-neuraminidase Protein Genes Research Articles

Reverse transcription PCR-based detection of matrix and hemagglutinin-neuraminidase genes among Avian orthoavulavirus 1 clinical isolates in the Philippines, 1991-2017

Newcastle disease (ND) is a highly infectious disease that affects devastatingly the avian population worldwide. It is caused by Avian orthoavulavirus 1 (AOAV-1), or better known as Newcastle disease virus belonging to phylum Negarnaviricota, class Monjiviricetes, order Mononegavirales and family Paramyxoviridae. This virus consists of six principal structural proteins namely: the nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase protein (HN) and the large protein RNA dependent RNA polymerase (L).. The present study aimed to molecularly detect the M and HN gene segments of the AOAV-1 field isolates from clinical cases in the Philippines from 1991 through 2017. RT-PCR amplification and sequence analyses using primers NDV-For4359 and NDV-Rev4788 which anneal to the matrix gene and primers NDV-For6369 and NDV-Rev6598 targeting the HN genes, identified all isolates to be AOAV-1. Determining the different genes of the virus would greatly help scientists and researchers to accurately identify the viral isolates in order to improve epidemiological studies and surveillance of the disease in the country.

Genomic sequences of low-virulence avian paramyxovirus-1 (Newcastle disease virus) isolates obtained from live-bird markets in North America not related to commonly utilized commercial vaccine strains

Avian paramyxovirus 1 (APMV-1), also referred to as Newcastle disease virus (NDV), variants of low virulence were isolated from chickens, ducks and other unidentified species found in live-bird markets of the northeastern United States. These isolates were characterized as APMV-1 by the hemagglutination-inhibition (HI) assay utilizing NDV-specific polyclonal antisera. However, the isolates failed to react with a monoclonal antibody that has specificity for a wide variety of APMV-1 isolates. Although only highly virulent isolates require reporting to international regulatory agencies, the ability to correctly identify APMV-1 types is important for control and regulatory purposes. Protein gel patterns of the purified isolates resembled previously reported APMV-1 and anti-NDV polyclonal sera recognized the viral proteins. For three isolates oligonucleotide primers specific for the nucleoprotein, fusion protein and polymerase genes of NDV were utilized to synthesize cDNA using viral RNA as a template. Approximately 12 kb of the genome was subsequently sequenced for the three isolates that included the nucleoprotein, phosphoprotein, matrix protein, fusion (F) protein, hemagglutinin-neuraminidase protein genes and a 5′ portion of the polymerase gene. The isolates had an F protein cleavage site sequence of ERQER/LVG indicating low-virulence viruses that phylogenetically separated with other unique NDV isolates designated as a lineage 6 genotype. Additionally, a four amino acid insert was detected in the predicted phosphoprotein which complies with the “rule of six” among paramyxoviruses. These APMV-1 genotypes have not been previously reported in North America and further substantiate the heterogeneous genetic nature of these commercially important pathogens found worldwide.

Nucleotide and predicted amino acid sequence analysis of the fusion protein and hemagglutinin-neuraminidase protein genes among Newcastle disease virus isolates. Phylogenetic relationships among the Paramyxovirinae based on attachment glycoprotein sequences.

Highly virulent Newcastle disease virus (NDV) isolates are List A pathogens for commercial poultry, and reports of their isolation among member nations must be made to the Office of International Epizootes (OIE). The virus is classified as a member of the order Mononegavirales in the family Paramyxoviridae of the subfamily Paramyxovirinae. Two interactive surface glycoproteins, the fusion (F) and hemagglutinin-neuraminidase (HN) proteins, play essential roles in NDV attachment and fusion of cells during infection. Antibodies to the F or HN proteins are capable of virus neutralization; however, no full-length sequences are available for these genes from recently obtained virulent isolates. Therefore, nucleotide and predicted amino acid sequences of the F and HN protein genes from 16 NDV isolates representing highly virulent viruses from worldwide sources were obtained for comparison to older virulent isolates and vaccine strains. The F protein amino acid sequence was relatively conserved among isolates maintaining potential glycosylation sites and C residues for disulfide bonds. A dibasic amino acid motif was present at the cleavage site among more virulent isolates, while the low virulence viruses did not have this sequence. However, a Eurasian collared dove virus had a K114Q substitution at the F cleavage site unique among NDV isolates. The HN protein among NDV isolates maintained predicted catalytic and active site residues necessary for neuraminidase activity and hemagglutination. Length of the HN for the Eurasian collared dove isolate and a previously reported heat resistant virulent isolate were longer relative to other more recent virulent isolates. Phylogenetically NDV isolates separated into four groups with more recent virulent isolates forming a diverse branch, while all the avian paramyxoviruses formed their own clade distinct from other members of the Paramyxoviridae.

Interacting domains of the HN and F proteins of newcastle disease virus.

The activation of most paramyxovirus fusion proteins (F proteins) requires not only cleavage of F(0) to F(1) and F(2) but also coexpression of the homologous attachment protein, hemagglutinin-neuraminidase (HN) or hemagglutinin (H). The type specificity requirement for HN or H protein coexpression strongly suggests that an interaction between HN and F proteins is required for fusion, and studies of chimeric HN proteins have implicated the membrane-proximal ectodomain in this interaction. Using biotin-labeled peptides with sequences of the Newcastle disease virus (NDV) F protein heptad repeat 2 (HR2) domain, we detected a specific interaction with amino acids 124 to 152 from the NDV HN protein. Biotin-labeled HR2 peptides bound to glutathione S-transferase (GST) fusion proteins containing these HN protein sequences but not to GST or to GST containing HN protein sequences corresponding to amino acids 49 to 118. To verify the functional significance of the interaction, two point mutations in the HN protein gene, I133L and L140A, were made individually by site-specific mutagenesis to produce two mutant proteins. These mutations inhibited the fusion promotion activities of the proteins without significantly affecting their surface expression, attachment activities, or neuraminidase activities. Furthermore, these changes in the sequence of amino acids 124 to 152 in the GST-HN fusion protein that bound HR2 peptides affected the binding of the peptides. These results are consistent with the hypothesis that HN protein binds to the F protein HR2 domain, an interaction important for the fusion promotion activity of the HN protein.

Two distinct human parainfluenza virus type 1 genotypes detected during the 1991 Milwaukee epidemic.

The extent of genetic and antigenic variation found in a population of human parainfluenza virus type 1 (HPIV-1) during a single local epidemic was investigated. Fifteen HPIV-1 strains isolated from children in 1991 were analyzed. Nucleotide sequence variation in the hemagglutinin-neuraminidase protein (HN) gene demonstrated two distinct genotypes (genotypes C and D). Unique patterns were identified involving 62 nucleotide and 10 amino acid positions. These patterns represented 40% of all mutations within the HN gene. The remaining mutations were randomly distributed, and 74% involved only one (55%) or two isolates. Genotypes were statistically different from each other at both the nucleotide (P = 0.001) and amino acid (P = 0.001) levels and demonstrated unique potential N-linked glycosylation patterns. Thirty-eight monoclonal antibodies (MAbs) made to four different viral proteins (22 HN, 2 fusion [F], 1 phosphoprotein, and 13 nucleoprotein) (originating from two different genotypes [genotypes A and D]) were compared for their ability to bind to the clinical isolates in enzyme-linked immunosorbent assays (ELISAs) and hemagglutinin-inhibition (HI) assays. Twenty-one MAbs bound well to all clinical isolates in ELISAs and HI assays. The remaining 17 MAbs showed variation in all four structural proteins. Three HN MAbs demonstrated genotype C- and D-specific antigenic and neutralization differences. Evolutionary analysis using parsimony methods confirmed the differences between the two genotypes. No differences in either clinical presentation or disease severity between the two genotypes were found. Geographically localized HPIV-1 epidemics can be caused by at least two distinct genotypes with minor but specific antigenic changes. The clinical and immunologic roles of HPIV-1 genotypes have not been determined.

Sequence determination of the mumps virus HN gene

The hemagglutinin-neuraminidase protein (HN) of mumps virus was purified by immunoaffinity chromatography and fragmented by the combined action of CNBr and trypsin. The resulting peptides were separated by HPLC and sequenced by automated Edman degradation. Using this HN-specific amino acid sequence data, a degenerated oligo-nucleotide was produced and subsequently used to screen a mumps virus cDNA library to isolate HN-specific clones. The complete nucleotide sequence of the HN gene was determined. The monocistronic HN mRNA is approximately 1900 nucleotides long and encodes a single open reading frame of 582 amino acids. The HN protein has a unique hydrophobic stretch of 19 amino acids at its N-terminus that apparently anchors the protein in the viral envelope. A comparison of the mumps virus HN protein sequence with the sequences of the other known paramyxovirus HNs indicates that mumps virus is most closely related to SV-5, followed in decreasing order by NDV, parainfluenza virus 3, and Sendai virus.

Thermostabilities of virion activities of Newcastle disease virus: evidence that the temperature-sensitive mutants in complementation groups B, BC, and C have altered HN proteins.

Four virion activities of Newcastle disease virus (hemagglutinating, neuraminidase, hemolytic, and infectious activities) were examined before and after heat stress in low-salt buffer and physiological salt buffer (phosphate-buffered saline). The hemagglutinating and neuraminidase activities of the Australia-Victoria wild-type (AV-WT) strain were thermostable at both salt concentrations tested, whereas the thermostabilities of the hemolytic and infectious activities were salt dependent (thermostable in phosphate-buffered saline but not in low-salt buffer). Virions of RNA(+) temperature-sensitive (ts) mutants of AV-WT were tested for the stabilities of the four activities. Some mutants in groups B, BC, and C were as stable as AV-WT in all functions, but others were much less stable in all functions. The unstable mutants in groups B, BC, and C affirmed the assignment of the ts lesions of these mutants to the hemagglutinin/neuraminidase (HN) protein gene because HN function(s) are required for all four activities. The instability of these ts mutants was not related to their decreased virion HN protein content and was not due to physical loss of the HN protein from the virions. Three of four ts(+) plaque-forming revertants of the least stable mutant, BC2, coreverted for stability, confirming that the unstable phenotype is indeed the result of the mutation responsible for the ts phenotype. Group D mutants were approximately as stable as AV-WT in hemagglutinating, neuraminidase, and hemolytic activities; this is consistent with this group representing a lesion in a gene other than the HN protein gene. However, the infectivities of two of the three group D mutants were less stable than the infectivity of AV-WT in low-salt buffer.