A tiny viral time capsule, stored in a laboratory freezer for decades, was about to challenge what scientists knew about a dangerous bird pathogen.
By Enikő Fehér, Ádám Bálint, Szilvia Marton, Krisztina Bali, Sándor Belák, Krisztián Bányai
In 1933, a mysterious illness swept through poultry farms in Hertfordshire, England. The culprit, identified as Newcastle disease virus (NDV), was isolated and preserved—becoming the Herts'33 strain, a critical reference for research and vaccine testing for decades 1 .
For nearly a century, descendants of this original isolate have been used worldwide as challenge strains in vaccine efficacy trials. Scientists assumed they knew this virus well. That is, until recently, when a team of researchers decided to look closer—and discovered that the strain in their possession was unlike any NDV ever documented 1 .
Key Insight: The Herts'33 challenge strain used for decades in vaccine testing represents a potentially extinct genotype that differs significantly from all known NDV strains.
To appreciate this discovery, one must first understand the adversary. Newcastle disease virus is a significant avian pathogen that can cause devastating losses in domestic and wild bird populations 1 .
NDV strains are categorized into three main pathotypes based on their severity:
Low virulence, often used in live vaccines
Moderate virulence
The virus possesses a single-stranded RNA genome approximately 15,200 nucleotides long, arranged in six genes in the order 3'-NP-P-M-F-HN-L-5' 1 4 . These genes code for proteins that enable the virus to invade, replicate, and spread.
As more NDV strains were discovered worldwide, scientists developed a classification system to track their relationships. NDV strains are now grouped into two classes:
Generally avirulent strains found mainly in wild birds
This genetic diversity isn't just academic—it has real-world implications for disease control.
The pivotal experiment began when researchers obtained an aliquot of the Herts'33 challenge strain from the Veterinary Laboratories Agency in Weybridge 1 . This particular lineage, known as Herts'33(IVMP), had been maintained in eggs and used as a challenge strain at the Institute for Veterinary Medicinal Products in Budapest, Hungary 1 .
The original viral seed was passaged once in embryonated chicken eggs to obtain sufficient material for analysis 1 .
Viral RNA was carefully extracted using a specialized commercial kit 1 .
The RNA was converted to complementary DNA (cDNA) using reverse transcription 1 .
Researchers used random RT-PCR to amplify the viral cDNA, creating multiple copies for sequencing 1 .
The amplified cDNA was subjected to next-generation sequencing using an Ion Torrent PGM system 1 .
Sequence reads were assembled into a complete genome using bioinformatics software, producing a consensus sequence 1 .
| Research Tool | Specific Product/Technique | Function in the Experiment |
|---|---|---|
| RNA Extraction Kit | QIAamp Viral RNA Mini Kit | Isolate pure viral RNA from the sample |
| Amplification Method | Random RT-PCR | Create multiple copies of viral genetic material |
| Sequencing Platform | Ion Torrent PGM System | Determine the order of nucleotides in the genome |
| Assembly Software | CLC Genomic Workbench | Piece together sequence reads into a complete genome |
| Reference Sequences | NDV genomes from GenBank | Compare the new sequence to known viruses |
When the sequencing data emerged, the results were striking. The Herts'33(IVMP) genome consisted of 15,166 nucleotides with typical NDV organization 1 . However, comparative analysis revealed surprising truths.
BLAST searches and phylogenetic analysis showed that Herts'33(IVMP) shared ≤90% nucleotide sequence identity with all other NDV sequences in GenBank 1 . Its closest relative was the genotype I strain Ulster/67, with only 90.2% genome-wide identity 1 .
When focusing on the complete F gene sequence, the identity fell to ≤88.3%—well below the approximately 10% evolutionary distance cutoff established for genotype discrimination in NDV 1 .
| Comparison Strain | Genome-wide Nucleotide Identity | F-gene Nucleotide Identity | Genotype Relationship |
|---|---|---|---|
| Ulster/67 | 90.2% | Not specified | Closest known relative (Genotype I) |
| Herts/33 (AY741404) | 88.1% | 85.7% | Distinct lineage (Genotype IV) |
| Typical same-genotype strains | >90% | >90% | Would cluster together phylogenetically |
The distinctions weren't limited to nucleotide sequences. The researchers found notable variations in the viral proteins as well:
The M protein of Herts'33(IVMP) was 364 amino acids long—identical to most NDV isolates but shorter than the 380-amino-acid M protein of the Herts/33 genotype IV strain 1 .
The HN protein of Herts'33(IVMP) was 574 amino acids—three residues longer than the HN protein of other NDV strains, including the genotype IV Herts/33 1 .
Discovery: These findings at the protein level provided additional evidence that Herts'33(IVMP) and the previously known Herts/33 genotype IV strain have distinct evolutionary origins.
Why does this genetic detective work matter beyond academic curiosity? The answer lies in the practical application of this knowledge in protecting global poultry flocks.
Newcastle disease is primarily controlled through preventive vaccination of poultry 2 . However, a significant challenge arises from the limited compatibility between commercial vaccines and emerging novel NDV strains 7 .
When circulating field strains are genetically distant from vaccine strains, vaccine-induced protection may be incomplete, leading to outbreaks even in vaccinated flocks 7 . The discovery that a commonly used challenge strain like Herts'33(IVMP) represents a potentially extinct genotype highlights the gaps in our understanding of NDV diversity.
This research also underscores the value of historical virus isolates. By sequencing "old" NDV strains from early outbreaks, scientists can refine phylogenetic grouping schemes and better understand the evolution of viral genomes over time 1 .
| Characteristic | Herts'33(IVMP) | Contemporary Genotype VII Strains |
|---|---|---|
| Genotype | Potentially extinct unique genotype | Currently dominant worldwide |
| Pathology in Spleen | Induces atrophy and mild lymphoid depletion | Causes extensive necrosis and severe damage |
| Genetic Features | Distinct M and HN protein lengths | Characteristic genotype-specific signatures |
| Vaccine Challenge | Historically used for vaccine testing | Circulating strains that may evade vaccine protection |
Recent studies have shown that the M, F, and HN genes collectively influence the severity of pathological changes in infected birds 3 . Genotype VII viruses, currently circulating worldwide, induce more severe tissue damage in lymphoid organs compared to older strains like Herts'33 3 . Understanding these genetic determinants of virulence is crucial for developing more effective vaccines.
The sequencing of Herts'33(IVMP) represents more than just the characterization of a single viral strain—it illustrates how advanced genomic technologies are reshaping our understanding of pathogens. What was once considered a known quantity turned out to be a genetically distinct entity, potentially representing an extinct genotype that no longer circulates in nature 1 .
This discovery emphasizes the importance of continuous molecular characterization of viral strains, including those maintained in laboratory collections 1 . As one recent review noted, "Monitoring circulating NDV genotypes is crucial to reducing the impact of ND" 6 .
Implication: "The high genetic variability of NDV" means "virulent strains that may evade vaccine protection are more likely to emerge" 6 . The development of a new generation of vaccines using advanced technologies may substantially improve the efficacy of Newcastle disease prevention and control 7 .
The story of Herts'33(IVMP) reminds us that even well-studied pathogens can hold surprises—and that scientific curiosity, coupled with modern genomic tools, continues to reveal crucial insights for protecting animal health and global food security.