Highly divergent virulent isolates of Newcastle disease virus from the Dominican Republic are members of a new genotype that may have evolved unnoticed for over 2 decades.

A Newcastle disease virus (NDV) outbreak in chickens was reported in the Dominican Republic in 2008. The complete genome of this isolate, chicken/DominicanRepublic(JuanLopez)/499-31/2008 (NDV-DR499-31/08), and the fusion proteins of three other related viruses from the Dominican Republic and Mexico were sequenced and phylogenetically analyzed. Genetically, these four isolates were highly distinct from all other currently known isolates of NDV, and together, they fulfill the newly established criteria for inclusion as a novel genotype of NDV (genotype XVI). The lack of any reported isolation of viruses related to this group since 1986 suggests that virulent viruses of this genotype may have evolved unnoticed for 22 years. The NDV-DR499-31/08 isolate had an intracerebral pathogenicity index (ICPI) score of 1.88, and sequencing of the fusion cleavage site identified multiple basic amino acids and a phenylalanine at position 117, indicating this isolate to be virulent. These results were further confirmed by a clinicopathological assessment in vivo. In 4-week-old chickens, NDV-DR499-31/08 behaved as a velogenic viscerotropic strain with systemic virus distribution and severe necrohemorrhagic lesions targeting mainly the intestine and the lymphoid organs. The clear phylogenetic relationship between the 2008, 1986, and 1947 ancestral viruses suggests that virulent NDV strains may have evolved in unknown reservoirs in the Caribbean and surrounding regions and underlines the importance of continued and improved epidemiological surveillance strategies to detect NDV in wild-bird species and commercial poultry.

Isolation of a virulent Newcastle disease virus from confiscated LaSota vaccine.

Vials of Newcastle disease vaccine labeled as LaSota were confiscated by the Arizona Division of Customs and Border Protection officials. Two different avian type 1 paramyxoviruses were isolated from all three vials of vaccine submitted to the National Veterinary Services Laboratories. The LaSota strain of avian paramyxovirus type 1 virus was isolated from all three vials and analyzed by nucleotide sequence analysis. A virulent Newcastle disease virus was also present in all three vials, but in low concentration. The virulence of the Newcastle disease virus was characterized by the intracerebral chicken pathogenicity index chicken inoculation assay but could not be determined by nucleotide sequence analysis from the virus isolated from embryonating chicken eggs. The intracerebral chicken pathogenicity index value for the isolated Newcastle disease virus was 1.55. Strains of Newcastle disease virus with intracerebral pathogenicity indexes significantly above 1.0 have been found to selectively kill many types of cancer cells while not affecting normal nonneoplastic cells and are considered to be a viable option for cancer treatment in humans by alternative medical researchers; however, the treatment is not approved for use in the United States by the Food and Drug Administration. Customs and Border Protection officials have been notified of an increased risk of Newcastle disease virus entering the United States for use as a nonapproved cancer treatment. Illegal importation of Newcastle disease vaccine for vaccination of backyard poultry is also a threat. This case report emphasizes the importance of conducting chicken inoculation for complete virus pathotyping and demonstrates the need for stringent security procedures at U.S. borders to detect known livestock pathogens that may be smuggled in for use in animal agriculture and reasons unrelated to animal agriculture.

An rRT-PCR assay to detect the matrix gene of a broad range of avian paramyxovirus serotype-1 strains.

The current U.S. Department of Agriculture (USDA)-validated real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay designed to detect the matrix gene of avian paramyxovirus serotype-1 (APMV-1) is the primary screening assay used in the United States. It has previously been shown to be unable to consistently detect all members of class I APMV-1. Diagnostic testing relies on rRT-PCR to quickly detect APMV-1 in wild birds, backyard flocks, live bird markets, commercial poultry, and for export testing. Limitations of the current USDA assay have raised concerns about the potential for some strains of APMV-1 to remain undetected by the primary screening assay. Mismatches in the probe were shown to cause a loss in template binding efficiency, resulting in lack of detection by the assay. Here, we describe the development and analytical validation of a new rRT-PCR assay designed to target a highly conserved region of the matrix gene across a wide range of APMV-1 strains. Limit of detection testing revealed a 3 log10 decrease in sensitivity for one low-virulence strain when compared to the USDA validated assay. Conversely, the assay showed increased sensitivity for a class I isolate and two virulent strains of APMV-1 that were not detected by the USDA-validated assay. The new assay also demonstrated a high degree of specificity by the lack of detection of 43 non-APMV-1 viruses.

Avian paramyxovirus serotype-1: a review of disease distribution, clinical symptoms, and laboratory diagnostics.

Avian paramyxovirus serotype-1 (APMV-1) is capable of infecting a wide range of avian species leading to a broad range of clinical symptoms. Ease of transmission has allowed the virus to spread worldwide with varying degrees of virulence depending on the virus strain and host species. Classification systems have been designed to group isolates based on their genetic composition. The genetic composition of the fusion gene cleavage site plays an important role in virulence. Presence of multiple basic amino acids at the cleavage site allows enzymatic cleavage of the fusion protein enabling virulent viruses to spread systemically. Diagnostic tests, including virus isolation, real-time reverse-transcription PCR, and sequencing, are used to characterize the virus and identify virulent strains. Genetic diversity within APMV-1 demonstrates the need for continual monitoring for changes that may arise requiring modifications to the molecular assays to maintain their usefulness for diagnostic testing.