Leonardo Susta

Newcastle disease a review of field recognition and current methods of laboratory detection

Newcastle disease (ND) remains a constant threat to the poultry industry and is a limiting disease for poultry producers worldwide. The variety of clinical presentations and the emergence and spread of new genetic variants make recognition and diagnosis challenging. The current review details the pertinent features of the clinicopathologic disease in the main susceptible species, including chicken, turkey, duck, goose, pigeon, and other birds such as cormorants, psittacines, and canaries. Furthermore, the available and emerging laboratory diagnostic methodologies for the detection and typing of the virus are reviewed, including traditional techniques such as virus isolation and immunohistochemistry as well as rapid procedures based on molecular tools, such as real-time polymerase chain reaction, gene sequencing, and microarrays. The relevant genetic variability of ND viruses probably represents the major limitation in the validation and application of the current, advanced diagnostic molecular techniques. This underscores the importance of a multidisciplinary and comprehensive diagnostic approach, which should include, next to the new generation assays of the genomic era, the more traditional techniques such as histopathology, immunohistochemistry, and virus isolation.

Role of Poultry in the Spread of Novel H7N9 Influenza Virus in China

ABSTRACT The recent outbreak of H7N9 influenza in China has resulted in many human cases with a high fatality rate. Poultry are the likely source of infection for humans on the basis of sequence analysis and virus isolations from live bird markets, but it is not clear which species of birds are most likely to be infected and shedding levels of virus sufficient to infect humans. Intranasal inoculation of chickens, Japanese quail, pigeons, Pekin ducks, Mallard ducks, Muscovy ducks, and Embden geese with 106 50% egg infective doses of the A/Anhui/1/2013 virus resulted in infection but no clinical disease signs. Virus shedding was much higher and prolonged in quail and chickens than in the other species. Quail effectively transmitted the virus to direct contacts, but pigeons and Pekin ducks did not. In all species, virus was detected at much higher titers from oropharyngeal swabs than cloacal swabs. The hemagglutinin gene from samples collected from selected experimentally infected birds was sequenced, and three amino acid differences were commonly observed when the sequence was compared to the sequence of A/Anhui/1/2013: N123D, N149D, and L217Q. Leucine at position 217 is highly conserved for human isolates and is associated with α2,6-sialic acid binding. Different amino acid combinations were observed, suggesting that the inoculum had viral subpopulations that were selected after passage in birds. These experimental studies corroborate the finding that certain poultry species are reservoirs of the H7N9 influenza virus and that the virus is highly tropic for the upper respiratory tract, so testing of bird species should preferentially be conducted with oropharyngeal swabs for the best sensitivity. IMPORTANCE The recent outbreak of H7N9 influenza in China has resulted in a number of human infections with a high case fatality rate. The source of the viral outbreak is suspected to be poultry, but definitive data on the source of the infection are not available. This study provides experimental data to show that quail and chickens are susceptible to infection, shed large amounts of virus, and are likely important in the spread of the virus to humans. Other poultry species can be infected and shed virus but are less likely to play a role of transmitting the virus to humans. Pigeons were previously suggested to be a possible source of the virus because of isolation of the virus from several pigeons in poultry markets in China, but experimental studies show that they are generally resistant to infection and are unlikely to play a role in the spread of the virus.

Virulent Newcastle disease virus elicits a strong innate immune response in chickens

Newcastle disease virus (NDV) is an avian paramyxovirus that causes significant economic losses to the poultry industry worldwide. There is limited knowledge about the avian immune response to infection with virulent NDVs, and how this response may contribute to disease. In this study, pathogenesis and the transcriptional host response of chickens to a virulent NDV strain that rapidly causes 100% mortality was characterized. Using microarrays, a strong transcriptional host response was observed in spleens at early times after infection with the induction of groups of genes involved in innate antiviral and pro-inflammatory responses. There were multiple genes induced at 48 h post-infection including: type I and II interferons (IFNs), several cytokines and chemokines, IFN effectors and inducible nitric oxide synthase (iNOS). The increased transcription of nitric oxide synthase was confirmed by immunohistochemistry for iNOS in spleens and measured levels of nitric oxide in serum. In vitro experiments showed strong induction of the key host response genes, alpha IFN, beta interferon, and interleukin 1β and interleukin 6, in splenic leukocytes at 6 h post-infection in comparison to a non-virulent NDV. The robust host response to virulent NDV, in conjunction with severe pathological damage observed, is somewhat surprising considering that all NDV encode a gene, V, which functions as a suppressor of class I IFNs. Taken together, these results suggest that the host response itself may contribute to the pathogenesis of this highly virulent strain in chickens.

Clinicopathological Characterization in Poultry of Three Strains of Newcastle Disease Virus Isolated From Recent Outbreaks

Newcastle disease is a severe threat to the poultry industry and is caused by Newcastle disease virus, a member of the genus Avulavirus, family Paramyxoviridae. The virus is rapidly evolving, and several new genotypes have been discovered in the past few years. Characterization of these strains is important to evaluate field changes, anticipate new outbreaks, and develop adequate control measures. Three Newcastle disease isolates (APMV-1/duck/Vietnam, Long Bien/78/2002, APMV-1/chicken/Australia/9809-19-1107/1998, and APMV-1/double-crested cormorant/USA, Nevada/19529-04/2005) from recent outbreaks were investigated via clinicopathological assessment, immunohistochemistry (IHC), in situ hybridization, virus isolation, and serology in experimentally infected 4-week-old chickens. Phylogenetic studies showed that Australia isolate belongs to class II genotype I, Long Bien to class II genotype VIId, and Nevada cormorant to class II genotype V. Even though all 3 viruses had a virulent fusion protein cleavage site and ICPI values greater than 1.5, they all differed in their ability to cause clinical signs, in their lesions, and in their viral distribution in body tissues. The Long Bien isolate showed the most severe clinicopathological picture and the most widespread viral distribution. The Australia and Nevada cormorant isolates had a milder pathological phenotype, with viral replication restricted to only a few organs. The variability in clinicopathological characteristics despite the similarity in ICPI suggests that full clinicopathological assessment is necessary to fully characterize new isolates and that there are differences in pathogenesis among viruses of different genotypes.

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

ABSTRACT 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.

Complete Genome and Clinicopathological Characterization of a Virulent Newcastle Disease Virus Isolate from South America

ABSTRACT Newcastle disease (ND) is one of the most important diseases of poultry, negatively affecting poultry production worldwide. The disease is caused by Newcastle disease virus (NDV) or avian paramyxovirus type 1 (APMV-1), a negative-sense single-stranded RNA virus of the genus Avulavirus, family Paramyxoviridae. Although all NDV isolates characterized to date belong to a single serotype of APMV-1, significant genetic diversity has been described between different NDV isolates. Here we present the complete genome sequence and the clinicopathological characterization of a virulent Newcastle disease virus isolate (NDV-Peru/08) obtained from poultry during an outbreak of ND in Peru in 2008. Phylogenetic reconstruction and analysis of the evolutionary distances between NDV-Peru/08 and other isolates representing established NDV genotypes revealed the existence of large genomic and amino differences that clearly distinguish this isolate from viruses of typical NDV genotypes. Although NDV-Peru/08 is a genetically distinct virus, pathogenesis studies conducted with chickens revealed that NDV-Peru/08 infection results in clinical signs characteristic of velogenic viscerotropic NDV strains. Additionally, vaccination studies have shown that an inactivated NDV-LaSota/46 vaccine conferred full protection from NDV-Peru/08-induced clinical disease and mortality. This represents the first complete characterization of a virulent NDV isolate from South America.

In vivo transcriptional cytokine responses and association with clinical and pathological outcomes in chickens infected with different Newcastle disease virus isolates using formalin-fixed paraffin-embedded samples

Little is known about the host response of chickens infected with Newcastle disease virus (NDV) and the relationship between the innate immune response and the severity of clinical disease. Measurement of cytokine responses during infection in vivo can help to elucidate the mechanisms of virus pathogenesis. The transcriptional response of several cytokines from paraffin-embedded, formalin-fixed spleen of chicken naturally infected by NDV velogenic viscerotropic viruses was compared to the responses of atypical velogenic, velogenic neurotropic, and mesogenic strains during the first five days after infection. The RNA expression for IFN-γ and IL-6 was enhanced at day two in the highly virulent velogenic viscerotropic viruses (California and rZJ1 strains) and corresponded with the presence of the virus in tissues. However, in one atypical velogenic viscerotropic virus (Australia strain), two velogenic neurotropic viruses (Turkey ND and Texas GB) and, a mesogenic virus (Anhinga strain) the cytokine responses to infection were delayed or reduced. Increased levels of IFN-β RNA expression were only detected in the velogenic viscerotropic virus infected chickens (California and rZJ1 strains) at 3 days post-infection and one mesogenic strain (Anhinga) early in infection. The RNA expression levels of IL-2 did not increase upon infection with any of the viruses. A pronounced increase of RNA expression levels of IL-6 and IFN-γ was detected simultaneously with infiltration of macrophages and/or lymphoid necrosis in the histopathological analysis of the spleen and cecal tonsils. The differences in the RNA expression levels may help explain possible underlying mechanisms of clinical disease and/or immune responses in birds infected with strains of APMV-1 that cause distinct pathologic changes.

Evolutionary Changes Affecting Rapid Identification of 2008 Newcastle Disease Viruses Isolated from Double-Crested Cormorants

ABSTRACT A morbidity-mortality event involving virulent Newcastle disease virus (NDV) in wild double-crested cormorants (Phalacrocorax auritus) occurred in North America in the summer of 2008. All 22 viruses isolated from cormorants were positively identified by the USDA-validated real-time reverse transcription-PCR assay targeting the matrix gene. However, the USDA-validated reverse transcription-PCR assay targeting the fusion gene that is specific for virulent isolates identified only 1 of these 22 isolates. Additionally, several of these isolates have been sequenced, and this information was used to identify genomic changes that caused the failure of the test and to revisit the evolution of NDV in cormorants. The forward primer and fusion probe were redesigned from the 2008 cormorant isolate sequence, and the revised fusion gene test successfully identified all 22 isolates. Phylogenetic analyses using both the full fusion sequence and the partial 374-nucleotide sequence identified these isolates as genotype V, with their nearest ancestor being an earlier isolate collected from Nevada in 2005. Histopathological analysis of this ancestral strain revealed morphological changes in the brain consistent with that of the traditional mesogenic pathotypes in cormorants. Intracerebral pathogenicity assays indicated that each of these isolates is virulent with values of >0.7 but not more virulent than earlier isolates reported from Canada.

Pathologic Characterization of Genotypes XIV and XVII Newcastle Disease Viruses and Efficacy of Classical Vaccination on Specific Pathogen-Free Birds

To characterize the clinicopathologic features of recently described genotypes of Newcastle disease virus (NDV), 1 representative strain of genotype XIV and 2 of genotype XVII, all isolated from West Africa, were used to infect groups of ten 4-week-old specific pathogen-free chickens. The pathobiology of these 3 strains was compared to a South African NDV strain classified within genotype VII. All chickens infected with the 4 viruses died or were euthanized by day 4 postinfection due to the severity of clinical signs. Gross and histologic lesions in all infected chickens included extensive necrosis of lymphoid tissues (thymus, spleen, bursa of Fabricius, cecal tonsils, gut-associated lymphoid tissue), gastrointestinal necrosis and hemorrhages, and severe hemorrhagic conjunctivitis. Immunohistochemical staining revealed systemic viral distribution, and the most intense staining was in the lymphoid organs. Results demonstrate that the 3 West African strains from the previously uncharacterized genotypes XIV and XVII are typical velogenic viscerotropic NDV strains with lesions similar to the South African strain. Under experimental conditions, QV4 and LaSota NDV vaccine strains successfully protected chickens from morbidity and mortality against the genotype VII and one genotype XVII NDV strain, with no significant differences in the amount of virus shed when 2 vaccine schemes were compared.

Early Occurrence of Apoptosis in Lymphoid Tissues from Chickens Infected with Strains of Newcastle Disease Virus of Varying Virulence

Newcastle disease virus (NDV), the causative agent of Newcastle disease, is a prevalent problem in the poultry industry and often the cause of severe economic loss. There are many strains of the virus and these have varying virulence. The most virulent strains cause systemic lesions of lymphoid tissues, with necrosis and severe lymphoid depletion. Less virulent strains do not cause as much necrosis, but may predispose to secondary infection with other pathogens. Apoptosis or programmed cell death, has been demonstrated to play a role in the pathogenesis of other paramyxovirus infections, notably those caused by measles and canine distemper viruses. To investigate the role of apoptosis in lymphoid organs during NDV infection, immunohistochemistry for determination of expression of caspase-3, a marker of imminent apoptosis, was performed on formalin-fixed paraffin wax-embedded tissues (spleen, thymus, caecal tonsils and bursa of Fabricius) from 4-week-old chickens infected with NDV strains of varying virulence 2 days previously. The amount of apoptosis was proportional to the severity of the clinical disease elicited by the strains.