Pathogenesis of Kasokero virus in experimentally infected Egyptian rousette bats (Rousettus aegyptiacus).

Egyptian rousette bats (ERBs; Rousettus aegyptiacus; family Pteropodidae) are associated with a growing number of bunyaviruses of public health importance, including Kasokero virus (KASV), which was first identified as a zoonosis in Uganda in 1977. In this study, formalin-fixed paraffin-embedded tissues from a previous experiment in which KASV infection was confirmed in 18 experimentally infected ERBs were used for an in-depth analysis using histopathology, in situ hybridization (ISH) for detection of viral RNA, immunohistochemistry (IHC) to assess the mononuclear phagocyte system response, and quantitative digital image analysis to investigate virus clearance from the liver and spleen within a spatial context. Significant gross and histological lesions were limited to the liver, where KASV-infected bats developed mild to moderate, acute viral hepatitis, which was first observed at 3 days postinfection (DPI), peaked at 6 DPI, and was resolved by 20 DPI. A subset of bats had glycogen depletion (n = 10) and hepatic necrosis (n = 3), rarely with intralesional bacteria (n = 1). Virus replication was confirmed by ISH in the liver, spleen, lymph nodes, and tongue. In the liver, KASV replicated in the cytoplasm of hepatocytes, to a lesser extent in mononuclear phagocytes, and rarely in presumptive endothelial cells. Most KASV RNA, as detected by ISH, was cleared from the spleen and liver by 6 DPI. It is concluded that ERBs have effective mechanisms to respond to this virus, clearing it without evidence of clinical disease.

Early pathogenesis in rabbit hemorrhagic disease virus 2.

To detail early tissue distribution and innate immune response to rabbit hemorrhagic disease virus 2 (RHDV2), 13 rabbits were orally (Oryctolagus cuniculus) inoculated with liver homogenate made from a feral rabbit that succumbed to RHDV2 during the 2020 outbreak in Oregon, USA. Rabbits were monitored regularly, with euthanasia and collection of tissues and swabs, at 12, 24, 36, 48, 96, and 144 h post inoculation. Livers from these rabbits were positive by RT-rtPCR for presence of the virus. Using RNAscope for viral and replicative intermediates, rabbits had detectable viral genomic RNA at each time point, initially within the gastrointestinal tract, then in the liver by 36 h post inoculation. Also using RNAscope, there were increasing amounts of mRNA coding for TNF-α, IL-6, and IL-1ß within the liver and spleen through 48 h post inoculation. The results of this study aided our understanding of the local innate immune response to RHDV2, as well as aspects of pathogenesis.

Enhanced phylogenetic resolution of Newcastle disease outbreaks using complete viral genome sequences from formalin-fixed paraffin-embedded tissue samples.

Highly virulent Newcastle disease virus (NDV) causes Newcastle disease (ND), which is a threat to poultry production worldwide. Effective disease management requires approaches to accurately determine sources of infection, which involves tracking of closely related viruses. Next-generation sequencing (NGS) has emerged as a research tool for thorough genetic characterization of infectious organisms. Previously formalin-fixed paraffin-embedded (FFPE) tissues have been used to conduct retrospective epidemiological studies of related but genetically distinct viruses. However, this study extends the applicability of NGS for complete genome analysis of viruses from FFPE tissues to track the evolution of closely related viruses. Total RNA was obtained from FFPE spleens, lungs, brains, and small intestines of chickens in 11 poultry flocks during disease outbreaks in Pakistan. The RNA was randomly sequenced on an Illumina MiSeq instrument and the raw data were analyzed using a custom data analysis pipeline that includes de novo assembly. Genomes of virulent NDV were detected in 10/11 birds: eight nearly complete (> 95% coverage of concatenated coding sequence) and two partial genomes. Phylogeny of the NDV complete genome coding sequences was compared to current methods of analysis based on the full and partial fusion genes and determined that the approach provided a better phylogenetic resolution. Two distinct lineages of sub-genotype VIIi NDV were identified to be simultaneously circulating in Pakistani poultry. Non-targeted NGS of total RNA from FFPE tissues coupled with de novo assembly provided a reliable, safe, and affordable method to conduct epidemiological and evolutionary studies to facilitate management of ND in Pakistan.

Tropism of Newcastle disease virus strains for chicken neurons, astrocytes, oligodendrocytes, and microglia.

BACKGROUND: Newcastle disease (ND), which is caused by infections of poultry species with virulent strains of Avian orthoavulavirus-1, also known as avian paramyxovirus 1 (APMV-1), and formerly known as Newcastle disease virus (NDV), may cause neurological signs and encephalitis. Neurological signs are often the only clinical signs observed in birds infected with neurotropic strains of NDV. Experimental infections have shown that the replication of virulent NDV (vNDV) strains is in the brain parenchyma and is possibly confined to neurons and ependymal cells. However, little information is available on the ability of vNDV strains to infect subset of glial cells (astrocytes, oligodendrocytes, and microglia). The objective of this study was to evaluate the ability of NDV strains of different levels of virulence to infect a subset of glial cells both in vitro and in vivo. Thus, neurons, astrocytes and oligodendrocytes from the brains of day-old White Leghorn chickens were harvested, cultured, and infected with both non-virulent (LaSota) and virulent, neurotropic (TxGB) NDV strains. To confirm these findings in vivo, the tropism of three vNDV strains with varying pathotypes (SA60 [viscerotropic], TxGB [neurotropic], and Tx450 [mesogenic]) was assessed in archived formalin-fixed material from day-old chicks inoculated intracerebrally. RESULTS: Double immunofluorescence for NDV nucleoprotein and cellular markers showed that both strains infected at least 20% of each of the cell types (neurons, astrocytes, and oligodendrocytes). At 24 h post-inoculation, TxGB replicated significantly more than LaSota. Double immunofluorescence (DIFA) with markers for neurons, astrocytes, microglia, and NDV nucleoprotein detected the three strains in all three cell types at similar levels. CONCLUSION: These data indicate that similar to other paramyxoviruses, neurons and glial cells (astrocytes, oligodendrocytes, and microglia) are susceptible to vNDV infection, and suggest that factors other than cellular tropism are likely the major determinant of the neurotropic phenotype.

Whole-genome sequencing of genotype VI Newcastle disease viruses from formalin-fixed paraffin-embedded tissues from wild pigeons reveals continuous evolution and previously unrecognized genetic diversity in the U.S.

BACKGROUND: Newcastle disease viruses (NDV) are highly contagious and cause disease in both wild birds and poultry. A pigeon-adapted variant of genotype VI NDV, often termed pigeon paramyxovirus 1, is commonly isolated from columbids in the United States and worldwide. Complete genomic characterization of these genotype VI viruses circulating in wild columbids in the United States is limited, and due to the genetic variability of the virus, failure of rapid diagnostic detection has been reported. Therefore, in this study, formalin-fixed paraffin-embedded (FFPE) samples were subjected to next-generation sequencing (NGS) to identify and characterize these circulating viruses, providing valuable genetic information. NGS enables multiple samples to be deep-sequenced in parallel. When used on FFPE samples, this methodology allows for retrospective studies of infectious organisms. METHODS: FFPE wild pigeon tissue samples (kidney, liver and spleen) from 10 mortality events in the U.S. between 2010 and 2016 were analyzed using NGS to detect and sequence NDV genomes from randomly amplified total RNA. Results were compared to the previously published immunohistochemistry (IHC) results conducted on the same samples. Additionally, phylogenetic analyses were conducted on the complete and partial fusion gene and complete genome coding sequences. RESULTS: Twenty-three out of 29 IHC-positive FFPE pigeon samples were identified as positive for NDV by NGS. Positive samples produced an average genome coverage of 99.6% and an average median depth of 199. A previously described sub-genotype (VIa) and a novel sub-genotype (VIn) of NDV were identified as the causative agent of 10 pigeon mortality events in the U.S. from 2010 to 2016. The distribution of these viruses from the North American lineages match the distribution of the Eurasian collared-doves and rock pigeons in the U.S. CONCLUSIONS: This work reports the first successful evolutionary study using deep sequencing of complete NDV genomes from FFPE samples of wild bird origin. There are at least two distinct U.S. lineages of genotype VI NDV maintained in wild pigeons that are continuously evolving independently from each other and have no evident epidemiological connections to viruses circulating abroad. These findings support the hypothesis that columbids are serving as reservoirs of virulent NDV in the U.S.

Challenges in Preparing Veterinarians for Global Animal Health: Understanding the Public Sector.

Understanding of global systems is essential for veterinarians seeking to work in realms outside of their national domain. In the global system, emphasis remains on the public sector, and the current curricular emphasis in developed countries is on private clinical practice for the domestic employment market. There is a resulting lack of competency at graduation for effective engagement internationally. The World Organisation for Animal Health (OIE) has created standards for public sector operations in animal health, which must be functional to allow for sustainable development. This public sector, known as the Veterinary Services, or VS, serves to control public good diseases, and once effectively built and fully operational, allows for the evolution of a functional private sector, focused on private good diseases. Until the VS is fully functional, support of private good services is non-sustainable and any efforts delivered are not long lasting. As new graduates opt for careers working in the international development sector, it is essential that they understand the OIE guidelines to help support continuing improvement. Developing global veterinarians by inserting content into the veterinary curriculum on how public systems can operate effectively could markedly increase the potential of our professional contributions globally, and particularly in the areas most in need.

Expression of chicken interleukin-2 by a highly virulent strain of Newcastle disease virus leads to decreased systemic viral load but does not significantly affect mortality in chickens.

BACKGROUND: In mammals, interleukin 2 (IL-2) has been shown to decrease replication or attenuate pathogenicity of numerous viral pathogens (herpes simplex virus, vaccinia virus, human respiratory syncytial virus, human immunodeficiency virus) by activating natural killer cells (NK), cytotoxic T lymphocytes and expanding subsets of memory cells. In chickens, IL-2 has been shown to activate T cells, and as such it might have the potential to affect replication and pathogenesis of Newcastle disease virus (NDV). METHODS: To assess the effect of IL-2 during NDV infection in chickens, we produced a recombinant virulent NDV strain expressing chicken IL-2 (rZJ1-IL2). The effects of IL-2 expression were investigated in vivo using the intracerebral pathogenicity index (ICPI) in day-old chicks and pathogenesis experiments in 4-week-old chickens. In these studies, rZJ1-IL2 was compared to a control virus expressing the green fluorescent protein (rZJ1-GFP). Assessed parameters included survival curves, detailed histological and immunohistochemical grading of lesions in multiple organs, and virus isolation in blood, spleen and mucosal secretions of infected birds. RESULTS: At the site of infection (eyelid), expression of IL-2 was demonstrated in areas of rZJ-IL2 replication, confirming IL-2 production in vivo. Compared to rZJ1-GFP strain, rZJ1-IL2 caused milder lesions and displayed decreased viral load in blood, spleen and mucosal secretions of infected birds. In the rZJ1-IL2-infected group, virus level in the blood peaked at day 4 post-infection (pi) (10(3.46) EID50 /0.1 ml) and drastically decreased at day 5 pi (10(0.9) EID50/0.1 ml), while in the rZJ1-GFP-infected group virus levels in the blood reached 10(5.35) EID50/0.1 ml at day 5. However, rZJ1-IL2-infected groups presented survival curves similar to control birds infected with rZJ1-GFP, with comparable clinical signs and 100 % mortality. Further, expression of IL-2 did not significantly affect the ICPI scores, compared to rZJ1-GFP strain. CONCLUSIONS: Increased expression of chicken IL-2 during virulent NDV replication in naïve chickens decreased viral titers in blood, spleens, oral and cloacal secretions on day 4-5 post infection. This is consistent with the previously described role of IL-2 in enhancing the clearance of viruses in mammals, such as human respiratory syncytial virus.

Development of an improved vaccine evaluation protocol to compare the efficacy of Newcastle disease vaccines.

While there is typically 100% survivability in birds challenged with vNDV under experimental conditions, either with vaccines formulated with a strain homologous or heterologous (different genotype) to the challenge virus, vaccine deficiencies are often noted in the field. We have developed an improved and more stringent protocol to experimentally evaluate live NDV vaccines, and showed for the first time under experimental conditions that a statistically significant reduction in mortality can be detected with genotype matched vaccines. Using both vaccine evaluation protocols (traditional and improved), birds were challenged with a vNDV of genotype XIII and the efficacy of live heterologous (genotype II) and homologous (genotype XIII) NDV vaccines was compared. Under traditional vaccination conditions there were no differences in survival upon challenge, but the homologous vaccine induced significantly higher levels of antibodies specific to the challenge virus. With the more stringent challenge system (multiple vaccine doses and early challenge with high titers of vNDV), the birds administered the homologous vaccine had superior humoral responses, reduced clinical signs, and reduced mortality levels than those vaccinated with the heterologous vaccine. These results provide basis for the implementation of more sensitive methods to evaluate vaccine efficacy.

Separate evolution of virulent newcastle disease viruses from Mexico and Central America.

An outbreak of Newcastle disease (ND) in poultry was reported in Belize in 2008. The characteristics of three virulent Newcastle disease virus (NDV) isolates from this outbreak (NDV-Belize-3/08, NDV-Belize-4/08, and NDV-Belize-12/08) were assessed by genomic analysis and by clinicopathological characterization in specific-pathogen-free (SPF) chickens. The results showed that all three strains belong to NDV genotype V and are virulent, as assessed by the intracerebral pathogenicity index and the polybasic amino acid sequence at the fusion protein cleavage site. In 4-week-old SPF chickens, NDV-Belize-3/08 behaved as a typical velogenic viscerotropic NDV strain, causing severe necrohemorrhagic lesions in the lymphoid organs, with systemic virus distribution. Phylogenetic analysis of multiple NDV genotype V representatives revealed that genotype V can be divided into three subgenotypes, namely, Va, Vb, and Vc, and that all tested Belizean isolates belong to subgenotype Vb. Furthermore, these isolates are nearly identical to a 2007 isolate from Honduras and appear to have evolved separately from other contemporary viruses circulating in Mexico, clustering into a new clade within NDV subgenotype Vb.

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.

Expression of interferon gamma by a highly virulent strain of Newcastle disease virus decreases its pathogenicity in chickens.

The role of interferon gamma (IFN-γ) expression during Newcastle disease virus (NDV) infection in chickens is unknown. Infection of chickens with highly virulent NDV results in rapid death, which is preceded by increased expression of IFN-γ in target tissues. IFN-γ is a cytokine that has pleiotropic biological effects including intrinsic antiviral activity and immunomodulatory effects that may increase morbidity and mortality during infections. To better understand how IFN-γ contributes to NDV pathogenesis, the coding sequence of the chicken IFN-γ gene was inserted in the genome of the virulent NDV strain ZJ1 (rZJ1-IFNγ), and the effects of high levels of IFN-γ expression during infection were determined in vivo and in vitro. IFN-γ expression did not significantly affect NDV replication in fibroblast or in macrophage cell lines. However, it affected the pathogenesis of rZJ1-IFNγ in vivo. Relative to the virus expressing the green fluorescent protein (rZJ1-GFP) or lacking the IFN-γ insert (rZJ1-rev), expression of IFN-γ by rZJ1-IFNγ produced a marked decrease of pathogenicity in 4-week-old chickens, as evidenced by lack of mortality, decreased disease severity, virus shedding, and antigen distribution. These results suggest that early expression of IFN-γ had a significant protective role against the effects of highly virulent NDV infection in chickens, and further suggests that the level and timing of expression of this cytokine may be critical for the disease outcome. This is the first description of an in vivo attenuation of a highly virulent NDV by avian cytokines, and shows the feasibility to use NDV for cytokine delivery in chicken organs. This approach may facilitate the study of the role of other avian cytokines on the pathogenesis of NDV.

Comparing presence of avian paramyxovirus-1 through immunohistochemistry in tracheas of experimentally and naturally infected chickens.

Tracheas from chickens infected both in the field and experimentally with lentogenic Newcastle disease virus (also known as avian paramyxovirus-1 [APMV-1] and referred to here as "lentogenic NDV") were examined histopathologically to score degree of pathologic changes and by immunohistochemistry to determine presence of viral protein. In the field cases there was often a striking lack of correlation between severity of tracheal lesions and amount of immunohistochemical signal for APMV-1 protein. Experimental cases had minimal pathologic changes and also minimal immunohistochemical signal. Positive cells were often associated with surface deciliation. It may be that lentogenic NDV has only a minor role as a respiratory pathogen, merely compromising the mucosa to allow other respiratory pathogens to infect and worsen the clinical and pathologic presentation.

A Novel Hypomorphic Apex1 Mouse Model Implicates Apurinic/Apyrimidinic Endonuclease 1 in Oxidative DNA Damage Repair in Gastric Epithelial Cells.

Aims: Though best known for its role in oxidative DNA damage repair, apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional protein that regulates multiple host responses during oxidative stress, including the reductive activation of transcription factors. As knockout of the APE1-encoding gene, Apex1, is embryonically lethal, we sought to create a viable model with generalized inhibition of APE1 expression. Results: A hypomorphic (HM) mouse with decreased APE1 expression throughout the body was generated using a construct containing a neomycin resistance (NeoR) cassette knocked into the Apex1 site. Offspring were assessed for APE1 expression, breeding efficiency, and morphology with a focused examination of DNA damage in the stomach. Heterozygotic breeding pairs yielded 50% fewer HM mice than predicted by Mendelian genetics. APE1 expression was reduced up to 90% in the lungs, heart, stomach, and spleen. The HM offspring were typically smaller, and most had a malformed tail. Oxidative DNA damage was increased spontaneously in the stomachs of HM mice. Further, all changes were reversed when the NeoR cassette was removed. Primary gastric epithelial cells from HM mice differentiated more quickly and had more evidence of oxidative DNA damage after stimulation with Helicobacter pylori or a chemical carcinogen than control lines from wildtype mice. Innovation: A HM mouse with decreased APE1 expression throughout the body was generated and extensively characterized. Conclusion: The results suggest that HM mice enable studies of APE1's multiple functions throughout the body. The detailed characterization of the stomach showed that gastric epithelial cells from HM were more susceptible to DNA damage. Antioxid. Redox Signal. 38, 183-197.

Complete genome and clinicopathological characterization of a virulent Newcastle disease virus isolate from South America.

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.

Detection of rabbit hemorrhagic disease virus 2 in formalin-fixed, paraffin-embedded tissues via in situ hybridization.

Formalin-fixed, paraffin-embedded tissues from European rabbits (Oryctolagus cuniculus) that succumbed to rabbit hemorrhagic disease virus 2 (RHDV2; Lagovirus GI.2) during the 2019 outbreak in Washington, USA, were utilized for in situ hybridization via RNAscope (ACDBio). This detection method was both sensitive and specific, with no staining in tissues from RHDV- (Lagovirus GI.1) and RHDV2-negative rabbits, and only slight background staining of RHDV-positive rabbits; RHDV2-positive tissues had bright-red cytoplasmic staining. Although much of the viral mRNA detection was consistent with previously described antigen detection via immunohistochemistry of the liver, lungs, and spleen, there was also significant glomerular staining in the kidneys, and endothelial staining within blood vessels of almost all organs. We validated the RNAscope technique for detection of RHDV2 mRNA in formalin-fixed, paraffin-embedded tissues, with increased sensitivity from previous techniques, and identified additional affected cell types that may contribute to the understanding of pathogenesis.

Viral replication site distribution for rabbit hemorrhagic disease virus 2 in formalin-fixed, paraffin-embedded tissues via in situ hybridization.

We made 2 Z-based in situ hybridization (ISH) probes for the detection of rabbit hemorrhagic disease virus 2 (RHDV2; Lagovirus GI.2) nucleic acid in formalin-fixed, paraffin-embedded tissues from European rabbits (Oryctolagus cuniculus) that had died during an outbreak of RHD in Washington, USA. One probe system was made for detection of negative-sense RNA (i.e., the replicative intermediate RNA for the virus), and the other probe system was constructed for detection of genomic and mRNA of the virus (viral mRNA). Tissue sets were tested separately, and the viral mRNA probe system highlighted much broader tissue distribution than that of the replicative intermediate RNA probe system. The latter was limited to liver, lung, kidney, spleen, myocardium, and occasional endothelial staining, whereas signal for the viral mRNA was seen in many more tissues. The difference in distribution suggests that innate phagocytic activity of various cell types may cause overestimation of viral replication sites when utilizing ISH of single-stranded, positive-sense viruses.

Histopathologic and Immunohistochemical Evaluation of Induced Lesions, Tissue Tropism and Host Responses following Experimental Infection of Egyptian Rousette Bats (Rousettus aegyptiacus) with the Zoonotic Paramyxovirus, Sosuga Virus.

Ecological and experimental infection studies have identified Egyptian rousette bats (ERBs; Rousettus aegyptiacus: family Pteropodidae) as a reservoir host for the zoonotic rubula-like paramyxovirus Sosuga virus (SOSV). A serial sacrifice study of colony-bred ERBs inoculated with wild-type, recombinant SOSV identified small intestines and salivary gland as major sites of viral replication. In the current study, archived formalin-fixed paraffin-embedded (FFPE) tissues from the serial sacrifice study were analyzed in depth-histologically and immunohistochemically, for SOSV, mononuclear phagocytes and T cells. Histopathologic lesion scores increased over time and viral antigen persisted in a subset of tissues, indicating ongoing host responses and underscoring the possibility of chronic infection. Despite the presence of SOSV NP antigen and villus ulcerations in the small intestines, there were only mild increases in mononuclear phagocytes and T cells, a host response aligned with disease tolerance. In contrast, there was a statistically significant, robust and targeted mononuclear phagocyte cell responses in the salivary glands at 21 DPI, where viral antigen was sparse. These findings may have broader implications for chiropteran-paramyxovirus interactions, as bats are hypothesized to be the ancestral hosts of this diverse virus family and for ERB immunology in general, as this species is also the reservoir host for the marburgviruses Marburg virus (MARV) and Ravn virus (RAVV) (family Filoviridae).

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 1ß 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.