Genetic stability of a Newcastle disease virus vectored infectious laryngotracheitis virus vaccine after serial passages in chicken embryos.

Previously, we have demonstrated that the recombinant Newcastle disease virus (NDV) expressing the infectious laryngotracheitis virus (ILTV) glycoprotein D (gD) conferred protection against both virulent NDV and ILTV challenges in chickens. In this study, we evaluated the genetic stability of the recombinant vaccine after eight serial passages in embryonated chicken eggs (ECE). The vaccine master seed virus at the original egg-passage level 3 (EP3) was diluted and passaged in three separate repetitions (A, B and C) in ECE eight times (EP4 to EP11). RT-PCR analysis of the vaccine seed and egg-passaged virus stocks showed that there was no detectable insertion/deletion in the ILTV gD insert region. Next-generation sequencing analysis of the EP3 and EP11 virus stocks confirmed their genome integrity and revealed a total of thirteen single-nucleotide polymorphisms (SNPs). However, none of these SNPs were located in the ILTV gD insert or any of the known critical biological determinant positions. Virological and immunofluorescent assays provided additional evidence that the EP11 virus stocks retained their growth kinetics, low pathogenicity, and robust level of gD expression comparable to that of the vaccine master seed virus. This indicated that the SNPs were non-detrimental sporadic mutations. These results demonstrated that the insertion of ILTV gD gene into the NDV LaSota backbone did not significantly affect the genetic stability of the recombinant virus and that the rLS/ILTV-gD virus is a safe and genetically stable vaccine candidate after at least eight serial passages in ECE.

Efficacy of an adenovirus-vectored foot-and-mouth disease virus serotype A subunit vaccine in cattle using a direct contact transmission model.

BACKGROUND: A direct contact transmission challenge model was used to simulate natural foot-and-mouth disease virus (FMDV) spread from FMDV A24/Cruzeiro/BRA/55 infected 'seeder' steers to naïve or vaccinated steers previously immunized with a replication-deficient human adenovirus-vectored FMDV A24/Cruzeiro/BRA/55 capsid-based subunit vaccine (AdtA24). In two independent vaccine efficacy trials, AdtA24 was administered once intramuscularly in the neck 7 days prior to contact with FMDV A24/Cruzeiro/BRA/55-infected seeder steers. RESULTS: In Efficacy Study 1, we evaluated three doses of AdtA24 to estimate the 50%/90% bovine protective dose (BPD50/90) for prevention of clinical FMD. In vaccinated, contact-challenged steers, the BPD50/90 was 3.1 × 1010 / 5.5 × 1010 AdtA24 particles formulated without adjuvant. In Efficacy Study 2, steers vaccinated with 5 × 1010 AdtA24 particles, exposed to FMDV A24/Cruzeiro/BRA/55-infected seeder steers, did not develop clinical FMD or transmit FMDV to other vaccinated or naïve, non-vaccinated steers. In contrast, naïve, non-vaccinated steers that were subsequently exposed to FMDV A24/Cruzeiro/BRA/55-infected seeder steers developed clinical FMD and transmitted FMDV by contact to additional naïve, non-vaccinated steers. The AdtA24 vaccine differentiated infected from vaccinated animals (DIVA) because no antibodies to FMDV nonstructural proteins were detected prior to FMDV exposure. CONCLUSIONS: A single dose of the AdtA24 non-adjuvanted vaccine conferred protection against clinical FMD at 7 days post-vaccination following direct contact transmission from FMDV-infected, naïve, non-vaccinated steers. The AdtA24 vaccine was effective in preventing FMDV transmission from homologous challenged, contact-exposed, AdtA24-vaccinated, protected steers to co-mingled, susceptible steers, suggesting that the vaccine may be beneficial in reducing both the magnitude and duration of a FMDV outbreak in a commercial cattle production setting.

Engineered Newcastle disease virus expressing the F and G proteins of AMPV-C confers protection against challenges in turkeys.

Avian metapneumovirus (AMPV) infects the respiratory and reproductive tracts of domestic poultry, resulting in substantial economic losses for producers. Live attenuated vaccines appear to be the most effective in countries where the disease is prevalent. However, reversion to virulence has been demonstrated in several studies. Therefore, the development of a stable and safe next generation vaccine against the AMPV disease is needed. In the present study, we generated a recombinant Newcastle disease virus (NDV) vectoring the fusion (F) protein and glycoprotein (G) genes of AMPV subtype-C (AMPV-C) as a bivalent vaccine candidate using reverse genetics technology. The recombinant virus, rLS/AMPV-C F&G, was slightly attenuated in vivo, yet maintained similar characteristics in vitro when compared to the parental LaSota virus. Vaccination of turkeys with rLS/AMPV-C F&G induced both AMPV-C and NDV-specific antibody responses, and provided significant protection against pathogenic AMPV-C challenge and complete protection against velogenic NDV challenge. These results suggest that the rLS/AMPV-C F&G recombinant virus is a safe and effective bivalent vaccine candidate and that the expression of both F and G proteins of AMPV-C induces a protective response against the AMPV-C disease.

Newcastle disease virus vectored infectious laryngotracheitis vaccines protect commercial broiler chickens in the presence of maternally derived antibodies.

Newcastle disease virus (NDV) recombinants expressing the infectious laryngotracheitis virus (ILTV) glycoproteins B and D have previously been demonstrated to confer complete clinical protection against virulent ILTV and NDV challenges in naive chickens. We extended this study to assess whether maternally derived antibody (MDA) against NDV and ILTV would interfere with protection in vaccinated broiler chickens. Chickens with a mean NDV MDA hemagglutination inhibition (HI) titer of 6.4 (log2) and detectable ILTV neutralization (VN) antibodies at hatch were vaccinated with rLS/ILTV-gB or rLS/ILTV-gD at 1 or 10day of age (DOA) or on both days. Groups of birds vaccinated with the commercial ILT vaccines (FP-LT and CEO) or sham inoculated were also included in this study. All vaccinated birds were challenged with virulent ILTV strain at 21 DOA. By that time, NDV HI titers declined to 2.6 (log2) in unvaccinated birds, whereas the HI titers in NDV vectored vaccine groups increased to 3.5-6.3 (log2). At standard dosages, both vaccine candidates conferred significant clinical protection; however, the protection elicited by the rLS/ILTV-gD was superior to that of rLS/ILTV-gB. Recombinant rLS/ILTV-gD reduced ILTV shedding from tracheal and ocular tissues by approximately 3 log10 TCID50. Notably, there was no improvement in protection after booster vaccination at 10 DOA. Overall results indicate that the presence of maternal antibodies to NDV and ILTV did not significantly interfere with the ability of the NDV LaSota strain-vectored ILTV gB and gD vaccine candidates to elicit protective immunity against infectious laryngotracheitis.

Molecular Characterization of Enteric Picornaviruses in Archived Turkey and Chicken Samples from the United States.

Recent metagenomic analyses of the enteric viromes in turkeys and chickens have revealed complex viral communities comprised of multiple viral families. Of particular significance are the novel avian picobirnaviruses (family Picobirnaviridae), multiple genera of tailed phages (family Siphoviridae), and undescribed avian enteric picornaviruses (family Picornaviridae). In addition to these largely undescribed-and therefore relatively poorly understood-poultry enteric viral families, these metagenomic analyses have also revealed the presence of well-known groups of enteric viruses such as the chicken and turkey astroviruses (family Astroviridae) and the avian rotaviruses and reoviruses (family Reoviridae). The order Picornavirales is a group of viruses in flux, particularly among the avian picornaviruses, since several new genera have been described recently based upon community analysis of enteric viromes from poultry and other avian species worldwide. Our previous investigation of the turkey enteric picornaviruses suggests the avian enteric picornaviruses may contribute to the enteric disease syndromes and performance problems often observed in turkeys in the Southeastern United States. This report describes our recent phylogenetic analysis of turkey and chicken enteric samples archived at the Southeast Poultry Research Laboratory from 2004 to present and is a first step in placing these novel avian picornaviruses within the larger Picornaviridae family.

Generation of Newcastle Disease Virus (NDV) Recombinants Expressing the Infectious Laryngotracheitis Virus (ILTV) Glycoprotein gB or gD as Dual Vaccines.

Infectious laryngotracheitis (ILT) is a highly contagious acute respiratory disease of chickens caused by infection with infectious laryngotracheitis virus (ILTV), a member of the family Herpesviridae. The current commercial ILT vaccines are either unsafe or ineffective. Therefore, there is a pressing need to develop safer and more efficacious vaccines. Newcastle disease (ND), caused by infection with Newcastle disease virus (NDV), a member of the family Paramyxoviridae, is one of the most serious infectious diseases of poultry. The NDV LaSota strain, a naturally occurring low-virulence NDV strain, has been routinely used as a live vaccine throughout the world. This chapter describes the generation of Newcastle disease virus (NDV) LaSota vaccine strain-based recombinant viruses expressing glycoprotein B (gB) or glycoprotein D (gD) of ILTV as dual vaccines against ND and ILT using reverse genetics technology.

Multiple efficacy studies of an adenovirus-vectored foot-and-mouth disease virus serotype A24 subunit vaccine in cattle using homologous challenge.

The safety and efficacy of an experimental, replication-deficient, human adenovirus-vectored foot-and-mouth disease virus (FMDV) serotype A24 Cruzeiro capsid-based subunit vaccine (AdtA24) was examined in eight independent cattle studies. AdtA24 non-adjuvanted vaccine was administered intramuscularly to a total of 150 steers in doses ranging from approximately 1.0×10(8) to 2.1×10(11) particle units per animal. No detectable local or systemic reactions were observed after vaccination. At 7 days post-vaccination (dpv), vaccinated and control animals were challenged with FMDV serotype A24 Cruzeiro via the intradermal lingual route. Vaccine efficacy was measured by FMDV A24 serum neutralizing titers and by protection from clinical disease and viremia after challenge. The results of eight studies demonstrated a strong correlation between AdtA24 vaccine dose and protection from clinical disease (R(2)=0.97) and viremia (R(2)=0.98). There was also a strong correlation between FMDV A24 neutralization titers on day of challenge and protection from clinical disease (R(2)=0.99). Vaccination with AdtA24 enabled differentiation of infected from vaccinated animals (DIVA) as demonstrated by the absence of antibodies to the FMDV nonstructural proteins in vaccinates prior to challenge. Lack of AdtA24 vaccine shedding after vaccination was indicated by the absence of neutralizing antibody titers to both the adenovector and FMDV A24 Cruzeiro in control animals after co-mingling with vaccinated cattle for three to four weeks. In summary, a non-adjuvanted AdtA24 experimental vaccine was shown to be safe, immunogenic, consistently protected cattle at 7 dpv against direct, homologous FMDV challenge, and enabled differentiation of infected from vaccinated cattle prior to challenge.

Investigating Turkey Enteric Picornavirus and Its Association with Enteric Disease in Poults.

Previous research into the viral community in the poultry gastrointestinal tract has revealed a number of novel and partially described enteric viruses. It is evident that the poultry gut viral community remains minimally characterized and incompletely understood. Investigations into the microbiome of the poultry gut have provided some insight into the geographical distribution and the rapidly evolving taxonomy of the avian enteric picornaviruses. The present investigation was undertaken to produce a comparative metagenomic analysis of the gut virome from a healthy turkey flock versus a flock placed in the field. This investigation revealed a number of enteric picornavirus sequences that were present in the commercial birds in the field that were completely absent in the healthy flock. A novel molecular diagnostic assay was used to track the shedding of field strains of turkey enteric picornavirus in commercial poults inoculated with picornavirus-positive intestinal homogenates prepared from turkeys that were experiencing moderate enteric disease. The propagation of this novel enteric picornavirus in commercial poults resulted in significant reduction in weight gain, and suggests that this common inhabitant of the turkey gut may result in performance problems or enteric disease in the field.

Host Specificity and Phylogenetic Relationships of Chicken and Turkey Parvoviruses.

Inoculation of specific-pathogen-free chickens and turkeys with five chicken parvoviruses (ChPV) and five turkey parvoviruses (TuPV) resulted in productive virus replication only in the homologous host species. A phylogenetic tree based on nucleotide sequences of the VP1 gene segment revealed a host-specific clustering of the virus strains. These results suggest that the VP1 gene plays an essential role in host specificity of ChPV and TuPV strains and could be a relevant target sequence for strain classification.

Development of a Newcastle disease virus vector expressing a foreign gene through an internal ribosomal entry site provides direct proof for a sequential transcription mechanism.

In the present study, we developed a novel approach for foreign gene expression by Newcastle disease virus (NDV) from a second ORF through an internal ribosomal entry site (IRES). Six NDV LaSota strain-based recombinant viruses vectoring the IRES and a red fluorescence protein (RFP) gene behind the nucleocapsid (NP), phosphoprotein (P), matrix (M), fusion (F), haemagglutinin-neuraminidase (HN) or large polymerase (L) gene ORF were generated using reverse genetics technology. The insertion of the second ORF slightly attenuated virus pathogenicity, but did not affect ability of the virus to grow. Quantitative measurements of RFP expression in virus-infected DF-1 cells revealed that the abundance of viral mRNAs and red fluorescence intensity were positively correlated with the gene order of NDV, 3'-NP-P-M-F-HN-L-5', proving the sequential transcription mechanism for NDV. The results herein suggest that the level of foreign gene expression could be regulated by selecting the second ORF insertion site to maximize the efficacy of vaccine and gene therapy.

Comparative analysis of the intestinal bacterial and RNA viral communities from sentinel birds placed on selected broiler chicken farms.

There is a great deal of interest in characterizing the complex microbial communities in the poultry gut, and in understanding the effects of these dynamic communities on poultry performance, disease status, animal welfare, and microbes with human health significance. Investigations characterizing the poultry enteric virome have identified novel poultry viruses, but the roles these viruses play in disease and performance problems have yet to be fully characterized. The complex bacterial community present in the poultry gut influences gut development, immune status, and animal health, each of which can be an indicator of overall performance. The present metagenomic investigation was undertaken to provide insight into the colonization of specific pathogen free chickens by enteric microorganisms under field conditions and to compare the pre-contact intestinal microbiome with the altered microbiome following contact with poultry raised in the field. Analysis of the intestinal virome from contact birds ("sentinels") placed on farms revealed colonization by members of the Picornaviridae, Picobirnaviridae, Reoviridae, and Astroviridae that were not present in pre-contact birds or present in proportionally lower numbers. Analysis of the sentinel gut bacterial community revealed an altered community in the post-contact birds, notably by members of the Lachnospiracea/Clostridium and Lactobacillus families and genera. Members of the avian enteric Reoviridae and Astroviridae have been well-characterized and have historically been implicated in poultry enteric disease; members of the Picobirnaviridae and Picornaviridae have only relatively recently been described in the poultry and avian gut, and their roles in the recognized disease syndromes and in poultry performance in general have not been determined. This metagenomic analysis has provided insight into the colonization of the poultry gut by enteric microbes circulating in commercial broiler flocks, and has identified enteric viruses and virus communities that warrant further study in order to understand their role(s) in avian gut health and disease.

P and M gene junction is the optimal insertion site in Newcastle disease virus vaccine vector for foreign gene expression.

Newcastle disease virus (NDV) has been developed as a vector for vaccine and gene therapy purposes. However, the optimal insertion site for foreign gene expression remained to be determined. In the present study, we inserted the green fluorescence protein (GFP) gene into five different intergenic regions of the enterotropic NDV VG/GA vaccine strain using reverse genetics technology. The rescued recombinant viruses retained lentogenic pathotype and displayed delayed growth dynamics, particularly when the GFP gene was inserted between the NP and P genes of the virus. The GFP mRNA level was most abundant when the gene was inserted closer to the 3' end and gradually decreased as the gene was inserted closer to the 5' end. Measurement of the GFP fluorescence intensity in recombinant virus-infected cells demonstrated that the non-coding region between the P and M genes is the optimal insertion site for foreign gene expression in the VG/GA vaccine vector.

Newcastle disease virus (NDV) recombinants expressing infectious laryngotracheitis virus (ILTV) glycoproteins gB and gD protect chickens against ILTV and NDV challenges.

UNLABELLED: Infectious laryngotracheitis (ILT) is a highly contagious acute respiratory disease of chickens caused by infectious laryngotracheitis virus (ILTV). The disease is controlled mainly through biosecurity and vaccination with live attenuated strains of ILTV and vectored vaccines based on turkey herpesvirus (HVT) and fowlpox virus (FPV). The current live attenuated vaccines (chicken embryo origin [CEO] and tissue culture origin [TCO]), although effective, can regain virulence, whereas HVT- and FPV-vectored ILTV vaccines are less efficacious than live attenuated vaccines. Therefore, there is a pressing need to develop safer and more efficacious ILTV vaccines. In the present study, we generated Newcastle disease virus (NDV) recombinants, based on the LaSota vaccine strain, expressing glycoproteins B (gB) and D (gD) of ILTV using reverse genetics technology. These recombinant viruses, rLS/ILTV-gB and rLS/ILTV-gD, were slightly attenuated in vivo yet retained growth dynamics, stability, and virus titers in vitro that were similar to those of the parental LaSota virus. Expression of ILTV gB and gD proteins in the recombinant virus-infected cells was detected by immunofluorescence assay. Vaccination of specific-pathogen-free chickens with these recombinant viruses conferred significant protection against virulent ILTV and velogenic NDV challenges. Immunization of commercial broilers with rLS/ILTV-gB provided a level of protection against clinical disease similar to that provided by the live attenuated commercial vaccines, with no decrease in body weight gains. The results of the study suggested that the rLS/ILTV-gB and -gD viruses are safe, stable, and effective bivalent vaccines that can be mass administered via aerosol or drinking water to large chicken populations. IMPORTANCE: This paper describes the development and evaluation of novel bivalent vaccines against chicken infectious laryngotracheitis (ILT) and Newcastle disease (ND), two of the most economically important infectious diseases of poultry. The current commercial ILT vaccines are either not safe or less effective. Therefore, there is a pressing need to develop safer and more efficacious ILT vaccines. In the present study, we generated Newcastle disease virus (NDV) recombinants expressing glycoproteins B (gB) and D (gD) of infectious laryngotracheitis virus (ILTV) using reverse genetics technology. These recombinant viruses were safe, stable, and immunogenic and replicated efficiently in birds. Vaccination of chickens with these recombinant viruses conferred complete protection against ILTV and NDV challenge. These novel bivalent vaccines can be mass administered via aerosol or drinking water to large chicken populations at low cost, which will have a direct impact on poultry health, fitness, and performance.

Molecular and phylogenetic analysis of a novel turkey-origin picobirnavirus.

A previous metagenomic analysis of the turkey gut RNA virus community identified novel enteric viruses that may play roles in poultry enteric diseases or in performance problems noted in the field. As part of the molecular characterization of these novel enteric viruses, a reverse transcriptase-PCR diagnostic assay was developed, targeting a novel turkey-origin picobirnavirus (PBV) initially identified in a pooled intestinal sample from turkey poults in North Carolina. Little detailed molecular information exists regarding the family Picobirnaviridae, particularly for the PBVs that have been described in avian species. This diagnostic assay targets the turkey PBV RNA-dependent RNA polymerase gene and produces an 1135-bp amplicon. This assay was validated using in vitro transcribed RNA and was tested using archived enteric samples collected from turkey flocks in the southeastern United States. Further, a phylogenetic analysis suggests the turkey PBV is unique because it does not group closely with the recognized PBV genogroups circulating in mammalian hosts.

Recent progress in the characterization of avian enteric viruses.

Despite the importance of the poultry gut, remarkably little is known about the complex gut microbial community. Enteric disease syndromes such as runting-stunting syndrome in broiler chickens and poult enteritis complex in young turkeys are difficult to characterize and reproduce in the laboratory. A great deal of work has been done to characterize the bacterial population in the poultry gut, leading to useful performance-based interventions such as direct-fed microbial preparations. Advances in the application of rapid molecular diagnostics and the advent of the next generation of nucleic acid sequencing have allowed researchers to begin to decipher the microbial community in complex environmental samples. Researchers have made great strides recently in placing names to some of the unknown and undescribed small viruses in the poultry gut such as parvoviruses, picornaviruses, picobirnavirus, and calicivirus. Investigation into the novel avian astroviruses continues, and recent progress has been made in the molecular characterization of the avian rotaviruses. This review will focus on the recent advances that have been made in the discovery, description, and characterization of the multitude of viruses that reside in the poultry gut.

Effects of the HN gene C-terminal extensions on the Newcastle disease virus virulence.

The hemagglutinin-neuraminidase (HN) of Newcastle disease virus (NDV) is a multifunctional protein that has receptor recognition, neuraminidase, and fusion promotion activities. Sequence analysis revealed that the HN gene of many extremely low virulence NDV strains encodes a larger open-reading frame (616 amino acids, aa) with additional 45 aa at its C-terminus when compared with that (571 aa) of virulent NDV strains. Therefore, it has been suspected that the 45 aa extension at the C-terminus of the HN may affect the NDV virulence. In this study, we generated an NDV mesogenic strain Anhinga-based recombinant virus with an HN C-terminal extension of 45 aa (rAnh-HN-ex virus) using reverse genetics technology. The biological characterization of the recombinant virus showed that the rAnh-HN-ex virus had similar growth ability to its parental virus rAnh-wt both in embryonating chicken eggs and DF-1 cells. However, the pathogenicity of this recombinant virus in embryonating chicken eggs and day-old chickens decreased, as evidenced by a longer mean death time and lower intracerebral pathogenicity index when compared with the parental virus. This is consistent with our previous finding that the recombinant LaSota virus with a 45-aa extension at its HN C-terminal was attenuated in chickens and embryonating eggs. These results suggest that the HN protein C-terminal extension may contribute to the reduced virulence in some low virulence NDV strains.

Application of the ligation-independent cloning (LIC) method for rapid construction of a minigenome rescue system for Newcastle disease virus VG/GA strain.

Newcastle disease virus (NDV) can cause serious diseases and substantial economic losses to the poultry industry. To gain a better understanding of NDV pathogenesis, several reverse genetics systems for different NDV strains have been established. However, the construction of infectious cDNA clone by conventional restriction digestion/ligation cloning methods is a time-consuming process and has many drawbacks by its nature. To address the problems, we employed a novel and robust ligation-independent cloning (LIC) method for efficient assembly of multiple DNA fragments. Using this method, we successfully generated a NDV minigenome construct within three weeks by assembling RT-PCR products of the VG/GA strain genomic termini and a cDNA coding for the green fluorescence protein (GFP), as a reporter, into a modified pBluescript vector. Co-transfection of the NDV minigenome with three supporting plasmids expressing the N, P, and L proteins into MVA-T7 infected HEp-2 cells and followed by infection with NDV VG/GA resulted in the minigenome replication, transcription, and packaging as evidenced by the reporter gene GFP expression. These results suggest that this LIC approach is a powerful tool for all sequence-independent DNA cloning and multi-DNA fragment assembly, which has a potential application for rapid development of gene therapy and recombinant vaccines.

Expression of chicken parvovirus VP2 in chicken embryo fibroblasts requires codon optimization for production of naked DNA and vectored meleagrid herpesvirus type 1 vaccines.

Meleagrid herpesvirus type 1 (MeHV-1) is an ideal vector for the expression of antigens from pathogenic avian organisms in order to generate vaccines. Chicken parvovirus (ChPV) is a widespread infectious virus that causes serious disease in chickens. It is one of the etiological agents largely suspected in causing Runting Stunting Syndrome (RSS) in chickens. Initial attempts to express the wild-type gene encoding the capsid protein VP2 of ChPV by insertion into the thymidine kinase gene of MeHV-1 were unsuccessful. However, transient expression of a codon-optimized synthetic VP2 gene cloned into the bicistronic vector pIRES2-Ds-Red2, could be demonstrated by immunocytochemical staining of transfected chicken embryo fibroblasts (CEFs). Red fluorescence could also be detected in these transfected cells since the red fluorescent protein gene is downstream from the internal ribosome entry site (IRES). Strikingly, fluorescence could not be demonstrated in cells transiently transfected with the bicistronic vector containing the wild-type or non-codon-optimized VP2 gene. Immunocytochemical staining of these cells also failed to demonstrate expression of wild-type VP2, indicating that the lack of expression was at the RNA level and the VP2 protein was not toxic to CEFs. Chickens vaccinated with a DNA vaccine consisting of the bicistronic vector containing the codon-optimized VP2 elicited a humoral immune response as measured by a VP2-specific ELISA. This VP2 codon-optimized bicistronic cassette was rescued into the MeHV-1 genome generating a vectored vaccine against ChPV disease.

Chicken parvovirus-induced runting-stunting syndrome in young broilers.

Previously we identified a novel parvovirus from enteric contents of chickens that were affected by enteric diseases. Comparative sequence analysis showed that the chicken parvovirus (ChPV) represented a new member in the Parvoviridae family. Here, we describe some of the pathogenic characteristics of ChPV in young broilers. Following experimental infection, 2-day-old broiler chickens showed characteristic signs of enteric disease. Runting-stunting syndrome (RSS) was observed in four of five experimental groups with significant growth retardation between 7 and 28 days postinoculation (DPI). Viral growth in small intestine and shedding was detected at early times postinoculation, which was followed by viremia and generalization of infection. ChPV could be detected in most of the major tissues for 3 to 4 wk postinoculation. Immunohistochemistry staining revealed parvovirus-positive cells in the duodenum of inoculated birds at 7 and 14 DPI. Our data indicate that ChPV alone induces RSS in broilers and is important determinant in the complex etiology of enteric diseases of poultry.

HN gene C-terminal extension of Newcastle disease virus is not the determinant of the enteric tropism.

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) plays an important role in virus pathogenicity and tissue tropism. Sequence analysis revealed that the HN gene of many asymptomatic enteric NDV strains encodes a larger open reading frame (616 amino acids, aa) with additional 39 aa at its C-terminus when compared with that (577 aa) of respirotropic NDV strains. Therefore, it has been suspected that the HN C-terminal extension may contribute to the enteric tropism. In the present study, we generated a NDV respirotropic strain LaSota-based recombinant virus with a HN C-terminal extension of 39 aa derived from an enterotropic NDV strain using reverse genetics technology. The biological characterization of the recombinant virus, rLS-HN-ex, showed that the HN C-terminal extension slightly attenuated the virus pathogenicity in embryonated eggs and in day-old chicks when compared to the parental LaSota virus. However, the HN C-terminal extension did not alter virus tissue tropism. In infected chickens, the recombinant virus was detected and re-isolated from the tracheal tissue, but not from the intestinal tissue, exhibiting a similar respirotropic tissue preference as its parental LaSota strain. These results demonstrated that the HN protein C-terminal extension of NDV is not the determinant of the virus enteric tropism.