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Infectious laryngotracheitis (ILT) is a highly contagious acute respiratory disease of chickens caused by infectious laryngotracheitis virus (ILTV). Currently, modified live ILTV vaccines are used to control ILT infections. However, the live ILTV vaccines can revert to virulence after bird-to-bird passage and are capable of establishing latent infections, suggesting the need to develop safer vaccines against ILT. We have evaluated the role of three major ILTV surface glycoproteins, namely, gB, gC, and gD in protection and immunity against ILTV infection in chickens. Using reverse genetics approach, three recombinant Newcastle disease viruses (rNDVs) designated rNDV gB, rNDV gC, and rNDV gD were generated, each expressing gB, gC, and gD, respectively, of ILTV. Chickens received two immunizations with rNDVs alone (gB, gC, and gD) or in combination (gB+gC, gB+gD, gC+gD, and gB+gC+gD). Immunization with rNDV gD induced detectable levels of neutralizing antibodies with the magnitude of response greater than the rest of the experimental groups including those vaccinated with commercially available vaccines. The birds immunized with rNDV gD showed complete protection against virulent ILTV challenge. The birds immunized with rNDV gC alone or multivalent vaccines consisting of combination of rNDVs displayed partial protection with minimal disease and reduced replication of challenge virus in trachea. Immunization with rNDV gB neither reduced the severity of the disease nor the replication of challenge virus in trachea. The superior protective efficacy of rNDV gD vaccine compared to rNDV gB or rNDV gC vaccine was attributed to the higher levels of envelope incorporation and infected cell surface expression of gD than gB or gC. Our results suggest that rNDV expressing gD is a safe and effective bivalent vaccine against NDV and ILTV., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The role of N-linked glycosylation of the Newcastle disease virus (NDV) fusion (F) protein in viral replication and pathogenesis was examined by eliminating potential acceptor sites using a reverse genetics system for the moderately pathogenic strain Beaudette C (BC). The NDV-BC F protein contains six potential acceptor sites for N-linked glycosylation at residues 85, 191, 366, 447, 471, and 541 (sites Ng1 to Ng6, respectively). The sites at Ng2 and Ng5 are present in heptad repeat (HR) domains HR1 and HR2, respectively, and thus might affect fusion. Each N-glycosylation site was eliminated individually by replacing asparagine (N) with glutamine (Q), and a double mutant (Ng2 + 5) involving the two HR domains was also made. Each mutant was successfully recovered by reverse genetics except for the one involving Ng6, which is present in the cytoplasmic domain. All of the F proteins expressed by the recovered mutant viruses were efficiently cleaved and transported to the infected-cell surface. None of the individual mutations affected viral fusogenicity, but the double mutation at Ng2 and Ng5 in HR1 and HR2 increased fusogenicity >12-fold. The single mutations at sites Ng1, Ng2, and Ng5 resulted in modestly reduced multicycle growth in vitro. These three single mutations were also the most attenuating in eggs and 1-day-old chicks and were associated with decreased replication and spread in 2-week-old chickens. In contrast, the combination of the mutations at Ng2 and Ng5 yielded a virus that, compared to the BC parent, replicated >100-fold more efficiently in vitro, was more virulent in eggs and chicks, replicated more efficiently in chickens with enhanced tropism for the brain and gut, and elicited stronger humoral cell responses. These results illustrate the effects of N-glycosylation of the F protein on NDV pathobiology and suggest that the N-glycans in HR1 and HR2 coordinately downregulate viral fusion and virulence.

The nine serotypes of avian paramyxoviruses (APMVs) are frequently isolated from domestic and wild birds worldwide. APMV-1, also called Newcastle disease virus, was shown to be attenuated in non-avian species and is being developed as a potential vector for human vaccines. In the present study, we extended this evaluation to the other eight serotypes by evaluating infection in BALB/c mice. Mice were inoculated intranasally with a prototype strain of each of the nine serotypes and monitored for clinical disease, gross pathology, histopathology, virus replication and viral antigen distribution, and seroconversion. On the basis of multiple criteria, each of the APMV serotypes except serotype 5 was found to replicate in mice. Five of the serotypes produced clinical disease and significant weight loss in the following order of severity: 1, 2>6, 9>7. However, disease was short-lived. The other serotypes produced no evident clinical disease. Replication of all of the APMVs except APMV-5 in the nasal turbinates and lungs was confirmed by the recovery of infectious virus and by substantial expression of viral antigen in the epithelial lining detected by immunohistochemistry. Trace levels of infectious APMV-4 and -9 were detected in the brain of some animals; otherwise, no virus was detected in the brain, small intestine, kidney, or spleen. Histologically, infection with the APMVs resulted in lung lesions consistent with broncho-interstitial pneumonia of varying severity that were completely resolved at 14 days post infection. All of the mice infected with the APMVs except APMV-5 produced serotype-specific HI serum antibodies, confirming a lack of replication of APMV-5. Taken together, these results demonstrate that all APMV serotypes except APMV-5 are capable of replicating in mice with minimal disease and pathology.

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is a multifunctional protein that plays a crucial role in virus infectivity. In this study, using the mesogenic strain Beaudette C (BC), we mutated three conserved amino acids thought to be part of the binding/catalytic active site in the HN protein. We also mutated five additional residues near the proposed active site that are nonconserved between BC and the avirulent strain LaSota. The eight recovered NDV HN mutants were assessed for effects on biological activities. While most of the mutations had surprisingly little effect, mutation at conserved residue Y526 reduced the neuraminidase, receptor binding, and fusion activities and attenuated viral virulence in eggs and young birds.

Newcastle disease virus (NDV) expressing HIV-1 BaL gp160 was evaluated either alone or with monomeric BaL gp120 and BaL SOSIP gp140 protein in a prime-boost combination in guinea pigs to enhance envelope (Env)-specific humoral and mucosal immune responses. We showed that a regimen consisting of an NDV prime followed by a protein boost elicited stronger serum and mucosal Th-1-biased IgG responses and neutralizing antibody responses than NDV-only immunizations. Additionally, these responses were higher after the gp120 than after the SOSIP gp140 protein boost. [ABSTRACT FROM AUTHOR]

Infectious bronchitis virus (IBV) causes a major disease problem for the poultry industry worldwide. The currently used live-attenuated vaccines have the tendency to mutate and/or recombine with circulating field strains resulting in the emergence of vaccine-derived variant viruses. In order to circumvent these issues, and to develop a vaccine that is more relevant to Egypt and its neighboring countries, a recombinant avirulent Newcastle disease virus (rNDV) strain LaSota was constructed to express the codon-optimized S glycoprotein of the Egyptian IBV variant strain IBV/Ck/EG/CU/4/2014 belonging to GI-23 lineage, that is prevalent in Egypt and in the Middle East. A wild type and two modified versions of the IBV S protein were expressed individually by rNDV. A high level of S protein expression was detected in vitro by Western blot and immunofluorescence analyses. All rNDV-vectored IBV vaccine candidates were genetically stable, slightly attenuated and showed growth patterns comparable to that of parental rLaSota virus. Single-dose vaccination of 1-day-old SPF White Leghorn chicks with the rNDVs expressing IBV S protein provided significant protection against clinical disease after IBV challenge but did not show reduction in tracheal viral shedding. Single-dose vaccination also provided complete protection against virulent NDV challenge. However, prime-boost vaccination using rNDV expressing the wild type IBV S protein provided better protection, after IBV challenge, against clinical signs and significantly reduced tracheal viral shedding. These results indicate that the NDV-vectored IBV vaccines are promising bivalent vaccine candidates to control both infectious bronchitis and Newcastle disease in Egypt. [ABSTRACT FROM AUTHOR]