Detection of Newcastle disease virus using nucleic acid sequence-based amplification.

Newcastle disease (ND) is a contagious and widespread avian disease affecting most species of birds. ND virus (NDV) is the only member of the avian paramyxovirus serotype 1 (APMV1) causing ND outbreak in bird flocks. The technique of nucleic acid sequence-based amplification (NASBA) is a potential method to rapidly and reliably detect NDV isolates. Here, we describe an effective and unprecedented method for detecting NDV strains of all pathotypes. A conserved region of the fusion protein gene was used for designing oligonucleotides specific to all NDV pathotypes. The dynamic range of this NDV NASBA detection method is comparable to virus culture and therefore the NDV NASBA method is a potential alternative for NDV screening and surveillance.     

4株鹅源新城疫病毒融合蛋白基因的克隆及序列分析

测定了4株鹅源新城疫病毒(NDV)融合蛋白(F)基因5’端1700核苷酸片段的序列,并由此推导了F蛋白氨基酸序列,并对鹅源NDV的基因型分类地位进行探讨。结果表明,4株病毒F基因的同源性大于97%,与DNV标准强毒株F48E8 F基因的同源性为860%～868%,F基因转录起始序列及起始密码子位置与已知NDV完全相同;F蛋白具有和已知NDV相似的各种功能区,F蛋白前体F0裂解位点附近的氨基酸序列为112RRQKRF117,符合NDV强毒株的特征。对F基因第334～1682位核苷酸之间3种限制性内切酶HinfⅠ、BstoⅠ\,\%Rsa\%Ⅰ酶切图谱的分析表明,4株病毒的基因型与文献报道的I～Ⅷ型有明显差异。     

Genotypic and pathotypic characterization of Newcastle disease viruses from India

Newcastle disease virus (NDV) is an avian paramyxovirus that causes significant economic losses to the poultry industry in most parts of the world. The susceptibility of a wide variety of avian species coupled with synanthropic bird reservoirs has contributed to the vast genomic diversity of this virus as well as diagnostic failures. Since the first panzootic in 1926, Newcastle disease (ND) became enzootic in India with recurrent outbreaks in multiple avian species. The genetic characteristics of circulating strains in India, however, are largely unknown. To understand the nature of NDV genotypes in India, we characterized two representative strains isolated 13 years apart from a chicken and a pigeon by complete genome sequence analysis and pathotyping. The viruses were characterized as velogenic by pathogenicity indices devised to distinguish these strains. The genome length was 15,186 nucleotides (nt) and consisted of six non-overlapping genes, with conserved and complementary 3' leader and 5' trailer regions, conserved gene starts, gene stops, and intergenic sequences similar to those in avian paramyxovirus 1 (APMV-1) strains. Matrix gene sequence analysis grouped the pigeon isolate with APMV-1 strains. Phylogeny based on the fusion (F), and hemagglutinin (HN) genes and complete genome sequence grouped these viruses into genotype IV. Genotype IV strains are considered to have "died out" after the first panzootic (1926-1960) of ND. But, our results suggest that there is persistence of genotype IV strains in India.     

Oncolytic Newcastle disease virus for cancer therapy: old challenges and new directions

Newcastle disease virus (NDV) is an avian paramyxovirus, which has been demonstrated to possess significant oncolytic activity against mammalian cancers. This review summarizes the research leading to the elucidation of the mechanisms of NDV-mediated oncolysis, as well as the development of novel oncolytic agents through the use of genetic engineering. Clinical trials utilizing NDV strains and NDV-based autologous tumor cell vaccines will expand our knowledge of these novel anticancer strategies and will ultimately result in the successful use of the virus in the clinical setting.     

Newcastle disease virus-vectored rabies vaccine is safe, highly immunogenic, and provides long-lasting protection in dogs and cats

Effective, safe, and affordable rabies vaccines are still being sought. Newcastle disease virus (NDV), an avian paramyxovirus, has shown promise as a vaccine vector for mammals. Here, we generated a recombinant avirulent NDV La Sota strain expressing the rabies virus glycoprotein (RVG) and evaluated its potential to serve as a vaccine against rabies. The recombinant virus, rL-RVG, retained its high-growth property in chicken eggs, with titers of up to 10^9.8 50% egg infective doses (EID₅₀)/ml of allantoic fluid. RVG expression enabled rL-RVG to spread from cell to cell in a rabies virus-like manner, and RVG was incorporated on the surface of the rL-RVG viral particle. RVG incorporation did not alter the trypsin-dependent infectivity of the NDV vector in mammalian cells. rL-RVG and La Sota NDV showed similar levels of sensitivity to a neutralization antibody against NDV and similar levels of resistance to a neutralization antibody against rabies virus. Animal studies demonstrated that rL-RVG is safe in several species, including cats and dogs, when administered as multiple high doses of recombinant vaccine. Intramuscular vaccination with rL-RVG induced a substantial rabies virus neutralization antibody response and provided complete protection from challenge with circulating rabies virus strains. Most importantly, rL-RVG induced strong and long-lasting protective neutralization antibody responses to rabies virus in dogs and cats. A low vaccine dose of 10^8.3 EID₅₀ completely protected dogs from challenge with a circulating strain of rabies virus for more than a year. This is the first study to demonstrate that immunization with an NDV-vectored vaccine can induce long-lasting, systemic protective immunity against rabies.     

Ticks Associated with Macquarie Island Penguins Carry Arboviruses from Four Genera

Macquarie Island, a small subantarctic island, is home to rockhopper, royal and king penguins, which are often infested with the globally distributed seabird tick, Ixodes uriae. A flavivirus, an orbivirus, a phlebovirus, and a nairovirus were isolated from these ticks and partial sequences obtained. The flavivirus was nearly identical to Gadgets Gully virus, isolated some 30 year previously, illustrating the remarkable genetic stability of this virus. The nearest relative to the orbivirus (for which we propose the name Sandy Bay virus) was the Scottish Broadhaven virus, and provided only the second available sequences from the Great Island orbivirus serogroup. The phlebovirus (for which we propose the name Catch-me-cave virus) and the previously isolated Precarious Point virus were distinct but related, with both showing homology with the Finnish Uukuniemi virus. These penguin viruses provided the second and third available sequences for the Uukuniemi group of phleboviruses. The nairovirus (for which we propose the name Finch Creek virus) was shown to be related to the North American Tillamook virus, the Asian Hazara virus and Nairobi sheep disease virus. Macquarie Island penguins thus harbour arboviruses from at least four of the seven arbovirus-containing genera, with related viruses often found in the northern hemisphere.     

Spectrum of Newcastle disease virus stability in gradients of temperature and pH

Newcastle disease (ND) is one of the highly pathogenic viral diseases of avian species. The disease is endemic in many developing countries where agriculture serves as the primary source of national income. Newcastle disease virus (NDV) belongs to the family Paramyxoviridae and is well characterized member among the avian paramyxovirus serotypes. The failure of vaccination is one of the major causes of NDV outbreaks in field condition. The present study gives a brief picture about the biology of NDV genome and its proteins under different conditions of temperature and pH. Our results indicate that the NDV is non-infective above 42 °C and unstable above 72 °C. The study will be useful in defining an optimum storage condition for NDV without causing any deterioration in its viability.     

Recombinant newcastle disease virus expressing a foreign viral antigen is attenuated and highly immunogenic in primates

Paramyxoviruses such as human parainfluenza viruses that bear inserts encoding protective antigens of heterologous viruses can induce an effective immunity against the heterologous viruses in experimental animals. However, vectors based on common human pathogens would be expected to be restricted in replication in the adult human population due to high seroprevalence, an effect that would reduce vector immunogenicity. To address this issue, we evaluated Newcastle disease virus (NDV), an avian paramyxovirus that is serotypically distinct from common human pathogens, as a vaccine vector. Two strains were evaluated: the attenuated vaccine strain LaSota (NDV-LS) that replicates mostly in the chicken respiratory tract and the Beaudette C (NDV-BC) strain of intermediate virulence that produces mild systemic infection in chickens. A recombinant version of each virus was modified by the insertion, between the P and M genes, of a gene cassette encoding the human parainfluenza virus type 3 (HPIV3) hemagglutinin-neuraminidase (HN) protein, a test antigen with considerable historic data. The recombinant viruses were administered to African green monkeys (NDV-BC and NDV-LS) and rhesus monkeys (NDV-BC only) by combined intranasal and intratracheal routes at a dose of 10(6.5) PFU per site, with a second equivalent dose administered 28 days later. Little or no virus shedding was detected in nose-throat swabs or tracheal lavages following immunization with either strain. In a separate experiment, direct examination of lung tissue confirmed a highly attenuated, restricted pattern of replication by parental NDV-BC. The serum antibody response to the foreign HN protein induced by the first immunization with either NDV vector was somewhat less than that observed following a wild-type HPIV3 infection; however, the titer following the second dose exceeded that observed with HPIV3 infection, even though HPIV3 replicates much more efficiently than NDV in these animals. NDV appears to be a promising vector for the development of vaccines for humans; one application would be in controlling localized outbreaks of emerging pathogens.     

Strain variation and nuclear association of Newcastle disease virus matrix protein.

Five monoclonal antibodies to the matrix (M) protein of Newcastle disease virus (NDV) Australia-Victoria (AV) strain were generated and characterized. In competitive antibody-binding assays, the antibodies fell into three discrete groups. The antigenic sites described by these antibody groups were designated M1, M2, and M3. Each antibody reacted with a panel of NDV strains in a manner unique to its group, confirming the grouping by competitive antibody binding. Only site M1 was found on all 12 of the strains tested and may be a "pan-NDV" epitope. A large portion of the M protein of strain AV was detected in the nuclei of infected cells by all five monoclonal antibodies. In addition, the antibodies only stained the nuclei of cells infected with NDV strains expressing M protein containing the corresponding antigenic site. These results confirm that the immunoreactivity in the nucleus is actually caused by the M protein and not by a cross-reacting host protein induced by viral infection.     

Hematology, serum chemistry, and serology of Galápagos penguins (Spheniscus mendiculus) in the Galápagos Islands, Ecuador

The Galápagos penguin (Spheniscus mendiculus) is an endangered species endemic to the Galápagos Islands, Ecuador. In 2003 and 2004, 195 penguins from 13 colonies on the islands of Isabela and Fernandina in the Galápagos archipelago were examined. Genetic sexing of 157 penguins revealed 62 females and 95 males. Hematology consisted of packed cell volume (n = 134), white blood cell differentials (n = 83), and hemoparasite blood smear evaluation (n = 114). Microfilariae were detected in 22% (25/114) of the blood smears. Female penguins had significantly higher eosinophil counts than males. Serum chemistry on 83 penguins revealed no significant differences between males and females. Birds were seronegative to avian paramyxovirus type 1-3, avian influenza virus, infectious bursal disease virus, Marek's disease virus (herpes), reovirus, avian encephalomyelitis virus, and avian adenovirus type 1 and 2 (n = 75), as well as to West Nile virus (n = 87), and Venezuelan, western and eastern equine encephalitis viruses (n = 26). Seventy-five of 84 (89%) penguins had antibodies to Chlamydophila psittaci but chlamydial DNA was not detected via polymerase chain reaction in samples from 30 birds.     

Pasteurella multocida infections in rockhopper penguins (Eudyptes chrysocome) from Campbell Island, New Zealand

During an investigation into the population decline of rockhopper penguins (Eudyptes chrysocome) on Campbell Island, New Zealand, avian cholera (Pasteurella multocida) was found in dead adults and chicks. An RNA enveloped virus was isolated from Ixodes uriae, a tick which commonly parasitizes rockhopper penguins on the island. It is not known whether this virus is virulent for penguins. No evidence was obtained to suggest that avian cholera was the principal cause for the decline in the rockhopper penguin population.     

Incorporation of Host Complement Regulatory Proteins into Newcastle Disease Virus Enhances Complement Evasion

Newcastle disease virus (NDV), an avian paramyxovirus, is inherently tumor selective and is currently being considered as a clinical oncolytic virus and vaccine vector. In this study, we analyzed the effect of complement on the neutralization of NDV purified from embryonated chicken eggs, a common source for virus production. Fresh normal human serum (NHS) neutralized NDV by multiple pathways of complement activation, independent of neutralizing antibodies. Neutralization was associated with C3 deposition and the activation of C2, C3, C4, and C5 components. Interestingly, NDV grown in mammalian cell lines was resistant to complement neutralization by NHS. To confirm whether the incorporation of regulators of complement activity (RCA) into the viral envelope afforded complement resistance, we grew NDV in CHO cells stably transfected with CD46 or HeLa cells, which strongly express CD46 and CD55. NDV grown in RCA-expressing cells was resistant to complement by incorporating CD46 and CD55 on virions. Mammalian CD46 and CD55 molecules on virions exhibited homologous restriction, since chicken sera devoid of neutralizing antibodies to NDV were able to effectively neutralize these virions. The incorporation of chicken RCA into NDV produced in embryonated eggs similarly provided species specificity toward chicken sera.     

禽呼肠孤病毒感染对鸡法氏囊及免疫应答的影响

研究LY株禽呼肠孤病毒(ARV)感染1日龄SPF鸡后对法氏囊发育影响,对传染性法氏囊病毒(IBDV)、禽流感病毒(AIV)、新城疫病毒(NDV)疫苗免疫诱发的抗体的影响,及对强毒株IBDV致病作用的影响。结果表明,LY株ARV感染1日龄SPF鸡可引起法氏囊萎缩和部分淋巴细胞减少,但对增重及AIV和NDV疫苗免疫后抗体滴度却没有显著影响。ARV感染可降低弱毒IBDV疫苗免疫后的抗体反应,但对随后IBDV强毒株攻毒的抵抗力却与对照鸡无显著差异。经IBDV弱毒疫苗免疫后,再接种强毒株IBDV,不会引起死亡,但却仍能显著抑制对AIV、NDV疫苗免疫后的抗体滴度。然而,对于1~7日龄经ARV感染的鸡,IBDV强毒的这种免疫抑制作用又显著低于未经ARV感染的对照鸡。     

Assessment of newcastle disease vaccination of houbara bustard breeders (Chlamydotis undulata undulata)

The houbara bustard (Chlamydotis undulata undulata) is endangered in North Africa. Through a captive-breeding program established in Morocco by The Emirates Center for Wildlife Propagation, wild populations are being supplemented by the releasing of captive-reared birds. Newcastle disease, which is caused by Newcastle disease virus (NDV; Avian paramyxovirus type 1), can infect houbara bustards and is a significant threat through contact with backyard poultry and possibly wild birds. Three vaccination schedules for Newcastle disease were evaluated by serologic monitoring to assess the efficiency and safety of various types of vaccines (live vs. inactivated), vaccine strains (Hitchner B1 and Clone 30), and administration routes (intranasal vs. injection). We evaluated antibody titers in 211 adult houbara bustards for 10 mo. Antibody titers to NDV in both sera and egg yolks were monitored by hemagglutination inhibition test. The inactivated vaccine provided a high, homogeneous, and durable serologic response in breeders; titers were higher than log2 11 after 4 wk and remained higher than log2 7 after 10 mo. The response to the two live vaccines was similar, and antibody titers did not exceed log2 6 at sero-conversion. Maternally derived antibodies were efficiently transmitted in vitellus, further confirming that offspring of females hyperimmunized with the inactivated vaccine received high titers of maternal antibodies.     

Newcastle disease virus exerts oncolysis by both intrinsic and extrinsic caspase-dependent pathways of cell death

Newcastle disease virus (NDV), an avian paramyxovirus, is tumor selective and intrinsically oncolytic. Here, we present evidence that genetically modified, recombinant NDV strains are cytotoxic to human tumor cell lines of ecto-, endo-, and mesodermal origin. We show that cytotoxicity against tumor cells is due to multiple caspase-dependent pathways of apoptosis independent of interferon signaling competence. The signaling pathways of NDV-induced, cancer cell-selective apoptosis are not well understood. We demonstrate that NDV triggers apoptosis by activating the mitochondrial/intrinsic pathway and that it acts independently of the death receptor/extrinsic pathway. Caspase-8-methylated SH-SY5Y neuroblastoma cells are as sensitive to NDV as other caspase-8-competent cells. This demonstrates that NDV is likely to act primarily through the mitochondrial death pathway. NDV infection results in the loss of mitochondrial membrane potential and the subsequent release of the mitochondrial protein cytochrome c, but the second mitochondrion-derived activator of caspase (Smac/DIABLO) is not released. In addition, we describe early activation of caspase-9 and caspase-3. In contrast, cleavage of caspase-8, which is predominantly activated by the death receptor pathway, is a TNF-related, apoptosis-inducing ligand (TRAIL)-induced late event in NDV-mediated apoptosis of tumor cells. Our data, therefore, indicate that the death signal(s) generated by NDV in tumor cells ultimately converges at the mitochondria and that it acts independently of the death receptor pathway. Our cytotoxicity studies demonstrate that recombinant NDV could be developed as a cancer virotherapy agent, either alone or in combination with therapeutic transgenes. We have also shown that trackable oncolytic NDV could be developed without any reduction in oncolytic efficacy.     

Differentiation of velogenic, mesogenic and lentogenic strains of Newcastle disease virus by multiplex RT-PCR

A multiplex RT‐PCR technique has been developed for differentiation of velogenic, mesogenic and lentogenic pathotypes of Newcastle disease virus (NDV), using a set of three oligonucleotide primers designed from NDV genomic RNA (P1, P2 and P3). The primer pair P1 and P2 generated a RT‐PCR product of 204 bp, only with RNA from velogenic and mesogenic strains, whereas the P1 and P3 generated a 364 bp product only with RNA from mesogenic and lentogenic strains. Thirty four NDV strains, including some reference strains (known pathotypes), NDV field isolates and NDV vaccine strains, as well as other avian virus strains, were tested with multiplex RT‐PCR. All reference strains tested were differentiated in agreement with their intracerebral pathogenicity index (ICPI) values or with the pathotypes known in previous reports. The nucleotide sequence analysis of RT‐PCR products for four NDV strains was fully in agreement with the RT‐PCR characterisations of these strains. The RT‐PCR results of other avian RNA viruses further confirmed the reliability and specificity of this technique. However, the RT‐PCR failed to detect some other avian NDV, which may not originate from chicken. This multiplex RT‐PCR technique is simple and easy to perform. It could be applied not only to determine the origin of NDV, but also may be used diagnostically in molecular epidemiological analysis of ND and for prediction of pathotypes of NDV isolates.     

Molecular surveillance for avian influenza A virus in king penguins (<Emphasis Type="Italic">Aptenodytes patagonicus</Emphasis>)

An investigation of the presence of influenza A virus has been conducted in king penguins (Aptenodytes patagonicus) at the Possession Island in the Crozet Archipelago, Antarctica, using a rapid molecular diagnostic method based on real-time polymerase chain reaction. No evidence of outbreak or positive viral infection of influenza A virus was found in this study. We however recommend the implementation of long-term surveillance in seabird populations of polar ecosystems to detect the potential introduction of exotic strains and potential existence of a local epidemiological cycle for avian influenza viruses.     

Roles of the fusion and hemagglutinin-neuraminidase proteins in replication, tropism, and pathogenicity of avian paramyxoviruses

Virulent and moderately virulent strains of Newcastle disease virus (NDV), representing avian paramyxovirus serotype 1 (APMV-1), cause respiratory and neurological disease in chickens and other species of birds. In contrast, APMV-2 is avirulent in chickens. We investigated the role of the fusion (F) and hemagglutinin-neuraminidase (HN) envelope glycoproteins in these contrasting phenotypes by designing chimeric viruses in which the F and HN glycoproteins or their ectodomains were exchanged individually or together between the moderately virulent, neurotropic NDV strain Beaudette C (BC) and the avirulent APMV-2 strain Yucaipa. When we attempted to exchange the complete F and HN glycoproteins individually and together between the two viruses, the only construct that could be recovered was recombinant APMV-2 strain Yucaipa (rAPMV-2), containing the NDV F glycoprotein in place of its own. This substitution of NDV F into APMV-2 was sufficient to confer the neurotropic, neuroinvasive, and neurovirulent phenotypes, in spite of all being at reduced levels compared to what was seen for NDV-BC. When the ectodomains of F and HN were exchanged individually and together, two constructs could be recovered: NDV, containing both the F and HN ectodomains of APMV-2; and APMV-2, containing both ectodomains of NDV. This supported the idea that homologous cytoplasmic tails and matched F and HN ectodomains are important for virus replication. Analysis of these viruses for replication in vitro, syncytium formation, mean embryo death time, intracerebral pathogenicity index, and replication and tropism in 1-day-old chicks and 2-week-old chickens showed that the two contrasting phenotypes of NDV and APMV-2 could largely be transferred between the two backbones by transfer of homotypic F and HN ectodomains. Further analysis provided evidence that the homologous stalk domain of NDV HN is essential for virus replication, while the globular head domain of NDV HN could be replaced with that of APMV-2 with only a minimal attenuating effect. These results demonstrate that the F and HN ectodomains together determine the cell fusion, tropism, and virulence phenotypes of NDV and APMV-2 and that the regions of HN that are critical to replication and the species-specific phenotypes include the cytoplasmic tail and stalk domain but not the globular head domain.     

Thiol/disulfide exchange is required for membrane fusion directed by the Newcastle disease virus fusion protein

Newcastle disease virus (NDV), an avian paramyxovirus, initiates infection with attachment of the viral hemagglutinin-neuraminidase (HN) protein to sialic acid-containing receptors, followed by fusion of viral and cell membranes, which is mediated by the fusion (F) protein. Like all class 1 viral fusion proteins, the paramyxovirus F protein is thought to undergo dramatic conformational changes upon activation. How the F protein accomplishes extensive conformational rearrangements is unclear. Since several viral fusion proteins undergo disulfide bond rearrangement during entry, we asked if similar rearrangements occur in NDV proteins during entry. We found that inhibitors of cell surface thiol/disulfide isomerase activity--5'5-dithio-bis(2-nitrobenzoic acid) (DTNB), bacitracin, and anti-protein disulfide isomerase antibody--inhibited cell-cell fusion and virus entry but had no effect on cell viability, glycoprotein surface expression, or HN protein attachment or neuraminidase activities. These inhibitors altered the conformation of surface-expressed F protein, as detected by conformation-sensitive antibodies. Using biotin maleimide (MPB), a reagent that binds to free thiols, free thiols were detected on surface-expressed F protein, but not HN protein. The inhibitors DTNB and bacitracin blocked the detection of these free thiols. Furthermore, MPB binding inhibited cell-cell fusion. Taken together, our results suggest that one or several disulfide bonds in cell surface F protein are reduced by the protein disulfide isomerase family of isomerases and that F protein exists as a mixture of oxidized and reduced forms. In the presence of HN protein, only the reduced form may proceed to refold into additional intermediates, leading to the fusion of membranes.     

LTR point mutations in the Tax-responsive elements of HTLV-1 isolates from HIV/HTLV-1-coinfected patients

Background

Newcastle Disease Virus (NDV) has been considered to only infect avian species. However, one paramyxovirus named as Xiny10 was isolated from swine. The differences of Xiny10, another previous swine NDV (JL01) and vaccine strain La Sota were compared on the basis of sequences of the whole-lengthen Fusion (F) gene and biological characteristics.

Findings

Through serologic tests and sequence alignment, Xiny10 was proved as NDV. It has great differences with JL01 in virulence, biological characteristics, genotype and amino acid homology of F gene. The sequence alignment showed Xiny10 and La Sota both belonged to genotype II. It shared 97.3% to 98.7% identities with genotype II NDVs, which was higher than these strains from the other genotypes.

Conclusions

These above data suggested that the swine virus was NDV and it might be generated from La Sota.