ND belongs to the family Paramyxoviridae as the species Orthoavulavirus1 of birds within the genus Orthoavulavirus (Rima et al., 2019). Six open reading frames (ORFs) in its genome encode six major structural proteins: hemagglutinin-neuraminidase (HN), matrix protein (M), splicing protein (F), nucleoprotein (NP), and RNA-dependent RNA polymerase (L) (Ganar et al., 2014).
Definition of Newcastle Disease
Viral Agent and Ecology
In addition, several species of wild birds have been found to harbor APMV-1 strains with lower virulence, which may or may not cause clinical disease (Rosenberger et al., 1974; Wobeser et al., 1993). There is evidence of viral transmission at the poultry-wild bird interface, even though some viruses may be otherwise maintained in wild or domestic bird reservoirs (Kim et al., 2007a; Cardenas Garcia et al., 2013 ).
Pathogenicity
However, it is not clear whether these viruses are the result of transmission from poultry or are viruses regularly maintained in wild and domestic birds (Senne et al., 1983;. The potential pathogenicity of APMV-1 strains can be predicted from the nucleotide sequence of the cleavage site of the fusion gene , according to genetic analysis (Glickman et al., 1988; Collins et al., 1993).
History and Classification
At the site where the fusion protein cleaves, virulent APMV-1 strains have three or more basic amino acids (arginine or lysine) at positions 113 to 116 and a phenylalanine at position 117, while low virulence APMV-1 strains have less than have two amino acids. basicly derived amino acid residues and a leucine at position 117 (Glickman et al., 1988). According to a recent summary, NDV has been considered a useful laboratory virus for replication and virulence studies since the 1970s (Alexander et al., 2012).
Epidemiology and Emergence of the Virulent NDV........................................6-7
It appears that one of the main reservoirs for the establishment of virulent NDV in poultry are lentogenic strains transmitted mainly by wild birds (Ayala et al., 2016). However, continued lentogenic NDV replication in chickens represents a possible risk factor for the development of virulent NDV.
Pathotypes and Pathotyping of NDV
The M protein, which is located directly beneath the viral envelope and is known to assist in the packaging and release of freshly assembled viruses, is known to maintain the structure of the virus (Battisti et al., 2012) . During the infectious cycle, the L protein is an RNA-dependent RNA polymerase that functions as the viral replicase and transcriptase, while the P protein is a cofactor of the polymerase (Dortmans et al., 2010).
Genotypes of NDV..........................................................................................9-13
- Diagnostic Dilemma................................................................................13-14
- Differential Diagnosis
- Virus Isolation
- Serological Diagnosis
According to some in vivo pathogenicity evaluation procedures, NDV isolates can also be categorized as velogenic (extremely virulent), mesogenic (moderately virulent), and lentogenic (non-virulent) pathotypes (Brown et al., 1999). 14 being the most reliable pathogenicity test recommended by the OIE, may not accurately reflect the true virulence of the virus (Dortmans et al., 2011a; 2011b). The chemistry of the F cleavage site is the only factor that can be used to predict the virulence potential of NDV at the molecular level (Panda et al., 2004).
Five different types of ND are recognized based on the clinical and pathological findings (Marks et al., 2014). However, Purkinje fiber necrosis and perivascular cuffing are often seen on histology (Banerjee et al., 1994). Other primary cell cultures that are highly permissive to NDV include avian myeloblasts (QM5), chick embryo fibroblasts (CEF), DF-1, chick embryo kidney (CEK), chick embryo liver (CEL) and chick embryo kidney (CEK) ( McGinnes et al., 2006b ).
Molecular Based Assays
Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
These monoclonal antibody (Mab)-based ELISAs may not be able to detect specific strains of NDV that may have some mutation in the single epitope against which the monoclonal antibody was generated, in addition to being expensive and inappropriate in the field.
Quantitative Polymerase Chain Reaction (qPCR)
19 previously undiscovered NDV isolates but also cooperate with matrix gene analysis (Kim et al., 2008a). A qPCR assay based on the F gene was developed for pathotyping of NDV to distinguish between virulent NDV strains and viruses of low virulence (Aldous et al., 2001; . Farkas et al., 2009). With the exception of a few isolates with nucleotide changes in the probe binding sites, this assay, which was developed to identify NDV isolates in the United States, has proven to be effective in identifying most isolates worldwide (Terregino et al., 2003).
Notably, when a new probe that accounted for the above nucleotide variations was developed and tested, all isolates that had previously escaped detection by the previous F gene assay were identified (Kim et al., 2008b). The qPCR method can also be used to measure viral load in different organs (Niesters, 2001) or virus shedding from immunized animals after infection with a highly infectious strain of NDV (Rasoli et al., 2014; Roohani et al., 2015). Dilution endpoints such as egg median infective dose (EID50) and tissue culture mean infective dose (TCID50) or plaque assay are common techniques to measure viral shedding (Hu et al., 2011).
Sequencing of NDV genes.....................................................................19-20
The F protein cleavage site and HN protein both contribute to pathogenicity (Huang et al. 2004; de Leeuw et al. 2005). The fusion of the viral surface with the host cell membrane is caused by the F protein (Munir et al., 2012). Recent examination of whole genome sequencing of viral isolates and reverse genetics has shown that the fusion protein cleavage site (FPCS), covering amino acids 112 to 117 of the F protein, cannot confer virulence to an otherwise avirulent strain (Rout and Samal, 2008; Khattar et al., 2009).
Infections with low-virulence strains of ND are restricted to the mucosal tissues of the host, which are the only sites where trypsin-like enzymes can replicate (Samadi et al., 2014). The amino acid sequence of the FO protein cleavage site, which confers virulence, is used as a molecular marker of virulence in NDVs (Fuller et al., 2007). To study the phylogeny and molecular epidemiology of NDVs, the research community has agreed that the F and HN protein genes can be used (Xu et al., 2008).
Samples, Sample Collection sites and Sample Transportation
First, 150 g of brain heart infusion broth (BHIB) (Oxoid, UK) was completely dissolved in 1L of distilled water with progressive stirring and heating, autoclaved at 121℃ for 15 min, and then cooled to room temperature.
Isolation and Propagation of NDV in Embryonated Chicken Eggs…
- Collection of Embryonated Chicken Eggs
- Preparation of Viral Inoculum
- Inoculation of Embryonated Eggs
- Harvesting of Allantoic Fluid
- Collection Procedures of Allantoic Fluid
The eggs with dead embryo within 24 hours after inoculation were considered as having non-specific causes (bacterial contamination, hemorrhage, traumatic injury) and discarded. A biosafety cabinet was used to collect allantoic fluid from embryonated eggs after 3–4 days of inoculation. All eggs were cooled at 4°C to reduce contamination of the allantoic fluid with blood during harvest.
The tongs and spoons were dipped in the disinfectant and flamed by spirit lamp; then the eggshell above the airspace was removed with forceps and allantoic fluid was collected.
Hemagglutination (HA) and Hemagglutination Inhibition (HI) Test
Preparation of 1% Chicken RBC Suspension
Hemagglutination (HA) Test..................................................................24-25
25 were placed in the first well and a two-fold dilution of 50 µL volume of the viral suspension was made over the plate (Figure 3.4). Then we also made a control type using the same procedure only using PBS instead of allantoic fluid. The plate was then kept at room temperature for about 30 minutes so that the erythrocytes could calm down.
The collected serum was placed in the first well of the plate and two-fold dilutions were made across the plate. Finally, 50 µL of 1% chicken erythrocyte suspension was dispensed into each well and mixed by gently tapping the plate. Again, the plate was kept at room temperature for about 30 minutes to allow the erythrocytes to settle.
Molecular Identification
Viral RNA Extraction.............................................................................25-26
A total of 12 ml of 100% isopropanol was added to wash solution 1, mixed and kept at room temperature. Then 60 µL of the sample (allantoic fluid) to be tested and 156 µL of lysis/binding solution were also added to the first well. Afterwards, the results of the samples were validated and interpreted according to the threshold level of Cq (Table 3.2).
28 Positive control Cq values for both NDV in the FAM channel and virulence factor in the Texas Red channel were set between 22.0–33.0. Then, a Cq value below 22.0 or above 33.0 assays was considered invalid for both NDV and virulence factor. In the negative control well, the Cq value for NDV and virulence factor was set to N/A or above 38, and the internal control Cq value was set below 34 for the CY-5 channel.
Phylogenetic Analysis...................................................................................30-32
For this 5 µL of PCR product together with 1 µL of 6X DNA loading dye (Thermo Fisher Scientific, USA) were mixed and loaded on 1% agarose gel. Among the positive sample, thirteen were randomly selected for partial fusion protein (F) gene sequencing. List of sequences used to construct the phylogenetic tree (BD= . Bangladesh; V= Virulent; LV= Low Virulent).
Cloacal and oropharyngeal swab samples from backyard chickens were inoculated into 9-10 day old embryonated chicken eggs and HA and HI tests were used to identify NDV. Out of 1282 samples, 119 samples were found positive for HA test (Table 4.1) indicating the presence of any hemagglutinating viruses. Of these 119 samples, 55 were found to be HI positive indicating that suspected NDV was present in those samples (Table 4.1).
Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Test
Differentiating Virulent and Nonvirulent NDV by real-time Reverse
Characterization of non-virulent NDV strains by partial F gene sequencing Partial nucleotide sequences of F gene were analyzed by BLAST to find the corresponding protein (amino acid) sequences. The obtained protein sequences were then compared with NDV fusion (F) protein sequences of F strain (BCRDV, accession no. KC987036.1) and M strain (RDV, accession no. JF950509.1) obtained from the GenBank database of NCBI. All sequences were aligned using Molecular Evolution Genetics Analysis (MEGA) (Version 11) program and amino acid sequence no.
Amino acid F (phenylalanine) at position 117 indicates virulent strains of NDV and L (leucine) at 117 indicates nonvirulent strains of NDV. Therefore, the isolated strains can be categorized as non-virulent NDV as stated by Alexander Lee et al. Phylogenetic analysis to explore the genetic relatedness of NDV isolates The phylogenetic tree was constructed by obtaining all avian F gene sequences.
Phylogenetic Analysis to Explore Genetic Relatedness of Isolated NDV
Detection of Newcastle disease virus RNA in infected allantoic fluids by in vitro enzymatic amplification (PCR). Biological and molecular characterization of field isolates of Newcastle Disease Virus (NDV) with comparisons to reference NDV strains. Genomic characterization of the first class I Newcastle disease virus isolated from mainland China.
Proteolytic cleavage of the viral glycoproteins and its significance for the virulence of Newcastle disease virus. Molecular characterization of Newcastle disease virus from backyard poultry farms and live bird markets in Kenya. Characterization of Newcastle disease virus isolates obtained from Eurasian collared pigeons (Streptopelia decaocto) in Italy.
Determination of organ tropism of Newcastle disease virus (Slain I-2) by virus isolation and reverse transcription -. Rapid detection and differentiation of Newcastle disease virus isolates by a one-step triplicate RT-PCR.
