First phylogenetic analysis of new Newcastle virus strain isolates in Algeria

Newcastle disease in chicken is caused by an avian paramyovirus serotype 1 (APMV1), it is one of the most economically and prevalent pathology around the world.

It was first identified in Java (INDONESIA) in 1926 and 1927 in Newcastle-upon-Tyne, England. However, it may have been prevalent as early as 1898, when a disease wiped out all the domestic fowl in North West Scotland (Macpherson et al., 1956).

Currently, the circulating strains associated with disease outbreaks worldwide predominantly are from genotypes V, VI, and VII of class II (Aldous et al., 2003; Lin et al., 2003).

Genotype VII was initially divided into two subgenotypes: VIIa, representing viruses that emerged in the 1990 s in the Far East and spread to Europe and Asia and VIIb, representing viruses that emerged in the Far East and spread to South Africa (Aldous et al., 2003). The two sub-genotypes of VII are further divided into VIIc, d, and e, which represent isolates from China, Kazakhstan and South Africa (Bogoyavlenskiy et al., 2009; Wang et al., 2006), and VIIf, g, and h, which represent African isolates (Snoeck et al., 2009). In Egypt, The sub-genotype VIId is predominant causing several ND outbreaks in poultry (Radwan et al., 2013).

Algeria has declared 3 times the Newcastle disease to the OIE between 1990 and 2012 (1992, 1997 and 2012). The disease causes inflammation of the respiratory tract and breathing difficulty, resulting between 5 to 15% mortality. The disease was successfully controlled by vaccination with conventional vaccine used in the field administered by drinking water or nebulization.

The situation changed in 2012 when a new virus strain was isolated from vaccinated flocks both in the East and West regions of Algeria. This virus was causing nervous symptoms: petechia in the proventriculus as well as a high level of mortality up to 67 % in vaccinated flocks, and up to 45 % of drop in laying hens.

The aim of this study was the examination of the genetic diversity of Newcastle virus strain isolated in Algeria starting from 2013 to 2016 that causes a high level of mortality in flocks that use conventional vaccines.

Viruses pools of 2 chicken imprints on FTA cards were submitted from each 40 chicken flocks for ND investigation from Algeria during the period of 2014 to 2016.

The flocks were selected after calls from the veterinarian in charge to signal a high level of mortality or an egg drop with nervous symptoms or petechia in the proventriculous after necropsy.

After the prints were made, the FTA card was sent to PHYLAXIA Laboratory in Hungary in order to check by PCR the positivity of the samples and after a sequencing in order to identify the genotype of the virus if the results were positive.

Viral RNA purification from FTA card was performed by RNA rapid extraction solution (Life Technologies) and MagMax Viral RNA isolation kit (Life Technologies) according to the manufacturer instruction. Purified RNA was used as template for the reverse transcription using MMLV-reverse transcriptase (Promega) and random hexamer primers (Promega) according to the manufacturer’s description.

NDV PCR was performed using K1-K2 primer pair, which amplifies a 1008bp long region between M gene position 778 and F gene position 545. (Lomniczi et. al. 1998 Arh.Virol 143:49-64).

Sequence differences of the PCR products in comparison with reference strains (obtained from GenBank) were calculated by Kimura’s method and phylogenetic tree was constructed using the Neighbor-joining method by TREECON for Windows program.

Figure 1: Phylogenic analysis of Algerian Newcastle virus isolates using Sequence differences of the PCR products in comparison with reference strains (obtained from GenBank) were calculated by Kimura’s method and phylogenetic tree was constructed using the Neighbor-joining method by TREECON for Windows program, it shows also the genetic distance between the field isolates and the strain used in the vaccination program in Algeria. Based on the 374 base pairs long (47-420 bp) nucleotide sequence of the F gene of NDV.

Table 1: Summary results of Newcastle genotype identification (2014 to 2016)

Five from 8 field sample originated from 4 layer farms between 4 and 14 weeks and 3 broiler farms were positive for NDV by RT-PCR between 2014 and 2016. All the positive grouped together with Velogenic NDV genotype VII strain, this group of isolates showed the closest relation to the Turkish, Pakistani and Arab Emirates ND virus.

There is a large genetic distance between the genotype found in the field and the genotype of the vaccine strain usually used in prophylactic program in Algeria (La Sota and Clone 30).

All positive cases underwent a minimum of 32% mortality preceded by acute nervous symptoms and all of them were vaccinated at least twice with live vaccines (La sota or Clone 30) or live and inactivated vaccines.

Pictures 1,2,3 and 4: Acute nervous symptoms on broiler chicken (East region of Algeria)

 

The velogenic Newcastle genotype VII is present in Algeria causing considerable economical loses.

NDV genotype VII began to replace genotype V as the most prevalent genotype in domestic flocks in Algeria.

There is a large genetic distance between the genotype discovered in the field and the genotype of the vaccine strain usually used in prophylactic program (La Sota and Clone 30).

Simultaneous detection of velogenic strains of NDV from various regions of the country warrants continuous isolation and molecular epidemiological investigations involving layers and broilers. Vaccine strategies that can elicit high antibody response along with continuous monitoring from the laboratory coupled with improved biosecurity measures in the farm is suggested.

 

 

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