Newcastle Disease Virus Genotype Research Articles

Immunity Elicited by a Turkey Herpesvirus-Vectored Newcastle Disease Vaccine in Turkey Against Challenge With a Recent Genotype IV Newcastle Disease Virus Field Strain.

Newcastle disease (ND) is still a major poultry disease worldwide. Vaccination remains the principal method of controlling ND in endemic countries. Various vaccination strategies, including the use of recently developed recombinant vaccines, have been used to control it. Recombinant vaccines that use the herpesvirus of turkey (HVT) as a vector to express one of the key antigens of Newcastle disease virus (NDV) have been developed to overcome some of the drawbacks related to the use of conventional vaccines. HVT as a vector appears to have unique beneficial characteristics: it is extremely safe, it is not affected by the presence of maternally derived antibodies, and therefore can be applied in the hatchery either in ovo or to day-old chicks. Due to its persistence in the bird, the HVT vector can be expected to induce life-long immune stimulation. In the present study, the efficacy of an HVT-based vector vaccine expressing the F gene of NDV (rHVT-F) was tested against a velogenic genotype IV NDV challenge in commercial turkeys with high levels of maternal antibodies (8.7 ± 0.8 log2 hemagglutination inhibition titer). The birds were vaccinated on the day of hatch by the subcutaneous route. Development of a humoral immune response to vaccination was detectable from 4 weeks of age by ELISA. The challenge strain used represents recent NDV genotype IV field strains from Morocco. Challenge with this strain induced ND-specific clinical signs and stunting without subsequent mortality in the non-vaccinated birds, whereas the vaccinated turkey poults showed protection as early as 3 weeks of age based on lack of clinical signs, better body weight gain, and reduction of challenge virus shedding. This is the first reported efficacy study of an HVT-vectored ND vaccine against a velogenic NDV challenge in commercial turkeys.

Monitoring of Newcastle disease virus in environmental samples.

Newcastle disease virus (NDV) is an important pathogen in poultry. Waterfowl and a number of other avian species serve as the host for NDV. Severity of the disease is variable and infected animals mainly develop respiratory and neurological symptoms. Outbreaks of NDV in poultry are recorded regularly in the Republic of Kazakhstan despite the widespread use of vaccines. Here we present evidence that nucleic acid found in open water bodies in Kazakhstan can be detected by means of next-generation sequencing and belongs to at least three distinct genotypes of NDV.

Genetic, antigenic, and pathogenic characteristics of Newcastle disease viruses isolated from geese in China.

Four Newcastle disease virus (NDV) strains were isolated from domestic, commercial geese that showed clinical signs that were believed to be the result of NDV infections. The genetic, antigenic, and pathogenic characteristics of the 4 NDVs were compared with those of NDV strains that were isolated from chickens. The complete genomes of 2 of the NDV strains contained 15,186 nucleotides (nt); the other 2 contained 15,192 nt, and exhibited the typical genomic organization of genotype II NDV and molecular characteristics of VIId NDVs. Phylogenetic analysis confirmed that the genotype II and VIId NDVs that were isolated from geese belonged to the same clusters as the corresponding genotypes of the chicken isolates. A serologic assay demonstrated that the antigenic relatedness among the NDVs was associated with their genotypes, rather than their hosts, and that amino acid substitutions in the F and/or HN proteins may contribute to the antigenic differences among these NDV genotypes. Geese infected with genotype VIId NDVs that were isolated from geese and chickens showed similar pathologic characteristics. NDVs that were isolated from geese did not differ in genetic, serologic, and pathogenic characteristics from those isolated from chickens, indicating that these NDVs were derived from chicken NDVs. Given the significance of geese in NDV epidemiology, effective biosecurity measures should be adopted to prevent the interspecies transmission of NDVs.

Co-circulation of paramyxo- and influenza viruses in pigeons in Egypt

ABSTRACTIn recent years, avian influenza virus (AIV) and Newcastle disease virus (NDV) have caused large-scale outbreaks in many countries, including Egypt. The culling and vaccination strategies have failed to control both viruses in Egypt. In this study, we investigated the outbreaks of nervous manifestations and deaths in pigeons between 2013 and 2015. The H5N1 subtype of the highly pathogenic avian influenza virus and pigeon paramyxovirus-1, an antigenic variant of NDV, were found to be the cause; AIV and pigeon paramyxovirus-1 were isolated from 61.3% (19/31) and 67.8% (21/31) of tested pigeons, respectively. Co-infection with both viruses was detected in 51.6% of pigeons (16/31). The AIV sequences showed PQGEKRRKKR/GLF motif at the haemagglutinin gene cleavage site, which is typical of the highly pathogenic H5N1 subtype. The phylogenetic tree showed that the highly pathogenic avian influenza belonged to clade 2.2.1.2. The NDV sequences carried one of the three motifs, 112GKQGRL117, 112KRQKRF117 or 112RRQKRF117, at the fusion protein cleavage site and were classified as genotypes I, VI and II in NDV-class II, respectively. This indicated that different genotypes of NDV can circulate simultaneously among pigeons. Further analysis revealed the clustering of some sequences in sub-genotypes Ia and VIb.2. To the best of our knowledge, these sub-genotypes have not been previously reported from pigeons in Egypt. Our results should serve as a base for future studies on both viruses in Egypt.

Genetic diversity of the genotype VII Newcastle disease virus: identification of a novel VIIj sub-genotype.

Newcastle disease (ND) is a highly contagious disease of poultry caused by Newcastle disease virus (NDV). Multiple genotypes of NDV have been circulating worldwide and NDV is continuously evolving, resulting into more diversity. Of multiple viral genotypes, VII is particularly important given that it had been associated with most recent ND outbreaks worldwide. In this study, an epidemiological investigation performed in northeastern China during 2014-2015 showed that 11 genotype VII isolates amounted to 55 percent in a total number of NDV isolates. Therefore, to evaluate the genetic diversity worldwide and epidemiological distribution in China of genotype VII NDV, a phylogenetic analysis based on the 1255 complete F gene sequences showed that VII is the most predominant genotype worldwide. A further detailed characterization on genotype VII was conducted based on the 477 complete F gene sequences from 11 isolates and 466 reference viruses available in GenBank. The results demonstrated that VII can be further divided into 8 sub-genotypes (VIIb, VIId-VIIj), indicating its complex genetic diversity. It is worthy of note that the isolation rate of VIIj is increasing recently. It emphasizes the necessity to pay close attention to the epidemiological dynamic of genotype VII NDV and highlights the importance of vaccination program.

Efficacy of a Recombinant Genotype VII Vaccine against Challenge with Velogenic Newcastle Disease Virus

Background: Newcastle disease virus (NDV) genotype VII has become the dominant genotype in China. However, NDV genotype II was used to make current commercial NDV vaccines. The mismatch of genotypes between circulating and vaccine strains of viruses may compromise the efficacy of vaccines.

Rescue of duck-origin virulent Newcastle disease virus from cloned cDNA and stable expression of the red fluorescent protein

Ducks are generally considered as potential reservoirs for different genotypes of Newcastle disease virus (NDV) and to be resistant even to velogenic NDV strains. However, outbreaks of highly virulent genotype VII NDV lethal to ducks have been frequently reported in China in recent years. But until now, the pathogenesis and potential vaccine of duck-origin genotype VII NDV are not known. In this study, a reverse genetics system using the prevalent high virulence genotype VIId isolate SS1 was constructed. Based on this system, a red fluorescent protein (RFP)-expressing virus was generated by inserting an additional transcription cassette coding for the RFP between the noncoding region of P and M genes. The rescue of the recombinant viruses was confirmed by western blotting, fluorescence microscopy and genetic marker detection. In addition, the replication kinetics, biological characteristics and pathogenicity of the rescued viruses were indistinguishable from the parental wild-type virus. Moreover, the recombinant virus rSS1-RFP could efficiently replicate in most of the duck tissues, especially in duck immune organs. The results obtained suggest that this reverse genetics system will provide a useful tool for the analysis of duck-origin NDV pathogenesis and dissemination, as well as preparation for novel vaccine vector or genotype-matched NDV attenuated vaccines used in ducks.

Molecular characterization, isolation, pathology and pathotyping of peafowl (Pavo cristatus) origin Newcastle disease virus isolates recovered from disease outbreaks in three states of India

ABSTRACTDisease outbreak investigations were carried out in three states of Northern India namely Haryana (Rewari), Uttar Pradesh (Noida) and Delhi, where a total of 110 Indian peafowls (Pavo cristatus) showed sudden onset of nervous signs and died within a period of two weeks during June, 2012. The F (fusion) gene-based RT-PCR detection of Newcastle disease virus (NDV) in affected tissues confirmed the presence of the virus. Three NDV isolates were selected (one from each area under investigation) and further characterized. They were found to be of virulent pathotype (velogenic NDV) based on both pathogenicity assays (MDT, ICPI and IVPI) and partial F gene sequence analysis. Additionally, the phylogenetic analysis revealed that the isolates belonged to the genotype VIIi and XIII of class II avian Paramyxovirus serotype1 (APMV-1) and related closely to new emerging sub-genotypes. This is the first report regarding the presence of the fifth panzootic vNDV genotype VIIi from India. In this scenario, extensive epidemiological studies are suggested for surveillance of NDV genotypes in wild birds and poultry flocks of the country along with adopting suitable prevention and control measures.

Phylogenetic characterization of virulent Newcastle disease viruses isolated during outbreaks in northwestern Iran in 2010.

The northwest of Iran shares long borders with three neighboring countries; therefore, it is considered one of the main entry portals of Newcastle disease virus (NDV) into the country. Ten virulent NDVs were recovered from 19 poultry farms of various prefectures in northwestern Iran during Newcastle disease outbreaks in 2010. The isolates were genotypically analyzed using an F-gene-specific reverse transcription polymerase chain reaction (RT-PCR) assay. The amplified F gene (nucleotides 189-1666) sequences of the NDV isolates were compared phylogenetically with those of previously published strains in GenBank. All of the NDV isolates belonged to genotype VIIb and were closely related to some isolates from Iran, Russia, and Sweden. Therefore, it can be postulated that these isolates evolved from previously reported strains. The velogenic viruses carried the motif (112)R-R-Q-K-R/F(117) at the F0 cleavage site and a unique substitution of (190)L→F which had never been reported in any NDV genotype VIIb isolate. They shared high sequence similarity with each other but were distinct from current NDV vaccines and NDV strains reported from other countries. This information is fundamental for improving the efficacy of controlling strategies and vaccine development for NDV.

A Raf kinase inhibitor demonstrates antiviral activities both in vitro and in vivo against different genotypes of virulent Newcastle disease virus

Newcastle disease (ND) is still one of the major plagues of birds worldwide. Combat actions are limited to vaccines, highlighting the urgent need for new and amply available antiviral drugs. Previous results have shown that Newcastle disease virus (NDV) downregulates the intracellular Raf kinase inhibitor protein (RKIP) expression for efficient replication, suggesting that this molecular may be a suitable target for antiviral intervention. In the present work, we investigated whether or not the Raf kinase inhibitor V (RKIV), which functions in the same way as RKIP by targeting the intracellular Raf kinase, is able to suppress the propagation of enzootic virulent NDV in vitro and in vivo. In vitro antiviral activity of RKIV was assessed by cell-based assay, and in vivo activity was determined in the chicken model. Our results clearly showed that RKIV treatment protected the cells from NDV-induced CPE with the effective concentrations on nM level, and inhibited virus replication in the lungs of infected chickens in a dose-dependent manner and protected chickens from the lethal infection by NDV. Thus, we conclude that the Raf kinase inhibitor compound RKIV, by inhibiting the host cellular target Raf kinase, might be very promising as a new class of antivirals against the enzootic virulent NDV infection.

Molecular characterization of genotype XIIIb Newcastle disease virus from central India during 2006–2012: Evidence of its panzootic potential

Newcastle disease virus (NDV) is the causative agent of Newcastle disease (ND) in many avian species. ND is a serious problem in developing countries, causing huge loss in the poultry industry. Although there are reports of continuous outbreaks of ND leading to serious losses to the poultry farming, very less is known about the genetic characteristics of its strains circulating in different parts of India. In the present study, we have five isolates of NDV reported from different outbreaks in Central India between the years 2006–2012. Deduced amino acid sequence of the F protein cleavage site and phylogenetic analysis of all the five isolates showed circulation of NDV genotype XIIIb. All the isolates showed a unique virulent cleavage site 112RRQKR↓F117. The close genetic similarity of all the isolates suggested circulation of the virulent NDV strains of the same ancestor in and around central India. Continuous isolation of genotype XIIIb NDV strains within the country suggests its panzootic potential. The study will be useful to understand the circulating strains of NDV and plan a vaccination strategy for poultry in India.

PHYLOGENETIC AND HISTOPATHOLOGICAL CHARACTERIZATION OF NEWCASTLE DISEASE VIRUSES REVEALS HIGH PREVALENCE OF VERY VIRULENT STRAINS OF GENOTYPE VIID IN EGYPT

Newcastle disease virus (NDV) is a highly contagious disease in poultry, also considered as a major challenge for the commercial and traditional poultry industry in Egypt. In this study clinicopathological and immunohistochemistry examination of fourteen flocks from different provinces in Egypt during 2013 and 2014 revealed velogenic ND features and positive staining for ND virus (NDV) antigen in pantropic organs of all strains which also revealed positive NDV by RRT-PCR, as it is alreadyconvinced that the fusion protein cleavage site has been deemed as responsible for the pathogenicity of NDV and selected as a target to identify and type lentogenic and velogenic strains, viral Genomic RNAs were extracted from representing swabs and organs of examined flocks, and partially amplifiedfor fusion gene of NDV using RT-PCR, then forwarded to gene sequencing. Four chosen samples amplified for full fusion gene sequence using specific sets of newly designed primers from the available sequence data of NDV strains in Gen Bank, The resulted sequence showed motif 112RRQKRF117 which is indicative of velogenic character NDV strains, and indicate presence of 2 mutations E74D and D170N in two strains. Phylogenetic analysis revealed that Egyptian strains are closely related to NDV genotype VIId strains of south East Asian countries based on the high nucleotide and amino acid similarity.

Phylogenetic analysis of some Newcastle disease virus isolates from the Sudan.

A reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify 1412 bp of the fusion protein gene (F gene) of four Newcastle disease virus (NDV) isolates; two velogenic (TY-1/90 and DIK-90) and two lentogenic isolates (Dongla 88/1 and GD.S.1). Following sequencing, nucleotide sequences were annotated and 894 bp were compared phylogenetically with those from strains previously reported in the Sudan and the virus strains published on the GenBank. It could be demonstrated that TY-1/90 and DIK-90 strains belong to the genotype VI of NDV and are in close genetic relationship to sub- genotype VIb. TY-1/90 and DIK-90 strains were observed to be genetically unrelated to the earlier Sudanese isolates of 1970/80s and the late of 2000s suggesting a different origin. The close genetic relationship to the European and African pigeon paramyxovirus type 1 (PPMV-1) suggests a common ancestor. Dongola, GD.S.1 strains were classified into genotype II that comprises non-pathogenic lentogenic NDV strains. The present genetic classification of NDV isolates of the Sudan provides valuable information on genotypes of NDV. Further molecular epidemiological investigations of the recent outbreaks of Newcastle disease in the Sudan are needed in order to improve the efficiency of control strategies and vaccine development.

Isolation and Molecular Characterization of Newcastle Disease Virus Genotype VI from Pigeons in Egypt

A pool of brain sample was collected from three pigeons showing torticollis and whitish green diarrhea in Desouk, Kafr-elshiekh Province, Egypt,during January, 2014. Virus Isolation has been pursued by inoculation of brain homogenate into the allantoic sac of 9 day old specific pathogen free (SPF) embryonated chicken eggs and incubated at 37oC for 4 days. Identification of the isolate was carried out by haemagglutination inhibition test using specific polyclonal antisera against NDV and AIV ( subtype H5 and H9), RT-PCR and confirmed by sequencing. Partial deduced amino acid sequence of the fusion protein at the cleavage site revealed that the isolate possessed the motif 112 K-R-Q-K-R-F117 indicating velogenic entity and phylogenetic analysis showed clustering of the study sequence with other genotype IV F gene sequences available in GenBank.The study indicated that pigeons may play epidemiological role in the introduction of a new NDV genotype VI to commercials chicken.

EFFECT ON IMMUNE RESPONSE AND VIRUS SHEDDING IN THE CHICKEN VACCINATED AGAINST INACTIVATED LOCAL STRAIN OF NEWCASTLE DISEASE VIRUS GENOTYPE VII

The effectiveness of the new inactivated vaccine develop from local velogenic strain of Newcastle disease virus (NDV) Genotype VII and commercial vaccines LaSota were compared by determining the immune response and virus shedding of vaccinated chickens. Ten different groups of chicken consists of ten each, were vaccinated with and without adjuvant of the inactivated vaccine via intramuscular and subcutaneous, respectively. Three different adjuvants were used which include Oil-emulsion Complete Freund’s, Oil-emulsion Squalene-based, and aluminium hydroxide gel (Alum). As a comparison, a group of chicken was vaccinated with commercial vaccine and control group was not vaccinated. With 100% survival rate and highest mean haemaglutination-inhibition (HI) titre of log2 6, the inactivated vaccine with adjuvant Alum and Oil-emulsion Complete Freund’s surpassed the LaSota. In addition, the virus shedding was significantly reduced and comparable to LaSota vaccinated chicken. Whereas, without adjuvant, the chicken HI antibody titre is below log2 4 after vaccination and only 20-30% were survived. Based on the post-mortem findings on the survived chicken from each vaccinated group, their internal organs appeared normal and no sign of haemorrhage or pathognomonic signs of Newcastle disease (ND). Conclusively, vaccinated chicken are effectively protected from morbidity and mortality against virulent genotype VII challenge with the addition of adjuvant into inactivated local strain of NDV genotype VII vaccine. Thus, the development of inactivated local NDV genotype VII vaccine is a promising candidate to control the current ND endemic in Malaysia.

SEQUENCE AND PHYLOGENETIC ANALYSIS OF NEWCASTLE DISEASE VIRUS GENOTYPE VII ISOLATED IN MALAYSIA DURING 1999-2012

Newcastle disease virus (NDV) is a contagious viral disease of many avian species particularly domestic poultry, and is responsible for causing significant economic losses to the poultry industry in Southeast Asia including Malaysia. Here we report the sequence and phylogenetic analysis of NDV that has been circulating in Malaysia. A total of 151 NDV isolates were selected during 1999-2012 throughout Malaysia and were characterized phylogenetically. The partial region of matrix (M) and fusion (F) protein of NDV was amplified by reverse transcriptase PCR, directly sequenced and compared genetically to the published sequences obtained from GenBank. The deduced amino acid sequence of the F protein cleavage site revealed the presence of three different motifs; 112RRRKRF117, 112RRQKRF117 typical for velogenic strains while 112GKQGRL117 indicates it is from avirulent strain or lentogenic strain. The phylogenetic analysis revealed that 13 isolates belonged to genotype I, 2 to genotype III, 6 to genotype VI, 1 to genotype VIII and 129 to genotype VII. Isolates belonging to genotype VII were further divided into five subgenotypes; VIIa, VIIb, VIId, VIIe and VIIh. Based on the phylogenetic tree and geographical data, it is found that NDV genotype VIIb and VIIe were isolated in 1999 while in year 2000 to 2009, most of the NDV isolates were NDV genotype VIId originated from China. No subgenotype VIId viruses were recovered after 2009 in Malaysia. In 2010-2012, NDV outbreaks were caused by subgenotypes VIIa and VIIh in Peninsular Malaysia. Interestingly, these subgenotypes have been isolated in East Malaysia since 2002 but did not cause major outbreak. These information points to the existence of multiple genotypes of NDV in Malaysia especially genotype VII and these findings emphasize the importance of continuous surveillance of NDV in Malaysia.

Infectious bursal disease vaccine ameliorates velogenic Newcastle disease virus infection in immunopotentiated chickens

In the present study, the potential immunoprotective role of infectious bursal disease (IBD) vaccine on immunopotentiated chickens challenged with velogenic viscerotropic Newcastle disease virus (vvNDV) genotype VIId was investigated. The chickens were divided into five groups vaccinated with NDV vaccine, but group (G1) was kept as control unvaccinated birds. The G2 and G4 were immunopotentiated using mannan oligosaccharides (MOS) while G3 and G5 received MOS and IBD vaccine. The G1, G2, and G3 were infected with vvNDV while G4 and G5 served as uninfected birds. The humoral response to NDV vaccine was measured by hemagglutination inhibition test while cellular response was estimated by chicken interleukin-2 ELISA, total and differential leucocytic count, bursa body weight index, and histopathological examination of the bursae. The results revealed that the immunopotentiated groups possessed a good cellular and humoral response to vvNDV although the severity of histopathological lesions was variable. The G3 had the best cellular and humoral response, and IBD vaccine enhanced the immune response against the infection with vvNDV. Histopathologically, the typical bursal lesions of NDV were observed in G1 such as lymphocytic depletion, acute inflammatory reaction, and degenerative changes of the bursal follicles. In contrast, such histopathological changes in the immunopotentiated groups were less evident both in severity and prevalence, while the immune reactive lymphoid hyperplasia was a prominent finding in G4 and G5. In conclusion, the immunopotentiation of the chickens as well as NDV and IBD vaccination improved the bird immunity and diminished the immunosuppressive effect of the vvNDV.

Molecular and antigenic characteristics of Newcastle disease virus isolates from domestic ducks in China

Newcastle disease (ND) is one of the most devastating diseases to the poultry industry. The causative agents of ND are virulent strains of Newcastle disease virus (NDV), which are members of the genus Avulavirus within the family Paramyxoviridae. Waterfowl, such as ducks and geese, are generally considered potential reservoirs of NDV and may show few or no clinical signs when infected with viruses that are obviously virulent in chickens. However, ND outbreaks in domestic waterfowl have been frequently reported in many countries in the past decade. In this study, 18 NDV strains isolated from domestic ducks in southern and eastern China, between 2005 and 2013, were genetically and phylogenetically characterized. The complete genomes of these strains were sequenced, and they exhibited genome sizes of 15,186 nucleotides (nt), 15,192nt, and 15,198nt, which follow the “rule of six” that is required for the replication of NDV strains. Based on the cleavage site of the F protein and pathogenicity tests in chickens, 17 of our NDV isolates were categorized as lentogenic viruses, and one was characterized as a velogenic virus. Phylogenetic analysis based on the partial sequences of the F gene and the complete genome sequences showed that there are at least four genotypes of NDV circulating in domestic ducks; GD1, AH224, and AH209 belong to genotypes VIId, Ib, and II of class II NDVs, respectively, and the remaining 15 isolates belong to genotype 1b of class I NDVs. Cross-reactive hemagglutination inhibition tests demonstrated that the antigenic relatedness between NDV strains may be associated with their genotypes, rather than their hosts. These results suggest that though those NDV isolates were from duck, they still don’t form a phylogenetic group because they came from the same species; however, they may play an important role in promoting the evolution of NDVs.

Characterisation of genotype VII Newcastle disease virus (NDV) isolated from NDV vaccinated chickens, and the efficacy of LaSota and recombinant genotype VII vaccines against challenge with velogenic NDV

A Newcastle disease virus (NDV) isolate designated IBS002 was isolated from a commercial broiler farm in Malaysia. The virus was characterised as a virulent strain based on the multiple basic amino acid motif of the fusion (F) cleavage site 112RRRKGF117 and length of the C-terminus extension of the hemagglutinin-neuraminidase (HN) gene. Furthermore, IBS002 was classified as a velogenic NDV with mean death time (MDT) of 51.2 h and intracerebral pathogenicity index (ICPI) of 1.76. A genetic distance analysis based on the full-length F and HN genes showed that both velogenic viruses used in this study, genotype VII NDV isolate IBS002 and genotype VIII NDV isolate AF2240-I, had high genetic variations with genotype II LaSota vaccine. In this study, the protection efficacy of the recombinant genotype VII NDV inactivated vaccine was also evaluated when added to an existing commercial vaccination program against challenge with velogenic NDV IBS002 and NDV AF2240-I in commercial broilers. The results indicated that both LaSota and recombinant genotype VII vaccines offered full protection against challenge with AF2240-I. However, the LaSota vaccine only conferred partial protection against IBS002. In addition, significantly reduced viral shedding was observed in the recombinant genotype VII-vaccinated chickens compared to LaSota-vaccinated chickens.

Identification of new sub-genotypes of virulent Newcastle disease virus with potential panzootic features

Virulent Newcastle disease virus (NDV) isolates from new sub-genotypes within genotype VII are rapidly spreading through Asia and the Middle East causing outbreaks of Newcastle disease (ND) characterized by significant illness and mortality in poultry, suggesting the existence of a fifth panzootic. These viruses, which belong to the new sub-genotypes VIIh and VIIi, have epizootic characteristics and do not appear to have originated directly from other genotype VII NDV isolates that are currently circulating elsewhere, but are related to the present and past Indonesian NDV viruses isolated from wild birds since the 80s. Viruses from sub-genotype VIIh were isolated in Indonesia (2009–2010), Malaysia (2011), China (2011), and Cambodia (2011–2012) and are closely related to the Indonesian NDV isolated in 2007, APMV1/Chicken/Karangasem, Indonesia (Bali-01)/2007. Since 2011 and during 2012 highly related NDV isolates from sub-genotype VIIi have been isolated from poultry production facilities and occasionally from pet birds, throughout Indonesia, Pakistan and Israel. In Pakistan, the viruses of sub-genotype VIIi have replaced NDV isolates of genotype XIII, which were commonly isolated in 2009–2011, and they have become the predominant sub-genotype causing ND outbreaks since 2012. In a similar fashion, the numbers of viruses of sub-genotype VIIi isolated in Israel increased in 2012, and isolates from this sub-genotype are now found more frequently than viruses from the previously predominant sub-genotypes VIId and VIIb, from 2009 to 2012. All NDV isolates of sub-genotype VIIi are approximately 99% identical to each other and are more closely related to Indonesian viruses isolated from 1983 through 1990 than to those of genotype VII, still circulating in the region. Similarly, in addition to the Pakistani NDV isolates of the original genotype XIII (now called sub-genotype XIIIa), there is an additional sub-genotype (XIIIb) that was initially detected in India and Iran. This sub-genotype also appears to have as an ancestor a NDV strain from an Indian cockatoo isolated in1982. These data suggest the existence of a new panzootic composed of viruses of subgenotype VIIi and support our previous findings of co-evolution of multiple virulent NDV genotypes in unknown reservoirs, e.g. as recorded with the virulent NDV identified in Dominican Republic in 2008. The co-evolution of at least three different sub-genotypes reported here and the apparent close relationship of some of those genotypes from ND viruses isolated from wild birds, suggests that identifying wild life reservoirs may help predict new panzootics.