Cross-species transmission of poultry pathogens in backyard farms: ducks as carriers of chicken viruses

ABSTRACT

In backyard farms of Lao People’s Democratic Republic, mixed-species rearing of poultry is a breeding-ground for cross-species transmission. Here, the epidemiology of viruses circulating among backyard poultry in Vientiane Province was assessed to guide future control strategies. Oral/tracheal and cloacal swabs, collected from 605 poultry (308 ducks, 297 chickens) between 2011 and 2015, were screened by PCR for Newcastle disease virus (NDV), coronavirus (CoV) and chicken anaemia virus (CAV). Chicken sera were screened for anti-NDV antibodies by ELISA. Statistical and phylogenetic analyses revealed transmission patterns and relationships.

Closely related strains co-circulated in chickens and ducks. While CoV RNA was detected in oral/tracheal swabs of 9.3% of the chickens and 2.4% of the ducks, rates were higher in faecal swabs of both species (27.3% and 48.2%). RNA of infectious bronchitis virus (IBV) and duck CoV was found in faecal swabs of chickens (19.7% and 7.1%) and ducks (4.1% and 44.1%). Moreover, DNA of the generally chicken-specific CAV was detected in oral/tracheal swabs of chickens (18.1%) and, sporadically, of ducks (2.4%). Despite serological evidence of NDV circulation or vaccination (86.9%), NDV RNA was not detected. We found a high prevalence and indication for cross-species transmission of different CoV strains in backyard poultry. Interestingly, ducks served as biological, or at least mechanical, carriers of viral strains closely related not only to IBV, but also to CAV. Bird containment and poultry species separation could be first steps to avoid cross-species transmission and emergence of novel strains with broad host range and enhanced pathogenicity.

RESEARCH HIGHLIGHTS

• High rates of avian viruses were detected by PCR in backyard poultry from Lao PDR.

• Diverse coronavirus and chicken anemia virus strains co-circulated.

• Phylogenetic analyses suggested virus transmission between chickens and ducks.

• Serological evidence of Newcastle disease was found, but viral RNA was not detected.

     

Molecular characterization of new emerging sub-genotype VIIh Newcastle disease viruses in China

Newcastle disease (ND) has been enzootic in China for several decades since the first recognition of the disease in 1946 in China. Continuous surveillance revealed that the sub-genotype VIId Newcastle disease virus (NDV) has been predominantly responsible for most of ND outbreak in China in recent years. But in the present study, three virulent NDVs isolated from poultry in southern China were classified as sub-genotype VIIh, which is highly related to the viruses circulating in some Southeast Asia countries. Continuous isolation of genotype VIIh NDV strains in the region suggests its panzootic potential. This is the first report of the sub-genotype VIIh NDVs in domestic poultry in China. The complete genome length of the three isolates was 15,192 nucleotides, and the motif at the cleavage site of F protein was ¹¹²RRRRR/F¹¹⁷ or ¹¹²RRRKR/F¹¹⁷, which was typical of virulent NDV. Phylogenetic analysis based on the F gene revealed that the three viruses had close relationship with the sub-genotype VIIh virus isolated from wild bird in 2011 in China. These viruses might have formed a stable lineage in poultry during 2012–2016 and have the potential to cause enzootic in China. Our study revealed the genetic and phylogenetic characteristics of the three sub-genotype VIIh isolates, which could help us to better understand the epidemiological context of these viruses.

     

Separate Evolution of Virulent Newcastle Disease Viruses from Mexico and Central America

An outbreak of Newcastle disease (ND) in poultry was reported in Belize in 2008. The characteristics of three virulent Newcastle disease virus (NDV) isolates from this outbreak (NDV-Belize-3/08, NDV-Belize-4/08, and NDV-Belize-12/08) were assessed by genomic analysis and by clinicopathological characterization in specific-pathogen-free (SPF) chickens. The results showed that all three strains belong to NDV genotype V and are virulent, as assessed by the intracerebral pathogenicity index and the polybasic amino acid sequence at the fusion protein cleavage site. In 4-week-old SPF chickens, NDV-Belize-3/08 behaved as a typical velogenic viscerotropic NDV strain, causing severe necrohemorrhagic lesions in the lymphoid organs, with systemic virus distribution. Phylogenetic analysis of multiple NDV genotype V representatives revealed that genotype V can be divided into three subgenotypes, namely, Va, Vb, and Vc, and that all tested Belizean isolates belong to subgenotype Vb. Furthermore, these isolates are nearly identical to a 2007 isolate from Honduras and appear to have evolved separately from other contemporary viruses circulating in Mexico, clustering into a new clade within NDV subgenotype Vb.     

Complete genome and clinicopathological characterization of a virulent Newcastle disease virus isolate from South America

Newcastle disease (ND) is one of the most important diseases of poultry, negatively affecting poultry production worldwide. The disease is caused by Newcastle disease virus (NDV) or avian paramyxovirus type 1 (APMV-1), a negative-sense single-stranded RNA virus of the genus Avulavirus, family Paramyxoviridae. Although all NDV isolates characterized to date belong to a single serotype of APMV-1, significant genetic diversity has been described between different NDV isolates. Here we present the complete genome sequence and the clinicopathological characterization of a virulent Newcastle disease virus isolate (NDV-Peru/08) obtained from poultry during an outbreak of ND in Peru in 2008. Phylogenetic reconstruction and analysis of the evolutionary distances between NDV-Peru/08 and other isolates representing established NDV genotypes revealed the existence of large genomic and amino differences that clearly distinguish this isolate from viruses of typical NDV genotypes. Although NDV-Peru/08 is a genetically distinct virus, pathogenesis studies conducted with chickens revealed that NDV-Peru/08 infection results in clinical signs characteristic of velogenic viscerotropic NDV strains. Additionally, vaccination studies have shown that an inactivated NDV-LaSota/46 vaccine conferred full protection from NDV-Peru/08-induced clinical disease and mortality. This represents the first complete characterization of a virulent NDV isolate from South America.     

Antigenicity,pathogenicity and immunosuppressive effect caused by a South American isolate of infectious bursal disease virus belonging to the “distinct” genetic lineage

Infectious bursal disease virus (IBDV) is the causative agent of a highly contagious immunosuppressive disease affecting young chickens. The recently described “distinct IBDV” (dIBDV) genetic lineage encompasses a group of worldwide distributed strains that share conserved genetic characteristics in both genome segments making them unique within IBDV strains. Phenotypic characterization of these strains is scarce and limited to Asiatic and European strains collected more than 15 years ago. The present study aimed to assess the complete and comprehensive phenotypic characterization of a recently collected South American dIBDV strain (1/chicken/URY/1302/16). Genetic analyses of both partial genome segments confirmed that this strain belongs to the dIBDV genetic lineage and that it is not a reassortant. Antigenic analysis with monoclonal antibodies indicated that this strain has a particular antigenic profile, similar to that obtained in a dIBDV strain from Europe (80/GA), which differs from those previously found in the traditional classic, variant and very virulent strains. Chickens infected with the South American dIBDV strain showed subclinical infections but had a marked bursal atrophy. Further analysis using Newcastle disease virus-immunized chickens, previously infected with the South American and European dIBDV strains, demonstrated their severe immunosuppressive effect. These results indicate that dIBDV strains currently circulating in South America can severely impair the immune system of chickens, consequently affecting the local poultry industry. Our study provides new insights into the characteristics and variability of this global genetic lineage and is valuable to determine whether specific control measures are required for the dIBDV lineage.

Research Highlights

• A South American strain of the dIBDV lineage was phenotypically characterized.

• The strain produced subclinical infections with a marked bursal atrophy.

• Infected chickens were severely immunosuppressed.

• The dIBDV strains are antigenically divergent from other IBDV lineages.

     

Protection afforded by avian influenza vaccination programmes consisting of a novel RNA particle and an inactivated avian influenza vaccine against a highly pathogenic avian influenza virus challenge in layer chickens up to 18 weeks post-vaccination

The efficacies of an oil adjuvanted-inactivated reverse genetics-derived H5 avian influenza virus (AIV) vaccine and an alphavirus replicon RNA particle (RP) AIV vaccine were evaluated in commercial Leghorn chickens. Challenge utilized A/turkey/MN/12582/2015, an isolate representing the U.S. H5N2 Clade 2.3.4.4 responsible for the 2015 highly pathogenic avian influenza (HPAI) epornitic in commercial poultry the United States. As part of a long-term, 36-week study, chickens were challenged at seven weeks of age after receiving a single vaccination, at 18 weeks of age following a vaccine prime-single boost, and at 36 weeks of age after a prime- double-boost. All vaccine programmes reduced virus oropharyngeal and cloacal shedding and mortality compared to the non-vaccinated control birds; however, chickens receiving at least one administration of the RP vaccine generally had diminished viral shedding especially from the cloacal swabbings. A detectable serum antibody response and protection were observed through 18 weeks post-vaccination. Our data suggest that, in conjunction with a comprehensive eradication, enhanced biosecurity and controlled marketing plan, vaccination programmes of commercial layer chickens with novel RP vaccines may represent an important tool for preventing HPAI-related mortalities and decreasing viral load during a catastrophic influenza outbreak.

RESEARCH HIGHLIGHTS

• Immunization of poultry following a vaccination schedule consisting of inactivated and RNA particle vaccines offered significant protection against lethal disease following HPAIV challenge.

• Virus shedding was significantly (P?<?0.05) reduced in chickens vaccinated with either inactivated and/or recombinant vaccines.

• Serum antibody titres were not a reliable indicator of protection.

• An inactivated vaccine containing 384 HAU of the homologous antigen was unable to induce complete protection.

     

A novel genotype of beak and feather disease virus in budgerigars (<Emphasis Type="Italic">Melopsittacus undulatus</Emphasis>)

House sparrow (Passer domesticus) is one of the most widely distributed wild birds in China. Five Newcastle disease virus (NDV) strains were isolated from house sparrows living around the poultry farms in southern China. These isolates were characterized by pathogenic assays and phylogenetic analysis. The results showed that all NDV isolates except one were velogenic and virulent for chickens. These four virulent strains for chickens possess the amino acid sequence ¹¹²R/K-R-Q-K/R-R-F¹¹⁷ in the F₀ cleavage site which is typical of velogenic NDV. Phylogenetic analysis indicated that these isolates belong to genotype VII and were closely related to the strains which were isolated from NDV outbreaks in chickens since 2000. One isolate of NDV from house sparrow belong to genotype II and was proved to be vaccine strain (Chicken/U.S./LaSota/46). The result of this study proved that house sparrow can carry the virulent NDV strains and the same genotype of viruses that are circulating in poultry are existing in house sparrows living around poultry farm in southern China.     

Molecular Epidemiology of Outbreak-Associated and Wild-Waterfowl-Derived Newcastle Disease Virus Strains in Finland,Including a Novel Class I Genotype

Newcastle disease (ND) is a highly contagious, severe disease of poultry caused by pathogenic strains of Newcastle disease virus (NDV; or avian paramyxovirus-1). NDV is endemic in wild birds worldwide and one of the economically most important poultry pathogens. Most of the published strains are outbreak-associated strains, while the apathogenic NDV strains that occur in wild birds, posing a constant threat to poultry with their capability to convert into more virulent forms, have remained less studied. We screened for NDV RNA in cloacal and oropharyngeal samples from wild waterfowl in Finland during the years 2006 to 2010: 39 of 715 birds were positive (prevalence, 5.5%). The partial or full-length F genes of 37 strains were sequenced for phylogenetic purposes. We also characterized viruses derived from three NDV outbreaks in Finland and discuss the relationships between these outbreak-associated and the wild-bird-associated strains. We found that all waterfowl NDV isolates were lentogenic strains of class I or class II genotype I. We also isolated a genetically distinct class I strain (teal/Finland/13111/2008) grouping phylogenetically together with only strain HIECK87191, isolated in Northern Ireland in 1987. Together they seem to form a novel class I genotype genetically differing from other known NDVs by at least 12%.     

Recombinant Newcastle disease virus (NDV) La Sota expressing the haemagglutinin–neuraminidase protein of genotype VII NDV shows improved protection efficacy against NDV challenge

Intensive vaccination strategies against Newcastle disease (ND) have been implemented in many countries for a long time, but ND outbreaks still occur frequently, with most isolates belonging to genotype VII of Newcastle disease virus (NDV). Many researchers have revealed that vaccines closely matched to epidemic viruses provide better protection. Therefore, using a previously established reverse genetics system, we generated a recombinant NDV vaccine strain (rLa Sota-HN) based on the La Sota vaccine strain expressing the haemagglutinin–neuraminidase (HN) protein of genotype VII NDV. The pathogenicity of the recombinant virus was confirmed by the mean death time in 9-day-old specific-pathogen-free embryonated chicken eggs and the intracerebral pathogenicity index in 1-day-old specific-pathogen-free chickens. Subsequently, 1-day-old chickens were immunized with commercial vaccine La Sota and recombinant virus rLa Sota-HN and then challenged with virulent genotype VII NDV strain. The results indicated that recombinant virus rLa Sota-HN provided increased protection of vaccinated chickens from morbidity and mortality, and inhibited the shedding of virulent virus after challenging with genotype VII virus, compared with the conventional vaccine La Sota. Our findings indicated that rLa Sota-HN is a promising vaccine candidate to improve the protection efficiency against ND in chickens, thereby preventing frequent outbreaks of this disease.     

Highly virulent Marek’s disease virus strains affect T lymphocyte function and viability of splenocytes in commercial meat-type chickens

ABSTRACT

In previous studies, we have demonstrated that very virulent plus Marek’s disease viruses (vv+MDV) are highly immunosuppressive in commercial meat-type chickens. The specific objectives of this work were to evaluate if vv+MDV immunosuppression (MDV-IS) is induced by reduction of lymphocyte responsiveness and/or viability. Three experiments were conducted to (i) compare vv+MDV 686 with a partially attenuated 686-BAC; (ii) compare vv+MDV strains (648A and 686) with vMDV (GA) and vvMDV (Md5); and (iii) compare chickens vaccinated with Md5-BACΔMEQ and with CVI988?+?HVT. In each experiment, spleens were collected at 28–30 days post infection and lymphocytes were isolated and investigated in three ways: their proliferative response to Concanavalin A (ConA) was analysed by MTT proliferation assay; cell death, and expression of CD45 and MHC-I was studied by flow cytometry; and MHC-IA and β-2 microglobulin (B2M) expression was evaluated by real time RT-PCR. Splenocytes of chickens inoculated with vv+MDV were severely impaired to proliferate when exposed to ConA. Furthermore, vv+MDV induced severe splenocyte death that did not occur after infection with v or vvMDV strains. Vaccination with CVI988?+?HVT, and at less level with Md5-BACΔMEQ reduced these negative effects. This is in contrast to our previous results in which Md5-BACΔMEQ but not CVI988?+?HVT protected against MDV-IS suggesting that although cell death and decrease lymphocyte function seem to be related to MDV virulence and certainly will be associated with immunosuppression, they might not fully explain the previously reported MDV-IS.

RESEARCH HIGHLIGHTS

• vv+MDV induces extensive death in splenocytes in meat-type chickens 28–30?dpi.

• vv+MDV impairs lymphocyte function in meat-type chickens 28–30?dpi.

• Vaccination protects against splenocyte death and reduced lymphocyte function.

• Cell lysis and reduced lymphocyte function do not fully explain MDV-IS.

     

Phylogenetic study base on matrix gene of Iranian Newcastle disease virus isolates, 2011-2012

Newcastle disease (ND), caused by avian paramyxovirus type 1, is a highly contagious and devastating viral disease of poultry of worldwide distribution with an enormous economic impact. In this study, the sequences of matrix protein (M) gene of three ND virus strains isolated from outbreak in chickens in poultry farms of Iran in 2011–2012 were determined. Phylogenetic analysis revealed that both strains clustered with the class II viruses, with one phylogenetically close to the genotype VIIb Newcastle disease viruses and the other closer to genotype VIId. This finding is essential for improving the disease control strategies and development of vaccines for ND. The study results thus emphasize the importance of continuous surveillance of this disease and of sharing the information to the global scientific community, which would help to fill the epidemiological gaps in the regions and to validate the robustness of diagnostic screening.     

New PA/1220/98-like variant of infectious bronchitis virus in Poland

ABSTRACT

The aim of the present study was to report the first detection of a new infectious bronchitis virus (IBV) variant in Polish commercial flocks which is completely different to any previously known in this region. In 2018, samples from Ross 308 breeding hens aged 35 weeks were delivered for IBV diagnosis. IBV presence was detected, but all attempts to amplify the S gene fragment were negative. The field material was analysed using the Illumina MiSeq platform and a 1073-nt fragment of the S1 coding region was obtained. The gCoV/ck/Poland/516/2018 strain shared only 52.7–58.1% nucleotide identity to any known genotype of IBV and shared the highest identity of 81.4% to the unique North American PA/1220/98 variant. Based on the obtained sequence, a specific molecular test was constructed and used for screening of chicken samples from 35 field cases delivered to our laboratory between 2018 and 2019 for IBV diagnosis. Application of this test enabled detection of another three chicken flocks as positive for this new strain. All positives were identified in commercial layers with egg production problems. To date, the virus has not been detected in broiler chickens. Taking into account the proposed criteria for the definition of a new IBV genotype or lineage, it seems that the detected viruses in Poland, together with the unique North American PA/1220/98 variant, may be classified as separate lineages/genotype in the new IBV classification.

RESEARCH HIGHLIGHTS

• The new IBV variant is distantly related to other known GI–GVII IBV genotypes/lineages.

• It affects long-lived birds causing egg production problems.

• The detected IBV and the unique North American PA/1220/98 variant are candidates for separate lineages in the new GVIII genotype.

     

Genetic analysis of Newcastle disease virus from Punjab, Pakistan

The strains of Newcastle disease virus (NDV) were isolated from five suspected outbreaks of ND in broiler (n = 3) and layers (n = 2) poultry farms. The egg-isolated viruses were subjected to biological and genetic characterization. Based on the biological characterization, isolates showed haemagglutination titer ≥log 2⁷, mean death time <55 h, intracerebral pathogenecity index ≤1.8, and egg lethal dose 50 from 10^?7.15 to 10^?9.31/1 ml. Genetic characterization of the fusion (F) gene revealed that the isolates clustered with NDV strains of genotype VII (VIIf) within class II, which remained predominant genotype in the domestic poultry of Asia. The deduced amino acid sequence of the isolates confirmed virulent motif ¹¹²RRQKRF¹¹⁷ at the F protein cleavage site. A bioinformatics and pairwise comparison approach was applied to estimate the synonymous and non-synonymous substitution rates (dN/dS) and selective evolutionary pressure for the F protein. The dN/dS calculated for genotype VII indicate purifying selection, which resulted in a low evolution rate in F gene. The F protein shows a strong negative pressure throughout the length of the protein and no recombination event was determined. Collectively, the results suggest that very similar virulent strains of NDV are involved during current wave of disease outbreak throughout the country. From these results, in conjunction with our recent reports of NDV from Pakistan, it is possible to conclude that emergence of novel group may require revisiting the diagnostics and vaccine control strategies.     

Synergistic effect of a combination of nicarbazin and monensin against coccidiosis in the chicken caused by Eimeria spp.

ABSTRACT

A clinical study was made into the abilities of nicarbazin and monensin and a nicarbazin?+?monensin combination to control Eimeria acervulina, E. maxima, and E. tenella in chickens. When included in the feed, at concentrations of 40?ppm nicarbazin or 40?ppm monensin, these products showed partial efficacy evaluated by daily weight gain (DWG) but no activity judged by daily feed intake (DFI) or feed conversion ratio (FCR). By contrast, the combination of 40?ppm nicarbazin?+?40?ppm monensin provided complete control of infection judged by greater DWG and DFI, and lower FCR. Monensin at a concentration of 40?ppm was ineffective in preventing lesions caused by all three species. Nicarbazin at a concentration of 40?ppm was unable to suppress lesions of E. acervulina and E. maxima but was able to suppress lesions caused by E. tenella. Nicarbazin 40?ppm?+?monensin 40?ppm suppressed lesions of all three species.

RESEARCH HIGHLIGHTS

• Nicarbazin or monensin at 40 ppm gave only partial control of Eimeria spp.

• A combination of 40 ppm nicarbazin + 40 ppm monensin controlled DWG, DFI and FCR.

• Nicarbazin or monensin at 40 ppm did not suppress all Eimeria spp. lesions.

• Nicarbazin 40 ppm + monensin 40 ppm suppressed lesions of all three species.

     

Fatal necrotic tracheitis by Aviadenovirus in captive Alagoas curassows (Pauxi mitu) extinct from the wild

Extinct from nature, captive young Alagoas curassows (Pauxi mitu) were found agonizing or dead with respiratory disease. Intranuclear inclusion bodies were found in the epithelia of the trachea, associated with marked necrotic tracheitis. An Aviadenovirus was isolated in chicken eggs and characterized genetically with 99% identity to the fowl Aviadenovirus A, as based on the hexon protein gene. This is the first report of respiratory disease caused by Aviadenovirus in any cracid species in Brazil, recommending for stricter biosecurity in the conservation premises.

RESEARCH HIGHLIGHTS

• Fatal tracheitis in curassows extinct from nature was associated with Aviadenovirus A.

• Seven-month-old Alagoas curassows (Aves: Cracidae) died with haemorrhagic tracheitis.

• Aviadenovirus A with 99% identity to fowl adenovirus 1 was detected in dead curassows.

• Fatal tracheitis by Aviadenovirus was described in Pauxi mitu (Aves: Cracidae).

     

Characteristics of field isolates of Newcastle disease virus isolated in the course of outbreaks in the poultry plant in the Leningrad region in 2000

A field isolate of Newcastle disease virus (NDV) was isolated in the Russko-Vysotskaya poultry farm, Leningrad region. Within four days after infection, the isolate caused 100% mortality in 60-day-old susceptible chickens. The HA titer of the allantoic fluid samples collected after one passage in SPF-chicken embryos was 1:512, and it reacted only with the NDV specific antiserum in HI test. Intracerebral pathogenicity index and mean embryo death time were 1.97 and 49 hours, respectively. The isolate has the amino acid sequence of the protease cleavage site of the fusion protein F0 (112R-R-Q-R-R-F117), which is similar to that in the velogenic strains of NDV. Therefore, it was concluded that the virus isolated in this work was an ethiological agent of the ND outbreak in this poultry farm.     

Experimental model of oral transmissible AA amyloidosis in quails

ABSTRACT

In poultry and zoo birds, mass outbreaks of amyloid A (AA) amyloidosis are often reported, and horizontal transmission is considered as one of the causes. However, oral transmission of avian AA amyloidosis in nature has been unclear. In order to clarify the horizontal transmission of avian AA amyloidosis, basic research using an appropriate oral transmission model is necessary. In this study, we developed an oral transmission model of AA amyloidosis using quails, and assessed the oral transmission efficiency of AA amyloidosis in quails and mice. Young quails, adult quails, and young mice received inflammatory stimulation with lipopolysaccharide; simultaneously, homogeneous amyloid fibrils were orally or intravenously administered. By histological examination, induction of amyloidosis by oral or intravenous administration of amyloid was confirmed in all species. Furthermore, both quail and murine AA amyloidosis were orally transmitted in a dose-dependent manner. These results support the possibility of horizontal transmission of avian AA amyloidosis in nature. This model will be able to contribute to the elucidation of spontaneous horizontal transmission of avian AA amyloidosis in the future.

RESEARCH HIGHLIGHTS

• Quail AA amyloidosis was orally transmitted in a dose-dependent manner.

• Oral transmission was less efficient than intravenous transmission.

• In-cage horizontal transmission did not occur during 4-week cohabitation.

• Amyloid deposition in tissues of quail was grossly visible.

     

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.     

MinION sequencing to genotype US strains of infectious laryngotracheitis virus

Over the last decade the US broiler industry has fought long-lasting outbreaks of infectious laryngotracheitis (ILTV). Previously, nine genotypes (I-IX) of ILTVs have been recognized using the polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) method with three viral alleles (gB, gM and UL47/gG). In this study, the genotyping system was simplified to six genotypes by amplicon sequencing and examining discriminating single nucleotide polymorphisms (SNPs) within these open reading frames. Using phylogenomic analysis of 27 full genomes of ILTV, a single allele (ORF A/ORF B) was identified containing SNPs that could differentiate ILTVs into genotypes congruent with the phylogenetic partitioning. The allelic variations allowed for the cataloging of the 27 strains into 5 genotypes: vaccinal TCO, vaccinal CEO, virulent CEO-like, virulent US and virulent US backyard flocks from 1980 to 1990, correlating with the PCR-RFLP genotypes I/ II/ III (TCO), IV (CEO), V (virulent CEO-like), VI (virulent US) and VII/VIII/IX (virulent US backyard flock isolates). With the unique capabilities of third generation sequencing, we investigated the application of Oxford Nanopore MinION technology for rapid sequencing of the amplicons generated in the single-allele assay. This technology was an improvement over Sanger-based sequencing of the single allele amplicons due to a booster amplification step in the MinION sequencing protocol. Overall, there was a 90% correlation between the genotyping results of the single-allele assay and the multi-allele assay. Surveillance of emerging ILTV strains could greatly benefit from real-time amplicon sequencing using the single-allele assay and MinION sequencing.

RESEARCH HIGHLIGHTS

• A multi-allelic assay identified nine ILTV genotypes circulating in the US

• Single-allele genotyping is congruent with whole genome phylogenetic partitioning

• US ILTV strains can be grouped into five genotypes using the single-allele assay

• The single-allele assay can be done using MinION sequencing of barcoded amplicons