IBV Isolates Research Articles

Molecular characterisation of influenza B virus from the 2017/18 season in primary models of the human lung reveals improved adaptation to the lower respiratory tract

ABSTRACT The 2017/18 influenza season was characterized by unusual high numbers of severe infections and hospitalizations. Instead of influenza A viruses, this season was dominated by infections with influenza B viruses of the Yamagata lineage. While this IBV/Yam dominance was associated with a vaccine mismatch, a contribution of virus intrinsic features to the clinical severity of the infections was speculated. Here, we performed a molecular and phenotypic characterization of three IBV isolates from patients with severe flu symptoms in 2018 and compared it to an IBV/Yam isolate from 2016 using experimental models of increasing complexity, including human lung explants, lung organoids, and alveolar macrophages. Viral genome sequencing revealed the presence of clade but also isolate specific mutations in all viral genes, except NP, M1, and NEP. Comparative replication kinetics in different cell lines provided further evidence for improved replication fitness, tolerance towards higher temperatures, and the development of immune evasion mechanisms by the 2018 IBV isolates. Most importantly, immunohistochemistry of infected human lung explants revealed an impressively altered cell tropism, extending from AT2 to AT1 cells and macrophages. Finally, transcriptomics of infected human lung explants demonstrated significantly reduced amounts of type I and type III IFNs by the 2018 IBV isolate, supporting the existence of additional immune evasion mechanisms. Our results show that the severeness of the 2017/18 Flu season was not only the result of a vaccine mismatch but was also facilitated by improved adaptation of the circulating IBV strains to the environment of the human lower respiratory tract.

Evaluating Disease Threats to Sustainable Poultry Production in Africa: Newcastle Disease, Infectious Bursal Disease, and Avian Infectious Bronchitis in Commercial Poultry Flocks in Kano and Oyo States, Nigeria.

The growth of the poultry industry in Nigeria is constrained by major poultry diseases, despite the implementation of vaccination programs. This study aimed to assess the level of protection against Newcastle disease (ND), infectious bursal disease (IBD), and avian infectious bronchitis (IB) afforded by current vaccination schedules and characterize the circulating virus strains in commercial poultry flocks in Nigeria. A cross-sectional study was conducted on 44 commercial poultry farms in Oyo and Kano states of Nigeria. Serum and tissue samples and data on flock, clinical and vaccination records were collected on each farm. Farms were classified as being protected or not protected against ND, IBD and IB based on a defined criterion. Real-time reverse transcription polymerase chain reaction (rRT-PCR) testing was performed for each target virus on tissue samples and positive samples were sequenced. A total of 15/44 (34.1%), 35/44 (79.5%), and 1/44 (2.3%) farms were considered to be protected against ND, IBD, and IB, respectively, at the time of sampling. NDV RNA was detected on 7/44 (15.9%) farms and sequences obtained from 3/7 farms were characterized as the lentogenic strain. Infectious bursal disease virus (IBDV) RNA was detected on 16/44 (36.4%) farms tested; very virulent (vv) IBDV and non-virulent (nv) IBDV strains were both detected in 3/16 (18.8%) positive samples. Sequences of IBDV isolates were either clustered with a group of genotype 3 virulent IBDV strains or were related to vaccine strains MB and D78 strains. IBV RNA was detected on 36/44 (81.8%) farms, with variant02, Massachusetts, 4/91, and Q1 variants detected. Sequences of IBV isolates were either clustered with the vaccines strains Massachusetts M41 and H120 or were most closely related to the D274-like strains or a clade of sequences reported in Nigeria and Niger in 2006 and 2007. This study revealed that most study farms in Oyo and Kano states did not have adequate protective antibody titers against IBV and NDV and were therefore at risk of field challenge. Infectious bursal disease virus and IBV RNA were detected on farms with a history of vaccination suggesting potential vaccination failure, or that the vaccine strains used mismatch with the circulating strains and are therefore not protective.

Pathogenicity differences between a newly emerged TW-like strain and a prevalent QX-like strain of infectious bronchitis virus

TW-like IBV isolates have appeared frequently in recent years in mainland China. In this study, we compared the TW-like IBV GD strain and the predominant QX-like SD strain in terms of serology and pathogenicity to 3-week-old specific-pathogen-free chickens. Both strains could cause severe respiratory distress and renal lesions, with a mortality rate were approximately 20%. Virus were continuously shed via the respiratory tract and cloaca. However, the infection pattern of the two isolates were different. The GD strain persisted for a longer duration and caused extensive damages to the tracheas and lungs. Moreover, chickens infected with the GD strain showed inefficient recovery of damaged cilia after infection. Our findings suggested that the newly emerged TW-like IBV GD strain showed obvious differences in pathogenicity, tissue tropism and replication efficiency compared with the QX-like IBV SD strain, with the TW-like GD strain showing stronger tropism to the respiratory tract and a longer duration of clinical signs.

GENETIC CHARACTERIZATION OF AVIAN INFECTIOUS BRONCHITIS VIRUS ISOLATES RECOVERED IN CIS COUNTRIES IN 2015–2017

Avian infectious bronchitis virus is a cause of major economic losses in poultry industry. However, control of the virus is very complicated due to its high variability. The mutation frequency in the hypervariable region of the S1 gene of the virus isolated from the vaccinated birds annually amounts to 1.5%. Long-term observations of the circulation of IBV isolates detected in a number of poultry farms demonstrated that the virus genetic lineages circulating on the poultry farms could eventually change. This stipulates the need for the continuous monitoring of the virus isolates for the prevention schedule optimization. The paper demonstrates test results of 840 biological samples collected from chickens on the poultry farms in Russia and some CIS countries in 2015–2017. From 311 positive samples 147 IBV isolates were recovered, the majority of which belonged to eight genetic lines of GI genotype: GI-1, GI-12, GI-13, GI-14, GI-16, GI-19, GI-22, GI-23. Moreover, recombinant isolates were detected as well as variant isolates that belonged to none of the known genotypes.

Genetics, antigenicity and virulence properties of three infectious bronchitis viruses isolated from a single tracheal sample in a chicken with respiratory problems

Three different IBV genotypes/serotypes, designated ck/CH/LDL/150434–I (LDL/150434–I), ck/CH/LDL/150434–II (LDL/150434–II) and ck/CH/LDL/150434–III (LDL/150434–III), were detected in a single tracheal sample from a chicken showing signs of respiratory disease. The viruses were isolated using a cross-neutralization test and limiting dilution in embryonated specific-pathogen-free (SPF) eggs. Isolate LDL/150434–I was a re-isolation of H120 vaccine strain that was introduced into the chicken flock by vaccination, transmitted between chickens, and later accumulated several genomic mutations. Isolate LDL/150434–II was a novel variant that originated from recombination events between H120 and ck/CH/LDT3/03-like viruses. The widespread use of H120 vaccine, which offered incomplete protection against heterotypic IBVs in the fields, may play important roles in the emergence of such a novel genetic variant. Based on the analysis of S1 and complete genomic sequence, isolate LDL/150434–III was related genetically but distinct from the established strains of nrTW I type viruses of GI-7 lineage circulating in Mainland China since 2009. The three IBV isolates were avirulent when they infected SPF chickens. Furthermore, synergistic effects on pathogenicity were not observed when the different types co-infected the SPF chickens. However, the isolates persisted in the respiratory tracts longer in combined infected birds than those in individual infected birds. The results provide insights into the evolution of the viruses and co-infection of chickens with different virus serotypes.

Evaluation of Protection Spectrum Provided Against Two Infectious Bronchitis Virus Isolates Using Some Commercially Available Vaccines

Abstract: The protection of the commercially available vaccines was evaluated against two Egyptian variant IBV isolates that was found to be related to IS/885 variant II strain by 86% and 84% respectively in commercial broiler chickens. The protection assessment was detected by clinical signs, gross lesions, detection of viral shedding in tracheal and cloacal swabs, estimation of kidney function tests (Creatinine and Uric acid), detection of specific IBV antibody titer and histopathological examination. No significant protection could be provided against both challenging viruses using the classical live H120, Ma5 vaccines at day 1 and 14 of age respectively. Also no significant protection could be provided using 2 different inactivated vaccines at day 5 of age. The results indicated that the used vaccine strains and the challenging IBV field isolates are not the same protecteotype, therefore, change in the program of field vaccination is required to obtain a relative improvement of protection.

Isolation and Molecular Characterization of Infectious Bronchitis Virus from Broiler Chickens, Egypt during 2014 -2016

In this study,surveillance on infectious bronchitis virus strains in 75 Egyptian broiler chicken flocks was performed using real-time RT-PCR at eight Governorates during 2014-2016. The birds in the selected flocks were suffering from respiratory and renal manifestations. The results of the present study showed that 31 (41%) samples were positive for IBV. All isolates were classified as variants using sequencing analysis of the obtained data and can be divided into 2 groups. Group I (variant) Egyptian IBV included isolates, EG-F728-1-2015-SP1, EG-F728-7-2015-SP1, EG-F862-3-2015-SP1, EG-F862-4-2015-SP1, EG-F888-2015-SP1, EG-F777-2015-SP1 and EG-F183-1-2016-SP1 which were very close to IBV-IS-1494-06 and had amino acid sequence identities between 87.5% to 96.2%. Group II (variant) Egyptian IBV included isolates, EG-F183-2-2016-SP1 and EG-F183-3-2016-SP1. These two IBV isolates were very close to IBV-IS/885 (variant 2) and they shared amino acid sequence identities from 92.3% to 97.1% with each other. In conclusion, different variants of IBV are present in broiler farms in high prevalence rate even in vaccinated flocks.

Molecular characterization of recent Iranian infectious bronchitis virus isolates based on S2 protein gene

Background: Avian infectious bronchitis (IB), with avianinfectious bronchitis virus (IBV) as the causing agent, is a ubiquitous endemicdisease of the chicken with devastating effects on its industry. A viralmembrane surface protein called S notonly induces neutralizing antibodies but also plays an important role in virusbinding and entry to host cells. Technically, S1 protein gene sequencing alsohelps greatly in IBV genotyping. OBJECTIVES: The aim of this study wasto characterize Iranian IBV based on S2 gene. METHODS: After RT-PCRamplification, the S2 gene of nine Iranian IBV isolates were sequenced and thencompared with reference strains. RESULTS: The isolates were classifiedinto genotype I as Massachusetts like IB Vs, genotype VII which clustered intotwo branches, VIIa (IS-1494 like IB viruses), and VIIb, and was related to QX-like viruses and Genotype VIII as 793/B like IBVs. CONCLUSIONS: As faras we know, this is the first S2-based classification study on Iranian IBVisolates providing a firm experimental basis to correlate with genotypiccharacterization

Molecular Studies on Infectious Bronchitis Virus Isolated from Broiler Chickens in Damietta Governorate, Egypt

Fifty-two samples of tracheal swabs and organs were collected from broiler chickens at different districts from Damietta Governorate during December 2012 to August 2014. The samples were collected from chicken farms suffered from respiratory signs, diarrhea and high mortality. Thirty-six samples tested positive for IBV infection when screened by real time quantitative polymerase chain reaction (RT-qPCR). Seven of them were subjected for further isolation and characterization of the virus. The seven selected IBV isolates were propagated in embryonated chicken eggs (ECE) and were confirmed using PCR amplification of the partial sequence of the S1 spike gene. The amplified products were sequenced and the phylogenetic analysis was performed. The seven IBV viruses were closely related to each other with 94-100% nucleotide identity and clustered within the same group with IBV/Eg/CLEVB-2/IBV/012 and Eg/12120s/2012 strains (variant 2-like strain), while their identity with the vaccinal strains used in Egypt was ranged from 67-91%, particularly D274 (89%-91%). These results indicated the complexity of IBV control in relation to the current vaccination programs in Damietta.

Isolation and Molecular Identification of IBV Isolates in Different Governorates in Egypt

Tracheal swabs and different organs are collected from 17 chicken farms showing respiratory signs and variable mortalities in different governorates. Three successive blind serial passages were performed. Four IBV isolates are detected in vaccinated chickens by RT-PCR and are identified by sequence and phylogenetic analysis of portion of Si gene. Two IBV isolates, IBV S40 and IBV S61 are related to Mass reference strains (Egypt/F/03, M41, H120, Ma5, and M52). However, IBV S78 and IBV S82 are related to Egyptian variant 2 IBV strains Ck/Eg/BSU-2/2011 and Ck/Eg/ BSU-3/201 1.These results indicate the continuous evolution of Egyptian IBV circulating in chickens despite vaccination using H120 live attenuated vaccine.

Molecular and antigenic characteristics of Massachusetts genotype infectious bronchitis coronavirus in China

In this study, 418 IBVs were isolated in samples from 1717 chicken flocks. Twenty-nine of the isolates were classified as the Massachusetts genotype. These 29 isolates, as well as two previously isolated Massachusetts genotype IBV strains, were studied further. Of the 31 strains, 24 were H120-like and two were M41-like isolates as determined by complete genomic sequence analysis, indicating that most of the IBV isolates were likely the reisolated vaccine virus. The remaining five IBV isolates, ck/CH/LHB/111172, ck/CH/LSD/111219, ck/CH/LHB/130598, ck/CH/LDL/110931, and ck/CH/LHB/130573, were shown to have originated from natural recombination events between an H120-like vaccine strain and other types of viruses. The virus cross-neutralization test found that the antigenicity of ck/CH/LHB/111172, ck/CH/LSD/111219, and ck/CH/LHB/130598 was similar to that of H120. Vaccination with the H120 vaccine offered complete protection against challenge with these isolates. However, isolates ck/CH/LDL/110931 and ck/CH/LHB/130573 were serotypically different from their parental viruses and from other serotypes in this study. Furthermore, vaccination with the H120 vaccine did not provide protection against challenge with these two isolates. The results of this study demonstrated that recombination is the mechanism that is responsible for the emergence of new serotype strains, and it has the ability to alter virus serotypes. Therefore, IBV surveillance of chicken flocks vaccinated with IBV live vaccines, as well as the consideration of new strategies to effectively control IBV infection using inactivated or/and genetically engineered vaccines, is of great importance.

Phylogenetic analysis of avian infectious bronchitis virus S1 glycoprotein regions reveals emergence of a new genotype in Moroccan broiler chicken flocks.

BackgroundInfectious bronchitis virus (IBV), a major pathogen of commercial poultry flocks, circulates in the form of several serotypes/genotypes. Only a few amino-acid changes in the S1 subunit of wild-type IBVS proteins may result in mutants unaffected by current vaccines.MethodsPartial S1 gene sequences of 3 IBV isolates of the Moroccan Italy 02 genotype from vaccinated and unvaccinated broiler chicken flocks, located in southern and central regions of Morocco, were amplified by RT-PCR, sequenced, and aligned for phylogenetic and amino-acid similarity analyses.ResultsThe three isolates were found genetically highly distant from known avian IBV based on partial sequences of their S1 genes: gammaCoV/chicken/Morocco/I01/2011(IBV/Morocco/01), gammaCoV/chicken/Morocco/I30/2010 (IBV/Morocco/30), and gammaCoV/chicken/Morocco/I38/2013 (IBV/Morocco/38), nucleotide sequence identities reached 89.5 % to 90.9 % among the three isolates. The deduced protein sequence identities ranged from 29.7 % (between IBV/Morocco/38 and Egypt SCU-14/2013-1) to 78.2 % (between IBV/Morocco/01 and Spain/05/866). Amino acid sequence comparison and phylogenetic analysis indicated the emergence of a new Moroccan genotype, clustering with regionally related isolates from Spain (Spain/05/866) and belonging to a new sub-genotype.ConclusionOur sequencing results demonstrate a co-circulation of wild-type infectious bronchitis viruses in broiler chickens. These results justify permanent monitoring of circulating strains in order to rationally modify vaccination strategies to make them appropriate to the evolving field situation.

Isolation and molecular characterization of nephropathic infectious bronchitis virus isolates of Gujarat state, India.

Infectious bronchitis (IB) is a common, highly contagious, acute, and economically important viral disease of chickens caused by Infectious bronchitis virus (IBV, sp. Avian coronavirus). Five pooled tissue suspensions of 50 layer birds and one reference Massachusetts vaccine strain were inoculated into specific pathogen free (SPF) chicken egg for isolation of IBV. Reverse-transcription polymerase chain reaction (RT-PCR) was carried out using post inoculated allontoic fluid to amplify the spike (S) glycoprotein of S1 subunit of IBV. All the eggs inoculated with five pooled tissue samples and vaccine sample showed dwarfing and curling of SPF embryos indicative of IBV. All the five samples and the vaccine sample produced the expected amplicons of 466 bp by RT-PCR. The sequencing of five isolates revealed that all the five sequences were 99.09–100 % similar among themselves and showed 99.10–100 % nucleotide identity with the vaccine strain. On multiple sequence alignment it was found that our isolates were more similar at S1 subunit nucleotide level with the reference Ma5 and H120 vaccine strains than the reference Mass41 strain. The sequences of Anand isolates revealed further genetic changes in the circulating IBV in comparison to previous isolate of Gujarat as well as higher differences with the strains isolated in other states showing substantial changes at genetic level in Indian IBV isolates, which may partially explain the increasing incidences of IB in the country in spite of the vaccination.Electronic supplementary materialThe online version of this article (doi:10.1007/s13337-015-0248-x) contains supplementary material, which is available to authorized users.

Characterization and analysis of the full-length genome of a strain of the European QX-like genotype of infectious bronchitis virus

In recent years, strains of infectious bronchitis virus belonging to the QX-like genotype have been causing huge economic losses in commercial chicken flocks in different countries in Europe. In order to expand the knowledge of the molecular features of these viruses, we have sequenced and characterized the complete genome of European QX-like IBV strain CK/SWE/0658946/10, which was isolated in 2010 in Sweden. The genome is 27664 nucleotides in length, comprising six genes and 5' and 3' untranslated regions. The ORF1a, spike and nucleocapsid genes were under strong positive selective pressure that resulted in genetic diversity in relation to classical IBV isolates. The full-length genome of the CK/SWE/0658946/10 strain has the highest nucleotide sequence identity (93.18%) to ITA/90254/2005 and the lowest nucleotide identity (89.10%) to strain CQ04-1. Phylogenetic analysis of partial S1 gene sequences of IBV strains showed that the European QX-like genotype comprises strains that have been predominantly circulating in this continent for the past decade.

Phylogenetic distribution and predominant genotype of the avian infectious bronchitis virus in China during 2008-2009.

BackgroundThe nephropathogenic avian infectious bronchitis (IB) caused unprecedented economic losses to the commercial chicken industry of China in 2008-2009. To investigate the prevalence of nephropathogenic IB in China, eighty IBV isolates from different provinces during 2008-2009 were identified by dwarf embryo test and RT-PCR.ResultsThe strains were mostly isolated in winter and spring with a wide age range of IB outbreaks, from 4 to 69 days. By the virus recovery trials, 70/80 of the strains resulted in the deaths or distresses of birds from nephritis. To learn more about the molecular evolutionary characteristics of the circulating field strains, the coding region of major spike 1 (S1) protein gene of these strains was RT-PCR amplified and sequenced. Compared to the published representative strains, nucleotides and amino acids sequence analysis indicated that the S1 genes of these strains and the reference strains displayed homologies ranging from 75.1% to 99.8% and from 73.1% to 99.8% respectively. S1 protein of the major pandemic strains contained 540 or 542 amino acids with the cleavage site of HRRRR or RRFRR. Phylogenetic analysis revealed that recent field isolates of IBV in China were mostly belonged to A2-branch (QXIBV-branch) and HN08-branch, only one isolate was belonged to Gray-branch and M41-branch respectively. Most of the 80 strains showed evolutionarily distant from vaccine strains.ConclusionsThe results of this study suggested that nephropathogenic IBVs were mainly A2-like strains in China during 2008-2009.

Sequence and phylogenetic analysis of S1, S2, M, and N genes of infectious bronchitis virus isolates from Malaysia

Two Malaysian infectious bronchitis virus isolates, MH5365/95 and V9/04 were characterized based on sequence and phylogenetic analyses of S1, S2, M, and N genes. Nucleotide sequence alignments revealed many point mutations, short deletions, and insertions in S1 region of both IBV isolates. Phylogenetic analysis of S1 gene and sequences analysis of M gene indicated that MH5365/95 and V9/04 belong to non-Massachusetts strain. However, both isolates share only 77% identity. Analysis based on S1 gene showed that MH5365/95 shared more than 87% identity to several Chinese strains. Meanwhile, V9/04 showed only 67-77% identity to all the previously studied IBV strains included in this study suggesting it is a variant of IBV isolate that is unique to Malaysia. Phylogenetic analysis suggests, although both isolates were isolated 10 years apart from different states in Malaysia, they shared a common origin. Analysis based on S2 and N genes indicated that both strains are highly related to each other, and there are fewer mutations which occurred in the respective genes.

Produção e caracterização de anticorpos monoclonais contra o vírus da bronquite infecciosa das galinhas

Um painel de anticorpos monoclonais (AcM) específicos contra o vírus da bronquite infecciosa das galinhas (VBIG) amostra Massachusetts M41 foi desenvolvido com a finalidade de possibilitar ferramentas para futuros estudos de amostras locais de VBIG. Os clones produtores de AcM foram detectados e caracterizados por ELISA, e para a determinação quanto à especificidade ao componente de VBIG foi utilizado western blotting (WB). Os híbridos produtivos foram clonados por diluição limitante, expandidos in vitro e mantidos em nitrogênio líquido. As bandas protéicas reconhecidas em WB pelos AcM apresentaram pesos moleculares que variaram de 180 a 30 kDa. Oito AcM reconheceram apenas um polipeptídeo e quatro ligaram-se ao polipeptídeo S (3, 18, 52 e 57) inteiro, com a banda de peso molecular aproximado de 180 kDa. Cinco AcM (5, 12, 41, 70 e 72) ligaram-se em mais de uma banda de VBIG. Outros sete AcM não se ligaram a nenhum polipeptídeo de VBIG. Entre os AcM que apresentam perspectivas de utilização em pesquisa e diagnóstico de VBIG incluem-se os produzidos pelo clone 42, dirigido contra S1, os produzidos pelos clones 34, 43 e 71, dirigidos contra S2, pelos clones 7 e 22, dirigidos contra M e pelos clones 15 e 50, cujos AcM reagiram contra a nucleoproteína N.

Differentiation and Detection of Infectious Bronchitis Virus Subtypes by Immunofluorescence

A wide range of cross-reactions among strains was found when immunofluorescence was used as a method for differentiation of IBV strains. Only conjugates prepared against 97 and Massachusetts strains were able to detect all the strains of IBV included in this study. The fluorescence with heterologous strains was weaker than the one present when tested with homologous strains. When the rinsing period after staining was extended from 10 minutes to 2 hours, the cross-reactions were markedly diminished, and only 97 and Holte conjugates gave bright fluorescence with other than their homologous strains. Holte conjugate gave good fluorescence with virus 97, and the 97 and 609 conjugates with Gray virus. Thus, the fluorescent antibodies appear to offer possibilities for the differentiation of IBV isolates. When infected hens were examined, IBV antigen was detected by fluorescent antibodies only in the larynx, trachea, and lung. The conjugates contained antibodies against Marek's Disease agent. In addition they produced some fluorescence which behaved in the bird tissues as a specific reaction but was eliminated by absorption with chicken or rabbit liver tissue powder. Therefore, when diagnosing IBV or any other disease in chicken tissue one should be aware of the possibility of extraneous antibodies or antigens even in chickens which appear healthy and are from a specific pathogenfree flock.