Nephropathogenic Infectious Bronchitis Research Articles

Preparation of polyclonal antibodies to chicken P62 protein and its application in nephropathogenic infectious bronchitis virus-infected chickens

Preparation of polyclonal antibodies to chicken P62 protein and its application in nephropathogenic infectious bronchitis virus-infected chickens

Preparation of polyclonal antibodies to the chicken Beclin1 protein and its application in the detection of nephropathogenic infectious bronchitis virus

Beclin1, also known as ATG6, has been shown to be closely related to coronavirus, however, the link between Beclin1 and nephropathogenic infectious bronchitis virus (NIBV) has been poorly investigated and there are no available antibodies specifically targeting the chicken Beclin1 protein. The study aimed to prepare and assay a polyclonal antibody to Beclin1, enabling a deeper understanding of the mechanism of action of Beclin1 in NIBV. In this study, we amplified the chicken Beclin1 target gene and constructed a recombinant plasmid using prokaryotic expression techniques, then obtained the recombinant target protein by induced expression. Finally, the serum is obtained by immunizing rabbits with the purified and concentrated protein. The results show that the antiserum potency of the ELISA assay was >1:204800. By western blotting and immunofluorescence, the antibodies we prepared specifically recognized the chicken Beclin1 protein, which is mainly found in the nucleus of trachea, lung, kidney, spleen and fabricant cells. NIBV infection significantly decreased the expression of Beclin1 in the trachea, but increased in others. We have successfully prepared specific rabbit anti-chicken Beclin1 polyclonal antibodies, and detected changes in tissues of diseased chickens infected with NIBV, laying the foundation for further studies on the role of Beclin1 in avian diseases.

Baicalin mitigates nephropathogenic infectious bronchitis virus infection-induced spleen injury via modulation of mitophagy and macrophage polarization in Hy-Line chick

Nephropathogenic infectious bronchitis virus (NIBV) infections continue to pose a significant hazard in the poultry industry. Baicalin is a natural flavonoid that has been reported to have antiviral activity, but its function in NIBV infection largely remains unclear. In this study, the antiviral mechanism of baicalin in the spleen of NIBV-infected chicks was mainly elucidated in mitophagy and macrophage polarization. 28-day-old Hy-Line brown chicks were randomly divided into four groups: the group of chicks was treated intranasally (in) with normal saline (0.2 mL) and subsequently divided into two groups: the Con group (basic diet), the Con+BA group (basic diet+10 mg/kg Baicalin); another group of chicks was intranasally infected with SX9 (10−5/0.2 mL) and subsequently divided into two groups: the Dis group (basic diet), the Dis+BA group (basic diet+10 mg/kg Baicalin). Spleen tissues were collected at 3, 7, and 11 days post infection (dpi). NIBV copy number was strikingly decreased in the spleens under BA treatment with infectious time. Histopathological examination showed enlarged and hemorrhagic white pulp and no clearly defined boundary between white pulp and red pulp in the Dis group, which could be improved by BA treatment. Meanwhile, the loss of cristae structure and vacuolization in mitochondria caused by NIBV infection was repaired in the Dis+BA group by ultrastructure observation. In addition, BA treatment inhibited the induction of mitophagy by NIBV infection. BA treatment also promoted innate immunity by enhancing type I IFN levels. Moreover, BA treatment up-regulated M1-related cytokines (iNOS, TNF-α, IL-1β, IL-6) and inhibited M2-related cytokines (ARG2, IL-4, IL-10, Pparg) at the mRNA and protein levels. However, the results from the splenic tissues at 11 dpi are opposite results from 3 and 7 dpi. Immunofluorescence analysis for M1 macrophage marker iNOS and M2 macrophage marker CD163 further validated this result. Collectively, BA inhibited mitophagy and triggered IFN activation, and M1 polarization, which contributed to the inhibition of NIBV infection.

Chicken intestinal microbiota modulation of resistance to nephropathogenic infectious bronchitis virus infection through IFN-I

BackgroundMammalian intestinal microbiomes are necessary for antagonizing systemic viral infections. However, very few studies have identified whether poultry commensal bacteria play a crucial role in protecting against systemic viral infections. Nephropathogenic infectious bronchitis virus (IBV) is a pathogenic coronavirus that causes high morbidity and multiorgan infection tropism in chickens.ResultsIn this study, we used broad-spectrum oral antibiotics (ABX) to treat specific pathogen free (SPF) chickens to deplete the microbiota before infection with nephropathogenic IBV to analyze the impact of microbiota on IBV infections in vivo. Depletion of the SPF chicken microbiota increases pathogenicity and viral burden following IBV infection. The gnotobiotic chicken infection model further demonstrated that intestinal microbes are resistant to nephropathogenic IBV infection. In addition, ABX-treated chickens showed a severe reduction in macrophage activation, impaired type I IFN production, and IFN-stimulated gene expression in peripheral blood mononuclear cells and the spleen. Lactobacillus isolated from SPF chickens could restore microbiota-depleted chicken macrophage activation and the IFNAR-dependent type I IFN response to limit IBV infection. Furthermore, exopolysaccharide metabolites of Lactobacillus spp. could induce IFN-β.ConclusionsThis study revealed the resistance mechanism of SPF chicken intestinal microbiota to nephropathogenic IBV infection, providing new ideas for preventing and controlling nephropathogenic IBV.39561Y51b4Zm9kk2tsUN9pVideo abstract

Preparation of the RIPK3 Polyclonal Antibody and Its Application in Immunoassays of Nephropathogenic Infectious Bronchitis Virus-Infected Chickens.

Receptor interacting protein kinase 3 (RIPK3) is a vital serine/threonine kinase in regulating the programmed destruction of infected cells to defend against RNA viruses. Although the role of RIPK3 in viruses in mice is well characterized, it remains unclear where in nephropathogenic infectious bronchitis virus (NIBV) in chickens. Here, we use a self-prepared polyclonal antibody to clarify the abundance of RIPK3 in tissues and define the contributions of RIPK3 in tissue damage caused by NIBV infection in chickens. Western blot analyses showed that RIPK3 polyclonal antibody can specifically recognize RIPK3 in the vital tissues of Hy-Line brown chicks and RIPK3 protein is abundantly expressed in the liver and kidney. Moreover, NIBV significantly upregulated the expression levels of RIPK3 in the trachea and kidney of chicks in a time-dependent manner. In addition, the activation of necroptosis in response to NIBV infection was demonstrated by the coimmunoprecipitation (CoIP) experiments through RIPK3 in the necrosome, which phosphorylates its downstream mixed-spectrum kinase structural domain-like protein (MLKL). Our findings offered preliminary insights into the key role of RIPK3 protein in studying the underlying mechanism of organ failure caused by NIBV infection.

Nephropathogenic Infectious Bronchitis Virus Mediates Kidney Injury in Chickens via the TLR7/NF-κB Signaling Axis.

Nephropathogenic infectious bronchitis virus (NIBV) is one of the most important viral pathogens in the world poultry industry. Here, we used RT–qPCR, WB and immunofluorescence to explore the interaction between NIBV and the host innate immune system of the kidney. Multiple virions were found in the kidney tissues of the disease group under electron microscopy, and pathological changes such as structural damage of renal tubules and bleeding were observed by HE staining. In addition, we found that the mRNA levels of TLR7, TRAF6, and IKKβ were upregulated after NIBV infection. IRF7 mRNA levels decreased significantly at 5 dpi and increased significantly at 11 to 18 dpi. The NF-κB P65 mRNA level increased significantly at 5 to 18 dpi and decreased at 28 dpi. However, NIBV infection-induced NF-κB P65 protein levels were downregulated at multiple time points. Moreover, we demonstrated that the cytokine (IFN-γ, IL-8, and IL-6) mRNA and protein expression levels were increased significantly at multiple time points after NIBV infection. Furthermore, immunofluorescence analysis showed that NF-κB P65 and IFN-γ were mainly located in the nuclear or perinuclear region. The positive signal intensity of NF-κB P65 was significantly lower than that of the normal group at 1 to 5 dpi, and there was no significant change in the subsequent time period. The positive signal intensity of IFN-γ decreased significantly at 5 dpi, and increased significantly at 11 to 28 dpi. In conclusion, we found that NIBV promoted cytokine release through the TLR7/NF-κB signaling axis, thus causing kidney injury.

Investigation of the Crosstalk between GRP78/PERK/ATF-4 Signaling Pathway and Renal Apoptosis Induced by Nephropathogenic Infectious Bronchitis Virus Infection.

This study aims to explore the crosstalk between GRP78/PERK/ATF-4 signaling pathway and renal apoptosis induced by nephropathogenic infectious bronchitis virus (NIBV). Hy-Line brown chickens were divided into two groups (Con, n = 100 and Dis, n = 200). At 28 days of age, each chicken in the Dis group was intranasally injected with SX9 strain (10-5/0.2 ml). Venous blood and kidney tissues were collected at 1, 5, 11, 18 and 28 days postinfection. Our results showed that NIBV infection upregulated the levels of creatinine, uric acid, and calcium (Ca2+) levels. Histopathological examination revealed severe hemorrhage and inflammatory cell infiltration near the renal tubules. Meanwhile, NIBV virus particles and apoptotic bodies were observed by ultramicro electron microscope. In addition, RT-qPCR and Western blot showed that NIBV upregulated the expression of GRP78, PERK, eIF2α, ATF-4, CHOP, Caspase-3, Caspase-9, P53, Bax, and on the contrary, downregulated the expression of Bcl-2. Furthermore, immunofluorescence localization analysis showed that the positive expression of Bcl-2 protein was significantly decreased. Correlation analysis indicated that endoplasmic reticulum (ER) stress gene expression, apoptosis gene expression, and renal injury were potentially related. Taken together, NIBV infection can induce renal ER stress and apoptosis by activating of GRP78/PERK/ATF-4 signaling pathway, leading to kidney damage. IMPORTANCE Nephropathogenic infectious bronchitis virus (NIBV) induced renal endoplasmic reticulum stress in chickens. NIBV infection induced kidney apoptosis in chickens. GRP78/PERK/ATF-4 signaling pathway is potentially related to renal apoptosis induced by NIBV.

Pathology, tissue tropism and antibody response of nephropathogenic infectious bronchitis virus (IBV) Indian isolate in experimentally infected chicken.

The online version contains supplementary material available at 10.1007/s13337-021-00693-4.

Nephropathogenic Infectious Bronchitis Virus Infection Altered the Metabolome Profile and Immune Function of the Bursa of Fabricius in Chicken.

Infectious bronchitis is a highly contagious, acute viral respiratory disease of chickens, regardless of the strain, and its infection may lead to considerable economic losses to the poultry industry. New nephropathogenic infectious bronchitis virus (NIBV) strains have increasingly emerged in recent years; hence, evaluating their infection-influenced immune function changes and the alteration of metabolite profiling is important. Initially, chickens were randomly distributed into two groups: the control group (Con) and the disease group (Dis). Here, the partial cytokines were examined, and the metabolome alterations of the bursa of Fabricius (BF) in NIBV infections in chickens were profiled by gas chromatography time-of-flight/mass spectrometry (GC-TOF/MS). The results revealed that the NIBV infection promotes the mRNA expression of inflammatory cytokines. Metabolic profile analysis indicated that clustering differed between the two groups and there were 75 significantly different metabolites detected between the two groups, suggesting that the host metabolism was significantly changed by NIBV infection. Notably, the following 12 metabolites were identified as the potential biomarkers: 3-phenyllactic acid, 2-deoxytetronic acid, aminomalonic acid, malonamide 5, uric acid, arachidonic acid, 2-methylglutaric acid, linoleic acid, ethanolamine, stearic acid, N-alpha-acetyl-l-ornithine, and O-acetylserine. Furthermore, the results of the correlation analysis showed that a strong correlation existed between metabolic biomarkers and inflammatory cytokines. Our results describe an immune and metabolic profile for the BF of chickens when infected with NIBV and provide new biomarkers of NIBV infection as potential targets and indicators of indicating therapeutic efficacy.

Ultrastructural Changes Induced by Nephropathogenic Infectious Bronchitis Virus Isolate (IND/AHL/16/01) In Experimentally Infected Chicken.

Ultrastructural Changes Induced by Nephropathogenic Infectious Bronchitis Virus Isolate (IND/AHL/16/01) In Experimentally Infected Chicken.

16S rRNA gene sequencing reveals an altered composition of the gut microbiota in chickens infected with a nephropathogenic infectious bronchitis virus

Infectious bronchitis virus (IBV), a member of the Coronaviridae family, causes serious losses to the poultry industry. Intestinal microbiota play an important role in chicken health and contribute to the defence against colonization by invading pathogens. The aim of this study was to investigate the link between the intestinal microbiome and nephropathogenic IBV (NIBV) infection. Initially, chickens were randomly distributed into 2 groups: the normal group (INC) and the infected group (IIBV). The ilea were collected for morphological assessment, and the ileal contents were collected for 16S rRNA gene sequencing analysis. The results of the IIBV group analyses showed a significant decrease in the ratio of villus height to crypt depth (P < 0.05), while the goblet cells increased compared to those in the INC group. Furthermore, the microbial diversity in the ilea decreased and overrepresentation of Enterobacteriaceae and underrepresentation of Chloroplast and Clostridia was found in the NIBV-infected chickens. In conclusion, these results showed that the significant separation of the two groups and the characterization of the gut microbiome profiles of the chickens with NIBV infection may provide valuable information and promising biomarkers for the diagnosis of this disease.

A Multi-Omics Study of Chicken Infected by Nephropathogenic Infectious Bronchitis Virus.

Chicken gout resulting from nephropathogenic infectious bronchitis virus (NIBV) has become a serious kidney disease problem in chicken worldwide with alterations of the metabolic phenotypes in multiple metabolic pathways. To investigate the mechanisms in chicken responding to NIBV infection, we examined the global transcriptomic and metabolomic profiles of the chicken’s kidney using RNA-seq and GC–TOF/MS, respectively. Furthermore, we analyzed the alterations in cecal microorganism composition in chickens using 16S rRNA-seq. Integrated analysis of these three phenotypic datasets further managed to create correlations between the altered kidney transcriptomes and metabolome, and between kidney metabolome and gut microbiome. We found that 2868 genes and 160 metabolites were deferentially expressed or accumulated in the kidney during NIBV infection processes. These genes and metabolites were linked to NIBV-infection related processes, including immune response, signal transduction, peroxisome, purine, and amino acid metabolism. In addition, the comprehensive correlations between the kidney metabolome and cecal microbial community showed contributions of gut microbiota in the progression of NIBV-infection. Taken together, our research comprehensively describes the host responses during NIBV infection and provides new clues for further dissection of specific gene functions, metabolite affections, and the role of gut microbiota during chicken gout.

The first evidence of a new genotype of nephropathogenic infectious bronchitis virus circulating in vaccinated and unvaccinated broiler flocks in Algeria.

Background and Aim:Avian infectious bronchitis virus (IBV) frequently infects broilers and is responsible for severe economic losses to the poultry industry worldwide. It has also been associated with kidney damage in the broiler flocks. The aim of the present study is to determine the presence of IBV and its possible involvement in kidney damage of broiler chicks.Materials and Methods:14 clinically diseased broiler flocks from Western and Central Algeria were sampled and analyzed by hemagglutination inhibition (HI) test and reverse transcriptase-polymerase chain reaction (RT-PCR) followed by phylogenic analysis.Results:The QX (100%) and 4/91 (60%) IBV serotypes were the most prevalent in the kidney damaged broilers regardless of vaccination status. The molecular detection of avian IBV by RT-PCR identified six samples as positive, of which only two isolates were typable by sequencing. We identified a novel IBDZ13a genotype which showed 93% sequence homology to the partial-S1 gene sequence of the IB 4/91 commercial vaccine strain. Sequencing analysis characterized this virus as a novel and divergent IB 4/91 field virus with eight amino acid substitutions that might have resulted in altered immunogenicity.Conclusion:The isolation of a new IBV strain (IBDZ13a) from vaccinated broiler flocks may explain the failure of the vaccination programs against IBV field strains. Combination of the HI test and RT-PCR indicated that the nephropathogenic IB outbreaks in broilers are related to this novel strain.

Complete genome analysis of Iranian IS-1494 like avian infectious bronchitis virus.

The nephropathogenic infectious bronchitis virus (IBV) strain IS-1494 like (variant-2; GI-23) was first isolated in the Middle East (1998). Despite intensive vaccinations, IS-1494 like IBVs are still circulating in Iran (the dominant genotype) and spread to other countries. Here, the full-length genome of this Iranian IS-1494 like IBV was (Mahed) determined to understand its evolutionary relationships. The genome consists of 27,652 nucleotides, with mutations in most of the structural genes. Thirteen open reading frames (ORFs) were predicted in the Mahed isolate (5′ UTR-1a-1b-S-3a-3b-E-M-4b-4c-5a-5b-N-6b-3′ UTR). ORFs 4b, 4c, and 6b, which has rarely been reported, were present in the Mahed genome. According to phylogenetic analysis of the full-length genome, 1a, S2, M, E, N protein, Mahed isolate clustered with the QX type strain. Based on the partial 1b, S1, Mahed clustered with the Q1 strain. The full-length genome of Mahed isolate shared the highest sequence homology with Gray and JMK (90.06–90.07%) and was least related to the Vic-s (86.21%). These data show that evolutionary variation because of recombination in IBV plays a major role in the adaptation and origin of IBV leading to new genetic and types of the virus strain.Electronic supplementary materialThe online version of this article (10.1007/s13337-018-0462-4) contains supplementary material, which is available to authorized users.

English

English

Comparative transcriptome analysis reveals induction of apoptosis in chicken kidney cells associated with the virulence of nephropathogenic infectious bronchitis virus

Avian infectious bronchitis virus (IBV) that causes respiratory and nephritic diseases in chicken is a major poultry pathogen leading to serious economic loss worldwide. The nephropathogenic IBV strains cause nephritis and kidney lesions intrinsically and the pathogenic mechanism is still unclear. In the present study, SPF chicks were infected with three nephropathogenic IBVs of different virulence and their gene expression profiles in chicken kidney were compared at transcriptome level. As a result, 1279 differentially expressed (DE) genes were found in very virulent SCDY2 inoculated group, 145 in virulent SCK2 group and 74 in non-virulent LDT3-A group when compared to mock infected group. Gene Ontology (GO) and KEGG pathway enrichment analysis on SCDY2 group displayed that the up-regulated DE genes were mainly involved in cell apoptosis, and the down-regulated genes were involved in metabolic processes and DNA replication. Protein-Protein Interaction (PPI) analysis showed that DE genes in SCDY2 group formed a network, and the core of the network was composed by cell apoptosis and immune response proteins. The clustering of gene expression profile among the three virus inoculated groups indicated that the majority of up-regulated DE genes on apoptosis in very virulent SCDY2 group were up-regulated more or less in virulent SCK2 group and those down-regulated on innate immune response in SCDY2 group were also down-regulated differently in SCK2 group. In addition, the number of apoptotic cells detected experimentally in kidney tissue were very different among the three virus inoculated groups and were positively accordant with the viral titer, kidney lesions and viral virulence of each group. Taken all together, the present study revealed that virulent nephropathogenic IBV infection modified a number of gene expression and induction of apoptosis in kidney cells may be a major pathogenic determinant for virulent nephropathogenic IBV.

The relationship between liver-kidney impairment and viral load after nephropathogenic infectious bronchitis virus infection in embryonic chickens

To examine the relationship of impairments of the liver and kidney with viral load after nephropathogenic infectious bronchitis virus (NIBV) infection in embryonic chickens, 120 specific-pathogen-free Leghorn embryonated chicken eggs were randomly divided into two groups (infected and control), with three replicates per group and 20 eggs in each replicate. The eggs in the infected and control groups were challenged with 0.2 mL of 105.5 ELD50 NIBV and sterile saline solution, respectively. The embryonic chickens’ plasma and liver and kidney tissues were collected at 1, 3, and 5 days post-inoculation (dpi), the liver and kidney functional parameters were quantified, and the tissue viral loads were determined with real-time PCR. The results showed that plasma potassium, sodium, chlorine, magnesium, calcium, and phosphorus levels were increased. The infected group exhibited significantly higher plasma uric acid, blood urea nitrogen, and creatinine levels than the control group at 3 dpi. The plasma concentrations of aspartate aminotransferase and alanine aminotransferase were significantly increased in the infected group. The total protein, albumin, and globulin levels in the infected group were significantly lower than those in the control group. The liver-kidney viral load in the infected group peaked at 3 dpi, at which time the kidney viral load was significantly higher than that of the liver. Our results indicated that NIBV infection caused liver and kidney damage in the embryonic chickens, and the results also demonstrated that the liver and kidney damage was strongly related to the tissue viral load following NIBV infection in embryonic chickens.

Productive replication of nephropathogenic infectious bronchitis virus in peripheral blood monocytic cells, a strategy for viral dissemination and kidney infection in chickens.

In the present study, the replication kinetics of nephropathogenic (B1648) and respiratory (Massachusetts-M41) IBV strains were compared in vitro in respiratory mucosa explants and blood monocytes (KUL01+ cells), and in vivo in chickens to understand why some IBV strains have a kidney tropism. B1648 was replicating somewhat better than M41 in the epithelium of the respiratory mucosa explants and used more KUL01+ cells to penetrate the deeper layers of the respiratory tract. B1648 was productively replicating in KUL01+ monocytic cells in contrast with M41. In B1648 inoculated animals, 102.7–6.8 viral RNA copies/100 mg were detected in tracheal secretions at 2, 4, 6, 8, 10 and 12 days post inoculation (dpi), 102.4–4.5 viral RNA copies/mL in plasma at 2, 4, 6, 8, 10 and 12 dpi and 101.8–4.4 viral RNA copies/106 mononuclear cells in blood at 2, 4, 6 and 8 dpi. In M41 inoculated animals, 102.6–7.0 viral RNA copies/100 mg were detected in tracheal secretions at 2, 4, 6, 8 and 10 dpi, but viral RNA was not demonstrated in plasma and mononuclear cells (except in one chicken at 6 dpi). Infectious virus was detected only in plasma and mononuclear cells of the B1648 group. At euthanasia (12 dpi), viral RNA and antigen positive cells were detected in lungs, liver, spleen and kidneys of only the B1648 group and in tracheas of both the B1648 and M41 group. In conclusion, only B1648 can easily disseminate to internal organs via a cell-free and -associated viremia with KUL01+ cells as important carrier cells.

Emergence of novel nephropathogenic infectious bronchitis viruses currently circulating in Chinese chicken flocks

ABSTRACTThe emergence of novel infectious bronchitis viruses (IBVs) has been reported worldwide. Between 2011 and 2014, eight IBV isolates were identified from disease outbreaks in northeast China. In the current study we analysed the S1 gene of these eight IBV isolates in addition to the complete genome of five of them. We confirmed that these isolates emerged through the recombination of LX4 and Taiwan group 1 (TW1) viruses at two switch sites, one was in the Nsp 16 region and the other in the spike protein gene. The S1 gene in these viruses exhibited high nucleotide similarity with TW1-like viruses; the TW1 genotype was found to be present in southern China from 2009. Pathogenicity experiments in chickens using three of the eight virus isolates revealed that they were nephropathogenic and had similar pathogenicity to the parental viruses. The results of our study demonstrate that recombination, coupled with mutations, is responsible for the emergence of novel IBVs.

Genetic Characterization of the Belgian Nephropathogenic Infectious Bronchitis Virus (NIBV) Reference Strain B1648

The virulent nephropathogenic infectious bronchitis virus (NIBV) strain B1648 was first isolated in 1984, in Flanders, Belgium. Despite intensive vaccination, B1648 and its variants are still circulating in Europe and North Africa. Here, the full-length genome of this Belgian NIBV reference strain was determined by next generation sequencing (NGS) to understand its evolutionary relationship with other IBV strains, and to identify possible genetic factors that may be associated with the nephropathogenicity. Thirteen open reading frames (ORFs) were predicted in the B1648 strain (5′UTR-1a-1b-S-3a-3b-E-M-4b-4c-5a-5b-N-6b-3′UTR). ORFs 4b, 4c and 6b, which have been rarely reported in literature, were present in B1648 and most of the other IBV complete genomes. According to phylogenetic analysis of the full-length genome, replicase transcriptase complex, spike protein, partial S1 gene and M protein, B1648 strain clustered with the non-Massachusetts type strains NGA/A116E7/2006, UKr 27-11, QX-like ITA/90254/2005, QX-like CK/SWE/0658946/10, TN20/00, RF-27/99, RF/06/2007 and SLO/266/05. Based on the partial S1 fragment, B1648 clustered with the strains TN20/00, RF-27/99, RF/06/2007 and SLO/266/05 and, further designated as B1648 genotype. The full-length genome of B1648 shared the highest sequence homology with UKr 27-11, Gray, JMK, and NGA/A116E7/2006 (91.2% to 91.6%) and was least related with the reference Beaudette and Massachusetts strains (89.7%). Nucleotide and amino acid sequence analyses indicated that B1648 strain may have played an important role in the evolution of IBV in Europe and North Africa. Further, the nephropathogenicity determinants might be located on the 1a, spike, M and accessory proteins (3a, 3b, 4b, 4c, 5a, 5b and 6b). Overall, strain B1648 is distinct from all the strains reported so far in Europe and other parts of the world.