Molecular epidemiology of IBDV in Italy: emergence of Northwestern European reassortants (genotype A3B1) and co-circulation with local strains

ABSTRACTReassortment among genome segments of infectious bursal disease virus (IBDV) field isolates was reported frequently worldwide, however the pathogenicity of the reassortant field IBDV is poorly understood. In this paper, a pathogenicity study on four representative IBDV field strains isolated from Southern China between 2005 and 2011 was conducted. Twenty-eight-day-old Three-Yellow chickens were divided into four groups and were inoculated intraocularly with one of the four field IBDV strains, namely NN1172, NN1005, GD10111 and JS7, respectively. The mortality and relative weight of bursa and thymus were subsequently determined in the acute phase of infection. In addition, B cells, T cells (CD4+ and CD8+) and virus were quantified in the bursa of Fabricius and thymus, respectively, by flow cytometry and real-time reverse transcription-polymerase chain reaction. The results showed that isolate NN1172, of which parts of segment A and B encoding the hypervariable (v) region of viral protein (VP2) and VP1, respectively, derived from vvIBDV strains, showed the most severe pathogenicity, and caused the most severe bursal B cell depletion as well as CD4+ and CD8+ T cell infiltration in the bursa of Fabricius. However, the virus induced the strongest decrease in CD4+ and CD8+ T cells in the thymus and exhibited the most efficient viral replication in the target organs. Isolate NN1005, whose vVP2 derived from vvIBDV and VP1 from unidentified origin, exhibited relatively lower pathogenicity compared to NN1172. The other two isolates, JS7 and GD10111, of which the vVP2 derived from vvIBDV and intermediate IBDV, and VP1 from 002–73 and attenuated IBDV, respectively, showed the lowest level of virulence. Our results suggest that various IBDV field isolates with different natural segment reassortments exhibit differential pathogenicity after infection of commercial Three-Yellow chickens.

Outbreaks of infectious bursal disease (IBD) still continue to afflict the Tunisian poultry industry even in those flocks where the vaccination program is strictly applied. To characterize the viruses that circumvent protection provided by vaccination, field isolates of infectious bursal disease virus (IBDV) obtained from vaccinated flocks that have repeatedly experienced IBDV outbreak episodes were analyzed from bursal samples by reverse transcription coupled with polymerase chain reaction and dideoxynucleotide sequencing of the VP2 hypervariable region. Although sequence data were obtained from samples collected from three distinct flocks over a period of 3 years, only limited sequence variation has been observed. The few nucleotide changes were silent and the deduced amino acid sequences were identical. Thus, the virus population that predominates in the field seems to represent a homogeneous antigenic pool. Compared with the VP2 sequences of several IBDV strains, this predominant pool was found to be closely related to the very virulent (vv) IBDV viruses described in Europe and Asia. Sequence and phylogenetic analysis of the precursor polyprotein coding sequence of a representative Tunisian isolate further confirmed its assignment to the vv genotype. The deduced amino acid sequence of the whole polyprotein of the Tunisian isolate was found to be identical to a South Korean IBDV strain. Alignment of the polyprotein amino acid sequence of 35 IBDV strains identified additional mutations outside the VP2 variable domain and which occur frequently in vv strains. Based on this comparative analysis, the set of amino acid residues that should represent a typical vv profile involves Ala222, Ile242, Ile256, Ile294, Leu451, Tyr680, N685, Ser715, Asp751, Val990, and Ala1005. Such a combination of amino acid changes was observed for the majority of vvIBDV strains that define a distinct phylogroup.

The role of T cells in protection by an inactivated infectious bursal disease virus vaccine

IBDV is still one of the most important causes of significant economic losses in poultry industry in Egypt, although implementation of the intensive vaccination programs. A recent increase in IBDV related mortality in most of vaccinated chicken broiler farms in different governorates, prompted to investigate the molecular characters of the circulating IBDV. In present study, 32 bursa samples were assayed in Kaferelsheikh, Menofia, Gharbia and Behera. The virus was isolated by inoculating bursa suspension into embryonated specific pathogen-free (SPF) eggs. CAMs collected and the virus detected by reverse transcriptase-polymerase chain reaction (RT-PCR) for IBDV targeting VP2 gene. Out of the tested samples 20 were positives. Ten IBDV-positive samples were selected for further isolation and characterization. Sequence analysis of PCR products of 10 selected samples was carried out. Eight samples were characterized as very virulent (vvIBDV) and two samples were similar to classical IBDV and vaccine strains. The genotyping of Egyptian vvIBDV indicate progressive evolution of IBDV in Egypt.

Background: Infectious bursal disease (IBD) is an economically important disease of birds which is controlled largerly by vaccination with live attenuated vaccine. The virus is more pathogenic in birds above three weeks of age. The problem of immuno-suppression in birds is one of the leading cause of increase in incidences and intensity of already existing diseases and also of new emerging diseases. Vaccination being one of the important preventive measure for IBD has been implicated as a cause of immunosuppression in many instances. This study was designed to compare the haemato-biochemical and gross-histopathological observations after infection with field and vaccine strain in birds below and above three weeks of age. Methods: In birds below and above three weeks of age, field and vaccine strain of IBD virus was inoculated with subsequent study of haemato-biochemical and gross-histopathological changes. Result: Results indicated that vaccine strain used in this study (IV-95 strain) caused a comparable damage with field strain. The bursal body weight index was not a good indicator for IBD pathogenicity. However, the clinical signs were dependent and could be correlated to aspects such as haemato-biochemical alterations, gross and histopathological lesions in organs other than bursa of Fabricius (BF). Bursal changes could not be correlated to clinical signs as the birds lacking frank clinical signs were equally suffering from bursal damage and depletion of lymphocytes. Hence attenuated intermediate invasive strain of vaccine was capable of damaging BF and may be responsible for immunosuppression.

Control strategy of respiratory complex infections should address precipitating and predisposing causative agents in general and immunosuppressive agents in particular. In both clinical and subclinical forms, infectious bursal disease virus (IBDV) is one of the most immunosuppressive diseases of young chickens. This study aimed to investigate the concurrent occurrence of subclinical infectious bursal disease (IBD) and multicausal respiratory complex infections caused by Newcastle disease virus (NDV) and avian metapneumovirus (aMPV) in broilers. In this study, 800 tissue samples (e.g., trachea, cecal tonsil, bursa of Fabricius, and spleen) and 400 sera samples were collected from broilers with confirmed respiratory signs selected from 20 broiler farms in west Azerbaijan province, Iran, from October 2018 to February 2019. Pathogens in the tissue samples were detected using RT-PCR for the VP2 gene of IBDV, F gen of NDV, and N gene of aMPV. The amplified products were sequenced afterward. At the end of the husbandry period, sera samples were used to detect antibodies against IBDV, aMPV, and NDV using ELISA and HI tests. Molecular results showed that the 45% (9/20), 30% (6/20), and 15% (3/20) of tissue samples were positive for IBDV, NDV, and aMPV, respectively. Regarding co-infection, 5% (1/20) of farm isolates were positive for IBD and ND, while 10% (2/20) of farms isolates were positive for IBD and aMPV. Co-infection of IBD, ND, and aMPV was not detected in farm isolates. Serological results indicated that the IBD co-infected flocks had almost higher (P<0.05) antibody titers against IBD; however, IBDV-NDV co-infected flocks and IBDV-aMPV co-infected flocks had lower antibody titer against NDVand aMPV, respectively. It can be concluded that lower antibody titer against ND and aMPV in IBD-ND and IBD-aMPV co-infections indicated suppressive effects of IBD on these diseases. Therefore, vaccination against IBD even in regions without clinical form of IBD is inevitable for the reduction of immunosuppressive effects of subclinical IBD on immune responses against these diseases.

Infectious bursal disease virus (IBDV) causes an economically significant disease of chickens worldwide. The emergence of very virulent IBDV (vvIBDV) has brought more challenges for effective prevention of this disease. The molecular basis for the virulence of vvIBDV is not fully understood. In this study, 20 IBDV strains were analysed phylogenically and clustered in three branches based on their full-length B segments. The amino acid triplet located at positions 145/146/147 of VP1 was found highly conserved in branch I non-vvIBDVs as asparagine/glutamic acid/glycine (NEG), in branch II vvIBDVs as threonine/glutamic acid/glycine (TEG) and in branch III vvIBDVs as threonine/aspartic acid/asparagine (TDN). Further studies showed that the three amino acids play a critical role in the replication and pathogenicity of vvIBDV. Substitution of the TDN triplet with TEG or NEG reduced viral replication and pathogenicity of the vvIBDV HuB-1 strain in chickens. However, the replication of the attenuated IBDV Gt strain was reduced in chicken embryo fibroblast cells, whilst it was enhanced in the bursa by substituting NEG with TEG or TDN. The exchange of the three amino acids was also found to be capable of affecting the polymerase activity of VP1. The important role of segment B in the pathogenicity of IBDV was confirmed in this study. These results also provided new insights into the mechanism of the virulence of vvIBDVs and may offer new targets for their attenuation to develop potential vaccines using reverse genetics.

The coding regions of segment A of two recent Korean very virulent (vv) infectious bursal disease virus (IBDV) isolates (KK1 and KSH) and one atypical IBDV isolate (K310) were amplified by reverse transcriptase-polymerase chain reaction, sequenced, and compared with published sequences for IBDV. The overall amino acid sequence similarity of the KK1 and KSH strains compared with foreign vvIBDV strains was between 97.43% and 98.02%. The KK1 and KSH strains, like vvIBDV strains, share unique amino acid residues at positions 222(A), 256(I), 294(I), and 299(S). The sequence of K310 strain was markedly different from other IBDV strains. The K310 strain had 12, 2, and 1 unique amino acid substitutions in the VP2 hypervariable region, VP4, and VP3 gene, respectively, and 3 of 12 substitutions in a VP2 hypervariable region were found in two hydrophilic regions known to be involved in antigenic determination. Also, the K310 strain had 222(S) and 254(S), which were found in variant IBDV strains. The SWSASGS heptapeptide is conserved in all Korean IBDV isolates. By phylogenetic analysis, KK1 and KSH were categorized in one group with foreign vvIBDV isolates, but K310 isolate was categorized in a separate group that was differentiated from the other IBDV strains compared. The K310 strain seemed to be evolved from a separate lineage of IBDV strain.

We determined the sequence of the coding region of segment A, coding for the viral proteins (VPs) VP2, VP4, and VP3, of a very virulent (vv) infectious bursal disease virus (IBDV) isolated in Israel and named IBDVks. We compared the deduced amino acid sequences of the proteins of the new isolate with those of the same proteins from several IBDV isolates, as published in recent years. The amino acid sequences of VP3 and VP4 of the Israeli isolate were 1.9%-2.3% different from the sequences of their counterparts from classical strains. Thus, the stable region of VP2 of IBDVks was very similar (0-0.68% difference) to the same region of VP2 from vv strains from Europe and Japan but distinct from that of proteins from classical strains from Europe, the United States, and Australia (up to 9.42% divergence), showing that IBDVks is more closely related to the vv strains from Europe and Japan. We found that viruses isolated in recent years resemble each other more than isolates from the same areas isolated a few years earlier. Hence, IBDVks can be categorized in one group with vv new isolates from Europe and Japan. This group has been found to be distinct from new isolates in the United States and strains isolated before the IBDV epidemic during the late 1980s.

Infectious bursal disease virus (IBDV) isolates were recovered from outbreaks to initiate activities towards developing a local vaccine strain. Use of indigenous chicken embryos was exploited to determine their potential, promote utilization of local resources for research, and enhance household economic activities. Bursa of Fabricius (BFs) samples from outbreaks shown to be IBDV positive was homogenized and inoculated in 4-week-old specific pathogen-free (SPF) IBDV seronegative white leghorn chicks. The harvested virus was inoculated into 11-day-old indigenous chicken embryos that were IBDV seronegative and passaged serially three times after which they were inoculated into 4-week-old indigenous chicks to test for presence and virulence of propagated virus. Out of 153 BFs collected from outbreaks, 43.8% (67/153) were positive for IBDV antigen and 65.7% (44/67) caused disease in SPF chicks. The embryo mean mortalities were 88% on primary inoculation, 94% in 1st passage, 91% in 2nd passage, and 67% in 3rd passage. After the third passage in embryos all the 44 isolates were virulent in 4-week-old indigenous chicks. The results show that indigenous chicken embryos support growth of IBDV and can be used to propagate the virus as an alternative viral propagating tool for respective vaccine preparation.

Infectious bursal disease virus (IBDV) vaccines do not induce sterilizing immunity, and vaccinated birds can become infected with field strains. Vaccine-induced immune selection pressure drives the evolution of antigenic drift variants that accumulate amino acid changes in the hypervariable region (HVR) of the VP2 capsid, which may lead to vaccine failures. However, there is a lack of information regarding how quickly mutations arise, and the relative contribution different residues make to immune escape. To model IBDV antigenic drift in vitro, we serially passaged a classical field strain belonging to genogroup A1 (F52/70) ten times, in triplicate, in the immortalized chicken B cell line, DT40, in the presence of sub-neutralizing concentrations of sera from birds inoculated with IBDV vaccine strain 2512, to generate escape mutants. This assay simulated a situation where classical strains may infect birds that have suboptimal vaccine-induced antibody responses. We then sequenced the HVR of the VP2 capsid at passage (P) 5 and 10 and compared the sequences to the parental virus (P0), and to the virus passaged in the presence of negative control chicken serum that lacked IBDV antibodies. Two escape mutants at P10 had the same mutations, D279Y and G281R, and a third had mutations S251I and D279N. Furthermore, at P5, the D279Y mutation was detectable, but the G281R mutation was not, indicating the mutations arose with different kinetics.

Reverse transcription - polymerase chain reaction of the VP2 gene followed by restriction endonuclease analysis was used to detect genetic variation among 25 strains of both field and vaccine strains of infectious bursal disease virus (IBDV) in Taiwan. Restriction fragment profiles generated by Taql, Sspl, Sacl, Nael, Hinfl, BstEII and Maelll showed variation between strains. The IBDV strains were placed into four molecular groups. Group 1 consisted of 3 subgroups. Subgroup 1A (6 strains) consisted of serotype II strains, classic serotype I strains, variant strains and one vaccine strain. Subgroups 1B and 1C consisted of 2 and 5 vaccine strains, respectively. Group 2 contained 2 vaccine strains. Group 3A contained 7 Taiwanese strains of very virulent IBDV (vvIBDV), while a Japanese vvlBDV strain was assigned to group 3B. Group 4 contained a Taiwanese vvlBDV strain and an Israeli vaccine strain (MB). A phylogenetic study indicated that Taiwanese vvlBDV strains are very similar to French and Japanese vvlBDV strains. It is concluded that hypervariable region of the VP2 of vvlBDVs and an attenuated derivative are different from classic strains, variant strains , and attenuated strains derived from classic strains.

Construction and Efficacy of recombinant Newcastle disease virus co-expressing VP2 and VP3 proteins of Very Virulent Infectious Bursal Disease Virus

The full-length cDNA of genomic segment A of three infectious bursal disease viruses, two attenuated strains (HZ2 and JD1) and one virulent field strain (ZJ2000), was amplified in a single step by reverse transcription-polymerase chain reaction, cloned into pGEM-T Easy Vector, and sequenced. The full length of cloned segment A contains 3259 nucleotides, which includes two partially overlapping open reading fragments (ORFs) ORF1 and ORF2, flanked by 5' and 3' noncoding regions. These strains shared high sequence identity with each other either at the nucleotide or deduced amino acid level. Strains HZ2 and JD1 were highly related to two attenuated strains, CEF94 and P2, whereas ZJ2000 was closely related to two other virulent strains, Cu-1 and Harbin. Substitutions of four amino acids at positions 253, 279, 284, and 330, a common feature of attenuated and most virulent strains, were also observed in these three strains. Two major hydrophilic peaks were conserved in the three strains; however, there are two amino acid substitutions at positions 280 (N to S) and 290 (M to L) in the second minor hydrophilic peak for all three strains, which might have a critical influence on antigenicity. Two amino acid substitutions near the VP2-VP4 cleavage site were identified in virulent strain JZ2000, which might be involved in increasing the virulence of the virus. Phylogenetic analyses indicated that these three Chinese strains are most closely related to some European virulent strains but are distinct from very virulent infectious bursal disease virus and variant strains.

Three commercially available infectious bursal disease vaccines, designated BV, BV-M, and LKT, were evaluated in chicks that were specific-pathogen-free and free of antibodies to infectious bursal disease virus (IBDV). For comparison, a pathogenic field isolate of the IBDV (FV) was also inoculated into 1-day-old and 3week-old chicks. At 5 weeks old, the mean virus-neutralizing (VN) antibody titers of the 3 vaccinated groups did not differ significantly (P< 0.05). At 7 weeks old the BV-induced VN titers were significantly higher (P<0.05) than the LKTand the BV-M-induced titers. Differences remained similarly significant (P<0.05) at 9 weeks old. Precipitating antibodies were consistently demonstrable in the serums of BV-vaccinated chickens at 5, 7, and 9 weeks old by the agar-gel precipitin (AGP) test, whereas many serums from the BVMand LKT-vaccinated birds had no detectable AGP reaction at 9 weeks old. Field virus exposure at 1 day or three weeks old resulted in high VN titers as well as persistent AGP reaction. The vaccine virus persisted in the bursa of Fabricius until birds were 9 weeks old in groups receiving LKT and BV vaccines but not in the group given the BV-M vaccine. Upon challenge with FV at 9 weeks old, the LKT and the BV-M vaccinated birds were better protected against bursal atrophy than the birds vaccinated with BV-strain. None of the vaccine viruses, when used according to the manufacturers' recommendations, or the FV, when given at 3 weeks old, caused significant (P<0.05) immunosuppression.