Virulent vaccine strains of infectious bursal disease virus not distinguishable from wild-type viruses with marker the use of a molecular

Molecular techniques have not only made timely and accurate detection of infectious bursal disease viruses (IBDVs) possible but also have allowed the identification of viral strains. Previously, we identified a genetic marker that distinguished wild-type IBDV strains from vaccine strains of the virus. The marker was an NgoM IV restriction enzyme site in the VP2 gene that was present in 10 wild-type viruses but not 16 vaccine strains of IBDV. On the basis of that study, we concluded that the NgoM IV marker could be useful in the identification of wild-type potentially pathogenic strains of this virus. Because virulent (hot) vaccine strains of IBDV are used to vaccinate commercial poultry, it was important to determine if the NgoM IV marker was present in these virulent vaccines. The infectious bursal disease Blen and Bursa Vac virulent vaccines were examined and determined to contain the marker. We concluded that the presence of this marker was not unique to wild-type strains of the virus. The absence of the NgoM IV marker, however, was consistent with some level of attenuation, and its presence appears to be consistent with virulent IBDV strains.     

传染性法氏囊病病毒超强毒HZ株和XN株VP2基因的克隆和序列比较

根据已发表的５２／７０株ＩＢＤＶ基因组序列,设计并合成了一对特异扩增ＩＢＤＶ ＶＰ２基因的引物。以陕西地区分离的ＩＢＤＶ野毒ＸＮ株,ＨＺ株为材料,以其基因组为模板利用ＲＴ－ＰＣＲ技术扩增出了１．５ｋｂ的ｃＤＮＡ产物,将ＶＰ２基因克隆于ＰＵＣ１１９质粒上,得到重组ＰＵＣ１１９质粒。     

Differentiation of infectious bursal disease viruses by restriction enzyme analysis of RT-PCR amplified VP1 gene sequence

In order to differentiate infectious bursal disease virus (IBDV) isolates/strains, a quick method of RT-PCR followed by restriction enzyme analysis of VP1 gene sequence is being reported for the first time. A 480 bp fragment, comprising one of the RNA dependent RNA polymerase motifs of VP1 gene sequence of an Indian classical virus, an attenuated vaccine strain, Georgia and two Indian field isolates, genetically similar to reported very virulent strains of IBDV, was amplified by RT-PCR. Restriction enzyme digestion of PCR products with Taq1 enzyme generated distinct profile for field isolates, different from the classical and attenuated viruses, whereas restriction profile with BstNI restriction enzyme was similar in all the viruses, irrespective of the pathotype. Therefore, the present results suggest that Taq1 digestion can be taken up for the differentiation of field isolates from the classical and vaccine strains. The sequence analysis of VPI gene of reported very virulent IBD viruses from Europe and Japan, using 'MapDraw' programme of Lasergene software, revealed similar restriction enzyme profile as in Indian field isolates.     

三株鸡传染性法氏囊病毒弱毒株的分离与分子鉴定

本试验在江苏省鸡场分离获得3株鸡传染性法氏囊病病毒（IBDV）,采用RT—PCR法扩增VP2基因,将产物克隆入pMD18T载体,经测序,并与IBDV代表株VP2基因的高变区序列进行分析比较。结果显示,3个分离株与超强毒株、强毒株、突变株及弱毒株的核苷酸同源性在89．5％～98．9％之间,与弱毒株Cu-1和疫苗株PBG-98同源性最高,为98．9％;推导出的氨基酸序列与代表性毒株的同源性在98．2％～99．5％之间。其中,七肽区的第三个丝氨酸残基突变为精氨酸或苏氨酸,279和284位氨基酸残基突变为天冬氨酸和苏氨酸,222、294和299位氨基酸残基分别突变为脯氨酸、亮氨酸和天冬氨酸。上述试验表明3株分离株均为临床弱毒株。     

Biological properties of a naturally attenuated infectious bursal disease virus isolated from a backyard chicken flock

The biological properties of an infectious bursal disease (IBD) virus isolated from bursas collected during an outbreak in a village chicken flock in Macedonia are described. The mortality rate was 50%. Two viruses coexisted in the bursas of infected chickens (IBDVwt and IBDVtc). The virus termed IBDVtc grows on chicken embryo fibroblast (CEF) cells from the first passage. Specific pathogen free chickens inoculated with IBDVtc at passage level 4 did not develop any clinical signs of disease. Some discrete bleeding on the leg muscles was seen and the bursa of Fabricius revealed pathological lesions similar to those caused by classical strains. However, the bursa recovered quickly (bursa lesion score 2) by 14 days post infection (PI). We also found evidence of bursal repopulation by means of perinuclear antigen staining. Strong CD3 influx was evident at 4 days PI, and at 33 days PI the CD3+ cell finding was comparable to the control. The mean antibody titre was 9.2 log 2 at 14 days PI. The amino acid composition of VP2 in IBDVwt (222 Ala, 242 Ile, 253 Gln, 256 Ile, 279 Asp, 284 Ala, 294 Ile and 299 Ser) is described. The same sequence was found in IBDVtc, except for two point mutations, at Gln253→His and Ala284→Thr. Such amino acid substitution is responsible for partial attenuation and the ability of the strain to replicate in cell culture. None of the commercial vaccine viruses has a similar arrangement of amino acids in the variable domain of IBDV. This strongly suggests that IBDVtc originates from a very virulent strain. To the best of our knowledge, this is the first report of a concomitant infection of chickens with highly pathogenic IBDV and its mutant counterpart.     

Confirmation of the existence of two distinct genetic groups of infectious bursal disease virus in Australia

OBJECTIVE: To characterise infectious bursal disease viruses (IBDVs) prevalent at major commercial sites throughout Australia and to compare the nucleic acid sequences of local strains of IBDV with those of characterised overseas strains. DESIGN: Samples of bursae were collected from 20 broiler farms that belonged to different poultry companies in New South Wales (NSW), Queensland (Qld), Victoria (Vic), Westem (WA) and South Australia (SA). METHOD: Bursae were collected from broilers between 24 and 35 days of age. Bursal tissue was homogenised and tested for the presence of IBDV antigen using four monoclonal antibodies (Mabs) which detect antigenic variation in IBDV strains. The nucleotide sequences of the hypervariable region (HVR) within the VP2 gene of IBDVs was determined and the deduced amino acid sequences compared with three vaccine strains and six previously characterised Australian IBDV strains. The deduced amino acid sequences were also compared with the published amino acid sequences of overseas strains. The phylogenetic relationships between Australian strains and overseas strains were then determined. RESULTS: IBDV was detected in birds from 14 out of 20 farms sampled. Typing with four Mabs showed that all viruses from Vic (6) and SA (10) were antigenic variants, whereas all viruses from NSW (29), Qld (4) and WA (5) were classical-like strains. Nucleotide sequencing of one sample from each of the 14 farms on which IBDV was detected confirmed results obtained with Mabs. The amino acid sequences of all Australian viruses differed from the amino acid sequences of foreign IBDV strains. Phylogenetic analysis showed that Australian IBDV viruses belonged to two distinct genetic groups. Very virulent (vv) IBDV strains belonged to a third genetic group, and overseas classical and variant strains belonged to a fourth genetic group. CONCLUSIONS: The results confirmed previous findings that there are two groups of IBDV strains circulating in commercial broilers in Australia. The majority are classical-like strains that are antigenically and genetically similar to vaccine strains 002/73 and V877. These classical strains were prevalent in broilers in three states, NSW, Qld and WA. The second group of strains are antigenic variants that were only found in broilers in two states, Vic and SA. All Australian IBDVs characterised to date are genetically distinct and can be differentiated from all other overseas strains. This enables identification of incursion of any exotic strain into Australian poultry, be it classical, US variant or wIBDV strains.     

Sequence analysis of both genome segments of three Croatian infectious bursal disease field viruses

In order to determine the mutations responsible for virulence, three Croatian field infectious bursal disease viruses (IBDV), designated Cro-Ig/02, Cro-Po/00, and Cro-Pa/98 were characterized. Coding regions of both genomic segments were sequenced, and the nucleotide and deduced amino acid sequences were compared with previously reported full-length sequenced IBDV strains. Phylogenetic analysis, based on the nucleotide and deduced amino acid sequences of polyprotein and VP1, was performed. Eight characteristic amino acid residues, that were common to very virulent (vv) IBDV, were detected on polyprotein: 222A, 256I, 294I, 451L, 685N, 715S, 751D, and 1005A. All eight were found in Cro-Ig/02 and Cro-Po/00. C-Pa/98 had all the characteristics of an attenuated strain, except for glutamine on residue 253, which is common for vv, classical virulent, and variant strains. Between less virulent and vvIBDV, three substitutions were found on VP5: 49 G --> R, 79 --> F, and 137 R --> W. In VP1, there were nine characteristic amino acid residues common to vvwIBDV: 146D, 147N, 242E, 390M, 393D, 511S, 562P, 687P, and 695R. All nine residues were found in A-Ig/02, and eight were found in B-Po/00, which had isoleucine on residue 390. Based on our analyses, isolates Cro-Ig/02 and Cro-Po/00 were classified with vv IBDV strains. C-Pa/98 shared all characteristic amino acid residues with attenuated and classical virulence strains, so it was classified with those.     

The use of gamma irradiation for the inactivation of infectious bursal disease viruses

The maximum dosage of gamma irradiation approved by the U.S. Food and Drug Administration (FDA) for poultry is 3.0 kiloGrays (kGy). This treatment is designed to reduce bacterial contamination on uncooked poultry carcasses and meat products. The possible presence of infectious bursal disease virus (IBDV) on poultry postharvest has prompted some countries to study the risk associated with introducing nonnative strains of the virus from imported commodities. The goal of this study was to determine if this risk could be reduced using gamma irradiation to inactivate IBDV. At the dosage approved by the FDA, the titers of IBDV vaccine strains were reduced between 0 and 1 log10. Titers of the pathogenic IBDV strains tested were not reduced after the 3.0 kGy exposure. Furthermore, titers of pathogenic viral strains were not reduced following exposure up to 5.0 kGy. As the exposure to gamma irradiation increased, the titers of the vaccine strains decreased. At the maximum dosage tested (10 kGy), the 89/03 variant virus vaccine was completely inactivated. Titers of the three classic IBDV vaccine strains were reduced between 1.6-2.0 logs after the 10 kGy exposure; however, these viruses remained viable after this treatment. Gamma irradiation is not an effective intervention to reduce the risk of IBDV introduction via processed poultry.     

广西梧州地区近十年传染性法氏囊病病毒vVP2变化特征分析

为了解近10年来广西梧州地区鸡传染性法氏囊病病毒(IBDV)分子进化情况,对2013年—2014年间来自该地区传染性法氏囊病(Infectious bursal disease,IBD)的法氏囊样品进行IBDV的分离鉴定,并对分离株以及课题组2006年—2013年间分离的毒株,共24株的VP2高变区(vVP2)进行序列分析和遗传进化分析。结果表明,QX0601等23个分离株在关键氨基酸位点上具有256I、284A、294I等超强毒株(vvIBDV)的分子特征,遗传进化分析表明,这23株分离株与UK661、HK46等超强毒参考株同处一个分支中,亲缘关系较近;QX110603在关键性氨基酸位点上则具有256V、284T、294L等弱毒株的特征,遗传进化分析显示,其与BJ836等致弱株处于同一分支,亲缘关系较近。对所有分离株进行氨基酸位点分析发现,该地区IBDV进化出现了新的特点,212D-212N符合国内近年来的分离株的变化趋势,209T-209A、338R-338H、359T-359R则表现出地域特点,未曾见过相似报道。研究结果表明,具有vvIBDV分子特征的分离株是该地区近10年来主要流行毒株,该地区IBDV毒株在vVP2序列上仍处于不断进化中,且带有地域特点。