Detection of RHDVa on the Iberian Peninsula: isolation of an RHDVa strain from a Spanish rabbitry

Rabbit haemorrhagic disease virus (RHDV), genus Lagovirus, family Caliciviridae, causes a large number of deaths in wild and domestic adult European rabbits (Oryctolagus cuniculus). The first documented outbreak dates from 1984 in China, but the virus rapidly dispersed worldwide. In 1997, an antigenic variant was detected in Italy and designated RHDVa. Despite causing symptoms similar to those caused by classic RHDV strains, marked antigenic and genetic differences exist. In some parts of Europe, RHDVa is replacing classic strains. Here, we report the presence of RHDVa on the Iberian Peninsula, where this variant was thought not to contribute to viral diversity.     

Phylogenetic analysis of rabbit hemorrhagic disease virus in China and the antigenic variation of new strains

This study aimed to investigate rabbit hemorrhagic disease virus (RHDV) in China. VP60 sequences of five RHDVs collected by our team, as well as those of 16 other published Chinese RHDV strains, were analyzed. Polygenic analysis using MEGA 4 software showed that 20 of the 21 Chinese strains could be clustered in the RHDVa subgroup, and WX/China/1984 was different from them. The Chinese RHDV strains were further classified into four subgroups, CH1 to CH4. Subgroup CH1, represented by the WX/China/1984 strain, was not prevalent in China after the first RHDV epidemic strain was reported. The CH2, CH3, and CH4 subgroups were far different from the CH1 subgroup, formed three separate clusters, and were distributed according to the time the strains were collected. Recently collected strains formed a new subgroup (CH4), represented by new RHDV varieties identified by challenging immunized rabbits and by comparison of genomic sequences. The present work is the first comprehensive analysis of Chinese RHDV and reveals a new RHDV variation that should be carefully monitored.     

Pathogenic, antigenic and molecular properties of rabbit haemorrhagic disease virus (RHDV) isolated from vaccinated rabbits: detection and characterization of antigenic variants

Summary.  Rabbit haemorrhagic disease virus (RHDV) isolates were obtained from several animals previously vaccinated with an inactivated vaccine. Seven isolates were analyzed by immunological and molecular biological methods and compared to reference strains. Antigenic characterization with monoclonal antibodies as well as haemagglutination assays demonstrated considerable differences between individual isolates. However, sequencing of the capsid protein genes revealed a high degree of homology between five of these isolates and the reference strain FRG. In contrast, two isolates specified remarkably different capsid proteins with a degree of variation not observed so far in RHDV. Amino acid alterations were found clustered between residues 301 and 328 (region C), 344 and 434 (region E) and also in the 3′ region of the capsid protein gene. Interestingly, experimental vaccination of rabbits followed by challenge with the heterologous variant strains showed restricted cross-protection against one of the strains. In summary, we found a level of antigenic variation not detected in RHDV so far, and describe two distinct new antigenic variants. Received November 3, 1997 Accepted October 19, 1998     

Phylogenetic analysis of rabbit haemorrhagic disease virus in France between 1993 and 2000, and the characterisation of RHDV antigenic variants

Summary.  The first molecular epidemiological study of Rabbit haemorrhagic disease virus undertaken in France between 1988 and 1995, identified three genogroups, two of which (G1, G2) disappeared quickly. We used immunocapture-RT-PCR and sequencing to analyse 104 new RHDV isolates collected between 1993 and 2000. One isolate was obtained in 2000 from a French overseas territory, the Reunion Island. The nucleotide sequences of these isolates were aligned with those of some French RHDV isolates representative of the three genogroups previously identified, of some reference strains and German and American RHDV antigenic variants. Despite the low degree of nucleotide sequence variation, three new genogroups (G4 to G6) were identified with significant bootstrap values. Two of these genogroups (G4 and G5) were related to the year in which the RHDV isolates were collected. Genogroup G4 emerged from genogroup G3, which has now disappeared. Genogroup G5 is a new independent group. The genogroup G6 contained an isolate collected in mainland France in 1999 and the isolate collected from the Reunion Island, as well as German and American RHDV variants. Multiple sequence alignments of the VP60 gene and antigenic analysis with monoclonal antibodies demonstrated that these French isolates are two new isolates of the RHDV variant. Received June 7, 2002; accepted August 12, 2002     

Phylogenetic analysis of rabbit haemorrhagic disease virus (RHDV) strains isolated between 1988 and 2003 in eastern Hungary

Summary. To define the genetic variability of RHDV strains collected in eastern Hungary, liver samples from rabbits that had died of RHD were collected between 1988 and 2003. The phylogenetic analysis of a 528-nucleotide-long portion of the gene encoding the VP60 capsid protein assigned the strains into three genogroups. The first group contained viruses from 1988–1993, and a second group comprised isolates from 1994–2002. A third group comprised all of the tested representatives of the RHDVa subtype and a Hungarian isolate from 2003. These findings were supported by the alignments of the deduced amino acid sequences of the VP60 gene and strongly suggest the presence of the RHDVa subtype in Hungary.     

Limited antigenic variation among recent infectious bursal disease virus isolates from France

Summary.  Seven neutralizing monoclonal antibodies (Mabs) to infectious bursal disease virus (IBDV) were used in an antigen-capture ELISA for the antigenic characterization of 58 IBDV isolates obtained in France since 1989. Fifty-six isolates exhibited an antigenic profile which was different from reference strain Faragher 52/70, and similar to French very virulent IBDV strain 89 163 (no binding of two Mabs). Two strains (3.4%), isolated in 1991 and 1994, showed additional antigenic modifications resulting in suppressed or reduced binding activity of three other Mabs. The two atypical viruses were not re-identified in field samples subsequently collected, hence showing that antigenic variants of IBDV do not tend to replace the antigenically dominant 89 163-like strains that have prevailed in France since 1989. Received March 14, 1997 Accepted June 2, 1997     

Numerical comparative serology of the Bhanja antigenic group(Bunyaviridae)

Summary Antigenic relationships between the two known members of the Bhanja serogroup (Bhanja virus, 7 strains; Kismayo virus, two strains) were studied by a plaque-reduction cross-neutralization test in Vero cells, using hyperimmune mouse sera. The results were then subjected to a cluster analysis, which showed (in the form of a dendrogram) a considerable antigenic difference between the two viruses. Within Bhanja virus, the European isolates fromHaemaphysalis punctata orH. sulcata ticks were antigenically indistinguishable, while the strains isolated in Africa and Asia from other tick species differed slightly from the European strains and also from each other.With 1 Figure     

Benign circulation of rabbit haemorrhagic disease virus on Lambay Island, Eire

With the exception of virus strains Ashington and RCV, other recognised strains of Rabbit haemorrhagic disease virus (RHDV) share relatively close genetic homology. Using serology and phylogenetic analysis, we have identified a third disparate virus lineage in healthy rabbits on Lambay Island off the east coast of Eire, where disease due to RHDV has never been observed. ELISA tests revealed high titre RHDV-specific antibodies in 81% of the sera from 11 healthy rabbits captured on this island, indicating that the virus is actively circulating amongst these rabbits. Nevertheless, infectious virus has not been isolated from rabbits living on this island. The detection of antibodies and the disparate Lambay virus lineage in an apparently healthy and isolated wild rabbit population provides the most convincing evidence yet that at least some strains of RHDV can circulate harmlessly for long periods of time in wild rabbits possibly by producing persistent or latent infections.     

Mumps virus strains isolated in Croatia in 1998 and 2005: Genotyping and putative antigenic relatedness to vaccine strains

Two mumps virus strains 9218/Zg98 and Du/CRO05 were isolated in two locations in Croatia in 1998 and 2005, respectively. Genetic characterization of these temporally distinct mumps virus isolates was carried out in order to determine their genotype and putative antigenic relatedness to mumps virus vaccine strains. Sequence analysis of the small hydrophobic (SH) gene revealed that isolate 9218/Zg98 shows less than 95% of similarity to any reference strain, thus representing a potential reference strain for a new genotype. Isolate Du/CRO05 clearly belongs to genotype G with the 97% of homology to the reference strain Glouc1/UK96. When compared to each other, the two Croatian strains have extremely low level of homology of only 89% indicating no relatedness between them. Putative antigenic properties of the HN protein of these two isolates were compared to different vaccine strains. The results reveal a higher level of homology of antigenic determinants to non-A genotype vaccine strains than to A genotype vaccine strain.     

Recombination in rabbit haemorrhagic disease virus: possible impact on evolution and epidemiology

Rabbit haemorrhagic disease (RHD) was first recognised in 1984 following the introduction of apparently healthy rabbits into China from Germany. The aetiological agent Rabbit haemorrhagic disease virus (RHDV) has subsequently killed hundreds of millions of domestic and wild rabbits particularly in Europe, China and Australia. Previously, using phylogenetic analysis we have attempted to understand the underlying factors that determine why this virus emerged, and why it has such an unpredictable epidemiology. Here we report the use of tree congruency supported by bootscanning analysis to detect recombination amongst both closely related, and widely divergent strains of RHDV. We show that recombination occurs commonly and in several different regions of the RHDV genome. Moreover, the first identified strain of RHDV, i.e. from China in 1984, showed evidence of recombination in the capsid gene, with a virus or viruses containing lineages in German strains. These observations imply that recombination may play a significant role in the evolution, epidemiology and diversity of RHDV.     

Characterisation of a non-pathogenic and non-protective infectious rabbit lagovirus related to RHDV

The existence of non-pathogenic RHDV strains was established when a non-lethal virus named rabbit calicivirus (RCV) was characterised in 1996 in Italy. Since then, different RNA sequences related to RHDV have been detected in apparently healthy domestic and wild rabbits, and recently a new lagovirus was identified in Australia. We have characterised from seropositive healthy domestic rabbits a non-lethal lagovirus that differs from RHDV in terms of pathogenicity, tissue tropism and capsid protein sequence. Phylogenetic analyses have revealed that it is close to the Ashington strain and to the RCV, but distinct. We proved experimentally that it is infectious but non-pathogenic and demonstrated that, contrary to the other described non-pathogenic lagoviruses, it induces antibodies that do not protect against RHDV. Our results indicate the existence of a gradient of cross-protection between circulating strains, from non-protective, partially protective to protective strains, and highlight the extent of diversity within the genus Lagovirus.     

Intragroup antigenic diversity of human respiratory syncytial virus (group A) isolated in Argentina and Chile

The intragroup antigenic diversity of the G glycoprotein of 226 human respiratory syncytial virus (HRSV) strains isolated in Buenos Aires (Argentina) and Santiago (Chile) between 1995 and 2002 was evaluated by ELISA with a panel of 14 anti-G monoclonal antibodies (MAbs). Out of 226 strains characterized, 172 (76%) belonged to group A and 54 (24%) to group B. Strains from both groups cocirculated throughout the study period in both countries, except in 1996, 2000, and 2002 when only group A strains were isolated. Within group A 23 different antigenic patterns were found as defined by the combination of reactivities with eight strain-specific anti-G MAbs. These antigenic patterns showed different behavior regarding their circulation. Some major patterns were observed in most years with variable proportions; other minor patterns were present in low proportions during 1 or 2 years and then were apparently replaced by new patterns. Some antigenic patterns occurred both in Argentina and Chile during the same epidemics. Since no strain-specific MAbs were available for group B, we could not evidence the antigenic variability within group B. These are the first data on antigenic characterization of HRSV strains isolated in Argentina and Chile. It is shown that the ELISA with MAbs directed against the G protein of RSV is a valuable tool. These results will also provide useful information for further studies to evaluate the antigenic variability of HRSV strains in relation with genetic characteristics.     

Detection of antigenic variants of the influenza B virus by melting curve analysis with LCGreen

Automated, high-throughput detection methods for single-nucleotide polymorphisms have been applied to the routine genotyping of genetic polymorphisms influencing drug metabolism. Melting curve analysis with LCGreen was introduced recently as one such technique which can be performed rapidly and easily. This technique was used to detect antigenic variants of the influenza B virus. The antigenic variants and vaccine-type strains of the influenza B virus are isolated from clinical specimens of one epidemic season, and they usually differ in one nucleotide in the HA1 gene, corresponding to one amino-acid substitution. By means of melting curve analysis with LCGreen, an antigenic variant clone and a vaccine-type clone were clearly distinguished. In addition, the proportions of the antigenic variants in the mixture-type isolates were estimated. The clinical isolates were detected as the vaccine-type strains, antigenic variants, or a mixture of both. It became clear that humans were infected with a mixture of the vaccine-type strains and the antigenic variants for a certain period after which the viral antigenicities vary. This technique will contribute to the analysis of antigenic shifts in influenza B virus.     

Identification of three distinct antigenic sites in parapoxviruses

Summary.  Monoclonal antibodies (Mabs) were generated in BALB/c mice immunized with gradient-purified particles, envelopes and cores of intracellular mature orf virus D-1701. Three distinct antigenic sites were identified in this virus strain. Their topographical relationships was determined by pairwise epitope specificity studies in competition ELISAs. One MAb (class IgM) neutralized virus infectivity. Four μg/ml purified IgM gave a 50% reduction of 100 PFU of orf virus D-1701. As shown by immunogold electron microscopy (ELMI), all MAbs reacted with epitopes localized on the virus surface. Western blotting analysis demonstrated that two proteins of a Mr of 39kDa and 22kDa were the main targets for the Mabs. Cross-reactivity studies of several parapoxviruses (PPV) differentiated stomatitis papulosa virus strains from orf virus and milker’s node virus (MNV) by a missing antigenic site. Received May 31, 1996 Accepted September 4, 1996     

Bovine herpesvirus 1: Molecular and antigenic characteristics of variant viruses isolated from calves with neurological disease

Summary This report presents data showing that several virus isolates recovered in Argentina, mainly from calves with non-purulent meningo-encephalitis, represent a hitherto unrecognized antigenic variant of BHV-1. The following experimental approaches have been adopted to demonstrate both the unique features among and the relatedness with BHV-1 of these isolates: i) crossed serum neutralization test with rabbit immune sera, ii) analysis by SDS-polyacrylamide gel electrophoresis of radio-labeled virus induced polypeptides and glycoproteins, iii) discriminating reactivity of a panel of monoclonal antibodies which recognize known virus types, and iv) restriction endonuclease analysis of viral DNA. Another strain of BHV-1, which exhibits a specific neuropathogenic potential [Hall et al., Austral. Vet. J.42, 229–237 (1966)] shares all major features with the viral strains originating from Argentina.Our results imply that antigenic variants of BHV-1 exist and that they can be accurately and easily identified and differentiated by the available methods.With 4 Figures     

The antigenic characteristics of the influenza virus subpopulations isolated from a single patient]

Examinations by HI and EIA of influenza A (H3N2) virus isolates of 1985-1990 showed the strains derived from nasopharyngeal washings from patients to present very frequently as phenotypic mixtures of stable virus variants. Immunological analysis with monospecific antibody to hemagglutinin antigenic sites revealed a wide spectrum of antigenic activity based on the degree of relationship with viruses of previous years. By means of the immune pressing with antibody of different specificity the isolated strains could be divided into 2 subpopulations each characterized by the presence of only three antigenic sites. The subpopulations homogeneous by the antigenic composition of hemagglutinin represented the strains with antigenic markers of hemagglutinins of previous variants with drift variants of epidemic nature.     

Genetic mechanisms of antigenic variation in infectious bursal disease virus: Analysis of a naturally occurring variant virus

The major immunogenic protein VP2 from a pathogenic field isolate (variant A virus) of infectious bursal disease virus (IBDV) was cloned and sequenced to examine antigenic variations. The VP2 open reading frame consists of 1509 nucleotides and codes for a 503 amino acid protein. Overall, the VP2 amino acid sequence of the variant A virus shares 98.6% identity with VP2 genes from other published IBDV strains. However, within the central region of VP2 (amino acids 222–334) lies a highly divergent area that we have termed thevariable domain. Relative to five other IBDV isolates, a total of six amino acid changes occur within the variable domain of the variant A virus. At positions 284–288, a substitution of isoleucine to threonine, a decrease in the number of Chou and Fasman turns, and a switch from a hydrophilic to a hydrophobic region are found only in the variant A virus. Together these changes predict a decrease in antigenicity as determined by calculation of potential antigenic sites. This suggests that only minor changes within VP2 contributed to the emergence of a variant virus that can cause disease in immunized birds.     

Possible relationship between antigenic properties and isolation history of HSV-1 strains

29 monoclonal anti-herpes simplex virus (HSV)-1 antibodies were produced and characterized with regard to virus neutralizing activity, intracellular or cell-surface location of viral antigens and, where possible, molecular weight of the viral protein recognized. 13 antibodies recognized viral antigens expressed on the surface of infected cells and 16 were directed to intracellular viral components. Only two antibodies exhibited virus neutralizing activity. Application of these antibodies to an antigenic comparison of standard laboratory HSV-1 strains F, HFEM, mP, Glasgow-17 and MAC revealed unique antigenic differences among these strains. The antibodies were further used in an antigenic comparison of 45 human HSV-1 isolates with defined isolation history. Except for two paired isolates from left and right trigeminal ganglia of two human cadavers, the antibody panel revealed antigenic differences among all isolates, including paired isolates from three additional cadavers. Overall, isolates from different human donors showed greater antigenic dissimilarity from each other in cell-surface associated than in intracellular antigens. The data suggest the possibility of a correlation between antigenic and biologic properties of HSV-1.     

武汉市2003年流感监测及当前优势流行毒株的抗原性和基因特性分析

目的 探讨2003年武汉市流感流行概况，分析优势流行毒株抗原性和基因特性变异特征。方法 每周在流感监测哨点医院统计流行病学资料，采样分离流感病毒标本，对其中3株H3病毒做血凝抑制试验并对其编码HA1区基因进行序列测定和分析。结果 从418份咽拭标本中分离到流感病毒58株，其中5，7株为H3亚型，1株为乙型；全年月分离率和门诊流感样病例数的变化有冬、夏两个高峰，最高峰均出现在7月；新分离到的3株H3病毒的抗原性与本年度疫苗组分株A／Panama／2007／99有较大差异，编码HA1区的氨基酸序列有14个位点发生了变异，基因种系发生树分析也证实他们的HA1基因存在较大差异。结论 2003年武汉市流感活动呈双峰型增强，H3病毒活动显著加强，其抗原性和基因特性与疫苗株相比均发生了较大的变异。     

Phylogenetic analysis of rabbit haemorrhagic disease virus strains from the Arabian Peninsula: did RHDV emerge simultaneously in Europe and Asia?

Rabbit haemorrhagic disease virus (RHDV) emerged in 1984 in China and subsequently a single strain apparently dispersed worldwide killing millions of rabbits. Two isolates that caused outbreaks in Saudi Arabia and Bahrain have been sequenced and analysed phylogenetically. The Saudi Arabian lineage is directly descended from the Chinese strain, but the Bahrain isolate occupies a distinct and more divergent lineage than the Chinese virus implying that epidemic RHDV strains have emerged at least twice during the past 20 years and are co-circulating in both domestic and wild rabbits.