Identification of the viral haemorrhagic disease virus of rabbits as a calicivirus.

Liver tissue from rabbits which had died of viral haemorrhagic disease (VHD) was used to identify the causative agent of the disease. After extraction of liver homogenates and density gradient ultracentrifugation, viral particles with buoyant densities between 1.32 to 1.38 g/ml and estimated sedimentation coefficients between 100 S and 175 S were obtained. The isolated virions were determined to have a diameter of 40 nm. The particles morphologically resembled those of the Caliciviridae family, had positive reactions both in ELISA and haemagglutination assays, and were infective for rabbits. By immunoblotting, a major structural protein with a molecular weight of 60 kDa was identified. RNA of high purity and of approximately 8 kb was isolated from virions. Labelled cDNA of virion RNA detected two RNAs of 8 kb and 2 kb in Northern blots of liver RNA from animals infected with the VHD virus (VHDV). Finally, isolated virion RNA injected directly into the liver or rabbits produced a disease with clinical symptoms and pathological findings typical of VHD. A calicivirus originally designated "rabbit haemorrhagic disease virus (RHDV)" was thus identified as the causative agent of VHD. The agent causing the European brown hare syndrome (EBHS) is also a calicivirus (EBHSV). VHDV and EBHSV are different members of the Caliciviridae family; their relationship can be clearly demonstrated by using different approaches of investigation.     

Detection of rabbit haemorrhagic disease virus (RHDV) in nonspecific vertebrate hosts sympatric to the European wild rabbit (Oryctolagus cuniculus)

Since its detection in China in 1984, rabbit haemorrhagic disease (RHD) has been the subject of numerous studies. Yet, the evolutionary origin of rabbit haemorrhagic disease virus (RHDV) is still under debate. For example, some aspects related to the epidemiology of the disease are still unknown, such as where the virus is hosted between RHD outbreaks. To detect the presence of RHDV in rabbit-sympatric micromammals, 51 rodents (29 Mus spretus and 22 Apodemus sylvaticus) and 31 rabbits (Oryctolagus cuniculus) from the same location in central Spain were analyzed. In those samples in which the virus was detected, a fragment of the VP60 protein gene from the RHDV capsid was sequenced and the phylogenetic relationships between them and other strains of RHDV in the Iberian Peninsula were analyzed. In total, five viral strains were identified in A. sylvaticus, M. spretus and O. cuniculus. All strains were found to be well supported within the clade of RHDV found in rabbits in the Iberian Peninsula. Moreover, one of the strains was found in all three species under study, which suggests the capability of RHDV to infect other mammals apart from the rabbit which have not yet been investigated. The transmission of the virus is discussed as well as its ecoepidemiological implications.     

Rabbit haemorrhagic disease virus 2 (RHDV2) outbreak in Azores: Disclosure of common genetic markers and phylogenetic segregation within the European strains

Rabbit haemorrhagic disease virus 2 (RHDV2) is widespread in several countries of Western Europe, but it has not been introduced to other continents. However, between late 2014 and early 2015, the presence of RHDV2 was confirmed outside of the European continent, in the Azores, initially in the islands of Graciosa, Flores, S. Jorge and Terceira. In this study we report the subsequent detection of RHDV2 in wild rabbits from the islands of Faial, St. Maria and S. Miguel, and display the necropsy and microscopic examination data obtained, which showed lesions similar to those induced by classical strains of RHDV, with severe affection of lungs and liver. We also disclose the result of a genetic investigation carried out with RHDV2 positive samples from wild rabbits found dead in the seven islands. Partial vp60 sequences were amplified from 27 tissue samples. Nucleotide analysis showed that the Azorean strains are closely related to each other, sharing a high genetic identity (>99.15%). None of the obtained sequences were identical to any RHDV2 sequence publically known, hampering a clue for the source of the outbreaks. However, Bayesian and maximum likelihood phylogenetic analyses disclosed that Azorean strains are more closely related to a few strains from Southern Portugal than with any others presently known. In the analysed region comprising the terminal 942 nucleotides of the vp60 gene, four new single nucleotide polymorphisms (SNP) were identified. Based on the present data, these four SNPs, which are unique in the strains from Azores, may constitute putative molecular geographic markers for Azorean RHDV2 strains, if they persist in the future. One of these variations is a non-synonymous substitution that involves the replacement of one amino acid in a hypervariable region of the capsid protein.     

Emerging rabbit haemorrhagic disease virus 2 (RHDV2) at the gates of the African continent

Until the beginning of this decade, the genetic characterization of rabbit haemorrhagic disease virus (RHDV) from Iberian Peninsula had revealed the existence of two genogroups, G1 and sporadically G6. In 2010, the new emerging rabbit haemorrhagic disease variant, RHDV2 or RHDVb, was described in France, from where it has rapidly spread throughout Europe, including Iberian Peninsula countries. Nevertheless, although cases of rabbit haemorrhagic disease (RHD) have been reported in the Canary Islands, a Spanish archipelago located 100 km off the coast of Morocco, no genetic characterization of RHDV had been carried out. Consequently, in order to identify the circulating RHDV strains in this archipelago, liver samples of six farm rabbits and fifteen wild rabbits were collected from several areas of the largest island, Tenerife, and analyzed for the presence of RHDV by antigen capture double antibody sandwich ELISA. In case of positive ELISA result, we amplified and sequenced two fragments of the vp60 gene, which were concatenated for phylogenetic purposes. The sequences analysis revealed the presence of RHDV2 in both farm and wild rabbits from several areas of Tenerife. This result constitutes the first finding of RHDV2 in the Canary Islands. These RHDV2 strains found in Tenerife shared two exclusive SNPs that have not been observed in the rest of RHDV2 strains. The identification of RHDV2 and the absence of classic RHDV strains in this study suggest that RHDV2 may be replacing classic strains in Tenerife, as has been also proposed in Iberian Peninsula, France and Azores. Given the proximity of the Canary Islands to the African continent, this result should raise awareness about a possible dispersal of RHDV2 from the Canary Islands to the North of Africa.     

Safety evaluation of a recombinant myxoma-RHDV virus inducing horizontal transmissible protection against myxomatosis and rabbit haemorrhagic disease

We have recently developed a transmissible vaccine to immunize rabbits against myxomatosis and rabbit haemorrhagic disease based on a recombinant myxoma virus (MV) expressing the rabbit haemorrhagic disease virus (RHDV) capsid protein [Bárcena et al. Horizontal transmissible protection against myxomatosis and rabbit haemorragic disease using a recombinant myxoma virus. J. Virol. 2000;74:1114-23]. Administration of the recombinant virus protects rabbits against lethal RHDV and MV challenges. Furthermore, the recombinant virus is capable of horizontal spreading promoting protection of contact animals, thus providing the opportunity to immunize wild rabbit populations. However, potential risks must be extensively evaluated before considering its field use. In this study several safety issues concerning the proposed vaccine have been evaluated under laboratory conditions. Results indicated that vaccine administration is safe even at a 100-fold overdose. No undesirable effects were detected upon administration to immunosuppressed or pregnant rabbits. The recombinant virus maintained its attenuated phenotype after 10 passages in vivo.     

A new recombinant Orf virus (ORFV, Parapoxvirus) protects rabbits against lethal infection with rabbit hemorrhagic disease virus (RHDV)

This report describes the generation of a new recombinant Orf virus (ORFV; Parapoxvirus) expressing the major capsid protein VP1 (VP60) of the calicivirus, rabbit hemorrhagic disease virus (RHDV). Authentic expression of VP1 could be demonstrated in cells infected with the recombinant D1701-V-VP1 without the need for production of infectious ORFV progeny. Notably, infected cells also released empty calicivirus-like particles (VLPs). Challenge experiments showed that even a single immunization with ≥10⁵ PFU of D1701-V-VP1 protected rabbits against lethal RHDV infection. ELISA tests indicated that the protective immunity mediated by D1701-V-VP1 did not strictly depend on the presence of detectable RHDV-specific serum antibodies. The induction of interleukin-2 found only in the sera of rabbits immunized with the D1701-V-VP1, but not in sera of rabbits immunized with the inactivated commercial vaccine RIKA-VACC, might indicate also some involvement of T-cells in protection. Collectively, this work adds another example of the successful use of the ORFV vector system for the generation of a recombinant vaccine, and demonstrates its potential as an alternative vaccine to protect rabbits against RHDV infection.     

博尔纳病病毒感染与病毒性脑炎发病的关系

目的 检测宁夏地区不明原因病毒性脑炎患者外周血单个核细胞(PBMCs)博尔纳病病毒(BDV)携带情况,分析BDV与标准病毒株之间的同源性.探讨BDV感染性疾病与不明原因病毒性脑炎之间的关系,同时也为部分病毒性脑炎的病因学诊断及其防治提供实验依据.方法 采用荧光定量巢式反转录酶聚合酶链反应(FQ-nRT-PCR)检测宁夏地区不明原因病毒性脑炎患者及正常人PBMCs BDV p24基因片段,并对其中阳性扩增产物进行基因序列测定,然后采用BLAST软件以及DNAsist 5.0软件对测序结果进行分析.结果 病毒性脑炎组样本BDVp24基因片段FQ-nRT-PCR阳性率(10.17%)显著高于正常对照组(0%)(P＜0.05).病毒性脑炎患者检出BDVp24基因片段的核苷酸序列与马BDV标准病毒株H3915序列同源性为97.67%,与H1766株比较在3个位点出现一致性突变(nt1659 T→C,nt1668 A→G,nt1674 T→C;突变率为3.49%);与strain V株比较在4个位点出现一致性突变(nt1648 C→T,nt1658 G→A,nt1667 A→G,nt1673 T→C;突变率为4.65%),但编码的氨基酸相同.证实扩增所得目的 基因片段确系BDV p24的相应片段.结论 宁夏地区不明原因病毒性脑炎患者中存在BDV感染,提示部分不明原因病毒性脑炎的发生可能与BDV感染有关.且该BDVp24扩增产物核苷酸序列与标准病毒株高度同源性,与马源strain H3915同源性最高.     

Survival of rabbit haemorrhagic disease virus (RHDV) in the environment

A study was conducted to investigate the persistence of rabbit haemorrhagic disease virus (RHDV) in the environment. Virus was impregnated onto two carrier materials (cotton tape and bovine liver) and exposed to environmental conditions on pasture during autumn in New Zealand. Samples were collected after 1, 10, 44 and 91 days and the viability of the virus was determined by oral inoculation of susceptible 11- to 14-week-old New Zealand White rabbits. Evidence of RHDV infection was based on clinical and pathological signs and/or seroconversion to RHDV. Virus impregnated on cotton tape was viable at 10 days of exposure but not at 44 days, while in bovine liver it was still viable at 91 days. The results of this study suggest that RHDV in animal tissues such as rabbit carcasses can survive for at least 3 months in the field, while virus exposed directly to environmental conditions, such as dried excreted virus, is viable for a period of less than 1 month. Survival of RHDV in the tissues of dead animals could, therefore, provide a persistent reservoir of virus, which could initiate new outbreaks of disease after extended delays.     

Exposure of rabbits to ultraviolet light-inactivated rabbit haemorrhagic disease virus (RHDV) and subsequent challenge with virulent virus

This study investigated whether exposure to inactivated rabbit haemorrhagic disease virus (RHDV) can produce an antigenic response in rabbits and protect them from a subsequent challenge with virulent virus. The aim was to determine if the spreading of baits containing RHDV, which is a common management practice in New Zealand to reduce rabbit numbers, could result in protective immunity in wild rabbits. RHDV was inactivated by ultraviolet (UV) light using an electronic UV crosslinker with a UV dose of 168.48 W-s/cm2 and a UV intensity of 0.0078 W/cm2. Two groups of four rabbits were then inoculated with inactivated virus via oral and intramuscular routes. Rabbits were monitored for 30 days post-inoculation and then challenged orally with virulent virus. No rabbit exposed to inactivated RHDV developed clinical signs of RHD or had antibodies at day 30 post-infection and all animals died within 82 h after challenge with virulent virus. No antibodies were detected at the time of death. These findings suggest that exposure to virus completely inactivated by UV light in the field or on baits will not protect rabbits against challenge with virulent virus.     

Isolation of Tioman virus from Pteropus giganteus bat in North-East region of India

Bat-borne viral diseases are a major public health concern among newly emerging infectious diseases which includes severe acute respiratory syndrome, Nipah, Marburg and Ebola virus disease. During the survey for Nipah virus among bats at North-East region of India; Tioman virus (TioV), a new member of the Paramyxoviridae family was isolated from tissues of Pteropus giganteus bats for the first time in India. This isolate was identified and confirmed by RT-PCR, sequence analysis and electron microscopy. A range of vertebrate cell lines were shown to be susceptible to Tioman virus. Negative electron microscopy study revealed the “herringbone” morphology of the nucleocapsid filaments and enveloped particles with distinct envelope projections a characteristic of the Paramyxoviridae family. Sequence analysis of Nucleocapsid gene of TioV demonstrated sequence identity of 99.87% and 99.99% nucleotide and amino acid respectively with of TioV strain isolated in Malaysia, 2001. This report demonstrates the first isolation of Tioman virus from a region where Nipah virus activity has been noticed in the past and recent years. Bat-borne viruses have become serious concern world-wide. A Survey of bats for novel viruses in this region would help in recognizing emerging viruses and combating diseases caused by them.     

First field trial of a transmissible recombinant vaccine against myxomatosis and rabbit hemorrhagic disease

As a novel approach for immunisation of wild rabbits, we have recently developed a transmissible vaccine against myxomatosis and rabbit hemorrhagic disease (RHD) based on a recombinant myxoma virus (MV) expressing the RHDV capsid protein [J. Virol. 74 (2000) 1114]. The efficacy and safety of the vaccine have been extensively evaluated under laboratory conditions. In this study, we report the first limited field trial of the candidate vaccine that was undertaken in an island of 34 Has containing a population of around 300 rabbits. Following administration by the subcutaneous route to 76 rabbits, the vaccine induced specific antibody responses against both myxomatosis and RHDV in all the inoculated rabbits. Furthermore, the recombinant virus exhibited a limited horizontal transmission capacity, promoting seroconversion of around 50% of the uninoculated rabbit population. No evidence of undesirable effects due to the recombinant virus field release was detected.     

Epidemiological consequences of a pathogen having both virulent and avirulent modes of transmission: the case of rabbit haemorrhagic disease virus

A number of pathogens cause chronic infection in survivors of acute disease and this is believed to be a common means of persistence, including for highly virulent agents. We present a model in which transmission from chronically infected hosts causes chronic infection in naive individuals, without causing acute disease--indeed 'protecting' against it. Thus the pathogen obtains the benefit of virulence (high transmission rate), but mitigates against the cost (high host mortality). Recent findings suggest that rabbit haemorrhagic disease virus (RHDV), a highly contagious and virulent pathogen, may also utilize this alternative, 'avirulent', mode of transmission. The model may resolve the paradox of how RHDV can be highly prevalent in some populations, in the absence of mortality. Differences in host demography determine whether avirulent transmission prevents large-scale mortality (as in most UK populations) or not. Other pathogens may exhibit similar behaviour and the implications for emerging diseases in general are discussed.     

Incidence, epizootiology and control of viral haemorrhagic disease of rabbits and the European brown hare syndrome in Germany.

Viral haemorrhagic disease (VHD) among domestic and wild rabbits and European brown hares have been observed in most of the Federal states of Germany. Acute outbreaks of viral haemorrhagic disease are most prevalent in small, fancy domestic rabbitries, with mortality varying between 5 and 90%, while mostly sporadic losses due to VHD are seen among wild rabbits. In hares, accumulated losses from the European brown hare syndrome (EBHS) can occasionally be observed in areas where fresh green feed, such as young O.O-rape plants, is available. VHD of rabbits and EBHS are caused by calicivirus strains. The natural virus reservoirs are wild rabbit and hare populations. Mutual infection of rabbits and hares seems to be possible. Transmission and spread of infectious virus occurs by direct contact of animals, or indirectly by contaminated excrement, green feed or living vectors. Calicivirus infections are widespread in several states, with accumulation of losses among domestic and wild rabbits by VHD, or in hares by EBHS, within certain areas. Disease hygiene, together with vaccination, are the officially preferred control measures in domestic rabbitries.     

Genetic Evidence for a Tacaribe Serocomplex Virus, Mexico

We isolated arenavirus RNA from white-toothed woodrats (Neotoma leucodon) captured in a region of Mexico in which woodrats are food for humans. Analyses of nucleotide and amino acid sequence data indicated that the woodrats were infected with a novel Tacaribe serocomplex virus, proposed name Real de Catorce virus.     

Association between specific HIV-1 Env traits and virologic control in vivo

HIV RNA levels are influenced by genetic characteristics of both the host and the virus. Here we applied machine learning techniques to determine if plasma-derived HIV-1 amino acid sequences can be used to predict spontaneous virologic control. We studied the relationship between HIV-1 env genotype and viral load in 20 chronically infected patients undergoing treatment interruptions (SSITT, Swiss-Spanish Intermittent Treatment Trial) and in 104 primary HIV infected (PHI) patients before antiretroviral therapy (cART) and where applicable also after treatment stop. Extensive longitudinal sampling during the interruptions was performed in nine SSITT patients. Sequences obtained from these nine patients during the first virus rebound were used as a training data set and revealed a strong genetic signature (accuracy 98.6% in cross-validation) associated with control of viremia at levels below 5000 copies/mL of viral RNA maintained for at least 2 months after the final cART stop. The simple sequence pattern at gp120 positions 268E/358T was confirmed to be predictive of control in the clonal sequences originating from these patients during all subsequent rebounds. Sequences from the remaining 11 SSITT patients with less frequent sampling and from the PHI patients were used for external validation. High sensitivities (71–100%) and negative predictive values (80–100%) but low positive predictive values (12–40%) were achieved in the patient-wise analysis which was based on presence of the genetic pattern in all clones. These results suggest that presence of virus lacking the amino acid pattern 268E/358T is associated with VL >5000 at baseline of PHI and with low probability of spontaneous virologic control after treatment stop. Conversely, however, presence of 268E/358T does not predict control of viremia. These residues in HIV gp120 might affect in vivo HIV-1 fitness either at the level of Env function or influence susceptibility to adaptive or innate immune response.     

宁夏地区人畜BDV p24基因系统发生树构建和序列分析

目的 了解宁夏及其周边地区博尔纳病病毒(BDV)的感染状况和基因特征及其种系发生来源.方法 采用巢式反转录实时荧光定量聚合酶链反应(FQ-nRT-PCR),检测119例病毒性脑炎(VE)患者和其密切接触的患病或健康牛205头、绵羊978只及脑血管病患者46例、多发硬化患者13例的外周血单核细胞BDVp24片段,检出的阳性序列连同前期检出的宁夏绵羊、VE患者和抑郁症患者阳性BDVp24基因序列与GenBank中5个国家7个动物种属29例BDVp24基因序列进行比对,分析其核苷酸和氨基酸序列的同源性,重新构建基因系统发生树.结果 23例阳性检测标本连同3例前期检测序列基因聚合分析显示核苷酸之间的同源相似度为96.5%～100.0%,氨基酸的同源相似度为95.3%～100.0%.与德国马源性标准株HE80同源相似度较高.构建基因的系统发生树发现宁夏绵羊、VE患者和抑郁症患者同德国马和绵羊、奥地利马都各自形成了独立的支系,其余样本形成混合支系,其中宁夏的VE患者、牛、绵羊同德围马、日本绵羊形成德国-中国宁夏-日本混合支系.结论 宁夏及其周边地区人和动物BDV的感染,存在区域源性BDV独立株和国外传人基冈保守株的多源状态.     

Genetic characterization of Indian type O FMD virus 3A region in context with host cell preference

The 3A region of foot-and-mouth disease virus has been implicated in host range and virulence. For example, amino acid deletions in the porcinophilic strain (O/TAW/97) at 93–102 aa of the 153 codons long 3A protein have been recognized as the determinant of species specificity. In the present study, 18 type O FMDV isolates from India were adapted in different cell culture systems and the 3A sequence was analyzed. These isolates had complete 3A coding sequence (153 aa) and did not exhibit growth restriction in cells based on species of origin. The 3A region was found to be highly conserved at N-terminal half (1–75 aa) but exhibited variability or substitutions towards C-terminal region (80–153). Moreover the amino acid substitutions were more frequent in recent Indian buffalo isolates but none of the Indian isolates showed deletion in 3A protein, which may be the reason for the absence of host specificity in vitro. Further inclusive analysis of 3A region will reveal interesting facts about the variability of FMD virus 3A region in an endemic environment.     

Characterization of protective humoral and cellular immune responses against RHDV2 induced by a new vaccine based on recombinant baculovirus

Rabbit hemorrhagic disease (RHD) is a lethal disease in rabbits caused by RHD virus (RHDV). Protection is only possible through vaccination. A new virus variant (RHDV2) which emerged in 2010 in France differed from the classical RHDV1 variant in certain aspects and vaccines against RHDV1 induced limited cross protection only. In a previous study, we designed a recombinant baculovirus based RHDV2-VP1 vaccine, which provided a protective immunity in rabbits against RHDV2. In the present study this newly created vaccine is characterized with regard to onset and duration of protection, and possible cross protection against classical RHDV1. Furthermore, humoral and cellular immune mechanisms in vaccinated and infected rabbits were analyzed. In all experiments, the recombinant vaccine was compared to a conventional liver-based RHDV2 vaccine.The RHDV2-VP1 vaccine induced a protective immune response already seven days after single vaccination and fully protected for at least 14 months. A booster vaccination 21 days after the first had a negative influence on long-term protection. The cross protection provided by the RHDV2-VP1 vaccine against classical RHDV1 was limited since only 50% of vaccinated rabbits survived the infection. Conclusively, the new, baculovirus-based RHDV2-VP1 vaccine has the potential to protect rabbits against the infection with RHDV2, blocks completely the disease progression and prevents the spread of RHDV2 at the population level.     

A putative amino acid ABC transporter substrate-binding protein,NMB1612, from Neisseria meningitidis,induces murine bactericidal antibodies against meningococci expressing heterologous NMB1612 proteins

The nmb1612 (NEIS1533) gene encoding the ∼27-kDa putative amino acid ATP-binding cassette (ABC) transporter, periplasmic substrate-binding protein from Neisseria meningitidis serogroup B (MenB) strain MC58 was cloned and expressed in Escherichia coli, and the purified recombinant (r)NMB1612 was used for animal immunization studies. Immunization of mice with rNMB1612 adsorbed to Al(OH)₃ and in liposomes with and without MPLA, induced antiserum with bactericidal activity in an assay using baby rabbit complement, against the homologous strain MC58 (encoding protein representative of Allele 62) and killed heterologous strains encoding proteins of three other alleles (representative of Alleles 1, 64 and 68), with similar SBA titres. However, strain MC58 was not killed (titre <4) in a human serum bactericidal assay (hSBA) using anti-rNMB1612 sera, although another strain (MC168) expressing the same protein was killed (median titres of 16–64 in the hSBA). Analysis of the NMB1612 amino acid sequences from 4351 meningococcal strains in the pubmlst.org/Neisseria database and a collection of 13 isolates from colonized individuals and from patients, showed that antibodies raised against rNMB1612 could potentially kill at least 72% of the MenB strains in the complete sequence database. For MenB disease occurring specifically in the UK from 2013 to 2015, >91% of the isolates causing disease in this recent period expressed NMB1612 protein encoded by Allele 1 and could be potentially killed by sera raised to the recombinant antigen in the current study. The NMB1612 protein was surface-accessible and expressed by different meningococcal strains. In summary, the properties of (i) NMB1612 protein conservation and expression, (ii) limited amino acid sequence variation between proteins encoded by different alleles, and (iii) the ability of a recombinant protein to induce cross-strain bactericidal antibodies, would all suggest a promising antigen for consideration for inclusion in new meningococcal vaccines.