Genetic complexity and multiple infections with more Parvovirus species in naturally infected cats

ABSTRACT: Parvoviruses of carnivores include three closely related autonomous parvoviruses: canine parvovirus (CPV), feline panleukopenia virus (FPV) and mink enteritis virus (MEV). These viruses cause a variety of serious diseases, especially in young patients, since they have a remarkable predilection for replication in rapidly dividing cells. FPV is not the only parvovirus species which infects cats; in addition to MEV, the new variants of canine parvovirus, CPV-2a, 2b and 2c have also penetrated the feline host-range, and they are able to infect and replicate in cats, causing diseases indistinguishable from feline panleukopenia. Furthermore, as cats are susceptible to both CPV-2 and FPV viruses, superinfection and co-infection with multiple parvovirus strains may occur, potentially facilitating recombination and high genetic heterogeneity. In the light of the importance of cats as a potential source of genetic diversity for parvoviruses and, since feline panleukopenia virus has re-emerged as a major cause of mortality in felines, the present study has explored the molecular characteristics of parvovirus strains circulating in cat populations. The most significant findings reported in this study were (a) the detection of mixed infection FPV/CPV with the presence of one parvovirus variant which is a true intermediate between FPV/CPV and (b) the quasispecies cloud size of one CPV sample variant 2c. In conclusion, this study provides new important results about the evolutionary dynamics of CPV infections in cats, showing that CPV has presumably started a new process of readaptation in feline hosts.     

Comparisons of feline panleukopenia virus, canine parvovirus, raccoon parvovirus, and mink enteritis virus and their pathogenicity for mink and ferrets

Parvoviruses from mink (mink enteritis virus [MEV]), cats (feline panleukopenia virus [FPV]), raccoons (raccoon parvovirus [RPV]), and dogs (canine parvovirus [CPV]) were compared. Restriction enzyme analysis of the viral replicative-form DNA revealed no consistent differences between FPV and RPV isolates, but CPV and MEV isolates could be distinguished readily from other virus types. Feline panleukopenia virus, RPV, and MEV, but not CPV, replicated to high titers in mink. However, on the first passage, disease and microscopic lesions were observed only in mink inoculated with MEV. Feline panleukopenia virus and RPV isolates replicated in ferrets, but disease or microscopic lesions were not observed. Feline panleukopenia virus and RPV isolates could be passaged repeatedly in mink and ferrets. Virulence of FPV and RPV isolates was low compared with that of MEV, and only a single mink inoculated with FPV or with RPV developed clinical disease on the sixth passage of virus.     

Structural proteins of bovine viral diarrhea virus.

A procedure for the purification of radioactively labeled bovine viral diarrhea virus was critically evaluated. Purification of virus from artificial mixtures of unlabeled infected and labeled noninfected cells indicated that the extent of purification was approximately 100-fold with respect to host proteins. Residual host proteins were found to contaminate the viral preparation even after extensive purification by differential and isopycnic zonal centrifugation. Co-electrophoresis of 3H-labeled virus with 14C-labeled host cell material in neutral sodium dodecyl sulfate-7.5% polyacrylamide gels provided a means to distinguish viral specific proteins from host cell protein contaminants. Four major electrophoretic components were identified as being of viral origin; molecular weights of the components were estimated from their migration rates relative to protein markers of known molecular weight. Two viral components (VC), VC 1 and VC 3, migrated heterogeneously and had molecular weights of 93,000 to 110,000 and 50,000 to 59,000 daltons, respectively. Molecular weights of VC 2 and VC 4 were 70,000 and 25,000 daltons, respectively.     

A non-haemagglutinating isolate of mink enteritis virus

A virus was isolated from mink showing clinical and pathological signs of mink enteritis. This virus was identified as mink enteritis virus (MEV) from results of serological tests, determination of its density in CsCl (1.415 g cm^?3), and morphology, including size (20 nm in diameter). The isolate was designated MEV-S. In contrast to other known MEV strains, the MEV-S isolate has no haemagglutinating (HA) activity with swine red blood cells (RBCs) at 4°C and pH 6.8.Neither was there any HA at other pH values and temperatures, or when worse, bovine and rhesus monkey RBC's were used.     

Identification and characterization of fowlpox virus strains using monoclonal antibodies.

The use of 2 monoclonal antibodies (MAbs), P1D9 and P2D4, which recognize different fowlpox virus (FPV) antigens, for the identification and characterization of FPV strains was evaluated. Initially, the MAbs were used in conjunction with a dot blot assay that enabled FPV to be differentiated from the avian herpesvirus, infectious laryngotracheitis virus. Confirmation of the specificity of these MAbs was provided by the demonstration that only FPV antigens were recognized by a combination of both antibodies when used for immunoblotting proteins contained in various avipoxviruses. Later, an antigenic characterization of 11 FPV field isolates, 6 FPV vaccine strains, and 3 pigeonpox virus vaccines was performed by Western blotting with the individual MAbs. Whereas MAb P2D4 consistently recognized a protein with an apparent molecular weight of 60 kD, there was variability in the size of the antigen that was immunoreactive with the other MAb. For example, MAb P1D9 recognized an antigen of apparent molecular weight of 46 kD in all vaccine strains except 2 of FPV origin. In these exceptions, either only a 39-kD or both a 42- and 46-kD protein were immunoreactive. As for the field isolates, a 39-kD antigen was recognized in 8 of them, whereas a 42-kD antigen was detected in the remaining 3. Therefore, the more extensive immunoblotting technique may facilitate FPV strain differentiation, whereas routine diagnosis of fowlpox could be accomplished by using the MAb-based dot blot assay.     

Hemagglutination by canine parvovirus: serologic studies and diagnostic applications

Conditions for canine parvoviral hemagglutination (HA) and hemagglutination-inhibition (HI) reactions were defined. The HA phenomena were used to differentiate canine parvovirus (CPV) from feline panleukopenia virus (FPV), mink enteritis virus (MEV), and minute virus of canines. Serologic comparisons of the CPV, FPV, and MEV by HA-HI and serum-neutralization tests indicated that CPV, FPV, and MEV were antigenically similar but were different from minute virus of canines. Diagnostic application of HA tests to fecal samples from acute cases of enteritis was discussed. Combinating HA tests with HI tests on fecal samples provided a rapid and specific diagnostic method for CPV infection. Secular seroprevalence studies indicated the emergence of CPV infeciton in the United States dog population-at-large in 1978.     

Antigenic and genomic comparisons of some feline parvovirus subspecies strains.

Fourteen feline parvovirus (FPV) strains isolated from cats, mink and dogs were comparatively examined on their antigenic and genetic diversities by using monoclonal antibodies against feline panleukopenia virus (FPLV) and restriction enzyme analysis of viral DNA. Mink enteritis virus (MEV) strains recently isolated in the northeastern area of the People's Republic of China were found to possess more similar antigenic and genetic properties to the antigenic variant virus of canine parvovirus (CPV) ("new" antigenic type CPV), than to FPLV strains and MEV Abashiri strain of Japan. A feline isolate detected in normal cat feces was considered to be rather CPV because of its antigenic and genetic characteristics. An early isolate of "new" antigenic type CPV strains showed a similar cleavage pattern to those of "old" antigenic type CPV strains when digested with HinfI. The results including some features above-mentioned suggest the presence of antigenic heterogeneities and genomic polymorphisms among FPV subspecies viruses.     

水貂肠炎病毒分子检测技术与基因工程疫苗研究进展

水貂肠炎病毒（MEV）是一种自主复制性的最小的动物DNA病毒。研究表明,MEV是引起水貂病毒性肠炎的病原体,能够引起水貂剧烈腹泻,具有较高的发病率和死亡率。随着对水貂肠炎病毒研究的不断深入,目前已经在病毒病原学、结构与非结构蛋白、感染机制、诊断与疫苗防控等方面取得很大进展。文章主要针对MEV分子检测新技术与基因工程疫苗等方面进行了综述。     

抗猫泛白细胞减少症病毒单克隆抗体的制备及其生物学特性鉴定

用 PEG60 0 0沉淀和蔗糖密度梯度离心从细胞培养物中纯化猫泛白细胞减少症病毒 ( FPV) ,以纯化FPV免疫 BALB/c小鼠 ,运用淋巴细胞杂交瘤技术 ,获得了 4株抗 FPV的特异性单克隆抗体 ( Mc Ab)。其腹水 Mc Ab的 ELISA效价在 1 0 - 4～ 1 0 - 5之间 ,其中 1株具有血凝抑制能力。经 ELISA阻断试验及 ELISA交叉反应性试验测定 ,这 4株 Mc Ab可与 FPV、犬细小病毒 ( CPV)和水貂肠炎病毒 ( MEV)呈特异性反应 ,因此可作为检测 FPV、CPV和 MEV共同抗原的通用试剂     

Comparison of feline parvovirus subspecific strains using monoclonal antibodies against a feline panleukopenia virus

Four monoclonal antibodies (mAb) against a feline panleukopenia virus (FPLV) TU 1 strain, one of the host range variants of feline parvovirus (FPV), were produced and applied for antigenic analysis of FPLV, canine parvovirus (CPV) and mink enteritis virus (MEV). All mAbs were considered to be directed at epitopes on the virus capsid surface because they neutralized the infectivity and inhibited the hemagglutination (HA) of the homologous virus as well as other FPV strains. They were of the mouse IgG1 type. High antigenic homogeneity among FPLV strains was confirmed by HA-inhibition (HI) test with the mAbs and polyclonal immune sera against FPLV or CPV. But the TU 11 strain of FPLV was antigenically distinguished from the remaining 14 FPLV strains by both the HI test and the micro-neutralization test with one of the mAbs produced. MEV Abashiri strain was found to be antigenically indistinguishable from FPLV. Most of the CPV strains isolated after 1981 were considered to be antigenically different from earlier CPV isolates when some mAbs were applied in the serological tests, confirming the replacement of CPV by an antigenic variant in Japan. However, antigenically different CPVs were detected at the end of 1984 from unrelated epizootics occurred a month apart in the same area.     

Antigenic and genetic analysis of canine parvoviruses in southern Africa

Canine parvovirus (CPV) is a significant pathogen of domestic and free-ranging carnivores all over the world. It suddenly appeared at the end of the 1970s and most likely emerged as a variant of the well known feline panleukopenia virus (FPV). During its adaptation to the new host, the domestic-dog, the virus has changed its antigenic profile twice giving rise to two new antigenic types, CPV-2a and CPV-2b. These new types have replaced the original type CPV-2 in the United States of America, Europe and Japan. However, no data about the prevalence of the new antigenic types on the African continent are available. In this study, 128 recent parvovirus isolates from South Africa and Namibia were antigenically typed with type-specific monoclonal antibodies. No original CPV-2 viruses were found and its complete replacement by the new antigenic types conforms to the situation in other parts of the world. The predominant strain found in southern Africa was CPV-2b (66%), which differs from the situation in Europe and Japan where CPV-2a is the most prevalent type. Analysis of the capsid protein DNA-sequences of four selected African isolates gave no hint of a specific African parvovirus lineage.     

Vaccination with canine parvovirus type 2 (CPV-2) protects against challenge with virulent CPV-2b and CPV-2c

Mutations in canine parvovirus (CPV) field isolates have created concerns regarding the ability of vaccines containing CPV-2 to protect against infection with the newly identified antigenic types CPV-2b and CPV-2c. To address this concern, the efficacy of CPV-2 strain NL-35-D currently in use as a commercial vaccine was demonstrated against an oral challenge with CPV-2b and CPV-2c, respectively. Clinically healthy specific pathogen free Beagle dogs were either vaccinated or treated with water for injection first at 8-9 weeks of age and again at 11-12 weeks of age. All dogs were challenged either with CPV-2b or CPV-2c three weeks after the second vaccination. During the two week period following challenge, clinical signs, white blood cell counts, serology by haemagglutination inhibition (HI) and serum neutralisation tests, and virus shedding by haemagglutination test were assessed. All control dogs developed clinical signs of parvovirosis (including pyrexia and leucopenia) and shed virus. Vaccinated dogs seroconverted (HI titres > or =80), remained healthy throughout the study and shed more than 100 times less virus than controls. In conclusion, vaccination with the low passage, high titre CPV-2 strain NL-35-D cross-protects dogs against virulent challenges with CPV-2b or CPV-2c by preventing disease and substantially reducing viral shedding.     

MEV、ADV和CAV三种病毒多重PCR检测方法的建立

建立一种同时检测貂肠炎病毒(MEV)、貂阿留申病毒(ADV)和犬腺病毒(CAV)的多重PCR诊断方法.引用已有的CAV引物,并根据GenBank发表的MEV、ADV序列保守区域设计特异性引物进行PCR扩增,可同时得到扩增长度为795(MEV)、451(ADV)、1 019 bp(CAV)3奈特异性片段,对猪细小病毒(PPV),犬瘟热病毒(CDV)进行PCR检测结果为阴性.各种模板、引物之间相互不构成干扰.敏感性试验证明,可以检测到模板中MEV 101.5 TCID50和CAV 100.5 TCID50的病毒含量,对ADV检测的敏感性更高.     

Molecular and structural basis of the evolution of parvovirus tropism.

Parvoviruses have small genomes and, consequently, are highly dependent on their host for various functions in their reproduction. Since these viruses generally use ubiquitous receptors, restrictions are usually intracellularly regulated. A lack of mitosis, and hence absence of enzymes required for DNA replication, is a powerful block of virus infection. Allotropic determinants have been identified for several parvoviruses: porcine parvovirus, canine parvovirus (CPV), feline parvovirus (feline panleukopenia virus), minute virus of mice, Aleutian disease virus, and GmDNV (an insect parvovirus). Invariably, these identifications involved the use of infectious clones of these viruses and the exchange of restriction fragments to create chimeric viruses, of which the resulting phenotype was then established by transfection in appropriate cell lines. The tropism of these viruses was found to be governed by minimal changes in the sequence of the capsid proteins and, often, only 2 or 3 critical amino acids are responsible for a given tropism. These amino acids are usually located on the outside of the capsid near or on the spike of the threefold axis for the vertebrate parvoviruses and on loops 2 or 3 for the insect parvoviruses. This tropism is not mediated via specific cellular receptors but by interactions with intracellular factors. The nature of these factors is unknown but most data point to a stage beyond the conversion of the single-stranded DNA genome by host cell DNA polymerase into monomeric duplex intermediates of the replicative form. The sudden and devastating emergence of mink enteritis virus (MEV) and CPV in the last 50 years, and the possibility of more future outbreaks, demonstrates the importance of understanding parvovirus tropism.     

Surveillance activity for canine parvovirus in Italy

Recent identification of unusual canine parvovirus (CPV) mutants in cats and dogs suggests that CPV type 2 (CPV-2), which emerged suddenly in the late 1970s, is undergoing continual genetic and antigenic variations. A peculiarity of parvoviruses is that single-nucleotide substitutions may determine drastic phenotypic changes. The effects of either natural or artificial mutations on CPV phenotypic properties have been largely investigated, and this sets up CPV as an interesting model to study virus evolution. By monitoring the evolution of CPV-2 in Italy, we observed the onset and quick spread of a Glu-426 mutant, antigenically different from the pre-existing variants that were partially displaced within a few years of the initial identification of the new mutant. The identification of CPV-2 variants raises several questions concerning their impact on the efficacy of the current CPV-2 vaccines, based on the original CPV-2 strain that no longer exists in the field.     

Viral and viral protein specificity of antibodies induced in cows persistently infected with noncytopathic bovine viral diarrhea virus after vaccination with cytopathic bovine viral diarrhea virus

Neutralizing and nonneutralizing antibodies to bovine viral diarrhea (BVD) virus were detected in 3 cows persistently infected with noncytopathic BVD virus after vaccination with modified-live cytopathic BVD virus. Neutralizing antibodies detected in serum samples from each persistently infected cow at 3 weeks after vaccination were highly specific for certain isolates of cytopathic BVD virus and reacted only with a viral protein with a molecular weight of 53,000. Neutralizing antibodies to 1 of 3 isolates of noncytopathic BVD virus were detected in a serum sample obtained at 12 weeks after vaccination from 1 of 3 persistently infected cows. Nonneutralizing antibodies were detected in all cows at 7 to 12 weeks after vaccination. The nonneutralizing antibodies were less specific for isolates of BVD virus and reacted with viral proteins with molecular weights of 115,000, 80,000, 53,000, and 47,000.     

表达H5亚型禽流感病毒HA蛋白的重组禽痘病毒的构建

为构建表达H5亚型禽流感病毒(AIV)A/Duck/Anhui/1/06(H5N1)(简称AH/06)HA蛋白的重组禽痘病毒,本研究利用感染-转染的方法通过转移载体pSY681-gfp-gpt将AIV株AH/06的HA基因同源重组到禽痘病毒(FPV)中,并利用gfp和gpt双重筛选标记在鸡胚成纤维细胞中经过多轮筛选,得到纯化的重组禽痘病毒(rFPV-gfp-gpt-AHHA)。通过PCR、序列测定和western blot的方法对rFPV-gfp-gpt-AHHA进行鉴定和抗原活性分析。结果表明AH/06的HA基因稳定的重组到rFPV-gfp-gpt-AHHA中,其表达的HA蛋白能够与AH/06的阳性血清反应,显示出了良好的抗原性。同时病毒的生长曲线也显示外源HA基因的插入并没有影响亲本病毒的复制。这些结果为进一步的免疫效力研究奠定了基础。     

兔抗犬细小病毒2a型多克隆抗体交叉保护研究

为探究犬细小病毒（canine parvovirus,CPV）血清型只有一种的条件下,常用疫苗CPV-2型和CPV-2a病毒免疫血清抗体对国内流行CPV-2a病毒的中和率,试验将藏獒源CPV-2a毒株（10⁵ TCID₅₀/100 μL）和CPV-2（10^4.5 TCID₅₀/100 μL）疫苗接于F81细胞增殖,PEG6000法浓缩制成免疫原,各免疫新西兰长白兔3只,间隔2周1次,共免疫4次。收集血清纯化制备多克隆抗体,通过血凝抑制试验（HI）和血清中和试验（SN）分别用2种抗体中和CPV-2、CPV-2a病毒,对两者保护率进行初步评价,并对本地感染CPV-2a的4只犬,每组2只进行治疗,每日跟踪白细胞消长规律。结果显示,CPV-2a多抗中和国内流行病毒CPV-2a的HI抗体、中和抗体水平都极显著高于CPV-2多抗（P<0.01）,而中和CPV-2病毒的HI抗体、中和抗体水平两者差异不显著（P>0.05）,CPV-2a多抗组治疗能较快恢复正常值,2组白细胞数有显著差异（P＜0.05）。结果表明,CPV-2a多抗与常用CPV-2型疫苗免疫抗体相比能高滴度的中和CPV-2a,更适用于中国犬细小病毒的防制,对研发新型生物制品有一定意义。     

Nucleic acid and structural proteins of infectious bursal disease virus isolates belonging to serotypes I and II

The nucleic acid and structural proteins of infectious bursal disease virus serotype I (six isolates) were compared with those of serotype II (two isolates). Five of the serotype I isolates originated from chickens, whereas both serotype II isolates were from turkeys. The growth curves of representative isolates of both serotypes were similar, but the latent period and virus yield were different. The seven isolates tested had two segments of double-stranded genomic RNA. The RNA migration patterns of viruses belonging to each serotype were similar, but differences were noticed between the two serotypes. There were differences in the molecular weights of viral proteins (VP) 3 and 4 from the two serotypes, and serotype II isolates lacked VP-2.     

An annotated historical account of canine parvovirus

A brief annotated history of canine parvovirus-type 2 (CPV-2) and its variants is summarized with emphasis on the most significant contributions of individuals involved in the initial recognition of CPV-2 and subsequent discoveries that have advanced our knowledge of the nature and evolution of this novel canine virus. Time has obscured the observations of many veterinary clinicians and researchers throughout the world who sensed the presence of a new disease when CPV-2 first made its appearance in 1978 and then, within 1-2 years, spread worldwide. Since 1979, nearly 600 articles, papers, numerous text chapters and monographs have been published on the subject of CPV-2. The early history is well known by veterinary infectious diseases specialists and noteworthy publications are recorded on the National Library of Medicine (USA) website, PubMed and in review articles. Because of the great number of publications, it is not practicable to cite them individually; however, reference is made to certain individuals, reviews and selected papers that I consider particularly relevant to the history of progress in the understanding of CPV-2 and the disease it causes. The clinical disease caused by CPV-2 and its variants, the immune response to infection or vaccines, host range and the development of practical diagnostic assays are noted in historical context. The basic biological properties and the physical, molecular and antigenic structure of CPV-2 and its variants are also discussed briefly. Finally, key players who have contributed to the antigenic and DNA sequence (evolutionary) relationships between CPV-2 and the other autonomous parvoviruses of carnivores are noted and hypotheses regarding the origin and evolution of CPV-2 and its variants are mentioned.