The challenge for the next generation of feline calicivirus vaccines

Feline calicivirus (FCV) has been shown to evolve within individual cats and in the environment of colonies. This evolution and the diversity it creates has important clinical implications, not only for the disease in cats, but also for attempts to control disease by vaccination. Generally speaking, existing vaccines appear to be very effective at controlling the majority of clinical disease. However, some concerns remain including a failure to induce sterilising immunity, occasional vaccine breakdowns, and for live vaccines, occasional vaccine-induced disease. Key areas for future vaccine development include monitoring and broadening the cross-reactivity of vaccine immunity to field viruses, especially the recently evolved highly virulent strains, and attempting to reduce/eliminate field virus shedding by vaccinated cats.     

Evaluation of a feline viral rhinotracheitis-feline calicivirus disease vaccine.

An attenuated respiratory disease vaccine against feline viral rhinotracheitis (FVR) and feline calicivirus (FCV) disease was evaluated for safety and efficacy in specific-pathogen-free cats. Twenty cats were vaccinated twice intramuscularly, with 28 days between vaccinations. Ten unvaccinated cats were used as contact controls. Adverse effects were not noticed after vaccination, and the vaccinal virus did not spread to contact controls. Arithmetical mean serum-neutralizing titers against vaccinal FCV strain F9 and challenge FCV strain 255 were 1:13 and 1:15 at 28 days after the 1st inoculation. These titers increased to 1:45 and 1:196 after the 2nd inoculation. After challenge exposure of vaccinated cats to virulent FCV 255 virus, mean titers increased to 1:129 and 1:865, respectively for F9 and 255 viruses. The F9 postchallenge mean titer for vaccinated cats was 21.5 times higher than that for the 8 contact controls that survived challenge exposure. The arithmetical mean serum neutralizing titer for FVR was low (1:2) after the 1st vaccination, but increased to 1:35 after the 2nd vaccination. Challenge exposure to virulent FVR virus resulted in a marked anamnestic immune response (mean titer of 1:207, compared with 1:12 for contact controls). In general, vaccinated cats remained alert and healthy after challenge exposure with FCV-255, whereas unvaccinated contact control cats developed definite signs of FCV disease, including central nervous system (CNS) depression (6 of 10) and dyspnea indicative of pneumonia (5 of 10). Two controls died of severe pneumonia. A mild fibrile response was detected in 28% of vaccinated cats, compared with a more severe febrile response in 78% of control cats. Some vaccinated cats developed minute lingual ulcers that did not appear to be detrimental to the health of the cat. After FVR challenge exposure, vaccinated cats were free of serious clinical signs. Five of 18 vaccinated cats had mild signs of FVR, including an occasional sneeze, low temperature, and mild serous lacrimation for 1 or 2 days. Contact controls developed definite clinical signs of FVR. The combined FVR-FCV vaccine appears to be safe and reasonably efficacious. Vaccination against FCV disease and FVR should be part of the routine feline immunization program.     

Molecular virology of feline calicivirus.

Caliciviridae are small, nonenveloped, positive-stranded RNA viruses. Much of our understanding of the molecular biology of the caliciviruses has come from the study of the naturally occurring animal caliciviruses. In particular, many studies have focused on the molecular virology of feline calicivirus (FCV), which reflects its importance as a natural pathogen of cats. FCVs demonstrate a remarkable capacity for high genetic, antigenic, and clinical diversity; "outbreak" vaccine resistant strains occur frequently. This article updates the reader on the current status of clinical behavior and pathogenesis of FCV.     

Immunization against feline calicivirus infection.

Forty-three cats (experiments 1 and 2) were vaccinated (2 doses, 27 and 30 days between doses) with the F-9 strain of feline calicivirus by the intramuscular route. There was no untoward response in any of the cats to the administration of the vaccinal virus nor was there spread of the virus from 20 vaccinated cats to nonvaccinated cats held in contact during the next 6 months (experiment 2). The vaccinated cats developed serum-neutralizing antibodies that were increased further after the 2nd vaccination. The level of serum-neutralizing antibodies was related to the quantity of vaccinal virus administered. Twenty-three cats vaccinated with the F-9 strain were protected to a significant degree when challenge exposed to virulent calicivirus strain FPV-255 (experiment 1).     

携带IBV DNA疫苗重组减毒沙门氏菌的构建及免疫原性研究

为研究携带IBV DNA疫苗重组减毒沙门氏菌免疫原性,本研究将携带有禽传染性支气管炎病毒(IBV)S1、M、N基因的pVAX1-S1、pVAX1-M和pVAX1-N重组质粒转入减毒沙门氏菌X4550株,构建IBV S1、M、N基因DNA质粒重组减毒沙门氏菌X4550疫苗株(X4550/pVAX1-S1,X4550/pVAX1-M,X4550/pVAX1-N),用间接免疫荧光法(IFA)检测重组质粒体外表达,SPF鸡经口服免疫,测定体内组织分布及稳定性、特异性IgG、IgA、CD4+和CD8+T细胞含量并进行攻毒实验。结果表明,重组质粒可在体外细胞中表达目的蛋白;重组减毒沙门氏菌X4550疫苗株可在肠、脾、肝、心、肾、肺6个组织中分布并稳定存在;特异性IgG,IgA、CD4+和CD8+T细胞显著升高(p0.01),用104EID50IBV M41株攻毒,保护率达到73%(11/15)。本研究为研制IBV基因减毒沙门氏菌疫苗提供了依据。     

Results of molecular diagnostic assays targeting feline herpesvirus-1 and feline calicivirus in adult cats administered modified live vaccines

In this pilot study, 12 adult, gang-housed cats that were known to be previously exposed (n=12) to feline herpesvirus-1 (FHV-1) and/or vaccinated against (n=2) feline calicivirus (FCV) and FHV-1 were randomly assigned to one of two groups of six cats each. Nasal and pharyngeal samples were collected from each cat on days -7, -3, and 0 prior to vaccination and on days 3, 7, 10, 14, 17, 21, and 28 after vaccination with an FHV-1, FCV, and panleukopenia (FVRCP) vaccine developed for intranasal (six cats) or parenteral (six cats) use. FHV-1 DNA was amplified from 1/12 cats (1/69 samples; 1.4%) prior to vaccination and 2/12 cats after vaccination (2/154 samples; 1.3%). FCV RNA was amplified from 2/12 cats (2/69 samples; 2.9%) prior to vaccination and 7/12 cats (12/154 samples; 7.8%) after vaccination. Positive molecular diagnostic assay results for FHV-1 and FCV were uncommon prior to or after vaccination in these cats.     

Update on feline calicivirus: new trends.

In addition to being important upper respiratory tract pathogens of cats, FCVs are increasingly reported as a cause of a highly contagious febrile hemorrhagic syndrome. Strains causing this syndrome are genetically different from the vaccine strain and other nonhemorrhagic FCV isolates. They apparently differ from one outbreak to another. The syndrome is characterized variably by fever; cutaneous edema and ulcerative dermatitis; upper respiratory tract signs; anorexia; occasionally icterus, vomiting, and diarrhea; and a mortality that approaches 50%. Adult cats tend to be more severely affected than kittens, and vaccination does not appear to have a significant protective effect. Rapid recognition of the disease through identification of clinical signs and appropriate testing, followed by strict institution of disinfection, isolation, and quarantine measures, are essential to prevent widespread mortality resulting from the infection.     

Carrier-state infection of feline T-lymphoblastoid cells with feline calicivirus

The susceptibility of feline T lymphocytes to feline calicivirus (FCV) in vitro was investigated using feline T-lymphoblastoid cell lines, namely MYA-1 and FL74 cells. The virus titers of supernatants in FCV-infected MYA-1 and FL74 cell cultures increased rapidly, and FCV antigens were also detected in the FCV-infected cells. There were slight differences in the molecular weights of capsid proteins expressed in FCV-infected MYA-1, FL74 and Crandell feline kidney cells. MYA-1 and FL74 cells were productively and persistently infected with FCV, and FCV antigens were observed in the FCV-infected cells for more than one month. At 3 months post infection, FCV-infected FL74 cells that stopped producing infectious FCV could be reinfected with FCV. However, no cytopathic effects were observed.     

The preparation of a polyvalent feline calicivirus antiserum.

A polyvalent antiserum capable of neutralizing 82 isolates of feline calicivirus made from cats in various parts of North America was produced by the sequential inoculation of SPF cats at three-week intervals with feline calicivirus strains F-9, 68-2024 and FS, followed by a final booster inoculation two weeks after the third inoculation with all three strains combined. Sera raised against the same strains but individually and then pooled failed to show such broad cross-neutralizing capacity. The polyvalent serum should prove useful for the confirmation of an isolation of feline calicivirus.     

Sites of persistence of feline calicivirus

Various tissues were collected from eight cats persistently infected with feline calicivirus (FCV) strain 255 to determine the sites of viral persistence. Tissues were tested by virus isolation and an immunohistochemical technique in which infected cells were detected in formalin-fixed, paraffin-embedded tissue sections using rabbit antiserum to FCV 255, a biotinylated second antibody and streptavidin-peroxidase. Virus was detected by one or both techniques in tonsillar tissues of each animal, and not in other samples. Infected cells were detected in samples from six of eight kittens, and in each animal were few in number, and were cells of the superficial tonsillar epithelium or the stratum germinativum of the adjacent fossa mucosa. Transmission electron microscopic examination of tissues from three of the cats revealed calicivirus-like particles in cells similar to those identified immunohistochemically. These results confirm that the tonsillar region is the major site of FCV persistence and indicate that virus replication during persistence is confined to the surface epithelium of the tonsil and adjacent fossa mucosa.     

猫杯状病毒不同分离株致病性的比较

为确定猫杯状病毒(Feline calicivirus,FCV)SH、JL-1、JL-2分离株毒力强弱,将其分别人工接种6~11周龄健康非免疫家猫,分成接种组和对照组。接种前后分别测定各组猫体温、体质量,观察发病情况及临床症状,按照欧洲药典对症状进行计分。于感染后14d麻醉处死,制备病料组织切片,观察组织器官的病理变化。结果显示,家猫感染FCV后总发病率为100%,死亡率为66.7%;SH、JL-1、JL-2组临床症状平均得分分别为8、4、9,对照组得分为1。病理解剖观察发现,猫感染FCV后,以鼻、眼分泌物增加,口腔溃疡为特征,消化道病变较轻微,肺部出现不同程度的变性、出血,呈明显病理性"肉样变";病理组织学观察发现,肺脏有数量不等的肺泡上皮细胞和巨噬细胞脱落,伴随少量纤维蛋白渗出和弥漫性肺泡损伤。结果表明,FCV SH、JL-1、JL-2分离株均有一定的致病性,其中以JL-2株的毒力最强。     

猫杯状病毒的分子生物学

猫杯状病毒（FCV）是猫的上呼吸道感染、口腔水疱性疾病及慢性胃炎等疾病的重要病原。该病毒具有典型的杯状病毒结构。其基因组为单股正链含polyA的RNA，全长约7．6kb，编码3个开放读码框（ORFs)。其中ORF1约5．3kb，编码1763个氨基酸（aa)的非结构蛋白；ORF2长约2．1kb，编码671aa的结构蛋白，ORF3位于基因组3‘末端，长320bp，编码106aa的蛋白。该病毒结构蛋白分子量为58－76kDa，分为6个区，各区具有特殊功能。非结构蛋白包括3C多肽，3C半胱氨酸蛋白酶和3DRNA依赖的RNA聚合酶样区等结构。FCV的基因工程疫苗研究已有一定的进并取得了相应的应用价值。FCV分子生物学的研究有助于研究该病毒的毒力和致病机理以及新型疫苗的研制。     

Detection of protective antibody titers against feline panleukopenia virus, feline herpesvirus-1, and feline calicivirus in shelter cats using a point-of-care ELISA

Serum antibody titers are a useful measurement of protection against infection (feline panleukopenia virus [FPV]) or clinical disease (feline herpesvirus-1 [FHV] and feline calicivirus [FCV]), and their determination has been recommended as part of disease outbreak management in animal shelters. The objective of this study was to determine the sensitivity, specificity, and inter-observer and inter-assay agreement of two semi-quantitative point-of-care assays for the detection of protective antibody titers (PAT) against FPV, FHV and FCV in shelter cats. Low sensitivity for FPV antibodies (28%) rendered a canine point-of-care assay inappropriate for use in cats. The feline point-of-care assay also had low sensitivity (49%) and low negative predictive value (74%) for FPV PAT detection, but was highly accurate in the assessment of FHV and FCV PAT. Improvements in accuracy and repeatability of FPV PAT determination could make this tool a valuable component of a disease outbreak response in animal shelters.     

Characterization of the subunit particles of feline calicivirus

In the culture fluid from cells infected with feline calicivirus (FCV) F4 strain, the infectious and smaller non-infectious subunit particles were detected by complement fixation (CF) test after sucrose gradient centrifugation. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses confirmed the existence of the subunit particles of FCV, and showed that the infectious and subunit particles were mainly composed of 65K capsid protein. The subunit particles were further purified by ion-exchange chromatography and sucrose gradient centrifugation. The purified subunit and infectious particles had the same neutralizing epitope on 65K protein detected by immunoblot analysis with a neutralizing monoclonal antibody. Antigenic comparison between the infectious and subunit particles by the CF tests using an antiserum against heterologous strain of FCV F14 indicated that the subunit particles might have more highly conserved antigens of FCV than the infectious particles.     

Antigenic change in feline calicivirus during persistent infection.

To determine if antigenic variation occurred during persistent infection of cats with feline caliciviruses (FCV), nine persistent (progeny) isolates from nine different carrier cats were compared antigenically to the original infecting parent strain, FCV 255, by two-way cross-neutralization tests with rabbit antisera. Five of the nine progeny viruses isolated 35 to 169 days after initial infection were antigenically different from the parent strain. These five isolates represented four distinct antigenic phenotypes. The emergence of four distinctly different antigenic variants from a single parent strain indicates that FCV, like many other RNA viruses, exhibits considerable antigenic heterogeneity during replication in its natural host, and supports the hypothesis that antigenic variation contributes to chronic FCV infection.     

猫杯状病毒分子致病机制的研究进展

猫杯状病毒(Feline calicivirus,FCV)是引起猫科动物的一种急性、高度接触性传染病的病原,严重危害猫科动物的健康。本文从FCV的主要蛋白及其功能、病毒的复制与细胞的相互作用、病毒受体、病毒致细胞凋亡作用等方面综述了FCV分子致病机制的研究进展,以期为FCV的分子生物学特性、遗传与变异规律、新型疫苗的研发及抗病毒药物的筛选提供参考。     

Studies of parvovirus vaccination in the dog: the performance of live attenuated feline parvovirus vaccines

The performance of three live attenuated feline parvovirus vaccines licensed for use in the dog was studied. At the end of the primary vaccination course 67 per cent of dogs had inadequate antibody levels (less than or equal to 32) as measured by a haemagglutination inhibition test. Interference by maternal antibody accounted for some of the failures but the fact that there was no significant difference in performance between dogs vaccinated at 12 weeks or 16 weeks of age indicated that maternal antibody was not the only factor.     

Preparation and characterization of neutralizing monoclonal antibodies to feline calicivirus.

Fourteen neutralizing monoclonal antibodies (N-MoAbs) were prepared against the F4 strain of feline calicivirus (FCV), the prototype strain of FCV in Japan, and examined for their ability to neutralize FCV isolates. Neutralization-resistant variants of the F4 strain were selected under the presence of 4 individual N-MoAbs in cell culture systems and used in cross-neutralization tests and enzyme-linked immunosorbent assay with all of the 14 N-MoAbs. The results revealed the identification of at least two antigenic determinants on FCV F4: one being more broadly conserved among FCV isolates than the other. Usefulness of antigenic variants resistant to N-MoAbs for analysis of neutralization determinants on FCV was also demonstrated.